MMBIO 240 Exam Two

gene

gene

DNA sequence encodes a function at the level of RNA or protein

gene expression

DNA makes mRNA to make proteins; some RNA functional without need for protein, cell can start making protein and stop, RNA levels often correlate with protein levels

reverse transcription

enzyme reverse transcriptase from retroviruses to convert RNA to cDNA

cDNA

complementary DNA, antiparallel relative to template RNA

RNA dependent DNA polymerase

reverse transcriptase

reverse transcription primers

Poly dT: make cDNA of all mature mRNAs, eukaryotes have poly A tails on mature mRNAs

Random hexamers: random six nucleotide primers, make cDNA of ALL RNAs

Gene specific primers: primers specific to gene, makes cDNA of specific sequences

cDNA use

eukaryotic genes had introns that are nearly impossible to work with, can study alternative splicing; after splicing, reverse transcriptase can readily clone mRNA

cDNA cloning

clone cDNA into expression vector to make recombinant protein and use strong promotor to express at high levels

RT-PCR

semi quantitative, increased number of PCR cycles yield more product; relative, produces double stranded cDNA

Q-RT-PCR

only want to count one strand, only cDNA, avoid too many copies

microarrays

measure gene expression levels at RNA level, measures expression of thousands of genes at a time; relative to control sample

prepare fluorescently labeled cDNAs for microarray

isolate mRNA, convert to cDNA using reverse transcriptase, add cDNA to microarray, fluorescent spot represents a gene expressed in cells

microarray colors

red: gene more expressed in sample two

green: gene more expressed in sample one

Yellow: equal expression, experimental conditions did not affect gene expression

one sample is control, the other is experimental

RNA sequencing

extract RNA from cells, generate cDNA, multiple fragments from cDNA, sequence fragmented cDNAs, make sequence fragments to transcriptome, quantify results

polymorphism

differences in DNA sequences that occur in a population; coexistence in a population of multiple alleles (different version of same gene) at a given locus (genetic location); must occur in 3% of the population

wild type

attribute or sequence found in ‘normal’ genome

polymorphism results

no sequence change, sequence change but no change in protein function, sequence change causes change in protein function or makes nonfunctional

human genome

diploid, may have nonfunctional gene compensated for by second functional copy; males higher incidence for sex linked genetic disease

substitution mutant

one nucleotide replaces another, total number still the same

deletion mutant

some nucleotides are missing relative to wild type

insertion mutant

new nucleotides now present compared to wild type

wobble hypothesis

third codon base tends to code for same or similar amino acid

silent mutations

change in DNA sequence doesn’t result in amino acid change

frameshift mutations

insertions or deletions in coding regions of genes are not in multiples of three

delete three nucleotides: lose one amino acid, may impact protein function

insert three nucleotides: gain one amino acid, may impact protein function

insert or delete number other than three, entire downstream sequence is thrown out of frame with severe impact on protein function

SNP

can harbor rare palindromes or lack common palindromes which cause different size fragments after cutting DNA with restriction enzymes

variable number tandem repeats (VNTR)

noncoding regions of genome, separated with gel electrophoresis, needs lots of SNPs and VNTRs to find person of interest, needs less than one percept in human population

current method of RFLP

DNA based microarray focused on SNPs to screen many SNPs in hours

chromatin

only formed in eukaryotic not prokaryotic cells

metaphase chromosome

condensed to avoid breakage as it is pulled apart, DNA must be evenly split between two daughter cells

centromere

DNA sequence repeats where spindle fibers attach for pulling or separation

telomere

DNA sequence repeats to protect end of chromosome

level one

naked DNA, no compaction

level two

DNA associates with histone octomers

nucleosome: DNA and histone octomer, 1.6-2 DNA twists

linker region: area of DNA between histone octomers

net positive charge

C terminus globular for DNA wrapping, N terminus has linear tails available for modification

quantifying DNA around histone and in linker region

digest DNA with micrococcal nuclease, an enzyme with endonuclease and exonuclease, then after incubation, the undigested DNA is tightly wrapped around histone; dimer: two histones worth DNA

leve three

interactions between histone octomers and likely histone HI protein to result in greater compaction

level four

30 nm fiber associates with protein scaffold for further compaction, histone protein HI involved in forming structure

histone cores for DNA compression, histone tails regulate gene expression

dehistonized metaphase chromosome scaffold

protein skeleton where loops of 30 nm fibers attach

histone acetylation

decreases positive charge, DNA less attracted to histones and packing state of chromatin is looser

histone acetyltransferase (HAT)

adds acetyl groups, activates genes

histone deactylases (HDAC):

removes acetyl groups, low acetylation has more methylation and more deactivated genes

goals of DNA replication

make complete new copies of genetic material in preparation for cell division so daughter cells have same genes/traits as parent

maintain genetic information accurately

extra work to keep linear genome from shortening

pulse chase experiment

add a “pulse” of labeled substance, usually radioactive through liquid growth medium, which is then incorporated into DNA, RNA, or protein

remove pulse label and restore regular, unlaced molecules by replacing with nonradioactive medium

watch and track labeled molecules

shows where and how things move within cell, how things are made, and how long they last before they are degraded or recycled

meselson stahl

pulse chase experiment showed that DNA replication is semi-conservative

grows bacterial cells in medium with heavy (radioactive) nitrogen, then switch bacterial cells to normal medium and replicate. centrifuge to identify if cells have heavy, light or a mixture of DNA

semi conservative

new daughter strand made by hybridizing to one parent strand

dna replication direction

bidirectional in both prokaryotes and eukaryotes through a semi-discontinuous model of replication, where some new DNA molecules are continuous and others are not due to origins of replication

DNA synthesis must occur differently on different strands because its polymerized 5’-3’

dna origin of replication

A:T rich region of DNA where DNA replication begins

initiator (dnaA protein)

recognizes ORI sequences and binds to them, starts to unwind DNA and recruits other proteins to the site

helicase (dnaB)

breaks hydrogen bonds to open DNA template; template necessary to know which nucleotides are needed for the new strand

single stranded DNA binding proteins

keeps DNA template from reannealing

primase (dnaG)

synthesizes small RNA primers, provides 3’ -OH ends for extension, no preexisting 3’ -OH needed

DNA polymerase

uses template to add nucleotides onto preexisting 3’ -OH

DNA polymerase III

exists as a dimer, one copy does essentially all DNA synthesis on the leading strand and the other does most of the DNA synthesis on the lagging strand; detects mistakes, cuts them out, and fixes them

Rnase H

finds RNA primers that are now part of larger DNA molecules and removes nearly all RNA nucleotides

DNA polymerase I

one copy per replication fork, removes last RNA nucleotides, fills gaps created from removed RNA primers from Okazaki fragments on lagging strand, synthesizes minor amounts of DNA and proofreads like DNA polymerase III

DNA ligase

phosphodiester bonds to seal Okazaki fragments, requires ATP

topoisomerase I

makes temporary nick in one strand of DNA to relieve twisting tension, located upstream of replication fork where tension builds

linear DNA

primase cannot lay down RNA primer at very end, and even if it could the end would be RNA and would be degraded by Rnase H; both leading and lagging strands of DNA get shorter because both act as templates over time

telomeres

DNA repeat regions at ends of linear chromosomes

uses exonuclease to degrade lagging strand and extends leading strand by using internal RNA as template to synthesize new cDNA. Primase, DNA polymerase III and I now resume lagging strand synthesis, finished by ligase. End product has unequal ends.

Produced in some cells to keep chromosomes intact but not produced in most cells so that cells can only divide so many times before becoming nonviable

error rate of human DNA polymerase

1 in 10^4-10^5 nucleotides

DNA corrections

DNA polymerase corrects 99-99.99% of its own mistakes and mismatch DNA repair corrects another 99-99.99% of all errors and mutations

permanent errors

occurs 1 in 10^8-10^11 base pairs; total genome in one diploid cell has 6×10^9 base pairs

UV light

causes pyrimidine dimers, intra strand cross links

T:T most common; C:C, C:T, T:C; results in covalent bonds between base pairs

causes kinks and change DNA structure

X-rays and gamma rays

space or radioactivity, causes DNA insertions from double stranded DNA breaks and subsequent random joining

hydrolytic cleavage of water

causes depurination and deamination

Phosphodiester bonds: leads to single strand breaks, rare and fixed with ligase

N glycosyl bonds: leads to depurination, happens 10,000 times a day per cell

exocyclic amine groups to bases: leads to deamination, bases may change to different bases

oxidation from cellular processes

causes addition of -OH and =O to bases

oxygens with unpaired electrons, attack other molecules to fix problem; naturally produced during cellular respiration, antioxidants protect us

causes different bases to hydrogen bond and potentially permanent mutations

alkylating and chemical cross linking agents

natural and synthetic chemicals

addition of alkyl groups, can lead to direct or indirect DNA damage

viruses and transposons

causes DNA insertions, viral DNA modifies host DNA and viral DNA to ligate together covalently

mutations

Somatic mutations only affect individual but germline affect future offspring

mismatch

hydrogen bonding disrupted, results from base change

susceptible cells

rapidly dividing cells; mismatches, deletions, insertions may be perpetuated; double stranded breaks and bulky adducts can block DNA replication

intentional DNA damage

cancer therapy, immunosuppression, and proteins that cause hypermutation in retroviral genomes

loss of function mutation

wild type gene makes functional protein

after mutation, no functional protein is made

gain of function mutation

wild type gene makes a nonfunctional protein; DNA damage results in mutation that restores functionality or intentionally increases severity of human pathogens

ames test

identifies potential DNA mutagens

salmonella bacterial strain that requires histidine for growth due to a point mutation in gene related to biosynthesis; point mutation is a single nucleotide mutation and is more likely to be fixed by random mutagenesis; add His- bacteria, potential mutagen, and a small amount of histidine to agar; monitor development of bacterial colonies that are his+ , which is indicative of a point mutation that by chance repairs previous mutation

nonfunctional histidine biosynthesis gene

only grow with histidine; histidine production is mutated or faulty

functional histidine biosynthesis gene

cells grow with or without histidine, true antigen will cause mutation to be more frequent than without it

detecting DNA damage

damage often changes DNA structure, DNA replication and RNA transcription enzymes discover damage when DNA is used as template

direct repair

no DNA is removed and replaced, damage is reversed

Photolyase reversal of thymine dimer, methyltransferase removal of methyl

excision repair

bulky additions to nucleotides are removed and replaced with correct nucleotides; TWO TYPES

base excision repair

single damaged bases only, depurinations, deamination, oxidations, alkylations

step one: damaged base removed; sugar flips to outside of DNA

step two: cleave DNA backbone on 5’ side of damaged base

step three: fill in missing nucleotides

step four: DNA ligase seals remaining nick

transcription coupled nucleotide excision repair

DNA damage unnoticed until RNA polymerase transcribes DNA; RNA polymerase stalls at lesion, repair enzymes are recruited to site, lesion is fixed, and transcription resumes

mismatch repair

base pairing mismatches, short insertions or deletions following DNA replication are repaired using similar mechanisms to excision repair

most commonly by mistakes made by DNA polymerase during DNA replication

in some bacteria, methylation of parent DNA strand signals enzymes to repair the other strand

homologous DNA sequence

two DNA molecules with very similar DNA sequences and in same order, don’t need to be one hundred percent match but close

DNA recombination

process of introducing new DNA sequences into existing DNA

crossing over

homologous DNA sequences from different chromosomes line up next to each other and switch places

genetic linkage

method to determine how close two genes are to each other based on frequency of crossing over together vs crossing over separately; linked genes tend to move together when crossing over but CAN segregate independently

homologous recombination process

sister chromatids align during meiosis or mitosis, nicks in DNA required for dsDNA recombination, DNA strands dehybridize and reenneal with sister strand to similar sequences; DNA strands can displace each other for varying distances

resolving holliday junction

branch migrates to end of linear molecule OR a double stranded break occurs before reaching the end of the molecule

types of DNA sequences commonly used in recombination

repeat sequences because as DNA strands dehybridize, the likelihood of finding homologous sequences is much higher for a repeat region; retrovirus, LINES, SINES, DNA transposons

recombinant organism with large genome

clone subgeneric fragment that contains region you want to manipulate; cut fragment at desired location, two cuts for deletion and one cut for insertion; ligate for deletion, or insert fragment and ligate for insertion

recombinant large genome requirements

needs at least 0.5-1 kb of DNA on either side of insertion or deletion in order to promote double crossover events; inserts modified and wild type DNA sequences into living cells. One to five percent of cells undergo homologous recombination

time of recombination

spontaneously in cells, most efficient during meiosis and mitosis when homologous chromosomes line up prior to cell division

genome

genetic material required for organism to replicate; encodes all organismal functions and are unique to each organism

sequencing

PCR, reduce length of original DNA, put in sequencing machine, machine adds fluorescent bases individually into new strand, fluorescent signal is detected and tells computer order of bases

multiple sequence alignment

lines up homologous positions, allows comparison

align with computational methods

use math to find best alignment by assigning scores; match, mismatch, internal and terminal gaps

gap

lines bases even if sequences are different lengths

insertions and deletions: impossible to tell which sequence lost or gained info

terminal gaps: sequence is naturally shorter or artificially cut off

Nucleotide Alignment

custom scores, balance between mismatch and gap

match/mismatch

match: identical base

mismatch: nonidentical bases or substitution

gap opening penalty

penalized for not having a letter that begins gap or indel, not evolutionary favorable