Conjugation

Question 0

Name two groups of bacterial mutants.

Answer 0

Nutritional mutants - some can make amino acids, some can not. So loss of enzymes disables bacteria from growing in basic simple medium which means a particular nutrient must be added. These mutants are also called auxotrophs.

Antibiotic mutants - bacteria can be selected using antibiotics if they have certain genes.

Question 1

Why are bacteria useful?

Answer 1

Can be grown in vast numbers
Haploid - no dominant/recessive properties
Allow biochemical studies - enzyme reactions for example.
Mutations allow us to understand what genes do - genetic analysis.

Question 2

What is transformation?

Answer 2

DNA can be added to bacteria and taken up at low frequency. In nature, bacteria that spontaneously lyse, release DNA into the medium and this can be taken up by other bacteria. This DNA can recombine with existing DNA in the bacteria to produce new genotype/phenotype.

Question 3

What’s the frequency for transformation?

Answer 3

Transformation usually occurs for 1 in 1000 cells so at 10-3. If two genes are to be taken up then frequency is 10-3 x 10-3 = 10-6.

Question 4

Why do we need transformation?

Answer 4

Transformation allows us to find how near genes are to one another. This is called mapping.

Question 5

What is conjugation?

Answer 5

The unidirectional transfer of genetic information between cells by cell-to-cell contact. This uses genes and sequences present on primitive plasmids called F plasmids.

Question 6

Define ‘unidirectional’.

Answer 6

One copy of the DNA is transferred from one cell - the ‘donor’ to the ‘recipient’

Question 7

What is the site of initiation called?

Answer 7

oriT (origin of transfer) or mob region (mobility).

Question 8

What does the F factor in the plasmid containing the transfer genes code for?

Answer 8

Pilus formation draws the donor and recipient cells together, making a bridge. This also promotes transfer and replication.

Question 9

What F factor symbol is used for donor and recipient?

Answer 9

Donor: F+
Recipient: F-

Question 10

What are the two states the F factor can exist in?

Answer 10

F+ refers to a factor on a plasmid in an extrachromosomal state.
Hfr (high frequency recombination) state describes the situation when the factor has integrated itself into the chromosome.

Question 11

Describe the steps for plasmid transfer.

Answer 11

The F+ donor cell containing an F plasmid is capable of synthesising a sex pilus.
The sex pilus contacts the recipient F- cell.
The plasmid is activated for transfer when an endonuclease cleaves one strand of DNA at the origin of transfer.
The sex pilus retracts and pulls the donor and recipient cells together. The F plasmid is transferred as a single stranded DNA molecule.
The complementary strands to both F plasmid strands are synthesised in the donor and recipient cells. Both cells are F+ and synthesise the sex pilus.

Question 12

How are genes mapped?

Answer 12

By selecting each of the bacteria in medium showing transfer of the gene following growth in medium where nutrients are not supplied.

Question 13

What are the problems with mapping using F plasmids?

Answer 13

They are limited to few bacterial types such as E. coli and it’s close relatives.
Mapping of the whole chromosome is rare.
They can only transfer genes when next to each other and therefore genes that are distant from one another may not be mapped.
As F plasmids are large, low-copy and conjugative plasmids, although they are capable of chromosomal mobilisation they are too large to physically handle with any ease for genetic manipulation and gene cloning.

Question 14

Define transduction.

Answer 14

The transfer of genetic information between cells through the mediation of a virus (bacteriophage) particle.

Question 15

What is the life cycle of a virion?

Answer 15

The virions attach to the cell membrane. The proteins of the capsid inject the DNA core into the cell. Once within the cell, some of the bacteriophage genes (the early genes) are transcribed by the hosts RNA polymerase and translated to produce enzymes that will make many copies of the phage DNA and will turn off (even destroy) the hosts DNA. The bacteriophage cuts up the bacterial DNA. As fresh copies of phage DNA accumulate, other genes (the late genes) are transcribed and translated to form the proteins of the capsid. The stockpile of DNA cores and capsid proteins are assembled into complete virions. Another ‘late’ gene is transcribed and translated into molecules of lysozyme. The lysozyme attacks the peptidoglycan wall from the inside. The cell ruptures and releases it’s content of virions ready to spread to new host cells. Some bacterial DNA may be inserted into the DNA of the new host, replacing those already there and giving the host an altered phenotype - be able to grow in different media or in antibiotics.

Question 16

What are the two types of bacteriophage with different life cycles?

Answer 16

Lytic
When a lytic phage injects it’s DNA into a host cell, it begins an ordered sequence of gene expression that involves the generation of many copies of the phage DNA, typically in the form of long concatamers (attached phage genomes). The phage also direct the generation of proteinaceous ‘heads’ and pack their DNA into these heads. When ‘mature’ phage are assembled, they cause lysis and release approximately 102 to 103 progeny.

Lysogenic
Upon injecting their DNA into the host under some conditions, they follow a route similar to that chosen by lytic phage. However, they also possess the ability to repress their lytic functions and take up semi-stable residency in the host. This residency typically takes the form of an integration of the phage DNA into the host chromosome, usually by a site-specific recombination event. The resulting integrated phage is termed a prophage. This integration event is reversible and is driven largely by phage genes. If a prophage decides to lyse the cell, it excises itself from the chromosome, begins expression of DNA synthesis and other lytic functions, packages it’s DNA appropriately, and lyses the cell.

Question 17

Phages package DNA by ‘headfull packaging’. What does this mean?

Answer 17

Fill the head with a little more than one phage length of DNA. They typically recognise their own DNA as appropriate for packaging because of special sequences termed pac sites. Occasionally, the phage make an error and begin to package a host gene. The P1 phage does this. It occurs because a site on the host DNA is similar to the phage pac site.

Question 18

What is P22?

Answer 18

An example of a mutant which has completely lost the ability to recognise pac sites so that it packages bacterial DNA randomly. Since an infected cell contains about half lost and half phage DNA, the P22 mutant makes a generation of a phage in which half of the phage heads contain host DNA.

Question 19

What is a transductant?

Answer 19

A recipient cell whose genotype and phenotype have been changed by a transduction event.

Question 20

How can mapping be done?

Answer 20

By isolating host bacterial DNA, cutting it up then cloning the DNA onto plasmids. When DNA from transduction is identified then genes can be ordered and therefore mapped as those close to each other because they were found together on the same plasmids.

Question 21

Where two fragments of DNA are found with homology then the fragments are linked. True or False?

Answer 21

True.