BMS 503 - Virology/Mutagenesis (Exam III)

What is the lambda phage?

temperate phage with a dsDNA linear genome that circularizes upon cell entery with complex protein structure surrounding the genome (complex symmetry)

What is the lambda life cycle?

lambda absorbs to the LamB porin of the E. coli cell, injects genome, and goes down the lytic or lysogenic pathway

What is the lytic pathway?

new virions are made, the cell lyses into host cell genome

What is the lysogenic pathway?

phage genome integrates into host cell genome, replicates only when host genome divides but can reactivate to become lytic

During the lytic pathway, the phage replicates _____ and ____ the host cell

quickly, kills

Generally, temperate phages are ____ when cell conditions are good or when conditions are very bad

lytic

The lambda phage genome and gene expression is a ____ of gene regulation events

cascade

What happens in the early cascade of the lambda gene expression?

initiates at Pl and PR and terminates after N and Cro, N promotes expression of middle genes

What happens in the middle cascade of lambda gene expression?

initiates from Pl and Pr, N protein is a transcriptional anti-terminator, leftward transcription proceeds throug the int gene and produces Int (integrase), Xis, and CIII while rightward transcription proceeds through the Q gene producing CII

What happens during late cascade of the lambda gene expression?

initiates from PR', requires Q protein anti-terminator, encodes phage structural components and lysis enzymes

The lytic vs lysogenic switch is made of what 3 proteins?

CI, CII, CIII

What does CI do?

represses lambda phage gene expression

What do CII and CIII do?

asses host cell nutrition

What does CII do specifically?

positive regulator and promotes expression of CI (repressor)

What does CIII do specifically?

inhibitor of host cell protease that cleaves CII, is only effective against low levels of protease

Poor growth media will result in high ____ which will result in ______ level host cell protease that ____ CII

cAMP, low, degrades

What happens if the lambda phage is in poor growth media?

CIII inhibits remaining protease, CII promotes expression of CI repressor, CI repressor prevents further lambda gene ecpression and lysogeny is favored

Rich growht media results in _____ cAMP which results in a ____ level of protease which results in ____ CII

low, high, less

What happens if the lambda phage is in rich growth media?

CI repressor is not made and the lytic cycle is favored

Lambda integrase (INT) and Integration host factor (IHF) do what?

recognize the attP sequence on the phage DNA and attB sequence on the bacterial chromosome, then integrate the lambda genome onto the bacteria chromosome

After integration, the lambda is called what?

prophage/lysogen

CI does what when lambda becomes a lysogen?

represses transcription of all lambda genes except itself to maintain CI repressor concentration and keep lambda a lysogen

What must happen for a lambda prophage to become lytic?

CI repressor must be inactivated

Under what conditions does the CI repressor become inactivated?

DNA damage

What happens when the DNA is damaged?

activates the bacteria protein RecA, which binds CI and stimulates autocleavage, transcription of phage genes occur, INt and Xis act to recombine lambda genome out of the chromosome

The lambda genome, upon entering the host cell, is circularized via sticky ends called what?

cos sites

DNA is replicated via a rolling circle mechanism producing what?

long concatemer

The concatemer is cut by ______, creating ______

restriction enzyme, sticky ends

A single unit genome is package into what?

the lambda capsid

Assembly of lambda is similar to what?

T4

What happens during assembly of lambda?

capsid proteins self assemble into viral particles, viral DNA is packaged in head-full manner, tails are assembled to full heads

What happens during release of lambda?

makes protein to depolymerize peptidoglycan, bursts host cell to release progeny phage

Why is E. coli's genome rife with mutations?

horizontal gene transfer, recombination, and a variety of mutagenic and DNA repair strategies

Genome changes are selected for through what?

evolution

What is a mutation?

heritable change in genotype (DNA sequence) that can lead to a change in phenotype (observable properties of an organism)

What is a mutant?

strain of any cell or virus differing from parental strain in genotype

What is wild-type?

an organism with the typical genotype found in nature

What are silent mutations?

mutations that have no affect on the organism (changes regions between genes, 3rd base codon, or 1 amino acid into similar one with normal functioning protein)

A mutation is a change in ____ while the effect of the mutation on the organism is the change in _____

genotype, phenotype

Some mutations are _____, meaning they change the phenotype of the organism only under certain environmental conditions

conditional

What are selectable mutations?

mutations that give the mutant a growth advantage under certain conditions

What are nonselectable mutations?

mutations that usually have neither an advantage nor disadvantage over the parent

What are induced mutations?

occur due to agents in the environment or through deliberate manipulation by humans

What are mutagenic agents?

factors that include chemical and physical agens and sometimes even infectious agents

What are spontaneous mutations?

mutations that occur without external intervention (typically a result of DNA replication error)

What is an insertion?

addition of one or more bases

What is a deletion?

subtraction of one or more bases

What is an inversion?

DNA sequence is flipped in orientation

What is a point mutation?

mutations that change only one base pair

What is a silent mutation?

does not change amino acid sequence

What is a missense mutation?

changes the amino acid sequence to another

What is a nonsense mutation

changes the amino acid sequence to a stop codon

What is a frame-shift mutation?

changes the open-reading frame of the gene

DNA viruses have a mutation rate 10-100x ____ than that of most other microorganisms

greater

Why is it easier for viruses to mutate than other microorganisms?

they have a much faster muutation rate and fewer RNA repair mechanisms than DNA repair mechanisms

How can mutations be caused?

mistakes in DNA replication, HGT, physical or chemical mutagens

What are the three chemical mutagens?

base analogs (incorporated in place of ATCG), base modifiers (modify ATCG usually via oxidation) and intercalating agents (alter DNA structure by inserting itself between bases)

What are physical mutagens?

UV radiation or ionizing radiation (create breaks in DNA and generate thymine dimers)

How can you measure a mutagen?

Ames Test

What happens curing an Ames Test?

uses bacterial strain auxotrophic for histidine, has frameshift mutation in hisG gene so that it cannot grow unless histidine is supplied, is placed on medium with a chemical, changes mutation to normal form

An Ames Assay can be used to do what?

be modified to test for the mutagenicity of chemicals processed in the liver