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Prokaryotic Molecular Genetics > Plasmids > Flashcards

Flashcards in Plasmids Deck (85):
1

What are Plasmids?

small, circular, extra-chromosomal DNA which replicates independently of host chromosomal DNA

2

Are all plasmids circular?

No, they are not.

3

R-factors in plasmids give bacteria what?

Antibiotic resistance

4

How many proteins can plasmids encode for?

Depending on their size, they can encode either a few or hundreds of different proteins

5

Do plasmids give large amounts of advantages to bacteria?

No, they give bacteria selective advantage only in certain conditions

6

When purifying plasmids, after hours of ultracentrifugation, where in the tube would you find the plasma DNA?

Due to a formation of a gradient, the plasmid would be the lower band showing

7

The EtBr bound to DNA makes it ___________ in salt solution made with heavy atoms such as CsCL.

less dense

8

What are the 3 properties of plasmids?

1. Replicon (DNA molecules that can replicate autonomously in the cells
2. Plasmids has one origin of replication (oriV)
3. The ori region determines the mech. of replication

9

What are the two types of plasmid replication?

1. Theta replication
2. Rolling circle

10

What is Theta replication?

Replication that begins by opening the two strands of DNA at the ori region, creating a structure that looks like the Greek letter "Theta" (circle with line through it)

11

What is unidirectional replication?

Replication that terminates when the replication fork gets back to the origin

12

What is bidirectional replication?

Replication that terminates when the replication fork meets somewhere on the DNA molecule opposite the origin

13

Rolling circle plasmid replication for this mechanism are named ________.

RC plasmid

14

There are two stages to rolling circle plasmid replication. What are they?

1. Replicated and form double and single strand circular plasmid
2. The complementary strand is synthesized on the single-stranded DNA to make another double-stranded DNA

15

What kind of protein is encoded by the plasmid?

Rep protein

16

What are the functions of the Ori regions?

1. Host range
2. Regulation of copy number

17

When dealing with the Host range of functions, what are the ranges seen as functions of the Ori regions?

1. Narrow host range
2. Broad host range
3. Incompatibility
4. Plasmid replication control mechanism

18

What regulation of copy number functions of the Ori region is?

Relaxed plasmid, molecules which inhibit a plasmid replication when the number reach certain level (mechanism present in high copy plasmids

19

Plasmid incompatibility is when plasmids ____________, and one or the other will be lost at a higher than normal rate when the cells divide

can not coexist

20

What is Incompatibility group (Inc group)?

It is the plasmid members of the same Inc group that are not able to coexist

21

Why can't plasmids members of the same Inc group are not able to coexist?

Because they share the same replication control mechanism and/or the same partitioning (par) functions

22

When dealing with plasmid replication control mechanism, what type of plasmid is regulated by a ctrRNA and Protein?

Cole1-derived plasmid

23

R1 and ColB1-P9 plasmid does what in bacteria?

Regulation of Rep protein by antisense RNA

24

How are Iteron plasmids regulated?

Regulation by Coupling

25

What type of plasmid are ColE1-derived plasmid?

Endogenous plasmid

26

ColE1-derived plasmid makes _______ protein

Immunity

27

Many plasmid cloning vector are derived from what type of plasmid?

ColE1-derived plasmid

28

ColE1-derived plasmid replication is regulated by?

RNA I (small RNA encoded by plasmid)

29

In ColE1-derived plasmid, RNA I interfere with the processing of RNA II by?

forming a double strand RNA.

30

RNA I and RNA II are ___________.

complementary

31

What forms the primer for plasmid replication in regards to ColE1-derived plasmid?

RNA II

32

Do ColE1-derived plasmid require encoded proteins to initiate DNA replication at their oriV?

No, they do not require encoded protein.

33

Imm stands for?

Immunity protein

34

Kil stands for?

lyses protein

35

Inc stands for?

RNAI (ctRNA)

36

rnall stands for?

replication primer

37

oriV stands for?

origin

38

Rop does what?

regulates copy number

39

Bom stands for?

origin of transfer

40

Mob stands for?

transfer protein

41

Cer does what?

maintains monomers

42

What does Exc do?

excludes incoming plasmids

43

What does Cea mean?

Colicin toxin

44

What are the two mechanisms of regulation?

1. Stop replication
2. Initiate replication

45

How does stop replication work? (Mech. of Regulation)

Presences of RNA I --> Pairing of RNA I - RNA II (short sequence - kissing complex)-->Rop protein stabilize the kissing complex--> RNA I-RNA II form a double stranded RNA--> Prevent formation of secondary structure of RNA II required to hybridize DNA

46

How does initiate replication work? ( Mech. of Regulation)

Absences of RNA I--> RNA II form a hybrid RNA-DNA at ori--> RNA H cleaved RNA II--> 3'-OH is release serve as primer for first replication

47

More plasmid --> __________ --> stops replication

more Rop + RNA I

48

Less plasmid --> ____________ --> allows replication

les Rop + RNA I

49

Rop + RNA I ---> ?

no plasmid replication

50

RNA II -->?

plasmid replication

51

T or F: ColE1 plasmid do not require a plasmid-encode protein to initiate replication at their oriV, only an RNA primer synthesized from the plasmid

True

52

What type of plasmid-encoded protein do most plasmids require?

Rep

53

What does "Rep" do?

It separates the strand of DNA at oriV region

54

Is "Rep" specific?

It is very specific, only binds to oriV from the same plasmid --> recognize specific sequences

55

T or F: The synthesis of Rep fully controls the copy number of the plasmid

False: it only partially controls the number of plasmid copies

56

What is done to inhibit the synthesis of Rep protein?

An antisense RNA (called CopA)

57

How does antisense RNA work?

Antisense RNA forms a hybrid

58

Hybrid prevents translation of what?

Rep A

59

What does RNase III target?

The Hybrid

60

T or F: RNA III is a chromosomal enzyme

True

61

When does replication occur?

Replication occurs after plasmid enters the cell

62

What is "iteron sequence"?

It is an oriV region that contains several repeats of a certain set of DNA bases

63

The interon sequence has ___ to ___ bp long and exist from ___ to ___ copies in this region.

17 to 22; 3 to 7

64

What does repA gene encode for?

RepA protein

65

What makes RepA protein so important?

it is the only plasmid-encode protein required for replication

66

The host chromosome encodes the other proteins that bind to this region to allow initiation of the replication. What are they?

DnaA, DnaB, DnaC, and DnaG

67

RNA binding does what?

Stimulates replication and decreases replication

68

What stimulates replication?

Low concentration of plasmid

69

What decreases replication?

High concentration of plasmid; Plasmids coupled

70

What are the qualities of a good plasmid for cloning vectors?

1. ori
2. a selectable marker
3. a multiple cloning site (MCS) inside a scorable marker
4. small
5. high copy number

71

The concept of ____________ is important because it is a quick easy, 1-step process of determining whether a transformed bacterial colony has plasmid + insert or not.

Alpha-complementation

72

The key to alpha-complementation is?

The fact that the lac-Z gene product is a tetramer, and each monomer is made of two parts. lacZ-alpha, and lacZ-omega.

73

What is another method of alpha complementation without using a alpha- complement?

Completely delete the lac operon from the chromosome, but introduce lac-Z-omega fragment on an F' fertility factor

74

When a bacteria lose the plasmid during cell division, they are said to be what?

Cured of the plasmid

75

What are the 3 mechanisms to prevent curing of plasmids

1: Addiction systems (plasmid produce toxic protein that kills the cells that lose the plasmid)
2. Site-specific recombinases
3. Partitioning systems

76

Multimer resolving enzymes are what?

Site-specific recombinases

77

What normally acts at the dif site to resolve replicating chromosomes?

XerCD

78

XerCD can also act at the ________ site in ColEl plasmids to resolve plasmid dimers.

Xer

79

P1 plasmid system of E. coli is a prototype comprising a partition site and two proteins, ParA and ParB1. To form the partition complex necessary for segregation, what must happen?

P1 ParB must recognize a complicated arrangement of A-box and B-box DNA motifs located on opposite ends of a sharply bent parS partition site of 74bp.

80

A crucial step in partition is the initial formation of the so-called "__________" between P1 ParB and the partition site.

partition complex

81

Partition complexes are bound by what? (which delivers each plasmid to its correct cellular location)

ATPase ParA

82

ParB of E. coli consists of a proteolytically sensitive __________ (residues 1-141), which binds _______ and forms higher order oligomers and a carboxy-terminal dimerization region (residues 142-333), which contains all of the determinants required for DNA binding.

N-terminal region; ParA

83

The ________site is the minimal partition site required for segregation.

parS-small

84

Partition efficiency is increased when full-length ______ is used and the site is bent by the host auxiliary factor IHF.

parS

85

P1 plasmid system of E. coli is a prototype comprising a partition site and two proteins, ParA and ParB1. To form the partition complex necessary for segregation?

P1 ParB must recognize a complicated arrangement of A-box and B-box DNA motifs located on opposite ends of a sharply bent parS partition site of 74 bp.