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Molecular Biology > Molecular Biology Week 8 > Flashcards

Molecular Biology Week 8 Flashcards

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1
Q

What are the three ways in which bacteria can transfer genetic material?

A

Transformation, transduction and conjugation.

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2
Q

What does Transformation involve (1)?

A

This involves the uptake of naked DNA by competent cells.

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3
Q

What does Transduction involve (2)?

A

This involves the transport of bacterial DNA by bacteriophages.

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4
Q

What does Conjugation (3) involve?

A

This involves the temporary direct physical contact. Transfer of DNA by direct cell to cell contact.

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5
Q

Who discovered Transformation and describe the experiment which shows bacterial tranformation:

A

Discovered by Fred Griffith; the experiment involved the use of living (S) cells with a capsule which were virulent and living (R) cells without a capsule. S cells on their own killed the mouse, heat killed the mouse did not die. Heat killed S cells mixed with the non-virulent R cells caused the mouse to die as a blood sample of the mouse showed that bacterial genetic info transformation had occured.

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6
Q

What can the specific cells which can uptake a naked DNA molecule be called?

A

Competent cells. Naked DNA is incorporated into the recipient chromosome in a heritable form.

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7
Q

What is natural transformation?

A

This is when the process of transformation is random, the DNA comes from a donor bacterium and any portion of a genome may be transferred.

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8
Q

Describe DNA in nature:

A

Occurs when bacteria lyse and release DNA. Fragments can be large and contain several genes and DNA in nature can be bound by a competent call and taken inside.

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9
Q

Describe competency:

A

This is a complex phenomenon depending on several factors:

  • Bacteria need to be in a certain stage of growth.
  • Only certain GP and GN genera.
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10
Q

Can bacteria be artificially competent?

A

Bacteria can also be made competent artificially by chemical treatment and heat shock to make them transiently permeable to DNA.

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11
Q

What is an example of a bacterium that must be in a certain phase of growth for competency?

A

Streptococcus Pneumonia in exponential phase secretes a small protein called competence factor that stimulates 8-10 new proteins.

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12
Q

Describe the 4 steps to bacterial cell transformation of S.pneumonia?

A

Firstly, a competent cell binds a double stranded DNA molecule.

  • Cleaved by an ENDONUCLEASE to fragments 5 to 15kb.
  • Hydrolyzed by an EXONUCLEASE (Strand 1)
  • Move through MEMBRANE, align to homologous REGION and INTEGRATE by recombination (Strand 2)
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13
Q

Describe the 4 steps to ‘transformation’ of a bacterium such as Haemophilus which does NOT produce competence factor:

A

Haemophilus takes up DNA only from CLOSELY RELATED species.

  • Double strand DNA is complexed with protein which is taken inside in MEMBRANE VESICLES (ENDOCYTOSIS)
  • TRANSFERRED DNA MUST HAVE: a special 11bp sequence that is repeated higher than 1400 in Haemophilus infl. DNA.
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14
Q

What are the 2 methods for artificial transformation?

A
  • Heat shock with CaCl2 treatment of cells.

- Electroporation - electric field produces holes in the cell membrane perimeter. Exogenous DNA can enter.

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15
Q

What are 2 common techniques to the artificial transformation of DNA?

A

A high conc of DNA is used (insurance?)

DNA is inserted into plasmid (generally)

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16
Q

Who discovered conjugation ? (2)

A

Lederberg and Tatum.

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17
Q

Describe the Lederberg and Tatum experiment.

A

A - can produce some genes that B cannot, mutated
B - can produce some genes that A cannot
Mix of both A and B = prototrophic colonies, both A and B can produce genes

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18
Q

What did Davis’ experiment demonstrate?

A

That direct cell to cell contact was needed for conjugation

19
Q

What is an auxotroph?

A

Mutants that require added nutrients to grow

20
Q

Describe Davis’s U-tube experiment:

A

The filter allowed solution through, not the cells. Sequestered cells on either side. As cells could not touch - no prototroph could form therefore cells were separate. (with pressure/suction so solutes were mixed)

21
Q

What do donor cells contain, describe length;

A

A fertility factor plasmid (f-factor) It is 100kb long and is a tra operon with genes for pilus formation and gene transfer.

22
Q

What are the three types of donor describe them;

A 
F+ = F factor alone autonomous
F' = Factor autonomous with additional genes
HFr = integrated F factor plasmid
23
Q

What does a recipient have?

A

designated F-

24
Q

Describe the mating of a F+ and F-

A

Pilus forms, extending from F+
TDNA strand of F factor moves
Each strand replicated
F- becomes F+ = acquisition of F factor plasmid.

25
Q

Describe the Hfr and F- mating

A

Hfr has an integrated f plasmid with a functional tra operon.

  • DNA transfer begins with nick in the F factor at the ORIGIN OF TRANSFER
  • While REPLICATING The chromosome moves through pilus.
  • Only part of the F factor is transferred NOT ALL. THEREFORE F - does NOT become donor
26
Q

What does Hfr stand for?

A

This means high frequency of recombination because a very high efficiency of chromosomal gene transfer when compared with F+

27
Q

Describe F’ and F- mating;

A

F’ occurs due to an imprecise excision of integrated F plasmid from a chromosome.
Mating for this is pretty much identical to F+ and F-

28
Q

Does the chromosome move from the donor at a constant rate?

A

Yes it does at a constant rate.

29
Q

What is HFr frequently used for?

A

To map the relative location of bacterial genes.

30
Q

What happens in an interrupted mating experiment?

A

The conjugation bridge is broken at various intervals after the start of conjugation by mixing the culture vigourously.

31
Q

How can the order and timing of the gene transfer be determined?

A

These can be determined as they are a direct reflection of genes on the bacterial chromosomes.

32
Q

What does transduction involve?

A

The transfer of genes by bacterial viruses.
Bacterial genes are incorporated into a phage capsid due to errors in the virus life cycle.
These ‘defective’ viruses then inject them into another bacterium completing transfer.

33
Q

What are the two possible outcomes of the life cycle of a phage?

A
  • Reproductive-lytic cycle

- Dormancy-lysogenic cycle

34
Q

What are the steps involved in transduction (1) - reproductive-lytic cycle?

A

Absorption, DNA replication, protein synthesis, assembly and release.

35
Q

What is transduction (1) - reproductive-lytic cycle caused by?

A

Usually caused by a virulent phage.

36
Q

What are the steps involved in transduction (2) - dormancy-lysogenic cycle?

A

Absorption, penetration, integration into bacterial chromosome, multiplication along with chromosome.

37
Q

What is transduction (2) domancy-lysogenic cycle usually caused by ?

A

By temperate phages, bacteria is LYSOGEN.

38
Q

What is the integrated viral genome called?

A

Prophage

39
Q

What are the two type of transduction (not the life-cycles)?

A

Generalised and Specialised.

40
Q

What is generalised transduction?

A

This occurs as a result of the lytic cycle and any random segments of DNA can be transferred

41
Q

What is specialised transduction?

A

This occurs as a result of lysogenic cycle and only genes adjacent to integrated viruses are tranferred.

42
Q

There are four steps associated with generalised transduction, what are they starting with during maturation and ending with how bacteriophages deliver the donor bacterium’s DNA it is carrying into the recipient bacterium.

A

During maturation, a bacteriophage head or capsid assembles around a fragment of the donor bacterium’s degraded DNA.
The ‘defective’ bacteriophages are released.
The bacteriophage carrying the donor bacterium’s DNA absorbs to a recipient bacteria.
Upon infection the bacteriophage delivers the donor bacterium’s DNA it is carrying into the recipient bacterium.

43
Q

There are three steps associated with specialised transduction, sometimes occurring during spontaneous excision starting with a small piece of the donor bacterium’s DNA adjacent to the bacteriophage to the bacteriophage insertion site is picked up.

A

The bacterial DNA incorporated in the phage capsid replaces some of the phage genome.
These bacteriophages absorb to a recipient bacterium and inject its genome.
The bacteriophage carrying the donor bacterial DNA inserts into the recipient bacterium’s nucleoid.

Molecular Biology (8 decks)

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