Genetics Exam 3

spontaneous mutations

-occur at low rate 2-12 per every million

mutations that arise by mistake

-called microsatellites

-most common repeating units are one two or three base sequences

-account for 3% of the total DNA in the genome

-arise from rare, random events

-expanded by slipped mispairing/ stuttering

Simple Sequence Repeats (SSR)

Deletion Insertion Polymorphisms (DIP)

short insertions or deletions of genetic material

chemical mutagenesis

-used for genetic screens

mutations that arise from chemical agents that alter DNA

germ line mutations

occur in gametes or in gamete precursor cells

-transmitted to the next generation

-provide raw material for natural selection

Qualities of germ line mutations

-continuous introduction of new mutations

-loss of deleterious mutations due to selective disadvantage

-increase in the frequency of a few mutations with selective advantage

Genetic variation is due to a balance between

-male germ cells undergo continuous mitosis

-there are more mutations in sperm from older fathers

why is the mutation rate in sperm about 2-4 times higher than in eggs

S. Luria and M. Delbruck

Fluctuation Test infected wild type bacteria with phage

examined the origin of bacterial resistance to phage infection

-bactericide becomes a selective agent

-kills nonresistant cells

-allows survival of cells with pre existing resistance

Evidence of bacterial resistance arising from mutations that occurred before exposure to bactericide

-Bacterial resistance arises from mutations that occurred before exposure to bactericide

-mutations occur as the result of random processes

Interpretations of the Fluctuation test and replica plating

substitution

-transition and transversion

replacement of a base by another base

transition

purine replaced by another purine, or pyrimidine replaced by another pyrimidine

transversion

purine replaced by a pyrimidine or pyrimidine replaced by a purine

deletion

block of 1 or more base pairs lost from DNA

insertion

block of 1 or more base pairs added to DNA

10,000/cell/day

Depurination

100-500/cell/day

-C changed to U

-Normal C-G--> A-T after replication

deamination of C

x-rays

break the sugar phosphate backbone of DNA

Ultraviolet (UV) Light

causes adjacent thymines to from abnormal covalent bonds (thymine dimers)

8-oxodG mispairs with A

-Normal G-C--> mutant T-A after replication

Oxidative damage

exceedingly rare

Incorporation of incorrect bases by DNA polymerase is

proofreading

function of DNA polymerase that recognizes and excises mismatches

base tautomerization

results in replication mistakes

-base analogs

-hydroxylating agents

-alkylating agents

-deaminating agents

-intercalating agents

How mutagens alter DNA: chemical actions of mutagens

replace a base

-almost identical to normal base

How base analogs alter DNA

alter base structure and properties

-add an -OH group

How hydroxylating agents alter DNA

alter base structure and properties

-add ethyl or methyl groups

How do alkylating agents alter DNA

alter base structure and properties

-remove amine (-NH2) groups

How do deaminating agents alter DNA

insert between bases

How do intercalating agents alter DNA

-occur in non-germ cells

-not transmitted to the next generation of individuals

-can affect survival

-can lead to cancer

-FDA screens

Properties of somatic mutations

-chemical repair

-end joining

-repairing a single/stretch of bases

Major mechanisms of DNA repair

-reversal of DNA base alterations

-homology-dependent repair of damaged bases/nucleotides

-double strand break repair

-mismatch repair of DNA replication errors

Accurate repair systems

-base extension repair

-nucleotide excision repair

examples of homology-dependent repair of damaged bases or nucleotides

homologous recombination

nonhomologous end-joining

examples of double strand break repair

DNA glycosylases

remove altered nitrogenous base

-DNA glycosylase removes nitrogenous base

-nucleotides are removed

-new DNA fills gap

-removes uracil from DNA

base excision repair

UvrA-UvrB complex

scans for distortions to double helix

UvrB-UvrC Complex

nicks the damaged DNA

DNA polymerase

Fills the gap from damaged DNA

deletions and chromosome rearrangements

unrepaired double strand breaks can lead to

DNA-->RNA-->Protein

Central dogma of molecular biology

DNA to RNA

transcription

RNA to Protein

translation

-Ribose instead of deoxyribose

-nitrogenous base uracil instead of thymine

-most RNA are single stranded

Three major chemical differences between RNA and DNA

base pairs within other parts of the same molecule

Most RNAs are single stranded but can form

-many RNAs can be made from one gene

-many proteins can be made from one RNA

Describe how information stored in genes can be amplified

triplet codons of nucleotides

represent individual amino acids

3 nucleotides

How many nucleotides make up 1 amino acid

-a gene's nucleotide sequence is colinear with the amino acid sequence of the encoded polypeptide

-each nucleotide is only part of 1 codon (NO overlapping)

-codons consist of 3 bases

Properties of genetic code

reading frame

the beginning of a gene establishes a

-frame shift-scrambled protein sequence (mutant)

-normal protein-reading frame restored

Adding or deleting 1/2 bases vs adding/deleting 3 bases

-triplet codons

-codons are nonoverlapping

-3 stop codons dont encode an amino acid (UAA,UAG,UGA)

Key concepts of the genetic code

AUG

start codon for translation initiation

degenerate (more than 1 codon can specify an amino acid), yet unambiguous

Genetic code is

-frameshift

-missense

-nonsense

mutations can be created in three ways:

Almost

Is genetic code universal?

one of the two strands of the DNA double helix

what serves as a template for a single-stranded RNA transcript

complementary base pairing

RNA strand is formed through

-promoters

-RNA polymerase

-Terminators

Transcription process in prokaryotes

Promoters

DNA sequences that provide the signal to RNA polymerase for starting transcription

RNA Polymerase

catalyzes transcription and adds nucleotides in 5'-3' direction

ribonucleotide triphosphates (ATP CTP GTP UTP)

How are phosphodiester bonds formed?

hydrolysis of bonds in NTPs

what provides energy for transcription

terminators

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

RNA polymerase binds to promoter sequence

-DNA unwound to form open promoter complex

-phosphodiester bonds formed between 1st two nucelotides

Initiation of transcription

-Core RNA polymerase loses affinity for promoter and moves in 3'-5' direction on template strand

elongation of transcription

terminators

-form from harpin loops

RNA sequences that signal the end of transcription

processed to make an mRNA

-5' methylated cap

-3'poly-A tail

-introns removed by RNA splicing

In eukaryotes, the primary transcript is

capping enzyme

adds a "backward" G to the 1st nucleotide of a primary transcript

processing

adds a tail to the 3' end of eukaryotic mRNAs

RNA splicing

what removes introns

exons (expressed sequences)

sequences found in a gene's DNA and mature mRNA

Introns (intervening sequences)

-some eukaryotic genes have many

sequences found in DNA but not in mRNA

alternative splicing

produces different mRNAs from the same primary transcript

ribosomes

-coordinate movement of tRNAs carrying specific amino acids

Translation takes place on

tRNAs

-have complementary anticodons

-covalently coupled to a specific amino acid (charged tRNA)

short single-stranded RNAs of 74-95 nt

directs amino acid incorporation into a growing polypeptide

Base pairing between an mRNA codon and an anticodon of a charged tRNA

amino acid attached to the tRNA

will be attached to the growing polypeptide chain in translation

wobble

-why genetic code is degenerate

some tRNAs recognize more than one codon

-begin with Methionine

-ribosomal subunit binds to 5' cap and migrates to AUG codon

-initiator tRNA carries Met

Initiation of Translation

-addition of amino acids to C-terminus of polypeptide

-charged tRNAs ushered into A site by elongation factors

Elongation of Translation

-no normal tRNAs carry anticodons for stop codons

-release factors bind to stop codons

-release of ribosomal subunits, mRNA, and polypeptide

Termination of Translation

missense mutations

replace one amino acid with another

nonsense mutations

change codon that encodes an amino acid to a stop codon

frameshift mutations

-not if multiples of 3 are inserted/deleted

result from insertion or deletion of nucleotides with the coding region

silent mutations

-degenerate genetic code-most amino acids have > 1 codon

do not alter the amino acid sequence

null mutations

ex. deletion of an entire gene

completely block function of a gene product

hypomorphic mutations

gene product has weak, but detectable activity

Hypermorphic mutations

ex. Achondroplasia

generate more gene product or the same amount of a more efficient gene product

Neomorphic Mutations

ex. ectopic expression

generate gene product with new function or that is expressed at inappropriate time or place

an accurate sequence of the human genome that was completed in 2003

Human genome project

-fragmenting the genome

-cloning DNA fragments

-sequencing DNA fragments

-reconstructing genome sequence from fragments

General ideas behind genome sequencing

-restriction enzymes

-mechanical shearing

methods of fragmenting DNA

-recognizes a specific sequence of bases

-cuts sugar-phosphate backbones of both strands

-restriction fragments are generated

-makes hundreds of restriction enzymes available

Process of restriction enzymes

digestion of DNA with restriction enzymes

How are restriction fragments generated

4-8 bp of double strand DNA

-palindromic (identical when read)

-each cuts at same place relative to its recognition sequence

Recognition sites for restriction enzymes are usually

mechanical forces break phosphodiester bonds

-pass DNA through a thin needle at high pressure

-sonication (ultrasound energy)

-ends can be blunt or have protruding single stranded regions

mechanical shearing process

Gel electrophoresis

-place gel in buffered aqueous solution, remove comb, load DNA samples into walls, apply electric current

DNA has negative charge and moves towards positive charge

process that distinguishes DNA fragments according to size

staining gel with dye and photographing the gel under UV light

-migration distance depends on size

-determine size by comparing to DNA markers of known size

How to visualize DNA fragments after gel electrophoresis