Interrupted conjugation mapping and F’ PDF

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Interrupted conjugation mapping and F’...

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Interrupted conjugation mapping and F’ Interrupted mating is the cessation of conjugation by breaking the conjugation pilus. Experiments that test for gene transfer at timed intervals (time-of-entry mapping) are used to determine the distances between genes. A. Describe the process of an interrupted mating experiment B. Create a map of bacterial genes using data from interrupted conjugation with several Hfr strains. Remember that the F plasmid has insertion sequences that control insertion into bacterial chromosome and ultimately excision from it. F factor integration into the bacterial chromosome can take place at any IS element on a bacterial chromosome. At each IS sequence, the episome can be oriented in either of two directions, depending upon how the sequence is inserted. Different Hfr strains have the F factor in different places in the bacterial chromosome, but once F factor insertion has taken place, the location and direction of the F factor remains constant for that Hfr strain.

DNA transfer always begins with a nick in F at oriT, and the orientation and position of F determine the starting point of gene transfer- the gray arrowheads in this figure. Think of the arrows going into the bacterial cells- like the head of a snake with its body trailing along behind. The relative distance between any two pairs of genes, as measured in time of transfer, is constant.

Here is a set of 6 different Hfr strains all with the same 8 genes. At first it looks like the genes are in different orders in the different strains, but remember that start of transfer site and direction of transfer is important. When we line the genes up on top of one another, we can create a complete map of the genome, including the entry and direction of F for each strain.

You can allow cells to conjugate and separate them at specific time points- you basically just need to shake them up so that the pilus breaks- and then you can select to see which genes have transferred in those periods of time.

If you do this with lots of cells, you can plot out the data and determine the location, in minutes, of each gene. Here you can see that for this strain, azi transfers first, then tonA, then lac and finally galB. You can determine how far apart the genes are in minutes by referring to the X axis.

This slide summarizes interrupted conjugation analysis. For each strain, a number of interrupted matings are performed and their data plotted. The data are used to make maps for each strain, and then the strain maps are used to make a composite map of the genome. The yellow map on the outside is the composite map here.

Different Hfr strains transfer genes starting at different locations, depending upon where oriT is located, and in one of 2 different orientations (clockwise or counter- clockwise), depending on the orientation of the oriT sequence. The relative location of the genes (genetic map) remains the same

Here’s a problem for you to try. Make a composite map with the gene locations, distances between them, the oriT locations, and the direction of the oriTs- remember they are like the head of a snake and I’ve drawn them in for you.

Here is the solution. Note that we don’t know the distance between B and G because we have no data on those two genes being transferred together.

There’s one more type of F cell that we need to talk about- the F’. An Fdonor bacterium contains a functional F factor derived from imperfect excision of the F factor from an Hfr chromosome. The resulting Ffactor contains all its own DNA plus a

segment of the bacterial chromosome from which it is excised. Donor cells carrying such an F factor are called Fcells.

Cells that contain a complete Ffactor are partial diploids because they contain two copies of the bacterial chromosome genes found on the Ffactor. The partial diploidy is retained as a characteristic of the exconjugants and their descendants. Partial diploids can be used to examine the mode of action of bacterial genes, and their regulation.

The exconjugants of an F’ x F- mating become F’....