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Applied Genetics > Transposable genetic elements > Flashcards

Transposable genetic elements Flashcards

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Biology 101 flashcards
1
Q

what is a replicon

A

DNA that is replicated in a cell

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2
Q

examples of replicons

A

chromosome

plasmid

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3
Q

what are transposable elements

A

DNA sequences which can ‘hop’ from a site on one replicon to a site on another (or same) replicon

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4
Q

what did transposable elements used to be called

A

Insertion Sequences (IS) or transposons (Tn)

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5
Q

what is the simplest are smallest transposable elements

A

insertion sequences

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6
Q

how many insertion sequences are there in a bacterial chromosome

A

May be multiple copies of IS in a bacterial chromosome

Used to “type” bacteria

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7
Q

what is a transposase

A

Code only for the peptide responsible for their transposition

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8
Q

do transposases have an advantage

A

do not confer any particular phenotypic trait, (don’t have any advantage)

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9
Q

what are ITRs

A

inverted terminal repeats

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10
Q

what do ITRs do

A

flank the coding region

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11
Q

what do most transposable elements have

A

an ITR

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12
Q

what is the IS1 structure

A

host DNA
inverted repeats
base pairs
central region ‘base pairs’ encodes transposase

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13
Q

what is the target site in transposition of IS elements

A

site of integration

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14
Q

what happens in transposition of IS elements

A

Staggered cuts are made in DNA at target site by transposase
IS element inserts
DNA polymerase and ligase fill the gaps

(note—transposase behaves like a restriction enzyme)

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15
Q

what does transposition of IS elements create

A

Small direct repeats (~5 bp) flanking the target site are created

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16
Q

what can integration of IS element cause

A
  • Disrupt coding sequences or regulatory regions
  • Alter expression of nearby genes
  • Cause deletions and inversions in adjacent DNA
  • Result in crossing-over
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17
Q

what is a composite transposon

A

Code for selectable marker gene(s)

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18
Q

why are new composite transposons readily formed

A

As IS may be in either orientation

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19
Q

what flanks marker gene(s) in composite transposon

A

IS elements

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20
Q

what is the function on transposon carried by

A

IS

- one may be defective

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21
Q

what are selectable marker examples

A

lux genes (glows)
antibiotic resistance
ability to metabolise certain sugars

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22
Q

what happens in composite transposons

A

Two insertion sequences flanking internal DNA
Encode antibiotic resistance genes, etc.
IS can transpose independently, or composite Tn can transpose as a unit

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23
Q

what is the function of P out

A

can switch on chromosomal genes downstream of insertion site but is also used by Tn10 to regulate transposase activity

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24
Q

what can you insert if have a set of genes without promoter but want to turn them on

A

P out promoter and know works as this promoter has antibiotic
resistance so will survive

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25
Q

what enzyme is involved in tetracycline resistance

A

P out

prevents ribosomes binding to translation start on transposase mRNA, decrease transposase synthesis

26
Q

what do non-composite transposonscarry

A

Tn carries at least three genes:
Marker gene e.g. antibiotic resistance
Transposase
Resolvase

27
Q

what is the transposase in non-composite transposon for

A

for integration

28
Q

what is the resolvase for in the composite transposon

A

resolves the co-integrate in the second step of the transposition process via res site
i.e. has 2 separate proteins that allows non-homologous recombination events to occur

29
Q

what is resolvase

A

protein

30
Q

what don’t the Tn3 family have

A

IS at termini

31
Q

what do Tn3 family have

A

long inverted repeats at termini

32
Q

where are Tn3 found

A

both Gram-negative and Gram-positive bacteria

33
Q

where are the Tn insertion sites

A

they may not always be random
Tn5 and 7 have insertional bias
may favour GC rich

34
Q

what is Tn insertional bias

A

At certain points there is some bias, likes having more of these (Tn insertions) than less of them

35
Q

what does Tn insertions require

A

long inverted sequences

36
Q

what are Mariner transposons

A

Mariner-family transposable elements are a diverse and taxonomically widespread group of transposons occurring throughout the Animal kingdom

37
Q

what is the mariner transposable element known to yield

A

a random distribution of insertions

38
Q

where are mariner elements inserted

A

at TA dinucleotide pairs in the target sequence and transpose independently of any host factors

39
Q

what does the insertion of mariner transposons cause

A

adds another base

40
Q

what are the types of transposition

A

conservative

replicative

41
Q

what happens in conservative transposition

A

cut-and-paste

42
Q

what happens in replicative transposition

A

leaves an original copy and replicates itself​Transposon duplicated during transposition
Results in formation of cointegrate intermediate

43
Q

what resolves cointegrate

A

resolvase

44
Q

what does does resolvase do

A

cuts four DNA strands, conformational change, put the right ones together​

45
Q

what can go wrong in replicative transposition

A

may not properly separate, DNA cannot be replicated and the organism dies (catenated DNA)

46
Q

what are transposons used for

A
  • Tn insertions can be found in large number of sites on the bacterial chromosome
  • Tn insertion in a gene usually causes complete loss of function
  • phenotype of the insertion mutation is completely linked to antibiotic resistance
47
Q

what does Tn insertion cause

A

Disruption of open reading frame

Many Tns cause polar mutations

48
Q

where can Tn insertions be found

A

In principle it’s possible to find Tn insertions in any non-essential gene in the genome

49
Q

how is it possible to transfer a mutation into a new strain using transposons

A

by selecting for antibiotic resistance

50
Q

how are transposons made mutant

A

Tn must be delivered into cell

51
Q

how is Tn delivered into cell

A

Often achieved by transformation of plasmid containing Tn sequence

52
Q

what is needed for Tn delivery into cell

A

Only feasible if we have an organism that we can transform with high efficiency
e.g. need a high efficiency plasmid

53
Q

what must happen after plasmid has inserted DNA sequence in transposon delivery

A

Need to get the plasmid in to insert the DNA sequence, then need to lose the plasmid from the bacteria

54
Q

what does losing the plasmid from bactiera after insertion allow us to see

A

Allows us to distinguish cells with Tn on plasmid and Tn in chromosome

55
Q

what is a good method to use in transposon delivery

A

use special donor (mobilising) strains & conjugation

56
Q

where must the plasmid go to in transposon delivery

A

plasmid with Tn can replicate in donor strain (correct oriV site)

57
Q

what is used to transfer plasmid into cell in transposon delivery

A

Conjugation used to transfer the plasmid carrying Tn into target strains (broad host range)

58
Q

how will the plasmid with Tn replicate in the cell

A

Plasmid with Tn cannot replicate in this strain cell)

Only cells with Tn in chromosome will be resistant to antibiotic

59
Q

what do mini transposons have

A

transposase outside of the transposable element

60
Q

what happens to a plasmid that is taken from E. coli and put into Pseudomonas

A

plasmid that carries transposon can be mated into Pseudomonas but can only replicate in E. coli
following conjugation to insert Tn plasmid into Pseudomonas

61
Q

how are transposants selected for Pseudomonas when plasmid from E. coli insert

A

transposants selected for on Pseudomonas selective agar supplemented with kanamycin; so only Tn containing Pseudomonas can grow

62
Q

what do all mutants in Tn library ideally have

A

a single Tn insertion so any change in phenotype is linked to a single locus

Applied Genetics (20 decks)

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