Combo with "Genetics Ch 5 Genetic Linkage and Mapping in Eukaryotes" and 27 others

constitutive

A mutant with a gene that is always (inappropriately) turned on is a(n) ___ mutant.

Variance

A numerical measure of the spread of the distribution around the mean

Stabilizing selection

A pattern of natural or artificial selection that favors an intermediate phenotype over extreme phenotypes

Disruptive selection

A pattern of natural or artificial selection that occurs when both extreme phenotypes are favored over intermediate ones

Discontinuous variation

A phenotype distribution containing discrete or separable categories

Continuous variation

A phenotype distribution in which the phenotypic variation is distributed across a range of values in an uninterrupted continuum

Define plasmid

A plasmid is a self-replicating molecule of DNA that occurs in bacteria, separate from the main chromosome

Define 'Suicide Vector'

A plasmid that has a narrow host range replicon system

What is a genetic screen?

A procedure where a population is mutagenized and the progeny are examined for mutant phenotypes

(Lecture 16) Secretion Protein translocation

A protein can go to the cyto, inner membrane, periplasam, outer membrane, secreted into extracellular space. -Most proteins are directed outside of the cytoplasm by N-terminal signal sequence. (N is the start (first AA), C is the end). -Interact with the major transporter in inner membrane, which is the SEC system.

Functional domain

A protein region with a specific function or interaction

Activator

A protein that binds to DNA and stimulates transcription of a gene when cAMP binds to a regulatory protein, CAP assumes its active shap eand can attach to a speciic site on the upstream end of the lac promoter; this attachmetn increases the affinity of RNA polymerase for ht epromoter, amount of glucose rises as glucose rises, the cAMP falls, and with out cAMP CAP detaches from the operon and becomes inactive and RNA polymerase binds less efficiently to the promoter

Core promoter region

A region of DNA immediately adjacent to the start of transcription in eukaryotes where RNA polymerase II binds

operon

A region of DNA that codes for a series of functionally related genes under the control of the same promoter.

RNAi

A regulatory gene silencing mechanism based on double stranded RNA which can target complementary sequences for inactivation

RNA interference

A regulatory gene-silencing mechanism based on double stranded RNA which can target complementary sequences for inactivation

Physical map

A set of overlapping genomic clones assembled into contigs that, once assembled, cover the entire genome

Define 'Class I integrons'?

A single gene (int) that codes for a recombinate enzyme (integrase)

How transfer of DNA occurs during conjugation

A single strand must be nicked at the oriT site by an endonuclease. This endonuclease can be a RELAXASE, NICKASE or TRANSFERASE. This endonuclease (usually relaxase in this lecture) remains bound to the 5' end of the nicked strand through transesterification. The RELAXASE is also a helicase, and is docked to a type 4 protein secretion system. The RELAXASE threads the nicked single strand into a secretion pore for transfer into the recipient cell.

What does the phrase 'plasmids are replicons' mean?

A small number of genes on the replicon control when the DNA molecule replicate at the oriV site

Genetic chimera

A tissue or organism comprised of cells of two or more distinct genotypes

Quantitative trait

A trait exhibiting polygenic inheritance and displaying continuous phenotypic variation

Leader region (trpL)

A transcribed region upstream of the major enzyme encoding genes of a repressible operon that encodes a small peptide whose rate of translation reflects the concentration of the amino acid in the cell

Standard deviation

A value expressing the deviation from the mean in the same units as the scale of the measurement in the sample

Multiple cloning sites

A vector DNA sequence containing several unique restriction enzyme target sequences facilitating cloning of inserted DNA fragments

Shuttle vector

A vector that can replicate in two species due to the additions of another species's origin of replication and selectable markers into the vector

repressor; promoter

A(n) ___ is a protein that inhibits gene transcription. In prokaryotes, this protein binds to the DNA in or near the ___.

inducer

A(n) ___ is a specific small molecule that binds to a bacterial regulatory protein and changes its shape so that it cannot bind to an operator, thus switching an operon on.

operon; genes of the operon; promoter; operator; operator; regulatory gene

A(n) ___ is made up of a promoter and the ___. The ___, which includes a(n) ___, is the stretch of DNA where RNA polymerase binds. Regulatory proteins bind to the ___. The genes of the operon code for a related set of proteins. A(n) ___ located away from the operon codes for a protein that controls the operon.

polycistronic

A(n) ___ system is common in bacteria--the expression of many genes (i.e. lacZ and lacY) are turned up or down by a single regulatory factor (i.e. lacI).

QQ

A, purine to purine

QQ: what else would you get?

A: Penacillin resistant one -spores, persister cells: (cells that are alive but have stopped growing, "a dormant cell". can be reanimated.) Actually small fractions of bac. populations have persister cells, and a big problem in biofilms b/c cells aren't growing and not killed by antibiotics because full of dormant persister cells that can tolerate).

PROPS OF CELLS

ALL CELLS HAVE GENES, METABOLISM THAT TAKES UP NUTRIENTS, ALL GROW AT DIFFERENT RATES, EVOLVE, ACCUMLATE MUTATIONATION, SOMETIMES DIFFERENTIATE, COMMUNICATE AND ARE VERY ADAPTIVE. -bac have largest biomas in genome, single prokarackotes chromosome in NUCLEIOD** PATHOGENS: microorams that cause disease. inf diseases no longer major cause of death.

GyrB

ATP-ase

initiator codons

AUG

lots of RepA

According to the tests preformed, the overproduction of RepA from a gene on an unrelated plasmid has no effect on the copy number (replication of the plasmid), However, if you introduce iteron sequences (R1, R2, R3) to an unrelated plasmid, you lower the copy number of the plasmid, because you form the handcuffing between two plasmids.

Competent Factor

Accumulation of competent factors leads to the formation of a translocasome in the cell membrane, which facilitates uptake of DNA from the environment.

MreB/ParM

Actin homologues

SOS response (Bacterial)

Activated by DNA damage. Initiates transcription of xis protein for excision of the prophage from the bacterial genome.

Positive control of lac

Activated: by CAP after it binds cAMP (upstream of lacP)

A model for the action of enhancers and transcription activators

Activator proteins bind to distal control elements grouped as an enhancer in the DNA A DNA binding protein brings the bound activators closer to the promoter The activators bind to transcription factors and mediator proteins and help them form active transcription initiator complex on promoter

Both Eukaryotes and Prokaryotes use activators and repressors. But what is more common in eukaryotes?

Activators.

How to enhance curing of plasmids

Adding EtBr or proflavine to the medium

Euchromatin

Addition of acetyl groups to histones is typically associated with ______ (euchromatin or heterochromatin)?

DNA Methylated

Addition of methyl groups to certain bases in DNA after DNA is synthesized. In fact, the DNA of most plants and animals has methylated bases, usually cytosine. Inactive DNA, such as that of inactivated mammalian X chromosomes, is generally highly methylated compared with DNA that is actively transcribed

Heterochromatin

Addition of methyl groups to histones is typically associated with _______ (euchromatin or heterochromatin)?

Insertion

Addition of one or more bases.

What does Histone Acetylation do?

Adds acytal group to the H4 protein of the histone tail which weakens the interaction with DNA (DNA becomes looser around the nucleosome). This destabilizes the nucleosome (chromatin) structure. Which allows for transcription to take place.

Purine

Adenine, Gaunine Two rings

produce a complimentary strand to the target molecule and detect which bases are addded at each step. Millions of sequencing reactions can be run simultaneously, (high throughput) the DNA to be sequenced does not need to be known prior to sequencing. can sequence samples found in trace amounts, can sequence DNA that is difficult to clone

Advantages of next generation/third generation sequencing (4):

DNA doesn't need to undergo cloning or PCR, it is faster, it is cheaper

Advantages of third generation sequencing over next generation sequencing (3):

Primer walking or shotgun sequencing

After cloning a DNA fragment, one of these two methods can be used to sequence the DNA fragment to know what the nucleotide bases are:

What does 'curing agents' mean?

Agents that can increase the natural frequency of segregational loss

Why is Agrobacterium tumefaciens a different T4SS?

Agrobacterium tumefaciens delivers DNA into plant cells, not proteins

Animal axes system

All determined by cytoplasmic determinant shape of human established as soon as it is fertilized

What does the term 'compatible' mean in reference to unrelated plasmids?

All replicate cells would carry the same plasmids

Response elements to a particular stimulus can be associated with multiple genes. This allows what?

Allows a single stimulus to activate multiple genes.

What is the reason Prokaryotes have gene regulation?

Allows them to respond to their environment efficiently and economically.

A single gene may have multiple response elements. This allows what?

Allows this gene to be activated by many different stimuli.

What is 'signature tagged mutagenesis'?

Allows you to do a mutagenesis experiment where the product is mutants that have lost the capacity to cause disease (virulence mutants)

Catabolite repressor protein

Also called CAP (crp)

environmental

Altering patterns of gene expression in prokaryotes would most likely serve an organism's survival by allowing an organism to adjust to changes in ___ conditions.

Ionsine

Alternate purine, often found in the anticodon position of tRNA's. (the 5' base anticodon.) -Can base pair with A, C or U -Is a modified base, not put in by trx but one of those rnas that change as trx happens.

three sites in translation

Aminoacyl site Peptidyl site Exit site

What attaches the amino acid to tRNA?

Aminoacyl-tRNA transferases

Headfull

Amount of DNA packaged into virus head. Sequence for each DNA begins and ends at different points in different virions.

Whole genome shotgun sequencing

An approach to genome sequencing where the DNA of the entire genome is fragmented into smaller pieces and a large number of fragments are chosen at random and sequenced with the aim that all genomic regions will be sequenced multiple times

Clone-by-clone sequencing

An approach to genome sequencing where the chromosome is first broken into overlapping clones that are then arranged in linear order to produce a physical map of the genome. Each clone in the map is then sequenced separately

Nucleotide Excision Repair

An endonuclease removes a patch of single-stranded DNA containg certain types of damaged bases, including dimers.

end problem 2) TERMINAL INVERTED REPEATS (TIR) ex. barrelia, which has a linear genome

An enzyme forms a phosphodiester bonds to link TIRS on ends of linear chromosome. There are now 2 chromosomes, however enzyme RESOLVASE separates and resolves the ends to separate them. 5' GGAATT [] AATTCC- 3' 3' CCTTAA [] TTAAGG 3' -A inverted repeat *5'GGAATT* / AATTCC 3' 3'CCTTAA / *TTAAGG 5'* -5' phosphate, 3' oh, and barrelia has an enzyme that interacts with TIRS and forms an phosodiester bond to link the 2 strands together, and forms a CIRCLE, single stranded DNA circle. -GO FROM 1 single linear chromosome links together, separate it, replicate it, resolve it back to the original and now you have 2 copies of these. -now replication can proceed normal with primers all the way around, and now you have a dbs big circle of TWO CHROMOSOMES, however both in one big circle so need to separate. Thus, you have enzyme RESOLVASE and interacts with ends to resolve them and back to original chromosomes without the 2 ends linked together,

Node

An evolutionary branch point in a phylogenetic tree

trp operon

An example of a repressible operon

lac operon

An example of an inducible operon

Heritability

An important measure of the potential responsiveness of a trait to natural selection or artificial selection

Polycistronic mRNA

An mRNA molecule in bacteria that is a transcript of all of the genes in an operon

Catabolite Repression

An operon enabling the catabolism of one nutrient is repressed by the presence of a more favorable nutrient (commonly glucose).

promoter

An operon is a region of DNA that codes for a series of functionally related genes under the control of the same ___.

FtsA

Anchors Ftsz to membrane Mutation= no proper ring can form

Initiation of translation

Another opportunity for regulating gene expression; most commonly at hte initiation stage; initiation of translation of some mRNA can be blocked by regulatory proteins that bind to specific sequences or structures within the ultranslated region at the 5' cap and the poly-A tail of an mRNA molecules FINISH REVIEWING

RNA processing is another way to what?

Another way to regulate genes in eukaryotes.

RNA Interference or Silencing is another way to what? And what does it do?

Another way to regulate genes in eukaryotes. This is a precise mechanism used by eukaryotic cells to limit the invasion of foreign genes (viruses or transposons) and to censor the expression of their own genes.

RNA stability is another way to what?

Another way to regulate genes. The more stable a mRNA is, the longer it will be available to the protein -synthetic machinery.

What are some resistance phenotypes plasmids can have?

Antibiotic, ion, UV, phage-resistance, bacteriocin-resistance

Common Riboswitch features

Aptamer (Curvy thing binds ligand) Anti terminator (between pokey), expression platform, terminator

activator; repressor

AraC protein is a(n) ___ when bound to arabinose and a(n) ___ when arabinose is absent.

What does the phrase 'conjugative plasmid' mean?

Are able to exit one cell and enter another through the process of conjugation

Experimental

Are experimental or computational approaches to annotate a genome more successful?

Negative control, because they make DNA less accessible for transcription

Are histone deacetylases (HDACs) associated with negative or positive control of gene expression? Why?

Spontaneous Mutations

Arise in low rate in any cells in the absence of any added agent. Result from errors in DNA replication.

What are 'Multipurpose plasmid vectors'?

Artificially derived plasmids

HOW THE par system works

As one ParA-ATP binds to the genome, group binding of other ParA-ATP is initiated. ParA-ATP grows at both ends. ParB forms a comples on the parS site of the plasmid. The ParB-parS complex interacts with ParA-ATP and dephosphorylates it into ParA-ADP. As the ParA-ATP's turn to ParA-ADP's, they unbind from the DNA. As this continues over and over, the Plasmid is driven down the DNA. The ParA-ADP that comes off, ultimately becomes ParA-ATP. It binds elsewhere in the nucleoid, repeating the process.

how parA/parB/parS works

As one ParA-ATP binds to the genome, group binding of other ParA-ATP is initiated. ParA-ATP grows at both ends. ParB forms a comples on the parS site of the plasmid. The ParB-parS complex interacts with ParA-ATP and dephosphorylates it into ParA-ADP. As the ParA-ATP's turn to ParA-ADP's, they unbind from the DNA. As this continues over and over, the Plasmid is driven down the DNA. The ParA-ADP that comes off, ultimately becomes ParA-ATP. It binds elsewhere in the nucleoid, repeating the process.

Where do sigma factors bind?

At the -10 Pribnow Box and the -35 region of genes (promoters).

Auxotrophs: (2 types)

Auxotrophs(as opposed to prototrophs) -mutants that have some deficiency. 1) Anabolic auxotrophs ( synthesis pathways, taking nutrients to make building blocks), ex. amino acids, vitamins, cofactors, nucleotides. -Auxotrophic mutant cannot grown unless product of anabolic pathway is added. (cells deficient in making these metabolites required) -ex. Arg- (phenotype) cells cannot grown unless you add arginine. _____________________________________________________ 2)Catabolic pathways (Degradation pathways, ex. lactose, arabinose (sugars), nitrogen compounds to utilize the energy in their anabolic pathways) -Auxotroph in catabolic pathways means cells cannot utilize whatever that substance is, solely for their growth. (cell can't utilize a particular resource.) -ex. lac- mutant, cells cannot grow if lactose is the sole carbon source. (minimal media plus lactose, cells would not be able to grow).

QQ: Why is it generally easier to isolate temperature sensitive mutants rather than cold sensitive? types of proteins in cold sensitive?

B, heat destabilizes proteins. bonus, the types of proteins in cold sensitive mutants is ribosome proteins :)

Q1 What might be the effect of a mutation that increases DnaA expression?

B, premature initiation of replication. More dnaA, more will be bound to OriC thus premature initiation of replication.

"Safe sex" approach to gene exchange

Bacteria have protection system (restriction & modification). 1. Restriction endonucleases degrade foreign DNA. 2. Protective methylation (modification) of host DNA using matching methylation enzymes. Foreign DNA without DNA methylation is destroyed (unless comes from similar species and is also methylated for protection).

Lac+ phenotype

Bacteria that are able to grow on a medium containing lactose as the only sugar

Lac- phenotype

Bacteria that are unable to grow on a medium containing lactose as the only sugar

Natural selection of bacteria

Bacterial cells that conserve resources and energy have a selective advantage over cells that are unable to do so natural selection has favored bacteria that express only the genes whose products are needed by the cell Ex. E coli Such feedback inhibition, typical of anabolic pathways, allows a cell to adapt to short-term fluctuations in supply of a substance it needs (if eating the necessary amino acid, won't turn on the pathway to make that pathway- E. Coli thrive off tryptophan; but if not eating it, will turn pathway on)

What is the significance of T4SS?

Bacterial evolution & adaptation (via HGT systems) Bacterial pathogenesis

Hershey/Chase

Bacteriophage T4 -Label protein and DNA with isotopes -Infect with phage, blend to remove ghosts -Conclud that DNA is transmitted and not protein

Lytic Cycle

Bacteriophage quickly replicates, killing host cell.

Lysogen

Bacterium harboring prophage.

Chargraff

Base pairing rules

Why are fruit fly maternal effect genes also called egg-polarity genes?

Because their products, made and deposited into the egg by the mother, determine the head and tail ends as well as the back and belly of the embryo

Allolactose

Besides glucose and galactose, the breakdown of lactose also produces a small amount of:

To stop the spread of heterochromatin to keep the expression of critical genes from being blocked

Besides making sure enhancers act with the right promoter, what is a second function of insulator sequences?

Gene regulation in prokaryotes

Better understood & simpler than eukaryotic gene regulation •In prokaryotes, genes coding for proteins in a pathway are grouped together & controlled by the same regulation - called operons Main source of energy: ATP; (starting from glucose); First choice for bacteria is glucose (uses glycolysis and oxidative phosphorylation to make ATP)

DNA replication

Bidirectional Leading continuous strand and lagging discontinuous strand Ends with collision of complexes sometimes ter sites present

rpoC

Bind DNA template

Repressors

Bind to regulatory sequences in the DNA and prevent transcription of target genes. Some must first bind a small ligand.

Activators

Bind to regulatory sequences in the DNA and stimulate transcription of target genes. Most must first bind a small ligand.

Streptomycin

Binds to 16s rRNA of the 30s Subunit and interferes with binding of methionine tRNA

RNA 1

Binds to RNA 2 and interacts with Rop and forms a kissing complex, preventing the initiation of replication of the plasmid.

SYBR green

Binds to all DNA

Ciproflaxin

Binds/Inhibits DNA gyrase (Binds to gyrA)

single stranded binding protein

Bonds with ssDNA that has been opened by dnaB helicase and prevents it from reforming a double helix.

Cell type-specific transcription factors

Both liver cells and lens cells have the genes for making the proteins albumin and crystallin, but only liver cells make albumin (a blood protein) and only lens cells make crystallin (the main component of the lens of the eye). The specific transcription factors (activators and repressors) made in a particular type of cell determine which genes are expressed. Collection of their activator and repressor proteins Eye cells do not let albumin gene to express The differences between cell types, therefore, are due not to different genes being present, but to differential gene expression, the expression of different genes by cells with the same genome

Compare and contrast miRNAs and siRNAs

Both: are small, single-stranded RNAs that associate with a complex of proteins adn then can base-pair with mRNAs that have a complementary sequence; this pairing leads to degradation or an end of translation; processed from double-stranded RNa precrusors by the enzyme Dicer miRNAS are encoded by genes int eh cell's genome siRNAs arise form longer stretch of double stranded RNA

Vg/Vp

Broad sense heritability H2 =

In gene regulation for eukaryots each cell type is differentiated by?

By Activating a different subset of genes.

Okazaki fragments connected

By lig

dna pol III b, genes dnaX and holA/B/C/D

CLAMP loading

Yes

Can blunt end DNA molecules be recombined?

Repressor

Can switch off the operon (protein) blocks attachment of RNA polymerase to the promoter , preventing transcription of its genes; specific for the operator of a particular operon regulatory TrpR that is far away that makes a repressor (makes mRNA); made in an inactive form; if you eat trpR, it binds to the inactive repressor and and activates it which it then attaches to the operator and shuts it down)

100-200 kb

Capacity of a BAC

250-2000 kb

Capacity of a YAC

Under 15 kb

Capacity of a plasmid for insert sequences:

Under 23 kb

Capacity of linear phage chromosomes for insert sequences:

MTase

Carries SAM to A or C in the restriction site and protecting dna from cleavage

Composite Transposon

Carry functional genes between two different IRs.

Inducible system

Catabolic pathway; production of a specific enzyme in response to the presence of substrate; If lactose is present, the pathway turns on Usually off but cam be turned on

Co-repressor

Causes binding of aporepressor to DNA

Inducer

Causes release of aporepressor from DNA

Tumor-suppressor genes

Cells contain genes whose normal products inhibit cell division because the proteins they encode help prevent uncontrolled cell growth.

Cured cell

Cells that do not have a single copy of the plasmid.

As you learned in chapter 12, mitosis gives rise to two daughter cells that are genetically identical to the parent cell. Yet you, the product of many mitotic divisions are not composed of identical cells, why?

Cells undergo differentiation during embryonic development, becoming different from each other; in the adult organism, there are many highly specialized cell types

Francis Crick

Central Dogma, that Dna encoded info into RNA (Carrier molecule) to proteins (carries out function) -rna is reverse transcribed to dna, look at pic!

Point Mutation

Change in a single base. 1. Transition: Purine --> purine 2. Transversion: Purine --> pyrimidine

Frame-Shift Mutation

Changes the ORF of the gene.

Nonsense Mutation

Changes the amino acid sequence to a stop codon.

Missense Mutation

Changes the amino acid sequence to another.

Chaperone vs protoseomes

Chaperone=fold proteins Protoseomes=destroy proteins

Histone modification

Chemical modifications to histones play a direct role in the regulation of gene transcription. The N-terminus of each histone molecule in a nucleosome protrudes outward from the nucleosome. These histone tails are accessible to various modifying enzymes, which catalyze the addition or removal of specific chemical groups. In histone acetylation, acetyl groups (—COCH3) are attached to positively charged lysines in histone tails Deacetylation is the removal of acetyl groups. When the histone tails of a nucleosome are acetylated, their positive charges are neutralized and they no longer bind to neighboring nucleosomes Histone acetylation enzymes may promote the initiation of transcription not only by modifying chromatin structure, but also by binding to, and "recruiting," components of the transcription machinery. Acetylation enzymes may promote the initiation of transcription via binding and recruiting components of transcription.

Closed chromatin

Chromatin in which regulatory DNA is covered by nucleosomes, thus restricting access of regulatory proteins

Open chromatin

Chromatin in which the association of DNA with nucleosomes is relaxed in regions containing regulatory sequences, allowing access by regulatory proteins

Define 'gene cassette'

Circular DNA encoding a single, promoterless gene and an attc sequence

Insulator sequences

Cis-acting sequences that act to prevent cross-talk between regulatory elements of an adjacent gene

What does 'gene capture and expression system' and a 'gene release system' relate to?

Class I integrons

What are the 3 forms of conjugation?

Classical conjugation, mobilisation, and conduction

Bacteria artificial chromosome

Cloning vector used in bacteria that utilizes the F plasmid origin of replication; can accept DNA inserts up to 500 kb

Yeast artificial chromosome

Cloning vector used in yeast that utilizes an endogenous yeast origin of replication, centromere and telomere; can accept DNA inserts in excess of 1 megabase

Expression vectors

Cloning vectors possessing DNA sequences required for DNA fragments inserted into the vector to be transcribed and translated

Operons

Clusters of genes undergoing coordinated transcriptional regulation by a shared regulatory region are called:

What are 2 examples of non-conjugative plasmids?

ColE1 and RSF1010

What are 2 high copy number plasmids?

ColE1 and RSF1010

DNA Microarray

Collection of microscopic DNA spots attached to a solid surface. Measure expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome.

Knockout libraries

Collections of mutants in which most or all of the genes of a particular organism have been mutated by inactivating their expression

Interspecific comparison

Comparison of genomes between species

Intraspecific comparison

Comparison of genomes within a species

Cos sites

Complementary single stranded stretches found in temperate λ phage. Aid in circularizing DNA inside host and recognizing where genome is cut and packaged during replication.

cAMP-CRP

Complex that binds to the promoter and interacts with RNA polymerase to increase the rate of transcription initiation.

Noninducible

Condition in which transcription of bacterial genes or operons cannot be activated

What is 'conduction'?

Conduction is Tn-mediated mobilisation of a non-conjugative plasmid

Messleson and Stahl

Confim semiconservative replication 1. Ecoli grown with heavy N-15 2. Centrifugation after placing back into N14 and replicating 3. DNA before div = bands at bottom One rep = middle Two rep = Higher

Define 'Mobilisable plasmid vectors'

Conjugation can be carried out using a plasmid known as a mobilisable plasmid vector

What is an example of T4SS?

Conjugation, where DNA is transferred

Why were no bacteria recovered when mouse lives

Connections to antibody formation Knew that the result was transformation because the new cells have the SAME polysaccharide. If they were mutated IIR then they would have their own polysaccharide

griff. Why were no bacteria recovered when mouse lives

Connections to antibody formation Knew that the result was transformation because the new cells have the SAME polysaccharide. If they were mutated IIR then they would have their own polysaccharide

We now know that at least some of it is functional in terms of containing regulatory, centromeric and telomeric sequences

Conserved noncoding sequences are no longer called junk DNA because:

Delete N terminal arm

Constituative

Eukarytoic gene expression can be regulated at any stage!

Control of gene expression is in response to internal or external environments And to create specialized cells All cells have the same genome Differences in cells with the same genome are the result of differential gene expression (amount of functional protein that is made) A typical human cell expresses about 20% of its genes at a given time; highly differentiated cells, such as muscle or nerve cells express an even smaller fraction of their genes; the subset of genes expressed in cells of each type is unique, allowing these cells to cary out their spedific function

Riboswitching

Control of transcription of new RNA or translation of a full length RNA by switching RNA conformation

Coordination does?

Controls the initiation of rounds of replication

Holoenzyme

Core enzyme + sigma factor.

Proofreading

Correction of mismatch by DNA polymerase III during DNA replication.

Linear chromosome end problem, solutions

Covalently closed telomere Invertron reverse transcriptase circularized cohesive ends

Induced Mutations

Created by treating the organisms with added mutagens - mutagenesis.

crossing over occurs at a ____ rate between genes that are further apart [lower/higher]

Crossing over occurs at a higher rate between genes that are farther apart, and a lower rate between genes that are closer together -higher rate = higher frequency

How a cell knows where to go to do its job?

Cytoplasmic determinants and inductive signals both contribute to the development of a spatial organization (arrangement in space) in which the tissues and organs are all in their characteristic places This process called pattern formation Cytoplasmic determinants in egg are encoded by sets of genes called maternal effect genes Because these genes control orientation (polarity) of the egg and the organism, they are also called egg-polarity genes (establish anterior-posterior, dorsal-ventral and right-left axis in embryo)

Pyrimidine

Cytosine, Thymine, Uracil One ring

QQ: Why is it important that inner membrane proteins undergo cotranslational translocation rather than post-translational translocation? (why have these 2 systems?)

D, if hydrophobic inner membrane proteins being made outside of membrane, the whole thing would aggregate. (A: the hydrophobic regions of the inner membrane proteins would form aggregates if fully translated.) this why there is a special srp/cotranslation secretion to take care of hydrophobic residues.

ParB

DNA binding protein

Southern blot

DNA cut up with REase, gel, trans to membrane, complementary to probe is visible on the blot

What induces a lysogen to enter the lytic cycle?

DNA damage to the host cell.

DNA Probe

DNA fragment that is labeled with a radioactive or nonradioactive tag.

Cyclic di-AMP does waht

DNA integrity scanning protein. scans. Inhibit sporulation. bound cdi-AMP

Inversion

DNA is flipped in orientation.

Reversion

DNA mutates back to original sequence.

CpG Islands. Where are they found?

DNA regions with many CpG sequences. Found near transcription start sites.

Activator binding site

DNA sequence to which an activator protein binds to regulate gene expression in bacteria

trans

DNA sequences that affect other sequences at a relatively distant site or sites on a different chromosome

cis

DNA sequences that affect other sequences right next to them on the same chromosome

Insulator

DNA sequences that blocks or insulate the effect of enhancers in a position-dependent manner.

(topA) Topo 1

DNA supercoiling (packaging)

Transduction

DNA tranasfer mediated by a bacteriophage

Franklin and Wilkins

DNA xray diffraction revealing its shape

Franklin and Wilkins

DNA xray diffraction revealing its shape. phtograph

DnaA reactivation

Dars (dna reactivating sequence) makes ADP dissociate from DnaA which promotes binding of it

regulation by titration of DnaA

DatA binds DnaA and all Dna is taken up on those so there is none left to bind at oriC

What are fast stop mutants?

Defective in DNA synthesis such as DNA Pol III

What are slow stop mutants?*

Defective in Replication initiation such as DnaA

Restriction Endonucleases

Degrades foreign DNA.

DnaC

Delivers the DnaB to the replication complex

DnaC

Delivers the DnaB to the replication complex -clamp loader

dnaC

Delivers the dnaB (helicase) to replication complex.

Difference between ribose and deoxyribose

Deoxy loses O on 2'

Difference between ribose and deoxyribose

Deoxy loses O on 2' RNA HAS A OH ON 2' CARBOND!!!!

How does E Coli sense that the glucose concentration is low? and Relay it to the genome?

Depends on the interaction of an allosetic regulatory protein with a small organic moleucle (cyclic AMP=cAMP), which accumulates when glucose is scarce; the regulatory protein is called catabolite activator protein (CAP) is an activator

Shuttle vector

Designed to work in more than one species

Interrupted Mating Experiment

Determine frequency of gene transfer. 1. Hfr + F- cells mate. 2. Conjugation bridge is fragile, transfer frequencies of genes more distant from oriT are lower. 3. Interrupt mating at different intervals. 4. Transfer frequencies of genes in recipients is determined.

Where do the CpG islands sit?

Diagonally across from each other on opposing strands.

What generates the first differences among cells in an early embryo? How do different sets of activators come to be present in two cells?

Differences among cells arise from the egg's cytoplasm 1. Cytoplasmic determinants (maternal substances, regulate gene expression which determine its developmental fat) 2. Inductive signals and interaction between embryonic cells via cell surface molecules (cause changes in target cells, induction)

DnaB

Dna Helicase, hexamer

How can mutations be fixed? Replication fidelity 1) Proofreading

Dna pol 3 is very specific and incorrect base pauses the complex. - DnaQ will remove incorrect bases ONLYY on the 3' end. -DnaQ (3'-->5' exonuclease) pol III can only remove if its on the 3 END

Okazaki fragments synthesized by

DnaPol III (new one for each fragment)

Lagging strand initiated by

DnaPol III and RNA primer (3-OH end)

Leading strand is initiated by

DnaPol III with RNA primer (3-OH end)

Mismatches removal

DnaQ (3'-->5' exonuclease) pol III can only remove if its on the END

YAC's

Do BAC's or YAC's carry a larger insert size?

Silent Mutation

Does not change the amino acid sequence.

What does the phrase 'non-conjugative plasmid' mean?

Don't have the large number of genes required for the process of conjugation

Guanine

Double bonded O

Thymine

Double bonded oxygen and METHYL

MicroRNAs REVIEW pg. 365

During the past few years, another mechanism that blocks expression of specific mRNA molecules has come to light; researchers have found small-single stranded RNA molecules called microRNA's that can bind to complementary sequences in mRNA molecules The miRNAs are formed from longer RNA precursors that fold back on themselves, forming a long, double-stranded hairpin structure held together by hydrogen bonds. An enzyme called Dicer cuts the double-stranded RNA molecule into short fragments. One of the two strands is degraded, and the other strand (miRNA) associates with a large protein complex and acts as a homing device, directing the complex to any mRNA molecules that have the complementary sequence. The miRNA-protein complex then either degrades the target mRNA or blocks its translation.

Coordination of replication with Cell division How is that it takes E. coli 40 minutes to replicate DNA and 20 minutes to divide (no matter the media).... that equals 60 minutes, not 30 minutes when grown in LB!

E. coli doubling time in minimal media w/ succinate ~140 minutes (not good carbon source) E. coli doubling time in glucose minimal media ~70 minutes E. coli doubling time in rich Luria Bertani (LB) ~30 minutes -BUT it takes E coli 20 min to divide, and 40 minuets to replicate its dna.. 60 no equal 30 minuets. -OVERLAP occurs, new replication starts half way through first replication so doubling time is 30 minuets when whole process takes 60. -(happens in both cells)

galactoside permease

E. coli makes specific transport proteins called ___ in order to move lactose into the cell when glucose is low/absent.

cDNA

EXPERIMENTAL approaches to annotate a genome makes use of:

Additive trait

Each allele of additive genes can be assigned a quantitative value that indicates its contribution to a polygenic trait, and this quantitative value is known as:

Stages in gene expression of eurkaryotes

Each stage is a potential control point at which gene expression can be turned on or off or accelerated or slowed down

During which phase does competence in bacteria peak?

Early log phase.

Development from Egg to Larva

Egg develops in ovary Eggs are surrounded by nurse cells and follicle cells These cells supply the egg with nutrients, mRNA, proteins to make egg shell After the egg is fertilized, embryonic development results in the formation of a segmented larva until it develops into adult fruit fly

Plaques

Empty spaces amidst a bacterial lawn that indicate where E.coli were killed by phages. (Primarily when culturing bacteriophages)

Reporter Gene

Encodes an easily assayed protein. Used for regulation of a gene by fusion.

lacY gene

Encodes lactose permease. Allows lactose to pass through cell membrane.

lacZ gene

Encodes β- galactosidase.

Cis

Enhancer sequences are (cis or trans) regulatory sequences

Restriction enzymes

Enzymes cut dsDNA at specific neculeotide sequences (restriction site)

Genomic imprinting

Epigenetic phenomena that create differential expression of alleles depending on whether they were maternally or paternally inherited

2(n) +1

Equation for calculating the number of distinct phenotypic categories for a polygenic trait produced by segregation of additive alleles of a given number of genes (n)

DNA Mutases

Error prone DNA polymerases - repair DNA rapidly, but generate numerous mutations.

Essential Genes:

Essential genes are gene products required for growth. (replication, translation, trx, cell division, cell wall, central metabolism ect.) -all essential, can't knockout the function of any of these genes and have a surviving bac colony.

Genes with estrogen response element will have enhanced transcription in the presence of what>?

Estrogen.

Phylogenomics or comparative genomics

Evolutionary genomics is also known as: (2 things)

RNA interference may have evolved as what?

Evolved as an "immune system" for the genome.

Protein Processing

"After translation, proteins can be modified." -formyl group and N-methione can be removed. (often happens to proteins where very first AA is clipped off). -Signal Sequence can be cleaved (internal part of the protein that tells it where to go). -Modifications such as phosphorylation, acetylation, lipids added, lipoproteins, glycoproteins/sugars added. (methylation is in DNA YO, not proteins.) -DnaA-ADP back and forth to DnaA-ATP is an example of protein modification that impacts activity. -post-translational modification of a protein

(C)Chargraff

(1950) -knew dna consisted of d nucleotide monophospahtes. -UTILIZED CHEMICAL MEASUREMENT OF DIFFEREND DEOXYMUNOCLEOTIDE (dNMP). he chemicaally fragment, and then just quantified bases. 2 rules, dbs 1)A=T if found 15% A, in given piece of dna thus 15% T then 2_G=C, 35%G thus 35% C (ADDS UP TO 100%) 3)organims have different ratios of dna. futher evidence dna was info molcule Base pairing rules ANSWER, C. IT WOULD BE SINGLE STRANDED RNA PHAGE, WITH UTILIZE MUCH DIFFERENT THINGS FO RTHEIR GENOME. AS LONG AS DNA DOUBLE STRANDED, CHARGAFFS RULES APPLY. rna no uracil

Screens vs. selections (screen)

(2 ways of isolating mutants) Screen: every mutant grows and must be examined invidually. -if you have 4000 transposon mutants and looking for some phenotype, gotta screen all 4000. (ex. was before, ex. was not that bad because it was an obvious color phenotype. however tricky if ex, biochemical mech.)

QQ: which one of these is dnaB

(A) helicase, little ring guy preceding replication complex. d is the beta clamp (dnaN) b is the primase (dnaE) e is the clamp loader (Actually like 10 protiens so no given name) more advanced, there's a mutation is one of these WHAT IS THE EFFECT! (EXAM!!!)

Repressible system

(anabolic pathway); ceasing production of a specific enzyme, in response to the presence of a specific substrate (metabolite) in the environment If tryptophan is present (if it is bound allosterically to a regulatory protein) (in E. Coli) pathway is turned off It is always on, but can be inhibited

GreaB

(dentry channel blocked) nucleases that insert into the entry channel to degrade backracked RNA (activ

QQ: Calculate mutation rate

(empirically: by experimentation/observation) mutation rate= (m2-m1)/ (n2-n1) m=mutants @ time 1 or 2 n= cells at time 1 or 2

QQ mRNA is typically much less stable in bacteria compared with rRNA and tRNAs. Why? (Click in for credit)

-

(Lesson 6) Hemimethalated dna

-(DnaQ, dam and MDM GATC?) -(what is it?) When A's in GATC/CTAG sequence are methylated by SeqA -preferentially fix the new strand. 1)removes the mismatch from the new stand 2) DNA pol 3 fills in THUS, pol 3 located in replication fork, POL 3 actually fixes errors in Hemimethalated DNA therefore CLOSE TO THE REPLICATION COMPLEX. (continued) foreshadows that replication is not always smooth, and needs to be restarted.) -process of replication restart requires HOMOLOGOUS RECOMBINATION. -> main function for replication restart to get replication going. -two sequences of same sequence can be interchanged, a way to acquire dna however may function is replication restart..

(Lesson 6) Hemimethalated dna

-(DnaQ, dam and MDM GATC?) -(what is it?) When A's in GATC/CTAG sequence are methylated by SeqA -preferentially fix the new strand. 1)removes the mismatch from the new stand 2) DNA pol 3 fills in THUS, pol 3 located in replication fork, POL 3 actually fixes errors in Hemimethalated DNA therefore CLOSE TO THE REPLICATION COMPLEX. (continued) foreshadows that replication is not always smooth, and needs to be restarted.) -replication restart requires HOMOLOGOUS RECOMBINATION. -> main function for replication restart to get replication going.

spefific proteins

-(also dna/rna are neg, charged)

IS3

-(number is italicized for transposon) -1200-1500 bp's -IR (20-40 bps), then promoter, and 2 open reading frames ORFA and ORFB, IR. -20-40 bp, -GENES in same orientation, 3 RF's

how does a ribosome know not to encode UAG or UGA as a stop codon?

--structural elements in mRNA --structural elements in growing peptide chain

Pribnow Box

-10 region of gene.

Gram negative bacteria (outer surface main difference) periplasam:

-2 membranes, 1 is phospolipolayer and 1 is LPS. periplasam: has the cell wall with peptidoglycan with beta barrel proteins. -(we mostly do transformation?)

Sigma 70 structure

-35 region, -10 region, start site, transcription region

General Model

-All transposons have characteristic inverted repeats at ends of transposon, that all encode a transposase. -transposase function: to recognize the inverted repeats and cut them; either both strands (copy paste) or one strand (replicative)

parental ditypes (PD)

-Assuming no crossover between the homologs, ascospores produced by one of these arrangements contain the same alleles as the original parent strains -also 2-strand double crossover [no recombination]

DnaA cycles between.. (2)

-Beta clamps left on lagging strand to help process, and Beta clamps are a physical sign that replication just occurred. look at drawn figures. -So for DnaA-ADP to reactivate to Dna-ATP, uses DARS sites, which is dnaA reactivating sequence in the chromosome that binds dnaA-ADP. This causes ADP to come off and ATP replaces it, so it's the sequences in the chormosome that reactivate the dnaA.

siRNA's & miRNA's participate in what processes?

-Degradation of mRNA -Translation inhibition -Methylation of DNA -chromatin remodeling.

4. Titration of DnaA

-DnaA binding to OriC is concentration dependent. Lower conc. of dnaA, reduce the likelihood of chromonsonal replication initiation. DatA (opp. effect of dars) is a sequence that binds binds and sequesters dnaA. DnaA is no longer as a pool available to initiate replication, bound to DatA sites so it's a way of shutting down replication. -sites that bind dnaA and inhibit replication initiation, and going to happen right after replication intiated. so these sites located close to the oriC. and look what happens, right after repl initiation, datA had DOUBLED. (BECAUSE THERES 2 COPIES OF IT NOW SINCE THERE ARE 2 COPIES OF ORIC). so another way of slowing down replication right after replication initiation. location on the chromosome matters :)

IR vs. DR

-IR (same sequence but reverse compliment) -DR (same seq directly on same top or bottom strand)

#2 Non-complex transposons

-IS elements with more genes (IS element is transposase flanked by inverted repeats). these are Is elements except with some genes inserted inbetween IRs in add'n to tranposase.

3. Translational Coupling

-If you have the end A, stop codon and RBS of B (these are usually bound in a similar structure unless there is translation.) -"No translation of A means no translation of B!" -If A is being translated, here is B (with RBS), and if you have a ribosome interacting with the stop codon, the other ribosome binding site is freed up and another ribo can come and translate b. -"Mutations in A impact translation will affect translation of B." -where you have you have a structure at the end of gene A and B, and the stop codon of A is in a stem loop of RBS of gene b. so here the rbs of gene b is inaccessible to the ribosome, because the rbs base pairs with the 16s rRna, so if in a stem loop, that can't happen. so unless A is translated, B won't be translated thus they are coupled :) When A and B are translated, the ribosome goes all the way to the stop codon and disrupts the stem loop so B can be translated. Anything that affects the translation of A (mut, insertion, nonsense ect), will effect the translation of B.

second-division segregation pattern

-In a heterozygous meiocyte, if a crossover occurs between the gene and the centromere, the alleles segregate at meiosis II when sister chromatids segregate each ascus contains 4 parentals and 4 recombinant spores

(non-complex) TN3

-Ir, A, res site, R, Amp^r, IR A=transposase R=resolvase, represses A res site=site of recombination -R (resolvase gene) and res site important in replicative transposition. moves by replication not c/p.

Insertion mutations

-Mobile Genetic Elements are what cause insertion mutations. -transposons -phage (lysogenic), can insert into chromosome -genomic islands, large pieces of dna that move around. -insertions ALMOST ALWAYS NULL! -(one exception would be mobile introns, designed to jump in the gene but then splice out) -trying to knock out gene in the lab, insert antibiotic resistance gene (kan^r) to knock out function of yfg.

mut rates...

-Mutation rates that lead to phenotype (1 in ever 10^-10 bases, fixed) can vary from gene to gene. (has to do with number of potentional targets that can mutated) -you want a TrpA- mutation, (is~200 aa) any mutation in this 200 aa's can lead to a null phenotype) (loss of function mutation) -mut. rate for this relatively high, 10^-6/-7 -if you have something that more specfic, like streptomycin recitive, there are about 5-6 aa's in S12 ribosomal protein. the target is very small because mut have very spef mutation and cant completely knock out S12 because they cell wont have translation. so high mut. rate 10 -10/-11. -

Gram positive:

-NO OUTER MEMBRAINE -thicker -covalently attached surface proteins to peptoglycan

Conclusions of fredrick griffith

-Phenotypic traits can be passed between different bacteria -Dead cells contains substance that encodes information for a particular trait

(mechanism 3) Regulation of DnaA expression. -On old strand, you have a number of GATC sites that are methylated and the expression of the gene was repressed by protein (?guess) SEQA, which has a .... (dual)

-Repressed by SeqA, has dual function to bind to promoter of dnaA and shuts down dnaA expression. (represses dnaA expression) -less dnaA, chances of replication intiation are lower. another thing going on... -DnaA is also an auto repressor, once dnaA made it begins to repress itself. (like a thermostat, neg. feedback loop.)

Some sort of regulatory protein that has negative feedback (QQ what would a graph drawn when regulator is made and represses itself.?)

-Rid regulator, nothing inhibiting expression. Add more x, concentration of repressor, the expression of regulation curve would go down. (opposing forces which will push for a constant level of regulator and expression). -too much regulator, then the expression willl shut off. if conc. goes down, the expression will shift back up.

QQ2: Discuss why transposition is so rare? Why is IS3 putting the breaks on transposition, as general theme in all transposons?

-Transposition limited, vast majority of transposon hopping leads to null mutation. (used in screens/selections). push pull, tightly controlled process.

Avery, McLeod, McCarthy EXP 2

-Treated mix of killed IIIS and live IIR with ribonuclease -Treated mix of killed IIIS and live IIR with DNA-ase -Discover that DNA is the transformer

QQ. Discuss what would happen in a RF1 or RF2 deletion mutation, which one would be more severe and what info do you need to answer the question.

-What is the most common stop codon/one with the least disruptive effects. (the ones used the least would be. -UAA is the most common (64%). UGA is 29% of stop codons in cell and UAG is 7% of cell. -RF1 is the easiest to get rid of, cuz then RF2 is still able to do 93% of the stop codons in the cell. translation would continue, ribosome might get stuck and then recycled by next system.

when calculating recombination frequencies, for the larger distance, the double crossover progeny are-

-added twice because each represents 2 crossovers between them

three-point test-cross analysis

-allows you to map 3 linked genes simultaneously -

DNA polermase 3

-always moves and attaches nucleotides in 5' to 3' direction. -pol 3 collexts the free basescatalyzes bond -makes complimentary strandon the leading strand bu adding nucleotides to the 3' END OF THE COMPLIMENTARY (NEW) STRAND. (polymerization is condensation rxn)

Genetic linkage

-can be quantified to map the positions of genes on chromosomes

ex. TN10

-complex transposon -IR, IS10 element encodes transposes gene, IR, tet^r, IR, IS10, IR -2 IS10 elements flank internal sequence. -when the whole thing moves, get cutting of the transposes on the outside. cutting of transposon on the inside, leading to genome reagangement. IS elements move on their own as well.

Zmax

-corresponds to the recombination frequency that is most likely to be correct

exonuclease

-degrade DNA from the 3' to 5' direction an enzyme that removes nucleotides from the ends of DNA fragments. A 50-30 exonuclease removes nucleotides from the 50 end, while a 30-50 exonuclease removes nucle-otides from the 30 end. endonuclease enzyme can cut dna at internal postions.

#3 Complex (replicative?) Transposons

-dna flanked by 2 identical IS elements. (2 IS3's, generated complex tr)

mapping function [based on Poisson distribution]

-does not account for interference -can be used to correlate the frequency of crossover with the actual number of crossover events

what crossover event is least frequent in 3-point test-cross?

-double crossover -both events must occur

an organism that exhibits complete linkage [rare]

-drosophila males have no crossing over

(

-each strand has 3 potential reading frames, thus 6 in total. (6 potential open reading frames for every segment) different genes could be using different reading frames, all 6 used throughout the genome.

QQ: Think of other example of phenotypic lag

-enzymes that make membanes -type 3 secretion system/flagelum mutation (cells still have a flagella, just takes a while for it to get turned over) -not linksys experiment (more step-wse evolution)

QQ

-if all open reading fra

IS3 ORFA

-in 0 reading frame -has helix turn helix, direct transpoase to IR in order for B's catalytic activity, -Inhibits transposition; ORFA will bind to inverted repeats to prevent ORFAB binding.

pol 1 activity? replicaiton stuff

-leading strand and lagging strand, we have polymerase, helicase beta clamp ect. this leads to leading strands with contious replicaiton and lagging discontinuous replication. we have primer laid down, pol moving opp direction of replication fork. eventually new primer laid down, new beta clamp and new polmerase. bottom is lagging strand (3' to 5') and top is leading (5' to 3'), thus remember antiparaled orientation. -lagging stand we have rna primers, oks fragments going opposite direction. (all abover dna pol 3) -however we have probelm with oks fragmenets, thus in new strand we have a rna/dna hybrid. to fix use enzyme DNA Pol 1, and comes in to insert in nick to degrade rna and inserts proper dna behind it. theres a little gap that gets filled by dna ligase. thus 2 things that fix rna in oks fragments, pol 2 and ligase. coming in at the 5 prime end and removing the nucleotides. -pol 1 exonuclease activity is 5'(phosphate end) to 3'

lod score

-logarithm of the odds ratio -determines theta value -sum all lod scores for each theta value to get Zmax -higher than 3.0=linkage at theta value -less than -2.0=significant evidence against genetic linkage -between -2.0 and 3.0 are inconclusive

QQ:Why doesn't the cell simply replicate both DNA strands exclusively by the "leading-strand" mode around the entire chromosome? Discuss.

-more slowly, 2x as long. -lagging strand has repeat advantages because dicontious -other strand serves as backu (homologous recombination. -the whole lagging strand would be single stranded dna (no ssDNA problem.)

odds ratio

-numerator=likelihood that distribution of genotypes and phenotypes in pedigree is produced by genetic linkage -denominator=chance that it is produced by independent assortment

Creighton and McClintock

-obtained cytological evidence that recombination between the genes was accompanied by physical exchange between the homologs -fragment on one end of chromosome knob on other

Ex. we have an E.coli cell with system.. TanB

-outer-membrane protein called TanB -TanB is a receptor for T1 lytic phage (so it kills the cells) -say you have a mutant cell, it divides and you have a mutation TanB- (nonsense, so tanB not made) -when the cell divides, it's still going to have this tanB on the surface, division, each of the daughter cells have 3 now. there will be some protein turnover. -so even though these are tanB(-?_), it going to be the wild type and have 3 proteins in tonB and still can be infected by T1 phage. -This is lag, takes awhile for the outer-membrane protein to be cleared from the cell surface. -can be complicating when thinking of mutation rates. (muts don't instantaneously happen)

theta value

-range 0-.50

IS3 ORFAB

-requires frameshift during translation, which makes ORFAB (not so common) -when translation happening and ribosome gets to end of ORFA, it shifts frames and starts translating B to make HYBRID. -hybrid protein makes the transposase, must have both things together. -transposon is limiting how frequently is transposition, limiting how frequently transposase is made.

Why bacteria good systems to study genetics?

-short generation time (20 min) -control environemnt -smaller genomes/less comples. 95% is open reading frames and euks even more ocmplicated -genetic crosses -asexual -less paperwork/ethics haploid: one copy of each gene so no dominatn/ressive. whatever copy they have is the phenotype. -small, 1 mL is 10^9 CFUS

Other specialized transport systems, numbered Type 1-6 secretion systems.

-simple to complex, important for pathogensis. -can transport proteis from periplasam to the extracell, from cyto to extcell or from cyto to other cells. (many functions.)

#1 IS elements

-simple transpons -Inverted repeats flanking 1-2 genes (one of which is the tranposase, so perhaps solely) -Flanked by direct repeats (like all) (~3-9 bp small DR)

upper limit to recombinant gametes-single crossovers

-single crossover produces 50% recombinant gametes and 50% parental -all 4 possible single crossovers result in same recombinant gametes

haplotype

-specific array of alleles in a set of linked genes on a single chromosome -these alleles tend to be passed on together during meiosis -may consist of any combination of linked genes with genetic variation

transposase/ cut and paste

-staggered ;cuts DNA (knicks dna at end of IR) and left with transposon, ase and IRs that's capable of transposition into new site. -ds break leads to a staggered cut in the host dna -direct repeats is where transposon inserts -gaps filled in by normal repair mechanism, sealed by ligase and transpose moved to new site. -IRs flank transposon and IR is flanked by direct repeats.

allelic phase

-the arrangement of alleles of linked genes on parental chromosomes -obstacle for mapping human genes -can be determined when disease-causing allele segregates with known genetic marker

why are genes that are closer together less likely to recombine?

-the closer the genes are, the less likely a recombination nodule will form between them

✿✿✿✿✿✿Lec. 38 Transposons✿✿✿✿✿✿✿✿* Transposons

-type of non-homologous recombination (not in homology to insert into genome) -transposons are DNA elements that all encode transposase, allowing them to move. -Discovered by Barbara Mcclinktok in so's maize

...

...

Complete review

...

Mechanisms of Post-transcription regulation

...

Regulation of metabolic pathway in bacteria

...

What if?

...

add pgs. 358

...

RNAi

...injecting double stranded RNA molecules into a cell somehow turned off expression of a gene with the same sequence as the RNA

What is the multiplicity of infection required for culturing bacteriophage?

0.1

map units (m.u.) vs centiMorgan (cM) vs recombination frequency

1 m.u. = 1 cM = 1% recombination

Two ways for plasmid replication

1) Unidirectional replication 2) Bidirectional replication

Sec dependent translocation system (2 types, dependent on where protein is going to go) ?

1) post-translational, proteins not destined for the inner membrane (ie periplasam, outer, extra ect.). -They go through the Sec system after 'fully translated and then secreted' -Protein called SecB (chaperone), binds and controls folding. SecB will bind to these specific ones (?lol), and this is going to go to the SecYEG system. -YEG is like a pore in inner membrane, and SecB is going to direct it to the SecYEG system (uh seca?), and protein SecA provides energy (hydrolyzing ATP). -Signal sequence is cleaved, protein gets in the periplasam without signal sequence (?). SecB binding to it after it's made, directed to SecA and transported through sec system with energy from atp (proteins destined outside of inner membrane.

How to increase frequency?

1) use mutagen: Amp^s, plate with ampicillin and filter disk with mutagen , thus getting more with amp^r. -around the mutagen, get all the colonies with much higher frequency and away you get less colonies. -advantages: increase frequency -disadvantage: sometimes difficult to track down the cause of the mutation, if you have a bunch of muts in genome, hard to know which is responsible. "difficult to isolate casual mutant"

QQ: What are some way you can get silent mutations? supressor: second site mutation that compensates for the first one. (would mean your org mut has no phenotype?)

1)intron mutation (There are group 1 and group 2 introns) 2)synomous mutation (due to wobble codon) 3)functional robustness (proteins can tolerate some mutations) 4)non-coding region (however there are functional noncoding regions), 'nonfunctional region' -if you change the environment, might change whether or not a mutation is silent or not.

Ribosome Components

1. 2 subunits: 30s (bottom; 16s rRNA) + 50s (top; 5s and 23s rRNA) 2. 52 proteins 3. 3 rRNAs

subunits in 70S ribosome

1. 30S (contains 16S rRNA) 2. 50S (contains 23S and 5S rRNA)

Translation Initiation

1. 30s binds to Shine-Dalgarno sequence on mRNA (brought on by IF3) 2. IF1 blocks A site and IF2 escorts the Formylmethionine tRNA to the start codon. 3. fmet tRNA binds directly to the P site and releases IF3. 4. 50s subunit docks to the 30s subunit which causes GTP to be hydrolyzed on IF2, thus releasing the initiation factors.

T4 Phage Life cycle

1. Adsorption and penetration (penetrates outer membrane and cell wall using tail --> injects) 2. Early mRNA synthesis (after 2 min) 3. DNA replication (after 5 min) 4. Late mRNA synthesis + formation of new viral particles (after 12 min) 5. After life cycle is complete, host cell bursts and releases newly formed viruses (after 22 min).

Name 6 pathogens that use T4SS

1. Agrobacterium tumefaciens 2. Bartonella henselae 3. Bordetella pertussis 4. Brucella spp. 5. Helicobacter pylori 6. Legionella pneumophila

Small Regulatory RNAs (sRNA)

1. Antisense (complementary to mRNA sense). 2. Binding can either stabilize the target mRNA or make it susceptible to degradation.

What are the 6 features of 'pathogenicity islands'?

1. Are usually large 2. Encode virulence factors 3. Encode 'mobility' genes 4. Have DR borders 5. Are unstable 6. Have a G+C content different from host DNA

What are the 3 sub-types of T4SS?

1. Bacterial conjugation (classical Tra (transfer) systems) 2. DNA uptake and release 3. Effector translocator systems

Lysogenic Cycle

1. Bacteriophage is dormant. 2. Integrates into cell chromosome. 3. Can reactivate to become lytic.

Chemical Mutagens

1. Base analogs have similar structures to natural bases. Incorporated during DNA replication and causes point mutation because of incorrect base pair. 2. Intercalating agents (distort DNA to induce single nucleotide insertion or deletion).

λ Phage

1. Capsid head 2. Non-contractile tail 3. Lysogenic (temperate- shows restraint)

T4 Phage

1. Capside head 2. Contractile tail 3. Virulent/lytic (intermperate - unable to moderate)

Agents that cause mutation?

1. Chemical (compounds that interfere with DNA) 2. Physical (radiation or heat) 3. Biological (insertion of transposons)

Translation Elongation (5 steps)

1. Codon in A site, tRNA+EF-TU-GTP is bound to 30s 2. 23s rRNA cleaves carboxyl end of peptide in tRNA in P site (forms bond with amino group in amino acid in A site) 3. Ef-G-GTP attaches and causes ribosome to translocate 3 nucleotides forward toward 3-OH 4. uncharged tRNA is ejected 5. Repeat

What are the 3 steps of conduction?

1. Cointegrate formation in the donor cell 2. Conjugal transfer of the conintegrate to the recipient cell 3. Resolution of the cointegrate in the recipient cell

RNA Polymerase

1. Consists of ɑɑββ' subunits. 2. Synthesizes RNA.

Describe transcription elongation.

1. Core polymerase synthesizes RNA strand 5'-3'. 2. Added bases are complementary.

What are the 6 steps in the analysis of Tn-mutants

1. Cut DNA at restriction sites 2. Southern Hybridisation using Tn-probe 3. Clone Tn-containing fragment 4. Sequence DNA and analyse sequence 5. Identify the gene in the WT genomic sequence and PCR amplify and clone the gene 6. Further analysis

How do we get polarity (3 ways)

1. DNA insertion, if you have an operon (promoter, the gene A/b, then terminator. The most common way to have DNA insertion is by transposons. -Transposon insertion in A can impact expression of B,. QQ: how can transposon insertion in A influence expression of B. How could transposon in an operon prevent/alter expression in the downstream gene. -reading frame would be not affected because each would have it's own RBS and start codon. Whole point of operons is that translation is independently controlled. reading frame of A might be affected by insertion (not polar), but reading frame of B wouldn't be affected (polar). -structure/translation coupling, where structure of the upstream genes impacts the downstream genes. -Transposons have their owns sets of genes, -(trx termination, there is a stem loop that can terminate trx). So Factor-Independent trx termination -IF this transposon encodes trx terminator, then trx is going stop and fall off and gene never gets transcribed. (A main way to get polarity is transcriptional terminator. ********* -can lead to factor independent or dependent (rut/rho key. if we have a gene A being transcribed and translation is occurring, and rho can jump on the ra of the rut and blocked so it doesn't terminate (normal). however if you have a nonsense mutation, ribosome will be stopped by release factor ect. rho can now jump on the rna because nothing is blocking it, because no translation occurring thus factor dependent termination) termination, can mess up translational termination among others.?

What are the Neisseria gonorrhoeae DNA release systems?

1. DNA is released via a T4SS encoded by a genetic island 2. Autolysis occurs only in stationary phase

QQ Do bacteria evolve by

1. Directed Change (Lamarkian change, as organisms adapt to environment/they gain new targets ex. bac evolving resistance to antibiotics/plague to direct mutations) 2.Darwinian (higher organism) *****Darwinian evolution: Selection of pre-existing RANDOM mutations (no directed mutation). -random mutation rate and environment selects for the beneficial ones. experiments to show this

SOS Response (Regular)

1. Extensive DNA damage induces SOS regulatory system. 2. RecA activated by DNA damage to inactivate LexA (represses expression of SOS genes).

Hfr (High frequency recombination)

1. F factor is integrated into bacterial chromosome. 2.Transferred DNA is integrated into the chromosome of the recipient by recombination. 3. DNA is transferred in order (gene mapping).

(Lecture 9)** first one TWO SPECIFIC TRX TERMINATION MECHANISMS:

1. Factor-Independent Termination (remember all the information is coded in the sequence of the rna, no proteins or anything) -

F factor

1. Fertility plasmid containing extra chromosomal genes. 2. Encodes sex pilus. 3. Self-transmissible by way of starting at oriT site as single-stranded DNA.

Open Reading Frame (ORF)

1. Frame of reading for the mRNA. 2. 3 possible frames per transcript. 3. Stop codon is in the same frame as start codon.

Affinity Chromatography

1. Gene encoding the protein is fused to DNA encoding a peptide tag that has a strong affinity oa small ligand molecule. 2. Target ligand is attached to beads. Cell extract containing tagged protein is passed over beads. 3. Only tagged proteins will bind to the beads.

Transformation in gram positive bacteria vs gram negative bacteria

1. Gram positive bacteria: Secrete competent factor, accumulation of factor leads to translocasome formation in cell membrane which allows DNA through. (Quorum sensing) 2. Gram negative bacteria: Stress induces competence (ability to uptake foreign DNA). CaCl2, low temperature (4 C) increase competence by making the membrane more permeable to DNA. Starvation, heat shock (42 C) induce.

Polymerase Chain Reaction

1. Heat to 95C for 30s; Denaturation separates strands. 2. Cool to 55C for 30s; Primers annealed. 3. Heat to 72C for 60s; Taq polymerase replicates sequence. 4. Repeat steps 1-3 30 times.

Requirements for bacteriophage infection of a host?

1. Host recognition and attachment via cell-surface receptors. 2. Genome entry. 3. Assembly of virions. 4. Exit and transmission.

Sigma Factor (σ)

1. INITIATES RNA synthesis (NOT elongation). 2. Recognizes promoters by binding to -10 (Pribnow Box) and -35 regions of genes. 3. Guides the core enzyme to initiate transcription.

Inducer vs corepressor

1. Inducer binds to repressor and causes it to release from the operator (induction) 2. Corepressor binds to repressor which then binds to the operator. Releases from operator when corepressor releases. (Derepression)

Initiation of Translation (3 steps)

1. Initiation factor (and GTP) bind to 30s 2. Initiator tRNA and mRNA bind to 30s (utilizing shine dalgarno sequence) 3. 50s subunit is bound to 30s through GTP hydrolysis = 70s initiation complex

-3 types of transposons (simplest to complex)

1. Insertion Sequence (IS elements) 2. Non-complex transposons -basically more complicated IS elements 3. Complex transposons (2 IS elements close flanking sequences in-between them)

Describe λ phage DNA as it goes from the capsid head to inside the host.

1. Inside capsid head, the DNA is linear. 2. Once inside the host, cos site ligate to form circular DNA. 3. Cos recognition sites determine where the genome is cut and packaged during replication.

What are the 6 steps in random Tn-mutagenesis?

1. Isolate plasmid DNA and tag with a 'signature' sequence 2. Use the mutant collectively to infect an animal 3. Recover bacteria from the deceased animal spleen 4. Compare the 'output' tag sequences with the 'input' tags 5. Missing 'output' tags indicate avirulent Tn-mutants 6. Confirm the avirulence phenotype and identify the Tn-inactivated gene

Recombinant DNA Technology

1. Isolate plasmid. 2. Enzymatically cleave DNA into fragments. 3. Isolate fragment with gene of interest. 4. Insert gene into plasmid. 5. Insert plasmid with gene into bacterium. 6. Culture bacteria. 7. Harvest copies of genes or proteins coded by gene.

Bicoid research

1. Led and proved the presence of proteins required for some of the earliest step in pattern formation 2. Increased our understanding of the mother's critical role in initial phases of embryonic development 3. Gradient morphogens that can determine polarity and position is a key developmental concept for a number of species

Quorum Sensing in A. Fischeri

1. LuxI protein synthesizes acyl homoserine lactone autoinducer (AI). 2. AI accumulates outside cell; When concentration reaches a certain level, diffuses back into cell. 3. AI binds to activator protein (LuxR) --> Increased transcription of lux operator --> Luciferase. 4. Luciferase --> bioluminescence.

How to map genome by conjugation?

1. Mate an Hfr cell with an F- cell. 2. Use markers to detect transfer ( catabolic - sugar, biosynthesis - amino acids or nucleotides, antiobitics - kanamycin, ampicillin) 3. Periodically plate cells on different selective medium (ability to grow determines the order of the genes).

Steps of nucleoid occlusion

1. MinD-ATP binds to membrane, MinE binds to complex and cleaves phosphate = energy 2. MinD-ADP dissociates from membrane and binds ATP in the cytoplasm inserts into membrane farthest from MinE 3. MinC binds to MinD-ADP inhibiting FtsZ formation 4. Gradient of MinC is centered at poles 5. SlmA binds to palindromes in DNA sequence reduces ability of FtsZ polymers to form nucleoid

DNA Mobility Shifts

1. Mix DNA fragments with proteins and run on gel. 2. DNA-protein complexes run slower than DNA alone due to size. 3. Allows identification of DNA-binding proteins and the conditions for when they bind.

What does it mean when it is said that the genetic code is degenerate?

1. More than one codon may code for a specific amino acid. 2. More codons exist than encodable amino acids.

What are the 3 features of transposable elements?

1. Move by insertional integration 2. Random insertion sites 3. Insertion can cause mutation

Prokaryotic Viruses

1. Must bind + cross cell wall without hurting host cell. 2. Uses host's nucleotides, amino acids, ATP to replicate genome and build more viruses. 3. Exit through cell wall (lyse host).

Transformation

1. Natural transformation: Free DNA taken up from environment and then integrated into genome. 2. Plasmid transformation

What are the steps in 'targeted gene disruption using an SV plasmid'

1. PCR-amplify an internal fragment of the target gene 2. Clone the PCR-fragment into the MSC of the SV 3. Transfer the SV-construct to the non-permissive host 4. Compare the phenotype of the disruptant with that of the WT

How does Hfr conjugation occur?

1. Pilus connects. 2. Donor DNA replicated by rolling-circle + transferred. 3. Connection breaks, fragment of donor DNA incorporated into recipient chromosome.

Plate hybridization

1. Plate bacteria 2. Transfer to membrane 3. Denature DNA on membrane 4. Add probe 5. Wash of unbound probe/view x-ray 6. Compare to plate

Rho (ρ) Independent Termination

1. Polymerase slows at pause site. 2. GC-rich sequence forms stem loop. 3. Series of U residues are weakly bonded to A's of DNA --> Become weak with stalling. 4. mRNA breaks off of DNA and polymerase is released.

σ 70 Factor

1. Primary sigma factor. 2.Considered the "housekeeping" sigma factor because it transcribes most genes in the cell and keeps essential genes and pathways functional.

Two scenarios for repressors?

1. Repressor binds to DNA and repressed target gene; inducer causes repressor to release. 2. Repressor + ligand (corepressor) complex bind to DNA and represses target gene.

RT-PCR

1. Reverse transcriptase creates cDNA from RNA 2. Same process as PCR but begin with RNA and RNA primers

Rho (ρ) Dependent Termination

1. Rho factor binds to mRNA. 2. Rho pulls itself to the paused RNA polymerase. 3. Breaks polymerase and mRNA off of DNA.

Transposons

1. Segments of DNA that are able to transposition (hop from one place to another in DNA) 2. Contain transposases (enzymes that promote transposition). 3. Large amounts exist in all living organisms.

Two Component Signal Transduction

1. Sensor kinase binds to signal, and self-activates by phosphorylation. 2. Response regulator gets phosphate from sensor and binds to chromosome --> Alters transcription rate of multiple genes.

What are the 6 steps in conjugation process?

1. Sex pilus attaches to receptors on the recipient cell 2. Contraction of the pilus draws the two cells together 3. Formation of mating pore through which DNA can pass from donor to recipient 4. The plasmid is nicked at the oriT and the 5' end begins to transfer 5. Replication of DNA 6. Recipient becomes donor

Describe transcription initiation.

1. Sigma factor binds to DNA and recruits the core enzyme and scans for the promoter region. 2. Core enzyme unwinds DNA at the promoter. 3. Sigma factor is released.

Insertion Sequence (IS)

1. Simplest transposable element. 2. Encodes transposase flanked by inverted repeats (IR).

What are the 4 features of prokaryotic plasmid vectors?

1. Small and multicopy 2. Amplifiable 3. Have a selectable gene/marker 4. Have one or more unique RE sites

Base Excision Repair

1. Specialized enzymes recognize specific bases and remove them without breaking the phosphodiester bonds. 2. Results in abasic site (AP), which is recognized by AP endonuclease. 3. AP site allows DNA polymerase I to synthesize a replacement strand containing the proper base.

Translation Termination

1. Stop codon on mRNA enters the A site (no tRNA) 2. Protein releasing factor enters the A site. 3. Peptidyltransferase is activated and releases the complete protein. 4. Ribosome recycling factor and EF-G enter the A site causing 50s to undock from 30s.

Gene replacement using homologous recombination.

1. Targeted mutagenesis. 2. Knockout gene of interest with another gene.

Draw how transposition + duplication happens.

1. Transposase cuts DNA at the inverted repeat sequences at both ends of the transposable element and the target sequence. 2. Ligate transposable element with target DNA.

Transposon Marking

1. Transposons easy to identify (contain antibiotic resistance genes --> cells with transposons grow with antibiotics present) 2. Create large insertion mutations (knock out gene function) 3. Easy to identify sequence (mutated gene is located next to transposon)

trp Operon Control

1. TrpR: aporepressor 2. Tryptophan: corepressor 3. TrpR holorepressor =TrpR + tryptophan Holorepressor binds to trp operon and represses transcription; blocks RNA polymerase.

Physical Mutagen - Radiation

1. UV and ionizing radiation cause formation of toxic oxygen radicals. 2. Radicals cause two adjacent pyrimidines to dimerize (thymine dimers) 3. Prevents DNA replication and gene transcription.

What are the 6 features of TE's?

1. Unique size 2. Unique IR 3. Encode transposition functions 4. Move by insertional integration 5. Random insertion sites 6. Insertion can cause mutation

T4 Genome Replication

1. Upon entry, genome becomes circle. 2. Early genes transcribed take control of cell and destroy cell chromosomes. 3. Rolling circle replication of genome. 4. Progeny genomes are linked in concatemer (several genomes linked together). 5. Cut with an offset so individual genomes have slight overlaps.

Describe Transduction

1. Virus injects viral DNA into cell. 2. At end of life cycle, viral DNA packaged into viral capsid before cell lysis. 3. Sometimes package host DNA by accident --> Results in virus carrying host DNA. 4. Old host DNA transferred to new host via virus.

Specialized Transduction

1. Viruses integrated their genome into the bacterial chromosome (lysogeny). 2. When entering lytic cycle, adjacent bacterial genes (next to viral att sites) are sometimes mistakenly picked up due to excision error.

AraC

1. When arabinose is absent, AraC is rigid and elongated; Represses expression of genes that break down arabinose. 2. When arabinose is present, AraC is more compact; Stimulates binding of RNA polymerase, which transcribes the genes.

Translation Elongation

1. aminoacyl-tRNA binds to the A site (acceptor site) 2. Peptide bond forms between the new amino acid and the growing peptide chain in the P site. 3. EF-G (with GTP) binds to the ribosome, GTP is hydrolyzed and shifts the ribosome to the next codon.

Pheremones in Enterococcus

1. cAD1 pheremone binds to TraC of donor 2. Binds to TraA repressor, induces TraE (negative control) 3. Activates transcription of tra genes (Positive control) 4. Inhibitorautoinducer peptide produced TraB inhibits production of cAD1

Gene regulation results in dramatic changes that require three processes

1. cell division (increase in cell number) 2. cell differentiate (cell specialization in structure and function) 3. Morphogenesis (creation of form or body shape) All cell activities depends on gene expression on production of proteins all three have their basis in cellular behavior;

2 mechanisms:

1. cut and paste (ase cuts one strand) 2. replicative transposition (transposase cuts both strands)

two ways RNA can be processed

1. digestion with Rnases 2. modification (isomerization)

two ways to terminate transcription

1. factor-independent 2. factor-dependent

two parts needed for factor-independent

1. inverted repeat 2. short string (>4) A's to synthesize into U's

Attenuation trp operon

1. low conc. tryptophan; ribosomes pause at trp codons and transcription continues. 2. High conc. they do not pause at the codons and the transcription stops by factor-independant termination

tRNA

1. tRNAs bind individual amino acids. 2. Cloverleaf structure. 3. Anticodon that matches with codon on mRNA.

Helix structure minor groove

1.2 nm

Helix structure minor groove

1.2 nm smaller part. some proteins do interact with smaller minor groove. what proteins see is other chemical components sticking out of the bases and interactig with them.

How genes regulated?

1.Chromatin Histone acetylation DNA methylation 2. Transcription Alternative RNA splicing mRNA degredation (sequences of 3' end of RNA) 3. Translation blocking binding of mRNA to ribosome microRNA (miRNA), inhibits initiation stage interference RNA (siRNA) Chromatin remodeling 4. Protein Post translational modification

DNA cloning

1.Use REase to cute DNA into pieces 2. Mix and ligate 3. New part has no ori c but is replicated when plasmid vector does everytime

DNA Polymerase III

11 GENES, attaches nucleotides in 5' to 3' direction.

DNA pol III

11 genes

GalR

110bp apart, opposite sides dna two operator sites it binds and bends dna so promotor site is mushed

what part of the ribosome bind to the RBS?

16S rRNA

In which component of the ribosome is there the complementary sequence?

16s rRNA of 30s subunit.

Conclusions of fredrick griffith

192-Phenotypic traits can be passed between different bacteria -Dead cells contains substance that encodes information for a particular trait

(G) Griffith

1928 Transformation with steptococcus pneumoniae 1. Rough (non path) and Smooth (path) 2. Live IIR injected: No bacteria recovered 3. Live IIIS injected: Type IIIS recovered, dead mouse 4. Heat killed IIIS: none recovered, lives 5. Mix DEAD IIIS, LIVE IIR = recover IIIS mouse dies -USED T2 PHAGE, (1952) *PHAGE dna Alone is sufficient to direct syntheisis of more phage. -Mixed rough with heat killed smoothe infected mice and died. HOWEVER got smoothe bacteria. CONCLUDED THAT PHENOTYPIC TRAIGHS CAN BE PASSED NETWEEN BACTERIA, DEAD CELLLS CONTAIN A SUBSTANCE THAT ENCODES INFO FOR A PARTICULAR TRAIGHT. -further info that dna was informaion molecule

Avery, McLeod, McCarthy EXP 2

1944 -Treated mix of killed IIIS and live IIR with ribonuclease -Treated mix of killed IIIS and live IIR with DNA-ase -Discover that DNA is the transformer why s, onumonie only work? they take up dna and intergrate it into teir genome, thus NATURAL TRANSFORMATION (JHORIZANTAL TRANFORMATION).

(A)Avery, McLeod, McCarthy EXP 1

1944 (just on plates, looking for colony morpholgy) AVERY FOLLOWS GRIFFITH -more strep -Extraction of components from HEALT KILLED STEP, MIXED rough cells with each category of polysacs, lipids, proteins and nucleic acids. WHEN mixed with nuc. acids, got BOTH ROUGH AND SMOOTH , THUS DNA IS TRANSFORMING PRINCIP;E. -then took nuc acids and dreated sample with RNASES AND DNASES,.*rnases got mixed, dnases got non-mixed. howeverrr, skeptimism could not be repeated in all orgs. -Discover that Nucleic acids transform

(H)Hershey/Chase

1952 another way to show dna was herediatar material, following up on Aver.mcloud. -"Hershey Chase Demonstrated (supported Avery experiment results) that DNA is in fact the genetic material of bacteriophage T2. Phage proteins contain sulfur and no phosphate, while phage DNA contains phosphate but no sulfur. Phages were allowed to infect ecoli, then put in medium containing either Phosphorus32 or Sulfur35, were then blended briefly, and then the progeny of the phage were examined. ~40% of input P32 was found to be in the DNA of the progeny." -used a diff, kind of system, a T2 phage. (BASIC STRUCTURE HAD DBS DNA IN CENTER) -dna has phosphorous inside, and proteins have sulfur!! (differentially labeling the phage and the info molcule would be carried out into new phage. so put into flask with e coli and made new phage -ONE SET OF radiolabeled phage CONDTIONS USING phosphorous P32 (RADIO LABELED PHAGE) AND ANOTHER conditions of sulufre, S35, THUS LABELING PROTEIN. thus grew culture in radio labeled molecules, and phage coat -RADIO LABELED 32P PHAGE, RECOVERED 40% OF RADIOATIVITY VS. 0% OF PROTEIN(35S)----FURTHER EVI THAT DNA IS INFO MOLECULE -Label protein and DNA with isotopes -Infect with phage, blend to remove ghosts -Conclud that DNA is transmitted and not protein -is it proteins or dna q

(W) Watson and Crick meausrments

1953 -used chargaff rules, took wilkins X-RAY photograph 51 to understrand that it's helical in nature, very regular molecule. develop dna stucture/base [airing rules. Semiconservative replication prediction 34 angstrom in 1 turn, 3.4 nm (10 ANGSTROM=1nm) turn of the dna helix aka 10 bases. width 3.4 ANGSTROM -DNA DOUBLE HELIX WITH PHOS ON OUTSIDE, BASES ON INSIDE. -Linus Paulling came up with tripple helix, but didnt work because phosphate in middle of structure, thus negativey charged so WRONG BROOOOOO wouldnt hold. 1953 Wats and crick looked at widths of the bases, and AT together and GC together, which happen to be H bond and exact right lengths. fits chargaffs rules, -Crick decided it was antiparallel, 2dna strands running in opposite directions. -10 bases in helical turn

(M) Messleson and Stahl

1958 Confim semiconservative replication 1. Ecoli grown with heavy N-15 2. Centrifugation after placing back into N14 and replicating 3. DNA before div = bands at bottom One rep = middle Two rep = Higher

DnaA cycles between.. (1) (mech. 1)

2 forms: DnaA-ADP (high affinity sites REP. STOPS) ⇌ DnaA-ATP (high/low affinity sites) REP. GOES (ADP-Hda deactivates dnaA-ATP by removing a phosphate for dnaA-ADP) *dnaA-ADP can only bind to high affinity sites at OriC. *dnaA-ATP can bind to both low and high affinity sites at OriC. -OriC must be completely bound by dnaA-ATP (active form), for replication to go. coordinate the transition of dnaA to determine when replication occurs. *DnaA-ATP with ADP-HDA to deactivate DnaA to remove phosphate from dnaA. -On lagging strand, we have rna primers and okasaki fragments (dna pol 1 will eventually come in to clean up), however initially at Okz ends we have Beta clamps (B clamp around the dna, that dnaE/pol 3 binds to help process along the dna, and pol 3 come off and clamps left). -Protein ADP-HDA associates with Beta clamps and interacts with DnaA-ATP, the phosphate comes off and now you have DnaA-ADP. (So dnaA-ATP deactivated by HDA protein bound to the beta clamps, occurring only after replication). -HDA

➙

2) Rely on fact that auxotrophs don't in these select conditions. -utilize that auxotrophs can't grow (lolz) -some antibiotics only kill GROWING cells (Ex. PENICILLIN!, will only kill kill growing cells by inhibiting cell wall synthesis). -can use penicillin to enrich for auxotrophs , or anything really that doesnt grow in a certain condition. experiment: Looking for an E.coli Bio- (can't make biotin, need biotin to grow) -grow one of these cells in Bio+ with a mutagen first, you make a bunch of mutants. (if try w/o mutagen, bad chances). *key is, bio+. your auxotrophs can grown on this __, you need something to enrich your auxotrophs. you put this culture in a test tube that doesn't have biotin. (will biotin auxotrophs grow? NO! but anything that's not an auxo will grow...) -Unless we add,,, penicillin, anything that is growing is going to die :3. Auxotrophs aren't growing but aren't killed, however our PROTOTROPHS (bio+ are growing are they are killed), getting rid of everything we don't want and what we want isnt being killed. Bio-/pencillin+ ➙➙(grow, Bio- doesn't grown cuz no added biotin, bio+ killed)➙➙now we have a flask enriched for Bio- (killed off all the bio+). -take this and plate on bio+, screen colonies (like was shown) and try to get auxotroph. -will work for any sort of thing where cell's growth is slowed down, a nice counter solution by selecting for things that don't grow. (but, enrich is not 100%)

Major groove

2.2 nm

Major groove

2.2 nm, wide part of dna helix where most proteins interact with major groove!!!!

number of tRNA synthetases

20

Rate of pol 3 adding nucleotides??? (help) EXAM!!

2000 nt/second KNOW MATH PROBLEM FOR EXAM!!

number of tRNAs

27

(Lecture 11) How many tRNA's?

27 tRNA's, but 61 codons. This discrepancy is accounted for by wobble base pairing, third base in the codon has a little less strict base pairing so that 1 tRNA can interact with multiple codons.

suppressors

2nd site mutation that overcomes the mutation (restores the phenotype) example: if have a +1 mutation, then add a -1 mutation... the -1 mutation is the suppressor!

Chromosomal Segregation 1. Partitioning of E coli (plasmids are similar) -partitioning means one copy in each daughter

3 components: 1) Dna site called parC on the chromosome 2)parB: proetin that binds parC DNA sequence site. 3)parA is a filament like structure that binds parB at parC When parB is bound to parC, parA binds to it and this leads to the filament being formed and it pushes the chromosomes to each cell. -the filaments will polymerize and chromosomes segregated into each daugher cell.

Polypeptides are processed and modified in the golgi body before transportation out of the cell, regulatory molecules bind to a polypeptide to alter its function, and regulation of protein stability

3 ways eukaryotic genes are post-translationally regulated:

DnaQ

3' --> 5' Editing endonuclease of RNAIII

what part of the tRNA are amino acids added to by aminoacyl tRNA syntheses?

3' CCA

dnaQ

3' to 5' exonuclease editing in E coli.

Dna III AND Dna I read in which direction

3'-->5'

codon

3-nucleotide sequence

One turn

3.4 nm, 10bp

(Mechanism 2 SeqA Sequestration) On OriC, we have the sites DV?,dnaA binding sites, low affinity sites, high affinity sites.

3rd componenet: # OF GATC SITES (its on both strands, site where DAM actually methylates) -say this is a newly replicated oriC, old strand is methylated ORiC is site of intiation of replication, so if physically blocked then that blocks replication. Right after replication, (MDM) one strand is methylated and one strand is not. (HEMIMETHLATED, SIGH THAT REPLICATION JUST OCCURERED. ,3rd component, # of GATC sites (on both sites), which are the methlated sites by DAM. -We have hemimethalated dna, and then our friend SeqA protein that binds to hemimethalted dna @ oriC (and some other places). -SeqA comes along to bind to hemimethlated sites and this is blocking oriC, thus dnaA cannot bind and replication intiation is BLOCKED. -dna will be hemimethalted for a certain amount of time after replication and new round of replicatin cannot be initiated until you have fully methylated dna. Once dna fully methylated, SeqA no longer binds

Capping, polyadenylation, and intron splicing modify pre-mRNA, alternative capping and polyadenylation can be used in different cell types, alternative splicing to produce different mature mRNA molecules, and RNA editing to modify base sequences of the RNA

4 ways eukaryotic genes are regulated through mRNA processing:

Translational regulatory proteins bind to delay translational initiation, RNA silencing by RNA interference so translation doesn't occur, regulation of mature mRNA transport to the cytoplasm, and regulation of mature mRNA stability

4 ways eukaryotic genes are regulated through regulation of mature mRNA

Leading strand extended in direction

5 --> 3'

Transcription factors bind to promoter regions, regulatory regions like enhancers and silencers bind regulatory proteins, open chromatin structure is formed by protein action, alternative promoters used in different cell types produce different pre-mRNA molecules, methylation of DNA inhibits transcription

5 ways eukaryotic genes are TRANSCRIPTIONALLY regulated

Bonds between nucleotides

5' and 3'

What can affect the stability of mRNA

5' cap Poly (a) Tail 5' UTR Coding region 3' UTR

Blunt ends

5' or 3' ends of double stranded DNA lacking any single stranded overhangs

DnaIII and Dna I assemble in which direction

5'-->3' extend towards the 3-OH end

what is recombination limit? why?

50% -Once genes are far enough apart to produce recombination frequencies of 50%, the pattern of segregation is that expected of independent assortment

Ribosomal subunits

50s (23s+5s=34 proteins L1-34) + 30s (16s = 21 proteins S1-21) = 70s Ribosome

how many reading frames could a genetic sequence have?

6! (three different potential reading frames on each template and codon strand)

Where is the enhancer located for the Alpha Chain of Tcell Receptor?

69,000 bp downstream of promoter.

complete ribosome

70S

Steps of a forward genetic screen

:Choose an organism, choose a mutagen, mutagenize the organism, screen for phenotypic defects, identify the gene sequence responsible for the mutation, analyse the molecular function of the gene

Operators

A bacterial regulatory DNA sequence to which repressor proteins and activator proteins bind

Vector

A carrier fragment of DNA with attributes that will allow amplification in a biological system

steroid receptor

A category of transcription factors that responds to steroid hormones. An example of a glucocorticoid receptor.

DNA library

A collection of cloned fragments of DNA (single source

helix-turn-helix motif

A common structural design in the lac repressor protein is the ___, which fits easily into the major groove of the DNA double helix.

lactose

A compound known as X-gal is widely used in molecular genetic research. When wild-type (normal) E. coli is grown on medium containing X-gal, the bacterial colonies turn blue. In contrast, when lacZ mutants are grown on medium containing X-gal, the bacterial colonies remain their normal white color. X-gal is likely to be a compound chemically similar to ___.

Corepressor

A compound that binds to the allosteric site of a repressor causing it to activate the DNA binding domain and repress transcription

Inducer

A compound that binds to the allosteric site of a repressor causing it to inactivate its DNA binding domain and no longer repress transcription

Inhibitor

A compound that binds to the allosteric site of an activator and causes the DNA binding domain to be inactive so that the activator cannot bind and help initiate transcription

Effector

A compound that binds to the allosteric site of an activator causing the formation of a functional DNA binding domain so that the activator can bind to the DNA and help initiate transcription

Organizaiton of typical eukaryotic gene

A eukaryotic gene adn the DNA elements that control it are organized like 18.8 Transcription initiation complex: assembles on the promoter sequence at het upstream end of the gene; one of these proteins, RNA polymerase II, then proceeds to transcribe the gene, synthesizing a primary RNA transcript RNA processing includes enzymatic addition of a 5' cap and a poly-A tail Control elements Associated with most eukarytoic genes are multiple control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins

DNA clone

A fragment of DNA that is inserted into a vector

Reporter

A gene can act as this if its product can be detected directly or is an enzyme that produces a detectable product

Attenuation

A gene regulatory mechanism that fine-tunes transcription to match the momentary requirements of the cell, achieving a steady state of compound availability

Modifier gene

A gene that has a lesser effect on the phenotype but also makes contributions

Transgene

A gene that has been modified in vitro by recombinant DNA technology and introduced into the genome via transformation

Major gene

A gene that has substantial effect on phenotypic variation

Inducible operon

A general term for an operon that is NOT EXPRESSED under one set of environmental conditions but whose transcription is ACTIVATED under an alternative environmental condition

Population

A group of interbreeding organisms

Ti plasmid

A large circular plasmid of Agrobacterium tumefaciens that harbors genes for transfer of DNA into plant cells

Genomic imprinting

A mechanism that resets epigenetic patterns in meiosis in certain mammalian genes

Square root method

A method for estimating allele frequencies in a population based on manipulation of the frequency of the homozygous genotype

Genotype proportion method

A method for estimating allele frequencies in a population by manipulation of genotype frequencies

Allele counting method

A method for estimating allele frequencies in a population by tabulating the number of alleles of each type

Exon shuffling

A molecular mechanism for formation of new genes either by duplication, translocation, or inversion of chromosomal segments

mazF

toxin

Reg. of translation by sRNA

trans-acting Bind RNA chaperone proteins (Hfq)

Common control point for transcription

transcription Regulation at thsi stage is often in response to signals coming from outside the cell like hormones or other signaling molecules; gene expression is equated with trnascription for both bacteria and eukaryotes

****************************************

transcription termination (all processes, have initiation /elongation/termination) and remember in dna rep, termination was not absolutely essential but in trx is more specifIC process.

negative control

transcriptional regulation by repressor proteins

LuxR

transcriptional regulator

tra genes

transfer genes on the plasmids. Encode Tra proteins.

cut and paste mechanism

transposase cuts both strands of the dna, tr come out and move to another site.

what happens when anticodon matches the codon in translation

triggers tRNA in the A site to interact with tRNA already present in P site

How to do genetics on essential genes? (issue: you can't make a null mutation and see what happens..)

typically what's done is a class of mutant called a CONDITIONAL LETHAL MUTATION: "mutation only relevant under certain conditions" -(your mutation only verts it's effect under certain conditions, a way of turning your mutation 'on or off', [still always there, but only during affect of certain conditions] [temp. typically used, you can change temperature of mutation and then observe it's effect]

genetic code

universal assignment of codons -64 codons ~61 encode for amino acids 20 standard amino acids (two rarely used)

constitutive genes

unregulated genes, which have constant levels of expression

UTR

untranslated region 3'--->degradation 5'---> inhibition of translation (in order to shorten RNA this 5' cap needs to be removed; nuclease enzyme can rapdily chew up the mRNA

ethidium bromide

use to label DNA -intercalating agent

Pyrrolysine

used in methanogens and is inserted in UAG

Selenocysteine

used in methanogens for certain enzymatic rxns --in rare occasions leads UGA to this - not the stop codon

Phage

viruses of bacteria, can infect a cell and inject genome

exon skipping

when an exon is spliced out of a pre-mRNA.

heterodimer

when two polypeptides encoded by different genes bind to each other to form dimer.

homodimer

when two polypeptides encoded by the same gene bind to each other to form a dimer.

BLOCKS to replication -a solution?

with ssGap/lession in lagging, repair can fix. HOWEVER, top leading strand is stuck with no way to restart because there is no 3' end, THUS lays down NEW PRIMER WITH DnaG! (continue) Pol 3 is a highly specific active site, thus low mutation and can only recognize proper base pairs. (POL 3 CANT RECOGNIZE DAMAGED BASES.) solution: TRANSLESION SYNTHESIS (TRANSLESION DNA POLMERASES. -Dna pol's 2, 6 and 5 can swap with Dna pol 3 to replicate OVER LESIONS. -However, they have a HIGH ERROR RATE than pol 3. (trade off). -SOS RESPONSE!!!

BLOCKS to replication (cases where rep doesnt need to re-started, however blocks need to be overcome) -a solution?

with ssGap/lession in lagging, repair can fix. HOWEVER, top leading strand is stuck with no way to restart because there is no 3' end, THUS lays down NEW PRIMER WITH DnaG! (continue) Pol 3 is a highly specific active site, thus low mutation rate and can only recognize proper base pairs. (POL 3 CANT RECOGNIZE DAMAGED BASES.) if base is damaged, methyl added ect. it can't recognize and it's stuck. how is it overcome? -(lagging strand) not that difficult, if we have a rna primer here and (opp dirction) and dna pol 3 moving along to get stuck at ssGap, replication complex can still proceed because another primer can be laid down and repair processes can come and fix that. (not discontinuous) HOWEVER, top leading strand is stuck with no way to restart because there is no 3' end, THUS lays down NEW PRIMER WITH DnaG! (solution!) dnaG can come ahead of damage, lay down new primer so dna pol 3 can jump ahead and continue replication. the ssgap lesion can be fixed later) -this is how these lesions kind of 'jumped' over. ANOTHER THING THAT CAN HAPPEN IS: -3 NEW dna polymerases that the cell uses, 2,4 and 5 to swap out with dna pol 3 when there is a lesision solution: TRANSLESION SYNTHESIS (TRANSLESION DNA POLMERASES. -Dna pol's 2, 6 and 5 can swap with Dna pol 3 to replicate OVER LESIONS. -However, they have a HIGH ERROR RATE than pol 3. (trade off) because their active sites less speficic. -also seen in sos response, where there is so much damage that just make a bunch of translesions polymerases to replicaite over and deal with the mutations so rep. doesnt stop

Technique for isolating auxotrophs

would be the same for temperature sensitive (same kind of condition). would be looking for something that grew at 28, but didnt grow at 37. (this would be a temp. sensitive mutant). -where is the mutant? no idea, so must do more work to figure out why it's not growing at 37 but is at 28.

1) confusion? lol

you take a flask of strep sensitive e.coli and plate on strep, you may get a few colonies. -however if you take same strep sensitive and then grow in presence of strep, before you plate, and then plate this on strep, you get a ton of colonies. -interpret as bac adapting to 'presence' of strep, leading to more strep resistance mutants. -exp. disproving this is Lurai Delbrok fluctuation test -T1 phage interacts with TonB receptor -E coli tonB+ are killed, so they're asking for the evolution of TonB-. 1) Took large flask of tonB+ (sensitive to phage) E coli and plated aliquots/samples on plates with T1 phage. -get a mutants in roughly equal number 2) same exp. except plated independent cultures on phage. -saw some cases with very few mutants, and some cases with a ton of mutants. numbers were very different, not due to random change. -very good evidence the bac were NOT ADAPTING to the phage. -if lamarkian evolution directed mut, the adaption would be happening on these plates -if darwinian, random mutation would then lead to the number of . (it's selection for pre-existing mutation)

QQ: nomenclature ?? ask

~hisA: gene (or rna) ~hisAT98A: mutant designation, gene HisA: protein HisA+: phenotype HisAK98A: mutation, gene His+: protein designation His, His-: phenotype

α dnaE ε dnaQ θ holE β dnaN dnaX dnaX holA holB χ holC φ hol D

α dnaE- Polmerization ε dnaQ- 3' to 5' editing θ holE- present in core β dnaN -beta sliding clamp dnaX- organizes complex; joins leading and lagging strand dnaX- Binds clamp loaders and SSB protein holA- Clamp loading holB- Clamp loading χ holC- Bind SSB φ hol D- Holds χ hol C (which binds SSB) to the clamp loader

Scenario 2: Presence of Lactose

β- galactosidase (LacZ), when at low concentrations, cleaves and rearranges lactose to make the inducer allolactose. Allolactose binds to LacI, reducing its affinity to the operator and thus allows induction of the operon.

λ vs T4

λ /T4 1. Lysogenic / lytic 2.Circle via cos/ Circle via terminal repeats. 3. theta replication + rolling / rolling only. 4. Rolling circle makes concatemers/ Concatemers formed via recomb. 5. Cut concatemer at cos sites/ cut concatemers according to 103% length.

Reverse transcriptase in eukaryotes

Extend the 3' end with it it has its on RNA template

When in the same cell, R100 blocks transfer of F (reduced the fertility of F). How does this work?

F and R100 have interchangeable tra genes

What are 2 low copy number plasmids?

F and RP1

What are 3 examples of conjugative plasmids?

F, RP1, R100

What is 'fertility inhibition'?

Fertility inhibition refers to a phenomenon where when one plasmid is in a cell with another, it can block the fertility function of one of the plasmids

Slow Release

Filamentous phages can extrude individual progeny through cell envelope.

What is the function of FinO?

FinO codes for a repressor that inhibits traJ that is needed to switch on genes

What is the main reason eukaryotes have gene regulation?

For Cell Specialization. They are multicellular and consist of hundred of different cell types, each differentiated to serve a different specialized function.

Western Blots

For identifying a specific protein product from a mixture using proteins. 1. Primary antibody specifically binds to target protein. 2. Secondary antibody binds to the primary antibody. 3. Tag on secondary antibody for easy detection. 4. Run thru electrophoresis + 5. probe membrane with primary antibody; after, probe with secondary antibody.

Maternal

For normal development, the H19 gene must be active on the ____ (maternal or paternal) chromosome only and silenced on the other

Paternal

For normal development, the IGF2 gene must be active on the ___ (maternal or paternal) chromosome only and silenced on the other

Dominance variance (Vd)

For quantitative traits, the portion of genetic variance attributed to the dominance effects of contributing genes

Interactive variance (Vi)

For quantitative traits, the proportion of the genetic variance derived from epistatic interactions between the alleles of genes that influence a quantitative phenotype

Additive variance (Va)

For quantitive traits, the component of genetic variance contributed by genes having an additive effect on phenotypic variance

Body plan

For the tissues to function effectively all over, its three d arrangement must be established and superimposed on the differentiation process

inducer

For transcription to occur in the lac operan, a(n) ___ molecule (a derivative of lactose) must bind to the repressor, causing it to change shape and release from the operator.

2-3 stem loop

Formation of this stem loop allows RNA polymerase to continue transcription through the leader region and into the structural genes of the operon

3-4 stem loop

Formation of this stem loop in the trpL mRNA terminates transcription in the leader region before it reaches the structural genes of the operon

Region 2:3 anti-attenuator stem loop

Forms when trp levels are low. Scarce tRNA trp makes ribosomes stall at trp codons in region 1. Ribosome covers region 1 and allows region 2 & 3 to form stem loop. Less energetically favorable 3:4 stem loop cannot form.

Region 3:4 attenuator stem loop (terminator stem loop)

Forms when trp levels high --> RNA polymerase falls off RNA before it transcribes structural genes. Ribosome translates through trp codons & encounters translation stop codon between regions 1 & 2. Ribosome stops & covers regions 1 & 2, allowing 3&4 to form 3:4 loop which binds RNA polymerase and causes it to release before reaching trpE.

What is the difference between forward and reverse genetics?

Forward genetics begins investigation with a mutant phenotype and proceeds toward the identification of the gene sequence, while reverse genetics begins with a gene sequence and seeks to identify the corresponding mutant phenotype

Why are plasmids ubiquitous?

Found in every ecological niche on the planet - e.g. soil, body

Bernard Davis

Found out that no contact between the two sides of the U tube led to no recombinants.

Frameshift mutation:

Frameshift mutation: reading frame disrupted by addition or subtraction of bases. (Crick/Brenner used this, EtBr) if rf mutation, -bac. evolved if theres some sort of mutation, these other frames dont really code for long stretches. -nice b/c, for any segment of dna, there's just one open reading frame (typical). (cuz lots of stop codons, if all just oprf's without stops, head to tell which gene is which.

Trans acting factor

From a seperate source (protein, gene, whatever, sequence)

Cis acting factor

From the same source, gene, place protein sequence

Gene

Functional unit of nucleic acid. Can encode catalytic rna.

conclusions from Franklin and Wilkins

G-C three bonds A-T two bonds AntiPolarity

What happens when there is an absence of galactose?

GAL80 protein binds to GAL4 and prevents the activation of transcription.

UV light

GFP fluoresces when exposed to:

Negative control of gal

GalR repressor, two P two O, UdP-galactose involved in LPS and capsule synth

What happens when there is a presence of Galactose?

Galactose binds to GAL80 and prevents the GAL80 from binding to GAL4. Thus GAL4 then activates the transcription of galactose metabolizing genes by interacting with BAT.

Operons

Gene expression in bacteria is controlled primarily by regulating these:

Gene knockouts

Gene insertions that result in a deletion of the entire coding region of the gene creating null alleles that produce no protein product are known as:

Repressible operon

General term for an operon that is EXPRESSED under one set of environmental conditions but whose transcription is REPRESSED under an alternative environmental condition

Compare the roles of general and specific transcription factors in regulating gene expression.

General transcription factos function in assembling the transcription initiation complex at the promoters for all genes; specific transcription factos bind to control elements associated with a particular gene and either decerase or increase transcription of the gene

Homologous genes

Genes descended from a common ancestral gene

Repressible

Genes involved in anabolism are typically this type of operon:

Inducible

Genes involved in catabolism are typically this type of operon:

syntenic genes

Genes located on the same chromosome -so close together that alleles cannot assort independently

constitutively (constantly)

Genes that code for regulatory proteins are examples of genes that are ___ expressed.

Horizontal Gene Transfer

Genes transferred from one organism to another (donor to recipient)

Paralogous genes

Genes whose origin lies in a gene duplication event within an extant or ancestral species

Orthologous genes

Genes whose origin lies in a speciation event in two different species

Prophage

Genetic material of a virus integrated into a bacterium. Able to reactivate at any time.

Inherited Predisposition to Cancer

Geneticists are devoting much effort to identifying inherited cancer alleles so that predisposition to certain cancers can be detected early in life. About 15% of colorectal cancers, for example, involve inherited mutations.

Genome of prokaryotes vs. eukaryotes

Genome of prokaryotes •In prokaryotic genomes, most of the DNA codes for protein, tRNA, or rRNA •The small amount of noncoding DNA consists mainly of regulatory sequences, such as promoters. •The coding sequence of nucleotides along a prokaryotic gene proceeds from start to finish without interruption by noncoding sequences (introns). Genome of eukaryotes •In eukaryotic genomes most of the DNA does not encode protein or RNA, and it includes more complex regulatory sequences. •In fact, humans have 10,000 times as much noncoding DNA as prokaryotes. •Some of the noncoding DNA in multicellular eukaryotes is present as introns within genes. Indeed, introns account for most of the difference in average length between human genes and prokaryotic genes.

Reverse genetics

Genotype (Gene) ➜Phenotype (Mutation to phenotype) -start with gene make specific mutations in gene to create mutants

SgrS

Glucose 6 PO4 toxicity Activated to induce transcription when its present. SgrS binds to Hfq inhibiting ptsG

Inducer Exclusion

Glucose transport by PTS causes catabolite repression of lacY permease activity.

GFP

Green fluorescent protein 1. GFP fused to protein. 2. Ask where protein ends up.

Coordinated Gene Regulation in Eukaryotes.

Groups of genes that are able to turn off and on together but are not clustered together in an operon.

Gene families

Groups of genes that are evolutionarily related

GyrA

Gyrase Nick closing

what's a mutation? lesion?

HERITABLE CHANGE in the genome of an organism. (DNA OR RNA[genome of viruses] CAN BE CHANGED, HOWEVER UNTIL THEY GET HERITABLE, NOT CONSIDERED MUTATIONS. different from a lesion... LESION: which is a mistake or damage to a genome. (not yet a mutation.) because they havent been 'fixed yet', can still be repaired. -mutations dont have to be a phenotype, you can have changes in genome with no observable phenotype and still a mutation. (mutant usually an observabl phenotype) -go over 'hard example" -we have an AC mismatch error after a round of replication, and doesnt get fixed. (NOT YET MUTATION) then we have another round of replication, top strand goes back to wild type however bottom strands get replicated before it gets fixed, now we can see that where the mistake was is now the proper base pair which looks like normal dna and cell cant recognize mistake was there. MUTATION ALL NOT MUTATIONS: -wrong NTP in mRNA (mRNA not in the genome, not a heritable change;maybe mutant protein) -methylation of DNA(DNA is highly methylated and this can be reversed this not a mutation) -wrong dNTP by DnaPOL (if not fixed a mutation). (dna pol often does but in wrong dntp, however could be fixed)

Abortive Intiation

Happens between open complx and promoter escape. -Sigma leaves after 12 bases added. then elongation? -trx can be discontious, bumps and pauses (backtracking)

DNA polymerase 1

Has 5' to 3' exonuclease activity. -'repair polymerase,; which fills in single-stranded gaps; -REMOVES RNA PRIMERS (?) -also involved in repair of the gaps formed on the lagging strand during replication. - Also possesses both 50-30 and 30-50 exonuclease activity.

Global systems

Heat shock response, nitrogen metabolism, spore formatoin in b. sub

dnaB

Helicase. It unzips the DNA is a hexamer (donut ring of 6 tiny balls!) and breaks H bonds between base pairs to destablize double helix and create replication fork. -Seperates the DNA strands acting like a wedge wrapping itself around one strand and DnaB is main one in E-Coli. -uses energy from ATP to break h bonds holding bases together, allows 2 parental stands to begin unwinding creating 2 replication forks. (each parental strand has own helicase. -E.coli has at least 6 diff. types of helicases, some involved in repair/or conjuction, but principal helicase in dna replication is DNAB!!! - ie. in conjugal plasmid transfer.

Regulatory Proteins

Help a cell sense internal changes and alter its gene expression to match.

Epigenetic

Heritable patterns or changes in gene expression that are not associated with any change in DNA sequence

What does 'HFT' stand for?

High Frequency plasmid Transfer

In general, what is the effect of histone acetylation and DNA methylation on gene expression?

Histone acetylation is associated with gene expression; DNA methylation is associated with lack of expression

chromatin remodeling

Histone modifications and DNA methylation affect ___, which can affect whether particular genes are transcribed. (eukaryotes)

histone variants

Histones with a slightly different composition than the standard histone; Exist for all histones except H4; created by an accumulation of mutations in one or several of the 70+ genes that encode for histones

RNApol + sigma70

Holoenzyme

Most important question in biology

How a fertilized egg develops into a full-term animal or human? Somatic cell division should produce genetically identical cells; but how come the cells in our body have different structures and function

Using molecular probes

How are DNA libraries screened for specific DNA sequences?

Both require fragments to overlap for contig assembly and sequences are required to be annotated after contig assembly

How are clone-by-clone sequencing and whole genome shotgun sequencing similar?

Masking of mRNA delays or prevents translation

How are eukaryotic genes TRANSLATIONALLY regulated?

By duplication of the genomic DNA or by unequal crossing over at prophase I of meiosis

How do duplicate genes arise?

Can be upstream, downstream or even within the gene

How do enhancer sequences differ from proximal elements and the core promoter?

Knockout mutants have mutations in their genetic code that make their genes lose proper function, whereas RNAi uses double stranded RNA complementary to the target gene to degrade the mRNA of the target gene

How do knockout mutations and RNA interference differ?

Animals do not exhibit totipotency, so the transgenes must be injected into eggs, embryos, or cells that give rise to gametes in order to create a fully transgenic animal

How do the methods of producing transgenic animals differ from producing transgenic plants?

By direct entry to the DNA not bound by histones, by chromatin remodelers that change the distribution or composition of histones, or by chromatin modifiers that add methyl or acetyl groups to modify histones

How do trans-acting proteins access specific regulatory DNA sequences in eukaryotic chromosomes (3 ways)?

Calculate the survivors using relative fitness values, and then use the allele counting method

How do you calculate the allele frequencies in a next generation after natural selection occurs?

Multiply the original number of each genotype by each genotype's relative fitness

How do you calculate the survivors of each genotype after natural selection acts on a population?

Provides information that helps align the physical map with the known chromosomes of the organism

How does comparison of the physical map to a genetic map in clone-by-clone sequencing aid in the sequencing?

More glucose causes less cAMP to be made

How does glucose effect levels of cAMP in the bacterial cell?

Enters a plant through wounds and then the virulence genes encode proteins that aid in the transfer of another portion of the Ti plasmid into the genome. This portion, known as TDNA, is inserted into the plant genome causing the plant to produce proteins that make the cells multiply in large numbers, creating crown galls

How does the Ti plasmid act in a natural system?

Any gene of interest can replace the TDNA portion and be integrated into the plant genome

How has the Ti plasmid been re-engineered to create transgenic plants?

It is expressed in the same manner as the transgene of interest and allows you to non-invasively visualize gene and protein expression

How is GFP used as a reporter gene?

Sequences of cDNA clones are compared with genomic sequences to identify the parts of the genome that undergo transcription

How is cDNA used in annotation?

Depending on the tissue type, different regulatory proteins are present that bind different enhancer sequences near the gene

How is tissue dependent transcription possible regarding the sonic hedgehog gene?

1000 bp

How many base pairs can dideoxy sequencing typically sequence?

What is the 'copy number'?

How many copies of the plasmid on average are inside that bacterium

5

How many structural genes are there in the trp operon?

3

How many structural genes make up the lac operon?

Q-RT-PCR

How much RNA copies are in a cell, pcr based on number of starting pieces, SIGMOIDAL, run out of something is a level off in graph

interference [I]

I=1-c -the double crossovers expected but not produced -negative interference produces more double crossovers than expected

QQ1

INVERTED REPEATS travel with the transposon or IS element when it hops. -IR is where cut site happens, DR are generated by where transposon inserts. -IR are constant locations, DRs are not cuz depends on where transposon hops.

QQ3:

IS elemts just encode transposases (own replication). neg impact is host has to replicatice transposon...(?)

QQ: Where does this variability arise from? (mutation rate not changing between cultures, where does variability come from?)

If a beneficial mutation occurred earlier for whatever you're selecting for, it comes down to chance. 'If you have a beneficial mutation early (high reproduction), this is called a jackpot mutation)' (look at line picture) -still only 1 mutation, just by change one happened earlier thus more TonB- reproduction. -so if calculation mutation rate, youd come up with very different rates thus calculating is tricky.

Yes

If a gene for antibiotic resistance is taken from one E. coli and introduced into the genome of another E. coli, would the recipient cell be correctly referred to as a transgenic organism?

QQ

If a mutation deletes the HDA locus, (which converts DnaA-ATP to DnaA-ADP and inactivates it, slowing down replication.) Get rid of HDA, state is ATP bound. Thus the logical answers is B, premature of DNA however actual answer is D because if done all the time, the cells cant properly segregate the chromosomes. -however you can rid dars sites, thus there is another mechanism for generate dnaA-ATP.

Clone by clone sequencing or whole genome shotgun sequencing

If an entire genome is too large to be sequenced by dideoxy sequencing, what two techniques can be used?

Post-translational

If an enzyme cannot produce a proper product, this is an example of _____ control

20%

If an organism has a relative fitness of 80%, what is its selection coefficient?

mazE/F method of operation

If anything in the cell effects the transcription or translation of mazE or mazF, then mazE would be degraded since it is an unstable antitoxin. This leads to MazF accumulating in the cells, which eventually leads to Cell Death (or inhibition of growth, if there is low levels of toxin present).

Complementation

If the mutations complement each other (Lac+) then they are on different genes

Translational

If there is no enzyme synthesis, this is an example of ______ control

Transcriptional

If there is no mRNA synthesis, this is an example of _____ control

On top of region 1, so regions 2 and 3 will pair, so the RNA polymerase continues to transcribe through the structural genes

If there is no tryptophan in the environment, the ribosome will stop here on the trpL mRNA:

On top of region 2, so regions 3 and 4 will pair causing RNA polymerase to fall off

If there is plenty of tryptophan in the environment, the ribosome will stop here on the trpL mRNA:

Both environmental variance and genetic variance

If two pure breeding lines of organisms are crossed and then the F1 selfed, the phenotypic variance of the F2 will be due to:

Environmental variance (Ve) only

If two pure breeding lines of organisms are crossed, the F1 phenotypic variance will be due to:

Jacob; Monod

In 1961 ___ and ___ proposed the operon model of gene regulation.

lacZ

In E. coli, beta-galactosidase is coded by the ___ gene.

lacZ

In E. coli, the gene that codes for beta-galactosidase is called ___.

lacY

In E. coli, the gene that codes for galactoside permease, the membrane protein that allows lactose into the cells, is called ___.

lacI

In E. coli, the gene that codes for the regulatory protein that shuts down lacZ and lacY is called ___.

Enhancer sequences

In bacteria, activator proteins bind to the activator binding site. However, in eukaryotes, activator proteins bind to:

Silencer Sequences

In bacteria, repressor proteins bind to the operator. However, in eukaryotes, repressor proteins bind to:

Directly prevent enhancer-mediated transcription

In bacteria, repressor proteins work by binding to an operator and overlapping the promoter so RNA polymerase can't access it. How do repressor proteins act differently in eukaryotes?

regulon

In cell biology and genetics, a(n) ___ is a collection of genes or operons under regulation by the same regulatory protein.

holE

In core

A program of differential gene expression leads to the differential cell types in a multicellular organism

In embryonic development of multicellular organisms, a fertilized egg (zygote) gives rise to cells of many different types, each with a different structure and corresponding funciton; cells are organized into tissues, organs, organ system, and then the whole organisms Any developmental program must produce cells of different types that form higher-level structures arranged in a particular way

chromatin remodeling

In eukaryotes, the first step in the control of gene expression is ______.

Combinational control of gene activation

In eukaryotes, the precise control of transcription depends largely on teh binding of activators to DNA control elemnts; considering the great number of genes that must be regulated ina typical animal or plant cell, the number of completely different nucelotide sequences found in control elements is small Combination of control elements in an enhancer associated with a gene turns out to be more important than the presence of a single unique control element

Near promoters

In mammalian genomes, CpG islands are clustered here:

transcription factor

In molecular biology and genetics, a(n) ___ (sometimes called a sequence-specific DNA-binding factor) is a protein that binds to specific DNA sequences, thereby controlling the flow (or transcription) of genetic information from DNA to messenger RNA. Transcription factors perform this function alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes.

beta-galactosidase

In order to cleave lactose into glucose and galactose, E. coli needs an enzyme called ___.

Threshold trait

In polygenic and multifactorial inheritance, a trait with different phenotypes that are determined by whether individual organisms are above or below a critical value in the phenotypic scale

Double digest

In restriction mapping, in order to determine which order of restriction sites is correct, this must be performed:

cis

In the lac operon, the promoter and operator are ___ acting (cis or trans?)

activator; repressor

In the presence of arabinose, the AraC protein is a(n) ___; in the absence of arabinose, the AraC protein is a(n) ___.

Corepressor

In the trp operon, tryptophan acts as a ______

Gene Amplification

In this very unusual process, the normal DNA replication process is seriously flawed. The result is that instead of making a single copy of a region of a chromosome, many copies are produced The genes on each of the copies can be transcribed and translated, leading to an overproduction of the mRNA and protein corresponding to the amplified genes as shown below. The squiggly lines represent mRNA being produced via the transcription of each copy of the gene.

Prokaryotes

In which organisms has lateral gene transfer been an important evolutionary mechanism?

Negative Regulation

Inactivation of transcription from a promotor by a REPRESSOR (cis acting)

Additive genes

Incremental differences are observed in phenotype when these types of genes are involved:

Allolactose

Inducer of the lac operon.

Inducible vs. Repressible operons

Inducible are catabolic and Repressible are anabolic (use energy)

The genes that control galactose metabolism with GAL4 is considered to be what?

Inducible.

Epigenetic inheritance

Inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence The chromatin modifications that we have seen do not entail a change in the DNA sequence; yet they may be passed along to future generations of cells; inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance DNA mutations are permanent Chromatin can be reversed May explain why one identical twin gets a genetically based disease and the other one doesn't;

Rifampicin

Inhibits bacterial RNA polymerase by binding and creating a physical block

Actinomycin

Inhibits transcription, binds to DNA

dnaA

Initiator Protein, Primisome (priming complex formation). Starts the whole replication process and is the central regulator of replication. -promotes unwinding at oriC

What are the categories of TE's?

Insertion sequences, transposons, mutator phages

Single

Introducing a circular molecule of DNA into a genome favors retrieval of recombinants produced by (single or double) crossover

Double

Introducing a linear molecule of DNA into a genome favors retrieval of recombinants produced by (single or double) crossover

Site-directed mutagenesis

Introduction of specific nucleotide changes in a DNA molecule in vitro

Inversions

Inversions do not delete DNA however swap the order. -we are having homologous recombination between INVERTED repeats. -instead of repeats going in same direction, they're point towards one another. -you don't really lose any dna

Inversion mutation

Inversions do not lose any dna, genes are intact, so these are often LEAKY. (Can have a larfe scale effect) -don't see this often because chromosome have structure and direction. (ter sites, oriC, dif sites, cop sites that interact with ftsk that are directional) even though genes may be intact, youd be messing up a info thats directional in the chromosome structure.

parM and ParAB proteins

Involved in plasmid partitioning.

What is the 'coupling system'?

Involves traG protein that aids transfer of plasmid, where the end result is both donor and recipient containing the plasmid

No

Is a genetic variance of zero ever found in nature?

No

Is an environmental variance of zero ever found in nature?

What happens when glucose and lactose are present in E. Coli enviornment?

It prefers glucose; the enzymes for glucose breakdown in glycolysis are continually present. Only when lactose is present and glucose is in short supply does E. Coli use lactose and only then does it synthesize enzymes necessary for lactose breakdown

What does ribonucleo reductase do?

It's an enzyme catalyst that make DNA precursors

TraI

Know as relaxase. It is an endonuclease that nicks DNA. RELAXASE pushes the DNA into the recipient cell, then comes into the recipient cell after.

(M)Mendel ***************** start a chapter 1

L

Mendel ***************** start a chapter 1

L

pET vector

LOOK OVER AGAIN. LYTIC PHAGE SLIDE 19

LacO c

Lac+ (Changed lacO and its not recognized by LacI+) Cis acting; DOMINANT

LacI -d

Lac+ (Forms inactive dimer with LacI+) trans acting; DOMINANT

LacI mutation (-)

Lac+ (LacI cant binds to the operator) Recessive to (+) Repressor gene that encodes for a diffusible product (turning of Y and Z)

LacY mutation

Lac- Trans complementation; Recessive

LacI s mutation

Lac- (LacI binds to LacO but is not released by the inducer) Trans acting; DOMINANT

LacZ mutation

Lac- (beta galactosidase inactive) Trans complementation; Recessive

LacP mutation

Lac- (different) (Changed promoter, not recognized by sigma70) Cis acting; Recessive

Lac Presence of lactose

LacI becomes allolactose, RNA polymerase binds to operator, ON

Scenario 1: Absence of Lactose

LacI binds as a tetramer to the operator region. It represses the lac operon by preventing open complex formation by RNA polymerase.

Lac Absence of lactose

LacI binds to operator, OFF

Uninducible Lac- mutants

LacZ, LacY, LacP, LacIs (Superreppressor)

First Primer located on

Lagging strand

What is the lambda phage receptor in E.coli? What is the receptor for T4?

Lambda = maltose porin ; T4 = OmpC porin

Genetic Island

Large insertions of sequence

LUCA

Last universal common ancestor. alpha proteobacteria, symbiosis with bacteria became mitochondria.

lig

Ligase, seals nicks

Describe the characteristics of the genome of T4 and lambda bacteriophages.

Linear, double-stranded DNA genome.

Regulatory Gene

Located some distance from the operon it controls and has its own promoter; regulatory genes are expressed continuously at a low rate Why not turned off permanetely? The binding of repressors to operators is reversible (an operator vacillates between two states-one with out the repressor bound and one with the repressor bound; the relative duration of each state depends on the active number of repressors) and trp the repressor is an allosteric protein with two altnernative shapes (active and inactive); the trp repressor is synthesized in an inactive form with little affinity for the trp operator

The CpG islands are often methylated. CpG methylation is associated with what?

Long term gene repression. Such as the inactivation of the X chromosome in female mammals.

Replication fidelity 2) MDM

METHYL DIRECTED MISMATCH REPAIR. dam-deoxyadenosine methylase. GATC -thus new delay in methykation, ch3 to distinguish between old and new. MutS, MutL, MutH endonuclease bind to mismatch MutH cuts 5' side of unmethylated GATC yrD helicase unwinds in front of mismatch New strand degraded by special proteins

Shigella in the body

MORE IN NOTES has a type 3 secretion system to kill macrophages. It has lost and gained a lot of things to evolve from nonpathogenic E.coli into shigella.

BioRad Gene Pulser

Machine used typically for electroporation.

Temperate Phages

Maintain stable relationship with host cell; Neither multiply nor are lost from cell.

DNA protein interaction

Major grooves, Helix turn Helix motifs of proteins (dimers bind inverted repeats)

Lysis

Makes protein to depolymerize peptidoglycan --> Bursts through cell wall.

Luciferase

Makes theee light from LuxR!! after phosphorylation :]

What is the process that involves the formation of sex pili and a channel through which DNA passes from donor to recipient?

Mating Pair Formation (Mpf)

What are T4SS?

Method by which bacteria can deliver macromolecules from the cell

Dam Methylase

Methylate "A" in the 5'-->3' GATC sequence (One side)

Cytosines adjacent to guanines

Methylation involves attaching CH3 groups to which bases?

Genomic imprinting

Methylation permanently regulate gene expression of either maternal or paternal allele of a particular gene at the start of development

Mosaic Nature of Genomes

Microbial genomes have undergone extensive gene loss and gain (deletions, insertions, mutations, etc.)

Proteins in cytokinesis of Ecoli

MinD/MinC: move back and forth MinE: drives pole to pole oscilation of mind/c SlmA: associate with nucleoid so ftsz cant bind when its present

What is the 'mobilisation' process?

Mobilisation is when a conjugative plasmid assists the transfer of a NC plasmid into a cell

______ suggested that nonparental allele combinations resulted from recombination between the X chromosomes of the heterozygous female parent

Morgan -X linked gene for eye/wing in Drosophila

Where is DNA methylation most common? What is this called?

Most common on the cytosine bases adjacent to the Guanine Nucleotides called "CpG Islands"

Mutants in DnaQ are called what?

MutD

Methyl Directed DNA mismatch repair

MutS, MutL, MutH endonuclease bind to mismatch MutH cuts 5' side of unmethylated GATC yrD helicase unwinds in front of mismatch New strand degraded by special proteins

(continuing lecture 12) Mutation: Silent

Mutation: heritable change to the genome, doesnt have to have function/protein/coding. Silent mutation: mutation that is heritable but no observable phenotype.

Cytosine

NH2 Group and Db oxygen

Adenine

NH2 group

Are eukaryotes genes organized into operons?

NO -Typically have separate mRNA's

QQ: can a deletion be reverted? (think for inversions too)

NO, because the info sequence is gone. deletion mutation never reverted. -could be suppressed? -can you suppress that deletion of Z so that the cells can still grow with you add them with sugar A? YEAH, if you suppress it, get rid of X. then A is no longer toxic to cells. so even though thats a deletion of Z, youre able to supress it by making a mutation in a. -one example of how youre able to suppress a deletion.

Resolution

NOT magnification, ability to distinguish between 2objects as separate; dictated by light property. limits the size of can been clearly seen by microscopy

Additive variation

Narrow sense heritability estimates the proportion of phenotypic variation that is due to what?

Va/Vp OR R/S

Narrow sense hertitability h2 = (2 things)

Allolactose/IPTG

Natural inducer of the lac operon/gratuitious inducer

Directional selection

Natural or artificial selection that continuously changes the frequency of an allele in a particular directoin

Sigma Factor Regulation

Need to coordinately activate genes, operons, and regulons of seemingly disparate function. Regulate by synthesis or activity of sigma factor that directs the expression of all those genes.

Repressor

Negative control utilizes this type of protein

What does SeqA do?? (uhm)

Negatively regulates DNA replication at OriC by binding to hemimethylated sequences and sequestering it to prevent further replication by binding it to cell membrane....

AraC

No arabinose N terminal arm binds dna, Dimer binds, Forming a dna loop that represses expression

araCc/araC+

No inducer = araC+ acts as a repressor in absences of arabinose

Lack of gene expression

No mRNA and no protein

RNA synthesis requirements

No primer Synthesized 5'-->3' Read 3'-->5'

proofreading in transcription compared with RNAP mutation generation

No proofreading or editing in transcription doesn't lead to more mutations from RNAP because mutations in RNA aren't hereditary ---any mistake wouldn't be considered a mutation yet!

Does gene regulations stop?

No- Even when an organism is fully developed, gene expression is fully regulated in a similarly fine-tuned manner Any changes in the expression of one or few genes can lead to development of cancer

Proteins of cytokinesis in Bacillus

Noc =SlmA (nucleoid association) DIVIVA: Fixes MinD/C to poles

Nonsense mutation: mut. that causes a

Nonsense mutation: mut. that causes a STOP codon. -special class of missnese muts (base change) -usually NULL. if you make a nonsense mutation in a coding sequence, translation stops (partial protein, function, uh release factor?) -may be leaky if close to the C-terminus of a modular protein, the internal domain in the n-terminus making the protein would not be affected. -or a nonsense repressor

**Termination

Not essential or as precise. 2 proteins called TerA and TerB in specific region of the chromosome, that act like one way gate in automobile parking lot, allowing replication forks to pass one direction but not another. -Reason it stop is because of Tus protein (Terminus utilization substance) binds to Ter sites and kicks off replicating helicase (dnaB in E coli). -Tus protein interacts with one strands. -knock out ter sites, cell does okay without them. -ter sites are site specific dna sites in the genome with a direction to them, and the replication complex can proceed in one direction. -this is theta replication

Homologous nucleotides

Nucleotides descended from a common ancestral nucleotide

post-translational control

Of the three modes of gene regulation, which is the fastest in response time (in terms of regulating the activity of the gene product)?

transcriptional control (can stop gene expression at the earliest possible step, saving the most energy and materials for the cell)

Of the three modes of gene regulation, which is the most efficient in resource use?

Auto-inducer in Gram positives

Oligopeptides, to be cleaved and or modified

Regulation of Transcription initiation

Once chromatin has been modified for expression, the initiation of transcription is the next major step at which gene expression is regulated

Design of PCR primers

One for sequencing, Two for PCR Extension is toward 3' end

Bicoid gene

One maternal effect gene (egg- polarity gene, because they determine axes) is called Bicoid gene (two-tailed) encodes a protein (morphogen) that determine head structures Hypothesis: Bicoid normally encodes for a morphogen that specifies head (anterior) end of the embryo Prediction: If this is true, then the Bicoid mRNA and its encoded protein should be localized in the anterior of the egg Results: only find the Bicoid mRNA where the head should be! Make a mutant, where the mother is defective for this bicoid gene; the mutant has two tails =no headdevelopment Indicates morphigenous define the polarity of the egg

What happens in 'segregational loss'?

Only 1 of the 2 daughter cells receives a copy of the plasmid after replication

Operons

Operons are multiple coding sequences on one transcript -Remember there is one promoter, (trx controlled by one promoter, one information), however each gene is controlled by it's own ribosome binding site that can be different. -Ex., A is very good, you get high levels of translation of A, so base pairs well with 16s rRNA. -Thus the translation of these are all INDEPENDENTLY CONTROLLED (different) -Transcription is controlled together (same)

1

Organisms of the genotype with the highest reproductive success are given a relative fitness (w) of:

Transgenic organisms

Organisms that are the recipients of genes inserted into their genomes

DnaX

Organizees complex, joins leading and lagging DNApolIII complexes, clamp loading

DNA replication Begins

OriC

itiation

OriC, dnaA

What does 'oriT' stand for?

Origin of conjugal DNA transfer

what site carries the growing polypeptide chain?

P site!

DnaG

PRIMASERna Primer synthesizer

Proteins of cytokinesis in caulobacter

ParB: at one pole (MipZ), Links Mipz to polar DNA loci (centromeric DNA near oriC) Ftsz: at other end. Polymerizes position furthest from loci of parb/mipz complex (middle) replication of dna creates ParB at other pole

Caulobacter crescentus mech nucleoid occlusion

ParS: (near ori-rep) ParB: (binds to parS spreads to neighbor chromo) ParB+ParS: localized to old cell before rep. by PopZ ParA: binds ATP forms filaments TipN: fixes filaments ParB+ParS ratchet along

Where did the extra hershy and chase phage dna go

Phage dna make more than needed and some done even bind to share

Forward Genetics

Phenotype (how trait physically observed ➜ Genotype (actual DNA sequence). (Phenotype to Mutation) -do a mutagenesis, map gene sequence and got strong with WGS (whole genome sequencing which made sequencing cheaper and mapping takes too long). -contrasts with classical genetics which relies on selecting mutants on the basis of their phenotype and then studying the nature of the mutation -start with mutants with desired phenotype localize and identify gene with mutation *study the properties of the mutant to understand the function of the gene

***(Lecture 17) One thing that confused mutation rates in.. Phenotypic lag:

Phenotypic lag: if a mutation is not an instantaneously phenotype, and it takes awhile for the mutation to take effect. -ex. if a cell divides, we have a wt and one cell become a mutant, changes so that one protein is not made, the protein that was already there doesn't instantly disappear. -'"time delay in some mutations"

Vibrio Harveyi pathway QS

Phosphorelay 1. High density is light 2. Low density is dark

Endospore formation in Gram+

Phosphorelay systems activate relay of sigma factors

LuxU

Phosphotransfer protein (Step two)

What are the 2 methods of plasmid detection?

Physical and genetic

Temperature sensitive mutant

Pick E.coli essential gene rpoA^ts (temperature sensitive). -If we had our wild type rpoA growing @28C and @37, both grow. -However if we had (temp sensitive) rpoA^ts, it grows @28 but now @37 it's lethal. -so we can propagate mutation at 28, and when we want to study the function we ship the temp up to 37, where rpoA function is lost, and we can see affect on cell for limited time (cuz cell doesn't divide/propagate.) -this is a temp. sensitive, sort of like auxotrophs

PFU

Plaque forming units

What does 'narrow host range' mean?

Plasmids can replicate in a related group of bacteria

What does 'broad host range' mean?

Plasmids can replicate in a unrelated group of bacteria

Mutation Rate

Pol 3 errors 1/10^5 nt's Proofreading fixes 99% MDM fixes add'n 99.9% =1 mutation for ever 10^10 base pairs formed thus 1/2 However if dnaQ mutation (ridding it), then 1 mutation/ 10^8 bps, thus 1/20 progeny. However if also (double mutant) an MDM mutation, 1 mutation/ 10^5 bps, thus 50 mutations per progeny.

Degredation of primers and replacement with DNA by extending 3' end of okazai fragment

Pol I (PolA)

5' --> 3' exonuclease

Pol I (degrade primers, widen gaps)

3' --> 5' exonuclease

Pol I and Pol III

Polarity

Polarity means that you have upstream mutations (towards 5' end) that impact downstream gene expression. (big concern talking about operons) -mutation in A (mut 1, phenotype biofilms), we don't know if it's due to defect in A,B, or C. The mut. in A could be affect B and C in the operon. (polarity)

PolA

Polymerase I DNA, Primer removal, gap filling,

Quorom sensing

Population density dependent regulation of gene expression -coordinates group behavior, regulates traits like biofilm, competence, virulence

activator

Positive control of transcription occurs when a regulatory protein called a(n) ___ binds to a regulatory sequence in DNA.

Activator

Positive control utilizes this type of protein

Sequestering Ori-C

Prevent replication 1. Binding needs fully methylated dna 2. hemimehtylated dna binds SeqA and those ind regoin overlap dnaA binding sites blocking the binding of DnaA

Restart replication forks

Primosome and D loop formation or Proteins assemble relisome using 3'-OH as a primer

DnaA

Primosome, Initiator protein, priming complex formation

Whole genome shotgun sequencing

Prior construction of a physical map is not needed in this genome sequencing approach:

LESSON 5: Linear chromosome End problem -can apply to phages as well

Problem: rna ends on linear chromosome that need to be dna (5' rna ends), and (in circular chromosome, dna pol 1 and fixes that rna,) *however here there is NO PRIMER on end for dna pol, which always need a primer*, which always need a 3' end primer. (essentially gaps of single stranded rna (Dna?)). solution: 1) PHAGE LAMDA 2) TERMINAL INVERTED REPEATS (TIR)

Transduction (two types)

Process by which bacteriophages carry host DNA from one cell to another. 1. Generalized (can transfer any) 2. Specialized (can transfer only a few closely linked genes)

Quorum Sensing

Process where bacterial cells work together at high density. 1. Autoinducer is secreted by cells; At certain extracellular secretion, reenters cells. 2. Binds to regulatory protein (in this case, LuxR) and complex activates transcription of target gene.

transcription; translation

Prokaryotic processing features simultaneous ___ and ___.

Covered promoters

Promoter in which nucleosomes are found adjacent to the transcription start site, preventing efficient transcription initiation, and characterizes genes whose transcription is regulated

Open promoter

Promoters that reside in open chromatin resulting in constitutive transcription

2-D Gels

Proteins are separated in two dimensions. Can be isoelectric point, protein complex mass in the native state, or protein mass.

domains

Proteins have ___ that have a distinctive structure and function.

motif

Proteins have domains that have a distinctive structure and function. A domain that is observed in many different proteins is called a(n) ___.

What is the 'dtr' system?

Proteins locate a particular DNA sequence, form a nucleoprotein complex, cut DNA to start process of DNA transfer from donor to recipient

Trans-acting regulatory proteins

Proteins that are able to bind target regulatory sequences on any chromosome

Chromatin remodelers

Proteins that enzymatically change the distribution or composition of histone octomers (nucleosomes)

Chromatin modifiers

Proteins that enzymatically modify histones by adding or removing methyl or acetyl groups at specific amino acid residues

Transcriptional activators

Proteins that stimulate and stabilize the basal transcription apparatus (BTA) at the core promotor. Stimulate transcription.

Release Factors

Proteins whose structure resembles tRNA, and bind to stop codons. If the A site is empty, and translation cannot proceed any further, so the release factor comes and to bind, to destabilize the complex and mRNA/protein released and everything falls apart. then ribosome can go translate something else. -There are 2 different RF's the recognize different stop codons, and it's useful for genome engineering. RF1, UAG/UAA. RF2 UAA, UGA.

Name a bacteria that does not have a plasmid

Pseudomonas aeruginosa

Electroporation

Pulsing of high-voltage electricity makes cell membrane more permeable.

Tranversion

Purine to pyridmine (vice versa) -A to C, C to T (vice versas)

Transistion

Purine ➙ Purine -less disruptive to structure (A to/ G, C to/C)

Key components of a cloning vector

REase sites OriC Selectable markers (antibiotic resistance, lac Z)

process of translation

RNA --> protein

dnaG/ Primase

RNA primer synthezier/polymerizer. -LEAVES RNA PRIMERS ON LAG STRAND (?) -a rna polymerase that uses a dna template to make rna.

Alternative RNA splicing

RNA processing in the nucleus and the export of mature RNA to the cytoplasm provide several opportunities for regulating gene expression that are not available in prokaryotes. Different mRNA moleucles are produced from the same primary transcript, depending on which RNA segments are treated as exons and which are introns; The primary transcripts of some genes can be spliced in more thanone way, generating different mRNA molecules. Notice in this example that one mRNA molecule has ended up with the green exon and the other with the purple exon.

rut

RNA sequence (on mRNA) that rho binds to

difference between RNA and DNA

RNA: Uracil DNA: Thymine

digestion of RNA with Rnases

Ranges cuts up ribosomal RNA to give different products of 16S RNA, tRNA, 23S, and 5S

Proteolysis

Rapidly removes σ factors. Rapid turnover allows more exact control.

F+ Cell

Recipient cell that receives a copy of F factor.

Transformation

Recombinant DNA molecules are introduced into the bacteria by the process known as:

Gene Fusion (or translational fusion)

Reflects transcriptional and translational control of subject gene. Inserts reporter gene between target gene --> Produces target protein combined with reporter protein.

Operon Fusion (or transcriptional fusion)

Reflects transcriptional control of a subject gene. Reporter gene and ribosome binding site inserted into middle of target gene. --> Produces reporter protein and diminished target protein.

Regulation of Chromatin structure

Regulation of chromatin Structure (Histone Code Hypothesis: change in chromatin pattern, shape and transcription chromatin modification includes DNA unpacking which involves histone acetylation CO-CH3 (increase gene expression) and DNA methylation (inhibit gene expression) Heterochromatin (highly condensed chromatin) Methylation of histones condenses the chromation but phosphorylation next to methylation undoes that effect chromatin modification includes DNA unpacking which involves histone acetylation and DNA methylation 6 billion base pairs in our genome DNA of eukartyoic cells is packaged with proteins in an elaborate complex known as chromatin (basic until which is the nucleosome; packs cell's DNA into a compact form, and regulates gene expression Location of genes protmoter's relative to nucleosomes and to the sites where the DNA attaches to the chromosomes scaffold or lamina can affect whether the gene is transcribed or not If have heterochromatin=not expressed! (repressive) Certain modifications to the histones and DNA of chromatin can influence both chromatin structure and gene expression

negative

Regulation of the gene products of lacZ, lacY, and lacI is an example of ___ (positive/negative) control.

c; a; b

Regulation of the lac operon (matching): 1. lactose present, glucose absent 2. lactose absent 3. lactose present, glucose present a. operon is not transcribed b. operon is transcribed, but NOT sped up through positive control c. operon is transcribed quickly through positive control 1. ___ 2. ___ 3. ___

Silencer sequences

Regulatory DNA sequences that can repress transcription of specific genes that may be located distantly from the sequence

Cis-acting regulatory elements

Regulatory elements that are only able to regulate transcription of genes on the same chromosome and do not influence transcription of genes on other chromosomes

Attenuation

Regulatory mechanism in which translation of a leader peptide affects transcription of a downstream structural gene.

operator

Regulatory proteins bind to the ___ to control expression of the operon.

Enhancer Sequences

Regulatory sequences in eukaryotes that are located a greater distance from the promoter and bind regulatory proteins

Proximal elements

Regulatory sequences in eukaryotes that lie in close proximity to the promoter and bind regulatory proteins and are essential to promoter recognition and transcription

Translation Termination

Release factors RF2 : UAG, UAA RF3, RF4: interact with and activate hydrolase mimic tRNA cannot form peptide bonds

Stalled Ribosomes

Remember that Rna pol can get backtracked/stalled and factors such a GREB system can help. -Ribosomes are energy expensive (>90% of rna in a cell in ribosomal, rRNA. Also, majority of protein is in ribosomes.) -If ribosomes stuck on a mRNA, it needs to be recycled because much energy was invested.

RNaseH

Removes RNA primers. Endonuclease that cleaves rna in rna/dna duplexes. primer removal in eukaryotes. -a specific RNase which cuts RNA-DNA hybrids; involved in replication of ColE1- like plasmids. -

rnhA

Removes rna primers, endonucease cleaves rna

Invertron

Replication begins at both ends Promed by terminal protien attached to 5' ends

Telomere

Replication inititiated internally producing a circular dimer Telomere resolvase cleaves and ligates ends to hairpins

Replicative Transposition vs Non-replicative Transposition

Replicative: Transposon element is replicated to occur in both the original site and the target site. Non-replicative: Transposon element jumps from original site to the target site.

&&&&&&&&&&&&&&&&&&&&&& Puromycin

Resembles tRNA and enters the 'A' site = Premature chain release

Puromycin

Resembles tRNA and enters the 'A' site = Premature chain release

Topo IV (Ecoli) Topoisomerase II

Resolves catamers (cut and pass through method)

Heat-shock response element will respond to what?

Respond to heat and other stress.

LuxO + Sigma factor

Response regulator + sigma54

R = S(h2) OR R = mean of offspring - mean of population

Response to selection (R) = (2 things)

Pseudogenes

Reverse transcription of RNA and its insertion into the genome leads to the formation of:

Revertant:

Revertant:

Shine-Dalgarno Sequence

Ribosome binding site. Complementary sequence to 3' end of 16s rRNA.

Stringent response

Rich to minimal media accumulation of ppGpp (Some pppGpp)

DnaG

Rna Primer synthesizer

Northern blot

Rna extraction, electrophoreses, useful for questions about gene expression

Electrophoresis

Run to red 1. Seperation with electric field bc DNA is negative charged 2. Dyed with ethidium bromide and exposed to UV light 3. Small fragments go farther, compared to known ladder

what sets reading frame?

START CODON and RBS (ribosome binding site)

Rate of DNA replication

Same in all media

How do you find an auxotrophic mutant? (auxos easy phenotypes to measure, a lac- won't grown lactose. )

Say you had a plate of 8 colonies (His+/Bio+) (rich media, has histidine and biotin, cells don't have to make them just take from media), and you picked colonies and plate. -So to look for histidine auxotrophic mutant, (Can't make it's own histidine), would not give cells histine and look for things that couldn't grow. "Histidine auxotroph thus His-, Bio+." (requires extra histinde to be added to grow cuz cant make it's own, while wild type can) -Looking for Biotin auxotroph, His+, Bio- is a Biotin auxotroph. in reality would never get 2 colonies thatd work..qq

Ligase

Seals DNA nicks yo -forms phospodiester bond between 2 polynucleotide chains. -Seals single-stranded gaps (nicks) in double-stranded DNA. Also used for the forma- tion of recombinant DNA molecules in gene cloning. -ATTACHES OKASAKI FRAGMENTS TOGETHER BY FORMING COVALENT PHOSODIESTER BONDS BETWEEN NUCLEOTIDES.

Mean of parents - mean of population

Selection differential (S) =

Gene regulation through RNA splicing

Selection of Alternative splicing sites of pre-mRNA leads to the production of different proteins.

two rarely used amino acids

Selenocysteine Pyrrolysine

Watson and Crick

Semiconservative replication prediction

The nuclear membrane of eukaryotes does what?

Separates transcription and translation in time and space.

Pseudogenes

Sequences recognizable as mutated gene sequences often derived from gene duplication or retrotransposition events

Open reading frames

Sequences that have the potential to code for polypeptides in a DNA strand

Cis-acting regulatory sequences

Sequences to which proteins bind to regulate transcription of genes located on the same chromosome as the sequences

dars starts with (2.)..

Sequestion of OriC (intiation of DNA replication) by SeqA protein. -Hemimethylation/ MDM important -SeqA binds to hemimethylated DNA at OriC. -Site methylation by DAM, [GATC-Ch3]

Regulon

Set of genes and operons scattered around the chromosome with related functions. Controlled by a single repressor or activator.

Enhancer sequences

Sets of regulatory sequences that bind specific transcriptional proteins that can elevate transcription of eukaryotic genes

RNA binding site

Shine dalgarno

Promotor

Short DNA sequences recognized by the sigma factor that bind to RNAPol and recruit its core enzyme to the promotor for transcription at transcription start sites (part of the promotor sequence +1)

Promotors

Short nucleotide sequence recognized by sigma factors

Sticky end

Short single-stranded overhangs created by the cleavage of DNA by specific restriction endonucleases, which can potentially base pair with complementary single stranded sequences

CpG dinucleotides

Side by side cytosine and guanine nucleotides in the same DNA strand to which methyl groups can be added in nucleotide methylation

Consensus sequences of σ 70 promoters

Sigma factors contain similar sequences in -35 region / -10 region. Vary by only two bp in each one depending on the type of σ factor.

What do the genes on plasmids control?

Significant phenotypes

Cis

Silencer sequences are (cis or trans) regulatory sequences

Draw Rolling Circle Replication

Single strand nick at ori-T.

Factor-Independant termination

Site included in the region of two fold symmetry (like an inverted repeat) followed by four A's in the template strand

XerCD resolvase

Site specific recombination durning replication that resolves dimers by matching up dif sequences

Factor-Dependent termination

Site specifing a sequence in RNA (rut) recognized and bound by a protein (rho) that chases the RNA and releases it and the script at transcription pause sites (G-C rich)

Iteron sequences (IS sites)

Sites where RepA binds to do handcuffing. If IS sites are deleted, RepA can't bind, and the plasmid cannot replicate.

DnaN

Sliding Clamp

Two RNA's that trigger RNA interference. What do they do? How are they produced?

Small interfering RNA (siRNA) & MicroRNA (miRNA) They trigger RNA interference. They are tiny RNA's produced when larger double-stranded RNA molecules are cleaved by the enzyme dicer.

Autoinducer

Small molecule synthesized by LuxI that binds to regulatory protein and activates transcription of a target gene.

Effectors

Small molecules act as inducers, and co-repressors of transcription

end problem 1) PHAGE LAMDA

Solution is to make linear chromosome into a circle using COS SITES that are reverse complements. -after replication have these linear ends problem, lamda after replication has these sites with different overhangs called cos sites. they are REVERSE COMPLIMENTS, and one another to bind to one another. 5' ATGCAT' 3' reverse 5' GCATGC 3' (start at 3') these 2 overhanging complimentary single stranded pieces base pair and bind to form circle with cos in middle. this is replicated form of lamda.

Phase Variation

Some microbes use gene regulation to periodically change their appearance. Gene regulation depends on situation --> Regulation changes expression which changes appearance.

Define 'Broad Host Range plasmid vectors'

Some plasmids have replicons that allow them to replicate in bacteria other than E. coli

Mechanisms of Action of Repressors

Some specific transcription factors function as repressors to inhibit expression of a particular gene. Eukaryotic repressors can cause inhibition of gene expression in several different ways. block the binding of activators either to their control elements or to components of the transcription machinery. Other repressors bind directly to their own control elements in an enhancer and act to turn off transcription even in the presence of activators

Allele

Specific sequence variant of a given gene. IE a mutant of a wild type gene. Also, alleles would be the different possible flavors of ice cream :3

FtsK

Spools DNA through itself into daughter cells is a hexamer and two are needed

Phosphorelay of Spo0A B. subtilis

Sporulation: Kinase A-E phophorylates spo0F then Spo0B, then Spo0A (big 400+ genes)

How to replication start? Coordination Mechanisms

Starts at OriC, then DnaA protein binds to OriC to start bacterial replication (major point of E.coli coordination.) 4 mechs: 1. Inhibition of DnaA activity, 2. Sequestration of OriC by protein called SeqA -site of dna rep, oriC is physically sequestered/blacked thus blocking replication. This mechanism (right after replication dna state is one strand methylated, one stand not (HEMITHYLATED-MDM) 3. Repression of DnaA (Graph 4. Sequestration of DnaA

RNA pol backtracking

Stem-loop or hairpin at 3' end GreA/B exonucleases degrade rna until rearanged and properly prepared elongation continues

Name 4 gram positive bacteria?

Streptococcus, Enterococcus, Staphylococci, Bacillus

Genetics

Study of Discrete Nucleic Acids

Deletion

Substraction of one or more bases.

Nucleoside

Sugar+Base

Nucleoside

Sugar+Base WITH NO PHOSPHATE

DNA gyrase α gyrA ε gyrB

Supercoiling -DNA gyrase (UNWINDING) α gyrA- Nick closing ε gyrB- ATPase *gyrase is a specific Type II topoisomerase which adds negative supercoils into dna and uses ATP -stablizes dna as it's unwond. -topoisomerase's unwinds dna supercoiling in front of rep. fork.

Condensation of DNA

Supercoiling of DNA, over or under twisted ~10 bp/turn. - done by gyrases and toposimerases.

(****LECTURE 19)⇆ Suppressors:

Suppressors: are second sight mutation(not just one mutation) that RESTORES a phenotype. (mutant ⇆ phenotype) (reverting the phenotype, going either way mutant ⇆ wildtype ) -say you have a wildtype phenotype (can form biofilms), then you mutation that you cant form biolfilms. supressor would be mutation that now RESTORES the ability to form biofilms. -if you have a situation that can suppress that, and go back to biofilms-. (you're just reverting the phenotype, going either way!) ex. say you have an operon here with 3 genes, and a mutation in A. your wildtype is Arg+, the ability to make agrmine -now after a C➡T mutation in A is now Arg-. (this is called an AUXTROPHIC mutant). (anaboilc pathway). 6

Operator

Switch segment of DNA Turned on: RNA polymerase can bind to the promote rand transcribe the genes of the operon, but can be turned off by a repressor

Synomous mutation:

Synomous mutation: mutation that doesn't affect the AA(signal sequence?) -due to wobble codon

Features of secondary messanger systems

Synth enzymes, Degradation enzymes, Receptor protein, Output response, Thought to respond to change in envirionment

LuxI

Synthesizes acyl homoserine lactone autoinducer (AI).

TAT (general way for proteins to go across inner membrane

TAT: is Twin arginine transport system. -Signal sequence of proteins going to TAT has twin arginines. -Used for some proteins that can only be properly folded in the CYTOPLASAM. (don't function in cyto but need to be made there.) -Transports these fully folded proteins, ones that must be folded in the cytoplasm. -pretty analagous to sec system, as you have a pore structure, (3 proteins), you have a signal sequence with two aragnines, ss directs it to TAT, ss cleaves, protein goes through and now protein in PERIPLASAM.

TRNA

TRANSFER RNA, brings AA to ribosome. size: SHORT, HIGHLY ORDERE is <100 bp STABILITY: LONGGG generation time, no rapid turn over Number: varibaleee, less than or equa to 27 trnas in E coli (61) codons

true or false: The two parental types are approximately equal in frequency, as are the two recombinant types

TRUE

true or false: homologous crossover occurs sometimes during mitosis

TRUE

mrna

TYPE:carries info, substrate for translation size: is about small 200 base pairs to HUGE over 5 kb. (VARIABLE SMALL TO HUGE) STABILITY: SHORT stabIITY, MINUET/SECONDS NUMBER:~4000 (MANYYY) (E. COLI, WIL VARY WITH ORG.)

Chromatin remodeling

Term that refers to chromatin modifications that reposition nucleosomes in such a way as to open or close promoters and other regulatory sequences

What does the Hardy Weinberg Equilibrium principle state?

That in a population practicing random mating and in the absence of natural selection, migration, genetic drift, and mutation, allele frequencies are stable at p + q = 1

Additive variance, dominance variance, and interactive variance

The 3 different kinds of allelic effects that genetic variance can be divided into:

Promoter (trpP), operator (trpO), and leader region (trpL)

The 3 parts of the regulatory region of the trp operon:

activator

The AraC protein is a(n) ___ when bound to arabinose.

activator

The AraC protein is a(n) ___ when it is bound to arabinose.

regulatory gene (lacI)

The ___, while not a part of the lac operon, plays a role in regulating the expression of the genes of the operon.

Define transduction

The ability of a plasmid to be transferred from one cell to another via the intermediator of a bacteriophage

Totipotent

The ability of an entire normal plant to regenerate from a single isolated plant cell

parS or parC

The actin like filaments form and push the plasmids to opposite sides of the cell

p + q = 1

The allele frequency equation

2-3 stem loop

The antitermination stem loop of trpL mRNA

AraC

The ara operon (which codes for genes required for the metabolism of the sugar arabinose) is controlled by the ___ regulatory protein.

arabinose

The ara operon is activated only when ___ is present.

positive control

The arabinose operon (ara) provides a particularly interesting example of ___________ in that when arabinose is present in the environment, the operon is transcribed.

ara initiator; RNA polymerase

The arabinose-AraC homodimer binds to a regulatory sequence of DNA called the ___; it also binds to ___.

Diauxic Growth

The biphasic curve of a culture growing on two carbon sources.

Fruit fly pattern formation

The body consist of a series of segments grouped into the head, thorax and abdomen Determined by positional information provided by cytoplasmic determinants in unfertilized egg When you build a building, first you have to decide where will be the front and where will be the back of the building. multi-cellular organisms must have a body plan Anterior-posterior (head-to-tail) axis Dorsal-ventral (back-to-belly) axis Right-left axis

Central dogma

The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. •It states that information cannot be transferred back from protein to either protein or nucleic acid. DNA information always transferred to RNA to protein

What affects gene expression in eukaryotic cells? and how?

The chromatin structure affects the gene expression. -The DNA must unwind from the histone proteins (to become looser) which makes it more accessible for transcription to take place.

Gene pool

The collection of genes and alleles found in the members of a population is known as a:

Differential reproductive success

The consequence of natural selection that leads individuals of a particular phenotype to be more successful than individuals with other phenotypes at producing offspring for the next generation

Cis

The core promoter is a (cis or trans) regulatory sequence

On

The default position of a repressible operon is typically (on or off?):

Off

The default position of an inducible operon is typically: (on or off?)

Paralogous

The delta globin gene and beta globin gene in the human are known as ______ (paralogous or orthologous) genes

Catabolite repression

The effect of glucose blocking the lac operon gene transcription is known as:

Photoreactivation

The enzyme photolyase binds to the pyrimidine dimer and cleaves the cyclobutane ring.

Beta-galactosidase

The enzyme that breaks down lactose into glucose and galactose

Response to selection (R)

The extent to which the difference between the mating trait mean value and the population mean value can be passed on to progeny

Protein processing and degradation

The final opportunities for controlling gene expression occur after translation; Many proteins undergo chemical modifications that make them functional Regulatory proteins are commonly activated or inactivated by the reversible addition of phosphate groups, and proteins destined for the surface of animal cells acquire sugars; Cell surface proteins and many others must also be transported to target destinations in the cell in order to function Even if you make the protein, there is a way to stop; proteasome is like a blender it breaks down the protein and stops them Proteasome works like a blender!= denatures it A protein must be tagged or labeled with a structure like ubiquitin (small protein), so the proteasome knows to destroy it

Yeast artificial chromosome

The first artificial chromosomes developed:

tetrad

The four ascospores in an ascus

lacZ

The gene of the lac operon that encodes the enzyme beta-galactosidase

lacY

The gene of the lac operon that encodes the enzyme permease

lacA

The gene of the lac operon that encodes the enzyme transacetylase

Types of cells associated with cancer

The genes that normally regulate cell growth and division during the cell cycle include Genes for growth factors Growth factor receptors And the intracellular molecules of signaling pathways. Mutations that alter any of these genes in somatic cells can lead to cancer.

How might a proto-oncogene—a gene that has an essential function in normal cells—become an oncogene, a cancer-causing gene?

The genetic changes that convert proto-oncogenes to oncogenes fall into three main categories: movement of DNA within the genome amplification of a proto-oncogene and point mutations in a control element or in the proto-oncogene itself

Angelman syndrome

The genetic condition that results from partial deletion of the portion of the maternal copy of chromosome 15 containing H19 and IGF2 or if this chromosome is not properly imprinted

Prader-Willi syndrome

The genetic condition that results from partial deletion of the portion of the paternal copy of chromosome 15 containing H19 and IGF2 or if this chromosome is not properly imprinted

p^2 + 2pq + q^2 = 1

The genotype frequency equation

lacY-

The genotype of a mutated E. coli whose cells cannot accumulate lactose in them (no galactoside permease membrane protein is needed for lactose to be imported) is inferred to be ___.

lacZ-

The genotype of a mutated E. coli whose cells cannot cleave the indicator molecule even if lactose is present as an inducer (does not make beta-galactosidase) is inferred to be ___.

lacI-

The genotype of a mutated E. coli whose indicator molecule is cleaved even if lactose (the inducer) is absent (constitutive expression of lacZ and lacY; gene for regulatory protein that shuts down lacZ and lacY is defected--does not need to be induced by lactose) is inferred to be ___.

Multiple gene hypothesis

The hypothesis that alleles of multiple genes contribute to the production of certain traits

Allolactose

The inducer compound of the lac operon system

Recombinant clone

The joining together of a cloning vector and donor DNA fragment produces a:

repressor; promoter

The lac operan ___ exerts negative control over three protein-coding genes by binding to the operator site in DNA, near the ___.

lactose

The lac operan is not transcribed when glucose is available because glucose prevents ___ transport into the cell.

negative

The lac operan is under ___ control.

trans

The lacI gene can act in ____ (cis or trans?)

Constitutive

The lacI gene undergoes _____ transcription

Gene duplication

The major source of new genes (about 80% of the time) is:

Relative fitness (w)

The measurement of the reproductive fitnesses of organisms in a population relative to one another

Molecular cloning

The method for DNA sequence amplification that does not require prior knowledge of the DNA sequence

PCR

The method that requires some prior knowledge of a DNA sequence that needs to be amplified

Disruptive

The mode of selection (out of directional, stabilizing and disruptive) that results in increased phenotypic variation

Lateral gene transfer

The movement of genes from one species into the genome of another species is known as:

Within the lacZ gene

The multiple cloning site is often located here in a bacterial plasmid:

lacP-

The mutant that fails to bind RNA polymerase

lacOc

The mutant that fails to bind the repressor protein

lacIs

The mutant that is unable to bind the inducer (allolactose)

lacI-

The mutant that is unable to bind to the operator

lacZ-

The mutant that produces a non-functional beta-galactosidase

lacY-

The mutant that produces a non-functional permease

lacIs

The noninducible mutant of the lac operon

DNA binding domain

The part of a repressor or activator protein responsible for locating and binding operator DNA sequences

Allosteric site

The part of a repressor or activator protein that binds a molecule which causes a change in the conformation of the DNA binding domain

What is the 'replicative transposition pathway'?

The pathway highlights the fact that in the process of moving, there is a replication of TE through the act of replication, both cells have a copy

Directional natural selection

The pattern of natural selection that favors one phenotype that has the higher relative fitness than other phenotypes in the population, so that there is a directional change in allele frequencies

1-2 stem loop

The pause stem loop of trpL mRNA

Toxin/antitoxin in persister cells

The persister cells have more toxins, so that growth is inhibited, and this results in the cell not dying due to the antibiotics.

Tree of life

The phylogenetic tree depicting the evolutionary relationships between organisms

What does the term 'cryptic' mean?

The plasmid is hidden due to not having an associated phenotype

Environmental variance (Ve)

The portion of phenotypic variation due to variability of the environments inhabited by individual members of a population

Bacteria artificial chromosome

The preferred artificial chromosome cloning vector

Glucose

The preferred energy source of E. coli

Positive

The presence of the activator (CAP) on the lac operon that allows more efficient transcription when glucose is absent in the environment represents this control of the lac operon:

Negative

The presence of the repressor on the lac operon that blocks transcription initiation unless lactose is in the environment represents this control of the lac operon:

To silence repetitive DNA

The primary role of the RNAi gene silencing mechanism

Feedback inhibition

The principle mechanism turning on and turning off trp operon gene transcription

Cis-dominant

The principle that the operator can only influence the transcription of adjacent downstream genes

Mutagenesis

The process by which an organism is treated with a mutagen to create mutations throughout the genome

Annotation

The process of attaching biological functions to DNA sequences

Gene annotation

The process of identifying the biochemical, cellular, and biological function of each gene product the genome encodes

Genome annotation

The process of identifying the location of genes and other functional sequences within the genomic sequence

Neofunctionalization

The process, following gene duplication, whereby a mutation in one of the two duplicates provides a function not performed by the original gene

Subfunctionalization

The process, following gene duplication, whereby mutations in each of the two copies can result in the two genes having complementary activities such that their combined activity is the same as the activity of the gene before duplication

Allostery

The property belonging to some enzymes of changing conformation at the active site as a result of binding a substance at a different site

Genetic variance (Vg)

The proportion of phenotypic variance due to differences among genotypes

Cis

The proximal elements are (cis or trans) regulatory sequences

DNAase I Hypersensitivity regions

The regions of the genes near transcriptionally active genes in the chromatin structure that become highly sensitive to "DNAaseI" (which digests DNA).

lacI

The regulatory gene that produces the lac repressor protein

lacI

The repressor protein is a product of the ___ gene.

Genomics

The scientific study of biological processes from the perspective of the whole genome

Shotgun sequencing

The sequencing method that relies on redundant sequencing of fragmented DNA in the hope that all regions will be sequenced at least a few times

Primer walking

The sequencing method that relies on the successive synthesis of primers based on the progressive attainment of new sequence information

Recombinant DNA technology

The set of techniques developed for amplifying, maintaining, and manipulating specific DNA sequences

What is the minimum replicon?

The smallest amount of plasmid DNA that is required to allow that plasmid to replicate

Population Genetics

The subfield of genetics that studies the genetic structure and evolution of populations

Define 'conjugation'

The temporary union of two bacteria or unicellular organisms for the exchange of genetic material

3-4 stem loop

The termination stem loop of trpL mRNA

Suppose you compared the nucleotide sequences of the distal control elements in the enhancers of three genes that are expressed only in muscle tissue. What would you expect to find? Why?

The three genes should ahve some similar or identical sequences in the control elements of their enhancers; because they are simliar the same specific transcription factors could bind to the enhancers of all three genes and stimulate their expression coordinately

Promoter, operator (lacO), and CAP-cAMP region

The three protein-binding regulatory sequences of the regulatory region of the lac operon:

Phenotypic variance (Vp)

The total variance observed for a trait is known as:

DNA binding domain and allosteric site

The two active sites of a repressor protein:

Knockout libraries and interference RNA

The two approaches widely used in reverse genetic analysis

Orthologous

The two beta globin genes between the human and the chimp are known as____ (paralogous or orthologous) genes

Transcription initiation and amount of transcription

The two levels of regulated transcription:

Directional and stabilizing

The two modes of selection (out of directional, stabilizing, and disruptive) that result in decreased phenotypic variation

Corepressor (- control) and inhibitor (+ control)

The two regulatory compounds involved in a repressible operon:

Inducers (- control) and effectors (+ control)

The two regulatory compounds involved in an inducible operon:

3-4 stem loop and 2-3 stem loop

The two stem loop structures of the trpL mRNA central to attenuation

Those derived from the genomic DNA of an organism (genomic lib), and those derived from mRNA

The two types of DNA libraries:

Inducer and corepressor

The two types of compounds that can bind to a repressor

Effector and inhibitor

The two types of compounds that can bind to an activator

Broad sense heritability and narrow sense heritability

The two widely used measures of heritability to assess different components of the contribution of genetic variation to phenotypic variation

Genomic library

The type of DNA library consisting of genomic DNA

Complementary DNA library (cDNA library)

The type of DNA library consisting of mRNA

cDNA library

The type of DNA library that represents a subset of all the genes in the genome; represents only the expressed genes

Forward genetic analysis

The type of genetic analysis that starts with a genetic screen to identify specific phenotypic abnormalities in a population of organisms that have been mutagenized.

Define transformation

The uptake of naked DNA

Bioinformatics

The use of COMPUTATIONAL approaches to annotate a genome is called:

Gene therapy

The use of genes as therapeutic agents to cure or alleviate disease symptoms is termed:

Selection coefficient (s)

The value of the reduction in reproductive fitness for an organism

DNAaseI hypersensitivity regions structure. What does this allow?

Their DNA configuration is more open during transcription. This relaxed chromatin structure allows for regulatory proteins to access the Binding sites on DNA

What happens if an enhancer is moved to another location in the genome?

Then transcription of the new adjacent gene is increased.

Termination

There are 3 stop codons, UAG, UAA or UGA (recognize) -There's no tRNAS that have anticodons that can interact with these stop codons, so the A site is empty, and then a protein called a release factor comes and and binds to A site and disrupts the whole processes.

Why is there looping of DNA in lagging strand?

There is looping in the lagging DNA strand because it allows DNA polymerase to rapidly loop ahead to next Okazaki fragment

Enhancers

These affect transcription at distant promoters. Stimulate promoter.

Insulator sequences

These direct enhancers to interact with the intended promoter and that block communication between enhancers and other promoters

Genotype proportion method and allele counting method

These two methods of determining allele frequencies can be used if the population is NOT in hardy weinberg equilibrium

DNA methylation and histone deacetylation

These two result in lack of transcription = more condensation and less transcription Comparison of the same genes in different tissues shows that the genes are usually more heavily methylated in cells in which they are not expressed. Removal of the extra methyl groups can turn on certain of these genes. Researchers have discovered that certain proteins that bind to methylated DNA recruit histone deacetylation enzymes. Thus, a dual mechanism, involving both DNA methylation and histone deacetylation, can repress transcription.

RNA polymerase and transcription factors

These two things are examples of trans-acting regulatory proteins

Define the phase 'TE's act as 'hot-spots' for recombination events'

They can transpose, form co-integrates, can cause deletions (& inversions) of DNA adjacent to the TE

What does the term 'incompatible' mean in reference to related plasmids?

They cannot stably coexist in the same cell

How are the eukaryotic genes coordinately expressed?

They have "response elements" which are common short consensus sequences in the promoters or enhancers that are shared by sets of genes, all of which respond coordinately to the same stimulus.

Green fluorescent protein

This allows a noninvasive means of visualizing gene and protein expression patterns in living organisms

YAC

This cloning vector has the highest capacity

BAC

This cloning vector has the second highest capacity

Narrow sense heritability

This estimates the proportion of phenotypic variation that is due to additive variation only

Broad sense heritability

This estimates the proportion of phenotypic variation that is due to total genetic variation

GAL4

This is an example of a transcriptional activator protein. This activates transcription of several yeast genes whose products metabolize galactose.

Nonrecombinant DNA

This is created when the sticky ends of rejoin rather than incorporating a donor insert

Antibiotic resistance like ampicillan resistance

This is often the selectable marker in a plasmid vector:

Histone Acetylation

This is the addition of acetyl groups (CH3CO) to histone proteins. -Another type of histone modification: Gene Regulation. Gene structure

DNA Methylation (what does it do?)

This is the methylation of the Cytosine bases. -causes repression in transcription.

parS/ parC

This is the plasmid partitioning system we learned for exam 1. Actin like filaments are formed and push the plasmids to opposite sides of the cell.

Selection differential (S)

This measures the difference between the population mean value for a trait and the mean trait value for the mating portion of the population (ie. average plant population height and average height of plants selected for mating)

Square root method

This method of determining allele frequencies can only be used if a population IS in hardy weinberg equilibrium

Recombinant DNA technology

This provides the tools for proceeding from mutant phenotype to DNA sequence, so that alleleic differences can be examined at the molecular level

Restriction map

This shows the numbers and relative positions of target sites for restriction enzymes of a DNA molecule

Frequency distribution

This shows what proportion of the population exhibits each measured value of a trait or falls into each category defined for the trait

Intraspecific comparison

This type of evolutionary genomics identifies sequence polymorphisms that are responsible for the genetic differences within populations of a single species

Interspecific comparison

This type of evolutionary genomics identifies sequences conserved over evolutionary time and thus facilitate the annotation of genomes and provide insight into the evolution of genes and organismal diversity

The transcriptional activators may interact with the BTA directly. How do they interact with the BTA indirectly?

Through protein co-activators.

TN-Seq (-if we do have the genome sequenced, we can use more powerful tn-seq)

Tn-Seq: combines transposon mutagenesis with deep sequencing. (relies on knowing genome sequence of your organism) -You are create a library of transposon mutants that just totally saturate the genome, multiple transposons in each gene. (E.coli with 4000 genes, want 100,000 transposons). -then take this mixed population library of a 100,000 mutants and you're going to grow that in the condition you're interested in. -then take whatever survives, (transposon mutants that hop into genes that are really essential wont survive that condition because they're mutant, so anything that survives is nOT ESSENTIAL.). so we isolate all the genomic dna from whatever survives, sequence it to get where all of these transposons hopped, and then we can map back which genes were important). -a way of on a global scale, do a transposon mutagenesis! "start with large library of transposon mutants (100,000-200,000). This is a pooled, mixed community. one culture of zoo of mutants. 1. grow in NON selective environment (lab media), and this is our control. 2. then isolate genomic dna 3. then sequence every transposon junction, (and getting 20/30 million sequences). 4. then map sequences back to genome. ➙make graph of genes A,B,C vs # of transposon junctions (Each one represents a spef. trans junction and how representative it is in the population, and this is your CONTROL.) (pencil). 5. Grow in your selective environment you care about (ie biofilms wtv) and then repeat 1-5 to take this pop that grows after 23 hours, isolate the genomic dna, sequence everyone transposon junction and then map the sequence back to genome. -doing this in parallel with your control/non-selective condition and your selective. (blue selective) -if no difference (A), means this dream is not very important in the selective environment -if selective is very low, means that C is important in the selective environment. "why? because any cell that had a transposon in C is now underrepresented, didn't grow well in that mixed condition. means that gene is IMPORTANT for growth in that condition :)" -if your selective condition does better (B), you have more transposons in your selective than your non-selective. -"Why? Selective greater, means that B inhibits growth in selective environment" -now if there's cells that have transposons in B, knocked out the function of B, those cells actually did better in the selective environment than they did in the lab.-thus B INHIBITS GROWTH in that selective environment.

Molecular cloning or PCR

To reproduce large amounts of individual pieces of DNA created by restriction enzymes, these two different processes can be used:

topA

Topo I DNA supercoiling

Topoisomerase (site speficic dna recombination)

Topoisomerase type 2 associated with FTSK, and separates DNA. Topo type 2 cuts two strands and nulls other through to close gap.

Polygenic traits

Traits resulting from the influence of multiple genes

Multifactorial traits

Traits whose phenotypic variation is the result of polygenic inheritance and environmental influences

What is the main mode of controlling gene expression?

Transcription

operator

Transcription is inhibited when a regulatory protein binds to the lac operon ___.

AraC

Transcription of the ara operon (which only takes place when arabinose is present) is turned on by an activator protein called ___.

Lactose is present and glucose is absent

Transcription of the lac operon genes is induced only under these two circumstances:

Negative control

Transcriptional control that involves the binding of a repressor protein to a regulatory DNA sequence to prevent transcription

Positive control

Transcriptional control that involves the binding of an activator protein to regulatory DNA to initiate transcription

(How to get polarity) 2. Factor Dependent Transcriptional termination

Transcrit rut sites, down by RHO. If the mRNA is being translated, RHO cannot get to the RNA polermerase. -Translation of the upstream gene can have affect on the downstream gene by factor dependent trx. termination. -So on DNA, we have rna polermase moving along making mRNA, with rut sites that clee down by rho so rho can get on. However if a ribosome is actually translating this mRNA (making protein), rut is going to move up the mRNA and be blocked. -"rho cannot contact RNA polermase if blocked by ribosome". -imagine there is a mut that impacts translation., (nonsense mutation: stop codon introduced). IF we look at a rna pol moving, the stop codon stops the ribosomes at this point (because RF ect). So if there is a rut site here, so RHO can get on and move up because no translation blocking it and lead to termination. -"This upstream (stop codon) mutation (in A) affecting translation of A is going to block TRANSCRIPTION of B. "(mech of polarity). --(doesn't have to be nonsense mut, anything that prevents trans of the gene, to open up region of untranslated mRNA to rho initiated transcriptional termination, will impact b because not even transcribed.

Frederick Griffith

Transformation with steptococcus pneumoniae 1. Rough (non path) and Smooth (path) 2. Live IIR injected: No bacteria recovered 3. Live IIIS injected: Type IIIS recovered, dead mouse 4. Heat killed IIIS: none recovered, lives 5. Mix DEAD IIIS, LIVE IIR = recover IIIS mouse dies

Heterologous transgenes

Transgenes that are transferred from one species to a different species

Reporter genes

Transgenes used to investigate gene regulation

Cotransional (2) -post (energy from SecA/ATP) -co (ribosome energy) -both cases sig sequence gets cleaved, but in co, the protein actually goes into the inner membrane rather than the periplasam (?). translating as it's attached

Translocation #2 Co-translational means transport while translation is happening. (for proteins destined for inner membrane) -You have a ribosome making a polypep, and the terminal signal sequence is recognized by protein SRP. -SRP: binds the signal sequence, stops translation and directs the ribosome to the Sec system. -In this case, the proteins are NOTT fully translated first, initially translated to (Sig sequence) and then stopped (?) -Same Sec system (secYEG), and once the ribosome gets to the sec system, the SRP comes off. Now the polpep is being fed into the sec system by the ribosome, and the energy for this is being provided by TRANSLATION. -Signal sequence is cleaved again, but instead of going to periplasam, protein is fed into inner membrane. -ultimately, the inner membrane (you have these hydrophobic transmembrane domains, about 20 AA's in alpha helices)

What are 'transposable elements'?

Transposable elements are sequences of DNA that are able to transpose to new positions within the genome of a single cell

What does 'DNA deletion' and 'DNA inversion' events relate to?

Transposition

Transposition

Transposition are rare events; frequencies range from 10^-3 to 10^-8 per generation.

How is RNA interference triggered?

Triggered by a double stranded RNA molecule

Corepressor

Trp functions in this system; a small molecule that cooperates with a repressor protein to switch an operon off; as tryptophan accumulates, more tryptophan molecules associate with trp repressor molecules, which can then bind to the trp operator and shut down production of the tryptophan pathway enzymes

True or false: PAI's are examples of a genomic island

True

True/False: Transposition is usually an inter-molecular event

True

Enhancers can stimulate any promoter in its vicinity. (T/F)

True.

Ftsz

Tubulin homologue that aggregates and forms a ring, landing pad for other chromosomes Filamenting temperature sensitive mutant z

Repression

Turn down rate of transcription or translation

Induction

Turn up or on rate of transcription or translation

Uracil

Two Double bonded oxygen

Nitrogen metabolism

Two component system, alternative sigma factor 54 NrtC protein (monomer), active form is multimer

Metabolic control

Two levels: First, cells can adjust the activity of enzymes already present; fairly fast response that relies on sensitivity of many enzymes to chemical cues that increase or decrease their catalytic activity Second, cells can adjust the production level of certain enzymes; they can regulate the expression of the genes encoding the enzymes; operon model

Gene duplication and lateral gene transfer

Two mechanisms that stand out as being major mechanisms responsible for generation of genes in eukaryotes and prokaryotes

Genetic variance (Vg) and Environmental variance (Ve)

Two things that contribute to phenotypic variance:

Produce a gated channel to allow lactose to enter the cell and produce an enzyme that breaks down lactose

Two things that lac+ bacteria are able to do to utilize lactose:

Directly by entering the nucleus and bind DNA to block transcription OR by binding to mRNA to block translation or signal the destruction of the mRNA

Two ways that RNAi can silence gene expression:

Variance and standard deviation

Two ways to describe the nature of a distribution around its mean

Paralogs

Type of homologous genes that perform biologically distinct but biochemically related functions

Orthologos

Type of homologous genes that usually have equivalent functions

Inducible operon system

Type of operon system that the lac operon is under

Both positive and negative

Type of transcriptional control the lac operon is under:

no glucose, high lactose

Under which conditions are the lac structural genes expressed most efficiently?

An enhancer can be located where in the gene it regulates.

Upstream, Downstream and within a gene it regulates.

Methyl Mismatch Repair

Use DNA methylation as an indicator for an error on the newly synthesized strand (during replication).

Southern Blot

Used for DNA fragments. 1. Run digested DNA on gel (generated via restriction digest). 2. Separates based on size. 3. Blot to membrane 4. Probe membrane with labeled DNA sequence (radioactive or other). 5. DNA probe will form hybrid with complementary sequences detected --> Determine if gene was correctly interrupted or replaced.

Northern Blot

Used for mRNA. 1. Run mRNA on gel by electrophoresis. 2. Separate based on size. 3. Transfer (blot) to membrane. 4. Probe with labeled DNA. 5. ID mRNA transcribed from a gene.

Alternative σ factors & regulation.

Used for special cases (Sigma regulation). Control of ratio of σ factors determines global control of protein synthesis.

Vg

Va + Vd + Vi =

Shotgun cloning

Vector + geonomic DNA mixed + rest enzyme +ligated

Viruses in cancer

Viruses seem to play a role in about 15% of human cancer cases worldwide. Viruses contribute to cancer development by integrating their genetic material into the DNA of infected cells. By this process, a retrovirus may donate an oncogene to the cell.

Transducing Particles

Viruses that are carrying portions of host DNA.

Bacteriophage

Viruses that attack bacteria. Do not harm eukaryotes.

Vg + Ve

Vp =

Whole genome shotgun sequencing

What approach to genome sequencing out of the two is applicable to any genome because it doesn't rely on the availability of specific genetic resources?

Clone-by-clone sequencing

What approach to genome sequencing out of the two relies on the availability of specific genetic resources and is thus only applicable to some model organisms?

Genetic maps made by recombination frequencies

What are the "available genetic resources" that are useful when using clone-by-clone sequencing?

Functional genomics, structural genomics, and evolutionary genomics

What are the 3 major subdivisions of genomics?

Transcriptional regulation, mRNA processing, regulation of mature mRNA, translational regulation, and post-translational regulation

What are the 5 levels of eukaryotic gene expression regulation?

Physical map does not need to be constructed in WGS sequencing, WGS sequencing can be applied to any genome

What are the differences between clone-by-clone sequencing and whole genome shotgun sequencing?

Genotype proportion method, allele counting method, and square root method

What are the three methods to calculating allele frequencies?

Coupling of transcription and translation: availability of tryptophan to be added to the nascent polypeptide chain

What determines the type of stem loop a trpL mRNA will form?

QQ:

What distinguishes a complex virus from a simple bacteria is that they CANNOT PERFORM TRANSLATION.

Uses algorithms to identify open reading frames, sequences that have the potential to code for polypeptides

What do COMPUTATIONAL approaches to annotate a genome do?

These sequences are more likely to be functional and encode genes than sequences that are not conserved

What do conserved coding sequences likely indicate?

Facilitates RNA polymerase binding at promoter to help initiate transcription

What does activator binding to an activator binding site do?

Blocks transcription by RNA polymerase

What does repressor protein binding to an operator do?

LacZ gene

What feature of the plasmid vector selects for only those bacteria that contain a recombinant plasmid and selects against nonrecombinant plasmids?

Repressor cannot bind to operator and lac operon structural genes are produced constitutively

What happens if there is a mutation in the lacI gene near the lac operon causing it to produce a mutant inactive repressor? (lacI- mutation)

Repressor binds to operator but cannot recognize allolactose so transcription does not occur even when lactose is in the environment

What happens if there is a mutation in the lacI gene near the lac operon causing it to produce a superrepressor? (lacIs mutation)

Repressor cannot recognize operator and lac operon structural genes are produced constitutively

What happens if there is a mutation in the operator region of the lac operon causing the operator region to be unrecognizable to the repressor? (lacOc mutation)

When the presence of a preferred catabolite (like glucose) represses the transcription of genes for an alternative catabolite (like lactose)

What is catabolite repression?

Genes that are transcribed continuously with no regulatory control

What is constitutive transcription?

The comparison of genomes both within and between species

What is evolutionary genomics?

Uses genomic sequences to understand gene function

What is functional genomics?

Transcription of products needed in changing conditions

What is regulated transcription?

The sequencing of the whole genome and the cataloging of sequences within the genome

What is structural genomics?

To determine the effects of losing gene function

What is the common goal of creating knockout mutations and using RNA interference in animals

Add a Poly-dt primer for reverse transcriptase to use

What is the first step in making a cDNA library from mRNA?

Allow you to investigate gene regulation by telling you where, when, and how much of a gene is expressed.

What is the function of reporter genes?

To produce 3 polypeptides that allow E. coli to utilize lactose as a carbon source

What is the function of the lac operon in bacteria?

Facilitates entry of lactose into the cell at the cell membrane

What is the function of the permease enzyme produced by the lac operon?

To synthesize tryptophan

What is the function of the trp operon?

Depending on the organism, gene families may be expanded because of their importance in biological function to the organism

What is the importance of gene families in reconstructing the evolution of genomes?

Forming a DNA loop containing enhancers and their intended promoter

What is the mechanism of insulator sequences?

The middle value of the distribution

What is the median of a distribution?

The most common value in the distribution

What is the mode of a distribution?

AraC

What is the protein that positively regulates transcription of the ara operon?

Selects for only bacteria containing the plasmid

What is the purpose of a selectable marker in a plasmid vector?

What is the rate limiting step in DNA replication?

What is the rate limiting step in DNA replication? Initiation of DNA replication

Can produce genes with novel functions and often lead to the formation of gene families

What is the significance of gene duplications?

What is the use of a shuttle vector?

What is the use of a shuttle vector?DNA sequences can be manipulated in bacteria where manipulation is easier and then shuttled into yeast for protein expression

TDNA (transfer DNA)

What part of the Ti plasmid is actually transferred to and incorporated into the plant genome?

Virulence genes

What part of the Ti plasmid is not transferred to the plant genome, but rather encodes for proteins that carry out the transfer of the other portion of the Ti plasmid?

Structural genomics

What type of genomics gives a parts list of the genetic tool kit of an organism?

Evolutionary genomics

What type of genomics illuminates the genetic bases of similarities and differences between individuals or species?

inducer

What, when taken up by a cell, binds to a repressor so that the repressor no longer binds to the operator?

Constitutive

When a gene is always expressed.

negative

When a regulatory protein shuts down transcription, it is an agent of ___ control.

positive

When a regulatory protein triggers transcription, it is an agent of ___ control.

does

When a repressor protein is not present, and lactose is present or absent, transcription ___ occur.

repressor; DNA (operator region); does not

When a repressor protein is present and lactose is absent, ___ binds to ___ and transcription ___ occur.

lactose; repressor (making it fall off the DNA); does; allosteric

When a repressor protein is present and lactose is present, ___ binds to ___. Transcription ___ occur. This is an example of ___ regulation.

inducer; allosteric

When a repressor protein is present and lactose is present, lactose acts as a(n) ___, binding to the repressor and causing it to fall off of the DNA, allowing for transcription of lacZ and lacY. This is an example of ___ regulation.

cis-antisense RNA (asRNA)

When bound to mRNA counterpart, asRNAs can stop transcription, translation, or trigger mRNA degradation.

In highly inbred populations in which all individuals are homozygous for alleles controlling a quantitative trait

When can Vg equal zero?

Exon shuffling

When exons from two or more genes are combined in a new genomic context, this is known as

Additive genes

When genes contributing to polygenic traits each contribute about an equal share incrementally to total phenotypic variation, they are known as:

When genetic variance cannot be partitioned into its separate components

When is it more beneficial to use broad sense heritability

repressor; operator

When lactose is present, it binds to the ___ and causes it to fall off the ___, allowing transcription to occur.

Directional

When only one extreme of the phenotype distribution is favored, this results in _____ selection

Constitutive

When products are needed continuously to perform routine tasks, the DNA for these products undergoes ______ transcription

Double stranded RNA homologous to the gene

When ridding an experimental organism of the function of a gene using RNAi, what would you inject into the organism?

CAP-cAMP complex binds to the operon and increases the ability of RNA polymerase to transcribe

When the activator CAP binds cAMP in the lac operon, what happens?

When would eukaryotic expression vectors need to be used

When the protein intended to be made needs to undergo eukaryotic post-translational modifications that bacteria cannot do

Repressor-inducer complex falls off and allows RNA polymerase to access and initiate transcription

When the repressor binds allolactose in the lac operon, what happens?

Lysogeny vs lysis decision

When there is a high amount of phages infecting a single bacteria, lysogeny is preferred because there is a small ratio of bacteria to phage density. Thus, it is more advantageous to wait until there are more bacteria to infect. When there is a low amount of phages infecting a single bacteria, lysis is preferred because there are plenty of bacteria available to be infected, thus reproducing more phages is more advantageous.

Lysogeny

When viruses integrate their genome into bacterial chromosomes.

trp operon

Which bacterial operon uses attenuation?

alternative RNA splicing

Which of the following allows more than one type of protein to be produced from one gene? (eukaryotes)

Because only needed when something is in the environment that needs to be broken down

Why are inducible operons typically off?

Because only needed when it needs to make something that the environment lacks

Why are repressible operons typically on?

Can only read about 1000 bp and most DNA regions of interest are much larger than this

Why can dideoxy sequencing not be used to sequence many DNA regions of interest?

Because upon infecting the plant with the transgene, not all of the plant cells have the transgene inserted into their genome

Why does a genetically engineered plant cell need to be regenerated into an entire plant in order to observe the effects of the transgene on the plant phenotype?

The repetitive sequences can be mapped to several places in the genome

Why does repetitive DNA, especially seen in eukaryotes, make whole genome sequencing difficult?

Complete sets of cDNA clones are not available

Why is complete annotation of the transcribed regions of the genome using cDNA not possible?

Ensure expression of genes in an accurate pattern during different developmental stages AND to form differences among different cell types

Why is gene expression regulation important in eukaryotes?

Because some genes must be expressed on the paternal chromosome and silenced on the maternal or vice versa for normal development

Why is genomic imprinting biologically important?

To ensure that each parent passes a properly imprinted chromosome during reproduction

Why is it important that some epigenetic patterns are erased in germ line cells?

allosterically

With arabinose present, the AraC protein binds to arabinose; the protein is ___ regulated by arabinose.

operator

Without lactose to act as an inducer, the repressor/regulator protein (coded for by the lacI gene) would be located attached to the ___ region of the DNA.

Clone-by-clone because they can map to multiple locations in the genome

Would it be more beneficial to use clone-by-clone or WGS sequencing for sequences with repeats? Why?

is RNA modified in translation?

YES! heavily modified -- uses tRNAs

Is there an advantage to having a low vs. no mutation rate? (10^12 > 10^10?)

YES, evolution can act to change if random mutations are selected in fitness environment. however if none-changing env, more mutation

Can an IS sequence relocate itself?

Yes

Can one bacteria have more than one plasmid?

Yes

Does DNA pol require a primer

Yes

Two strains of bacterium, each with a plasmid and that produce pili, is it possible for the two strains to conjugate?

Yes

In prokaryotes, do translation and transcription co-occur?

Yes. Ribosomes translate mRNA while it is still being synthesized. Multiple ribosomes per mRNA ensure rapid translation.

Spo0A

Z ring formation: SigmaH to SigmaE; F in spore To Sigma G in spore To Sigma K in mother Cortex and core formation then spore

translational

___ control allows quick change regarding which proteins are produce.

post-translational

___ control is quick but energetically expensive.

transcriptional

___ control is slow, but efficient.

negative

___ control occurs when something must be taken away for transcription to occur.

regulons; regulon

___ coordinate the expression of different genes by using a shared regulator that acts on a regulatory sequence found in all genes of the ___.

constitutive

___ expression occurs in genes whose products are required at all times, such as genes that encode glycolytic enzymes.

allosteric regulation

___ occurs when a regulatory molecule binds to a protein to change its shape and activity

enhancer

a DNA sequence that functions as a regulatory element. the binding of a regulatory transcription factor to the enhancer increases the level of transcription.

silencer

a DNA sequence that functions as a regulatory element. the binding of a regulatory transcription factor to the silencer decreases the level of transcription.

phage λ

a bacteriophage that infects E. coli

temperate phage

a bacteriophage that usually exists in the lysogenic cycle

transcription factors

a broad category of proteins that influence the ability of RNA polymerase to transcribe DNA into RNA.

ATP-dependent chromatin remodeling

a change in chromatin structure that alters the degree of compaction and/or the spacing and histone composition of the nucleosomes. Requires ATP.

housekeeping genes

a gene that encodes a protein required in most cells of a multicellular organism.

induced

a gene that has been transcriptionally activated by the inducer

tissue-specific genes

a gene that is highly regulated and is expressed in a particular cell type.

repressible genes

a gene that is regulated by a corepressor or inhibitor, which are small effector molecules that cause transcription to decrease.

inducible genes

a gene that is regulated by an inducer which is a small effector molecule that causes transcription to increase

maternal effect genes

a gene that when a mutant in the mother, results in a mutatn phenotype in the offspring, regardless of the offspring's own genotype When a mother has a mutation in such a gene, she makes a defective gene product and her egg is defective=can fail to develop properly

activator protein (CAP)

a genetic regulatory protein found in bacteria

CpG island

a group of CG sequences that may be clustered near a promoter region of a gene. the methylation of the cytosine bases usually inhibits transcription.

open conformation

a loosely packed chromatin structure that is capable of transcription.

attenuation

a mechanism of genetic regulation, seen in the tcp operon, in which a short RNA is made but its synthesis is terminated before RNA polymerase can transcribe the rest of the operon.

ChIP-Seq (chromatin immunoprecipitation sequencing)

a method for determining whether proteins bind to a particular region of DNA. this method analyzes DNA-protein interactions as they occur in the chromatin of living cells.

merozygote

a partial diploid strain of bacteria containing F' factore genes

mediator

a protein complex that interacts with RNA polymerase II and various regulatory transcription factors. Depending on its interactions with regulatory transcription factors, mediator may stimulate or inhibit RNA polymerase II.

regulatory transcription factor

a protein or protein complex that binds to a regulatory element and influences the rate of transcription via RNA polymerase.

Ccdb

a protein that alters DNA gyrase such that dsDNA breaks are not re-sealed.

Activator

a protein that binds to DNA and stimulates transcription of a gene

methyl-CpG-binding proteins

a protein that binds to a CpG island when it is methylated.

translational repressors

a protein that binds to mRNA and inhibits its ability to be translated

polyA-binding proteins

a protein that binds to the 3' end polyA tail of mRNAs and protects the mRNA from degradation.

lac repressor

a protein that binds to the operator site of the lac operon and inhibits transcription

trp repressor

a protein that binds to the operator site of the trp operon and inhibits transcription

splicing factor

a protein that regulates the process of RNA splicing.

translational regulatory protein

a protein that regulates translation

NFR (nucleosome-free region)

a region within a chromosome where nucleosomes are not found.

repressor

a regulatory protein that binds to DNA and inhibits transcription

repressor

a regulatory protein that binds to DNA and inhibits transcription.

trans-acting factor

a regulatory protein that binds to a regulatory element in the DNA and exerts a trans effect

CREB protein (cAMP response element-binding protein)

a regulatory transcription factor that becomes activated in response to specific cell-signaling molecules that cause the synthesis of cAMP.

oriT

a second origin found in transmissible plasmids

insulator

a segment of DNA that insulates a gene from the effects of nearby regulatory elements such as enhancers.

domain

a segment of a protein that has a specific function.

attenuator sequence

a sequence found in certain operons in bacteria that stops transcription soon after it has begun.

ARE (AU-rich element)

a sequence found in many short-lived mRNAs that contains the consensus sequence AUUUA.

regulatory element (control element)

a sequence of DNA (or possibly RNA) that binds a regulatory protein and thereby influences gene expression. Bacterial operator sites and eukaryotic enhancers and silencers are examples.

cis-acting element

a sequence of DNA, such as a regulatory element, that exerts a cis-effect

operator site

a sequence of nucleotides in bacterial DNA that provides a binding site for genetic regulatory protein

promoter

a sequence within a gene that initiates transcription

terminator

a sequence within a gene that signals the end of transcription

operon

a set of bacterial genes that are regulated together and transcribed into a single RNA

CRE (cAMP response element)

a short DNA sequence found next to certain eukaryotic genes that is recognized by the cAMP response element-binding (CREB) protein.

inducer

a small effector molecule that binds to a genetic regulatory protein and thereby increases the rate of transcription

corepressor

a small effector molecule that binds to a repressor protein, thereby causing the repressor protein to bind to DNA and inhibit transcription

inhibitor

a small effector molecule that binds to an activator protein, causing the protein to be released from the DNA, thereby inhibiting transcription.

arabinose

a sugar present in the cell walls of leaves

closed conformation

a tightly packed conformation of chromatin that cannot be transcribed.

activator

a transcriptional regulatory protein that increases the rate of transcription

activator

a transcriptional regulatory protein that increases the rate of transcription.

IRP (iron regulatory protein)

a translational regulatory protein that recognizes iron response elements that are found in specific mRNAs. It may inhibit translation or stabilize the mRNA.

TFIID

a type of general transcription factor in eukaryotes that is needed for RNA polymerase II function. It binds to the TATA box and recruits RNA polymerase II to the core promotor.

lytic cycle

a type of growth cycle for a phage in which the phage directs the synthesis of many copies of the phage genetic material and coat proteins. These components then assemble to make new phages. When synthesis and assembly is completed, the bacterial host cell is lysed, and the newly made phages are released into the environment.

lysogenic cycle

a type of growth cycle for a phage in which the phage integrates its genetic material into the chromosome of the bacterium. this integrated phage DNA can exist in a dormant state for a long time, during which no new bacteriophages are made.

SR proteins

a type of splicing factor.

glucocorticoid receptors

a type of steroid receptor that functions as a regulatory transcription factor.

Transcription block a)same b)opposite

a) Dna pol uses RNA as a primer when both rna polymerase AND Dna pol 3 are going in the SAME DIRECTION. b) When rna pol/dna pol 3 are going in different directions/towards one another, MFD remover portein RNA polmerase during head on collsions with POL 3. *RNA Polymerase can cause replication arrest. How can it be removed? ? MFD REMOVER PROTEIN

a) What are Okazaki fragments? b) How are gaps between Okazaki fragments filled?

a) they are short pieces of DNA produced from lagging strand template. b) They are filled by removing RNA primers, using exonuclease 5' and then DNA Pol 1 synthesizes DNA.. **********************************

Transcription block to replication a)trx moving in the direction of the complex b)opposite, trx head on with replication complex.

a)-dna pol 3 is about 10x faster than rna pol, so rna pol is removed however dna pol 3 hops over and use the rna that was being made for transcription as a primer. leaves a ssGAP but can be filled by dna pol 1. " Dna pol uses transcribed RNA as a primer when both rna polymerase AND Dna pol 3 are going in the SAME DIRECTION. " b) When rna pol/dna pol 3 are going in different directions/towards one another, MFD remover protein RNA polymerase during head on collisions with POL 3. MFD: protein that removes rna polymerase during head on collisions with dna polymerase, leads to a pausing of replication which recruits MFD protein. *RNA Polymerase can cause replication arrest. How can it be removed? ? MFD REMOVER PROTEIN

Positive Regulation

activation of transcription from a promotor by an ACTIVATOR (cis acting)

In eukaryotes transcription is controlled by what?

activators, co-activators and repressors.

nucleotide triphosphate

active molecules inserted into dna, with 3 phosphate alpha beta gamma.

what can affect recombination frequency?

age, environment, and sex -dietary levels of metal ions can affect crossover in flies

Shuttle vectors

allow plasmids to replicate in more diverse organisms

Missense mutation

an AA change.( to synomous/non-synomous) -base change can lead to an AA change (transition or tranversion) -silent (no observable phenotype) -null (nonfunctional) -leaky (partially functional) -gain of function (ex. lux to luxOD47E, "mutation makes this regulator constitutively active." mutation makes this activity no longer regulated by phosphosphorlation). -biofilms-, make this mutation (change 1 base of 5 million) to biolfilm+ gain of function).

IRE (iron response element)

an RNA sequence that is recognized by the iron regulatory protein

antisense RNA

an RNA strand that is complementary to a strand of mRNA

operon

an arrangement in DNA in which two or more structural genes are found within a regulatory unit that is under the transcriptional control of a single protein

cis-effect

an effect on gene expression due to genetic sequences that are within the same chromosome and often immediately adjacent to the gene of interest.

trans-effect

an effect on gene expression that occurs even though two DNA segments are not physically adjacent to each other. Trans-effects are mediated through diffusible genetic regulatory proteins.

Dicer

an endonuclease that makes a cut in double-stranded RNA.

histone acetyltransferases

an enzyme that attaches acetyl groups to the amino terminal tails of histone proteins.

allosteric enzyme

an enzyme that contains two binding sites: a catalytic site and a regulatory site.

constitutive exon

an exon that is always found in mRNA following splicing.

alternative exons

an exon that is not always found in mRNA. it is only found in certain types of alternatively spliced mRNAs.

enzyme adaptation

an inheritance pattern in which a modification to nuclear gene or chromosome alters gene expression in an organism but the expression is not changed permanently over the course of many generations

polycistronic RNA

an mRNA transcribed from an operon that encodes two or more proteins

Environment

another major source of development information is the enviornment around a particular cell; most influential are the signals impinging on an embryoinc cell from other embryonic cells int eh vicinity, including contact with cell-surface molecules on neighboring cells and the binding of growth factors secreted by neighboring cells ; such signals cause changes in target cells (induction)

mazE

antitoxin

dna parent strands are

are copied simultaneously at replication forks, although they run in opposite directions.

dna parent strands are

are copied simultaneously at replication forks, although they run in opposite directions. -The 2x strands run in opposite directions - The nucleotide of the 3' end of one strand pairs up with the nucleotide of the 5' end of the adjacent strand. - Strands are not identical, but are complementary. -Unwinding is due to action of enzymes such as helicase and DNA gyrase which break the hydrogen bonds between bases -DNA Polymerase - Also edits for errors in base pair replication (1 error out of 1x billion base pair replications)

Phospholipids

are made of fatty acids linked to glycerol phosphate by an ester bond. -Some phospholipids contain charged groups on the phosphate. -Phoslips forms a lipid bilyer. -phoslips are 'AMPHIPATHIC' (have hydrophobic and hydrophillic parts

ordered ascus

arrangement of daughter spores reflects the orientation of alleles carried by each chromatid in metaphase I

rpoA

assembles RNAP

egg-polarity genes

because maternal effect genes also control the orientation of hte egg and consequently of hte fly, maternal effect genes are also called egg-polarity genes One group of these genes sets up the anterior-posterior axis of the embryo, while a second group establishes the dorsal-ventral axis; mutations in maternal effect genes are generally embryonic lethals

QQ IN E COLI, when base pair mismatched during replicaiton escape the editing of dna pol 3, a mismatch repair system can still recongnize and recmove the nucleotides incorretly added to the new strand because..

becuase the A in GATC remains UN-METHYLATED for a breif time in newly synthized dna strands.

pLysS or pLysE

bind to T7 RNA pol and inhibit its activity. These are used if you are expressing genes that are toxic to the host.

coactivators

bind to other proteins like basal factors, transactivators, or chromatin remodeling complexes

How does binding of the trp corepressor and the lac inducer to their respective repressor proteins alter repressor function and transcription in each case

binding by the trp corpresspor activates the trp repressor, shutting off the trp operon; binding by the lac inducer inactivates the lac repressor; leading to transcription of the lac operon

ppGpp in e.coli

binding to the core RNA polymerase and sigma factors affecting expression of genes binds to betasubunits of RNA polymerase core enzyme

Topoisomerase resolvase

binds XerCD and resolves catamers/dimers

parA

binds the genome.

SSB

binds to single stranded dna

SSB (single stranded binding protein

binds to single stranded dna - keeps the DNA strands from binding back together

parA

binds to the genome

antitoxin method of operation. Stability of toxin vs. Antitoxin

binds to the toxin and inactivates the toxin. If a cell looses the plasmid that has the genes that code for the toxin and the Antitoxin, the toxins and antitoxins proteins are still there. Since the antitoxin is unstable, it will degrade, while the stable toxin stays and kills the cell. The toxin is STABLE, while the antitioxin is UNSTABLE.

Cyclic di-GMP sms

biofilm, motlity (oxygen, light, more)

The signaling molecules released by an embryonic cell can induce changes in a neighboring cell without entering the cell. How?

by binding to a receptor on the receiving cell's surface and triggering a signal transduction pathway that affects gene expressions

SeqA

can condense hemimethylated DNA

XerC/D

can resolve plasmid or chromosomal dimers. XerC/D recognizes cer sites on the chromosome. Site specific recombination happens at these cer sites, and the concatamer is resolved by one plasmid splitting into two plasmids.

oncogenes

cancer causing genes Subsequently, close counterparts of these oncogenes were found in the genomes of humans and other animals.

miRNAs

capable of binding to complementary sequences in mRNA molecules ; miRNAs are formed from longer RNA precursors that fold back on themselves, forming one or more short double-stranded hairpin structures held together by hydrogen bonds; after each hairpin is cut away from the precursor, it is trimmed by an enzyme into short double-stranded fragment of about 20 pairs miRNA protein complex either degrades the target mRNA or blocks its translation

where does incoming amino acid attach on growing polypeptide in translation?

carboxyl end

aminoacyl tRNA synthetase

carries out reaction: amino acid + tRNA-ATP --> aminoacyl-tRNA + AMP + Pi -attaches free amino acid onto tRNA

Cyclic AMP sms

catabolite repression (nutrient source)

rpoB

catalysis chain initiation/elongation

DNA translocases

catalytic ATPase subunit found in all chromatin remodeling complexes, similar to other motor proteins.

Cyclic di-AMP sms

cell division checkpoint, DNA damage repair, cell wall modification (DNA damage)

A certain mutation in E. Coli changes the lac operator so that the active repressor cannot bind. How would this affect the cell's production of B galactosidase ?

cell would continuously produce B galactosidase adn the two other enzymes for lactose utilization even in the absence of lactose thus wasting cell resources

Conjugation

cell-cell contact (sex pilus)

Fertilized egg to a catepole

cells go through mitotic cell divisions, cell differentiation, and morphogensis; why fertilized egg goes to this structure

linkage disequilibrium

certain haplotypes are maintained in a population, usually due to natural selection

response to environmental stress

certain proteins help a bacterium to survive environmental stress such as osmotic shock or heat shock. these proteins are required only when the bacterium is confronted with stress.

isomerization of RNA

change from uridine --> pseudourine --- chemical makeup doesn't change, just where the bonds are placed are different!

induction

changes in target cells; the molecules conveying these signals within the target cell are cell-surface receptors and other proteins expressed by the embryo's own genes Normally signaling molecules send a cell down a specific developmental path by causing changes in its gene expression that eventually result in observable cellular changes Thus, interactions between embryonic cells help induce differentiation of the many specialized cell types making up a new organisms

Plasmid sRNA

cis acting

Enhancers are ______- acting regions of DNA that interact with _______- acting _____________. ANd increase what?

cis acting regions of DNA that interact with Trans-Acting Regulatory Proteins (transcription activators). They increase the efficiency of transcription initiation.

parS-

cis acting site on plasmid that binds parB

parS

cis acting site on the plasmid that binds parB

Inhibit DnaA activity

clamp complex binds and promotes ADP-DnaA and it is not active in promoting unwinding of complex

HolA-D

clamp loading

protoncogenes

code for proteins that stimulate normal cell growth and division

Thousands of genes, but only a small amount of DNA

codes for proteins (1.5% in humans) ; rest codes for RNA products or isnt trascribed at all; the transcription factos of a cell must locate the right genes at the right time; when this goes array serious problems can happen

replisome replicon:

combo of preotiens that function at rep. forknd undertake synthesis of dna replicon:each region served by one dna orgin, any peice of dna that replicates as single unit.

MukBEF condensin

condenses and relaxes DNA, is a dimer with a hinge and dna arms

initiator tRNA

contains initiation codon - methionine

homeotic genes

control pattern formation in the late embryo, larva, and adult

Nirenberg and Matthaei (1961)

cracked genetic code to show RNA controlled the production of specific proteins ---translation in vitro (test tube)

morphogenesis

creation of form the physical process that gives an organism its shape

CAP

cyclic AMP; (accumulates when glucose is scarce) How does ecoli cell sense the glucose concentration and relay this information to the genome?: depends on interaction of an allosteric regulatory protein with a small organic molecule, cAMP regulatory protein is called CAP=activator

Cyclic GMP sms

cyst development (?)

What are the substances that initially determine the axes of the body?

cytoplasmic determinants; these substances are encoded by genes of the mother, fittingly called maternal effect genes

dars=

dars= DnaA Reactivating Sequence; -The sequence in chromosome that binds dnaA-ADP, and causes ADP to come off and ATP repalces it. 2. Starts with...

Once mRNA encoding a particular protein reaches the cytoplasm, what are four mechanisms that can regulate the amount of the protein that is active in the cell?

degradation of the mRNA, regulation of translation, activation of the protein, and protein degradation

(Lecture 18:)** Deletions

deletions can happen by homologous recombination between direct repeats. (same sequence repeated, homologous -sequence of A1=A2 (in circle is deleted part of genome) (deletion happens when homologous recombination/crossing over event between homologous sequences(direct repeats)(same sequence repeated). -circle has no origin so can't replicate. -genes bcd, between the direct repeats will be deleted. -typically at IS elements, and insertion sequences is where you these direct repeats/homologous recombination. -(sort of a type of transposon) -reverted goes back to the original sequence, suppressed is like a second site mutation that overcomes the original mutation.

posttranslational

describes events that occur after translation has occurred

Hok toxin

destroys cell membrane potential

Hok toxin

destroys membrane potential.

Khorana (1962)

developed a way to synthesize specific RNAs

Direct Repeats

direct repeats flank inverted repeats, with transposase in the middle. DR is b/c you have staggered cut.

lagging

discontinuous copying of the parental strand that runs 3′ to 5′ away from the replication fork IS BOTTOM STRAND 3' TO 5'

(X) pasteaur/ROBERT COCH

disproved theory of spontaneoug reneration. with swan neck flask, Rober Coch was first to demonstrate link betwee nMICROBES AND INFECTIOUS DISEASE. -devoloped techniques (semi solid media) for obtaining pure '__' of microbes. -'a group of cells that arise from a cell', USED A SEMI SOOLID MEDIA FOR GROWTH AND ISOLATION OF PURE CULTURES.

enhancers

distant distal control elements, may be thousands of nucleotides upstream or downstream of a gene or even within an intron a given gene may have mutliple enhancers each active at different times or in a different cell type ; Each enhancer is associated with only one gene Rate of gene expression can be increased or decreased by the binding of proteins, either activators or repressors to the control elements of enhancers

small, noncoding rna

diverse regulation and cofactors of proteins. less than SMALL 200-200 base pairs VARIABLE STABILITy HUNDREDS OF THEM!!!

_________ can be used to determine gene order

double recombinants, or double crossover -only one allele is different; this is the middle gene -list in each of 3 possible orders

DNA qq2

double stranded, abtiparalled, phosphates on outside and bases on inside. -DNA IS REPLICATED IN SEMICONSERVATIVE MANNER, 1 STRAND IS HELD AS TEMPLATE STRAND ANSWER, C REVERSE COMPLIMENT. 3 PRIME REV. COMP. GOES TO THE 5' OF THE DNA.

Q: approx how many genes does each org posses in their genome.

e.coli=4000 genes oryza=50000 homosapiens=20,000

shine-dalgarno sequences

element to TIR along with initiation codon (AUG) to help trigger initiation located 5-10 nucleotides upstream of initiation codon

example of frameshift mutation

ethidium bromide store in DNA by binding in the base pairs and cause a frameshift

Every restriction enzyme

every restriction enzyme (dna scissors that cut a specific sequence) has a corresponding methylase. -methylase methlyate RE target sequence to prevent cutting, why?? -because defends against foreign dna (innate immune system. -Genome protected by methylase LOOK AT NOTES

leading

formed by continuous copying of the parental strand that runs3′ to 5′ toward the replication fork. IS TOP STRAND 5' TO 3' ANTIPARALLEL oopsie picky is leading

beta clamp

foundation of the mobile platform that keeps DNA Pol from falling off

DNA sequencing

four different reaction mixes (one ddNTP, other three dNTP) with radioactive primer. Pieces separated by electrophoresis

The diploid cells undergo meiosis to produce four haploid ______ in a sac called an _____

four haploid ASCOSPORES in a sac called an ASCUS

muscle cell developments

from myoblasts; from embryonic precursor cells that have the ptoential to develop into a number of cell types

repressible enzyme

function in anabolic pathways, which synthesize essential end products from raw materials (precurosrs); by suspending production of an end product, when it is already present in a sufficient amont, the cell can allocate its organic precursors and energy for other uses) = avoid wasting energy!

ccdaB

gene that codes for a toxin/antitoxin. It inhibits host cell division.

Maternal effect genes

genes of the mother that code for proteins or mRNA that are deposited in unfertilized egg

Nomenclature

genes: first letter lowercase, italicized. (~rpoA) (~dnaA) RNA classic bacteria: 3 letters followed by big letter ______________________ Genomic era: genes referred by genomic designation -v. cholera. ~VCA0939 -easy to figure out the sequence. -if given function, often renamed _______________ Protein: -no italics -first letter UPPERCASE! -(RpoA) (DnaA) _______________________ Mutants: designated by AA change (or maybe if rna, would be nucleotide change) -ex. luxOD47E (referring to an allele (flavor) of luxO) _______________________ Name protein or genes by function: -in histidine utilization, hisA+, hisA- -E. coli (hisA) :if specified, implices that's it's negative/lost function mutant. ____________________ Phenotype: no italics, first letter upper case. -His+, His_ or just His. -again if just His, implies negative function. (not able to make it's own histidine, thus an auxotroph) -Lac+, Lac-, Lac is a phenotype.

positive control

genetic regulation by activator proteins

down regulation

genetic regulation that leads to a decrease in gene expression.

up regulation

genetic regulation that leads to an increase in gene expression.

linkage equilibrium

genotype for a chromosome at one gene is expected to be independent of its genotype for other genes

Operon

group of genes regulated together and controlled by an on/off switch called operator operator, the promoter (several genes under 1 control), and the genes they control (the entire stretch of DNA required for enzyme producting) E Coli growth depends on amino acid of trypthophan; if absent, repressor inactive, and opeon is on Picture: repressor example Downstream: end of genes Upstream: promoter; regulatory TrpR that is far away that makes a repressor (makes mRNA); made in an inactive form; if you eat trpR, it binds to the inactive repressor and and activates it which it then attaches to the operator and shuts it down)

tmRNA

has BOTH transfer and messenger RNA function. (trna first then mrna) -Interacts with A site of stalled ribosomes -@1, we have a ribosome, trna in the p site and a growing peptide. RNA got cleaved before the stop codon (RNases), and in this case, ribosome now stalled because nothing in the A site. (if there were a stop codon, would be okay because RF's would come in) -The tmRNA is what will recycle the stalled ribosome. tmRNA is bound to AA alanine (tRNA function of this, structure resembling.) This can be recruited to the A site, the alanine can be transferred to this peptide and a new rna can be used a template for more peptide synthesis. This a codon, this gets transferred and recruited by EF-TU, EF-G shifts this 1 codon and now the tmRNA is in the p site but the mrna part becomes the new template. now a new codon here provided by the tmrna, that can recruit another tRNA (appropriate one), and it can come in with peptide transfer. Normal translation occurs, except it's not the mrna being read, it's the tmRNA. -Goes on a specific tail, with it's own stop codon and release mechanism, then it falls apart and ribosome recycled. -tmRNA's has recycled the ribosome, as well as (This is not a random sequence of AA's), it's a specific tag that leads to specific degradation of this peptide chain. -Sort of a partial protein, thus this protein needs to be recycled and the tag it what recycles it. It goes to the cook system, cookxp with degrades this partially made protein -Thus 1) relieves stalled ribosome and 2) tags partial protein fragment for degradation. -

editing function of tRNA synthetase

has domain that is able to recognize incorrect amino acid and cleave it off **only editing step in translation!

Lactose

has glucose, E. coli can break it down into glucose and galactose-uses an inducable system

ex. of Site specific Homologous recombination, that only happens at a specific sequence, and here at dif sites.

have 2 chromosomes, with like sequences the same and leads to chromosomal dimers. (dimer is 2 copies of same thing) -Resolved through sites called dif sites on the chromosome positioned at the poles, so that the protein complex XerCD recombines these two things. mech: they are positioned at the septum (middle of cell), which a protein called FTSK does. (it's a dna translocase aka 'pump', that is a protein that binds around dna and moves it around.) Located at the septum in the cell is the FTSK protein, binding to proteins around septum. The junction of these, are the dif sites and there is actually sequences (KOPS) in the chromosome that gives direction. And these KOPS sequences allow FTSK to pump the DNA in the right direction, sequences called KOPS sequences. Until these dif sites are located right in the septum.

Self transmissible/ conjugative plasmids

have an oriT, and have all the components needed to move from donor cell to a recipient cell.

relaxed copy number

high copy number

LuxN/LuxQP

histidine kinases step one

how might a tRNA be rejected in the A site during translation

if anticodon doesn't match the codon

how is factor-dependent termination impacted by translation?

if ribosomes are already on mRNA downstream of rut/rho, then transcription termination is blocked!

cyclic AMP

in bacteria, a small effector molecule that binds to CAP. In eukaryotes, cAMP functions as a second messenger in a variety of intracellular signaling pathways; in some cases it binds to transcription factors such as the CREB protein.

inducer

inactivates the repressor

inducer

inactivates the repressor (tryptophan no longer present) This system saves energy!

Magnification

increases apparent size. Total mag= objective lens X ocular lens

Intragenic recombination

infrequent event in which crossing over occurs within a gene

Negative Regulation

inhibits

SgrT

inhibits glucose transport

RNA 2

initiates replication of the plasmid. It interacts with RNase H to begin initiation.

lysogenic

integrates genome

mediator protein

interacts with proteins at the promoter; protein-mediated bending of the DNA is through to bring bound activators in contact with mediator proteins mutliple protein to protein interactions help assemble and position the initiation complex on the promoter

DnaA

intiation. Primosome, Initiator protein, priming complex formation

Toxin/ Antitoxins

is a way for the plasmid to avoid curing by the cell. If the cell gets rid of the plasmid, it will be killed. This gets the cell "addicted" to the plasmid.

sigma factor

is that bind to RNAPol and recruit its core enzyme to the promotor for transcription at transcription start sites (part of the promotor sequence +1)

transcription factor

lacI is an example of a(n) ___.

lac operon

lactose (milk sugar) is available to E. Coli in the human colon, if the host drinks milk; lactose metabolism begins with hydrolysis of the disaccharide into its component monosaccharides, glucose and galactose, a reaction catalyzed by B-galactosidase Only a few moleucles of htis enzyme are present in an E Coli cell growing with out lactose normally ; if lactose is added to the bacterium's environment, however, the number of B-galactosidase molecules int eh cell increases thousandfold in 15 minutes. Gene for B galactosidase is part of the lac operon, which includes two other genes coding for enzymes that function in lactose utilization; entire transcription unit is under the command of a single operator and promoter LacI, located outsie the operon, codes for all allosteric repressor protient aht can swtich off lac operon (in active form) lac repressor is in active form (different) binding to the operator and switching the lac operon off; in this case a specific small molecule inactivtes the repressor (inducer) With out being bound to the repressor, the lac operon is trascirbed into mRNA for lactose-utilizing enzymes

elongation

leading/lagging

tetratypes are [more/less] frequent than PD

less frequent -->NPD are even less frequent

mRNA degradation

life span of mRNA molecules in the cytoplasm is important in determining the pattern of protein synthesis in a cell Bacteria mRNA molecules are typically degraded by enzymes within a few minutes; such short life span of mRNA is one reason bacteria can change their patterns of protein synthesis so quickly in response to environmental changing mRNAs in eukartyoes can last up to weeks

Refractive Glass Lenses used for.. Electromagnet lenses used for..

light microscopes electron microscopes duh

Bacterial morphologies

listen to which ones

dnaC

loads dnaB helicase onto dna.

acetylation

loosen chromatin structure and enhance transcription decetylation=decrease transcription

Low and high levels of RepA

low levels of RepA leads to plasmid replication. high levels of RepA leads to handcuffing.

stringent copy number

low or fixed copy number

Gene expressed

mRNA made-→protein produced pic repressor example Tryptophan is made at the end of the pathway at A Genes of operon lead up until

Cytoplasmic determinants

maternal substances in the egg that influence the course of early development After fertilization, early mitotic divisions distribute the zygote's cytoplasm into separate cells; the nuclei of these cells may thus be exposed to different cytoplasmic determinants , depending on which portions of zygotic cytoplasm was received The combination of cytoplasmic determinants in a cell helps determine its development fate by regulating expression of the cell's genes during the course of cell differentiation

mazEF toxin/ antitoxin system

mazEF cleaves rna at certain sites

Suicide vector

means that this vector cannot replicate in cell. (key) -but have mobilizing ability, transposon must introduced into host on a suicide vector.

SAM

methylase group

Attenuation

modulation of the rate of transcription by the rate of translation of a leader sequence of an mRNA

Avery, McLeod, McCarthy EXP 1

more strep -Extraction of components from IIIS and injected into IIR -Discover that Nucleic acids transform

operon

multiple genes (coding sequences) on same transcript -links genes of similar function **transcription of all in operon is the same BUT translation is different!

QQ: Estimate how many colonies you have to screen to find a His auxotroph?

mutation rate: (10^-10) mutation/base. for every base that gets added, 10^-10 chance that it's a mistake. -(1000 changes to get a histidine auxtroph estimation) ~1000/(10^-10)=10^7=10,000,000 bases to get auxotrophic mutant relying on random mutations. 10^13/10^6 bases in the cell=10,000,000 bottom line: must screen a lot of colonies, thus need other ways to get auxotrophs. -INCREASE mutant rate

embyronic lethals

mutations with phenotypes causing death at the embryonic or larval stage

DisA

no damage = c-di amp, senses holiday junction at stalled rep forks, snth inhibited, repair starts

cold sensitive

non-functional @ lower temperatures.

Non-Synomous mutation:

non-synomous mutation: mut that does affect the AA sequence. -'neutral' mutations, can actually have phenotype and considered 'functional' mutations.

The ascospores produced by the second arrangement will contain alleles in different combinations from the original parent strains, called a ______

nonparental ditype (NPD) -forming only when 4-strand double crossovers take place

Discovery of single mRNA lac operon

nonsense mutations

IS3 ORFB

not expressed alone, catalytic activity. -in -1 reading frame

coefficient of coincidence [c]=

observed double crossovers/expected double crossovers

coordinated control of dispersed genes

often occurs in repsonse to chemical signals from outside the cell ex. steroid hormones operons that work this way (genes being expressed together) have been been found in eukartyotic cells

inducable genes

on normally, but no reason for them to be on normally

GTF (general transcription factor)

one of several proteins that are necessary for basal transcription at the core promotor.

oriV

origin of replication of plasmids. A cis element.

Homologs that do not reshuffle alleles under study are called-

parental chromosomes or nonrecombinant chromosomes

FtsW/FtsI

peptidoglycan synthesis

Persister cells

persister cells have more toxins, so that growth is inhibited, and this results in the cell not dying due to the antibiotics.

prophage

phage DNA that has been integrated into the bacterial chromosome

F plasmid

plasmid in E. coli. Has a tra region that encodes for numerous transfer functions, including the sex pili. It also has IS2 and IS3 regions. These regions are insertion sequences (transposons) that are involved in the integration of the F plasmid into the host cells.

Genetic linkage mapping-

plots the positions of genes on chromosomes

dnaE

polmerization, catalytic subunit.

DnaE

polymerization, catalytic subunit of RNAIII

Maltose operon

positive regulation

SpoT

ppGTP synthetase, also degrades ppGpp

upper limit: Three-strand double crossovers

produce 50% parental and 50% recombinant gametes

upper limit: Two-strand double crossovers

produce ONLY PARENTAL gametes

upper limit: Four-strand double crossovers

produce all recombinant gametes -recombination LIMIT is 50%

lytic phage:

programs cell to be a phage factor (kills cell/host)

crossing over occurs during-

prophase I of meiosis

tissue-specific proteins

proteins are found only in a specific cell type and give the cell its chracteristic structure and function; first evidence of differentiation is the apperance of mRNAs for these proteins; different sets of gene are sequentially expressed in a regulated manner as new cells arise form divisions of their precursos

r [recombination frequency]=

r= # recombinant gametes / total # meioses

recombination frequency=

r=[.5TT]+[NPD]/total tetrads -only half TT spores are recombinant

(X)Rabies/

rabies bite exposure then 9 months, you ha e disease. however if vaccinate maybe exposed. (nerve tissue spread(. rabies animal, move spinal tissue, dry in flask. virus DEAD.

Crick and Brenner (1961)

reading frames and triplet codon -used T4 phage

Proteasomes

recognize ubiquitin-tagged proteins and degrade them.

Hemimethylated

recognized as newly synthesized DNA

Alleles of syntenic genes can be reshuffled when crossing over occurs between homologs to produce ______________

recombinant chromosomes

DNA methylation

reduces transcription

orientation-independent

refers to certain types of genetic regulatory elements that can function in the forward or reverse direction. certain enhancers are orientation independent.

alternative splicing

refers to the phenomenon in which a pre-mRNA can be spliced in more than one way.

5' untranslated region (leader region)

region between 5' end of mRNA and TIR -translation has not happened yet

Theta replication

regular replication. there are two strands involved.

Positive gene regulation

regulatory protein interacts directly with the genome to switch transcription on

RNA Polymerase can cause replication arrest. How can it be removed?

removed by protein Mfd

Negative control of arg

repressed by arginine (co-repressor) to repressor on the arg operon

Silencing

repressors recruting proteins that deacetylate histones, leading to reduced transcription

What does the clamp loader do?

responsible for loading Beta Clamp proteins onto DNA

revertants

restoration of original sequence

rpoZ

restores denatured enzymes to correct promotors

two parts needed for factor-dependent

rho rut

mechanism of factor-dependent

rho binds to rut, and rho makes its way up to the RNA Pol to terminate transcription -RNA Pol is faster than rho, so needs a rho-dependent termination site on the template to cause RNA Pol to pause and rho can catch up

rRna

ribosmal part of ribosme size: 23S 16S 5S LONG genreation time ONLY 3 OF THEM

translation location

ribosome

Qrr

sRNA (+Hfq)

Phage advantages

see bacteria; except even smaller genome, a trillion per mL -teach us much about the host since infects it.

Control elements

segements of noncoding DNA that help regulate transcription by binding certain proteins

selection

selection:(more powerful) only the mutants of interest can grow. (design environment so only things you want grow) -if you have 4000 transposon mutants, only the 5 or 10 that have your phenotype of interest will grow). -ex. antibiotic resistance (selection ex.) you have e.cole to mutagenize, and plate on kanamycin, only the kanamycin resistant colonies grow (any kanamycin sensitive dies) thus "EVERY COLONY is a mutant" -on each plate, you can screen a billion mutants

Transposon Mutatgensis:

semi-randomly hop around in genes in genome -almost ALWAYS PRODUCE NULL PHENOTYPE -great way to generate null mutation to screen for whatever you want. (looking for biofilm deficient mutants, do a transposon screen and find transpons that when hop into genome knock out biofilm. -transponson here is Kan^r, thus cell is kan senstive. key to understand that this is usually a sucicde vector/ -because we are going to select for kan resistant colonies, and if vector were to replicate then everything would be kanr and no mutants. we want the transposon to hop somewhere in the genome. only way to if you have this cell to be kanr, is to have this transposition event because the vector itself isnt going to be able to replicate. (cell: Kan^s, transposon: Kan^4) -select for kanr colonies, each colony has a unique transposition event (transposon somewhere unique in the chromosome). (IMPORTANT) lol. if you have 4000 colonies, 4000 different transposon hopping events). -tr inserted (kan resistant). -now either select or screen for mutant of interest. (ex. we have one mutant, but where is transposon? it hopped somewhere into the gene thats making these green cells go read, but what is this gene? -(SCREEN) isolate the genomic dna of the mutant, (has kanr and origin of replication so can replicate on own.)then cut genome with restriction enzymes that don't cut in transposon. -now we have transposon, with flanking sequences (want to know what these flank sequences are because tells us where the transposon hopped/what gene it knocked out.) -with enzymes, use dna ligase to get a plasmid like thing that has our transposon, RE, to isolate this sequence off of transposon (use primers), we can get flanking sequences. -if we have a specific gene to target (dont use this),

how would you distinguish a suppressor from a revertant?

sequence the genome!

translational initiation regions (TIRs)

sequences on mRNAs that flag the correct first codon for the ribosome

mechanism of factor-independent

series of A's cause RNA Pol to pause and allows time for hairpin formation. this causes mRNA to be dissociated form the RNA Pol and transcription terminates

QQ what would be a better antibitic, ciproflaxin or mitocycin C??

shares methyl transferase? ciprofloxacin -bacterial gyrase and eukaryotic gyrase are very different - won't affect our gyrase -both bacteria and eukaryotes have DNA, so mitomycin C would be toxic to us! -would crosslink our DNA too!

how does translation know to code start codon or methionine?

shine delgarno sequence! (RBS binding site)

Lederberg and the Tatum experiment

showed that sexual recombination occurs between cells of E. coli. They observed recombinant types when two auxorophic mutants were mixed.

Two component reg system + signal transduction

signals detected by sensors and transmitted in changed form; 1. Sensor protein: kinase autophosphorylation (histidine) 2. Response-regulator protein: trans. phosphate to group (aspartic acid) Phosphorelay (bind to DNA)

miRNAs (microRNAs)

small RNA molecules that can inhibit the expression of specific mRNAs via RNA interference

siRNAs (short-interfering RNAs)

small RNA molecules that silence the expression of specific mRNAs via RNA interference.

siRNAs

smaller than miRNAs,formed by much longer double-stranded RNA molecules, each of which give rise to many si RNAs

cell division

some proteins are needed for ____ ________. these are necessary only when the bacteria cell is getting ready to divide.

metabolism

some proteins function in the __________ of small molecules. for example, certain enzymes are needed for a bacterium to __________ particular sugars. these enzymes are required only when the bacterium are exposed to sugars in its environment.

motif

specific common molecular structure with common function

Enhancers for specific transcription factors

specific transcription factors that function as repressors can inhibit gene expression in several different ways; some repressors bind directly to control element DNA, blocking activator binding or, in some cases, turning off transcription even when activators are bound Some activators and repressors act indirectly by affecting chromatin structure silencing: repressors recruting proteins taht deacetylate histones, leading to reduced transcription

Replication Fidelity

spontaneous mut. (don't know the cause for, ) is 1 mutation in every 10^10 bases inserted, thus 1/2000 offspring have 1 mut. How does e coli do this? 2 mechs 1) PROOFREADING 2) METHYL DIRECTED MISMATION REPAIR (MDM)

Vibrio Fischerii

squid light organs; LuxIR

mutation rate example:

start a culture, (time 1) ➙ (time 2) look for the number of trp A mutants and we get the nuber of CFUs (viable bac.), grow for awhile, and at time two we do the same thing. (N1=1x10^6 N2=1x10^9 M1=5 M2=203) m=203-5/(1x10^9-1x10^6)=198/100=198/10^9... =1.98x10^-7 mutation rate

ppGpp sms

stringent response, alarmomone (growth rate)

homodimer

structure formed when two copies of a protein interact

inducible enzymes

synhtesis is induced by chemical signal (ex. allolactose)

EPA sites

tRNA exit site Peptide site Amino acid site

how can there be 64 codons with only 27 tRNAs and 20 tRNA synthetases?

tRNAs can recognize multiple codons (wobble base pair) and tRNA syntheses can recognize multiple tRNAs

Molecular Beacon

taq man probe taq removes probe when extending in PCR = fluorescence

temperature sensitive

temperature sensitive typically refers to heat sensitive. (non functional at higher temperatures. -the majority of temp. sensitive mutants are heat sensitive. (cold is rarer but can still be isolated)

rho

termination factor that acts as.. 1. ATPase to cleave ATP and get energy 2. RNA-DNA helicase

In lab studies, the ascospores can be removed from the ascus and cultured to determine their genotypes; this process is called-

tetrad analysis

If a single crossover occurs between either of the homolog pairs, a _______ is produced, containing two parental spores and two nonparental spores

tetratype (TT)

RISC (RNA-induced silencing complex)

the complex that mediates RNA interference.

posttranslational covalent modification

the covalent attachment of a molecule to a protein after it has been synthesized via ribosomes

Determination

the events that lead to the observable differentiation of a cell (before scientists knew about molecule changes occuring in embryos) ;once it had undergone determination, an embryonic cell is irreversibly committed to its final fate we understand determination in terms of molecular changes make tissue-specific proteins

Differential gene expression

the expression of different genes by cells with the same genome; differences between cell types are not due to different genes, but due to differential gene expresison

antitermination

the function of certain proteins, such as N protein in bacteria, that prevents transcriptional termination.

histone code hypothesis

the hypothesis that the pattern of histone modification acts much like a language or code in specifying alterations in chromatin structure.

Role of transcription factor

the initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors General transcription factors: essential for the transcription of all protein-coding genes Protein-protein interactions are crucial to the initiation of eukarytoic transcription Only when the complete initiation complex has assembled can the polymerase begin to move along the DNA template strand, producing a complementary strand of RNA specific transcription factors: high levels of transcription of particular genes at the appropriate time and place depend on the interaction of control elements with another set proteins

what establishes the reading frame

the initiation codon

de novo methylation

the methylation of DNA that has not been previously methylated. This is usually a highly regulated event.

maintenance methylation

the methylation of hemimethylated DNA following DNA replication.

positional information

the molecular cues that control pattern formation; are provided by cytoplasmic determinants and inductive signals; these cues tell a cell its location relative to the body axes and to neighboring cells and determine how the cell and its progeny will respond to future molecular signals Scientists studied Drosophilia, and studied mutants to look at development established that genes control development and have led to an understanding of the key roles that specific moleucles play in defining position and directing differential

motif

the name given to a domain or amino acid sequence that functions in a similar manner in many different proteins.

Negative controls of genes

the operons are switched off by active form of the repressor protein

combinatorial control

the phenomenon common in eukaryotes in which the combination of many factors determines the expression of any given gene.

catabolite repression

the phenomenon in which a catabolite represses the expression of certain genes

allosteric regulation

the phenomenon in which an effector molecule binds to a noncatlytic site on a protein and causes conformational change that regulates its function

DNA methylation

the phenomenon in which an enzyme covalently attaches a methyl group to a base in DNA.

feedback inhibition

the phenomenon in which the final product of metabolic pathway inhibits an enzyme that acts early in the pathway

gene regulation

the phenomenon in which the level of gene expression can vary under different conditions

gene regulation

the phenomenon in which the level of gene expression can vary under different conditions.

bidirectionally

the phenomenon in which two replication forks move in opposite directions outward from the origin.

cell differentiation

the process by which cells become specialized in structure and function; the different kinds of cells are not randomly distributed but are organized into tissues and organs in a particular three-arrangement

CAP site

the sequence of DNA that is recognized by CAP

allosteric site

the site on a protein where a small protein effector molecule binds to regulate the function of the protein

What generates the first differences among cells in early embryo?

the specific genes expressed in any particular cell of a developing organism determine its path; two sources of information tell a cell which genes to express at any given time during embryonic development One important source is the egg's cytoplams (contains RNA and proteins encoded by mother's DNA); cytoplasm of an unfertilized egg is not homogeneous; mRNA, proteins, other substances, and organelles are distributed unevenly in the unfertilized egg, and this unevenness has a profound impact on the development of the future embryo in many species

Pattern formation

the two processes of cytoplasmic determinants (protein and RNA) and induction contribute to pattern formation (morphogenesis) For animals, begins in early embryo, when the major axes of an animal are established; before tissues and organsappear, the relative positions of the animals head and tial etc are set up, thus establishing the boyd's three major body axes

3'-UTR (3'-untranslated region)

the untranslated region of mRNA

immunoprecipitation

the use of antibodies to cause other molecules, such as proteins, to precipitate, which allows them to be collected by centrifugation.

Rolling circle replication (sigma replication)

there are two types of sigma replication: SSO and DSO. you can have single strand replication or double strand replication.

proofreading and editing in transcription

there is NONE!