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