Genetics Chapter 18

What happens in BER for E. Coli?

- Nick Translation - DNA polymerase I uses 5' to 3' exonuclease activity to remove the damaged region and fills the region with normal DNA - DNA ligase then fills the region

What happens during a tautomeric shift?

- Occurs immediately prior to DNA replication because DNA in double helix holds bases in most stable form - First round of DNA replication will have T/G base pairing mismatch - Second round will have G/C pairing when it should have been A/T which causes the mutation. - Very temporary, eventually shifts back to old form. - Mismatch could be repaired by proofreading function of DNA polymerase

Germ Line Mutation

- Occurs in sperm or egg cell or precursor cells - After fertilization all resulting offspring of cell will have the mutation so mutation is found throughout entire body - Half the gametes will have the mutation

Base substitution

- One base is substituted for another

Mechanisms of NER

- Protein complex of two UvrA and one UvrB molecule tracks along and recognizes the distorted double helix and damaged DNA - UvrA is released and UvrC binds - UvrC makes cuts on both sides of the damaged DNA - UvrD (a helicase) separates the two strands and damaged DNA segment is released - UvrC and Uvr B are also released - DNA polymerase fills the gap and DNA ligase seals the strand

Ionizing Radiation

- Radiation of short wavelength and high energy - Creates chemically reactive molecules - Can cause base deletions, oxidized bases, single nicks in DNA strands, chromosomal breaks, etc. - Penetrates deeply (ex. X- Rays, Gamma rays)

Deanimation

- Removal of an amino group from cytosine base which produces uracil - DNA repair enzymes can recognize uracil as an incorrect base but if not it will incorporate adenine into new strand instead of guanine which would lead to another mutation - Removal of an amino group from 5-methyl cytosine produces thymine - DNA repair enzymes cannot recognize whether the thymine from deanimation or the guanine in the opposite strand is incorrect which makes it a hot spot for mutation

Nucleotide Excision Repair

- Repairs helix distorting regions - Several nucleotides in damaged strand are removed - The intact strand serves as the template to synthesis a new complementary strand - Found in Eukaryotes and Prokaryotes

Base Excision Repair

- Repairs non helix distorting changes - Uses the enzyme DNA N-Glycosylase - Recognize multiple abnormal DNA bases but are especially important for oxidative repair

Lethal mutation

- Results in death to the cell or organism

In addition to point mutations what else can occur that would cause a mutation?

- Short sequences of DNA may be deleted or added to the DNA.

Transversion

- The change of a purine to a pyrimidine and vice versa - Less common than transition

DNA sequences known as _______ are hotspots for mutation

Trinucleotide repeats

Point mutation

- A change in a single base pair

Mutation

- A heritable change in genetic material (DNA base sequence) - The gene is altered in a permanent way

Conditional Mutation

- Affects the phenotype only under specific conditions (Heat, etc.)

Genetic Mosaic

- An individual that has somatic regions that are different from each other

Base analogs

- Analogs become incorporated in daughter strands during DNA replication - Tautomeric shifts cause TG anc CA base pairs - Sometimes used in chemotheraphy because the mutations cause the cancer cell to die - Also causes other cells to die as well (hair, skin, etc.)

TNRE cont. In a coding sequence?

- CAG repeats in a coding sequences causes the proteins to aggregate or combine as a whole and carry long tracts of glutamine - This causes the progression of disease symptoms

Somatic Mutation

- Can occur later in life too - Only a portion of the offspring of the cell will have the mutation - Affected region depends on the timing (earlier the mutation, the bigger the mutation) - Cause genetic Mosaic - None of the gametes have the genetic mutation

Induced Mutations

- Caused by environmental agents

Down promoter mutations

- Causes the promoter to become less like the consensus sequence - Decreases the affinity of transcription factors and transcription rate

Nonionizing radiation

- Causes thymine dimers - Thymine dimers interfere with transcription and DNA replication - Does not penetrate as deeply (ex. UV light)

Germ Line

- Cells that give rise to gametes such as eggs and sperm

Nonsense

- Change from a normal codon to a stop codon - Translation is terminated earlier than expected

Reversion

- Changes a mutant allele back to the wild type

Spontaneous Mutation

- Changes in DNA structure as a result from natural biological or chemical processes

Oxidative DNA Damage

- Changes in DNA structure caused by an over accumulation of ROS - Guanine is especially susceptible, G ---> 8-oxoG which base pairs with Adenine not cytosine - After replication there will be an AT base pair where there should have been a GC base pair - A transversion muation - can be caused by environmental agents or spontaneously

Mutant Allele/Forward Mutation

- Changes wild type genotype into some new variation - Change in allele

Base Modification

- Chemical mutagens can covalently modify the structure of bases - Deanimation = modified bases cause A/G and T/C base pairing - Alkylating Bases = addition of methyl/ethyl groups to bases (ex. mustard gas)

Breakpoint

- Chromosomal rearrangement - The region where two chromosome pieces break and rejoin with other chromosome pieces - Can occur within a gene and inhibit gene function

What else can effect the expression of genes other than small changes in DNA sequence?

- Chromosomal structure changes

What happens in BER for Eukaryotes?

- DNA polymerase beta removes the site and can replace it with the correct nucleotide - Then DNA ligase will seal the region

Or what else can happen in BER for Eukaryotes?

- DNA polymerase delta synthesizes short strand of DNA - This makes a flap - Flap endonuclease removes the flap and DNA ligase seals the region

TNRE and DNA replication

- DNA replication proceeds just past the repeat - The hair pin forms in daughter strand and DNA polymerase slips of the template strand - DNA polymerase backs up and starts synthesis again from the end of the hairpin, this synthesizes the repeat twice - The hairpin spreads out and leaves a gap in the template strand - The gap is filled in by DNA polymerase and ligase which causes an even longer repeat region.

Deleterious Mutation

- Decreases the chances of survival and reproduction

Common Pathway

- Two or more proteins are involved in a pathway - Mutation causes a defect in one protein - The function of a different protein in the pathway may be altered to compensate

Steps of the Ames Test

1. Mix the potential mutagen with rat liver extract (which has cellular enzymes to activate the mutagen) and the Salmonella that cannot synthesize histidine 2. Have a control tube with no mutagen added 3. Put the two mixtures on separate growth plates that both do not contain histidine ( no growth) 4. The salmonella strain by itself shouldn't grown. If the agent is a mutagen then the mutation should allow growth 5. There is expected to be some growth on the control group colonies because spontaneous mutations can occur.

Suppressor Mutation

- A mutation at a second site that affects the phenotypic expression of a first mutation

Neutral Mutation

- A mutations that has no detectable effect on protein function - Silent and sometimes missense

Position Effect due to regulatory sequences

- A piece of one chromosome is inverted or translocated to a different chromosome - The gene is moved next a regulatory sequence for a different gene (enhancers or silencers) and it now regulated by that sequence.

Trinucleotide repeat expansion

- A repeated sequence of three nucleotides are repeated in tandem - Can increase from one generation to the next - When they increase above a critical size then they cause disease

Position Effect due to chromosome chromosomal rearrangment

- A translocation can move a gene from a tightly condensed heterochromatic structure to a loser condensed Euchromatic structure - In the heterochromatic structure the gene is not expressed and in the Euchromatic structure the gene can be expressed. - Creates a variegated gene expression

Frameshift

- Addition or deletion of nucleotides that is not divisible by three - Codons are read in three so this causes different amino acids down the entire reading frame.

Mutagens

- Agents known to alter the structure of DNA which lead to mutations - Can be chemical or physical - PERMANENTLY alter the structure of DNA

Somatic Cells

- All the cells of the body excluding the germ line cells

Oxidative Stress

- An imbalance between synthesis and removal of ROS

Are mutations good or bad?

- Depends, mutations can be positive, neutral, or detrimental. - Takes generations for natural selection to act on beneficial mutations.

Silent

- Do not alter the amino acid sequence of the polypetide - Because genetic code is degenerate mutatations can occur in the third base of the codon.

Missense

- Does alter the amino acid sequence of the polypetide due to the base substitution - ex. Sickle cell changes the sixth amino acid from glutamic acid to valine. - Causes blood cells to have sickle shape in low oxygen

Neutral Mutation

- Does not alter protein function - Does not affect survival or reproductive success

Beneficial Mutation

- Enhances the survival and reproductive success of an organism

Redundant Function

- First mutation inhibits function of protein - Second mutation alters a different protein to carry out function of first protein

Position Effect

- Gene is left intact but it's expression is altered when it is moved to a new location

Lederbergs Replica Plating

- Grew bacteria on master plate with no T1 phage - Velvet cloth was touched to plate and picked up a few bacteria from each colony. - Bacteria was transferred to two secondary plates that contained the T1 phage - Only mutant cells that are resistant to T1 could grow

Mutations in Nucleotide Excision Repair Enzymes

- In ability to repair UV induced regions due to inherited defects in NER genes - Xeroderma Pigmentosum (XP) and Cockayne Syndome (CS) - Pigmentation abnormalities, lesions, skin cancer, increased sensitivity to sunlight.

How big are mutations?

- It varies - They can be a single base or an entire change in chromosome structure

Depurination

- Most common - Removal of purine (A or G) from the DNA - Covalent bond between deoxyribose and a purine base is somewhat unstable - Can undergo a spontaneous reaction with water and produce an apurinic site

Intercalating Agents

- Mutagens contain flat structures that insert themselves between base pairs - Interrupts the helical structure causing single nucleotide additions/deletions - This causes frameshift mutations

Multimeric Protein

- Mutation in gene that encodes a protein subunit inhibits function - A mutation in a different gene suppresses this mutation and the function is restored.

Are mutations totally random

- Mutations can occur randomly but natural selection can act and select for organisms that carry them - Some genes mutate at higher rates than others because they are larger and there location may be a hot spot making them more likely to mutate.

Up promoter mutations

- Mutations that make a sequence more like the consensus sequence - Increase transcription

Mechanisms of BER

- N Glycosylase recognizes the abnormal base and cleaves the bond between the base and it's sugar - This leaves an apurinic/apyrimidinic site -AP endonuclease recognizes the missing base and cleaves the DNA backbone at the 5' end of the site

Physiological Adaptation

- Physiological events, such as the use or disuse of muscles, determined what genes were passed down to the next generation - Jean-Baptiste Lamarck, 1940s/50s

Wild type

- Prevalent genotype or phenotype in a natural population

Reactive Oxygen Species

- Products of oxygen that are generated by normal metabolism - If they accumulate then they can damage cellular molecules like DNA , proteins, and lipids

Anticipation or Dynamic Mutation

- TNRE disorders can get progressively worse in severity as they are passed down to future generations due to the lengthening of repeats - Depends on whether they are passed down from mother or father and on the gene.

Tautomeric Shift

- Temporary change in base structure - Tautomers = bases that exist in ketol and enol or amino and imino forms - The keto form is the stable form of G and T - The common form of A and C is the amino form - At low rates the enol form of G/T and imino form of A/C can interconvert - This promotes G/A and A/C base pairing

Ames test

- Test where an agent is a mutagen - Uses Salmonella Typhimurium = Cannot synthesize the amino acid histidine - There is a point mutation in a gene that encodes an enzyme in the pathway for histidine syntheses - There is a second mutation that can cause a reversion mutation and restore the activity of the enzyme to produce histidine - The Ames test determines if an agent increases the rate of this mutation (mutagen) or does not.

Transition

- The change of a pyrimidine to another pyrimidine (C to T) or a purine to another purine (A to G) - More common than transversions because of the spacing and the way a big purine and small pyrimadine fit into the backbone of DNA. - Type of point mutation/base substitution

Mutation Rate

- The likelihood that a gene will be altered by a new mutation - Where mutations occur is almost random - The number of new mutations in a given gene per cell generation (typically 10^-5 to 10^-9 per cell generation) - Varies due to the type of gene, the species, and environmental agents.

TNRE cont. In a noncoding sequence?

- The repeats produce CpG islands that become methylated which can silence genes - Expansions in the repeats cause change in RNA structure and cause disease symptoms

Intragenic Suppressor

- The second mutation is in the same gene as the first - The first mutation disrupts normal protein function - A suppressor mutation effects the same protein and restores the function

Intergenic Suppressor

- The second mutation is on a different gene than the first - common pathway, Multimeric protein, redundant function.

Mechanisms of TNRE

- The triplet repeat forms a hairpin or a stem loop - Triplet sequences contain atleast one C and one G - The hairpin forms due to C and G base pairing

What happens if the apurinic site is not recognized by DNA base excision repair before replication?

- There is complementary base to specify which incoming nucleotide should be attached to the new strand - Therefore any base can be added to the new strand which can lead to another mutation (75% chance)

Random Mutation

- There is variation in the population - Individuals by chance gain beneficial mutations - Natural Selection then acts on these mutations

What can NER repair?

- Thymine Dimers caused by UV rays - Chemically modified bases - Missing bases

Calculating Mutation Rate

= The amount of bacterial colonies with the mutagen added/ the amount of bacterial colonies originally plated and = The amount of bacterial colonies with no mutagen added/ the amount of bacterial colonies originally plated

What are the three types of Spontaneous mutations?

Deanimation, Depurination, Tautomeric Shift

What are the common diseases caused by TNRE?

Huntingon's, Fragile X, Muscular Dystrophy

Whose experiments were consistent with the random mutation theory?

Lederbergs