Genetic Exam 4 (chp 10, 11, 14)

phases of DNA replication

1. Initiation 2. Elongation 3. Maturation of okazaki fragment 3. Replication of telomeres (euk only)

Eukaryotes use protein called _______ to organize and pack their DNA

Histones

Lamarck theory of evolution

If an organism changes during life in order to adapt to its environment, those changes are passed on to its offspring.

Alkylating agents

MMS, EMS, ENU, mustard gas, cisplatin, cyclophamide

What are the difference between DNA Pol I and DNA Pol III structure and function?

Pol III is the primary polymerase used for replication. - pol I is positive in 5'-3' exonuclease activity, molecules in poly/cell 400 - pol III is neg in 5'-3' exonuclease activity, molecules in poly/cell is 15

How is a D loop formed?

The 3' G strand extension invades the duplex telomeric repeats

hypomorph

reduce function/partial function

depurination is

removal of A or G from strand of DNA. 1000/hr in every cell - deletions of bp

SINEs (short interspersed nuclear elements)

repetitive DNA sequences, approximately 500 bp in length on average, interspersed throughout the genome - The second most abundant class of transposable elements in human genomes; can create copies of itself and insert them elsewhere in the genome.

LINEs (long interspersed nuclear elements)

repetitive DNA sequences, approximately 6500 bp in length on average, interspersed throughout the genome - Long DNA sequence repeated many times and interspersed throughout the genome.

Telomerase is a

reverse transcriptase

What kind of enzyme is telomerase?

reverse transcriptase synthesizes DNA from RNA

circular genome replicated by

rolling circle replication

What are Okazaki fragments?

short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication

chromosomal copies are

spliced and packaged

mutation can be

spontaneous or induced

What are the function of proteins?

storage structural components transport molecules defense against foreign substances (antibodies) movement cellular communication

How do we keep the telomeres from shortening after every mitosis?

telomerase will bind to the overhanging 3' end of the chromosome. telomerase extends the end of chromosome.

How does telomerase work to prevent DNA loss at telomerase?

the enzyme binds to a special RNA molecule that contain seq complementary to the telomeric repeat. it extend to overhanging strand of the telomere DNA using complementary RNA as a template.

mutation frequency

the observed number of mutation per unit/population

mutation rates

the probability of a mutation occurring over a period of time

bidirectional replication

the process in which DNA replication proceeds outward from the origin in opposite directions

Transponsons

transposable elements of DNA excise themselves from a chromosome and reinsert themselves at another location

fusion

two pieces put together that should not be. can be at gene level or chromosome level.

deamination of cytosine to

uracil

sequence composition: eukaryotes

varying ratio; humans have 55-60% unique sequence

duplication

whole or part of chromosome duplicated

Deletion

whole or part of chromosome missing

After replication are histones all new and all old, or a mixture?

yes

Leading strand

The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5' to 3' direction.

Why can DNA Pol III not replace telomerase?

The primer may not be positioned right at the chromosome end and cannot be replaced with DNA so an overhang will still be present.

chemicals

- base analogs - intercalating agents - alkylating agent

neomorph

- cause the protein to have a new function

types:

- coding or noncoding mutation - point mutation affecting 1 basepair

how to label old and new strands?

- grow e.coli in media with heavy N15 DNA - then transfer to N14 media and grow to see what happens to the DNA after replication

Histones

- histone core for nucleosome - basic nucleosome structure in bead on a string chromatin - chromatin condensation by H1 binding

gain of function mutation

- hypermorph - neomorph

loss of function mutation

- hypomorph - null or amorph - antimorph/dominant negative

delta phage life cycle

- in viral heads, DNA is linear - sticky ends allow circularization after infection - during lytic pathway, I-DNA is excised and recircularized - the ter gene encodes an endonuclease that cuts fragment into linear unit chromosome at the cos sit for packaging.

Hypermorph

- increase protein function - may include mutation that create constitutively active molecule

supercoiling

- linear DNA with 20 turns -circular DNA with 20 turns - 20 turn linear DNA unwound 2 turns - circular DNA with 18 turns and short unwound region - supercoiled DNA with 20 helical turns and 2 superhelical turn

What challenges do eukaryotes have that are different than prokaryotes?

- linear genomes - bigger genomes - multiple chromosomes

Meselsohn and Stahl method

- mix DNA made of N14 (light) and N15 (dark) - centrifuge in a CsCl gradient

packing new chromatin

- number of histones must double so many new subunits must be made during S phase. - nucleosomes are disassemble as the replication fork approaches - daughter histones are typically a mix of old and new histones

The problem of ends: replication at the telomere

- overhang left after primer remove - sequence can be lost after many rounds of replication

special problem multiple chromosome: mitotic segregation

- protein attach to specific heterochromatin structure called centromeres to segregate chromosome in mitosis - each species has its own centromere seq, so they functionally conserved but not conserved at the level of seq - centromeres can vary in length and seq composition

causes of spontaneous mutations

- replication error due to tautomeric shift - replication error due to looping out - spontaneous chemical changes: deamination

What are the function of enzymes?

- speeds up chemical reactions and breaks down nutrient molecules - they control the speed of chemical reactions like digestion and metabolism

similarities with prokaryotic replication

- there are leading and lagging strand - lagging strand is made through creating a series of okazaki fragments - a helicase, gyrase, SSBs, polymerase, primate - replication is bidirectional

Differences with prokaryotic replication

- there are many Ori's per chromosome - there are more kinds of polymerase - chromosome have ends - chromatin is packed around histones - multiple replicants per chromosome

eukaryotic chromosome have special problem at the ends

- they deal with this by having special dedicated heterochromatin structures at the end of chromosome called telomeres

detecting mutation (replica plating)

- to recover auxotroph - mutation detected by inability to grow in some conditions

What is the Ames test for?

-uses direct selection to determine effect of a test chemical on reversion rate of histidine-requiring auxotroph Salmonella ***relies on the fact that mutagens increase frequency of reversions (if chemical is mutagenic, reversion rate will increase relative to a control) - to detect mutagens

consequence of unrepaired non waston crick base pairing

1. G pairs with T 2. mismatched G-T bp after replication 3. GC to AT transition mutation produced after next DNA replication

What is the function of a D-loop?

acts as a promoter for both the heavy and light strands of the mt DNA and contain essential transcription and replication elememt

frameshift mutation

addition or deletions of one or a few base pair leads to a changes in reading frame

chromosomal mutation

affect chromosome structure (part or whole)

Darwin's Theory of Evolution

all species of organisms arise and develop through the natural selection of small, inherited variations that increase the individual's ability to compete, survive, and reproduce. - variation is inherited

sequence composition: prokaryotes

almost all unique sequence some moderately repetitive DNA: rRNA, tRNA genes

temperate

alternates between the lytic and lysogenic pathway

unique sequences

appear once in the haploid genome - often protein coding

DnaA

binding induces a local supercoil

antimorph/dominant negative

causes a protein with opposite function or a subunit failure that poisons a whole machine

Nonsense mutation

change an amino acid codon into a stop codon, AT-TA transversion mutation changes the codon from lysine to UAA stop codon

Neutral mutation

change from an amino acid to another amino acid with similar chemical properties

Missense mutation

change from one amino acid to another, an AT to GC transition mutation changes the codon from lysine to glutamic acid

silent mutation

change in codon such that same amino acid is specified

linear replicates with many

chromosomal copies

null/amorph

complete loss of protein function

Capsule

consist of a head and a sheath only

Nucleotide repeat expansion

copies of a set of nucleotides increase in number with each generation

ionizing radiation

creates ions which can cause base changes or breaks in DNA or create free radicals that can attack DNA, modifying bases, causing depurination or cause backbone breaks

base modifying agent - nitrous acid is a

deaminating agent

essential control

demonstrate that "heavy" and "light" DNA can be separated N 14 and N15

Dna of different sizes separate by

density

Arthur Kornberg

discovered DNA polymerase I, achieve DNA replication in virto. demonstrates that DNA precipitate include radiolabel

In most eukaryotic and prokaryotic cells, the genome is

dsDNA

Darwin

evolution through natural selection on natural variation

Lamarck

evolution through use/disuse acquired traits are heritable

DNA polymerases

extend existing strands of DNA using a complementary strand as a template - req single strand/double strand interface - diverse in prok and euk

The amount of DNA in a cell is a

genome

semi-conservative

half of new DNA would be old

Why is the function of telomerase important?

helps to provide genomic stability in highly proliferative normal, immortal, and tumor cells by maintaining integrity of chromosome end, the telomeres.

base modifying agent - hydroxylamine is a

hydroxylating mutagen

Meselson-Stahl Experiment

inverted a technique called cesium chloride gradient centrifugation

Radiation

ionizing (alpha, beta, gamma, x-ray) nonionizing (UV)

solenoid

is a coil of wire that produces a magnetic field when carrying an electric current. coil wound into a tightly packed helix

In eukaryotic chromatin, what is a D-loop?

is a non-coding region

C-value paradox

lack of correlation between genome size and the biological complexity of an organism

Where is the D-loop located?

mitochondrial DNA

Eukaryotic genomes

multiple linear chromosome

detecting mutation (selection)

mutant detected by their ability to survive

detecting mutation (visible phenotype)

mutant detected by visible changes in appearance or behavior

base-modifying agents - intercalating agents wedge themselves between bases

mutation by addition and deletion

spontaneous mutation

mutation due to organic chemistry or replication error

Germline mutation

mutation in germ cells. Heritable

induced

mutations due to a mutagen

conservative

new DNA would be all new

non-waston crick base paring between normal pyrimidines and rare form of purines

normal T - rare enol form of guanine normal C - rare imino from of adenine

somatic mutation

occur in body cell that are not passed on. only affects that individual. Not heritable

ORC

origin recognition complex

inversion

part of a chromosome is backward

translocation

part of one chromosome in the wrong place

lysogenic pathway

phage chromosome becomes integrated in bacterial chromosome

lytic pathway I

phage chromosome excised from bacterical chromosome

UV raditation causes the formation if _____ _____.

pyrimidine dimers T + T = thymine dimers T + C C + C

non-waston crick base paring between a rare form pyrimidines and normal purines

rare imino form of C - normal A rare enol of T - normal G

transition mutation

A-T to G-C

Genome

All the genetic information in an organism; all of an organism's chromosomes.

Dna polymerases can only add a _____ to an existing free _____. This leads to the necessity of _________

Base 3' OH Primers

What enzymes are involved in the maturation of Okazaki fragments?

DNA polymerase III leaves 3' end of new okazaki fragment is next to 5' end of previous okazaki fragment. DNA polymerase I binds and replaces RNA in okazaki fragment with DNA

Elongation

DNA replication is semidiscontinuous

What protein interact with oriC in prokaryotes?

DnaA protein

What enzyme/proteins are involved in the replisome?

DnaB Helicase, pol III, primases

Topoisomerase

Enzyme that functions in DNA replication, helping to relieve strain in the double helix ahead of the replication fork.

Describe the different forms of chromatin including "beads on a string"

- Euchromatin: chromatin that is loosely packed and is stained light. Transcriptionally active. - Heterochromatin: chromatin that is tightly packed and stains dark. Transcriptionally inactive.

lagging strand

- The strand in replication that is copied 3' to 5' as Okazaki fragments and then joined up. - The strand that is synthesized in fragments using individual sections called Okazaki fragments

Properties of oriC

- 245 bp long - recognized by initiator proteins - which are produced by the dnaA locus - sequence region

DNA polymerase quick facts

1. addition of nucleotides to existing strand creates new phosphodiester bond: release two of three phosphates of nucleotide 2. template strand direct each new complementary base 3. only extend DNA strand in the 5' -3' 4. 3' - 5' exonuclease activity; incorrect bp is fixed

Initiation in Prokaryotes

1. replicator binds initiator protein (DnaA) 2. helicase elongate replication fork 3. DNA primase bind each helicase forming primosome 4. DNA pol III extend primer in 5' to 3' direction

Dispersive

1/2 old and 1/2 new - old DNA get diluted over many generation

highly repetitive DNA

10,000 to 10,000,000 copies

know the nm width and which histones are involved

11 nm wide x 5.7 nm thick Linker Histone H2A, H2B, H3, H4

in diploid cells there are how many copies of the genome?

2 copies

Human have approx meters of DNA per cell while human

2 meters.

base paring 5-bromouracil (behaves like thymine normal) in its normal state

5-bromouracil pair to adenine

Base-pairing of 5 bromouracil in its rare state (behaves like cytosine rare) in its rare state

5-bromouracil pair to guanine

Supercoiling

A method of DNA protection utilized by prokaryotes in which their large circular chromosome is coiled upon itself.

transversion mutation

C-G to G-C

gene mutation

a mutation that affect the expression of a gene or the function of its product

ORC binds ___ in ____

Ori in G1

DNA replication only occurs in

S phase of cell cycle

How do eukaryotes overcome each challenge?

They use telomeres

Deamination of 5-methylcytosine to

Thymine

Mutation

a change in DNA that alter the sequence

moderatly repetitive DNA

a few to 10,000 copies