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Mapping the Bacterial Chromosome Help (page 3)

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By — McGraw-Hill Professional
Updated on Aug 23, 2011

Establishing Gene Order

Mapping small regions in microorganisms has revealed that multiple crossovers often occur with much greater than random frequency, a phenomenon called "localized negative interference." One unambiguous method for determining the order of very closely linked sites is by means of three-factor reciprocal crosses. Suppose that the location of gene a is known to be to the left of gene b but that the order of two mutants with in the adjacent b gene is unknown. Reciprocal crosses will yield different results, depending upon the order of the mutant sites.

EXAMPLE 10.17   Assume the order of sites is a-b1-b2.

Establishing Gene Order

In the original cross, prototrophs (+ + +) can be produced by crossovers in regions (1) and (3). In the reciprocal cross, prototrophs arise by crossovers in regions (3) and (4). The numbers of prototrophs should be approximately equivalent in the two crosses.

Complementation Mapping

An F particle that carries another bacterial gene other than the sex factor produces a relatively stable F+ merozygote. These partial diploids can be used for complementation tests of mutants affecting the same trait.

EXAMPLE 10.18   An Hfr strain of E. coli is unable to ferment lactose (lacZ1) and can transfer the (lacZ1) gene through conjugation to a mutant (lacZ2) recipient, forming the heterogenote (lacZ2)/(F-lacZ1). If (lacZ1) and (lacZ2) are mutations in the same gene (i.e., functional alleles), then complementation does not occur and only mutant phenotypes are produced. If (lacZ1) and (lacZ2) are mutations in different genes, complementation could produce wild types able to ferment lactose.

Intragenic complementation may sometimes be possible when the enzyme product is composed of two or more identical polypeptide chains. Experimental evidence has shown that an in vitro mixture of inactive enzymes from some complementing mutants can "hybridize" to produce an enzyme with up to 25% normal activity. Mutants that fail to complement with some but not all other mutants are assumed to overlap in function. A complementation map can be constructed from the experimental results of testing all possible pairs of mutants for complementary action in bacterial merozygotes or in fungal heterokaryons. A complementation map cannot be equated in any way with a crossover map, since the gene is defined by different criteria. A complementation map tells us nothing of the structure or location of the mutations involved. Complementation maps are deduced from merozygotes or heterokaryons; crossover maps are deduced from recombination experiments.

Complementation Mapping

This indicates that mutants 1 and 2 are complementary and do not overlap in function. Hence, 1 and 2 are nonallelic mutations by this criterion. Mutant 3 fails to complement with either 1 or 2 and hence must overlap (to some degree) with both 1 and 2. Hence, 3 is functionally allelic with both 1 and 2.

Mapping By Deletion Mutants

A deletion in some segment of a functional gene cannot recombine with point mutations in that same region even though two point mutations at different sites within this region may recombine to produce wild type. Another distinctive property of deletion mutants is their stability; they are unable to mutate back to wild type. The use of overlapping deletions can considerably reduce the work in fine structure analysis of a gene.

EXAMPLE 10.20   Determining the limits of a deletion. Suppose that a series of single mutants (l, 2, 3, 4) has already been mapped as shown below:

Mapping By Deletion Mutants

A deletion that fails to recombine with point mutants 1 and 2 but does produce wild type with 3 and 4 extends over region X. A deletion that yields no recombinants with 3 and 4 has the boundaries diagrammed as Y. A deletion mutant that produces wild type only with point mutants 1 or 4 has the limits of Z.

EXAMPLE 10.21   Assigning point mutations 1–4 to deletion regions R, S, and T.

Mapping By Deletion Mutants

Given the deletions R, S, and T as shown above, the point mutation that recombines to give wild type with deletions S and T, but not with R, is 1. Number 3 is the only one of the four mutants that fails to recombine with one of the three deletions.

Practice problems for these concepts can be found at:

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