genetics 3, bio exam 4, genetics 2, genetics test 1
CRISPR stand for
"Clustered Regularly Interspaced Short Palindromic Repeats"
nanopore sequencing
"Third generation sequencing" Released in 2014 "Long reads" span thousands of nucleotides
Several additional proteins collaborate with the Hox proteins in repressing Distal-less
Two are proteins encoded by segment-polarity genes: sloppy-paired (Slp)-->Mark the anterior compartments (of each segment) engrailed (En)-->Mark the posterior compartments (of each segement)
How does a cell determine which of its thousands of genes to transcribe?
Two components: 1)short stretches of DNA of defined sequence 2)gene regulatory proteins that recognize and bind to this DNA
SWI/SNF complex
Two genetic screens in yeast for mutants in seemingly unrelated processes led to the discovery of the same gene whose product plays a key role in chromatin remodeling. In both cases, yeast cells were treated with agents that would cause mutations In one screen, these mutagenized yeast cells were screened for cells that could not grow well on sucrose (sugar nonfermenting mutants, snf). In another screen, mutagenized yeast cells were screened for mutants that were defective in switching their mating type (switch mutants, swi) One mutant gene was found to cause both phenotypes: could neither utilize sucrose effectively nor switch mating type. swi2/snf2 locus ("switch-sniff")
Double Helix Model of DNA: Six main features
Two polynucleotide chains wound in a right-handed (clockwise) double-helix. Nucleotide chains are anti-parallel: 5' → 3' 3' ← 5' Sugar-phosphate backbones are on the outside of the double helix, and the bases are oriented towards the central axis. Complementary base pairs from opposite strands are bound together by weak hydrogen bonds. A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds). Base pairs are 0.34 nm apart. One complete turn of the helix requires 3.4 nm (10 bases/turn). Sugar-phosphate backbones are not equally-spaced, resulting in major and minor grooves.
Model Organism Yeast
Two yeast regulatory systems in detail: 1. galactose-utilization pathway 2. Control of mating type
Some transposable elements are targeted to specific safe havens
Ty3 inserts exclusively near but not in tRNA genes (do not interfere with the production of tRNAs) Ty3 proteins recognize and bind to subunits of the RNA polymerase complex that have assembled at RNA promoters document
A pure line of plants of the genotype a/a; b/b; c/c; d/d; e/e (all recessive to wild type) are crossed with a wild type. One F1 individual expressed the recessive alleles d and 3. This individual arose most likely from
a deletion in the wild-type parent
Hybridization of single-stranded wild-type DNA with DNA from mutations caused by IS elements characteristically shows (through electron microscopy)
a single-stranded loop representing IS DNA
DNA elements that prevent the spreading of heterochromatin into adjacent euchromatic regions are called:
barrier insulators.
The "domain-swapping" experiment that grafts the Gal4 activation domain to the LexA DNA-binding domain generates a chimeric protein that will:
bind to the LexA site.
GAL3 promotes GAL gene expression by:
binding galactose and ATP, and subsequently binding to Gal80.
Hershey-Chase experiment
confirmed that DNA is the genetic material because only radiolabeled DNA could be found in bacteriophage-infected bacteria
intercalating agents
consequences: base insertions and deletions Planar molecules that mimic base pairs and can slip themselves in (intercalate) between the stacked nitrogen bases can cause insertion or deletion of a single nucleotide pair
A null repressor mutation (I-) results in:
constitutive transcription.
Chromatin made up of nucleosomes that are methylated at H3meK9 and bound up with HP-1 protein
contain epigenetic marks that are associated with heterochromatin.
Mixing bacterial genotypes produces rare recombinants
cross feeding? Some form of recombination of genes had taken place between the genomes of the two strains to produce the prototrophs
A plaque is a clear area in which all bacteria have been lysed by phages
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A single Tc1 element can repress transposition
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Admixture: mix of genes that results when individuals have ancestry from more than one subpopulation
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Allele frequency may vary along a gradient
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Allopolyploidy in Brassica
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Ames test of aflatoxin B1 mutagenicity
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An R factor may contain several transposons carrying resistance genes
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An aminoacyl-tRNA synthetase attaches an amino acid to its tRNA
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Aneuploidy affects the balance of gene dosage
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Area of concern in modern genetics
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Bacteria conjugate by using pili
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Bacteria exchange DNA by several processes
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Butterfly mimicry is facilitated by chromosome inversions
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Chemical mutagens induce mutations: base analog
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Chromosome Constitutions in a Normally Diploid Organism with Three Chromosomes (Identified as A, B, and C) in the Basic Set
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Coactivators bridge interactions between transcription factors and RNA polymerase II
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DNA transcription regulatory sequences and factors
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Demonstration of transposition through an RNA intermediate
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Different effects of inversions
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Double-stranded cDNA is synthesized from mRNA
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Electron micrograph of phage infection
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F plasmids transfer during conjugation
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Finding the clone of interest by using DNA or RNA probes
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Finding the clone of interest by using antibody
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Genetic drift can lead to fixation
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Histone acetylation, deacetylation, and gene expression acetyl coa lysine hat hdac coenzyme positive charge neutral charge acetylated lysine
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Human genes contain many transposable elements
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Inactivated endonuclease attached to a transcriptional activator domain to enable transcription This is basically the opposite of CRISPRi
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Inactivation of TEs following insertion into pi-clusters
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Indel mutations result in frameshifts
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Ionization of bases brought about by proton exchange between water and hydrogen bonds can cause DNA polymerase to insert a mismatch
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Knock out of Ins1 via deletion
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Life cycle of a retrovirus
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Maize mutator transposon used for knock out
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Meiotic nondisjunction generates aneuploid products
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Meiotic pairing in triploids often leads to aneuploidy
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Methods of introducing a transgene
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Nucleotide diversity among diverse organisms
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Nucleotide variation at the G6PD gene in humans
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One of the reactions catalyzed by HMTase
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Opposite DNA strands can serve as template for RNA
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Overview of DNA Damage and Repair
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P elements can be engineered to transfer genes
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Pedigrees show when genes are identical by descent
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Primary structure
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Probable origins of Williams syndrome
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Producing cells containing a targeted gene knockout
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RNAi Mechanism
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Reactions in exon splicing
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Real-Time PCR (qPCR)
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Replacement of Ins1 with GFP, Knock out of Ins1 via HR
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Replication slippage causes repeat expansion
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Repression and activation compared
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Retrotransposons have features in common with retroviruses
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Robertsonian translocation creating Down syndrome in certain progeny
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Signal-dependent regulation of gene expression
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Signal-dependent regulation of translation
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The logic of obtaining a genome sequence
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The pre-mRNA transcript of the rat-tropomyosin gene is alternatively spliced in different cell types.
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The repressor contains a
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Trinucleotide repeats in the FMR-1 gene hinder transcription
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Two results of transformation by simple yeast vectors
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Using CRISPR-mediated gene drive to wipe out mosquitos
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methylation phosphorylation acetylation ubiquitylation
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other modifications
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• The addition of a single ubiquitin (Ub) monomer to the sliding clamp (PCNA) allows the bypass polymerase to bind to PCNA and begin replicating
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targeted insertion of transgene by homologous recombination
document 1)Homologous recombination between the homologous regions on the vector and target gene in chromosome--->knockout 2) Full vector insert ectopically 3) No insertion
The Ds transposable element helps cause breakage
document Breakage of chromosome 9 was due to the presence of two genetic factors: • Ds (Dissociation): Located at the site of the break • Ac (Activator): Hard to map and was found at different locations in different plants document • C' (colorless) is dominant to C (blue) • Ac causes breakage at Ds in some cells leading to the production of blue spots • Acentric fragment produced document • Ds can also jump and cause breakage in new areas • Location of breakage determines overall phenotype
Alkylation-induced specific mispairing
document Some mutagens do not get incorporated into the DNA but instead alter a base in such a way that it will form a specific mispair.
An inserted element is flanked by a short repeat
document Virtually all transposable elements (in both prokaryotes and eukaryotes) are flanked by a target-site duplication, indicating that all use a similar mechanism Target site duplication • Transposase makes a five-base pair staggered cut
Unusual inheritance of imprinted genes
document mutations
Making mice where genes are knocked out is now super easy and cheap
document Some of the offspring will hopefully be CRISPR edited
Mechanism of transposition in prokaryotes: replicative
document e.g. Tn3 • a new copy of the transposable element is generated in the transposition event • One copy appears at the new site and one copy remains at the old site • Often uses transposase and resolvase to form and resolve a cointegrate
Three experiments demonstrating gene silencing
document jorgensen fire/mello baulcombe
The dideoxy sequencing method
document x3
Enhanceosomes recruit chromatin remodelers
enhanceosome forms a binding site for GCN5, which binds and adds acetyl groups to nuc 1,2 gcn5 complex chromatin remodelers 2 more pictures on document
The Proteome
Proteome is the complete set of proteins in an organism, organ, tissue, or cell Proteome is enriched by 2 cellular processes: 1)Alternative splicing of pre-mRNA 2)Posttranslational modifications of protein
Eukaryotes possess three forms of RNA polymerase, each of which transcribes different types of genes
RNA polymerase II transcribes all protein-encoding genes, for which the ultimate transcript is mRNA, and transcribes some snRNAs.
Transcription initiation in prokaryotes
RNA polymerase from E. coli contains five subunits of the core enzyme 2a, ß, ß ′, ω, and a σ factor The two α subunits: help assemble the enzyme and promote interactions with regulatory proteins, β subunit: is active in catalysis: β' subunit binds DNA; ω subunit has roles in enzyme assembly and the regulation of gene expression The σ subunit: binds to the −10 and −35 regions, thus positioning the holoenzyme to initiate transcription correctly at the start site. The σ subunit also has a role in separating (melting) the DNA strands around the −10 region
telomerase
RNA-containing enzyme RNA acts as a template for the addition of a telomeric repeat sequence, which is added to the 3' overhang
Even before the structure of DNA was elucidated, genetic studies indicated that the hereditary material must have 3 key properties
Replication Informational content Mutation
Eukaryotes
Structure of genome Size of genome Location of gene transcription and translation Gene clustering Default state of transcription DNA structure
Prokaryotes
Structure of genome Size of genome Location of gene transcription and translation Gene clustering Default state of transcription DNA structure
The telomeric cap structure
Telomeres also associates with proteins to form protective cap structure: hides the chromosome ends from the cell's DNA repair machinery "Cap" protects the telomere at the end of a chromosome
How does turning on and off toolkit genes build animal form? How is it choreographed during development?
The genetic control of development is fundamentally a matter of gene regulation in space and over time
How does histone acetylation alter chromatin structure and facilitate changes in gene expression?
histone deacetylation is the reverse arrow of the picture The addition of acetyl groups to histone residues neutralizes the positive charge of lysine residues and reduces the interaction of the histone tails with the negatively charged DNA backbone. This results in more open chromatin as the electrostatic interactions between adjacent nucleosomes and between nucleosomes and adjacent DNA are reduced In addition, histone acetylation, in conjunction with other histone modifications, influences the binding of regulatory proteins to the DNA. A bound regulatory protein may take part in one of several functions that either directly or indirectly increase the frequency of transcription initiation.
Dual control of the ara operon: AraC serves as an activator and as a repressor
in the presence of arabinose: activation -AraC protein binds to araI region -the CAP-cAMP complex binds to a site adjacent to araI in the absence of arabinose: repression -AraC protein assumes a different conformation and binds to both the araI and the araO regions, forming a DNA loop--->prevents transcription of the ara operon
Jacob and Monod (1960) proposed the operon model
in which a group of genes is regulated and expressed together as a unit The lacI gene regulates transcription of the structural genes by producing a repressor molecule The repressor is allosteric (it interacts reversibly with another molecule, causing both a conformational change in three-dimensional shape and a change in chemical activity). The lac operon is subject to negative control transcription occurs only when the repressor fails to bind to the operator region
stage 1: DNA sequencing library construction
isolated DNA--> fragment--> end repair--> adaptor ligation
Inversions
paracentric and pericentric
The ________________ is a cis-acting element that binds RNA polymerase II.
promoter
Building blocks of DNA
purine: adenine and guanine- two pyrimidine: cytosine and thymine- one
Base pairing in DNA
pyrimidine+ pyrimidine:DNA too thin purine+purine:DNA took thick purine+pyrimidine:thickness compatible with x-ray data
rRNA folds up by intramolecular base pairing
rRNAs assisted by the ribosomal proteins, carry out most of the important steps in protein synthesis document
Which of the following describes the histones associated with the nucleosomes of active genes?
rich in acetyl groups (i.e., hyperacetylated)
Compared to heterochromatin, euchromatin is:
rich in genes and comprised of loosely packed nucleosomes.
Genomics:
the study of genomes in their entirety
Repressors are trans-acting
the top one the expression is blocked the bottom one the expression is blocked Repressor is trans-acting: gene product can regulate all structural lac operon genes, whether in cis or in trans document
Class 2 DNA transposons
p-element structure document
Change in chromosome structure
Also known as rearrangements Deletion Duplication Inversion Translocation
Obtaining the sequence of a Genome
(1) break the genome up into thousands of more or less random little pieces (2) read the sequence of each little piece (3) overlap the little pieces where their sequences are identical (4) continue overlapping ever-larger pieces until you've accounted for all the little pieces
DNA transposons have been modified and used by scientists in two important ways:
(1) to make mutants that can be identified molecularly by having a transposon tag (2) as vectors that can introduce foreign genes into the chromosome.
reciprocal cross: white-eyed female x red-eyed male
(red female and while male)= white-eyed female x red-eyed male F1 : all females are red-eyed but all males are white-eyed Every female inherited the dominant w+ allele from the father's X-chromosome F2 ratio ½ red-eyed and ½ white-eyed of both sexes (because in this case white-eyed allele is present on the male X-chromosome)
2. Induced mutations
- Result from the influence of an extraneous factor, either natural or artificial Radiation UV light Natural and synthetic chemical - Production of mutations in the laboratory through exposure to mutagens is called mutagenesis
Genetic evidence for the operator and repressor
- Synthetic inducer - Not hydrolyzed by β galactosidase -IPTG
Annotation is the process of identifying
- genes - their regulatory sequences - their functions
accuracy of DNA polymerase is essential
--Less than 1 error per 1010 nucleotides is inserted --Due in part to "reading" of complementary bases --also contains its own proofreading activity (3'-to-5' exonuclease activity)
Replication of alleles during the S phase
-Each chromosome divides in to two sister chromatids -The single DNA molecule of each chromosome replicates producing two DNA molecules, one for each chromatid
Most toolkit proteins are:
-Transcription factors -components of ligand-mediated signal transduction pathways
Toolkit genes mutations from two sources have yielded most of our knowledge
1)Spontaneous mutations that arise in laboratory populations 2)Induced mutations at random by treatment with mutagens (chemicals or radiation)
Glucose levels control the lac operon
1. -Lactose: the lac operon is switched off? 2.+Lactose: 3. +Lactose +Glucose: the lac operon is switched on Mechanisms have evolved that prevent the cell from synthesizing the enzymes for lactose metabolism when both lactose and glucose are present together ---> Catabolite repression (glucose is a breakdown product/catabolite of lactose) ---> Works through an activator protein
Two mechanisms for regulating transcription of the tryptophan operon
1. A repressor protein binds an operator, preventing the initiation of transcription. 2. Attenuation : mRNA production is attenuated (decreased) when tryptophan is plentiful. ❖ Unlike other bacterial control mechanisms attenuation acts at a step after transcription initiation
x-linked dominant disorders
1. Affected males pass the condition to all their daughters but to none of their sons. 2. Affected heterozygous females married to unaffected males pass the condition to half their sons and daughters. e.g. 1.Hyphophosphatemia (type of Vit D resistant rickets) 2. Some forms of hypertrichosis excess body and facial hair
Types of Point Mutation
1. Base Substitution: one base pair is replaced by another Transition: Purine is replaced by a purine (A to G or G to A) or Pyrimidine is replaced by a pyrimidine (C to T or T to C) Transversion: Pyrimidine is replaced by a purine or purine is replaced by a pyrimidine 2. Base insertions or deletions (indel mutation) Addition or deletion of a single base pair or multiple base pairs
Homology-dependent repair systems
1. Base-excision repair • After DNA proofreading by DNA polymerase, this is the most important mechanism used to remove incorrect or damaged bases • The main target is nonbulky damaged bases- result from variety of causes: methylation, deamination, oxidation or the spontaneous loss of a DNA base 2. Nucleotide-excision repair • Can correct bulky adducts and correct damage to more than one base • Relieves stalled replication forks and transcription complexes
The DNA sequence of the genome becomes the starting point for a whole new set of analyses aimed at the structure, function, and evolution of the genome and its components.
1. Bioinformatics analyses the information content of entire genomes. 2. Comparative genomics considers the genomes of closely and distantly related species for evolutionary insight 3. Functional genomics the use of expanding variety of methods, including reverse genetics to understand gene and protein function
Cotranscriptional processing of RNA steps
1. Capping 2. Splicing 3. Cleavage and Polyadenylation
Large-Scale Chromosomal Changes
1. Change in chromosome number 2. Change in chromosome structure
Transposable elements in eukaryotes class 1
1. Class 1 retrotransposons: Transpose through RNA intermediates Encode a reverse transcriptase that produces double-stranded DNA copy (from an RNA intermediate) 2. Class 2 DNA transposons Encode a transposase that cuts the transposon from the chromosome and catalyze the reinsertion at other chromosomal locations
Recombinant DNA Techniques
1. Cleavage of DNA at specific sites - restriction nucleases 2. DNA ligation- to design and construct DNA molecules that are not found in nature. 3. DNA cloning- cloning vectors or the polymerase chain reaction (PCR) in which a portion of DNA is repeatedly copied to generate many copies of identical molecules 4. Nucleic acid hybridization- find a specific sequence of DNA or RNA with great accuracy and sensitivity on the basis of its ability to selectively bind a complementary nucleic acid sequence. 5. DNA sequencing- Rapid determination of the sequence of nucleotides of any DNA (even entire genomes) 6. Microarrays- DNA bound to glass chip
Base-excision repair in Bacteria
1. DNA glycosylases: cleave base-sugar bonds, thereby liberating the altered base & generating apurinic or apyrimidinic (AP) site 2. AP endonuclease: nicks the damaged strand upstream of the AP site 3. Deoxyribophophodiesterase (dRpase): removes the AP sugar-phosphate 4. DNA polymerase: fill up the gap with nucleotide 5. DNA ligase: seals the new nucleotide into the backbone document
Base-excision repair in Eukaryotes
1. DNA glycosylases: cleave base-sugar bonds, thereby liberating the altered base & generating apurinic or apyrimidinic (AP) sites 2. AP endonuclease: nicks the damaged strand upstream of the AP site 3. DNA Polymerase β: inserts a single nucleotide (short patch) or 2-10 nucleotides (long patch) 4. Flap Endonuclease: in long patch BER, removes flap with AP site 5. DNA ligase: seals the new nucleotide into the backbone document
Several Molecular Processes Lead to Epigenetic Changes
1. DNA methylation 2. Chemical modification of histone proteins 3. RNA molecules that affect chromatin structure and gene expression
Eukaryotic gene regulation must be able to;
1. Ensure that the expression of most genes in the genome is off at any given time while activating a subset of genes 2. Generate thousands of patterns of gene expression
Finding a specific clone of interest
1. Finding specific clones by using probes a)Those that recognized a specific nucleic acid sequence (DNA or RNA) Probes for finding DNA • Probing for DNA makes use of the power of base complementarity. •Two single-stranded nucleic acids with full or partial complementary base sequence will "find" each other in solution by random collision. b)Those that recognize a specific protein 2. Finding specific clones by functional complementation/mutant rescue
Overview of transcriptional regulation
1. In bacteria all genes are transcribed into RNA by the same RNA polymerase, whereas 3 RNA polymerases function in eukaryotes. 2. RNA transcripts are extensively processed during transcription in eukaryotes; the 5' and 3' ends are modified and introns are spliced out 3. RNA pol II is much larger and more complex than its bacterial counterpart.
Stages of transcription
1. Initiation 2. Elongation 3. Termination
Types of transposable elements in the human genome
1. LINE (Long Interspersed Elements): move like a retrotransposon with the help of an element encoded reverse transcriptase Autonomous, e.g. L1 elements 2. SINE (Short Interspersed Elements): Do not encode their own reverse transcriptase Nonautonomous, e.g. Alu
Inheritance of an X-linked recessive disorders
1. Many more males than females show the rare phenotype 2. None of the offspring of an affected male show the phenotype, but all daughters are "carriers," who bear the recessive alleles masked in the heterozygous condition. 3. No male-to-male transmission
Different Cell Types Synthesize Different Sets of Proteins
1. Many processes are common to all cells, and any two cells in a single organism therefore have many proteins in common 2. Some proteins are abundant in the specialized cells in which they function and cannot be detected elsewhere, even by sensitive tests. Hemoglobin, for example, can be detected only in red blood cells. 3. At any one time, a human cell expresses 30-60% of its approximately 25,000 genes
There are three major mechanisms to alter chromatin structure
1. Moving nucleosomes along the DNA, also called chromatin remodeling. 2. Histone modification in the nucleosome core. 3. Replacing the common histones in a nucleosome with histone variants.
Approaches for deducing the total polypeptide parts
1. ORF (Open Reading Frame) detection 2. Direct evidence from cDNA sequences 3. Predictions of binding sites 4. Using polypeptide and DNA similarity 5. Predictions based on codon bias
Mechanism of transposition in prokaryotes
1. Replicative method 2. Conservative method
Probes for finding proteins
1. Requires an expression library, made by using expression vectors that will produce the protein. 2. Requires an antibody to the specific protein product of the gene of interest
Transposable elements in prokaryotes
1. Short sequences called IS elements -Can move themselves to new positions -Do not carry genes other than those needed for their movement 2. Longer sequences called transposons
Base insertions or deletions (indel mutations)
1. Simplest is the addition or deletion of a single base pair. 2. Sometimes multiple additions or deletions of single base pairs at once: cause for certain human genetic diseases Frameshift mutations result from insertions or deletions of a base pair -Can be very severe, especially if they occur early in the coding sequence -Except multiples of three, which would reestablish the initial frame of reading document
Gene Mutations
1. Spontaneous Mutations 2. Induced Mutations
Gene Mutations hehe
1. Spontaneous Mutations 2. Induced Mutations
Regulatory components of the lac system
1. The I gene : encodes the Lac repressor protein (diffusible protein) can block the expression of Z, Y and A genes 2. The lac promoter site/P : site on the DNA to which RNA polymerase binds to initiate transcription of the lac structural genes 3. The lac operator site/O: is the site on DNA to which the Lac repressor binds
How do bacteria survive the onslaught of bacteriophages?
1. The classical defense most bacteria have is the restriction endonuclease system. 2. 40% of bacteria have a highly targeted adaptive immune system that uses mechanisms found in DNA in the CRISPR region of the genome to grab bits of the DNA of bacteriophages. These are used as a guidance system to take DNA cutting enzymes that the bacteria makes and target these specifically to the bacteriophages DNA and chop it up.
Eukaryotic gene regulation
1. Transcriptional gene regulation 2. Post-transcriptional gene regulation- gene silencing: ncRNA (siRNA, miRNA)
order
1. bioinformatics 2. comparative genomics 3. functional genomics
Spontaneous mutations caused by the cellular environment
1.Depurination (loss of a purine base) - N-glycosyl linkages to deoxyribose hydrolyze -DNA of each human cell loses about 5000 purine bases (A&G) every day
Self-splicing introns
1981 Tom Cech • Pure rRNA isolated from Tetrahymena: could splice itself without any added protein • He found that the molecular mechanism of splicing was very similar to that of splicing of a protein coding gene. •This discovery and others that demonstrated enzymatic activity of RNAs (ribozymes) have led to the theory of the RNA world:
Transposable elements in eukaryotes class 2
2. Class 2 DNA transposons Encode a transposase that cuts the transposon from the chromosome and catalyze the reinsertion at other chromosomal locations e.g. P elements (first DNA transposons to be molecularly characterized).
Spontaneous mutations caused by the cellular environment 2
2. Deamination: -The major type of deamination converts cytosine to an altered base, uracil. -Rate is ~ 100 bases per cell per day - Unrepaired U residues will pair with A in replication, resulting on the conversion of a G:C pair into an A:T pair
monohybrid cross has ___ genotype(s)
3
What is the S. Pyogenes CRISPR/Cas9 system type 2
3 different RNAs generated but only one of these goes on to make a protein. 1 protein generated document
Spontaneous mutations caused by the cellular environment 3
3. Oxidatively damaged bases - Oxygen species, such as superoxide radicals, hydrogen peroxide (H2O2), and hydroxyl radicals (·OH), are produced as by-products of normal aerobic metabolism. - These oxygen species can cause oxidative damage to DNA, as well as to precursors of DNA (such as GTP), resulting in mutation. document thymidine glycol: Blocks replication if unrepaired 8-oxo-7-hydrogeoxyguanosine: Mispairs with A (resulting high levels of G to T transversions)
Mechanism of transposition in prokaryotes: conservative???
??? •The DNA of the element is not replicated and the element is lost from the site of the original chromosome • Also called "cut and paste" mechanism • Reaction is initiated by the element encoded transposase document e.g. Tn10
Reverse Genetics
A DNA sequence, an mRNA or a protein----disrupt----> Assess the role of the normal gene product Reverse-genetic analysis starts with a known molecule—a DNA sequence, an mRNA, or a protein—and then attempts to disrupt this molecule to assess the role of the normal gene product in the biology of the organism. 1. Random mutagenesis 2. Targeted mutagenesis 3. Create phenocopies
Dihybrid Cross
A dihybrid cross involves two pairs of contrasting traits Mendel's dihybrid cross generated a unique F2 ratio To start a dihybrid cross, Mendel started with two pure parental lines round, green seeds R/R y/y wrinkled, yellow seeds r/r Y/Y 9:3:3:1?
Full or Complete Dominance and Recessiveness
A fully dominant allele will be expressed when only one copy is present (as in a heterozygote) Homozygous dominant cannot be distinguish from heterozygote e.g. at the phenotypic level A/A = A/a Fully recessive mutation Phenylketonuria (PKU) and many other single-gene human diseases are fully recessive, whereas their wild type alleles are dominant.
gene interaction
A gene does not act alone Acts in concert with many other genes in the genome The phrase gene interactions refers to the ways genes collaborate or interact to influence a phenotype To find it out: 1. Analyze protein interactions directly 2. Analyze mRNA transcripts 3. Genetic analysis Interactions between alleles of one locus (variations on dominance) Interactions between two or more loci - these interactions reveal the number and types of genes underlying a particular biological function
Construction of a human genomic DNA library
A genomic library is usually stored as a set of bacteria, each bacterium carrying a different fragment of human DNA How many plasmids are taken up by each cell is determined by transformation efficiency
E. coli cells that have null mutations in the gene encoding adenine methylase will have
A higher spontaneous mutation rate than wild-type E. coli.
recessive fly
A homozygous purple-eyed fly is crossed to a homozygous red-eyed stock carrying the suppressor. The recessive allele pd will result in purple eye color when unsuppressed. A recessive allele su has no detectable phenotype itself, but suppresses the unlinked recessive allele pd.
2. Functional RNA
A large group of functional RNAs suppress the expression of genes at many levels and also maintain genome stability MicroRNAs (miRNAs)- regulate gene expression Small interfering RNAs (siRNAs) & piwi-interacting RNAs(piRNAs)-protect the integrity of plant and animal genomes, and regulate gene expression Long noncoding RNAs (lncRNAs/ncRNAs)
epistatic interaction
A minimum of two genes are required for epistatic interactions; these usually participate in the same pathway Epistasis is readily detected among progeny of dihybrid crosses involving genes with both dominant and recessive alleles There are six ways epistasis could affect the predicted 9:3:3:1 dihybrid ratio
suppressors
A mutant allele of one gene that reverses the effect of a mutation of another gene, resulting in a wild-type or near wild-type phenotype. Suppression implies that the target gene and the suppressor gene normally interact at some functional level in their wild-type states e.g. an allele a+ normal phenotype recessive mutant allele a abnormality A recessive mutant allele s at another gene suppresses the effect of a Genotype a/a. s/s will have wild-type (a+-like) phenotype
peptide bond
A peptide bond forms by a dehydration reaction between the carboxyl group of one amino acid and the amino group of another document
pure line
A pure line is a population that breeds true for (shows no variation) in the particular character being studied
Enhancer-blocking insulators
A regulatory element, such as an enhancer, that can act over tens of thousands of base pairs could interfere with the regulation of nearby genes. To prevent such promiscuous activation, regulatory elements called enhancer-blocking insulators have evolved When positioned between an enhancer and a promoter, enhancer-blocking insulators prevent the enhancer from activating transcription at that promoter
Key sites of interaction in the ribosome
A site: binds an incoming aminoacyl-tRNA whose anticodon matches the codon in the A site of 30S subunit P site: tRNA in this position binds the growing peptide chain E site (exit site): contains tRNA lacking an amino acid (deacylated) The decoding center in the 30S subunit ensures that only tRNAs carrying anticodons that match the codon will be accepted into the A site. The peptidyltransferase center in the 50S subunit is the site where peptide-bond formation is catalyzed
The structure of transfer RNA
A tRNA has an anticodon that complementarily base-pairs with the codon in the mRNA The corresponding amino acid is covalently linked to the CCA sequence at the 3' end of all tRNAs
test cross
A test cross reveals the genotype of a dominant phenotype by crossing it with a homozygous recessive
pulse-chase experiment
A type of experiment in which a population of cells or molecules at a particular moment in time is marked by means of a labeled molecule (pulse) and then their fate is followed over time (chase). eukaryotic mRNA moves from nucleus
"Traditional" whole genome shotgun (WGS) sequencing employs which of the following techniques?
A) the construction of genomic libraries B) cloning of DNA in bacterial systems C) Sanger dideoxy DNA sequencing
Change in chromosome number
Aberrant euploidy: changes in whole chromosome sets Aneuploidy: changes in parts of chromosome sets
Alkyltransferases are enzymes that directly reverse lesions
Alkyltransferases remove alkyl groups, like those added by EMS document
2. Yeast two-hybrid test to study protein-protein interaction
Activate transcription but cannot itself bind to DNA Binds to the transcriptional start site
Before translation can proceed, tRNA molecules must be chemically linked to their respective amino acids
Activation (charging or aminoacylation) done by aminoacyl tRNA synthetase There are 20 different synthetases, one for each amino acid, and they are highly specific since they recognize only one amino acid
positive regulation
Activator protein must bind to its target DNA sites as a necessary prerequisite for transcription to begin
Maize transposable elements revisited
Activator transposase catalyzes excision and integration document
Gel electrophoresis
Agarose gel DNA migrates to the anode end (+) DNA fragments with distinct sizes will form distinct bands Bands can be visualized by staining the DNA with ethidium bromide
Many DNA-binding proteins that affect gene expression are allosteric. What is allostery and why is it such a common mechanism of gene regulation?
Allostery is the ability of signal molecules, known as effectors, to bind to proteins. This binding changes the shape of the protein, either enabling it to bind to, or preventing it from binding from, cis-acting binding sites. It is a common mechanism because it is reversible and is dependent on the concentration of the effector.
Proteins
Amino acids all have; -a carboxyl group (COOH) -an amino group (NH2) -an R group bound to a central carbon atom The R group of an amino acid confers specific chemical properties amino group:nh2
Describe how an Ames test can reveal if a chemical is mutagenic. Make sure to include all information that can be obtained from an Ames test
An Ames Test combines a chemical with His- mutants of Salmonella and rat liver enzymes to test if a chemical is mutagenic. If the chemical is mutagenic, then we would potentially expect mutations in the genes responsible for the production of histidine. Some of these mutations will cause reversions in the His- genes, resulting in these genes regaining wild-type function. The number of revertant colonies with respect to the chemical concentration determines the degree of mutagenesis. The strain of the Salmonella that is reverted determines the type of mutation caused by the chemical (indels, substitutions, etc).
Hfr strains
An Hfr (high-frequency recombination) strain has the F factor integrated An Hfr strain can donate genetic information to an F- cell, but the recipient does not become F+ ~F+ x F- recipient becomes F+ (low rate of recombination) ~Hfr x F- recipient remains F- (high rate of recombination)
Finding specific nucleic acids by using gel electrophoresis and blotting blot
An actual Northern blot, run with RNA isolated from the seeds of various plants. A single RNA probe is used to identify the presence of a single locus. The results show that maize is more closely related to rice, sorghum, and millet than it is to soybean or cotton.
Incomplete Penetrance
An organism is pentrant for a trait when the phenotype is consistent with the genotype An organism which does not produce the phenotype generally associated with the genotype is nonpentrant Traits for which nonpentrant individuals routinely occur are said to display incomplete penetrance Individual II 1 does not display the phenotype but passed the dominant allele to at least two progeny. Since the allele is not fully penetrant, the other progeny (for example, III 2) may or may not have inherited the dominant allele.
Overlapping versus nonoverlapping genetic codes
Analyses of mutationally altered proteins showed that only a single amino acid changes at one time in one region of the protein. document
The Genetic Approach to Development
Animal genomes contain tens of thousands of genes, but only a fraction of them control the developmental process Development is the attainment of a differentiated state by all cells of an organism The development of a multicellular animal from a fertilized egg demonstrates the power of controlled gene expression • Genes must be expressed carefully over time to bring about the specialization of the formation of the animal's body • Transplantation experiments played a central role in early embryology and demonstrated the long-range organization activity of embryonic tissues -amphibian embryo -developing chick vertebral limb bud -ZPA (zone of polarizing activity)
Transposable elements insert into safe havens
Another example is R1 and R2 elements of arthropods R1 and R2 are LINEs: Insert only into genes that produce ribosomal RNA (rRNA) There are hundred to thousands of rRNA genes in arthropods encoding the same product. Therefore the host can tolerate insertion into a subset. However too many insertions can interfere with ribosome assembly. document
replication fork
Another prediction of the Watson-Crick model of DNA replication is that a replication zipper, or fork, will be found in the DNA molecule during replication. This fork is the location at which the double helix is unwound to produce the two single strands that serve as templates for copying
Arg mutants
Arg-1 mutants grew when supplied with any one of the chemicals Arg-2 grew when given citrulline or arginine but not ornithine
Transcription Elongation in prokaryotes
As the RNA polymerase moves along the DNA, it unwinds the DNA ahead of it and rewinds the DNA that has already been transcribed. In this way, it maintains a region of single-stranded DNA, called a transcription bubble, within which the template strand is exposed
Mating Systems
Assortative mating: individuals choose mates based of resemblance to themselves Positive assortative mating: similar types mate Negative assortative mating (disassortative mating): unlike individuals mate
polygenes in progeny of a dihybrid self
Assume two independently assorting genes pairs R1/r1 and R2/r2 Both R1 and R2 contribute to wheat-seed redness. R1;R2 two doses of redness R1;r2 one dose r1;R2 one dose r1;r2 zero doses
Alleles at the Molecular Level
At the molecular level, the primary phenotype of a gene is the protein it produces
Describe the processes that result in autopolyploidy versus allopolyploidy.
Autopolyploidy is often the result of non-disjunction. Allopolyploidy is often the result of hybridization.
autosomal dominance inheritance
Autosomal dominant inheritance has six characteristics: Each individual who has the disease has at least one affected parent Males and females are affected in equal numbers Either sex can transmit the disease allele In crosses where one parent is affected and the other is not, approximately half the offspring express the disease Two unaffected parents will not have any children with the disease Two affected parents may produce unaffected children
Autosomal Recessive Inheritance
Autosomal recessive inheritance has six key features: Individuals who have the disease are often born to parents who do not If only one parent has the disorder the risk that a child will have it depends on the genotype of the other parent If both parents have the disorder, all children will have it The sex ratio of affected offspring is expected to be equal (The affected progeny include both male and female) The disease is not usually seen in each generation but if an affected child is produced by unaffected parents, the risk to subsequent children is ¼ If the disease is rare in the population, unaffected parents of affected children are likely to be related to one another
inducible enzymes
Bacteria adapt to their environment by producing certain enzymes (inducible enzymes) only when specific substrates are present
lexa lacking the activation domain
Bacterial LexA protein cannot activate transcription on its own
tautomeric forms
Bases may take on rare tautomeric forms prone to mismatch
Parental imprinting can greatly affect pedigree analysis
Because the inherited allele from one parent is inactive, a mutation in the allele inherited from the other parent will appear to be dominant, whereas, in fact, the allele is expressed because only one of the two homologs is active for this gene
Mutations in cis acting regulatory elements have two important properties that are distinct from mutations in coding regions
Because they affect regulation in cis, the phenotypes are often dominant Because only one of several cis-acting regulatory elements may be affected, other genes functions may be completely normal
Isolation By Distance
Bias in mate choice arises from the amount of geographic distance between two individuals Species or populations exhibiting such patterning of genetic variation are said to show population structure
In order to get proper expression of the even-skipped gene in the eve stripe 2 of a developing Drosophila larvae, high concentrations of bicoid and low concentrations of Giant and Kruppel are required. What does this tell us about how bicoid, Giant, and Kruppel interact with the regulatory element responsible for the proper expression of the even-skipped gene in the eve stripe 2?
Bicoid acts as an activator and Giant and Kruppel act as repressors of eve stripe 2.
Single-strand DNA-binding (SSB) protein
Bind tightly and cooperatively to exposed single-stranded DNA without covering the bases Unable to open a long DNA helix directly, but they aid helicases by stabilizing the unwound, single-stranded conformation
Three Distinct Types of Genetic Pathways
Biosynthetic pathways are networks of interacting genes that produce a molecular compound as their endpoint Signal transduction pathways receive chemical signals from outside a cell and initiate a response inside the cell Developmental pathways direct growth, development and differentiation of body parts and structures
Producing cDNA molecules with sticky ends
Blunt ends Double-stranded oligonucleotides (linkers/adapters) Sticky ends
Tertiary structure
By folding of secondary structures document
How are scientists easily able to observe (i.e. visualize) the expression of developmental genes in a developing embryo?
By linking these genes or their regulatory sequences to a reporter gene, such as LacZ or a fluorescent gene.
How is Distal-less (Dll) expression restricted to the more anterior segments?
By repressing its expression in the abdomen Several lines of evidence have revealed that the Distal-less gene is repressed by two Hox proteins Ultrabithorax Abdominal A
Describe the function of the CAP-cAMP system in bacteria. Why does it regulate several operons related to metabolism?
CAP-cAMP is an example of catabolite repression, acting as a signal for the presence of glucose. When there are low levels of glucose, cAMP is produced from ATP. When cAMP is present, it binds to CAP, allowing CAP to bind to the lac promoter, activating the transcription of the operon. It regulates several operons because there are many enzymes that cleave sugars into glucose, so it would make sense that these all need to be turned off in the presence of glucose.
no glucose present (cAMP high), lactose present
CAP-cAMP--->positive control document
Gal4-LexA hybrid
Can activate transcription when fused to the Gal4 activation domain
How does Cas9 work ?
Cas9 has a channel that DNA can fit into. It scans the DNA looking for sequence that match the guide sequence When a DNA sequence complementary to the crRNA is found the scanning stops There is one additional check The part of the RNA that came from the palindromic repeats of the bacteria has to also have a very short piece of RNA that is complementary to bit of the bacteriophage DNA. This is called the PAM sequence (Protospacer Adjacent Motif) Only when all this happens, and we have the crRNA bound, do we have a fully active enzyme. document Now the RNA binds to the complementary strand of the DNA and opens up the DNA helix Now the bacteriophages DNA gets cut very close to the PAM site document Now the bacteriophages DNA gets cut very close to the PAM site so now it looks like this and the bacteriophage is essentially dead document
Cas9
Cas9 is an endonuclease that can cut double stranded DNA Cas9 is only activated when the tracrRNA and the crRNA are associated with it. The tracrRNA and the crRNA have a short overlapping sequence, which means they have to bind to each other in order for this to work properly. document
Colchicine induces autopolyploidy
Caused by inhibiting spindle fibers document
The amplification of donor DNA inside a bacterial cell entails the following steps:
Choosing a cloning vector and introducing the insert Introducing the recombinant DNA molecule inside a bacterial cell Recovering the amplified recombinant molecules
1. Chromatin Remodeling
Chromatin can be dynamic; nucleosomes are not necessarily in fixed positions on the chromosomes. Chromatin remodeling changes nucleosome density or position and is an integral part of eukaryotic gene regulation Repositioned nucleosomes make regions of the chromosome accessible to transcription regulatory proteins (Transcription activators, RNA polymerase II etc) The SWI/SNF is one of the best-studied remodeling complexes
Position effect variegation (PEV) in Drosophila reveals genomic neighborhoods
Chromosomal neighborhoods exist that can silence genes that are experimentally relocated to adjacent regions of the chromosome Files---Irradiated with X-rays---> progeny screen for unusual phenotypes---> mutation in the white gene near the tip of the X-chromosome---> progeny with white eyes instead of wild type red color. some produce white and red patches Cytological examination: chromosomal rearrangement in the mutant flies
Bacteriophage vectors
Cloning in phage lambda •Harbors DNA as an insert "packaged" inside the phage particle • carry ~15 kb donor inserts • The central part of the phage genome is not required for replication or packaging of λ DNA molecules in E. coli and so can be cut out by restriction enzymes and discarded. •The deleted central part is then replaced by inserts of donor DNA.
Complementation Test
Complementation testing is when two pure breeding organisms with similar mutant phenotypes are mated If complementation occurs, wild type offspring are obtained and the mutations are known to affect two different genes When the mutations fail to complement, the offspring have the mutant phenotype and the mutations are known to affect the same gene Determines whether two distinct recessive mutations are of one gene or of two different genes Results show that the mutant condition is determined by the recessive allele of a single gene. $x£= white $xY=blue £xY=blue From this set of results, we can conclude that mutants $ and £ must be caused by alleles of one gene (say, w1) Why? But ¥ must be caused by a mutant allele of another gene (w2) because complementation is seen. When two independently derived recessive mutant alleles producing similar recessive phenotypes fail to complement, the alleles must be of the same gene.
Quaternary structure
Composed of 2 or more separate folded polypeptides
Geneticists use recombinant DNA and genomic technology to identify genes, diagnose and treat genetic disorders, produce commercial and pharmaceutical products, and solve crimes However, applications of the knowledge gained from the project raise ethical, social, and legal issues
Concerns about genetically modified foods center on consumer safety and environmental consequences The Ethical, Legal and Social Implications (ELSI) Program was established as an adjunct to the Human Genome Project. Address issues such as confidentiality of genetic information and implications for medical practice, genetic counseling, and reproductive decision making Genetic Information Nondiscrimination Act (2008): designed to inhibit improper use of genetic information in health insurance and employment Direct-to-consumer (DTC) genetic tests are offered by many companies These include diagnostic, carrier, pharmacogenomic, prenatal, genealogical, paternity, and many more kinds of testing -A saliva sample or cheek swab is mailed to the company for the test -Many questions arise as to the quality, effectiveness, and accuracy of the tests -Many tests are not FDA regulated Somatic gene therapy affects only the individual being treated Germ-line therapy can affect individuals in future generations Enhancement gene therapy is controversial and unapproved due to the issue of whether genetic technology should be used to enhance human potential In 2013, the Supreme Court of the United States ruled that naturally occurring genes cannot be patented
Two superimposed tRNAs
Conservation of structure tells us that shape is important for tRNA function.
Combinations of maternal-effect and gap proteins control individual pair-rule stripe formation
Consider the second stripe expressed by the even-skipped gene stripe 2 lies within the broad region of hunchback expression and on the edges of the regions of expression of two other gap proteins: giant and Kruppel within the area of the future stripe 2: large amounts of Hunchback protein small amounts of Giant protein and Kruppel protein certain concentration of the Bicoid protein no other stripe of the embryo will contain these proteins in these proportions
2. Comparative genomics:
Considers the genomes of closely and distantly related species for evolutionary insight • To identify conserved regions • To reveal how species diverge Features looked at when comparing genomes: -sequence similarity -gene location -length and number of coding regions within genes -amount of non-coding DNA in each genome -highly conserved regions maintained in organisms Computer programs that can line up multiple genomes and look for regions of similarity among them are used. Step 1: Decide which species' genomes to compare Step 2: Identify the most closely related genes, called homologs Genes that are homologs can be recognized by similarities in their DNA sequences and their amino acid sequences of the proteins they encode document
female sex chromosomes
Contain many hundreds of genes Most do not take part in sexual function
male sex chromosomes
Contain only a few dozen genes Mainly male-specific genes
The cis-acting regulatory element responsible for Distal-less expression in the embryo
Contains two binding sites for the Hox proteins (Hox1 and Hox2)
Long-term inactivation of genes in a chromatin environment
Control of mating-type switching in yeast
Genetic engineering in animals
Creation of Caenorhabditis elegans transgenes Transgenic DNA injected directly as plasmids, fosmids or other DNAs cloned in bacteria document
DNA Synthesis in Bacteria Involves Five Polymerases, as well as Other Enzymes
DNA Polymerases I, II, III, IV, V DNA polymerases I, II, and III can elongate an existing DNA strand (called a primer) but cannot initiate DNA synthesis All three possess 3' to 5' exonuclease activity, allowing them to proofread newly synthesized DNA and remove and replace incorrect nucleotides Only DNA polymerase I demonstrates 5' to 3' exonuclease activity, excising primers and filling in the gaps left behind DNA polymerase III is the enzyme responsible for the 5' to 3' polymerization essential in vivo Its 3' to 5' exonuclease activity allows proofreading DNA polymerases I, II, IV, and V are involved in various aspects of repair of DNA damaged by external forces such as UV light
Southern- and Northern-blot analysis of DNA or RNA
DNA analysis- Southern blotting RNA analysis- Northern blotting Protein analysis- Western blotting
Unwinding the double helix
DNA helicases -Hexameric ring -Unwind the DNA double helix by moving in the 5' to 3' direction along one strand or in the 3' to 5' direction along the other (both types of helicase exist) -Open up the helix at rates of up to 1000 nucleotide pairs per second Unwinding causes extra twisting and supercoiling DNA gyrase, a topoisomerase, removes extra twists during replication
The reaction catalyzed by DNA ligase
DNA ligase uses a molecule of ATP to activate the 5' end at the nick (step 1) before forming the new bond (step 2). The energetically unfavorable nick sealing reaction is driven by being coupled to the energetically favorable process of ATP hydrolysis. Primase synthesizes an RNA primer to initiate DNA synthesis DNA polymerase III is not very processive (i.e it falls off the DNA easily). A "ß clamp" is required to keep DNA pol III attached to the DNA template
Bacterial DNA polymerases
DNA polymerase I or pol I 1. Polymerase activity: catalyzes chain growth in the 5'-to-3' direction 2. 3'-to-5' exonuclease activity: removes mismatched bases 3. 5'-to-3' exonuclease activity, which degrades single strands of DNA or RNA DNA polymerase III or pol III -Catalyzes DNA synthesis at the replication fork
DNA polymerase III
DNA polymerase III is a complex enzyme (holoenzyme) made up of 10 subunits (will be in next PowerPoint) The holoenzyme and some other proteins at the replication fork form a complex called the replisome
Reaction catalyzed by DNA polymerase
DNA polymerase catalyzes DNA synthesis and requires a DNA template, a primer and all four deoxyribonucleoside triphosphates (dNTPs)
Organisms Use DNA Repair Systems to Counteract Mutations
DNA repair systems maintain the integrity of genetic material susceptible to spontaneous and induced damage These repair systems counteract genetic damage that would result in genetic diseases and cancer
DNA replication and the yeast cell cycle
DNA replication in most eukaryotic cells occurs only during a specific part of the cell division cycle, called the DNA synthesis phase or S phase In yeast, the method of control is to link replisome assembly to the cell cycle
Mendel's Second Law (The law of independent assortment)
Different gene pairs assort independently in gamete formation Gene pairs on different chromosome pairs assort independently at meiosis
Biological Repair Mechanisms
Direct reversal of damaged DNA • Cyclobutane pyrimidine dimer (CPD) can be repaired by: CPD photolyase • Enzyme binds to the photodimer and splits it to regenerate the original bases • Enzyme requires light to function: photoreactivation
Bacterial Conjugation
Do bacteria possess any processes similar to sexual reproduction and recombination? Joshua Lederberg and Edward Tatum, 1946 : discovered a sexlike process in bacteria document x2
Interactions between the alleles of a single gene: Variations on dominance
Dominance is a manifestation of how the alleles of a single gene interact in a heterozygote -Full or Complete dominance & recessiveness -Incomplete dominance -Codominance
Expression of Hox genes in the Drosophila embryo
Drosophila embryo showing eight regions where eight individual Hox genes are expressed Actual image of the expression of seven Hox genes visualized by in situ hybridization • In the developing embryo, the Hox genes are expressed in spatially restricted domains head-thorax-abdomen
epigenomes
During its life span, an organism has one genome, but this genome can be modified in diverse cell types at different times to produce many epigenomes
How can you change phenotype without changing the genome?
During its life span, an organism has one genome, but this genome can be modified in diverse cell types at different times to produce many epigenomes twins? genetically identical? When a mouse's agouti gene is completely unmethylated, its coat is yellow and it is obese and prone diabetes and cancer. When the agouti gene is methylated (as it is in normal mice), the coat color is brown and the mouse has a low disease risk. Human epidemiological studies have provided evidence that prenatal and early postnatal environmental factors influence the adult risk of developing various chronic diseases and behavioral disorders
If the DNA sequence of the gene does not correlate with activity, what does?
During the development of gametes, methyl groups are added to the DNA in the regulatory regions of imprinted genes in one sex only.
Genetic Engineering in Mice
Ectopic insertions: Transgenes are inserted randomly in the genome Usually as multicopy arrays Targeted insertion/ Gene targeting • Transgene inserted into a location occupied by a homologous sequence in the genome (transgene replaces its normal homologous counterpart).
messenger RNA (mRNA)
Encodes information necessary to make polypeptide chains (protein) Serve as the intermediary that passes information from DNA to protein
Enhancer-blocking insulators prevent enhancer activation
Enhancer-blocking insulators prevent gene activation when placed between an enhancer and a promoter. off
constitutive enzymes
Enzymes continuously produced regardless of chemical makeup of the environment are called constitutive enzymes
Mechanisms of spontaneous mutations Errors in DNA replication
Errors in DNA replication - Can also lead to frameshift mutations If a DNA strand loops out and becomes displaced small insertions and deletions can occur (indel mutations) -Replication slippage can occur anywhere in the DNA but is more common in repeat sequences Hot spots for hereditary diseases -Fragile-X, Huntington Disease
Mechanisms of spontaneous mutations
Errors in DNA replication During DNA replication, DNA polymerase occasionally inserts incorrect nucleotides -Although DNA polymerases can proofread and correct these errors, they may persist if not detected and corrected These types of errors, due to mispairing, predominantly lead to point mutations Purines and pyrimidines can exist in tautomeric forms-alternate chemical forms that differ by a single proton shift in the molecule Tautomeric shifts can change the bonding structure, allowing non-complementary base pairing -May lead to permanent base-pair changes and mutations
Replication in eukaryotic organisms
Eukaryotic chromosomes exist in the nucleus as chromatin. The basic unit of chromatin is the nucleosome. Eukaryotic replisome performs all the functions of the prokaryotic replisome; in addition, it must disassemble and reassemble the nucleosomes.
enhancers
Eukaryotic enhancers can act at great distances to modulate the activity of the transcriptional apparatus. Enhancers contain binding sites for many transcription factors, which bind and interact cooperatively. These interactions result in a variety of responses, including the recruitment of additional co-activators and remodeling of chromatin.
Translation initiation in eukaryotes
Eukaryotic initiation factors 5' end Cap 40S subunit Initiator tRNA--> Initiation complex--> • Complex moves in the 5' to 3' direction • Unwinds base paired regions • Exposed sequence is scanned for an AUG codon where translation begin--> • AUG align with initiator tRNA • Initiation complex is joined by 60S to form the 80S ribosome
Transcription initiation in eukaryotes
Eukaryotic promoters are first recognized by general transcription factors (GTFs). The function of GTFs is to attract the core RNA polymerase II so that it is positioned to begin RNA synthesis at the transcription start site. The GTFs and the RNA polymerase II core constitute the preinitiation complex (PIC)
Transcriptional activator proteins recruit the transcriptional machinery
Eukaryotic transcriptional activators often work by recruiting parts of the transcriptional machinery to gene promoters • One way that Gal4 works to activate gene expression is by binding to TBP at a site in its activation domain. • Through this binding interaction, it recruits the TFIID complex and in turn RNA polII document
What is the difference between euploids and aneuploids?
Euploids have the same number of each chromosome. Aneuploids do not have the same number of each chromosome.
Mutations of haplosufficient genes are recessive
Even though the mutated copy of the gene produces nonfunctional protein, the wild type copy generates enough functional protein (wild type phenotype)
Exd, Hth mutation
Exd and Hth proteins, which are broadly expressed in every segment, also bind to the distal-less cis acting regulatory element and are required for transcriptional repression in abdomen document
Repair of double-strand breaks
Exposure to X-rays and reactive oxygen species (ROS) can lead to both strands of DNA being cleaved, resulting in chromosomal rearrangements, cancers, or cell death Double-strand break repair (DSB repair) reattaches two broken DNA strands 1. Nonhomologous end joining (NHEJ) 2. Homologous recombination repair
Transcriptional activator proteins
Expression of GAL genes and other targets of transcription factors is typically monitored by using a reporter gene (e.g. lacZ) whose level of expression is easily measured.
Two features of epigenetic silencing:
Expression of a gene can be repressed by virtue of its position in the chromosome rather than by mutation in its DNA sequence Epigenetic silencing can be inherited from once cell generation to the next
codominance
Expression of both alleles of a heterozygote Parents with different phenotypes produce an offspring with a third phenotype Third phenotype will show both parental phenotypes simultaneously Andalusian chickens also show this pattern of inheritance If you cross a black (BB) chicken with a white (WW) chicken, you get black+white speckled (BW) chicken
Ectopic insertions
Expression pattern of the randomly inserted genes maybe abnormal (position effect) DNA rearrangements can occur (mutating the sequence)
Expressivity
Expressivity measures the degree to which a given allele is expressed at the phenotypic level; that is, expressivity measures the intensity of the phenotype. For example, "brown" animals (genotype b/b) from different stocks might show very different intensities of brown pigment from light to dark. Different degrees of expression in different individuals may be due to variation in the allelic constitution of the rest of the genome or to environmental factors. Each of these dogs has SP, the allele responsible for piebald spots in dogs.
Drosophilia cross
F1 progeny have red eye: Allele for white eyes is recessive Crossing red-eyed F1 males and females produce a 3:1 ratio red-eyed: white eyed (just like autosomal recessive traits) But all the white-eyed flies are males
The 9:3:4 F2 ratio: Recessive Epistasis
F2 9:3:4 is diagnostic of recessive epistatsis Double mutant must express only one of the two mutant phenotypes White must be epistatic to magenta A recessive phenotype (white) overrides the other phenotype: recessive epistatsis
Discovery of Transposable Elements
First identified by Barbara McClintock- 1940s McClintock's work was revolutionary in that it suggested that an organism's genome is not a stationary entity, but rather it is subject to alteration and rearrangement, a concept that was met with criticism from the scientific community of the time • McClintock worked with what is known as the Ac/Ds system in maize • Ac/Ds system was discovered by conducting standard genetic breeding experiments using plants with an unusual phenotype. • The first transposable element she discovered was a site of chromosome breakage, named "dissociation" (Ds). Although McClintock eventually found that some TEs can "jump" autonomously, she noted that the movements of Ds are regulated by an autonomous element called "activator" (Ac), which can also promote its own transposition.
a molecular mechanism for suppression
First mutation alters the binding site of one protein so that it can no longer bind to a partner A suppressor mutation in the partner alters the binding site so that both proteins are able to bind once again
Genetic variation and its measurement
Fixed (invariant) vs segregating (polymorphic) sites Number of halpotypes Gene Diversity: probability that two alleles drawn at random from a gene pool will be different Heterozygosity: expected proportion of heterozygotes Nucleotide diversity: gene diversity averaged over all nucleotide sites in a gene
The Gal pathway
GAL1, GAL2, GAL7 & GAL10 • Encode enzymes that catalyze steps in the biochemical pathway that converts galactose into glucose • Three additional genes- GAL3, GAL4 and GAL80 encode proteins that regulate the expression of the enzyme genes •The key regulator of GAL gene expression is the Gal4 protein: a sequence specific DNA-binding protein
cDNA is inserted into the vector in the correct triplet reading frame with a bacterial protein ( ß-galactosidase),---->
Fusion protein
Two pathways for nucleotide-excision repair in Bacteria
GG-NER is initiated when UvrA and UvrB recognize the bulky adducts or pyrimidine dimer. UvrB is a helicase and separates the strands. TC-NER is initiated when an RNA polymerase complex is stalled by a DNA lesion in the transcribed strand, which recruits UvrA and UvrB. document x2 UvrA is released and UvrB recruits UvrC, which cleaves phosphodiester bonds 8 nucleotides 5' and 4-5 nucleotides 3' of the DNA damage site UvrD (helicase) and DNA Pol I excise the 12-13 nucleotide region and fill in the gap DNA ligase seals the nick
Two pathways for nucleotide-excision repair in Eukaryotes
GG-NER is initiated when a protein complex of XPC and XPE recognizes a distorted double helix caused by a damaged base TC-NER is initiated when an RNA polymerase complex is stalled by a DNA lesion in the transcribed strand and CSA and CSB bind at this site to form a recognition complex. XPC/XPE and CSA/CSB attracts the multiprotein TFIIH complex XPB and XPD (helicases) unwind, XPA and RPA expand bubble XPF-ERCC1 and TFIIH subunit XPG endonucleases cleave and excise 27-nucleotide region Gap filled by DNA replication factors Nick sealed by ligase
Genomic Imprinting
Gender-specific silencing of genes and whole chromosomes • Genomic imprinting explains some unusual patterns of inheritance • Certain autosomal genes have unusual inheritance patterns e.g. an igf2 allele is expressed in a mouse only if it is inherited from the father: maternal imprinting because the copy of the gene derived from the mother is inactive. • Conversely, a mouse H19 allele is expressed only if it is inherited from the mother- paternal imprinting because the paternal copy is inactive • Imprinted genes are expressed as if there were only one copy of the gene present in the cell even though there are two. • No changes are observed in the DNA sequences of imprinted genes; that is inactive gene can be active or inactive in the progeny, depending on whether it was inherited from mom or dad.
2. The yeast two-hybrid system for detecting gene interaction
Gene for one protein under investigation is spliced next to the DNA-binding domain fusion protein:" bait" Gene for another protein under investigation is spliced to the activation domain and the resulting fusion protein: "target"
The Outside of the DNA Helix Can Be Read by Protein
Gene regulatory proteins recognize sequences specifically by binding to the outside of the DNA -specifically at major groove of DNA minor groove major groove: gene regulatory proteins
Difference between gene silencing and gene repression?
Gene silencing is a position effect that depends on the neighborhood in which genetic information is located e.g. position effects variegation in Drosophila melanogaster
Gene targeting
Gene targeting enables us to eliminate or modify the function encoded by a gene 1. Gene replacement • Mutant allele can be repaired by substituting a wild-type allele for the mutant one in its normal chromosomal location • Gene replacement avoids both the position effect and DNA rearrangements associated with ectopic insertion, because a single copy of the gene is inserted in its normal chromosomal environment 2. Gene Knockout A gene maybe inactivated by substituting an inactive gene for the normal gene Carried in embryonic stem cells (ES cells) cultures Two stages: 1)Produce ES cells containing a gene knockout 2)Transfer ES cells containing the inactive gene/knockout into mice embryos
Aneuploidy
Generally, an aneuploid organism differs from the wild type by only one chromosome or by a small number of chromosomes Trisomic: 2n+1 Monosomic: 2n-1
interaction of genes in pathway
Genes act by controlling cellular chemistry Early in the twentieth century, Archibald Garrod, an English physician, made the first observation supporting this insight He proposed that alkaptonuria was due to a missing enzyme, namely homogentisic acid oxidase
structural genes
Genes coding for the primary structure of an enzyme are called structural genes
The Genetic Toolkit for Drosophila Development
Genetic toolkit: The genetic toolkit for animal development is composed of a small fraction of all genes. Determines the overall body plan, number, identity, and pattern of body parts Toolkit Genes are involved in the building of organs and tissues and specification of cell types
Is there a link between telomere shortening and aging?
Geneticists studying human diseases that lead to a premature-aging phenotype have recently uncovered evidence that supports such a connection. People with Werner syndrome experience the early onset of many age-related events Werner Syndrome Afflicted people have shorter telomeres than those of normal people owing to a mutation in a gene called WRN, which encodes a protein (a helicase) that associates with proteins that comprise the telomere cap (e.g. TRF2) Progeria The researchers have found that short or dysfunctional telomeres activate production of progerin, which is associated with age-related cell damage
Barrier insulators stop the spread of heterochromatin
Genome contains DNA elements called barrier insulators that prevent the spreading of heterochromatin by creating a local environment that is not favorable to heterochromatin formation
Safe havens in small genomes
Genome of unicellular yeast very compact, with closely spaced gene and few introns (70% of the genome is exons)
Describe why genomic imprinting can increase the difficulty of pedigree analysis.
Genomic imprinting involves the silencing of one copy of a gene depending on which parent from which it was inherited. It can make recessive mutations look dominant because the wild-type version is silenced. Also, it can make autosomal genes appear sex linked.
Histone deacetylation can turn off gene transcription
Glucose + galactose •In the presence of glucose, GAL1 transcription is repressed by the Mig1 protein, which binds to a site between the UAS and the promoter of the GAL1 gene • Mig1 recruits the Tup1 repressing complex, which contains a histone deacetylase, turning gene transcription off. • The Tup1 complex is an example of a corepressor: facilitates gene repression but is not itself a DNA-binding repressor. tup1 mig 1 gal4 gal 1 uas
2. Next-generation WGS
Goal is to obtain large number of overlapping sequence reads that can be assembled into contigs 1. DNA molecules are prepared for sequencing in cell free reactions, without cloning in microbial reaction 2. Millions of individual DNA fragments are isolated and sequenced in parallel during each machine run 3. Advanced fluid handling technologies, cameras, and software make it possible to detect the products of sequencing reactions in extremely small reaction volumes • Started around 2005 and some of the first instruments were capable of producing data 200 times faster and cheaper than conventional Sanger approaches • 454 Life Sciences (now Roche) was the first company to commercialize a next generation sequencing technology
Mating-type switching is controlled by recombination of DNA cassettes
HMLα copied into the MAT locus HMRa is copied into the MAT locus
Mating-type switching and gene silencing process HO gene
HO gene: encodes an endonuclease, required for the initiation of switching Cleaves DNA only at the MAT locus (due to the DNA sequence specificity of the HO endonuclease). The interconversion of mating type then take place by a type of recombination between---> segment of DNA (a cassette) from one of the two unexpressed loci and the MAT locus----> Replacement of the old cassette at the MAT locus with a new cassette from either HMRa or HMLα
Haplotypes
Haplotypes are combinations of alleles at multiple loci on the same chromosomal homolog Often used for loci that are physically close Often grouped into classes A haplotype network shows the relationship among the haplotypes
Transcriptional activator proteins are modular
Have separable domains for DNA biding, activation or repression and interaction with other proteins
Gene silencing is caused by the spread of heterochromatin
Heterochromatin can "spread" to neighboring euchromatin and silences the white gene Patches of white tissue: descendents of a single cell in which white gene has been silenced and remains silenced Red patches: cells in which heterochromatin has not spread to the white gene . Gene remain active document
Gene regulation has been studied extensively in E. coli
Highly efficient genetic mechanisms have evolved to turn transcription of specific genes on and off depending on a cell's metabolic need for specific gene products These responses can be due to changes in the environment as well as nonenvironmentally regulated cellular activity and cell division
Features of the CRISPR/Cas9 system
Highly specific Tightly regulated Highly efficient
2. Modification of histones
Histones are known to be the most conserved proteins in nature; that is, histones are almost identical in all eukaryotic organisms from yeast to plants to animals • All four of the histones that make up the core of the nucleosome are relatively small proteins (102-135 amino acids) •These histone tails are subject to several different types of covalent modifications that in turn control critical aspects of chromatin structure and function document
Holoprosencephaly
Holoprosencephaly is associated with mutations in the human Shh coding region (result alterations in the Shh protein) Abnormalities occur in brain size, in the formation of the nose, and in other midline structures. One copy of a normal Shh gene appears to be insufficient for normal midline development (the gene is haploinsufficient). Human fetuses homozygous for loss-of-function Shh mutations very likely die in gestation with more severe defects. Holoprosencephaly is not caused exclusively by Shh mutations. Shh is a ligand in a signal-transduction pathway Mutations in genes encoding other components of the pathway affect the efficiency of Shh signaling and are also associated with holoprosencephaly.
Human sex chromosomes
Homologous region/pairing region/pseudoautosomal region: Genes present on both sex chromosomes Are inherited like genes on autosomal chromosomes Differential region: contain most of the genes, have no counterparts on the other sex chromosome Genes in the differential regions show inheritance patterns called sex linkage: X-linkage or Y-linkage A gene that is sex linked can show phenotypic ratios that are different in each sex
Making segments different: integration of Hox inputs
How are the different segmental identities established by Hox proteins? 1. The Hox genes are expressed in different domains along the anteroposterior axis 2. The regulation of target genes by Hox proteins
Phylogeny of living mammals and other amniotes
How the mammalian genome has evolved over the history of the group? Evolutionary relationships among three major groups of mammals Monotremes differ from other mammals in that they lay eggs. Inspection of the platypus genome revealed that it contains one egg-yolk gene called vitellogenin. Analyses of marsupial and eutherian genomes revealed no such functional yolk genes.
The Homeobox
Hox genes and their effects on fly development • The homeotic/hox genes in animals encode regulatory proteins that bind to DNA • Each gene contains a subset of nucleotides called a homeobox, that are similar in all homeotic genes • The homeobox encodes a 60 amino acids that serve as a DNA-binding domain • This domain is related to the helix-turn-helix motif • Edward Lewis: a pioneer in the study of homeotic genes • The clustering of Bithorax complex genes suggested that the multiple loci had arisen by tandem duplication of an ancestral gene • This idea led researchers to search for similarities in the DNA sequences of Hox genes •They found that all eight Hox genes of the two complexes were similar enough to hybridize to each other.
pku
Human disease phenylketonuria (PKU) -Is caused by a defective allele of the gene that encodes the liver enzyme phenylalanine hydroxylase (PAH) -Phenylpyruvic acid: Interferes with the development of nervous system, leading to mental impairment -Most of the mutations are small changes affecting only one nucleotide pair among the thousands that constitute the gene -null alleles: the proteins encoded by them completely lack PAH function -PKU heterozygous carriers have no problem metabolizing phenylalanine: haplosufficient
Mutations in Mitochondrial DNA Cause Human Disorders
Human mtDNA contains 16,569 base pairs coding for 13 of over 70 proteins required for aerobic cellular respiration mtDNA is very susceptible to mutations -Doesn't have histones to protect from mutations -Mitochondria have high concentrations of reactive oxygen species (ROS) generated by cell respiration --ROS damages organelle contents (proteins, lipids, mtDNA) For a human disorder to be attributed to mitochondrial DNA, -the inheritance must exhibit a maternal inheritance pattern -the disorder must reflect a deficiency in the bioenergetic function of the organelle -there must be a specific mutation in a mitochondrial gene
Huntington's disease
Huntington's disease is a late onset disease. If one parent has the disorder the child will have 50% chance of inheriting the disorder, since only one copy of the mutant allele is required. If one of the children is unaffected and has children with an unaffected partner the disease will not appear in later generations.
Genomic imprinting requires insulators
ICR (Imprinting control region)--> unmethylated in female gametes---> can bind a regulatory protein called CTCF---> CTCF act as enhancer blocking insulator---> Igf2 off H19 on paternal and maternal allele document
Initiation factors (IF)
IF3: keep 30S subunit dissociated from the 50S subunit IF1 and IF2: ensure that only the initiator tRNA enters the P site 30S subunit + mRNA+ initiator tRNA • Prokaryotic initiation complex is formed at a Shine-Dalgarno sequence near the 5' end of an RNA that is still being transcribed
During DNA replication, incorrectly inserted nucleotides are recognized and removed. The correct nucleotide is put in by DNA polymerase I and III (proofreading)
If proofreading fails, mismatch repair becomes activated -Mismatches are detected, cut, and removed (endonuclease and exonuclease). Correct nucleotide inserted by DNA polymerase A strong link between defective mismatch repair and cancer has been documented Postreplication repair responds after damaged DNA has escaped repair and failed to be completely replicated
hox mutation: cis-acting regulatory element and reporter-gene expression
If these two binding sites are mutated such that the Hox proteins cannot bind, Distal-less expression is derepressed in the abdomen document
We can also use CRISPR/Cas9 to "knockin" bits of DNA
If we make an artificial piece of DNA that is identical to the cleaved region of DNA then when the cell tries to repair its own chromosomal DNA it will sometimes accidentally incorporate this into its own DNA by homologous recombination. document Now the artificially produced piece of DNA is "knocked in" to the genome
How the activation of hunchback depends on the concentration gradient can be seen by performing some tests in vivo
Link gene regulatory sequences to a reporter gene: e.g Lac Z or GFP of jellyfish---> introduce the DNA construct into the fly germ line---> monitor reporter gene expression in the embryo offspring of transgenic flies
A replicating bacterial chromosome
In 1963, John Cairns tested this prediction by allowing replicating DNA in bacterial cells to incorporate tritiated thymidine ([3H]thymidine) Autoradiograph of a bacterial chromosome after one replication in tritiated thymidine. Interpretation of the autoradiograph Autoradiogram of the intact replicating circular chromosome of E. coli shows that -DNA synthesis is bidirectional -Replication starts from a single site called origin of replication (OR) The region in which parental strands are separating and new strands are being synthesized is called a replication fork
How can we use CRISPR/Cas9 for genetic engineering?
In 2012, Jennifer Doudna and Emmanuelle Charpentier demonstrated that combined crRNA and the tracrRNA into one artificial RNA called a single guide RNA (sgRNA) could be used for engineering. This means we can artificially make a sgRNA that can be designed to target any part of the genome (as long as it has an appropriate PAM sequence nearby) All we have to do is artificially express the Cas9 and the sgRNA together We can put two different sgRNA into the same protein and cut at 2 places in the genome we can cut out large regions of DNA
codominance blood groups
In Human ABO blood groups there is a codominance of antigen alleles The four different blood types: A, B, O, and AB Blood types are produced by three different alleles of one gene: IA, IB and i IA and IB are dominant over i IA and IB are codominant
Like those of Drosophila, vertebrate Hox genes are organized in clusters and expressed along the anteroposterior axis
In mouse: 4 complexes of Hox genes 39 genes present on 4 different chromosomes The order of Hox genes parallels the order of body parts in which they are expressed • The Hox genes are expressed in distinct domains along the anteroposterior axis of the mouse embryo
Fertility factor (F)
In bacterial conjugation in E. coli, F+ cells serve as DNA donors and F- cells are the recipients F+ cells contain a fertility factor (F factor) that confers the ability to donate DNA during conjugation Recipient cells are converted to F+
The 12:3:1 F2 ratio: Dominant Epistasis
In foxgloves, two genes interact in the pathway that determine petal coloration Two genes are unlinked One gene affects the intensity of the red pigment in the petal Allele d: light red color Mutant allele D: dark red color The epistatic W restricts pigment to the throat spots
Lethal Alleles: The Agouti Locus
In mice, wild type coat color is agouti, produced by a combination of yellow and black pigments along each hair A dominant allele of agouti, AY, causes yellow pigment to be deposited along the entire hair, resulting in a yellow coat The AY allele is recessive embryonic lethal, so all yellow mice are heterozygous Normal wild type mice coats dark pigmentation A mutation called yellow coats light coat color Yellow coat is a recessive lethal allele: An allele that is capable of causing the death of an organism yellow x wild type (homozygous) Progeny yellow: wild type (dark) 1 : 1 1:1 ratio is always observed the in the progeny Results suggest - Yellow mouse is always heterozygous for the yellow allele - Yellow allele is dominant over wild type However, if any two yellow mice are crossed with each other, the result is always as follows 2/3 yellow and 1/3 wild type (2:1 ratio) The AY allele produces effects on two characters: - Coat color (single dose) - Death (double dose) Pleiotropic: any allele that affects several properties of an organism
Autosomal Polymorphisms
In natural populations of organisms, a polymorphism is the coexistence of two or more common phenotypes of a character. The alternative phenotypes of a polymorphism (morphs) are often inherited as alleles of a single autosomal gene in the standard Mendelian manner. e.g. brown versus blue eyes pigmented versus blond hair attached versus free ear lobes
Integration of the F plasmid creates an Hfr strain
In some cases, an F factor is excised from the chromosome of an Hfr strain In the process, the F factor (referred to as F') often brings several adjoining genes with it The DNA of an F' plasmid is part F factor and part bacterial genome. They can be used to establish partial diploids for studies of bacterial dominance and allele interaction (Chapter 11).
Synthetic Lethals
In some cases, when two viable single mutants are inter-crossed, the resulting double mutants are lethal In a diploid F2, the ratio would be 9:3:3 because the double mutant would be absent
Beadle and Tatum experiment
In the early 1940s, George Beadle and Edward Tatum were also interested in the relationship among genes, enzymes and traits Their plan was to investigate the genetic control of cellular chemistry Their genetic model was Neurospora crassa (a common bread mold) -Their studies involved the analysis of simple nutritional requirements -Found numerous mutants that had defective nutrition: auxotrophic mutants Beadle and Tatum focused on arginine auxotrophs The genes that mutated to give arginine auxotrophs mapped to three different loci on three separate chromosomes: arg-1, arg-2 and arg-3 A key breakthrough was that the auxotrophs for each of the three loci differed in their response to the structurally related compounds ornithine and citrulline
Regulation of the lac operon: no lactose
In the no lactose present The lac operon is transcribed only in the presence of lactose The lac operon is switched off
Lactose Metabolism in E. coli Is Regulated by an Inducible System
In the presence of lactose, the concentration of the enzymes responsible for its metabolism increases rapidly from a few molecules to thousands per cell The enzymes responsible for lactose metabolism are inducible Allolactose is the inducer
variable expressivity
In variable expressivity individuals who carry the alleles for a trait show a phenotype but to a varying degree of severity
Eukaryotic initiation of replication
In yeast, three proteins are required to begin assembly of the replisome ORC- origin recognition complex Cdc6 Cdt1 Replication is linked to the cell cycle through the availability of Cdc6 and Cdt1.--> synthesized during late mitosis and G1 and destroyed by proteolysis after synthesis has begun
Inbreeding
Inbreeding: breeding among relatives Inbreeding depression: reduction in vigor or reproductive success often caused by the expression of deleterious recessive alleles
Translation steps
Initiation Elongation Termination
Use of a plasmid vector, pUC18
Insertion of DNA into pUC18 is detected by inactivation of the lacZ gene document
Some Genes Produce Variable Phenotypes
Interpretation of genotypic and phenotypic ratios is based on the assumption that there is a strict correlation between phenotype and genotype However in some cases different phenotypes can result from the same genotype Incomplete penetrance and variable expressivity can complicate interpretation of genotypic and phenotypic ratios
Deletions
Intragenic deletion- within a gene Multigenic deletion- several genes Pseudodominance- recessive alleles that seem to be showing dominance when deletions occur document
Arabinose Operon
Is an example in which a single DNA-binding protein (AraC) may act as either a repressor or an activator Map of the ara operon Control gene: encodes an activator protein structural genes: encode the metabolic enzymes that break down the sugar arabinose • Transcription is activated at araI : the initiator region (contains a binding site for an activator protein) • The araC gene: encodes an activator protein • When bound to arabinose, this protein binds to the araI site and activates transcription of the ara operon
The Genetic Code
Is written in linear form, using the ribonucleotide bases that compose mRNA molecules as "letters" In the mRNA, triplet codons specify one amino acid Reads three nucleotides at a time in a continuous, linear manner Thus, the code is nonoverlapping Is degenerate, in many cases, more than one codon is assigned to a single amino acid In addition, several codons can pair with more than one anticodon (wobble). The genetic code designates the amino acids specified by each codon.
What is CRISPR ?
It is a very efficient method of genetic engineering that allows precision cutting and DNA rearrangement (Usually) utilizes either: -Non-homologous end joining (NHEJ), which ligates double-stranded breaks -Homologous recombination
Phosphorylation
Kinase: attach phosphate groups to hydroxyl groups of amino acids (serine, threonine, tyrosine) Phosphatase: remove phosphate groups •The addition and removal of phosphate groups serves as a reversible switch to control a variety of cellular events, including enzyme activity, protein-protein interactions, and protein-DNA interactions document
Fosmids and BACs are cloning vectors that carry large inserts
Lambda phage+ plasmid DNA Replicate extrachromosomally BAC (Bacterial Artificial Chromosome) Used in large scale genome sequencing projects
The attenuation mechanism is common to several operons for enzymes responsible for synthesis of other amino acids
Leader peptides of amino acid biosynthesis operons document
Chromatin structure varies along the length of a chromosome two types
Least condensed chromatin: euchromatin Heterochromatin: coated with special proteins (HP1, HP2, etc.)
Secondary structure
Local regions of the polypeptide chain fold into specific shapes, called the protein's secondary structure. Each shape arises from the bonding forces between amino acids that are close together in the linear sequence.
Detecting Genetic Variation
Locus: location in the genome Single nucleotide polymorphism (SNP): variation at a single nucleotide site Microsatellites: repetitive sequences (2-6 bp) stars are SNPs
If we picture a three-dimensional embryo as a globe, then positional information must be specified that indicates:
Longitude: location along the anteroposterior axis Latitude: location along the dorsoventral axis Altitude or depth: position in the germ layers
A mutant search led to the genes required to repress transposition
Look for mutants that have regained normal movement twitching movement document
Deletion Loops
Loops created when chromosomes are pairing, either during meiosis or in polytene chromosomes
continous variation
Many cases of continuous variation are influenced by the environment -e.g. genetically identical plants grown in a plot show a bell-shaped curve for height Many cases of continuous variation do have a genetic basis -e.g. human skin color The interacting genes underlying hereditary continuous variation are called polygenes Locations on chromosomes are associated with trait are called quantitative trait loci (QTL)
Eukaryotic Gene Control Region Consists of a Promoter Plus Regulatory DNA Sequences
Many gene regulatory proteins also influence the chromatin structure of the DNA control region thereby affecting transcription initiation indirectly (not shown) • Mediator and the general transcription factors are the same for all polymerase II transcribed genes • The gene regulatory proteins and the locations of their binding sites relative to the promoter differ for each gene Mediator and the general transcription factors are the same for all polymerase II transcribed genes The gene regulatory proteins and the locations of their binding sites relative to the promoter differ for each gene
Classification of genes by developmental function
Many toolkit genes were classified according to their: 1. Function in controlling the identity of body parts (different segments or appendages) -Among the very first toolkit genes identified -most globally acting genes that affect animal form 2. The formation of the body parts 3. The number of body parts 4. The formation of cell types 5. The organization of the primary body axes (e.g. anteroposterior and dorsoventral)
Bacterial colonies, each derived from a single cell
Members of a colony that share a single genetic ancestor are known as a cell clone. document
mendels model of inheritance (most genes dont meet these criteria)
Mendel's model of inheritance assumes that: -each trait is controlled by a single gene -each gene has only 2 alleles -there is a clear dominant-recessive relationship between the alleles
Mendels first law or law of segregation
Mendel's view of equal segregation was that the members of a gene pair segregated equally in gamete formation Now we know that gene pairs are located on chromosome pairs and that it is the members of a chromosome pair that actually segregate, carrying the genes with them
Why areas of the genome with heavily-methylated cytosines are hot spots for mutations.
Methylated cytosines are deaminated into thymine, which is not recognized as a mutation by repair mechanisms.
Epigenomic Methods
Methylation -Bisulfite sequencing -Nanopore Chromatin accessibility ChIP-seq
Histone methylation can activate or repress gene expression
Methylation is another post-translational modification of arginine and lysine residues in the histone tails that can result in altered chromatin and gene expression. The enzyme histone methyltransferase (HMTase) adds one, two, or three methyl groups to a specific amino acid in the tail of histone H3.
Unlike acetylation, the addition of methyl groups can either activate or repress gene expression.
Methylation of specific lysine residues, does not affect charge, but creates binding sites for other proteins that either activate or repress gene expression depending on the residues modified. For example, methylation of H3 lysine residue 4 [H3K4(me)] is associated with the activation of gene expression and is enriched in nucleosomes near the start of transcription.
Organelle Genes: Inheritance independent of the Nucleus
Mitochondria and chloroplasts are specialized organelles located in the cytoplasm. They contain small circular chromosomes that carry a defined subset of the total cell genome. Mitochondrial genes: energy production Chloroplast genes: photosynthesis Neither organelle is genetically independent, because each relies to some extent on nuclear genes for function Each organelle is; present in many copies per cell contains many copies of the chromosome Mitochondrial or chloroplast genome often packaged into subcellular structures called nucleoids Replication in mitochondria is dependent on genes encoded by nuclear DNA Those shown entering the organelle are derived from the cytoplasm and encoded by nuclear genes.
Applications of PCR
Molecular Identification Sequencing Genetic Engineering
Sex-linked Single-Gene-Inheritance Patterns
Most animals and many (diecious) plants show sexual dimorphism: individuals are either male or female In most cases sex is determined by a special pair of sex chromosomes. Humans body cells have 46 chromosomes 22 homologous pairs of autosomes 2 Sex chromosomes Females : pair of identical X chromosomes Males : non-identical pair X and Y chromosomes At meiosis in females, the two X chromosomes pair and segregate like autosomes (each egg receives one X chromosome) Gametes are of only one type and the female is said homogametic sex in humans At meiosis in males, the X and Y separate so that there are two types of sperm, (half X and half Y): Male is called heterogametic sex in humans
inheritance of dimorphism
Most human populations are dimorphic for the ability to taste the chemical phenylthiocarbamide (PTC). People could either detect it as a foul, bitter taste or cannot taste it at all. Two tasters sometimes produce nontaster children-allele that confers ability to taste is dominant and that the allele for nontasting is recessive
What is heterochromatin if not genes ?
Most of the eukaryotic genome is repetitive sequences that do not make protein or structural RNA
Long-Term Inactivation of Genes in a Chromatin Environment
Most of the genes in eukaryotic genomes are off at any one time. One of the most surprising findings of the genomics era is that many eukaryotic genes are inactive for the life of the organism. Why do organisms have genes that are always inactive? How do organisms keep genes in an inactive state for their entire lifetime?
A mutation is an alteration in DNA sequence
Mutations may be -single-base pair substitutions -deletion or insertion of one or more base pairs -major alteration in chromosomal structure Mutations may occur in somatic or germ cells
conservative replication
NOT RIGHT
dispersive replication
NOT RIGHT
incomplete dominance
Neither allele is dominant Offspring from a cross between parents with contrasting traits may have an intermediate phenotype in the heterozygous individual The phenotypic ratio is identical to the genotypic ratio From this 1:2:1 ratio in the F2, we can deduce that the inheritance pattern is based on two alleles of a single gene The occurrence of the intermediate phenotype suggests an incomplete dominance
RNA polymerase directs the synthesis of RNA using a DNA template.
No primer is required for initiation, and the enzyme uses ribonucleotides instead of deoxyribonucleotides.
The molecular consequences of mutations at noncoding regions
Noncoding regions contain many crucial binding sites -At the DNA level: RNA polymerase and its associated factors binds, specific transcription regulators - At the RNA level, ribosome binding site, 5' and 3' splice sites for exon joining, sites that regulate translation and localize the mRNA to particular areas and compartments within the cell. document
Nondisjunction
Nondisjunction is a failure of disjunction, which is the normal segregation of homologous chromosomes or chromatids to opposite poles at meiotic or mitotic divisions
A mutation does not affect the length of a gene but results in an abnormally short protein. The mutation is most likely of a type called
Nonsense
The structure of chromatin and histones
Nucleosomes are composed of DNA wrapped around eight histones (an octamer made up of two copies of each of the 4 core histones (H2A and H2B and a tetramer of H3 and H4)
Regulation of gene expression through Alternative splicing
Number of genes in human genome : ~ 21,000 Spectrum of human proteins is in excess of 70,000 Alternative splicing: • Allows different but related proteins to be synthesized from the same primary transcript • One gene can encode more than one polypeptide when its pre-mRNA is alternatively spliced More than 70% of human genes are alternatively spliced Many mutations with serious consequences for the organism are due to splicing defects
Uracil DNA glycosylase removes uracil from DNA
Numerous DNA glycosylases exist Uracil-DNA glycosylase, removes uracil from DNA. Uracil residues, which result from the spontaneous deamination of cytosine, can lead to a C-to-T transition if unrepaired One advantage of having thymine (5-methyluracil) rather than uracil as the natural pairing partner of adenine in DNA is that spontaneous cytosine deamination events can be recognized as abnormal and then excised and repaired
Overdominance
Occurs when a heterozygote has a more extreme phenotype than that of either of its parents A trait that shows overdominance sometimes confers a survival advantage in the heterozygote
1. RNA-seq to study the transcriptome
Often completed as a comparison of gene expression document
Model for how enhancer-blocking insulators might work
One proposal is that enhancer-blocking insulators create new loops that physically separate a promoter from its enhancer.
y-linked inheritance
Only males inherit genes in the differential region of the human Y chromosome, with fathers transmitting the genes to their sons The gene that plays a primary role in maleness is the SRY gene, sometimes called the testis-determining factor. Hairy ears: A phenotype proposed to be Y linked
Relationships Between Genes of Gene Families
Ortholog Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Paralog Paralogs are genes related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one.
Which of the following statements is/are TRUE about P elements in Drosophila?
P elements are examples of eukaryotic DNA transposons
Insertion of P elements causes the mutations underlying hybrid dysgenesis
P elements in the newly formed zygote are in a silencing-free environment
PKU is an example of fully recessive mutation
PAH (phenylalanine hydroxylase) gene is haplosufficient One "dose" of the wild type allele P produces enough PAH to break down phenylalanine entering the body.
Scientists have noticed that about 40% of bacteria species contain 29bp palindromic repeats sequences in them - what did they do ?
Palindromic repeats (i.e. this is the same DNA sequence repeated in different places)
Inversions creates inversion loops during meiosis
Paracentric inversions can lead to deletion products document Pericentric inversions can lead to duplication-and-deletion products Document
Paralog
Paralogs are genes related by duplication within a genome.
How can defects in the same repair pathway lead to such different disease symptoms?
Patients with XP fall into 8 complementation groups, carrying mutations in one of 8 genes encoding proteins XPA through XPG Patients with Cockayne syndrome have a mutation in one of two proteins called CSA and CSB, which are thought to recognize stalled transcription complexes.
why peas
Peas were available from seed merchants in a wide array of distinct variant shapes and colors Peas can either self-pollinate (self) or cross-pollinate (cross/outcross) Inexpensive and easy to obtain Take up little space Have a short generation time Produce many offspring
autosomal dominant disorder
Phenotype tends to appear in every generation of the pedigree Affected mothers or fathers transmit the phenotype to both sons and daughters The equal representation of both sexes among the affected offspring rules out inheritance through the sex chromosomes
Hybrid dysgenesis in Drosophila
Phenotypes that are manifested in the germ line (sterility, a high mutation rate, and a high frequency of chromosomal aberration, etc.).
Cloning vectors
Plasmid vectors Bacteriophage vectors Fosmids Bacterial Artificial Chromosome (BAC)
pleiotropic
Pleiotropic: any allele that affects several properties of an organism
Consequences of mutations at splice sites
Point mutations can alter mRNA splicing (a) C to T Transition leading to a GT dinucleotide in the exon (new 5' splice site) (b) G to T transversion mutation would eliminate the 5' splice site so the intron would be retained in the mRNA
Promoter-proximal elements are necessary for efficient transcription
Point mutations in the promoter and promoter-proximal elements hinder transcription of the ß-globin gene
polygenic inheritance
Polygenic inheritance occurs when multiple genes are involved in controlling the phenotype of a trait The phenotype is an accumulation of contributions by multiple genes These traits show continuous variation and are referred to as quantitative traits A large proportion of variation in natural populations takes the form of continuous variation, which is found in characters that can take any measurable values between two extremes e.g. height, weight, skin color etc Many common human diseases such as atherosclerosis and hypertension are thought to have a polygenic component
Dominant negative
Polypeptides with this type of mutation act as "spoilers" e.g. mutations in the gene for collagen protein give rise to human phenotype brittle-bone disease
Polyploids
Polyploids: more than two chromosome sets Autopolyploid: multiple sets originating from within one species Allopolyploids: multiple sets from two or more different species
What is the primary benefit of positive assortative mating? What is the primary benefit of negative assortative mating? (
Positive assortative mating often results in the selection of beneficial traits. Negative assortative mating often helps maintain genetic diversity.
Prokaryotic Initiation of Replication
Precise time Fixed origin (ori) Bidirectional DNA synthesis is initiated at origins of replication in prokaryotes
Maternal effect genes
Products of maternal-effect genes (mRNA and proteins) are deposited in the egg cytoplasm -Many of these products are distributed in a gradient or concentrated in specific regions of the cell Maternal-effect genes encode transcription factors and proteins that regulate gene expression, the products of which activate or repress expression of the zygotic genome • Sets up the anteroposterior axis • A key member of this class is the Bicoid gene bicoid mutants are missing the anterior region
Posttranslational events
Protein folding inside the cell into its correct three-dimensional shape (native conformation) Nascent proteins are folded correctly with the help of chaperones Posttranslational modification of amino acid side chains -Phosphorylation -Ubiquitination
Synthesizing Pure Lines
Pure lines are among the essential tools of genetics These fully homozygous lines express recessive alleles Maintenance of stocks for research Pure lines are made through repeated generations of selfing Repeated selfing leads to an increased proportion of homozygotes, a process that can be used to create pure lines for research or other applications
Homozygous recessives from inbreeding
Recessive mutations of any particular type are generally fairly rare and are therefore seen more commonly when individuals are related. In this example cousins have married and have produced affected children.
2. Nucleotide-excision repair (NER) steps
Recognition of damaged bases Assembly of a multiprotein complex at the site Cutting of the damaged strand several nucleotides upstream and downstream of the damage site and removal of the nucleotides (~30) between the cuts Use of the undamaged strand as a template for DNA polymerase followed by strand ligation
Protein targeting
Recognize amino acid signatures in a protein sequence and target those proteins to places where their activity is required e.g. Signal sequences target proteins for secretion document
Generating Recombinant DNA molecules
Recombinant DNA refers to the joining of DNA molecules, usually from different biological sources, that are not found together in nature
A point mutation in a gene's promotor will most likely cause the production of
Reduced amounts of mRNA and protein
positive control or negative control
Regulation of the inducible or repressible type may be under positive control or negative control Negative control: genetic expression occurs unless it is shut off by some form of a regulator molecule Positive control: transcription occurs only if a regulator molecule directly stimulates RNA production
Describe the cause and effect of replication slippage during DNA replication. What types of sequences are most likely to be affected by slippage?
Repeat sequences are often affected by replication slippage. Mispairing between the daughter and template strands creates loops during DNA replication. This often leads to the expansion of repeat sequences.
epistasis
Replacement of a mutant phenotype produced by one mutation with a mutant phenotype produced by mutation of another gene The observation of epistatsis suggests a common developmental or chemical pathway
Generalized Translesion Synthesis Model in Bacteria and Eukaryotes
Replicative polymerase stalls at lesion (depurination, bulky adducts, pyrimidine dimers) Beta clamp (E. coli) or PCNA (eukaryotes) recruits TLS/bypass polymerases Bypass polymerase falls off quickly and is replaced by replicative polymerase document
An abundance of an end product in the environment represses gene expression
Repressible system
negative regulation
Repressor protein must be prevented from binding to its target DNA sites as a necessary prerequisite for transcription to begin
The metabolism of lactose
Requires two enzymes: 1. a permease to transport lactose into the cell 2. b galactosidase to cleave the lactose molecule to yield glucose and galactose
X-ray diffraction analysis of DNA
Rosalind Franklin's critical experimental result DNA is long and skinny and has two similar parts that are parallel to each other and run along the length of the molecule. Also showed that the molecule to be helical or spiral like. DNA is a helix of precise dimensions
In E. coli, mutations arising during repair are mostly caused by
SOS repair
Describe how the SWI/SNF complex uses chromatin remodeling to regulate gene expression
SWI/SNF remodels chromatin by moving histones, and therefore exposing cis-acting binding sites (like the TATA box) that were previously wrapped around histones.
• Coding strand/Nontemplate can be read 5'-to-3' just like the transcript except Ts will be in place of Us • Template strand is in the 3'-to-5' orientation • RNA is synthesized in the 5'-to-3' direction
Sequences of DNA and transcribed RNA document
selfing a monohybrid plant
Sequential generations of selfing increases the proportions of homozygotes, according to the principle of equal segregation and independent assortment (if the genes are on different chromosomes).
Why are genes in the HMR and HML cassettes not expressed?
Sir2 a histone deacetylase: facilitates the condensation of chromatin and helps lock up HMR and HML in chromatin domains where transcription cannot be initiated (sir: silent information regulators)
essential genes and lethal alleles
Some genes are required for life (essential genes), and mutations in them (lethal alleles) may result in death.
Cloning DNA fragments with blunt ends
Some restriction enzymes produce blunt ends rather than staggered cuts. In addition, cDNA and the DNA fragments that arise from PCR have blunt or near-blunt ends. While blunt end fragments from all these sources can be joined to the vector with the use of ligase alone, this is a very inefficient reaction because blunt ends cannot stick together
Generating Recombinant DNA molecules sources
Sources of donor DNA 1. Entire genome (digested with restriction enzymes) 2. PCR products 3. cDNA copies of mRNA
Attaching donor and vector DNA
Sources of donor DNA 1. Entire genome (digested with restriction enzymes) 2. PCR products 3. cDNA copies of mRNA document
Gender-Specific Silencing of Genes and Whole Chromosomes
Specific genes or even a whole chromosome are silenced for the entire lifetime of an organism. However, unlike the prior examples, these genes or chromosomes are silenced in males or females but not both.
3. Predictions of binding sites
Statistical "gene finding" computer programs can search for the predicted sequences of the various binding sites used for promoters, transcription start sites, 5' and 3' splice sites etc. document
PCR steps
Step 1: DNA denaturation: Expose the DNA template to high temperature to separate the two DNA strands and allow access by DNA polymerase and PCR primers. Step 2: Primer Annealing: Lower temperature to allow primers to anneal to their complementary sequence Step 3: Primer Extension: Adjust temperature for optimal thermostable Taq polymerase activity to extend primers 1. Primers 2. dNTPs 3. DNA polymerase (Taq polymerase) 4. Target DNA 5. Mg2+
Cloning DNA fragments with sticky ends
Sticky ends/staggered cuts
Expression of the vast majority of transposable elements is silenced by host RNAi pathway
Studied the difference between the mobility of a transposable element called Tc1 (DNA transposon) There are 32 Tc1 in the worm genome Tc1 transposes in somatic but not germ-line cells (transposition is repressed in the germ-line cells)
Safe havens
Successful transposable elements insert into so-called safe havens in the genome 1. For the grasses, safe haven: insert into other retrotransposons 2. Heterochromatin or centromeres (few genes but lots of repetitive DNA)
Genetic evidence for allostery
Super Is mutations : cause repression to persist even in the presence of an inducer
ZPA
Tabin and colleagues at Harvard found that the morphagen produced by the ZPA is the protein from the Sonic hedgehog (Shh) gene Shh protein also in developing feather buds and neural tubes developing chick vertebral limb bud ZPA (zone of polarizing activity): organize pattern along anteroposterior axis)
Reading the DNA sequence from an automatic sequencer
Tag is a fluorescent dye and a different fluorescent color emitter is used for each of the four ddNTP reactions Each colored peak represent a different size fragment of DNA, ending with a fluorescent base document
Transcription elongation in eukaryotes
Takes place inside the transcription bubble (similar to prokaryotes) Nascent RNA has very different fates in prokaryotes and eukaryotes In prokaryotes, translation begins at the 5' end of the nascent RNA while the 3' half is still being synthesized. In eukaryotes, RNA must undergo further processing before it can be translated.
Duplications
Tandem duplication- duplicate regions adjacent to each other Insertional duplication- extra copy is somewhere else in the genome Segmental duplication- large scale duplications, usually 10-50 kb
Which of the following statements is/are FALSE about Tc1 elements found in C. elegans?
Tc1 elements are retrotransposons.
The Ames Test Is Used to Assess the Mutagenicity of Compounds
The Ames test uses a number of different strains of Salmonella typhimurium selected for their ability to reveal the presence of specific types of mutations The Ames test is used extensively during the development of industrial and pharmaceutical chemical compounds In 1970s, Bruce Ames recognized that there is a strong correlation between the ability of compounds to cause cancer and their ability to cause mutations. • Many known carcinogens have been shown by the Ames test to be strong mutagens - More than 60 compounds found in cigarette smoke test positive in the Ames test and cause cancer in animals
What is the C-value paradox? What is the explanation for the C-value paradox?
The C-value paradox is the lack of correlation between genome size and biological complexity. The commonly accepted explanation is that larger genomes have more repetitive DNA, including transposable elements.
Finding specific nucleic acids by using gel electrophoresis and blotting
The DNA must be denatured first, which allows it to stick to the membrane
The 9:7 ratio
The F2 ratio from the harebell dihybrid cross shows both blue and white plants in a 9:7 ratio A plant will have white petals if it is homozygous for the recessive mutant allele of either gene or both genes To have a blue phenotype, a plant must have at least one copy of the dominant allele of both genes because both are needed to complete the sequential steps in the pathway Similar 9:7 F2 ratio can come from gene regulation
The structure of the Human Genome
The Human Genome Project (HGP) was a coordinated effort to sequence and identify all the genes of the human genome HGP's most valuable contribution will be the identification of disease genes and the development of new treatment strategies Extensive maps have been developed for genes implicated in human disease conditions
CAP and RNA polymerase bind next to each other
The base sequence shows that CAP and RNA polymerase bind directly adjacent to each other on the lac promoter
Regulatory proteins control transcription
The basics of prokaryotic transcriptional regulation: Genetic switches Regulatory proteins control transcription positive regulation and negative regulation
enhanceosome
The binding of multiple regulatory proteins to the multiple binding sites in an enhancer can catalyze the formation of an enhanceosome
DNA structure before Watson and Crick
The building blocks of DNA Chargaff's rules of base composition X-ray diffraction analysis
Whole-genome sequencing (WGS)
The current general strategy for obtaining and assembling the sequence of a genome is called whole-genome shotgun (WGS) sequencing. Two methods 1. Traditional WGS (Chapter 10) 2. Next-generation WGS
Many forms of evidence are integrated to make gene predictions
The different forms of gene-product evidence—cDNAs, ESTs, BLAST similarity hits, codon bias, and motif hits—are integrated to make gene predictions.
gal4 lacking the activation domain
The experimental removal of the activation domain shows that DNA binding is not sufficient for gene activation
Drawing stripes: Integration of gap-protein inputs
The expression of each pair-rule-gene in seven stripes is the first sign of the periodic organization of the embryo and future animal How are such a periodic patterns generated from a prior aperiodic information? The key discovery was that each of the 7 stripes that make up the expression patterns of the: even-skipped and hairy pair rule-genes is controlled independently.
Model for the Mismatch repair in E. coli
The first step in mismatch repair is the recognition of the damage in newly replicated DNA by the MutS protein. The binding of this protein to distortions in the DNA double helix caused by mismatched bases initiates the mismatch-repair pathway by attracting three other proteins to the site of the lesion MutH, which performs the crucial function of cutting the strand containing the incorrect base. document
inferring gene interaction
The genetic approach that reveals the interacting genes for a particular biological property follows Step 1. Obtain many single-gene mutants and test for dominance Step 2. Test the mutants for allelism-are they at one or several loci : Complementation test Step 3. Combine the mutants in pairs to form double mutants to see if the genes interact
3.Functional Genomics
The global approach to study the function, expression and interaction of genes products is termed functional genomics "Omics" The transcriptome: The sequence and expression patterns of all RNA transcripts (which kinds, where in tissues, when, how much). The proteome: The sequence and expression patterns of all proteins (where, when, how much). The interactome: The complete set of physical interactions between proteins and DNA segments, between proteins and RNA segments, and between proteins. 1. RNA-seq to study the transcriptome 2. Yeast two hybrid system: protein-protein interaction 3. ChIP (Chromatin immunoprecipitation assay): protein-DNA interaction
Structural Organization of Nucleosomes
The histone octamer forms a protein core around which the double-stranded DNA is wound
hybrid vigor
The hybrid shows greater size and vigor than do the two contributing lines This general superiority of multiple heterozygotes is called hybrid vigor Some hybrids between genetically different lines show hybrid vigor. However, gene assortment when the hybrid undergoes meiosis breaks up the favorable allelic combination, and thus few members of the next generation.
Intron Removal and Exon Splicing
The intron ends almost always have GU at the 5' end and AG at the 3' end (the GU-AG rule) The existence of conserved nucleotide sequences at splice junctions suggested that there must be cellular machinery that recognizes these sequences and carries out splicing. document
lac operon
The lac operon has three structural genes, lacZ, lacY, and lacA, with an upstream regulatory region consisting of an operator and a promoter The entire gene cluster functions in an integrated fashion to provide a rapid response to the presence or absence of lactose
Regulation of the lac operon: lactose present
The lac operon is switched on
monohybrid
The monohybrid cross reveals how one trait is transmitted from generation to generation Monohybrid crosses involve a single pair of contrasting traits
1. bioinformatics
The nature of the information content of DNA The information content of the genome: sum of all the sequences that encode proteins and RNA, plus binding sites document
Packing of Eukaryotic DNA
The packaging of eukaryotic DNA into chromatin means that much of the DNA is not readily accessible to regulatory proteins and the transcriptional apparatus. Therefore, the modification of chromatin structure is a distinctive feature of many eukaryotic processes including gene regulation, DNA replication and DNA repair.
sum rule
The probability of either of two mutually exclusive events occurring is the sum of their probabilities calculate the probability of either two 4's or two 5's
product rule
The probability of independent events both occurring together is the product of their individual probabilities e.g. The possible outcomes from rolling two dice. Calculate the probability, p, of rolling a pair of 4's
Recovery of amplified recombinant molecules
The recombinant DNA packaged into phage particles is easily obtained by collecting phage lysate and isolating the DNA that they contain. To obtain the recombinant DNA packaged in plasmids, fosmids, or BACs, the bacteria are chemically or mechanically broken apart. The recombinant DNA plasmid is separated from the much larger main bacterial chromosome by centrifugation, electrophoresis, or other selective techniques that distinguish the chromosome from the plasmid by size or shape.
Regulation of Transposable Element Movement by the Host
The repression of transposable elements was first investigated in the laboratory of Ron Plasterk in the late 1990s, using the model organism C. elegans
Chargaff's rules of base composition
The total amount of pyrimidine nucleotides (T + C) always equals the total amount of purine nucleotides (A + G). The amount of T always equals the amount of A, and the amount of C always equals the amount of G. But the amount of A + T is not necessarily equal to the amount of G + C
Spliceosome assembly and function
The spliceosome is composed of several snRNPs that attach sequentially to the RNA
Analyzing Double Mutants
The standard 9:3:3:1 F2 Mendelian ratio suggests NO gene interaction (if the two mutations under study are on different chromosomes) Inheritance of skin coloration in corn snakes Wild type: black-and-orange camouflage pattern. Phenotype is produced by two separate pigments; Orange pigment o+ Black pigment b+ These two genes are unlinked a. Genotype of camouflage: o+/-;b+/- b. Black genotype o/o;b+/- c. Orange genotype o+/-;b/b d. Albino genotype o/o;b/b The 9:3:3:1 ratio is produced because the two pigment genes act independently at the cellular level
Tailless cat
The tailless Manx phenotype in cats also is produced by an allele that is lethal in the homozygous state A single dose of the Manx allele, ML, severely interferes with normal spinal development, resulting in the absence of a tail in the ML/M heterozygote. But in the ML/ML homozygote, the double dose of the gene produces such an extreme abnormality in spinal development that the embryo does not survive.
Making Genomic and cDNA libraries
The task of isolating a clone of a specific gene begins with making a library of genomic DNA or cDNA
Wobble allows tRNA to recognize two codons
The third nucleotide of an anticodon (at the 5' end) can form hydrogen bonds either with its normal complementary nucleotide in the third position of the codon or with a different nucleotide in that position
Transcription Termination in prokaryotes
The two major mechanisms for termination in E. coli are called intrinsic and rho dependent. Intrinsic mechanism: the termination is direct. When polymerase encounters the stable hairpin loop it sets off the release the RNA Rho-dependent termination entails the binding of rho protein to rut, the pausing of polymerase, and rho-mediated dissociation of the RNA from the RNA polymerase.
genetic engineering techniques
The use of recombinant-DNA techniques to alter an organism's genotype and phenotype is termed genetic engineering, and its application to practical applications is called biotechnology Gene transferred: Transgene Engineered products: Transgenic organism
The human genome carries relics of our egg-laying ancestors
There are traces of vitellogenin gene sequences detectable in the human and dog genomes at positions that are in the same position as (syntenic to) the vitellogenin genes of the platypus and chicken These sequences are molecular relics of our egg-laying ancestors document
Describe the similarities and differences between IS elements, simple transposons, and composite transposons in prokaryotes.
They all utilize transposase in order to transpose. IS elements only contain what they need to transpose, while transposons are able to carry other genes such as antibiotic resistance genes. Composite transposons utilize IS elements for transposition, while simple transposons utilize their own transposase gene.
Heterochromatin may spread farther in some cells than in others
This could be what is happening to the white gene of Drosophila document
No recombinants are produced without cell contact
This possibility of cross feeding was ruled out by Bernard Davis document
This allows us to selectively "knock out" regions of the genome
This utilizes the natural repair mechanism, Non-homologous end joining (NHEJ), to ligate double-stranded breaks document
Describe why Jacob and Monod used IPTG as a synthetic inducer during their experiments investigating the genetic control of the lac operon.
To maintain consistent levels of activation of the lac operon. If they used lactose, activation of the operon would decrease as the lactose is cleaved.
Discovery of the lac system: Negative control
To study gene regulation, ideally we need three things: A biochemical assay that lets us measure the amount of mRNA or expressed protein or both Reliable conditions in which differences in the levels of expression occur in a wild-type genotype Mutations that perturb the levels of expression.
transcription
To utilize the information, an RNA copy of the gene must be synthesized in a process called transcription. • Transcription is asymmetrical: only one strand of the DNA of a gene is used as a template for transcription. • This strand is in the 3'-to-5' orientation • RNA is synthesized in the 5'-to-3' direction
Analysis of cis-acting regulatory elements with reporter genes
Toolkit loci often contain multiple independent cis-acting regulatory elements---> that control gene expression in different places or at different times during development or both (A,B, and C)
Prokaryotic and eukaryotic transcription and translation compared
Transcription is more complicated in eukaryotes
Gene Therapy
Transplantation of a gene to correct a genetic deficiency e.g. SCID: Severe Combined Immunodeficiency Disease Caused by a mutation in the gene encoding the blood enzyme adenosine deaminase (ADA) • As a result of the loss of this enzyme, precursor cells that give rise to one of the cell types of the immune system are missing • Unable to fight infection • Treatment: Stem cells can be removed from bone marrow and the wild type copy of the ADA gene is introduced using a retrovirus vector. • Transformed cells can then be infused back into the patients. • Caveat: Insertion of the viral vector can inactivate important genes and some of the treated patients have developed leukemia
Steps in a chromatin immunoprecipitation assay (ChIP)
Treat cells with a chemical that will cross-link proteins to DNA Add an antibody that reacts specifically with the encoded protein Immune complex can be purified
Ultrabithorax mutant embryos and Ultrabithorax/Abdominal-A double mutant embryo
Ultrabithorax mutant embryos Ubx-: Distal-less expression expands to the first abdominal segment DII depressed in A1 Ultrabithorax/Abdominal-A double mutant embryo Ubx-,abd-A-: Distal-less expression extends through the first seven abdominal segments (A1-A7)---->Both proteins are required for the repression of Distal-less expression in the abdomen DII depressed in A1-A7 document
uniparental inheritance
Uniparental inheritance: progeny inherit organelle genes exclusively from one parent but not the other -In most cases parent is mother, a pattern called maternal inheritance
CRISPR/Cas9 can also be used to switch on or off genes
Uses a mutant Cas9 that can bind everything but can't cut DNA This means it locks on tightly to the DNA that matches the guide sequence An example of how this can be used is by having a big Cas9 protein sitting at a transcription factor binding site we can block the transcription factor from coming into the gene promoter, switching off the expression of that specific gene in a highly targeted way. document
Unstable mutant phenotypes that revert to wild type are a clue to the participation of mobile elements
What is the relation between Ac and Ds? How do they interact with genes and chromosomes to produce these interesting and unusual phenotypes?
How do we decide between these alternatives?
When studying infrequent events such as the invention of a gene, evolutionary biologists prefer to rely on the principle of parsimony favor the simplest explanation involving the smallest number of evolutionary changes Preferred explanation for the pattern of vitellogenin evolution in mammals is that this egg-yolk protein and corresponding gene were present in some egg-laying amniote ancestor and were retained in the egg-laying platypus and lost from non-egg-laying mammals.
SIp, En mutation
When the binding sites for both proteins are mutated, reporter gene expression is derepressed in both compartments of each abdominal segment
allele lethal and environment
Whether an allele is lethal or not often depends on the environment in which the organism develops Whereas certain alleles are lethal in virtually any environment, others are viable in one environment but lethal in another. e.g. Human hereditary diseases (Cystic fibrosis and sickle-cell anemia are diseases that would be lethal without treatment).
Describe the process of gene silencing of transposable elements through the production of piRNAs in animals. In your answer, describe the process that determines which transposable elements are silenced by piRNAs and which transposable elements are not silenced by piRNAs.
Whether or not a transposable element is silenced by piRNAs is determined by whether or not that transposable element has jumped into a pi-cluster. These pi-clusters are often referred to as transposable element "traps." If a TE jumps into a pi-cluster, then single-stranded RNA is transcribed, which is recognized by piwi-Argonuate. If the piwi-Argonuate recognizes TE mRNA that is complementary to the piRNA, then it is degraded.
1944-Avery, MacLeod, McCarty
Which chemical component of the dead donor cells had caused this transformation ? -s strain extract, when dna destroyed, mouse lives, no live s strain recovered everything live s strain recovered and mouse dies
Penetrance and Expressivity
Why would an organism have a particular genotype and yet not express the corresponding phenotype? 1. The influence of the environment 2. The influence of other genes: Modifiers, epistatic genes, or suppressors in the rest of the genome may act to prevent the expression of the typical phenotype 3. The subtlety of the mutant phenotype: The subtle effects brought about by the absence of a gene function may be difficult to measure in a laboratory situation.
Bacterial mutants
Wild type bacteria are prototrophic A prototroph can synthesize all essential organic compounds and therefore can be grown on minimal medium Through mutation, an auxotroph has lost the ability to synthesize one or more essential compounds; it must be provided with them in the medium if it is to grow
A model for recessive epistasis
Wild-type alleles of two genes (w+ and m+) encode enzymes catalyzing synthesis of a blue petal pigment Homozygous m/m plants produce magenta flowers homozygous w/w plants produce white flowers double mutant w/w ; m/m also produces white flowers, indicating that white is epistatic to magenta
Discovery of the Fertility factor (F)
William Hayes, 1953 • Discovered that the conjugating patterns acted unequally. • One parent transfer some or all of its genome into another cell/acted as a donor, and the other cell acted as a recipient
Degeneracy Revisited
Wobble: The ability of certain bases at the third position of an anticodon in tRNA to form hydrogen bonds in various ways, causing alignment with several different possible codons
lacZ encodes the enzyme: ß galactosidase
X-Gal (artificial substrate)----B galactosidase---->blue dye (blue colonies) DNA inserts disrupts--->No B galactosidase--->white colonies lacZ gene document
Xeroderma pigmentosum and cockayne syndrome
Xeroderma pigmentosum (XP) is characterized by the early development of cancers, especially skin cancer and, in some cases, neurological defects. In contrast, patients afflicted with Cockayne syndrome have a variety of developmental disorders including dwarfism, deafness, and mental impairment. In broad terms, XP patients get early cancer, whereas Cockayne syndrome patients age prematurely.
Drosophila and vertebrate Hox protein show striking similarities
Yellow: residues in common Red: divergent residues
Synthesis of β-Galactosidase and Permease in Haploid and Heterozygous Diploid Operator Mutants
Z- and Y- are recessive to their respective wild-type alleles (Z+ and Y+). Oc is constitutive : caused the lac operon structural genes to be expressed regardless of whether inducer was present. Operator is cis-acting
Zygotically active genes
Zygotic genes are transcribed in the nuclei of the developing embryo -They are transcribed in specific regions in response to the distribution of maternal-effect proteins 2. gap genes: affects the formation of a contiguous block of segments 3. pair rule genes: act a double-segment periodicity 4. segment polarity genes: affect pattern within each segment segmentation-gene mutants are missing parts of segments
Consider an E. coli cell with the following mutations. What effect would each mutation have on the function of the lac operon (assuming no glucose is present)? (Answer parts a-d separately) (8 pts) a) a mutant lac operator that cannot bind repressor b) a mutant lac repressor that cannot bind the lac operator c) a mutant lac repressor that cannot bind to allolactose d) a mutant lac promoter that cannot bind CAP plus cAMP
a) The lac operon would always be turned on because the repressor cannot turn it off by binding to the operator. b) Same as answer a. c) The operon would be uninducible. The repressor would remain bound to the operator even in the presence of the inducer. d) The operon would be transcribed only weakly. Even in the presence of glucose, CAP plus cAMP would be unable to facilitate polymerase binding to the lac promoter.
A maize plant is homozygous for an allele Cm, caused by the insertion of a Ds element into the coding region of a gene (C) that encodes an enzyme necessary for the synthesis of anthocyanin in maize kernels. a) If this plant also contains an Ac element in its genome, what will be the phenotype of its kernels? Justify your answers. b) If this plant does not contain an Ac element, what will be the kernel phenotype? Justify your answers. c) If the plant does not contain an Ac element, but it does contain an autonomous element for another family of transposon, what will be the kernel phenotype? Justify your answers.
a) spotted (The Ac element provides a transposase that can move the Ds element during kernel development.) b) colorless (no transposase = no movement = no restoration of color) c) colorless (The transposase enzyme is family specific; similar to part b), there is no transposase that can recognize and move elements of the Ac/Ds family.)
structure of target DNA sites
a)lac operator: to which Lac repressor binds b)CAP-binding site: to which CAP-cAMP complex binds Many DNA binding sites are symmetrical (rotational twofold symmetry). This rotational symmetry corresponds to symmetries within the DNA-binding proteins, many of which are composed of two or four identical subunits
Enhanceosomes help recruit the transcriptional machinery
b-interferon enhanceosome dna-bending proteins Transcription factors recruit a co-activator (CBP): binds both to the transcription factors and to RNA pol II, initiating transcription
Cytological examination:
chromosomal rearrangement in the mutant flies
Genome
complete set of DNA in a single cell of an organism 1995: 1.8-Mb genome of the bacterium Hemophilus influenzae 1996: 12-Mb genome of Saccharomyces cerevisiae 1998: 100-Mb genome of C. elegans 2000: 180-Mb genome of Drosophila melanogaster 2001: 3000-Mb of the first draft of human genome 2005: the first draft of Chimpanzee genome 2010: the first draft of Neanderthal genome
cAMP-CAP complex activates transcription
complex binds to promoter cAMP molecules+CAP---->CAP-cAMP • CAP binds to specific DNA sequences of the lac operon (the CAP-binding site in the promoter) • The DNA-bound CAP is then able to interact physically with RNA polymerase and increase the enzyme's affinity for the lac promoter • By itself, CAP cannot bind to the CAP-binding site of the lac operon • By binding to cAMP, its allosteric effector, CAP is able to bind to the CAP-binding site and activate transcription by RNA polymerase • By inhibiting CAP when glucose is available, the catabolite-repression system ensures that the lac operon will be activated only when glucose is scarce
Determining the base sequence of a DNA segment
ddNTP dNTP (OH on the left instead of H)
Unbalanced rearrangements
deletion and duplication document
Types of chromosome mutations
deletion, duplication, inversion, translocation
Some of the genes associated with types of human cancers are shared members of the animal toolkit
e.g. Patched gene encodes a receptor for the Hedgehog signaling pathway. In addition to causing inherited developmental diseases, mutations in patched gene are associated with cancers. document
Allosteric effectors influence the DNA-binding activities of activators & repressors
e.g. allosteric effector in lactose metabolism is an isomer of the sugar lactose: allolactose ❖ Allosteric effectors control the ability of activator or repressor proteins to bind to their DNA target sites
The generation of a transgenic plant
e.g. crop plants carrying genes that confer resistance to certain bacterial or fungal pests document
Combinations of maternal-effect and gap proteins control individual pair-rule stripe formation
eve stripe 2 element contains multiple sites for the maternal Bicoid protein and the Hunchback, Giant and Kruppel gap proteins
Griffith's experiment
genetic material could be transferred between dead bacteria and living bacteria 1928 - Griffith's experiments on the bacterium Streptococcus pneumoniae r strain+ s strain heat-killed= mouse dies + s stain live cells
The basics of lac catabolite repression: choosing the best sugar to metabolize
glucose levels regulate cAMP levels High glucose: ATP--X-->no cAMP Low glucose ATP---->cAMP--->a high concentration of cAMP is necessary for activation of the lac operon Glucose breakdown product modulate the level of cyclic adenosine monophosphate (cAMP)
modification state
heterochromatin formation, gene silencing gene expression gene expression silencing of Hox genes, X chromosome inactivation
A number of mutations affect the expression of the lactose operon in E. coli. Complete the table given below. Use + to indicate that a functional enzyme is synthesized and - to indicate that a functional enzyme is not synthesized.
inducer iptg absent b-galactosidase permease iso+Y+ oc y-
glucose present (cAMP low); lactose present
inducer-repressor control of the lac operon--> negative control document
Balanced rearrangements
inversion and reciprocal translocation document
The lac repressor protein controls expression of the lac operon by binding to the:
lac operator site to repress expression.
The hierarchy of genes involved in establishing the segmented body plan in Drosophila
maternal effect genes--> zygotically active genes--> gap genes--> pair rule genes--> segment polarity genes--> homeotic genes (Hox genes) segmentation genes: gap-genes, pair rule genes, segment polarity genes • Gene products from the maternal genes regulate the expression of the first three group of zygotically active genes, which in turn control the expression of homeotic genes
Genes with products provided by the female to the egg are called:
maternal-effect genes.
A model for the inheritance of DNA methylation
methylated-->DNA replication divides into two paths--> DNA methyltransferase document
Inbreeding in populations that are normally outbreeding leads to which of the following?
more individuals affected by rare diseases
Transcriptional activator proteins may be activated by an inducer
no galactose: inactive Gal4 and GAL1 off • Gal80 protein binds to Gal4 with high affinity and directly inhibits Gal4 activity • Gal80 binds to regions specifically within Gal4 activation domain blocking transcription • Gal80 is expressed continuously hence always blocking Gal structural genes yes galactose and +Gal3: active Gal4 and GAL1 on document
plant forms sex cells, or gametes (eggs and sperm), only ____ copy of the gene enters into these reproductive cells
one
Base Substitution
one base pair is replaced by another document
Haploinsufficient
one wild type dose is not enough to achieve normal level function e.g. DiGeorge syndrome in humans (cardiovascular and craniofacial abnormalities) (not necessarily haploinsufficient) In the heterozygote (+/M), the spoiler polypeptide bind to the wild type polypeptide and distorts it/interferes with its function
Proposed Biochemical Pathway
one-gene-one-enzyme hypothesis: genes were responsible for the function of enzymes, and each gene apparently controlled one specific enzyme one-gene-one-polypeptide Some protein producing genes produce transport proteins, structural or regulatory proteins, rather than enzymes Some genes produce RNAs rather than proteins Some proteins (e.g. β-globin) must join with other proteins to acquire a function
Operators are cis-acting
operator acts simply as a protein-binding site and makes no gene product document
Genome surveillance in animals and bacteria
piRNAs in animals In the germ lines of several animal species including Drosophila and mammals, active transposons are repressed through the action of piRNAs (Piwi-interacting RNAs). Like siRNAs, piRNAs are short single-stranded RNAs (26-30 nt in mammals) that interact with a protein complex Once associated, piRNAs guide piwi-Argonaute (a protein complex) to degrade complementary mRNAs Unlike siRNAs, piRNAs do not originate from the double-stranded RNA pathway Instead animal genomes contain several long (often >100 kb) loci called pi-clusters that serve as traps to catch active transposons. A pi-cluster is comprised of remnants of many different transposons that represent prior insertions of active transposons into that locus.
Modes of delivering recombinant DNA into bacterial cells
plasmid,fosmids,bacteriophage vectors
Self-incompatibility leads to disassortative mating in Brassica
pollen inhibition and pollen tube growth document
glucose present (cAMP low); no lactose; no lac mRNA document
presence of glucose prevents lactose metabolism--> a glucose breakdown product inhibits maintenance of the high cAMP levels----> No CAP-cAMP complex. Which in turn is required for the RNA polymerase to attach at the lac promoter site
Drosophilia
red-eyed: wild w+ white eyed: mutant w Three pairs of autosomes plus a pair of sex chromosomes (X and Y) Female XX and male XY Number of X chromosomes in relation to the autosomes determine the sex X-linked inheritance
Epigenome:
refers to the epigenetic state of a cell
In the presence of the repressor molecule and free tryptophan, the trp operon is:
repressed.
A nonautonomous transposable element:
requires additional element(s) for its mobility.
Dominant lethal alleles
result in death of both homozygotes and heterozygotes, while recessive lethal alleles cause death only when homozygous.
Retroviruses replicate using:
reverse transcriptase.
The class of genes that affect patterning within each segment is the:
segment polarity genes.
sickle-cell anemia
sickle-cell anemia Disease of the red blood cells A single amino acid deletion causes a change in the conformation hemoglobin (red blood cells are elongated and somewhat curved, taking on a sickle shape) This change in shape makes the sickle red blood cells less efficient at transporting oxygen through the bloodstream. Two main alleles: HbA and HbS -Three genotypes have different phenotypes, as follows: -In regard to the presence or absence of anemia: the HbA allele (wild-type) is dominant -In the heterozygote, a single HbA allele produces enough functioning hemoglobin to prevent anemia -Heterozygotes are less susceptible to malaria
GAL80 inhibits GAL gene expression by:
specifically binding to Gal4 and preventing activation.
Bases may take on rare tautomeric forms prone to mismatch
tautomeric instead of mismatched
telomere
to compensate for over hang Add copies of a simple noncoding sequence (telomeres) to the DNA at the chromosome tips e.g. in humans tandem repeats of TTAGGG
For autosomes in diploid organisms, the aneuploid 2n+1 is
trisomic
Consequences of mutations on gene products
wild type missense nonsense frameshift
Polymerase Chain Reaction (PCR)
• 20-50 cycles • The products of each cycle serve as the DNA template for the next • Every cycle doubles the amount of DNA synthesized in the previous cycle document
Producing PCR products with sticky ends
• 3' ends of primers anneal to the target sequence • 5' ends contain sequences encoding the restriction enzyme site
Eukaryotic Gene Regulation
• A central component of gene regulation in eukaryotes is Mediator, a 24- subunit complex, which serves as an intermediary between gene regulatory proteins and RNA polymerase. • Mediator provides an extended contact area for gene regulatory proteins compared to that provided by RNA polymerase alone, as in bacteria • The packaging of eukaryotic DNA into chromatin provides many opportunities for transcriptional regulation not available to bacteria
Spontaneous mutations in humans: trinucleotide repeat disease
• A common mechanism responsible for many genetic diseases is the expansion of a three-base-pair repeat: trinucleotide repeat diseases e.g. human disease called fragile X syndrome -The most common form of inherited mental impairment, occurring in close to 1 of 1500 males and 1 of 2500 females. -It is manifested cytologically by a fragile site in the X chromosome that results in breaks in vitro.
Polydactyl
• A fairly common syndrome in humans • In 5-17 of every 10,000 live births • The discovery of the role of Shh in digit patterning led geneticists to investigate whether the Shh gene was altered in polydactlylous humans and other species. • In fact, certain polydactyly mutations are mutations of the Shh gene. • The mutations are not in the coding region of the Shh gene, rather, they lie in a cis-acting regulatory element, far from the coding region, that controls Shh expression in the developing limb bud
Defining the entire toolkit
• A few dozen genes are required for proper organization of the anteroposterior body axis of the fly embryo The anteroposterior and dorsoventral axes The relationship between adult and embryonic body axes
• Transposable elements in maize can inactivate a gene in which they reside, cause chromosome breaks, and transpose to new locations within the genome
• Ac: autonomous elements: - can perform these functions unaided • Ds: nonautonomous elements - can transpose only with the help of an autonomous element elsewhere in the genome.
Bulky adducts
• Aflatoxin B1 is a powerful carcinogen that attaches to guanine • The formation of the addition product leads to the breakage of the bond between the base and the sugar • Generate an apurinic site consequences: potentially mutagenic and blocked DNA replication and transcription
Hox proteins have a sequence in common
• All 8 Drosophila Hox genes encode proteins containing a highly conserved 60 amino acid domain: the homeodomain • The hybridization is possible due to 180bp length sequence in each gene: homeobox • The homeobox encodes a protein domain: homeodomain (60 amino acids) • Composed of three α helices: Helix 2 and 3 form a Helix-turn-helix motif similarly to the Lac repressor and other DNA binding proteins. • This suggested that Hox proteins are sequence specific DNA-binding proteins
Transposable Elements
• Also known as mobile elements, jumping elements, jumping genes, junk DNA, selfish elements etc. • Can move to new positions within the same chromosome or even to a different chromosome • Found in almost all organisms • Are the largest component of human genome, accounting for almost 50% of our chromosomes •Despite their abundance, the normal genetic role of these elements is not known with certainty
drosophilia and dysgenically induced mutations
• An important observation is that a large percentage of the dysgenically induced mutations are unstable; they revert to wild type or to other mutant alleles at very high frequencies. • This instability is generally restricted to the germ line of an individual fly possessing an M cytotype • The unstable Drosophila mutants had similarities to the unstable maize mutants characterized by McClintock • Hypothesis: Dysgenic mutations are caused by the insertion of transposable elements into specific genes thereby rendering them inactive • P element was found to be present in from 30-50 copies per genome in P strains but to be completely absent in M strains Why do P elements not cause trouble in P strains?
Distinguishing lac+ and lac- by using a red dye
• Another type of useful mutant differs from wild type in the ability to use a specific energy source. Example: the wild type can use lactose, whereas a mutant may not be able to
R plasmids
• Bacterial dysentery is caused by bacteria of the genus Shigella • This bacterium initially was sensitive to a wide array of antibiotics that were used to control the disease • Shigella isolated from patients with dysentery (In Japanese hospitals, 1950s) proved to be simultaneously resistant to many of these drugs, including penicillin, tetracycline, sulfanilamide, streptomycin, and chloramphenicol •This resistance to multiple drugs was inherited as a single genetic package, and it could be transmitted in an infectious manner • The vectors carrying these multiple resistances proved to be another group of plasmids called R plasmids • R plasmids confer antibiotic resistance • Transferred rapidly on cell conjugation, much like the F plasmid in E. coli • Genetically engineered derivatives of R plasmids, (e.g. pBR322 and pUC) have become the preferred vectors for the molecular cloning of the DNA of all organisms
Transposable elements in grasses are responsible for differences in genome size
• Barley and maize- genome size is large: genes are separated by large clusters of retrotransposons • Rice and sorghum- genome size is small
4. Using polypeptide and DNA similarity
• Because organisms have common ancestors they also have many genes with similar sequences in common • BLAST (Basic Local Alignment Search Tool) search • This is done by submitting candidate sequences as "query sequences" to the public databases (contain record of all known gene sequences). • Sequence can be submitted as a nucleotide sequence or protein sequence • The computer scans the database and returns a list of full or partial "hits," starting with the closest matches.
How does the binding of the cAMP-CAP complex to the operon further the binding of RNA polymerase to the lac promoter ?
• Binding of CAP bends DNA. This bending of DNA may aid the binding of RNA polymerase to the promoter. • CAP also makes direct contact with RNA polymerase that is important for the CAP activation effect.
3. By phenocopying
• Can be applied to great many organisms regardless how well developed the genetic technology is for a given species • RNAi mechanism: use for inactivating specific genes - Prepare double stranded RNA with sequences homologous to part of the gene under study - Introduce into cell - RISC (RNA induced silencing complex) degrades mRNA complementary to the dsRNA - Reduce gene expression Phenocopying: experimentally interfere with the function of a specific gene without changing its own DNA sequence.
gene knockout
• Cloned gene (Exon 2) inactivated by the insertion of neomycin-resistance gene (neoR) • Inserted neoR gene give selectable marker • Vector carry a second marker: herpes tk gene document
There are some detectable differences between the inventories of mouse and human genes
• Color vision: humans possess one additional paralog of opsins • Presence of this opsin give humans trichromatic vision: perceive colors across the entire spectrum of visible light • Mice have 1400 genes involved in olfaction
• Two Hox proteins and four other transcription factors act together to repress Distal-less expression and hence appendage formation in the abdomen
• Combinatorial and cooperative regulation of gene transcription imposes greater specificity on spatial patterns of gene expression and allows for their greater diversity
• Each gap gene contains cis-acting regulatory elements with different arrangements of binding sites, and these binding sites may have different affinities for the Bicoid protein.
• Consequently each gap gene is expressed in a unique distinct domain in the embryo, in response to different levels of Bicoid and other transcription-factor gradients. • The concentration dependent response of genes to graded inputs is a crucial feature of gene regulation in the early Drosophila embryo • • The cis-acting regulatory elements governing distinct responses contain different numbers and arrangements of transcription factor binding sites.
Postreplication repair: Mismatch repair
• Corrects errors in replication that are not corrected by the proofreading function of the replicative DNA polymerase. • Repair is restricted to the newly synthesized strand, which is recognized by the repair machinery in prokaryotes because it lacks methylation marker Mismatch-repair systems: 1. Recognize mismatched base pairs 2. Determine which base in the mismatch is the incorrect one 3. Excise the incorrect base and carry out repair synthesis
Large genomes are largely transposable elements
• DNA content varied dramatically in eukaryotes and did not correlate with biological complexity - Genome of salamanders are 20x times as large as the human genome - Genome of barley is more than 10x times as large as the genome of rice • The lack of correlation between genome size and biological complexity of an organism is known as the C-value paradox What is responsible for the additional DNA in the larger genomes?-Repetitive DNA Genome size usually corresponds to the amount of transposable-element sequences rather than to gene content
Genetic Engineering in plants
• Derived from a natural plasmid from a soil bacterium called Agrobacterium tumefaciens • Causes crown gall disease/tumors T-DNA: the region of Ti plasmid that inserts into host plant Ti plasmid vector document
• The paired limbs, mouthparts, and antennae of Drosophila each develop from initially small populations of about 20 cells.
• Different structures develop from the different segments of the head and thorax, whereas the abdomen is limbless. • The first sign of the development of these structures is the activation of regulatory genes within small clusters of cells, which are called primordia.
Why does HP-1 bind to some DNA regions and not to others?
• Discovery of another Su(var) gene encoded a methyltransferase that adds methyl groups to specific amino acid residue (lysine 9) in the tail of histone H3 (histone H3 methyltransferase or HMTase) • Chromatin modified in this way binds HP-1 protein, which then associates to form heterochromatin. document Chromatin made up of nucleosomes that are methylated at H3meK9 and bound up with HP-1 protein contain epigenetic marks that are associated with heterochromatin.
Functional RNA
• Does not encode information to make protein • RNA itself is the final product • Never translated into polypeptides Transfer RNA (tRNA)-bring correct amino acid to the mRNA (translation) Ribosomal RNA (rRNA)-major components of ribosomes, which are large macromolecular machines that guide the assembly of the amino acid chain by the mRNAs and tRNAs. Small nuclear RNAs(snRNAs)- form spliceosomes that removes introns from eukaryotic mRNAs
Promoter sequences in E. coli
• E. coli promoters have two consensus sequences, TTGACAT and TATAAT (Pribnow box), positioned at -35 and -10 with respect to the transcription initiation site • Mutations in any region diminish transcription, often severely
Producing a mouse containing the targeted gene knockout
• ES cells with the knockout gene inject into blastocyst stage embryo • Coat color of the future newborn is a guide to whether the ES cells have survived in the embryo. document
Steps in translation elongation
• Each amino acid is added to the growing polypeptide chain while the deacylated tRNA is recycled by the addition of another amino acid • Elongation factors: EF-Tu EF-G document
Termination of translation
• Elongation continues until the codon in the A site is one of the 3 stop codons: UGA, UAA or UAG • No tRNAs recognize these codons • Instead proteins called release factors (RF1, RF2 and RF3 in bacteria) recognize stop codons. • Stop codons are recognized by tripeptides in the RF proteins, not by an anticodon. • RFs fit onto the A site of the 30S subunit but do not participate in peptide-bond formation. • Instead, a water molecule gets into the peptidyltransferase center ---> Release polypeptide from the tRNA in the P site document
general transcription factors
• Eukaryotic promoters are first recognized by general transcription factors (GTF) e.g. TFIID, TFIIF • The function of GTFs is to attract the core RNA pol II so that it is positioned to begin RNA synthesis at the transcription start site
Homologous recombination repairs DSBs in dividing cells
• Exonucleases create 3' overhangs • 3' overhangs invade and displace a strand on the homologous chromosome, creating a displacement loops • Invading strand is extended document
Hox proteins repress appendage formation in the abdomen
• Expression of the Distal-less (DII) gene marks the positions of the future appendages • Expression of the Hox gene Ultrabithorax marks the position of the abdominal segments A1-A7 • Expression of the engrailed gene marks the posterior of each segment
1. Bacterial insertion sequences (IS)
• First found in E.coli in the gal operon • Encode a protein called a transposase (enzyme required for the movement of IS elements from one site in the chromosome to another) • All IS elements begin and end with a short inverted repeat sequences that are required for their mobility
Mating-type switching and gene silencing
• Haploid cells are able to switch their mating type • Haploid yeast switch mating type by replacing the information present at the MAT locus. e.g. an a cell will switch to an α cell by replacing the MATa allele with the MATα allele. document
Modifications of histone tails
• Histone proteins are organized into the core octamer with their amino-terminal ends making electrostatic contact with the phosphate backbone of the surrounding DNA. • Specific basic amino acid residues (lysine and arginine), as well as the uncharged serine and threonine, in the histone tails can be covalently modified by the attachment of acetyl and methyl groups. • These reactions takes place after the histone protein has been translated and even after the histone has been incorporated into nucleosome. •There are 44 histone lysine residues available to accept acetyl groups, so the presence or absence of these groups carry tremendous amount of information. • Information is stored in the patterns of histone modification rather than in the sequence of nucleotides. • With more than 150 known histone modifications, there are a huge number of possible patterns.
Acetylation of histone amino acids influences chromatin structure and gene expression
• Histones associated with the nucleosomes of: active genes: rich in acetyl groups (hyperacetylated) inactive genes: underacetylated (hypoacetylated) • Enzyme responsible for adding acetyl groups : Histone acetyltransferase (HAT) • Was an ortholog of a yeast transcriptional activator called GCN5 (encoded by the same gene in a different organism). • Various protein complexes that are recruited by transcriptional activators posses a HAT activity.
Organization and Expression of Hox genes
• Hox genes regulate the identity of body parts • Hox genes are clustered together in two gene complexes on the third chromosome of Drosophila antennapedia complex bithorax • The order of the genes in the complexes and on the chromosomes corresponds to the order of body regions from head to tail, that are influenced by each Hox gene
Homeotic genes (Hox genes)
• Hox mutants do not affect segment number • But alter the appearance of one or more segments • The structure formed in the mutant is a well-developed likeness of another body part •There are eight loci: Hox genes • Hox genes affect the identity of segments and their associated appendages in Drosophila • Generally the complete loss of any Hox-gene function is lethal in early development • The mutations that transform adults are viable in heterozygotes because wild-type allele provides normal gene function to the developing animal
Comparative genomics of Human Medicines
• Humans also differ in the number of copies of parts of individual genes, entire genes, or sets of genes: Copy Number Variation (CNVs) • CNVs include repeats and duplications that increase copy number and deletions that reduce copy number • Between any two unrelated individuals, there may be 1000 or more segments of DNA greater than 500 bp in length that differ in copy number. Some CNVs can be quite large and span over 1 million base pairs. • How copy numbers may play a role in human evolution and disease is of intense interest • People with high-starch diets have, on average, more copies of a salivary amylase gene than people with traditionally low-starch diets. • In other cases, copy number variations have been associated with syndromes such as autism.
Gal4 functions in most eukaryotes
• In addition to its action in yeast cells, Gal4 has been shown to be able to activate transcription in insect cells, human cells and many other eukaryotic species • This versatility suggest that biochemical machinery and mechanisms of gene activation are common to a broad array of eukaryotes • The ability of Gal4, as well as other eukaryotic regulators, to function in a variety of eukaryotes generally have the transcriptional machinery and mechanisms in common.
Polymerase switching in eukaryotes requires addition of a ubiquitin monomer
• In eukaryotes several bypass polymerases appear to be always present • Their access to DNA is regulated so that they are used only when needed • The addition of a single ubiquitin (Ub) monomer to the sliding clamp (PCNA) allows the bypass polymerase to bind to PCNA and begin replicating
Short term activation of genes in a chromatin environment
• In eukaryotes transcription levels are made finely adjustable in a chromatin environment by clustering binding sites into enhancers • Several different transcription factors or several molecules of the same transcription factor may bind to adjacent sites • The binding of these factors to sites that are the correct distance apart leads to an amplified, or superadditive, effect on activating transcription. • When the effect is greater than additive : Synergistic
Cancer as a developmental disease
• In long-lived animals development does not cease at birth or at the end of adolescence • Tissues and various cell types are constantly being replenished • Tissue and organ maintenance is generally controlled by signaling pathways • Inherited or spontaneous mutations in genes encoding components of these pathways: - can disrupt tissue organization and contribute to the loss of control of cell proliferation - Because unchecked cell proliferation is a characteristic of cancer, the formation of cancer may be a consequence.
Insertion into a new location
• In one of the first steps of insertion, the transposase makes a staggered cut in the target site DNA • Transposable element then inserts between staggered ends • Host DNA repair machinery fills in the gap opposite each strand overhang
• The spatial expression of toolkit genes is usually closely correlated with the regions of the animal affected by gene mutations
• In the absence of function of all Hox genes, segments form, but they all have the same identity: e.g. Limbs can form but they will have antenna identity Similarly wings can form, but they all have forewing identity etc.
2. Direct evidence from cDNA sequences (through the analysis of mRNA)
• In the cDNA, the ORF should be continuous from initiation codon through stop codon. • Thus, cDNA sequences can greatly assist in identifying the correct reading frame, including the initiation and stop codons. • EST (Expressed Sequence Tags): can be used to determine 5' and 3' ends of transcripts
Epigenetic Inheritance
• Inheritance of chromatin states from one cell generation to the next • In DNA replication, both the DNA sequence and the chromatin structure are faithfully passed on to the next cell generation Chromatin structure can be inherited
Translation initiation in prokaryotes
• Initiation codons are preceded by special sequences called Shine-Dalgarno sequences that pair with the 3' end of an rRNA (16S rRNA), in the 30S ribosomal subunit • rRNA performs the key function in ensuring that the ribosome is at the right place to start translation
The trp mRNA leader sequence
• Is at the 5' end of the trp operon mRNA before the first codon of the trp E gene • Is ~ 160 bases long • Part of this sequence works as an attenuator that governs the further transcription of trp mRNA The trp mRNA leader sequence contains an attenuator region and two tryptophan codons document
2. Targeted mutagenesis
• Is the most precise means of obtaining mutations in a specific gene Mutant version of gene A
extra sequence contains complex instructions that direct the intricate regulation of gene transcription
• It is estimated that the human genome encodes approximately 25,000 genes (about the same number as that for corn and nearly twice as many as that for the common fruit fly). • Even more interesting is the fact that those 25,000 genes are encoded in about 1.5% of the genome. • What exactly does the other 98.5% of our DNA do?
How did they deduce the mechanisms of gene regulation in the lac system?
• Jacob and Monod found that several different classes of mutations can alter the expression of the structural genes of the lac operon • They were interested in the interactions between the new alleles, such as an assessment of dominance. • They produced bacteria that are partially diploid and heterozygous for the desired lac mutations by using F' factors
Error-prone nonhomologous end joining (NHEJ) repairs double strand breaks in non-dividing cells
• Ku70-Ku80 heterodimer recognizes DSB • Recruits DNA-PKcs (kinase) and Artemis (nuclease) • Artemis is phosphorylated and trims overhangs • DNA pol fills in any small gaps • XRCC4, XLF, ligase ligate document
Dosage Compensation
• Makes the amount of most gene products from the two copies of the X-chromosome in females equivalent to the single dose of the X-chromosome in males • In mammals, dosages are made equivalent by randomly inactivating one of the two X-chromosomes in each cell at an early stage in development. •The inactivated chromosome, is called a Barr body, can be seen in the nucleus as a darkly staining, highly condensed heterochromatin structure.
Molecular Identification
• Molecular Archaeology • Molecular Epidemiology • Molecular Ecology • DNA fingerprinting • Classification of organisms • Genotyping • Pre-natal diagnosis • Mutation screening • Drug discovery • Genetic matching • Detection of pathogens
Choice of cloning vectors
• Must be small molecules for convenient manipulation • Some need to be able to replicate in living cells and others designed to be present in only a single copy to maintain the integrity of the inserted DNA • Must have convenient restriction sites • Must have a way to identify and recover the recombinant molecule
2. Transposons
• Not only carry the genes they need for their movement but also carry other genes • Transposons were originally detected as mobile genetic elements that confer drug resistance (R plasmids) • Many of these elements consist of recognizable IS elements flanking a gene that encodes drug resistance
Silencing an entire chromosome: X-chromosome inactivation
• Number of transcripts produced by a gene is proportional to the number of copies of that gene in a cell • This is true for autosomal genes • All individuals would not produce the same number of transcripts of genes located on the sex chromosomes if both X chromosomes were expressed in females
Double-stranded break repair (DSBR)
• Other 3' overhang invades, creating 4-branched double-crossover intermediate called a Holliday junction • Resolvases (endonucleases) cleave Holliday junctions to create noncrossover and crossover segments • DNA pol and ligase fills in gaps and ligate document
p elements
• P elements were discovered as a result of studying hybrid dysgenesis—a phenomenon that occurs when females from laboratory strains of Drosophila melanogaster are mated with males derived from natural populations. • In such crosses, the laboratory stocks are said to possess an M cytotype (cell type), and the natural stocks are said to possess a P cytotype.
Translation initiation
• Place the first aminoacyl-tRNA in the P-site of the ribosome and establish the correct reading frame of the mRNA • In most prokaryotes and all eukaryotes, the first amino acid in any newly synthesized polypeptide is methionine (codon AUG) • It is inserted by a special tRNA called initiator : tRNAMeti • In bacteria, a formyl group is added to the methionine while the amino acid is attached to the initiator, forming N-formylmethionine.
Deducing the protein-encoding genes from genomic sequence
• Protein present in a cell largely determine its morphology and physiological properties • One of the goals in genomic analysis and annotation is to determine an inventory of all of the polypeptides encoded by an organism's genome: PROTEOME
Restriction nucleases cut large DNA molecules into fragments
• Purified from bacteria • Cut the double helix at specific sites • Sequences are palindromic • Different species of bacteria make different restriction nucleases, which protect them from viruses by degrading incoming viral DNA document
Post-transcriptional Regulation
• RNA Splicing • RNA binding proteins and RNA recognition sequences • miRNA control
2. Nucleotide-excision repair (NER)
• Recognizes and corrects DNA lesions due largely to UV damage • Relieves stalled replication forks and transcription complexes • Interestingly, two autosomal recessive diseases in humans, xeroderma pigmentosum (XP) and Cockayne syndrome, are caused by defects in nucleotide-excision repair.
Ionizing radiation
• Results in the formation of ionized and excited molecules that can damage DNA •E.g. Reactive oxygen species: superoxide radicals, hydrogen peroxide (H2O2), and hydroxyl radicals (·OH) -Radiation may cause breakage of the N-glycosydic bond leading to formation of apurinic or apyrimidinic sites and cause strand breaks - Strand breaks are responsible for most of the lethal effects of ionizing radiation document x2
Genetic engineering in Yeast
• S. cerevisiae is the most sophisticated easily manipulated eukaryotic genetic model • Simplest yeast vectors are yeast integrative plasmids (Yips): derivatives of bacterial plasmids into which the yeast DNA of interest has been inserted • When transformed into yeast cells, these plasmids insert into yeast chromosomes by homologous recombination
A typical signal-transduction pathway
• Signaling begins when a ligand binds to a membrane-bound receptor • Leading to release or activation of intracellular proteins • Receptor activation often leads to the modification of inactive transcription factor (TF) • The modified TFs are translocated to the cell nucleus • Where they bind to cis-regulatory DNA sequences or to DNA binding proteins to regulate the level of target gene transcription
Clusters of Hox genes control development on most animals
• Similarities were found in the homeobox sequences from different species • Over 60 amino acids of the homeodomain, some mouse and frog Hox proteins were identical with the fly sequences at as many as 59 of the 60 positions • The extent of sequences similarity indicates very strong pressure to maintain the sequence of the homeodomain
Genetic Engineering
• Site-directed mutagenesis • Gene expression studies
Plasmid vectors
• Small, circular DNA molecules • Replicate their DNA independent of the bacterial chromosome (ori) • Restriction site (e.g. polylinker) • Carry a gene for drug resistance (e.g. ampR): select for bacterial cells transformed by plasmids • Carry a gene to distinguish plasmids with and without DNA inserts (e.g. lacZ gene): -Origin of replication (ori) -Polylinker (insert the DNA) -Drug resistance gene (ampR) lacZ gene
Bacterial Transformation
• Some bacteria can take up fragments of DNA from external medium (another way in which bacteria can exchange their genes). • The source of the DNA can be other cells of the same species or cells of other species • The DNA taken up integrates into the recipient chromosome • If this DNA is of different genotype from that of the recipient, the genotype of the recipient can become permanently changed: Transformation
Some genes enhance or suppress the spread of heterochromatin
• Suppressors of variegation called Su(var) are genes when mutated, reduce the spread of heterochromatin, meaning that the wild-type products of these genes are required for spreading. • e.g. HP-1 gene • Heterochromatin-1 (HP-1) protein: associated with heterochromatic telomere and centromeres. • Is required to generate higher order chromatin structure associated with heterochromatin drosophilia eye (chromosome inversion containing white+) second site mutations that affect the spreading of heterochromatin su(var) E(var)
Ubiquitination
• Targets the protein for degradation by a biological machine and protease called the 26S proteasome •The modification targeting a protein for degradation: addition of chains of multiple copies ubiquitin (ubiquitination). •Ubiquitin contains 76 amino acids and is found only in eukaryotes, where it is highly conserved in plants and animals document
• One major challenge facing a genome project is sequence assembly
• That is combining the individual reads into a consensus sequence: an authentic representation of the sequence for each of the DNA molecules in that genome
Maternal gradients and gene activation
• The Bicoid protein is a homeodomain type transcription factor that is translated from mRNA deposited in the egg and localized at the anterior pole. • Early Drosophila embryo is a syncytium (lacks any cell membranes) • Bicoid can diffuse through the cytoplasm and establishes a protein concentration gradient: -highly concentrated at the anterior -concentration gradually decreases as distance from that end increases (very little Bicoid protein beyond the middle of the embryo) Concentration gradient provide positional information about the location along the anteroposterior axis Ensure that a gene is activated in only one location along the axis (link gene expression to the concentration level)
A simplified lac operon model
• The P, O, Z, Y and A segments together constitute an operon -Repressor protein controls the lac operon -When the inducer binds the repressor, the operon is fully expressed
What is the connection between ability to utilize sugar and the ability to switch mating type ?
• The Snf2-swi2 protein is a part of a large, multi subunit complex called SWI/SNF complex • Activates transcription by moving nucleosomes that are covering TATA sequences • In this way complex facilitates the binding of RNA polymerase II • The SWI-SNF complex is a co-activator document
histone code
• The covalent modification of histone tails is said to be a histone code.
Development and Disease
• The discovery of the fly, vertebrate and human toolkit for development has also had profound effect on the study of the genetic basis of human diseases, particularly of birth defects and cancer • A large number of toolkit-gene mutations have been identified that affect human development and health.
Comparative genomics of humans
• The human species, Homo sapiens originated in Africa approximately 200,000 years ago • Around 60,000 years ago, populations left Africa and migrated across the world, eventually populating five additional continents •Any two unrelated humans' genomes are 99.9% identical • The difference of just 0.1% still corresponds to ~ 3 million bases • The challenge today is to decipher which of those base differences are meaningful with respect to physiology, development, or disease
The b-interferon enhanceosome
• The human ß-interferon gene encodes the antiviral protein interferon • Activated to very high levels of transcription on viral infection • The key to the activation of this gene is the assembly of transcription factors into an enhanceosome about 100bp upstream of the TATA box and transcription start site
The Bicoid protein activates zygotic expression of the hunchback gene
• The hunchback gene is a gap gene activated in the zygote in the anterior half of the embryo • This activation is through direct binding of the Bicoid protein to three sites 5' of the promoter of the hunchback gene.
lac operon is a cluster of genes
• The lac operon is a cluster of structural genes that specify enzymes taking part in lactose metabolism • These genes are controlled by the coordinated actions of cis-acting promoter and operator regions • The activity of these regions is, in turn, determined by repressor and activator molecules specified by separate regulator genes
Abundant tryptophan attenuates transcription of the trp operon
• The leader region can form two different conformations, depending on the presence or absence of tryptophan • In the presence of tryptophan, the hairpin structures formed act as a transcriptional terminator • In the absence of tryptophan, a different hairpin forms and acts as an antiterminator, and transcription proceeds. • The leader region contains two tryptophan codons • The antiterminator hairpin structure forms in the absence of tryptophan because the ribosome stalls at these codons (because there is not adequate charged tRNAtrp) • In the presence of tryptophan, the ribosome proceeds through this sequence and the terminator hairpin can form
Bacteria
• The most numerous organisms on our planet earth • Contribute to recycling of nutrients such as nitrogen, sulfur, and carbon in ecosystems • Some are agents of human, animal and plant disease • Others live symbiotically inside our mouths and intestines • Many are useful for industrial synthesis of a wide range of organic products
Comparative genomics of mice and humans
• The mouse and human lineages diverged ~ 75 million years ago • 99% of all mouse genes have some homologs in the human genome and at least 99% of all human genes have some homologs in the mouse genome • The mouse and human genomes contain similar sets of genes, often arranged in similar order
Bicoid and expression of genes
• The order of gene action further suggests that the expression of one set of genes might govern the expression of the succeeding set of genes e.g. In embryos from Bicoid mutants, the expression of several gap genes is altered, as well as that of pair-rule and segment polarity genes • This finding suggest that the Bicoid protein somehow (directly or indirectly) influences the regulation of gap genes. • Inspection of the Bicoid protein sequence reveals that it contains a homeodomain, related to but distinct from those of Hox proteins • Thus, Bicoid has properties of a DNA-binding transcription factor. • Each gap gene also encodes a transcription factor, as does pair rule, several polarity genes and as described earlier all Hox genes.
3. Studying the protein-DNA interactome using chromatin immunoprecipitation assay (ChIP)
• The sequence-specific binding of proteins to DNA is critical for correct gene expression • Using this technique can isolate DNA and its associated proteins in a specific region of chromatin (both can be analyzed together)
attenuator and attenuation
• The trp structural genes are preceded by a leader sequence containing a regulatory site called an attenuator • Transcription of the leader region of the trp operon can occur even when the operon is repressed in the presence of tryptophan (attenuation) • In the absence of tryptophan, transcription is not terminated in the leader region and proceeds through the entire operon
Model organism is Zea mays (maize)
• These plants produce variably colored kernels • Because each kernel is an embryo produced from an individual fertilization, hundreds of offspring can be scored on a single ear, making maize an ideal organism for genetic analysis. • Maize proved to be the perfect organism for the study of transposable elements (TEs)
Translesion or bypass polymerases differ from the main replicative polymerases in several ways
• They can tolerate unusually large adducts on the bases because their active site is larger than replicative polymerases • Have much higher error rate because they lack (or have poor) proofreading ability • Can only add few nucleotides before falling off (i.e. low processivity)
1. DNA Methylation
• This mark is not a histone modification; rather, it is the addition of methyl groups to DNA residues after replication. • An enzyme usually attaches these methyl groups to the carbon-5 position of a specific cytosine residue. picture: instead of sam, its DNA methyltransferase • In mammals methyl groups are usually added to the C in a CG dinucleotide • Pattern is symmetric: present in both strands in the same context C*G G C* • Most unmethylated are found in clusters near gene promoters: CpG islands
• Many toolkit genes encode transcription factors that control expression of other genes
• Thus the products of these remarkable genes function through principles that are already familiar-by binding to regulatory elements of other genes to activate or repress their expression
Spatial regulation of gene expression in development
• Toolkit genes are expressed in reference to spatial coordinates in the embryo • Spatial control of gene expression during development is largely governed by the interaction of transcription factors with cis-acting regulatory elements
Many roles of individual toolkit genes
• Toolkit proteins and regulatory RNAs have multiple roles in development e.g. Ultrabithorax protein: repress limb formation in the fly abdomen Promotes hind-wing development in the fly thorax • Most toolkit genes function at more than one time and place • Most may influence the formation or patterning of many different structures that are formed in different parts of the larval or adult body • Those that regulate gene expression may directly regulate hundreds of different genes
How does a cell determine which of its thousands of genes to transcribe ?
• Transcription of each gene is controlled by a regulatory region of DNA relatively near the site where transcription begins • Some regulatory regions are simple and act as switches thrown by a single signal and many others are complex • Two types of fundamental components: (1) Short stretches of DNA of defined sequence (2) Gene regulatory proteins that recognize and bind to this DNA.
Organizers
• Transplanted regions are called organizers: able to organize the development of surrounding tissues • Organizers are postulated to produce morphagens: molecules that induced various responses in surrounding tissue in a concentration-dependent manner
How do plants and animals survive and thrive with so many insertions in genes and so much mobile DNA in the genome?
• Transposable elements can insert into both introns and exons (subject to negative selection) • Only the insertion into introns will remain in the population (less likely to cause deleterious mutation) • Vast majority can no longer move (inactive) • A few elements remain active, and their movement into genes can cause disease e.g; Three separate insertions of LINEs have disrupted the factor VIII gene, causing hemophilia A. At least 11 Alu insertions into human genes have been shown to cause several diseases, including hemophilia B (in the factor IX gene), neurofibromatosis (in the NF1 gene), and breast cancer (in the BRCA2 gene).
5. Predictions based on codon bias
• Triplet code for amino acids is degenerate • Multiple codons for a single amino acid are termed synonymous codons. • In a given species not all synonymous codons for an amino acid are used with equal frequency e.g. Fruit fly; two codons for cysteine UGC 73% UGU 27%
dna history
• Until the early 1970s, DNA was the most difficult biological molecule to analyze • Today DNA has become the easiest macromolecule to study • It is now possible to: -to isolate a specific region of almost any genome -to produce virtually unlimited number of copies of it -to determine the sequence • In addition isolated genes can be altered (engineered) at will and transferred back into the germ line of an animal or plant
1. Random mutagenesis
• Use general mutagens: -chemical agents -radiation -transposable elements Focus on the gene in question 1. Focus on the map location of the gene. 2. Identify the gene of interest in the mutagenized genome and check for the presence of mutations
1. ORF (Open Reading Frame) detection
• Use the computational analysis of the genome sequence to predict mRNA and polypeptide sequences • Look for sequences that have characteristics of genes (gene size, start and stop codons etc) • Sequences with these characteristics typical of genes are called open reading frames (ORFs). • To find candidate ORFs, the computer scans the DNA sequence of both strands in each reading frame (Because there are 6 possible reading frames in all).
Cotranscriptional processing of RNA
• When nascent RNA first emerges from RNA pol II, a cap is added to the 5' end by several proteins that interact with the CTD • Cap consists of 7-methyguanosine residue linked to the transcript by 3 phosphate groups • Functions: 1. Protects RNA from degradation ; 2. Required for translation • Spilcing: The removal of introns and the joining of exons • RNA elongation continues until the conserved sequence AAUAAA or AUUAAA is reached, marking the 3′ end of the transcript • An enzyme recognize that sequence and cuts off the end of the RNA ~ 20 bases farther down. • To this cut end a stretch of 150-200 adenine nucleotides called a poly (A) tail is added.
En mutation
• When the engrailed-binding site is mutated, reporter expression is derepressed in the posterior compartments of each abdominal segment
SIp mutation
• When the sloppy-paired binding site is mutated, reporter expression is derepressed in the anterior compartments of each abdominal segment
The lac structural genes
• Z, Y and A are structural genes/segments encoding proteins • All three genes are transcribed into a single mRNA • Regulation of the production of this mRNA coordinates the synthesis of all three enzymes b-galactosidase and permease are required for lactose metabolism
Genomic DNA may not be a good starting point if the goal is to isolate and analyze genes that encode proteins (why?)
• mRNA is often a preferable starting point in the isolation of a gene. • Enzymatic conversion of mRNA into cDNA allows for the isolation of a gene copy without introns.
Physical mutagens induce mutations
•A large number of mutagens damage one or more bases in DNA - UV light - Ionizing radiation • Result in a replication block document
Error prone repair: Translesion DNA Synthesis
•A last resort mechanism, which spares the cell the worse fate of an incompletely replicated chromosome • Catalyzed by a specialized class of polymerases called bypass polymerases • Bypass polymerases are recruited to replication forks that have stalled because of damage in the template strand (a stalled replication fork can initiate a cell death pathway) • Bypass polymerases may introduce errors in the course of synthesis that may persist and lead to mutation or that can be corrected by other mechanisms such as mismatch repair In both prokaryotes and eukaryotes, replication blocks can be bypassed by the insertion of nonspecific bases. In E. coli, this process requires the activation of the SOS system. DNA synthesis becomes error-prone, inserting random and incorrect nucleotides in places that would normally stall DNA replication SOS repair can itself become mutagenic but allows cells to survive with DNA damage that would otherwise kill itself
Sequencing
•Bioinformatics • Genomic cloning • Human Genome Project
Synthesis-dependent strand annealing (SDSA)
•DNA helicases displace the extended invading strand •Annealing of the original broken chromosome pieces •DNA is extended and ligated document
Alternative splicing produces related but distinct protein isoforms
•FGFR2, a human gene that encodes the receptor that binds fibroblast growth factors and then transduces a signal inside the cell
•Gal3, Gal80 and Gal4
•Gal3, Gal80 and Gal4 are all part of a switch whose state is determined by the presence or absence of galactose • In this switch DNA binding by the transcriptional regulator is not the physiologically regulated step (as is the case in the lac operon), rather the activation of the activation domain is regulated The activity of eukaryotic transcriptional regulatory proteins is often controlled by interactions with other proteins
nucleosome packages
•Nucleosome packages are further wrapped around each other to condense the DNA. • Nucleosomes are spaced at intervals of about 200 nucleotide pairs, and they are usually packed together (with the aid of histone H1 molecules) to form a 30-nm chromatin fiber. document
genetic engineering
•Prior to genetic engineering, modifying the genetic makeup of plants and animals to enhance food production was restricted to selective breeding The selection and breeding of naturally occurring or mutagen-induced variants •Transgenic Atlantic salmon (bottom) overexpressing a growth hormone (GH) gene display rapidly accelerated rates of growth compared to wild strains and nontransgenic domestic strains (top). • GH salmon weigh an average of nearly 10 times more than nontransgenic strains. GloFish, marketed as the world's first GM-pet, are a controversial product of genetic engineering
Transcriptional activator proteins bind to UAS elements in yeast
•The Gal4 protein activates target genes through upstream-activating-sequence (UAS) elements. • Gal4 protein has 3 functional domains: DNA binding, dimerization, and activation
Disrupting gene function with the use of RNA interference
•The dsRNA will stimulate RNAi, degrading sequences that match those in the dsRNA document
presence of vitellogenins in the platypus but absence in other mammals
•The presence of vitellogenins in the platypus and its absence from other mammals could be explained in two ways 1) vitellogenin is a novel invention of the platypus, or 2) vitellogenin existed in a common ancestor of monotremes, marsupials, and eutherians but was subsequently lost from marsupials and eutherians. A simple pair-wise comparison between the platypus and another mammal does not distinguish between these alternatives. Compare to an outgroup Is vitellogenin likely to be present in the last common ancestor of the platypus, marsupials, and eutherians? Evolutionary relationships among three major groups of mammals
The tryptophan (trp) operon
•The trp operon contains five genes (trpE, trpD, trpC, trpB and trpA) that encode enzymes that contribute to the synthesis of the amino acid tryptophan
• The expression of the Distal-less (Dll) gene marks the start of the development of the appendages.
•This gene is one of the key targets of the Hox genes, and its function is required for the subsequent development of the distal parts of each of these appendages. • The small clusters of cells expressing Distal-less arise in several head segments and in each of the three thoracic segments but not in the abdomen.
MATa and MATα alleles:
•This replacement of one allele of MAT for the other is possible because yeast cells carry an additional silenced copy of both the MATa and MATα alleles: • HMRa and HMLα loci are on the same chromosome as the MAT locus, contain "cassettes" of the MATa and MATα alleles, respectively that are not expressed. • HMR and HML loci are thus "silent" cassettes. • HMRa and HMLα cannot be transcribed, and as such they are examples of transcriptional gene silencing. MATa=active HML (hidden MAT left) locus carries a silenced copy of the MATα HMR (Hidden MAT right) locus carries a silenced copy of the MATa allele
• Genetic variation among individuals provides the raw material for evolution.
•Two major processes are responsible for genetic variation (within a chromosome): - Mutation - Recombination
Ribozymes
❖ The first cells likely contained and used only RNA, not DNA or proteins. ❖ This became truly conceivable when ribozymes were discovered in 1980s.