BIOL 3010 exam 1

What are some of the major technological advances that have facilitated genetic and genomic analyses since ~1970?

DNA sequencing (Sanger Sequencing:

1) hybridization: recombinant DNA plasmid denatured into 2 single strands. The template strand is mixed with oligonucleotide primer complimentary to some of the vector bases -> template and primer anneal together

2) template-primer hybrid mixed with DNA pol, dNTPs, and ddNTPs with tags. DNA pol synthesizes a new strand- synthesis terminates when DDNTP added.

3) electrophoresis is run. Result: nested assay! Then detector reads color of tag on each fragment in assay

**ddTPs terminate synthesis because they are missing the 3' OH needed for chain elongation.

Human Genome Sequenced (1986-2000):

-shotgun strategy: determine DNA sequences, each about 1000 bases long, from both ends of random human genomic DNA inserts of BAC clones from a library.

-need to use paired end sequencing because of repeated sequences

-hierarchal strategy - separate genome into chunks by cloning fragments in BAC vectors, then determined the order of inserts and determined minimum tiling path.

-hierarchal and shotgun were used in parallel at NIH and private sites, respectively

Explain how PCR works.

-invented by Kary mullis and others

-denaturation (90 C): at high temp, DNA strands unwind

-anneal (50 C): at lower temp, added primers anneal to denatured strands

-extension (70 C): temp raised a little, so that DNA pol can extend

What is a gene? What is an allele?

gene: segments of DNA found on chromosomes

allele: different forms of a gene

Darwin described the process of natural selection. What is needed for this process to occur?

-variation among individuals in a population for some trait

-fitness differences - a consistent relationship between value of a trait and reproductive success (via fecundity or survivorship)

-inheritance - a consistent relationship for value of trait between parents and offspring

What are Mendel's "Laws" of Segregation and Independent Assortment and what analyses led him to these laws?

Law of Segregation: that two alleles from each parent separate during gamete formation, and then unite at random - one from each parent - at fertilization

Law of independent assortment - that different pairs of alleles segregate independently from one another during gamete formation

Describe advances in sequencing capability and how they parallel or exceed those observed for computing.

computing has led to rapid advances in efficiency of sequencing. For example, detectors are now able to be computerized to quickly perceive differences in fluorescence.

Describe the basic structure of nucleotides and DNA or RNA.

-Both have a sugar, phosphate, and base

-Bases can be ATGC (DNA) Or AUGC (RNA)

-RNA is usually single stranded

-DNA is missing a 2' OH (has H instead)

-nucleoside: sugar/base

-nucleotide: sugar, phosphate, and base

What observations suggested that particular bases pair in DNA?

-chargaff's observations that A and T are in the same ratio and G and C are in the same ration

What are examples of chemicals that interact with specific features of the DNA helix?

-transcription factors interact mainly with major groove

-DAPI binds to minor groove

-hogweed binds to major and minor groove -> when exposed to light, binds to DNA -> psoralen

Define "life," at least according to NASA.

a self-sustaining chemical system capable of Darwinian selection

What are the essential features of a genetic material and what makes RNA a particularly good candidate for being the first one?

-stores info

-expresses info

-replicate

-accomodate the introduction of new variation

RNA is good because....

1) encodes information

2) complex folding

3) some highly conserved across all life (deep evolutionary origins)

4) can act as an enzyme

5) self-replicating?

--> RNA pol ribozymeshave been evolved in lab, but RNA secondary structure blocks activity. Selection for use of RNA triplets alleviates this inhibition by secondary structure, though!

RNA World hypothesis:

1) RNA molecules with catalytic activities assemble themselves from primordial nucleotide "soup"

2) RNA molecules evolve and diversity by self-replication with mutation and recombination providing raw material for selection

3) RNA molecules begin to synthesize proteins which begin to improve on ribozyme-only functions

4) DNA appears with more stable information storage because two strands allow error correction

Are there any obstacles to the RNA World hypothesis, and, if so, how might they be accommodated?

-each nucleotide has 3 chemical moieties: complex chemistry

-contemporary nucleotides will not couple without chemical activation

-phosphates very limited (sequestered in minerals, not free) -> maybe aspartate instead?

-adenine possibly abundant, others not -> maybe hypoxanthene and other bases instead

-non-biotic synthesis of ribose not likely and ribose unstable anyway - threose/ glutamine/ aspartate instead?

-solvent problems: nucleotides do not self-assemble by Watson-Crick

hydrogen bonds in water; polymeric RNA unstable in water - maybe water evaporated into formamide

-different cation availability (maybe Mg++ instead)

-natural RNA polymerase ribozymes not known

What observations and experimental evidence suggest that self-replicating ribozymes might have existed in nature?

-ribonuclease P exists and is a ribosyme

-self-splicing introns exist

-in lab, got an RNA to have RNA pol activity

What early evidence showed that genetic material could be transferred between organisms, and that such material is DNA?

Griffith (1920s):

1) injected mice with live S form of bacteria -> dead

2) injected with mutated r form -> survived

3) injected with cell components of S -> survived

4) Injected with cell components of S and live R -> dead.

5) tissue analyzed after step 4 = live S recovered

--> showed phenomenon of transformation

Avery, McLeod, McCarty (1940s): DNA is the Genetic Material:

1) mixed R form with heat-killed S components

3) added protease / RNAse/ DNase/ Ultracentrifugation

4) found that only addition of DNase prevented recovery of transformed S cells

Explain the experimental results that led to the "one gene, one enzyme" hypothesis. What modern genetic and genomic data suggest the original hypothesis was framed too narrowly?

Beadle, Tatum (1941)

1) screened for yeasts unable to survive on minimal growth medium without nutrient supplementation

2) x ray muteginzed to create mutants

3) retested mutants for rescue with minimal medium and supplements

4) found mutants that needed arginine or its precursors

5) these mutants could be rescued by supplementing with arginine or precursor

6) different responses of different mutants implies that the corresponding genes encode proteins with distinct enzymatic activities and function in a linear pathway

What are the fundamental properties of the genetic code and how were they "deciphered"?

Crick/Brenner 1950s:

-frameshift mutations indicate a triplet code

-20 common amino acids but 1 or 2 nucleotide units insufficient to specify diversity of amino acids -> hypothesized that there was a triplet code

-added specific mutations (insertions and deletions) -> only insertions or deletions of three, or insertions or deletions that cancelled out, resulted in functional proteints

Nirenberg, Mattaei, Khorana, Leder 1960s:

1) used genetically altered mRNAs (ex: poly-U mRNA):

2) added to an in vitro translational system with radioactive amino acids

3) analyze radioactive polypeptides synthesized

4) deciphered the genetic code

properties:

1) triplet, non-overlapping codons

2) three stop ("non-sense") codons

3) degenerate (>1 codon can specify same amino acid)

4) initiation codon (AUG) marks start of reading frame coding for peptide

5) mutations can affect message (frameshift, non-sense, missense)

6) polarities of codons and amino acids correspond

Define the different elements comprising a eukaryotic "gene body" as defined in class. Does a gene consist of more than its gene body?

gene body:

-intron and exons

genes also consist of:

-UTRS (part of exons)

-repeated sequences

-transposons (change position within genome)

-promoter/ cis regulatory elements etc

Describe the different kinds of non-coding sequence found in the genomes of humans and other eukaryotes. How do these types of sequences compare to coding sequences as proportions of the total genome (very roughly, that is)?

-introns (26% of genome)

-repeated sequences (50% of human genome)

-transposons (45% of human genome)

-exons: 1.5% of genome!

Genome sizes do not correlate well with organismal complexity. What is an example of this surprising observation, and what seems to explain the absence of a correlation in many cases?

axolotl/salamander:

-humans and salamanders have about the same number of genes but salamanders have 10x larger genomes (3.6 Gb vs 32.4 Gb)

-due to repeated sequences!

What are "cis-regulatory elements," "trans-regulatory factors," "exons," "introns," "untranslated regions," "proximal promoters," "enhancers," "repressors," "activators," "caps," and "poly-A tails"? Which are protein coding sequence (or are actual proteins); which are not? Which make it into mature mRNA?

cis-regulatory elements (CREs) are regions of non-coding DNA which regulate the transcription of neighboring genes (cis bc on the same molecule of DNA as the gene being transcribed) --> include promoters

trans-regulatory factors: transcription factors: mostly DNA-binding proteins encoded by other genes

exons: coding sequence

introns: non-coding sequence that gets spliced out in the formation of mature mRNA

untranslated regions: part of exons that is not translated (where RNA pol binds??)

proximal promoters: CRE's located right next to genes

enhancers: CREs that are far from gene, increase gene expression by binding TFs

repressors: TFs

activators: TFs

caps: added to 5' end of mRNA

poly-A tail: added to 3' end of mRNA

exons are protein coding sequences (except UTRs)

What is a gene? Can one gene overlap another, and how does one know where a gene begins and ends in the genome?

An overlapping gene is a gene whose expressible nucleotide sequence partially overlaps with the expressible nucleotide sequence of another gene. In this way, a nucleotide sequence may make a contribution to the function of one or more gene products.

-genes begin with TSS and end at sites that attract termination elements

Define "chromatin" and its composition. What are some differences between heterochromatin and euchromatin?

-chromatin: DNA with protein (each chromosome is a single long molecule of DS DNA)

-1/3 each DNA, histone, non-histone proteins

-euchromatin: open, more accessible to RNA pol and TFs, more likely to be transcriptionally active

-heterochromatin (closed chromatin), tightly packed with nucleosomes, less accessible, less likely to be transcriptionally active

-heterochromatin often gene-poor and repeat-rich, dark-staining and localized to periphery of nucleus

What are the components and functions of nucleosomes? Do prokaryotes have them?

nucleosomes: core histones (H2A, H2B, H3, H4), linker histone (H1) wrap DNA

-prokaryotes do not have them

-histone tails exposed beyond nucleosome and can be post-translationally modified

-this can increase or decrease transcription

-histone mutations also associated with cancers

Histone modifications are essential for regulating chromatin state. What are major types of modifications and the enzymes that participate in them? What are their consequences for transcription?

-methylation - sometimes increases, sometimes decreases transcription (protein arginine methyltrasnferase, histone methyltrasnferases)

-acetylation: histone acetyl trasnferase (HAT), histone deacetylase (HDAC): acetylation increases transcription because the + charges interacts with DNA's - charge, resulting in looser interactions between DNA and histones

-other: phosphorylation, ubiquitination

consequences:

1) altered DNA Accesibility

2) Altered recognition by cofactors

What traits do cancer cells typically evolve that are different from normal cells?

-sustaining proliferative signaling

-evading growth suppressors

-activating invasion and metastasis

-avoiding immune destruction

-deregulating cellular energies

-enabling replicative immortality

-inducing angiogenesis

-resisting cell death

Are there particular aspects of cancer cells that allow facilitate the evolution of increasingly malignant phenotypes?

-grossly dysregulated gene expression

-growth unchecked and spread

-take resources from healthy cells

What evidence suggests an important role for H3K27 mutations in pediatric glioma?

-mutation found in almost all brains dissected after patients died of pediatric glioma

Distinguish ATP-dependent chromatin remodelers (e.g., SWI/SNF or chromodomain factors) from histone modifying enzymes (e.g., HDACs, HMTs). What are the shared features of ATP-dependent chromatin remodelers?

histone modifying enzymes: alter histones by acetylation, methylation, etc. Make DNA more / less available and make cofactors more/less likely to recognize

chromatin modelers: have an affinity for nucleosomes and recognize histone modifications, but overcome nucleosome-DNA interaction by nucleosome sliding, displacement, and modification.

Describe the various ways that ATP-dependent chromatin remodelers can affect DNA binding site accessibility.

1) nuc sliding

2) nuc dsiplacement

3) repositioning

4) ejection

5) unwrapping

6) dimer exchange

7) dimer ejection

What is a "pioneer" transcription factor and what distinguishes it from other types of transcription factors? What kinds of interactions can pioneer TFs have, and in what contexts have they been shown to be especially important?

-bind target DNA sequence EVEN in closed chromatin

-initiate chromatin remodeling

-permit bidning of other TFs, histone variants, chrom remodelers

-stabilize open chrom state

-play roles in cell programming and reprogramming

Several DNA motifs in proximal promoters are essential for the commencement of transcription from particular genes. What are two of these motifs and how do they function to promote transcription?

-TATA box: attracts TBP and associated factors

-initiator sequence - recognized by factors in PIC

-Downstream core promoter element - mau code for protein or be within UTR, recognized by PIC **also initiator sequence

How do "basal" or "general" transcription factors compare with tissue- or cell-type specific transcription factors?

-basal: general transcription factors

-tissue- or cell-type specific: exist in specific cells key in differentiation *include TBP (tata binding protein) that recruits other factors

What are the Pre-initiation and Mediator Complexes and how do they promote transcription? How do the proximal promoter and "distal" cis-regulatory elements (occupied or unoccupied) fit into the process?

PIC complex: recognizes promoter motifst that act cooperatively to promote transcription

MC: promotes pre-initiation complex assemble and Pol II localization. Integrates and communicates with TFs bound to Cis-reg elements and also non-coding RNAs that regulate transcription. Promotes looping of DNA resulting in TFs being close to transcriptional start sites, regulates Pol II pausing does not bind DNA directly

proximal promoter recognized by PIC

MC interacts with TFs bound to cis-reg elements

How does a single nucleotide mutation in a Duffy antigen gene confer resistance to malaria?

-GATA TFs regulate Duffy antigen (antigen recognized by parasite)

-GATA TFs bind cis-reg elements, acting as acitivators

-mutation T-46C alters GATA binding site and reduces transcription of Duffy antigen/ chemokine receptor

Explain one way in which a transcription factor can function as both an activator and a repressor.

-thyroid hormone: active form is T3

-T3 present - binds to TRs, converting them to repressors to activators for target genes

-with T3: activated TR rescruits MC and interacts with HAT. This turns on transcription.

-Without T3: TR cant recruit HAT, recruits HDA. HDA results in closed chromatin, prevents assembly of PIC = repression

Describe the possible steps by which a gene in a region of closed chromatin begins to be transcribed.

1) pioneer TFs bind target DNA even in closed chromatin

2) permits binding of other TFs, histone variants, chromatin remodelers

3) these result in chromatin being in stabilized open state

4) PIC forms as proteins recognize TATA box etc

5) mediator complex helps facilitate interactions between cis reg elements and promoter area and RNA pol

5) RNA pol binds

6) transcription initiated

What kind of post-transcriptional modifications are made to a primary RNA transcript during its maturation as mRNA?

-5' methyl guanine cap added during transcription

-introns spliced out

-poly A tail added

-alternative splicing results in one gene often times making multiple products

How does splicing work and how does it contribute to the diversity of mRNAs and proteins?

-cut at 5' end of exon

-lariat forms (2' and 5' bond)

-cut at 3' end of exon

-spliced section removed

-retains different exons or introns

Explain the mechanisms of cis and trans regulation that allow for normal splicing of eukaryotic pre-mRNAs.

1. cis and trans splicing regulators that influence spliceosome binding/activity

• cis regulators are RNA motifs: Exonic/Intronic Splicing Enhancers/Suppressors (e.g., ESE)

• trans regulators are proteins that bind cis motifs: serine/arginine-rich (SR) proteins promote splice site usage and compete with repressive, heterogeneous nuclear ribonucleoproteins (hnRNPs) and tissue-specific regulators

How might the speed of RNA pol II be regulated and what is a proposed mechanism by which this could result in alternative splicing to generate different mRNAs from a single gene?

2. Transcription rate differentially exposes exons to splicing factors

• histone methylation/ acetylation influence RNA pol II speed

• fast transcription results in bigger loops, more vulnerable to being spliced-out

• precise histone modification and chromatin state may favor some splice variants over others

What sorts of RNAs are regulated by degradation and in what contexts?

2. Transcription rate differentially exposes exons to splicing factors • histone methylation/ acetylation influence RNA pol II speed • fast transcription results in bigger loops, more vulnerable to being spliced-out • precise histone modification and chromatin state may favor some splice variants over others

targeted degradation: defective RNAs (unspliced, mis-spliced, containing mutations), non-coding RNAs transcribed by pol II

nucleus: marked by TRAMP complex for degredation

cytoplasm: NMD, NSD< or NGD

What explains the difference between the numbers of codons and the types of tRNAs found in eukaryotic cells?

Wobble pairing: the 5' base on the tRNA can bind to a number of different bases on the 3' end of the mRNA

What are the events of translation initiation, elongation and termination?

initiation:

-40s recognizes 5' cap on mRNA

-40s scans and finds start codon

-met tRNA binds start codon

-60s binds

elongation:

-another tRNA binds A site

-peptide bond formed between that AA and met

-met trna (empty) moved to e site and ejected

termination:

-ribosome encounters stop codon

-release factor binds A site so no tRNA can bind

-polypeptide released to be posttranslationally modified

What are two examples of ribosomopathies and what are their genetic bases?

1) diamond-blackfan anemia:

-mutation in RPS genes: defect in 40s subunit, 18s mRNA

-mutation in RPL genes: defect in 60s subunit

---> less complete subunits formed, particularly affects bone marrow cells

2) Treacher Collins syndrome

-mandibulofacial dystosis

-TCOF1 - localizes with RNA pol 1 to the nucleolus, where ribosomes are assembled, and when TCOF1 is prevented from being translated in vitro, RNA pol 1 fails to localize - ribosome assembly problems

-POLRIC or POLRID code RNA pol genes - mutations = no transcription of rRNA other than 5s rRNA