Bacterial viology

Question 1

define bacteriophage

Answer 1

obligate intracellular parasites that multiply inside bacteria making use of some or all of the host biosynthetic machinery

Question 2

what can bacteriophage genomes be? and where do bacteriophages bind to?

Answer 2

DNA or RNA both single or double stranded

they bind to specific receptors that are proteins or carbohydrates in the bacterial cell wall

Question 3

Why is cholera dangerous?

Answer 3

injection of bacteriophage causes protein to open up transmembrane protein and this causes diarrhea

Question 4

what is PhiX174? who discovered it? why was it significant? how big is its genome?

Answer 4

Fred Sanger. Extensively studied isometric virus with triangular faces. first DNA based genome to be sequenced -5386 nucleotides. Very small genome which is infectious by itself. Models for fundamentals of DNA replication. +sense ssDNA circular. 4 distinct intergenic regions.

Question 5

lytic /virulent phage?

Answer 5

PHAGE THAT CAN ONLY MULTIPLY WITHIN BACTERIA AND KILL THE CELL BY LYSIS

Question 6

lysogenc/ temperate phage?

Answer 6

phage that can either multiply via the lytic cycle or enter a quiescent state in the bacterial cell. Expression of most phage genes repressed as prophage.

Question 7

define prophage:

Answer 7

the genetic material of a bacteriophage, incorporated into the genome of a bacterium and able to produce phages if specifically activated.

Question 8

life cycle of PhiX174:

Answer 8

1) attachment of virus to the bacterial host folowed by injection of ssDNA
2) due to genomes’ positive polarity,DNA replication must commence before viral genes can be transcribed
3) DNA replication
- a)ssDNA converted to dsDNA
- b)amplification of the ds molecule
- c)ss genomic DNA synthesis and packaging

Question 9

What are m13/f1 phages?

Answer 9

ssDNa bacteriphages

Question 10

describe M13

Answer 10

filamentous phages that infect e.coli through pili, and are able to produce new virions without lysing the host cell.

Question 11

what are key M13 structural elements wrt genome organisation and size etc?

Answer 11

Circular, single-stranded DNA
~6.4 kb long
10 genes in the genome.

Question 12

why study bacteriophages?

Answer 12

Models for animal cell viruses
Gene transfer in bacteria
Medical applications
Lysogenic conversion

Question 13

can bacteriophages infect archaea?

Answer 13

yes

Question 14

how do bacteriophages infect cells?

Answer 14

Bind to specific receptors that are proteins or carbohydrates in the bacterial cell wall

Question 15

what type of virus is PhiX174?

Answer 15

a microvirus

Question 16

what is ds lamda bacteriophage replication cycle like? give specific times

Answer 16

very quick during lytic stage.
0mins-viruses absorb on lambda
30mins-viruses reproduced in cell
45mins-cell membrane breaks and releases phages

Question 17

what does the lamda cycle consist of?

Answer 17

eclipse period,intracellular accumulation phase, lysis and release phase

Question 18

what does the lamda lysogenic cycle consist of? (5)

Answer 18

1) phage DNA cirularizes (middle of both cycles)
2) phage DNA integrates into the bacterial chromosome this becomes a prophage
3) the bacterium reproduces normally and the prophage is copied too and transmitted to daughter cells
4) many cell divisions produce a colony of bacteria infected with prophages
5) occassionally the prophage exits the bacterial chromosome and this can initiate a lytic cycle.

Question 19

what does the lamda lysogenic cycle consist of? (5)

Answer 19

1) phage DNA circularises (middle of both cycles)
2) new phage DNA and proteins are synthesized and assembled into phages
3) cell lyses releasing phages
4) phage attches itsef to new host bacterium cell and injects its DNA
5) sometimes phage integrates into bacterial chromosome becoming a prophage and entering lysogenic cycle

Question 20

what is bacteriophage T4 made up of structurally?

Answer 20

head,collar,tail,base plate

Question 21

does the T4 bacteriophage encode an RNAP?

Answer 21

no. however it does encode proteins that modify host RNAP

Question 22

how long is T4 bacteriophage genome and how many products does it encode?

Answer 22

170,000bp and 300 gene products

Question 23

what system do bacteria use against bacteriophages?

Answer 23

a restriction system

Question 24

how does the bacterial restriction system work?

Answer 24

1) Entering DNA is methylated by modification enzyke and made resistant to degradation by restriction endonucleases
2) Restriction endonuclease can then cleave foreign DNA before it has a chance to be methylate

Question 25

how have some bacteriophages overcome the restriction system?

Answer 25

some modify their DNA. Uncut genome is then protected by methylation. Bacteriophage then replicates and eventually this leads to cell breakage and cell death

Question 26

Bacterial restriction system DETAIL

Answer 26

Bacteria have restriction endonucleases, which cleave double stranded DNA at specific points into fragments, which are then degraded further by other endonucleases.

This prevents infection by destroying the foreign DNA introduced by an infectious agent (such as a bacteriophage).

Given that the sequences of the restriction enzymes recognize are very short, the bacterium itself will almost certainly have many of these sequences present in its own DNA.

In order to prevent destruction of its own DNA by the restriction enzymes, the bacterium marks its own DNA by adding methyl groups to it. This modification must not interfere with the DNA base-pairing, and therefore, usually only a few specific bases are modified on each strand.

Endonucleases cleave internal/non-terminal phosphodiester bonds. Restriction endonucleases cleave internal phosphodiester bonds only after recognising specific sequences in DNA which are usually 4-6 base pairs long, and often palindromic

Question 27

what are CRISPR’s

Answer 27

Clusters of regularly interspersed short palindromic repeats in prokaryotes that are transcribed and processed into short RNAs that function in RNA interference

Question 28

what is the the CRISPR locus in E. coli transcribed to?

Answer 28

The CRISPR locus in E. coli is transcribed into a larger precursor RNA

Question 29

what organisms insert spacer sequences as a form of generating immunological memory

Answer 29

Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1–Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown.

Question 30

what do bacteria and archaea insert into CRISPR loci to generate immunological memory?

Answer 30

spacer sequences

Question 31

what mediates spacer acquisition in e.coli in vivo?

Answer 31

Cas1–Cas2

Question 32

what bacterium gene products to vibrio choler,e.coli and clostridium botulinum all produce?

Answer 32

toxins

Question 33

what bacterium has a CTX phage that infects it that produces a cholera phenotype?

Answer 33

vibrio cholera bacterium

Question 34

what can e.coli by infected by that causes hemorrhagic diarrhea?

Answer 34

lambder phage

Question 35

what phage can infect clostridium botulinum and what does it cause?

Answer 35

clostridial phage and causes botulism (food poisoning)

Question 36

what does the clostridial phage infect?

Answer 36

clostridium botulinum

Question 37

what are the 3 steps to phage display?

Answer 37

1) antibody library construction and display onto phage surface
2) selection by panning the library against Ag targets
3) screen for the desired specificty

Question 38

Human antibody library steps:

Answer 38

1) isolate B cells
2) PcR Vh and Vl genes
3) Display hmAbs fragments
4) visible on phage

Question 39

library selection steps:

Answer 39

1) Ag binding
2) epitopes, washing
3) elution –> amplification sometime
4) screening
5) cloning
6) antigens

Question 40

define antigen

Answer 40

a toxin or other foreign substance which induces an immune response in the body, especially the production of antibodies.

Question 41

define epitope

Answer 41

the part of an antigen molecule to which an antibody attaches itself