UVA BIOL3010 Genetics Quiz 1

Artificial Selection

purposful control over mating by choice of parents over next generation

technological advancements in genetic analysis

- invention of DNA sequencing

-invention of PCR

-human genome sequenced

-next generation sequencing

Next generation sequencing

can analyze base pairs of genome at very fast rate

lowered cost of identifying genome now is commercial industry

DNA sequencing (gel)

first sequencing was done by gel electrophoresis--smaller sements make it further through gell due to ability to make it though agar better

DNA Sequencing: Sanger Method

Mix DNA and DNA polymerase (func. only at high heat), primers, 4 types of nucleotides, and dye

denature DNA by heating

cool to allow primers to bind

heat up again- DNA polymerase will synthesize

eventually every length of strand will be synthesized

run strands of replicated DNA through gel electro. machine with a detector that can determine the base by the dye- label

strands sort by length because smaller and larger strands have same charge but different weights so small stands have greater speed.

Explain how PCR works

used in the lab to make millions of copies of a particular section of DNA

allows them to take tiny amounts of DNA and amplify them to have enough to do DNA sequencing or manipulate DNA for cloning

after PCR, electrophoresis can be used to check the quantity and size of DNA fragments

PCR steps

Denature DNA strand with heat

-add primers for area of interst

-taq-polymerase enzyme syntesizes two new strands of DNA using original stands as templates at high heat

-allow to cool DNA and new DNA will have double helix shape

-ad more taq poly and primers and repeat to yeild 4 strands of DNA

- after 20-30 cycles there are millions of copies of DNA synthesized from one double helix

-fist round or two the replicated DNA may be too long or have sticky stands but then they become more uniform

Denaturation

first step of PCR--A process in which a protein unravels, losing its specific structure and hence function; can be caused by changes in pH or salt concentration or by high temperature. Also refers to the separation of the two strands of the DNA double helix, caused by similar factors.

Annealing (PCR)

2nd step of PCR-- DNA primers attach to opposite ends of the target sequence

Extension

Third step of PCR-- when the temperature is raised and the new strand of DNA is made by the taq polymerase enzyme

Gene

sequence of nucleic acids that reside in chromosomes that encode for a protein that is transferred from a parent to offspring and is held to determine some characteristic of the offspring.

Allele

2 or more alternative forms of a gene.

version of a gene of a chromosome

arose initially from random mutation

examples of uses of allels

-pea colors: yellow and green- and shapes: wrinkled and round

-medel's monohybrind crosses- one allele was dom. one was recessive.

-----P generation- on parent carrien 2 dominant allels

-----F1 generation hybrids- carried one dominant and one recessive allele

What is needed for the process of natural selection to occur?

-Variation among indv. in a pop. of certain traits-genetic diversity

-fitness differences- consistant relationship b/t value of a trait- non-random mating

-reproductivve success- via survivorship or fecundity

-inheritance- consistant relationship for value of trait between parents and offspring

preformationism

inside sperm is a tiny version of an adult (homunculus)

Nicolas hartsoeker concluded that it was by the way of the male that there was a fully formed homunculus inside the sperm

disproven by microscope?

inheritance of aquired characteristics- Lamarck- (use and disuse)

Characteristics aquired through use and disuse can be passed on through genetics to offspring

mistaken idea of Jean Baptiste Lamarck that evolution occurs through the inheritance of traits that an organism develops in its own life time

if you stretch a lot to get a longer neck then children will have longer neck.

disproven by cutting off tails of mice and finding that the offspring always have tails.

Blending inheritance

parents with dif. phenotypes- progeny should be somewhere in middle- parental traits become mixed and forever changed in the offspring

offspring was 50/50 of parents

they couldnt figure out the obvious differences b/t biological brothers and sisters nor the persistance of variation w/in extended families.

disproven by mendels pea plants

Mendel's Law of Segregation

2 allels from each parent segregate during gamete formation and then unite at random at fertilization

in order to get 75%/25% ratio when crossing purebred yellow with purebred green there would have to be 2 allels present and they would had to segregate or some would be green

law of independent assortment

Each member of a pair of homologous chromosomes separates independently of the members of other pairs so the results are random

different alleles segregate independently from one another during gamete formation

mendel observed yellow wrinkly and green round bred together and saw that dif genes were not dependent on one another

Structure of DNA

-double helix with phosphate and deoxyribose backbone with base pairs hydrogen bonded on the center

-deoxyribose sugar, phosphate, nucleotides

-linear molecule

-A and T double H bond

-C and G triple H bond

-G and A are purines (double ring)

-T and C are pyrimidines (single ring)

-phosphates provide the energy for combining nucleotides

-nucleotides are linked by phosphodiester bonds

-stores biological information

-synthesized 5' to 3' with the phosphates binding to 3' of the sugar

-has minor and major grooves

RNA structure

-similar to DNA

-T is repalced with U

-Ribose sugar

-RNA is typically single stranded but can create complex secondary structures

-RNA is much more mobile and flexible in usage than DNA

what suggests that specfic bases pair in DNA

-A and T have a 1:1 ratio and C and G ahve a 1:1 ratio suggesting that they pair up- chargaff discovered this-- refects a significant aspect of the molecules inherent structure

-Watson's contributions- specific arrangements of purines and pyrimidines play crucial role in molecular intacrions- have a special role in formation of H bonds

-saw that A and T could be paired together such that 2 H bonds formed b/t them and is G and C were paired there were 3 H bonds between them

-2 pairs had same shape so they could fit anywhere without screwing up the stucture

-Crick's contributions- connected the chemical fact with X-ray data

-the bases in watson's apiring scheme could only happen if bases were attached to the backbone running in opposite directions

DAPI

a fluorescent stain that binds strongly to A-T rich regions in DNA (minor grooves

Furanocaumarins

-plant defense compounds that prevent unwinding of DNA helix prevention replication

-interact with indv bases and farm an adduct in the presence of UV light and form cross links

-results in painful leison and skin damage

Life defined by NASA

"life-sustaining chemical system capable of Darwinian Selection"

Essential features of genetic material

it must:

-store information

-express info

-replicate

-accomodate introduction of mutations and variation

Why is RNA a good genetic material?

-encodes info in 1D

-complex folding in 3D

-highly conserved across al life (rRNAs)

-organisms share common acestry based on RNA sequences

-its ancient

-can act as an enzyme and catalyze reactions on its own which could have evolved to allow it to replicate indp.

-simple in structure so it can be created in "primordial soup" of elemets and ions under right conditions

-can act as self-splicing introns: transcribed and the splice themselves out of initial transcript w/o proteins being present

RNA world hypothesis

hypothesis that RNA was the first nucleic acid to evolve and that early life was based on RNA, rather than DNA or protein

problems:

-contemporary nucleotides will not couple without chemical activation

-phosphate liker is very limited

How have self replication ribozymes maybe existed in nature?

replicases have been able to replicate RNA as long as 20 nucleotides in length

extant ribozymes do not have replicase activity needed for a RNA world

RNA ligases and replicases have been isolated

type 1 RNA replicator lost its replicating abilities and gained the ability to catalyze the RNA replication in other varieties

What showed that DNA could be transferred b/t organisms and that such material is DNA

transformation of dead bacteria genes to living bacteria genes giving non-lethal bacteria leathal capabilities

determined to be DNA by exposing bacteria to enzymes that destroyed proteins. DNA, RNA, and fats then combine with dead bacteria with smooth shape genes

all bacteria except bac. with destroyed DNA inc. the lethal bacteria's DNA after X amount of trials

Mouse experiment (S and R viruses)

-R form- lived

-S form- died

-S (heat killed) w/ R- died (R form got something from the dead S form) the mice had S morphology

one gene-one enzyme hypothesis

Idea proposed by Beadle and Tatum that each gene encodes a separate enzyme.

shows that there is a relationship b/t enzyme and proteins

gene body components

introns and exons including regions with the transcription start site and the end of the transcript

C-value paradox

The disconnect between genome size and organismal complexity (the C-value is the amount of DNA in a reproductive cell).

genome is incredibly dynamic and repeat sequences that can grow or shrink over evolutionary time

axolotl and human relations

gene structure is the same but introns are different

shit-ton more repetitive DNA which can grow or shirnk over evolutionary time

axolotl has a very large gene bodies that are few in number

non-coding area is expanded along with gene bodies

cis regulatory elements

A non-coding DNA sequence in or near a gene required for proper expression of that gene, often containing binding sites for transcription factors

trans regulatory elements (transcription factors)

DNA sequences that modify the expression or activity of genes that are not nearby on the chromosome, often by coding for transcription factors

Exon

expressed sequence of DNA; codes for a protein

Activators

drie translation can be regulated and have to be regulated precisely b/c of their role in transcription

co-activator

factor required for transcription that do not bind DNA but required for DNA-binding activator to interact with the basal transcription factors

bridge activators to basal transcritional apparatus

repressors and co-repressors

can diretly interfere with pol-2 complex binding at promoter

poly-A tail

a sequence of 50-250 adenine nucleotides added onto the 3' end of a pre-mRNA molecule

methylated cap

A modified guanine nucleotide that terminates a messenger ribonucleic acid molecule.

Promoters

DNA sequences near the beginning of genes that signal RNA polymerase where to begin transcription

terminators

RNA sequences that provide the signal to RNA polymerase for stopping transcription

insintric- do on their own

extrinsic- require additional proteins

mature mRNA contains

5' cap

5' UTR

exons/coding region

3' UTR

3' PolyA tail

Gene

A segment of DNA on a chromosome that codes for a specific trait

encodes a particular RNA or protein

they can overlap (different genes can code for the same phenotype)

gene begins at promoter sequence and ends when termination factor (rho protein) reaches RNA polymerase complex and dissociates the RNA polymerase from the DNA

Chromatin

Substance found in eukaryotic chromosomes that consists of DNA tightly coiled around histones

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

allows for parts of protein to be open and closed

Euchromatin

The less condensed form of eukaryotic chromatin that is available for transcription.

Heterochromatin

Eukaryotic chromatin that remains highly compacted during interphase and is generally not transcribed.

What kingdoms of life have nucleosomes?

Eukaryotes

components and functions of nucleosomes

made of 8 histones and DNA with possibility of other proteins or molecules binding to histones

main function is to reduce transcription by condensing and packing the DNA

enhanced DNA 7-fold by condensing naked DNA

UTRs (untranslated regions)

do not code for anything but are essential for proper protein synthesis

Introns

Noncoding segments of nucleic acid that lie between coding sequences.

are clipped out during transcript processing

5' cap

The 5' end of a pre-mRNA molecule modified by the addition of a cap of guanine nucleotide.

helps keep mRNA stable and facilitates transcription

3' poly-A tail

In eukaryotes, a series of 1-200 adenine residues added to the 3' end of an mRNA; the tail appears to enhance the stability of the mRNA by protecting it from degradation and facitilates transcription

SSR (Simple Sequence Repeat)

these is the most simple type of repetitive sequence; and most polymorphic

-These are tandem repeats of 2, 3 or 4 bp, repeated many times

Noncoding regions

make up most of the genome

only 1.5% of genome are exons

introns=26% genome

proximal promoter sequences

TATA box place that attracts the RNA pol II to the DNA and starts transcription

histone methlyation

the transfer of 1, 2, or 3, methyl groups to lysine or argenine residues of histone proteins by HMTs and PRMTs

can either increase or decrease transcription rates by condensing/ uncondensing

DNA methlyation decreases transcription

acetylation of histones

Unwinds DNA, adds actyl group- changes shape = genes turned ON

consequenses of histone modifications

- altered DNA accessibility

aletered recognition by cofactors- can result in particular targets that are recognized by other proteins

DNA breathing

nucleosomes loosen breifly and then tighten to allow for moments of transcription

ChIP-Seq

-isolate chromatin from genome

-fragment chromatin into small pieces

-incubate antibody to bind to protein of interest

-precipitate antibody-chromatin complex

-purify DNA from precipitate

-sequence DNA

-align sequences to genome to detect regions that have been enriched for protein of interest

histone marks= see where DNA associated proteins are active or bound

repressive marks are plentiful in regions where transcription is not occuring (away from RNA pol II)

Activating marks would be where transcription is occuring

RNA peak is due to transcription priming

Histone finctions beyond regulation of chromatin accessibility

-H2AZ- chromosome segregation

-H2AX- recuited to chromatin when DNA is damaged (exp where laser can damage DNA)

-protects DNA

-epigenetics turning on and off of DNA

cancer cell differences from normal cells

-genome is highly dysregulated

-have mutated histones typically

-resist cell death, draw blood vessels towards them, evade growth suppressors (tumor supressor genes can be turned off)

H3K17 mutations in pediatric glioma

H3 mutation is common in children with forms of brain cancer while rare in healthy cases

Shared features of ATP-dependent chromatin remodelers

- affinity for nucleosome

-domains to recognize histone modifications

-similar ATPase domains for overcoming nucleosome- DNA reactions

-Domains for interactions with other proteins

Affects of ATP- dependent chromatin remodelers on DNA binding site accessibility

-Nucleosome sliding

-Diplacement

-modification

Nucleosome sliding

remodeler attaches to the DNA and slides binding site so that the DBP will be blocked from the binding site by the remodeler

Displacement

DNA is unwrapped/rewrapped around nucleosome instead of b/t so that binding site cannot be accessed by the DBP

Modification

histone making the neucleosome must be modified hence "dimer exchange" and "dimer ejection"= altered composition

pioneer transcription

first transcription factor that attaches to chromatin and initiates process for transcription by attracting other needed TFs

-bind target DNA sequence even in close chromatin

-initiate chromatin remodeling

-permit binding of other TFs, histone variants, and chromatin remodelers

-stabalize open chromatin site

-play roles in cell programming and reprogramming

-include FoxA, FoxO, and GATA

TATA box

A promoter DNA sequence crucial in forming the transcription initiation complex.

7 nucleotides of the sequence located upstream of transcription site

attracts RNA polymerase weakly allowing for basal level of transcription

5'-TATAAA-3'

initiator

always found -2 to +4

helps with initiats of transcription

BRE

cis regulatory element found upstream of the TATA box

facilitates binding of TFIIB (part of the pre-initation complex of RNA pol-II

DPE (downstream promoter element)

works with initiator element to bind TFIID

motif

matching pattern associated with something

Basal factors

- found in most/ all locations where transcription is occuring

-binds RNA pol-II to the promoter

cell tissue/ specific transcription factors

unique to a certain cell or tissue transcription

pre-initiation complex (PIC)

macromolecular assembly of different transcription factors at the promoter for the start of transcription

mediator complex

A complex of proteins that interacts with the Pol II complex and allows transcription to begin.

helps get PIC to bind

Duffy antigen gene and resistance to malaria

-chemokine exploited by the plasmodium parasite and makes its way to blood cells causing malaria

BUT

with the mutation of the T-46C, there is an alteration of the GATA- binding site and thus reduces the transcription of the chemokine by 96%

-its programmed to look for specific binding site and if it cant find it then it cant infiltrate erythrocytes and replicte itself = no malaria

transcription factor actiing as and activator and repressor

--depends on the target gene

-T3, and active of TH, can bind to TH receptors and convert from repressor to activator

DNA methlyation

-happens in CpG islands or regions dense in C and G b/c transcription factir binding sites tend to be CG rich

-methlayed by DNA methlytransferase which adds methly comlex to the cytosine

-makes transcription difficult and promotes histone hethlation which makes nuclosomes which them makes the chromatin more tighly packed

phases of transcript elongation

-early elongation- 20- 60 bps and the RNA pol II stops

-productive elongation- continues rapidly through termination

-phases are regulated by recuitment of:

-escape from pausing (P-TEFb complex)

-enhance elongation efficiency (ELL)

-drive nucleosome dissassembly and displacement dowstream of RNA pol-II (which facilitates nucleosome transfer)

modifications to RNA as it matures into mRNA

-termination

-polyadenylation

-splicing

-addition of 5' guanine cap (prevents degredation)

-addition of poly A tail (protects from degredation

"AAUAAA" RNA transcript is cut and ~200 A's are added to the end , closely associated with termination)

Splicing

cut 1: splice donor site at 5' end of the intron (GU)

cut 2: splice acceptor sire at the 3' end of the intron, removing the intron (AG)

remaiining exons are then recombined

splicing can

alter promoter locations and transcription start sites

-retain different exons and introns

-select different locations for termination and poly A tails

-create alternative sites for translation initiation

speed of RNA pol-II could be modified by

histone aceetylation

methylation of histones

fast transcription results in

big loops and higher chance of exon being cut out along w/ introns

slow transcription results in

Smaller loops and lower chance of exons being cut out w/ introns

Cis regullatory elemets involved in splicing

-Exonic/ Intronic splicing supressor/ enhancers

trans regulatory elements involved in splicing-

hnRNP- inhibitory bind to ESS and ISS motifs DNA

SA-activitory bind to ESE and ISe motifs in DNA

RNA degradation

After serving ther purpose some last minutes, some last days' hours

degraded by ribonucleases

targeted degredation

defective RNA targeted by ribosome bc protein is id'ed as too short

RNA w/o proper stop codon is marked by ribosome

if RNA has a kink in it , it can be targeted bc it is unreadable to the ribosomes

difference b/t number of codons and the types of tRNAs found in eukaryotic cells

fewer types of tRNA than varieties of codons (61 codons)--its allowed bc the 3rd nucleotide of anticodon has wobble

wobble

allows for tRNA to recognize mult. codons and bring the proper amino acid corresponding with the codon

termination

occurs when a stop codon in the mRNA reaches the A site of the ribosome and brings over a release factor which disassembles the whole unit

zika virus

prevents normal mitotic cell division in fetuses resulting in cells with mult. or fewer copies of chromosomes. they are then detroyed by the body because of the irregularity

phenotypic consequences:

-small head, underdeveloped brain, eye damage, seizures, limited mobility, infect neural stem cells

replication of DNA semiconservatively

first theorized by watson and crick

shown by allowing bacteria to grow with nitrogen-14 and then allowed to grow with nitrogen-15 for a while

-when cells replicated there were two bands:

-14N+14N

-15N+14N