BIO 172 Test 3

basal transcription complex

(EUKS) contains promoter proximal elements, core promoter (General transcription factors) and RNA polymerase

what is needed for DNA sequencing? (sanger)

- template DNA - primer - DNA polymerase - deoxynucleotides - dideoxynucleotides (fluorescently labeled)

why do signal pathways have so many steps/so complicated?

1) signal amplification (turns a small signal into a large output) 2) signal branching to coordinate multiple responses for a signal and to coordinate multiple signals 3) signal can be subject to regulation and feedbacks at many steps 4) it may be easier to evolve elaboration of existing pathway than evolve a more "streamlined pathway"

two approaches of somatic cell transformation

1) use retrovirus vectors to deliver new genes to cells of patient. 2) isolate cells from individual (i.e. stem cells) and transform (with retrovirus or CRISPR) and amplify the cells and return to cells of patient

2 methods of gene therapy

1. germ-line- the functional gene is introduced into a fertilized egg or embryo. Every cell in the resulting individual has a copy of the function gene (transgene) 2. Somatic cell transformation- the transgene is only introduced into some somatic cells of the individual (ex: blood cells, intestinal cells)... less ethically controversial (you can get consent, no heritable changes, error damage cells not individuals) but also more difficult because it is hard to individually transform the trillions of cells in the body.

What happens to duplicated genes? (3)

1. if gene accumulates a non-functional mutation before it acquires a new function, it is lost 2. for 2 duplicated genes, if expression patterns become distinct, each is requires 4 organismal function and maintained 3. For 2 duplicated genes, if 1 gene acquires new biochemistry function that provides a selective advantage then both genes are maintained.

dif. with prok genome?? (% of genome is coding DNA, % of genome unique to species, % of genome acquired from other species and how?)

1. majority of genome is genes (some transposons but much better at getting rid of these because they are competing so heavily... selective advantage over slightly smaller genome) 2. about 15% of genes in a prok genome are unique to its own species... we see evolution in a genome metabolically and biochemically 3. 15-25% of genome appears to have been acquired from other, often distantly related species (this transfer of DNA from one microbe to another is called lateral/horizonatal gene transfer... with the pilus)... big role in spread of antibiotic resistance

two methods of sequencing? and explain

1.Limited targeted sequencing (Sanger sequencing). Approach: Use plasmid DNA (or PCR amplified DNA) as template, use specific primers to sequence the desired location on plasmid or PCR product 2. Large-scale genomic sequencing (High throughput). Approach: Randomly sequence fragments of genomic DNA. Must sequence very large number!!

What is the evidence prokaryotic genomes continue to evolve?

15% of genes in a prok genome are specific to that species (evolve not at morphological but at a biochemical, metabolic level)

exons constitute ___% of human genome

2%, coding, the rest are non coding introns

on any strand are there ________potential open reading frames

3

How frequently do stop codons occur in random non-coding DNA? How does this info help us?

3/64 (about 1 out of 20 in non-coding should be stop codon... THEREFORE if you have a protein coding gene that is 300 base pairs long... the likelihood of that happening is essentially 0 in non-coding DNA therefore you know that it is coding DNA... this makes it hard to distinguish smaller coding sequences as protein coding genes)

What is an example of nucleosomes that assemble into higher-ordered structures? and what does this do to the DNA?

30-nanometer fibers, it condenses the DNA to be even more compact but also makes much of the DNA inaccessible

what is an operator?

A regulatory region in DNA, binding site for the repressor

What technology is used to transform plant genomes?

Agrobacterium T-DNA

difference between signal amplification and signal branching?

Amplification turn a small signal into a big response. Branching takes one signal and turns it into many cellular responses.

consequences of sequencing an organism's genome

An individual's "personal genome" can be sequenced and compared to the species standard to determine potential for disease inheritance. A sequenced genome can be annotated and search for open reading frames. Genome sequences can be compared among species to determine evolutionary relationships.

open reading frame

An open reading frame is most likely protein coding. An open reading frame consists of a long string of codons. An open reading frame ends with a stop codon, but it has no additional stop codons.

What is necessary for RNA polymerase activation?

Chromatin opening

promoter-proximal elements

DNA elements immediately upstream of promoter. Proteins bind here and influence the rate of RNA polymerase initiation (EUKS)

How does enhancer function explain variable locations of enhancer?

DNA loops around (in the second functioning of enhancer) with the binding of transcriptional factors so that the enhancer can act on the promoter AND transcription factors bind to enhancers so that the DNA can be opened

what is next after the gel-electrophoresis is run of the nucleotides in the sanger method?

DNA may be run through the capillary tube... but ultimately you can see the sequence of bases (that terminated the strands) as they were incorporated in the newly synthesized strands (all the ones that terminated at same position have the same length and color) the order of colors and distances from the primer align with the order of the sequence associated with the colors and therefore you can determine the template sequence by looking at the sequence in fluorescence (the one closest to the primer will be the smallest strands... which traveled the farthest on the gel-electrophoresis

In what ways are DNA methylation and histone modification different?

DNA methylation is long-term and results in a chemical change in DNA (chemically modify DNA, permanently change DNA)... histone modification has circadian regulation it is a short term gene silencer Histone modification is a modification of the protein histone (do not permanently change the DNA)

How does the cell regulate the extent of chromatin packaging?

DNA methylation, modification of histones (acetylation) (EUKS)

Core promoter

DNA sequences that are bound by proteins that directly recruit RNA polymerase (EUKS)

Transposable elements are:

DNA sequences that can insert themselves into new positions within the genome.

enhancers

DNA sequences that control gene expression, can act a long distance away from the gene they regulate, they can be up or down stream or in coding/noncoding region. Many can be moved to a new location and still function, they effect the binding/activity of RNA polymerase at the promoter, they activate transcription of the corresponding gene, they determine when and where the gene is transcribed, every gene has a different suite of enhancers (EUKS)

why is methylation permanent?

DNMT adds methyl group to cytosine... covalently marks DNA/change in DNA, and it is inherited through cell division

How does DNA methylation effect transcription?

DNMT adds methyl groups to cytosines and: 1.DNA methylation changes the chemistry/chemical make-up and shape of the major groove therefore a DNA binding protein will not be able to bind to the methylated-cytosine (Transcription factors no longer recognize the now methylated sequences, only recognize when not methylated) 2 proteins with a methyl-cytosine binding domain (MBD) recruit chromatin remodelers like HDAC to the methylated DNA to pack up the chromatin (block gene transcription because it is packaged up)

In eukaryotic cells, why are certain genes expressed only in certain types of cells?

Different cell types have different regulatory transcription factors

The correct sequence of steps in the eukaryotic cell cycle is ____________ .

G1 → S phase → G2 → mitosis → cytokinesis

Which major checkpoint delays the cell cycle when DNA replication is incomplete?

G2 checkpoint

What is the purpose of having post-translational regulation? Why make proteins that are not active?

IT IS VERY IMPORTANT, vast majority of genes are regulated post-translationally it is important for speed and variety of responses and for interacting with the environment

What is the difference between Sanger sequencing and PCR with regard to the materials needed to perform these reactions?

In Sanger sequencing, dideoxynucleotides are needed, but not in PCR

How does protein phosphorylation regulate protein function?

It causes a conformational change in the protein.

why does alternative splicing increase the number of proteins encoded by a specific genome?

It produces more than one mature mRNA from the same gene. The splicing is done differently and different mature transcript... different exons are included or incorporated differently. mRNA will have a different coding sequence depending on which cell it is in (result in different protein isoforms and end up having different properties and functions important for the different cells)

Describe Sanger sequencing

Labeled dideoxynucleotides prevent the addition of another nucleotide after them. The resulting DNA fragments can then be run on a regular gel to determine the sequence.

How does an active CDK complex move a cell through a checkpoint?

MPF is a CDK complex (with cyclin) that drives entry into mitosis so therefore it is active it is controlled by cyclin accumulation, MPF CDK is present at all times so when the cyclin accumulates at a max (because the cell meets all the requirements for cell cycle progression) MPF triggers and controls the events that characterize mitosis i.e. : 1) it phosphorylates chromosomal proteins and causes chromosome condensation 2) it phosphorylates nuclear lamins and initiates nuclear envelope breakdown 3) it phosphorylates microtubule-associated proteins and activate mitotic spindle it triggers the events that occur during mitoses through protein phosphorylation

How high throughput sequencing work?

New technologies allow rapid, inexpensive sequencing of large amounts of DNA, entire genome is broken up into a bunch of random little fragments end up with 100 million small random pieces and then you sequence every one of the fragments and end up with 100 million short sequence reads... you use the sequence overlaps from individual sequence reactions to assemble the whole genome sequence (requires heavy computing)

What about PCR allows you to compare a disease allele to a normal allele?

PCR amplify the specific gene and then sequence the alleles

an operon has only one ____

Promoter and operator sequence

microRNA (miRNA) and small interfering RNA (siRNA) are incorporated into a protein complex called

RISC (RNA induced silencing complex)

in prokaryotes, transcriptional control is generally about controlling ___________

RNA polymerase binding to the promoter

example of signaling for lipid insoluble signals and how it works?

Ras signaling --> the receptor activates the Ras activating protein and binds to it changes its conformation that then activates a Ras protein (type of G protein) (Ras activating protein helps Ras bind GTP). Once Ras is activated, it activates a Kinase cascade called he MAPK pathway... Ras binds to MAPKKK and activates it by conformational change which phosphorylates MAPKK and becomes active and this phosphorylates MAPK and becomes active (signaling intermediates)... MAPK that is active and phosphorylated can move into the nucleus and activate transcription factors through phosphorylation and this can lead to a change in gene expression (response)

period of DNA replication

S phase

What is the most important feature of an STR that facilitates it use as a way to match DNA to a specific individual?

STRs are short tandem repeats, repeating sequences of up to 5 bp, they are hypervariable meaning that they vary among individuals and each individual has a unique trait... it is good because there are a lot of repeat regions and a lot of variations/many allelic combinations

What is the role of protein degradation in cell cycle control?

We need the cyclin levels to go down so that the cell does not immediately go into the next step in the cycle before it is ready to, as soon as the cell divided the next cell would be triggered for cell progression... daughter cells need to make own decisions on whether to divide

What would happen if all genes were constitutively transcribed?

You want cell differentiation because different systems have different functions and we need these different functions. This would be a waste of energy too.

What is methylation?

a chemical modification to the DNA which can influence gene expression

Long terminal repeats (LTRs) are

a class of transposable elements that uses RNA as an intermediate

each differentiated cell has _____

a different pattern of methylation

How might a misregulation of cyclin expression affect the cell cycle?

a lot of cyclins, a lot of active CDK, lots of protein phosphorylation, and cell cycle progression the opposite is true too

reporter genes and example

a nice way to test for enhancer activity... use gene that causes glowing... if it glows that means you have the transcription factors you need to open up the chromatin and interact with open DNA to cause transcription

Think of a gene in a specific cell type that is controlled by chromatin remodeling. Think of a gene in a specific cell type that is controlled by DNA methylation.

a photosynthetic related gene in a leaf cell (chromatin remodeling) a neuron silences muscle myosin (DNA methylation)

The difference between a single nucleotide polymorphism (SNP) and a point mutation is that:

a point mutation is when a base pair is changed to a different base pair, whereas a SNP is when the base pair differs among individuals in a population

What are the features of a complete ORF?

a start codon, a sequence of amino acid codons, and a stop codon; can have more than one ATG but only one stop codon

How do you do a genetic screening

add a compound to cells (ie. Ecoli) that induces mutations... mutagenize cells. Some cells will not be able to break down a certain substance and you can find which one these are by doing replica plating (first grow the cells on a media that they will grow on then transfer it/by stamping onto a new agar plate in the same location, this plate has the substance they can't break down/won't be able to grow on. With different growth patterns you can compare the plates).

putting a prok gene into a euk? (corn and cry protein example)

add maize promoter and enhancers (only want it expressed in the stalk) add 5' UTR sequences with the start AUG at the first AUG and NOT multiple AUGs. (because of difference in translation initiation), can have the same coding sequence, think about protein localization (need to add back on the signal peptide), transcription terminator from maize start with normal Ti plasmids then remove tumor inducing genes and incorporate Cry gene into plasmid SO now corn makes its own insecticide

when does post-translational control take place?

after a protein is synthesized

how do you manipulate mRNA transcripts in the lab for euks?

after mRNA is processed (one ORF in one molecule) a DNA copy of the mRNA is made which is a cDNA with the help of reverse transcriptase

sequence assembly is accomplished by

aligning the fragments by using a complex computer program

Sequence assembly is accomplished by:

aligning the fragments by using a complex computer program.

Where in the cell cycle are the checkpoints located and what are they checking for?

all control progression thru cycle, 1) before G1 (the cell size must be adequate an the nutrients must be sufficient and social signals must be present and the DNA undamaged) 2) before G2 (the chromosomes have to have replicated successfully, the DNA undamaged, and activated MPF is present) 3) b/w metaphase and anaphase (all chromosomes are attached to spindle apparatus)

how was insulin extracted to treat diabetes before? problems? solutions?

all insulin was isolated from human blood and plasma samples, there was a limited supply, it was very expensive and there were contamination issues solution: move the gene for insulin into the euk from the prok cell

Why does retroviral delivery not directly repair a mutated gene?

all it does is add another copy of a functional gene to override the mutant gene

Why does histone acetylation change the conformation of DNA?

amino acid being acetylated in histones tails is a lysine residue (it is positively charged). Ionic interaction b/w histone tails and DNA is one of attraction, causing the histones and DNA to package up with the bonding. Acetylation changes the positive charge to a partial negative charge so now what was attraction is repulsion. NO longer have higher order packaging. (EUKS)

interphase

any not in M phase, so it is in G1, S or G2

What technology is used to transform bacterial genomes?

bacterial transformation

Why is the cell cycle under tight control?

because if all cells divided, you would have cells blocking capillaries, neurons would get disrupted and there would be a huge biological problem, most of your cells do not and will never divide frequency of division varies greatly

why is it important to use multiple cDNA libraries to aid in the identification of genes in the human genome?

because libraries from different cell-types will express different genes

Why does selective pressure on recently duplicate genes drop?

because now there are two copies and less likely to have non-functional mutations on both of the gene copies

Why would your genome addition in a retroviral delivery vector be an RNA molecule?

because retroviruses do reverse transcription and therefore they only work on RNA genome

How is chromatin remodeling affect gene expression?

before gene expression can occur we need to open up the DNA to access the genes that are otherwise inaccessible... pre-requisite for other processes

lacI?

binds on operator but in presence of lactose it binds to lactose and can't bind to operator... repressor, prevents transcription of lacZ and lacY

Transcription factors bind to silencers...

bring in enzymes to close DNA (short term silencing)

Transcription factors that bind to enhancers...

bring in enzymes to open up and allow transcription (short term silencing)

what does cAMP do?

cAMP binds to CRP (transcription factor) when there is high glucose=CRP, low glucose=cAMP-CRP, CRP can't bind to DNA while cAMP-CRP can... without cAMP-CRP bound to lac operator RNA polymerase only has a low affinity for lac promoter. cAMP-CRP increases binding affinity for RNA polymerase @ lac promoter, thus increasing frequency of transcription

silencers

can be far away, up stream, down stream, in the gene but they repress rather than activate gene expression (EUKS

ability of glucose to repress lac operon transcription is called ________

catabolite repression

The technique of Sanger sequencing takes advantage of the fact that dideoxynucleotides (nucleotides in which the 3' hydroxyl group is absent) act as:

chain terminators

difference between activation by protein phosphorylation and Ras nucleotide exchange? G proteins versus proteins

change in protein conformation by covalent addition of a phosphate change in protein conformation by non-covalent binding of GTP BOTH cycle back and forth between an active and inactive state.

What are the special features of gene regulation?

chromatin remodeling through histone modifications by co-regulators, promoter and the basal transcription complex, enhancers/silencers and regulatory transcription factors that bind them, a complex set of interactions, and DNA methylation plays a role

Bioinformatics

combines math, biology and computer analyzing to analyze large sets of biological, biochemical and biophysical data

first sequence of human genome

completed by sanger sequencing and took 13 years and cost 3 billion dollars

gene therapy

correcting a defect by introducing a functional gene into the patient

what base is most often methylated?

cytosine

cytokinesis? how split? diff. for plants?

cytoskeletal ring of proteins that contract inside the cell membrane and creates a cleavage furrow. Plants build new plasma membrane in between the two cells and fill the space in between those

decision to divide depends on:

developmental state (stem cell?/cell type) nutritional and metabolic status wound response DNA damage pathogenesis senescence

In euk cells why are certain genes expressed only in certain types of cells?

different cell types have different regulatory transcription factors

what do the treatments do on the reporter gene? and example

different signals can turn on/activate transcription factors... ex: growth factors can activate a certain transcription factor that is bound to an enhancer

In comparing a leaf and root cell in the same plant, you are most likely to find the greatest difference in each cells content of:

enhancer binding transcription factors

enhancer and silencer examples

enhancer: HAT silencer: HDAC

some receptors have ___________ i.e. receptor with ________________

enzymatic activitites, cytoplasmic protein kinase domain

The promoter is _________ but _________ for gene expression.

essential insufficient

Why are promoters insufficient?

every gene has a promoter and promoters are largely similar from one gene to the next... if they didn't have other factors then every gene would be expressed in every cell

What are retrotransposons and how do they replicate?

ex: LINEs, a class of transposable elements, parasitic segments of DNA that are capable of replicating and moving within a genome, they are characterized by and RNA intermediate, they are likely evolved from retroviruses, largely a burden that your cells carry/ no evolutionary benefit known

a constitutively active gene is _______

expressed continuously

summarized process from signal to response

extracellular signal molecule, receptor protein, signaling intermediates, effector proteins

Why do multicellular organisms use DNA methylation?

for cell differentiation, some cells need to have genes permanently silenced... each cell has a different complement of silenced genes/and gene expression and different pattern of methylation

proteins that bind to a promoter to recruit factors needed to start transcription?

general transcription factors

What are Gene Families? (how do they arise?)

genes that are related, similar sequences that code for similar proteins or RNA products they can spread across many species or can all belong to the same species they arise through gene duplication (ex: globin gene family)

Genetic material transferred from parents to offspring is known as the:

genome

glucose and cAMP

glucose is inversely correlated with cAMP levels (glucose is a non-competitive inhibitor of enzyme adenyl cyclase which produces cAMP

what do proteasomes do?

help regulate protein levels

What is Highly condensed DNA? more open DNA?

heterochromatin, euchromatin

low glucose high lactose (lac operon transcription)?

high

what happens to cyclin levels?

high levels accumulate right before checkpoints, a lot of active CDKs and phosphorylation occurs and we get cell cycle progression cell size, nutrients, undamaged DNA and growth signals activates the cyclin expression peaks happen because the cell has achieved the criteria needed to move through a checkpoint then the levels go down quickly with the degradation of cyclins SO the cell division/cycle depends on synthesis of new cyclins

LINEs

highest portion of retrotransposons, they make up 20% of genome, they contain a promoter followed by genes that code for reverse transcriptase and integrase... when expressed reverse transcriptase generates a cDNA of the LINE sequence and integrase inserts the new LINE DNA into a new location in the genome. Thus when a LINE moves, a copy is left behind. Some retrotransposons encode the reverse transcriptase and integrase and those that do not depend on other retrotransposons for their expansion, the terminal repeats (LTRs) at end and beginning are recognized by the RTase and integrase

Role of histones in chromatin organization

histones form quaternary structure, histone tail of amino acids (side chains) are chemical modified to change their properties/chemical interactions (ex: histone acetylation)... chemical modifications change how histones interact with DNA and other molecules

Why do we do genome sequencing?

identify all genes/find genes to aid in the study of the genes and what goes wrong with those genes in diseases, predict protein sequence and function, investigate evolutionary questions, identify disease genes, identify cancer-causing mutations in individual tumors

How and when are post-transcriptional and post-translational regulation the same or different?

if it is after primary transcript is made it is post-transcriptional, all post-translational is post-transcriptional too... but there are things that are only post-transcriptional like alternative splicing

How does E. coli know if lactose is present?

if lactose is not present the lacI protein will bind to the operating sequence and will block transcription, if lactose is present lacI will bind to it, allowing transcription

How does receptor localization relate to the biochemical properties of the signal?

if receptor is usually localized in the cytoplasm then the signal is nonpolar if it has a transmembrane, extracellular, and cytoplasmic portion then the signal is polar

How can you infer the function of a gene based on its mutant phenotype?

in order to find the function of the normal gene through use of mutations, we need to know how the mutation perturbs the activity of the gene. You need to look at the phenotype in the absence of the gene (mutation where it doesn't get transcribed) versus the phenotype with the gene

What is difference between positive and negative gene regulation?

in positive regulation, the binding of a regulatory protein to the DNA is necessary for transcription to occur; in negative regulation, such binding prevents transcription.

difference between proks and euks? proks specifically... complication? euk complication? (related to identifying genes)

in proks there are no introns in bacteria and archaea, genes can be identified by promoter sequences associated with distinct ORFs. ONe complication is that genes are in polycistronic operons so there is one promoter for multiple open reading frames (euks have one promoter per ORF, genes contain introns and identification is more difficult, one strategy is to use cDNAs to identify mRNA sequences and locate the gene

targeted protein degradation

inactivating proteins is very important, proteolytic degradation can inactivate proteins through specific target

For the lactose operon, lactose is a(n) ______

inducer

key to cell cycle progression?

is control of cyclin accumulation

What is post-translational regulation? examples

it can regulate protein localization, activity and stability ex: folding, chaperones that assist proteins to fold correctly, complex assembly (a lot of proteins only function as protein complexes like ATP synthase which relies on PSI and PSII and cytochrome), targeted destruction of proteins (proteasomes degrade ubiquitin-tagged proteins and it targets misfolded proteins=error correction mechanism and controls lifespan of proteins=regulation of gene expression), chemical modification of proteins (acetylation, phosphorylation/dephosphorylation... proteins can be activated or deactivated by covalent modification such as addition of a phosphate group because of change in conformation of protein)

Once a gene is duplicated, there is a race that determines whether the duplicated genes are both retained. That race is between what two processes?

it depends on mutations and whether or not they accumulate mutations that provide fitness benefits it also depends on expression patterns and if they become distinct and each are required for organismal function or if the gene acquires a new biochemical function that provides an advantage 1. new biochemical functions 2. new patterns of expression

siRNA? what are they? what do they do?

it is a retrotransposon, breaks down double stranded RNA and recognizes retrotransposons to degrade, it is a defense mechanism against retrotransposons, joins with RISC complex in cytoplasm and will bind to retrotransposon RNA in cytoplasm and will cut it up before it can get translated or will sit to prevent translation

Illumina sequencing? what is it?

it is a type of high frequency sequencing allows you to get >billion basepairs of sequencing on something the size of a microscope slide. Illumina does several things to allow for this: 1. Microfluidics and very sensitive detection technology 2. PCR amplification on the substrate 3. Perform the sequencing reaction on the substrate and monitor the products in real-time

miRNA? what it does? where its from?

it is a way to turn off a gene, transcribed by a gene, post-transcriptional, doesn't break down retrotransposons, it breaks down a coding mRNA, it forms hairpin loops and the proteins bind to the stem loop and put it into RISC complex which will now target mRNA to degrade or sit and prevent translation

cry protein sprayed on corn kills the corn borer insect increases yield but... so...

it is expensive to make, store, and transport, uneven application; broad spraying can kill beneficial insects... so now we want to take the prok gene (that makes cry protein) and put it into the euk gene so it can be transcribed

Describe Histone Acetylation (how it changes histone interactions)... what are HATs and HDACs

it opens up the DNA/chromatin. HATs transfer acetyl group to lysine residues of a histone tail. HDACs remove the acetyl group and restore the structure of the DNA. (EUKS)

How does CRISPR target a specific genomic location? and what does it do? what is needed?

it uses an RNA molecule (guide RNA) that you design to base-pair with your sequence of interest needed: guide RNA, Cas9 protein that holds guide RNA and helps it basepair and cuts phosphodiester backbone and create a double strand break at that location, donor DNA template will have identical sequences to our location we are targeting on both sides of double strand break, THEN and there will be a crossing over event (homology directed repair) that uses the donor to repair the double strand break. STEPS: genomic sequence with mutation--> guide RNA and Cas9 cleave DNA at this specific location --> donor DNA with correct sequence (i.e. no mutation) provided for homology-directed DNA repair -->gene is now repaired

How is the activity of a G protein controlled?

its activity is controlled by whether it is bound to GDP (inactive) or GTP (active)... they cycle between active GTP and inactive GDP states

How does LacI repress lac operon transcription?

lacI is a repressor protein that binds to the operator sequence and blocks transcription. RNA polymerase (needed for transcription) can't bind because the promoter is too close to the operator sequence

How does a transcription factor find its regulatory DNA site?

lacI, like most transcription factors are proteins that must randomly diffuse around the cell and "bounce" into various cellular components.. if it it binds to something with which it has chemical affinity it will stay attached for a relatively long period of time.

which gene encodes the protein β-galactosidase?

lacZ

suppose there is a mutation to adenylyl cyclase such that it is no longer inhibited by glucose. How would this affect transcription of lacZ?

lacZ will be transcribed in high lactose conditions, regardless of glucose levels

Whole genome sequencing is often approached by a shotgun sequencing approach in which:

large genomes are randomly digested, short fragments are sequenced, and the overlapping sequences are assembled in order.

most common mechanism for regulating mRNA stability

length of polyAtail (longer, degraded less rapidly) sequences in UTR that bind proteins that can protect of degrade RNA (through RNases)

two categories of signals?

lipid soluble- rare, molecule can cross plasma membrane to enter cell, small nonpolar molecules, receptor proteins are inside the cell lipid insoluble- large hydrophilic molecules that are often proteins, carbs or other large macromolecules that can't pass through membrane and are the vast majority of signals

high glucose high lactose (lac operon transcription)?

low

high glucose low lactose (lac operon transcription) ?

low

low glucose low lactose (lac operon transcription)?

low

regulations of ribosome activity occur through_____

mRNA sequence, mRNA biding proteins, mRNA modification, codon usage, ribosomal protein modification

in cytoplasm, additional regulatory mechanisms controlling gene expression?

mRNA stability (it is very variable) in many cases the life span of mRNA is controlled by proteins binding to the 3' UTR of mRNAs and polyAtail... they can recruit RNases or protect from RNases

what do you need to make cDNA

mRNA, need cells of tissues or organs and isolate the mRNA and make cDNA's from mRNA; because of cell differentiation and different cells expressing different genes the cDNA's will be different depending on where you pull them from

goal of his operon regulation

maintain histidine homeostasis, maintain constant level of cytosolic

effector proteins examples and what the response from them

metabolic enzyme- altered metabolism, gene regulatory protein/transcription factor- altered gene expression, cytoskeleton protein- altered cell shape or movement

DNA methylation

methyl groups are most often added to cytosines and decrease the probability of gene expression

human insulin preproprotein changes when putting it in prok

modify sequences of the protein so that the signal peptide coding sequences are missing (this is used for bringing proteins to certain locations so we don't need this in prok) also have to engineer a new methianine to start of the insulin gene's coding sequence protease cuts out intervening sequences (c-chain) in the human ER so you end up with two peptide chains/ active insulin

Why is euk. post-transcriptional regulation very extensive and complex?

more steps involved going from a transcript to a protein (transcription and location are in different locations)

If you are expressing a foreign gene in a eukaryotic organism, how can you drive expression in specific tissues (e.g., how would you express the beta-carotene biosynthetic genes specifically in the seed?)?

move enhancer for the biosynthetic genes and put them into genome before the gene you want to transcribe

Why is Eukaryotic expression control more complicated than in prokaryotes?

multicellular organisms (eukaryotes) have many complex functions and cell differentiation which requires more complicated gene expression control... it is much more complex and more regulated steps too.

lacI ... _______ regulation and _______ expressed cAMP-CRP... __________ regulation

negative, constituitively (always) expressed positive

HAT's exert their effect on activity by ______

neutralizing the positive change of a lysine on histone tails

What is the purpose of transcriptional gene regulation?

not all genes are expressed in every cell, this leads to cell specificity/ cell differentiation

proteasome

not specific, will degrade any ubiquited protein it interacts with so there must be a machinery to target individual cyclins for ubiquitation and degradation during the cell cycle

chromatin remodeling

nucleosomes are modified to expose different stretches of DNA to the nuclear environment

Dosage

number of copies of each gene in a genome.

Describe the two mechanisms by which enhancers function

once bound to the specific DNA sequences at enhancers, Transcription Factors can promote transcription through: 1. recruit co-activators (HATs) or co-repressors (HDACs) to the enhancer or silencer and lead to chromatin remodeling 2. enhancers are bound by regulatory transcription factors that lead to interactions with the basal transcription complex through DNA looping and interacting proteins. These interactions promote RNA polymerase binding and transcription initiation. (EUKS)

how would you screen for E.Coli unable to transcribe histidine operon?

one media with high and one with low histidine, if unable to build histidine on the low media you know it's E.Coli unable to transcribe histidine.

How are the steps in the MAPK pathway linked? For example, why does MAPKKK specifically act on MAPKK? Why does MAPKK specifically act on MAPK?

one phosphorylates the other and this activates that protein which then does the same to the next with a series of phosphorylation and activation

What is hemi-methylated DNA?

one strand is methylated and the other strand is not (happens in DNA replication) DNA methyltransferase (DNMT) recognizes the hemi-methylated strand and methylates the new synthesized strand

it is essential for the piggy-back mechanism that the sites of methylation are _____________

palindromic

process of incorporating new line transposons into the genome

parent LINE is transcribed then mRNA of the LINE is brought to cytoplasm where it is translated and after that the mRNA and the reverse transcriptase and integrase that was translated are brought back into the nucleus where the cDNA is made with the help of the reverse transcriptase and then is made double stranded, the integrase makes a cut in the genome and then incorporates the daughter LINE into the genome (now two copies in the genome if you started with one)

how to doanalysis on str's?

pcr amplify the short tandem repeats and pcr products tell you how many repeats were in the sequence? primers will be outside of repeat sequence and present in all human populations genomes

An individual's genome sequence can reveal his or her disease susceptibilities and drug sensitivities, which allows treatments to be tailored to the individual. This approach is called:

personalized medicine.

what removes the phosphate?

phosphatase

SNPs arise from

point mutations

What kind of developmental or physiological situations require cell signaling?

post-translational responses and response to heat, or for embryos to develop, cells to differentiate, formation of tissues and organs during development regulates cell identity

What does chromatin remodeling do?

process that controls the extent of DNA packaging or condensation

why is it easier to identify genes and assemble genome sequences for proks?

prok. genomes are much smaller, no introns (just have to look for promoter sequences which are very conserved and orf which follow immediately after), and there is only 1 circular chromosome (vs. 23 linear ones)

an operon consists of what?

promoter, operator, coding sequence for structural protein

How can a signal be amplified?

protein kinase cascade is one example because one cell surface receptor activates many G protein molecules

How do kinases function?

proteins that phosphorylate other proteins, normally the kinases float around on their own and are inactive as enzymes. when it binds with the ligand, the receptor conformation changes and this leads to dimerization... the kinase domains of dimerized receptors phosphorylate both each other and other targets protein kinases are enzymes and they use ATP as a substrate and each have a specific set of substrates, phosphorylation (adding a phosphate group to a particular amino acid) acts to change substrate protein conformation and thus the activity of the protein. (adding or removing phosphate group can turn a protein on or off) kinases use ATP and protein as substrates and then takes a phosphate from the ATP and attaches it to the protein which then becomes active and this does the same to the next protein

gene regulation post-translational? post-transcriptional? transcriptional?

ptranslational: ubiquinimating proteins ptranscriptional: miRNA transcriptional: HDAC or HAT

how to find enhancers

reporter genes and gene expression profiling to identify enhancers

highly methylated... highly ____

restricted

how to edit genomes

retroviral complementation and CPRIPR gene editing

how do we get cDNA

reverse transcriptase will give you a DNA copy of mRNA (reverse transcriptase is an enzyme that uses RNA as a template for DNA synthesis) then the single stranded cDNA can be converted to a double stranded DNA by reverse transcriptase or DNA polymerase... REVERSE TRANSCRIPTASE requires a primer

euk ribosome regulation activity, how regulated?

ribosome small subunit assembly at 5' cap, ribosome scanning, ribosome translation elongation along the transcript (how often it is translated and how fast translation occurs is regulated by the cell)

pathway with CREB

signal molecule --> activation of G protein coupled receptor --> GTP bound G protein --> activation of adenylyl cyclase --> cAMP level increase --> activation of PKA --> activation of CREB --> target gene transcription

Consider the MAPK pathway. Identify the signal, the receptor, the signaling intermediates and the effectors.

signal: polar signaling molecule signaling intermediates: Ras activating protein, Ras protein, MAPKKK, MAPKK, MAPK effector: transcription factors

SNPs

single nucleotide polymorphism.... 1) different single base at genetic location 2) this variation exists in a significant portion of the population they may drive phenotypic differences (cystic fibrosis, eye color) while they may also have no effect on phenotype but provide hereditary info,

translation in euks (initiation)

small subunit of ribosome assembles here and scnas for AUG, @ AUG large subunit assembles and translation begins

protein transport

some proteins will be transported automatically, for some proteins the process is regulated, transcription factors in cytoplasm is inactive, if we regulate this then we can get transcription factors to move to the nucleus (where it needs to be to act)

example of lipid soluble signals? and explain the process?

steroid hormones (testosterone, cortisol, estrogen) are an example of lipid soluble signals that can pass through membrane (being hydrophobic), the receptor proteins are inside the cell most likely in cytoplasm, without signal present the receptor is stuck in the cytoplasm, when steroid binds to receptor it will change the receptors confirmation so the receptor now can move into the nucleus and act as a transcription factor in the case of steroids.

needed for PCR

template, thermal resistant DNA poly (taq DNA poly), dNTPs, and primers to anneal at a specific location

what is cell signaling?

the ability of cells to perceive signals from the surrounding environment

key to sanger method?

the ddNTPs lack the 3' OH and the DNA polymerase cannot extend the chain.... so when a dideoxynucleotide is incorporated (at a low frequency) the reaction on that strand is terminated) and since the dideoxys are fluorescently labeled you can identify which dd base terminated the synthesis

Why does genome analysis have to be done by computer?

the genome is sooooooo long

What might a cDNA tell you that a genomic sequence would not?

the introns are spliced out and you can now identify regions of the genome that are transcribed... very useful for identifying genes in a genome... you can compare the cDNA to genome sequence and when you compare the two there are gaps in alignment which represents introns ... the lines that match up represent exons and if you sequence many cDNAs you can: identify alternative splicing (if you see 2 different cDNAs for same genome location), get a picture of all genes expressed in an individual sample

how does histone acetylation cause DNA to open?

the lysine residues on histone tails are positively charged and therefore are attracted to the negative DNA and form tight coils.... when the negative acetyl group is added to the lysine group the lysine group no longer is attracted to the DNA because the charge is now slightly negative so the chromatin opens up

in lactose operon, what does repressor bind to?

the operator

how transposable elements spread?

the parent LINE is transcribed and brought to the cytoplasm and is translated (RTase, integrase, and mRNA transcript) they are brought to the nucleus and then RTase convert the mRNA into cDNA and the integrase makes a double strand break in genome and integrate the cDNA in this position.

How do transcription factors bind to DNA?

the transcription factors have a protein domain that chemically matches the DNA groove for that DNA sequence

advances in technology with sequencing

the use of fluorescent markers and capillary electrophoresis simplified DNA sequencing and allowed process to be automated... 23andme (and other companies) have been offering sequencing to people but it is controversial because it is a medical device that needs FDA approval... but they can now provide analysis of about 100 traits one at a time

Why don't transposable elements exponentially expand?

there is a defense mechanism against retrotransposon expansion in the genome... siRNAs function to silence retrotransposons... LINE elements are all over the place (in lots of transcribed regions and in multiple orientations)... your cells recognize the double stranded RNA and chop it up into pieces and incorporate it into the RISC complex in the cytoplasm and it is complementary to the retrotransposon RNA and will basepair with them and degrade it as soon as it gets out into the cytoplasm before it can translate and get copied and integrated into genome

where do miRNAs come from?

they are encoded for by a gene too, gene product isn't a protein but an RNA product

What is the role of the di-deoxy nucleotide in Sanger sequencing?

they are fluorescent and they will stop the synthesis of the synthesized strand because they lack the 3' OH

How do CDKs become active? and what do they do?

they are inactive on their own, they depend on cylcins that bind to CDKs and form a complex and it becomes active, catalytic site is exposed and will bind to proteins that carry out cell cycle progression. (CDKs phosphorylate target proteins involved in promoting cell division)

What are the key features of secondary messengers? and example

they are not proteins, G-protein-activated enymes catalyze the production of many small nonprotein signaling molecules called second messengers that diffuse rapidly and spread the signal message throughout the cell = massive amplification + branching. The same second messenger can trigger different events (e.g. open ion channels, activate protein kinases, etc.) in different cell types. cAMP

What are the roles of cyclins in cell cycle progression?

they are the most important regulators of the CDKs enzymatic activity... this will activate the CDK when it binds to it and this will then phosphorylate target proteins that promote cell division

problem with transposable elements

they can quickly expand to dominate a genome... a lot of energy and unneeded DNA

what happens to miRNA?

they fold back on eachother and form a hairpin loop structure on miRNA and is recognized by proteins that take out the sequence and produce mature miRNA and ultimately it is incorporated into RISC (RNA Induced Silencing Complex), this is brought to mRNA and will bind to the target by basepairing and either chops up/degrades mRNA (with RNase as part of its complex) or RISC blocks translatin of mRNA by sitting on mRNA and ribosomes can not come through (EITHER WAY turns off gene that mRNA represents) complementarity to a specific mRNA

How do miRNAs find their target mRNAs? and what do they do?

they have specific regulatory activity and target specific mRNAs for degradation or to block translation... interefere with mRNAs by direct base pairing

how do enhancers work?

they have to be bound by a protein (transcription factors containing DNA binding domains) because they are DNA sequences... transcription factors bind to specific DNA sequences (they match the DNA groove... typically at the major groove... each base pair combo has a unique chemical face in the major groove)

How do proteins bind specific DNA sequences?

they match the DNA groove... typically at the major groove... each base pair combo has a unique chemical face in the major groove)

what do polar signaling molecules rely on? why?

they rely on cell-surface receptors that have an extracellular, transmembrane and a cytoplasmic domain because polar signals cannot cross the plasma membrane.

pioneer transcription factors

they trigger the whole process, lead the way for all the other transcription factors (enhancer needs to be exposed for this transcription factor) usually this binding draws in a co-activator like HATs to open up the DNA to transcribe

How do miRNAs regulate a specific mRNA?

through complementary base pairing with the mRNA

What steps are involved in gene expression control?

transcription, translation, post-translational

ways of achieving gene duplication

unequal crossing over in recombination (one chromosome has 2 copies of a gene other chromosome has 0 copies as a result ... 2 copies are right next to eachother/tandem genes in genome), polyploidy (defects in chromosomal segregation during meiosis results in tetraploidy, which duplicates every gene in the genome ... does not result in tandem genes)

What distinguishes genes duplicated through unequal crossing over versus polyploidy?

unequal crossing over in recombination results in tandem genes in the genome on the chromosome polyploidy duplicates entire genome... the gametes are haploid and form a diploid sometimes there are mistakes and the two gametes are diploid and when they come together the results are a tetraploid which duplicates all genes in a genome (in this case duplicated genes end up on different chromosomes and this duplication does NOT result in tandem genes

translation in proks (initiation)

upstream of AUG are ribosomal binding sites to which rRNA base pairs and can translate from AUG down (ribosome will assemble upstream of all of the start codons on transcript simultaneously)

How do you find genes in a DNA sequence?

use properties of genes, look for potential promoter sequences in the genomic sequences, look for genes that encode proteins that have coding sequences (you can look for these by looking for start and stop codons)

retroviral complementation

use retroviruses to insert recombinant DNA into the human genome (transformation) to fix a genetic defect 1. recombinant RNA prepared and packaged into virus 2. Target cell infected 3.Reverse transcriptase makes a DNA copy 4. integrase inserts DNA into genome You have now added a NEW sequence into the human genome...you do NOT repair the mutant gene, but you complement it with a functional gene copy by transformation

GMO's , engineered traits?

used to help productivity, herbicide resistance, insect resistance, drought resistance, viral resistance, increased nutritional value

Short repeated sequences of DNA that vary in number from one chromosome to the next are called:

variable number tandem repeats.

What does a genetic screening do?

we can see the genes involved in a particular biological function/phenomenon

40% of the human proteins____

we don't know their biochem function or biological role

How does a ligand change the activity of the receptor it binds to?

when the signal binds to the receptor protein the protein changes confirmation and this as a result leads to the change in the receptor's activity.

How is a particular pattern of DNA methylation maintained through cell division?

when you replicate DNA you also replicate the DNA methylation pattern piggy-back model- methylated strand of DNA provides a marker on the newly replicated duplex for methylation of the other strand. The methylation status of genes from the previous generation is remembered. Template strand is methylated, new strand is un-methylated so regions of the DNA have one strand methylated and one strand is not (hemi-methylated DNA methyltransferase (DNMT) recognizes the hemi-methylated strand and methylates the new synthesized strand it is essential for the piggy-back mechanism that the sites of methylation are palindromic (proteins will recognize the methylated cytosine and they will bring in DNMT and they will methylate the un-methylated-cytosine on the opposite strand

promoter

where RNA poly associates and begins transcription

What happens when you remove the acetyl group?

with HDAC, restore the tight chromatid packing because you restore the ability of the DNA and the histone tail to have the ionic interaction

DNase 1 differing effects

with condensed chromatin (with histones) the chromatin do not break down because DNA is tightly packed and enzyme can't reach the phosphodiester bond to cleave it (DNA is highly inaccessible). with open chromatin, the DNase will chop everywhere except where nucleosomes are on the DNA and the DNA will be degraded

What can you do with cDNAs? What roles do cDNAs play in genome annotation?

you have a coding sequence with the introns spliced out, you can identify the regions of genome transcribed (important for identifying genes in the genome) you compare cDNA sequences to the genomic DNA because the introns are not in the cDNA sequences the cDNA will have gaps in alignments that represent introns and the lines that line up nicely and match the genome represent exons you also get to see which genes are transcribed you can identify alternative splicing because they will have different cDNA sequences for the same genomic location if you sequence enough cDNAs you get a good picture of the genes expressed and the level of expression in an individual sample (ie liver cell)

When you try to express a gene from one organism in another, what things do you need to change about that gene? What can stay the same? Does it depend on the expressing organism?

you will need to change all of the parts of gene expression control that differ between the two organisms to get the gene expressed properly - enhancers are only likely to work in the endogenous species (human enhancer can only be guaranteed to work in humans) -promoters often work in closely related species but best to use a promoter from the species -transcription termination- same as promoters -UTRs- likely to only get proper UTR function (ex: RNA stability) in endogenous species -translation initiation- very different in Proks vs. Euks going from euk to prok... need to take the introns out (easiest way to do this is use cDNA which has a copy of coding sequence without the introns), need to use promoter and transcription terminators from E.coli and fuse with insulin coding sequence, will have to add ribosome binding sites next to aug so that ribosome can bind there for translation initiation... things are fused with restriction enzymes then put into plasmid (with antibiotic resistance and an origin of replication

How would you identify all the enhancers for a new gene using reporters?

you would have to go through DNA surrounding a gene to identify functional enhancers. If you have ALL the enhancers, then your reporter will have the exact same expression as the gene in question.

Why do humans require clearing of DNA methylation during meiosis?

zygotes need to be able to transcribe all genes in order for an organism to become complex... methylation is maintained once it is set up... you want a zygote with potential to express all the genes in the genome... if you do not clear the DNA methylation you may have epigenetic traits where genome has normal sequence but its not functioning correctly without clearing methylation (autism is a cause)