Sections 1-2 Final Exam

Question 1

viruses can be grouped by:

Answer 1

host range, structure, genome, transmission mode

Question 2

The Baltimore scheme for classification of viruses has ____ categories

Answer 2

Seven (7)

Question 3

The Baltimore scheme categories:

Answer 3

1. +ve ssRNA
2. -ve ssRNA (goes to +ve by RdRp)
3. dsRNA (goes to +ve by RdRp)
4. dsDNA (goes to +ve by DdRp)
5. ssDNA (goes to +ve by DP then DdRp)
6. +ve ssRNA (goes through RT to make sDNA then Dp to dsDNA then DdRp)
7. ss/dsDNA circle?

Question 4

one of the most important and useful findings of bacteriophage research:

Answer 4

restriction enzymes

Question 5

How were restriction enzymes discovered?

Answer 5

Werner Arber- Noble Prize:
a host’s range changed depending on the host strain the viral particle came from

• observed that viruses can replicate In any permissive cell but can only replicate in restrictive cells if they came from that cell lineage
• observed that upon a viruses entry into a restrictive cell, its viral DNA was fragmented (its the target)
• Restorative cells can allow a virus to be able to infect a restricted cell

Question 6

2 key components to the finding of restrictive enzymes (by Werner Arber)

Answer 6

“r” restriction: cell has resistance to foreign DNA and produces enzyme that cuts it up
“m” modification= ability for a virus to confer resistance against “r” ad modify its DNA to protect it from the cutting enzyme

Question 7

The bacteriophage piX174 controls its transcript levels through _____

Answer 7

• differences in promotor strengths pB>pD>pA
• Terminators stop RNA pol so transcription doesn’t always go to the end

Question 8

The bacteriophage piX174 controls its protein levels through _____

Answer 8

• controlling transcript levels (amount of mRNA) through promoters and terminators
• Nested genes: have weak ribosomal binding sites so they are translated less often (non structural or replication)
  ex. non essential, lysis, scaffolding

Question 9

phage lambda discovery

Answer 9

Discovered by Esther Lederberg in 1951 from an E.coli strain (k-12)

Question 10

in E.coli strain (K-12), phage lambda is a

Answer 10

lysogen (able to generate lysis) and exist as a prophage (Ur-lambda primitive)

Question 11

phage lambda due to its two decision cycle it was one of the most well-studied, and they found that the structure of lambda virions ___

Answer 11

has an obvious pattern of gene organization verses where the proteins end up in the particles (head to tail)

Question 12

a recombinant lambda version was obtained from Paris and Pasadena (Papa) and it______

Answer 12

did NOT have tail fibers, moves easier in soft agar, and makes larger plaques

Question 13

phage lambda Papa has a ______-_____ ______ preventing its tail gene from making tail fibre proteins

therefore, not the mother of all lambda phages

Answer 13

frame-shift mutation

Question 14

it was found that Ur-λ adsorbs to the cell better than papa-λ, due to_____

Answer 14

a LamB maltose transporter receptor, only recognized by the J protein on the TAIL

Question 15

Which one can grow in glucose

Answer 15

Ur-λ (has the tail so can grow even with very little receptors

tails not essential but makes them efficient and better at binding to cells

Question 16

Phage λ strains WITHOUT tails, are incapable of attaching to cells when there is ____

Answer 16

low amounts of receptor present

Question 17

All siphoviruses have long non-contractile flexible tails of different sizes, why?

but all λs (a specific phage) have the same size

Answer 17

Hendrix showed that the lambda protein H is a molecular ruler to control tail length

If you delete parts of H, you get shorter tails
H size directly proportional to tail size

Question 18

Similar to H, different siphoviruses have different ___ _____Proteins showing a very strong relationship between gene size and tail sizeTape Measure Proteins

Answer 18

Tape Measure Proteins

Question 19

Phage λ DNA has 12 nt ssDNA ends called ____ _____ on the ends
Genome circularizes after entering the cell through the complimentary cos sequences

Answer 19

cos sites (cohesion)

Question 20

Phage λ: after infection and circularization of the DNA, 2 genes are expressed: ____ and _____

those help make more genes such as ___

Answer 20

N and cro

cll

Question 21

Phage λ: If lysis:

Answer 21

replication, structural and lysis genes turned on

Question 22

Phage λ: If lysogeny: only 2 genes subsequently expressed – cI and int

Answer 22

– cI and int
DNA integrates (by protein encoded by int), then only cI is expressed during lysogeny

Question 23

Phage λ: if lysis, activates expression of ____ and __ genes

Answer 23

structure & lysis

Question 24

Phage λ: if lysogeny: expresses genes ___ and ___

Answer 24

cI and int

Question 25

Immediately after infection, only the cro and N genes get transcribed because only the ___ and ____ are bound by the host's RNA pol

Answer 25

PL and PR

Question 26

transcription stops after cro and N because of the terminators

Answer 26

TL1 and TR1

Question 27

After N and Cro get translated, they bind to DNA and RNA pol at ___ sites

Answer 27

nut

Question 28

nut sites are named for N utilization as N is a ____ _______ protein

Answer 28

anti-terminator

Question 29

N is an anti-terminator so it leads to continuous RNA pol transcription past terminator to make __ gene

Answer 29

cll

Question 30

If cll is acted upon by proteases and inactivated, it means the cell is well growing and active and the _____ cycle occurs

Answer 30

lytic

Question 31

If cll is not inactivated, It means the cell is in poor state, and the _____ cycle occurs

Answer 31

lysogenic

Question 32

When cll gets degraded, activates replication and lysis genes and inhibits cl

Question 33

cll is a transcriptional regulator. It activates expression of __ (the repressor) and __ (integrate)

Answer 33

cl int

Question 34

cl protein function:

Answer 34

turns off all the replication and lysis genes, activates its own expression

Question 35

integrase

Answer 35

protein recombines the genome into the host genome

Question 36

Integration happens by recombination between the __ (attachment phage) and the ___(attachment bacterium) sites which are homologues sequences in the two DNA molecules

Answer 36

attP attB

Question 37

The organization of genes integrated is different than that in the linear virion form.

Answer 37

Genes that were next to each other in the circular form are now at opposite ends

Question 38

cl, the only gene expressed and the only protein made in the lysogenic state. cl is a ____-_____ protein that is only able to bind DNA as a dimer (binds to diff sites w diff affinities)

Answer 38

DNA-binding

Question 39

cl binds to 6 different sites on DNA: operators: (3) ________ which overlap promotors: (2) _____________ and operators: (3)________ which overlap promotor: (1)________

Answer 39

OR1,2,3 overlap PR and PRM OL1.2.3 overlap PL

Question 40

when there is ___ _____, PR and PL are left free and active so it allows for late gene expression Replication, structural, lysis

Answer 40

no cl

Question 41

when there is __ ___ ___, all 6 sites are occupied and PRM, PR, PL are all off so no expression

Answer 41

a lot of cl

Question 42

when there is cl, the cl proteins on the two sets of O sequences interact forming ____ _______ and makes the binding even stronger

Answer 42

DNA loops around

Question 43

preference for binding of cl is OR1>OR2>OR3 where ____ and ____ adjacent dimers interact and have a synergistic cooperate binding effect even stronger

Answer 43

OR1 and OR2

Question 44

The _____ is the weakest of all 6Os, last to be occupied and it overlaps PRM "Promotor for Repressor Maintenance"

Answer 44

OR3

Question 45

if cl levels decrease OR3 is the first to be vacant, frees up ____ and activates cl transcription

Answer 45

PRM This cells cell in lysogenic state

Question 46

cells carrying lambda integrated as a prophage are protected from super-infection by other lambda particles (and similar things) as there is a lot of ___ protein that can prevent expression of all incoming phage genes

Answer 46

cl

Question 47

lambda (and other prophages) can be included to enter lytic cycle by _____ of the cl protien from its DNA and prevention of its binding HOW??

Answer 47

REMOVAL OF cl protein TO ENTER LYTIC

Question 48

induction of the lytic cycle occurs when the cl protein is degraded by the bacterial protease ____

Answer 48

RecA cleaves and inactivates cl preventing its binding to DNA

Question 49

how does RecA work: (2 things)

Answer 49

- happens in response to hosts SOS response (bacteria response to stress such as DNA damaging agents) - cleaves and inactivates cl preventing its binding to DNA

Question 50

RecA is normally a _______ _____ but in response to DNA damage it becomes active as a protease

Answer 50

recombination enzyme

Question 51

RecA targets cellular repressors and removes them especially ______ which triggers the SOS response

Answer 51

LexA

Question 52

LexA normally inhibits expression of _____ _____ so when its removed all the DNA damage stress genes get expressed

Answer 52

stress genes

Question 53

LexA and cl look alike so RecA recognizes it by mistake and it is a way the phage co-opted the cell's damage control system as a cue for activating its lytic replication AKA:

Answer 53

things are going bad for this cell lets get out

Question 54

removal of cl repressor exposes PR and allows for transcription of cro protein

Answer 54

Right promotor

Question 55

Cro turns on expression of early lytic genes and inhibits ___ transcription

Answer 55

cl

Question 56

upon induction ______ occurs, where the phages genome comes out of its integrated into the cells chromosome state into a circle

Answer 56

excision

Question 57

integration requires ____ and excision requires __ and ____ both are translated from the mRNA at the end of the integrated genome

Answer 57

int int and xis (excisionsase)

Question 58

The genome must be in circular excised from for the lytic pathway to :(2 things)

Answer 58

1. replicate the DNA 2. express late genes

Question 59

During the lytic cycle: Integration into the host genome is prevented by a phenomenon called _____

Answer 59

retroregulation

Question 60

Retroregulation: anti termination by N allows PL transcription to proceed past int and xis to ____

Answer 60

sib

Question 61

sib does not encode a protein, so what does it do?

Answer 61

has a sequence that gets targeted by the cellular RNase enzymes, which degrade the mRNA in a 3' to 5' direction

Question 62

due to sib, more xis gets translated before the RNase makes it to it as compared to int, this disproportionate ratio keeps lambda DNA out of the chromosome ONLY WHEN

Answer 62

its in the excised circular form

Question 63

when its inside the chromosome to begin with, sib isn't downstream of int cuz of the attp, so int and xis get transcribed to allow excision, then sib prevents int translation so that the genome can't reintegrate

Question 64

why must the genome be In circular form for the lytic cycle?

Answer 64

1. cuz the structure gene promoter is at the other end of the linear version of the genome that is inside visions 2. cuz the mechanism for DNA replication for phage lambda is the rolling circle replication of a long concatemer that joins at cos sites

Question 65

SaPIs are

Answer 65

Staphylococcus aureus pathogenicity islands

Question 66

diff SaPIs are present in diff Staphylococcus aureus strains to encode various _____ factors that contribute to bacterial pathogenicity

Answer 66

virulence

Question 67

SaPIs are normally not active but they are induced for replication by ______ _____ that have a protein that induces SaPIs to make a protein that hijacks the phage capsid protein and packaging machinery (makes phages with shrunken heads and SaPI DNA)

Answer 67

replicating phages

Question 68

lysed cell releases SaPIs ____ _____and some of the original helper phage ____ _____

Answer 68

smaller heads larger heads

Question 69

released SaPI can

Answer 69

1- go and infect other cells 2- Integrate SaPI DNA into cells genome (recombination at att sites like lambda) 3- SaPI inactive for replication until helper gene comes Results in transfer of virulence factors from cell to cell

Question 70

SaPI encode repressor protein that keeps them inactive ____ (acts as sensor to see if helper is there) when helper phage comes, it encodes the antirepressor protien and SaPI is activated

Answer 70

Stl

Question 71

SaPI proteins inhibit

Answer 71

helper Phage gene replication shrink the head alter the terminase packaging enzymes so that SaPI genes get packaged

Question 72

SaPIs were the first to be discovered, now there is a lot of PICIs "___"

Answer 72

phage-inducible chromosomal islands

Question 73

CRISPR stands for

Answer 73

Clustered Regularly Interspaced Short Palindromic Repeats

Question 74

CRISPER was identified in bacterial genome sequences and it was shown that different isolates of the same species have different and that they are always associated with a set of genes:

Answer 74

sets of spacers cas: crisper assosicated

Question 75

They then noticed that those CRISPER spacers were similar to sequences found in:

Answer 75

plasmids and phages

Question 76

____________ ___________ is an important bacterium used in the production of yogurt and cheese

Answer 76

Streptococcus thermophilus (associated CRISPER by Canadian researcher in U LAVAL)

Question 77

took Streptococcus thermophilus and exposed it to phages and isolated cells that survived the infection, found out:

Answer 77

1- these cells are now more resistant to infection by one or both phages (immunity) 2-the resistant strains had additional new spacers in their CRSIPR region at the left end of the array and they matched sequences from phage genomes (less matches meant less resistance) 3- mutant phages were found with changes in the region corresponding to the resistance spacer, can replicate in cells resistant to the wild type

Question 78

New spacers in resistant LAB cells matched sequences from phage genomes, but partial match = partial (less) ____

Answer 78

resistance

Question 79

phages can bypass CRSIPR resistance by:

Answer 79

1. phages can become mutated (in regions that correspond to spacer making them unrecognizable) 2. phages can produced proteins that inhibit CRISPR-Cas systems

Question 80

some cas proteins: some cas proteins:

Answer 80

-acquiring the new spacers -carry out subsequent gene silencing task

Question 81

steps of CRISPR cas model of function

Answer 81

1. the repeat spacer region in transcribed into RNA 2. repeats fold into hairpins due to palindrome sequences 3. cas proteins process these repeats and cut them into small spacer containing RNAs (sRNA) 4. take the sRNA to target the matching phage or plasmid DNA

Question 82

V.C has SAPI-like island to protect it from phage BUT phage (new!) has Crisper cas, with spacers that match SAPI SO:

Answer 82

Absence of phage CRISPER cas: no viral replication as its blocked by SAPI-like islands Presence of CIRSPER-cas but no SAPI-like spacers, greatly reduced viral replication but some phages pick up the spacer and gain resistance Presence of CIRSPER-cas with SAPI-like spacers: good viral replication and protection from SAPI degredation