Segregation of Alleles

Segregation of Alleles

..... One of strong driving forces in biology is commonly called genetic mixing. ..While it is possible for the worst genes in each parent to come together to form a child, it is also possible for the best ones to get together also to form a superior child. ..By 'superior' is meant a child that is more fit - "survival of the fittest"!

..... But such genetic mixing requires a number of steps for diploid parents. ..They must first form haploid gametocytes, and that means their diploid chromosomes must partition themselves into two sets. ..This partitioning can be called genetic segregation. ..Thus one chromosome might have a brown eye color gene, while the other a blue eye gene. ..Placing the brown gene in one gamete and the blue one in another is the segregation of alleles.

..... Most commonly in labs, varieties of maize (corn) or fruit flies (Drosophila melanogaster [literally "black-throated dew lovers"]) are employed in making crosses that illustrate the segregation and recombining of various alleles and their linkages with other traits. ..While such experiments are considered fast in that they take only two weeks or so for the teacher to prepare and grow the organisms, there is a MUCH faster way to show the segregation of alleles. ..By fast, how about within a week!

..... ..There are interesting strains of the bacterium Escherichia coli which are partially diploid. ..And we know that they are diploid in the same sense as we usually speak of in eukaryotes because we can see the segregation of alleles. ..If this sounds opaque, consider that a cell might possess two alleles in either of two physical ways.

..... But we are interested in the first case, where they are on different chromosomes. ..So let us now begin "building" our model organism. ..First we start with an ordinary (prototrophic) strain of E. coli:

Genetic linkage map of E. coli

..... When this prototrophic or "wild" strain grows on MacConkey Agar, dark red colonies form overnight.

..... Then suppose we bombarded a few million of these "wild" cells with ultraviolet light, and then replated them on MacConkey agar. ..We would find that a few of the millions of colonies grew up pale in color because the UV light had damaged some component of their lac-operons so that the bacteria could not use the lactose in the medium to make acid that causes the colonies to grow up red. ..So now this is what we have:

Genetic map of E. coli lac<SUP>-</SUP>

..... Again: this grows up as pale colored colonies on MacConkey agar plates because it cannot use the lactose in the medium.

..... After some fancy tricks that will not be described here, this lac-operon mutant is mated with a male cell that possesses an interesting plasmid that is called "F-lac." ..This is a normal F-plasmid ("F" is for 'fertility') that has a normal lac-operon inserted in it. ..(Since all this can be done by natural means, the use of these organisms does NOT fall under the mandates and restrictions of the NIH Guidelines for the use of Recombinant DNA). ..What we have is this:

Genetic map of an F-lac cell

..... As you can see from the figure, this cell has two alleles of the lac-operon. ..One is normal and the other is mutant. ..In this case the normal one is dominant as it is expressed. ..Cells of this "F-lac" variety grow up as red colonies on MacConkey agar plates.


Segregation of the Lac-Operons

..... So long as we keep growing these bacteria in a medium containing only water, a few different minerals, a trace of thiamine (which E. coli make poorly), and LACTOSE, only those cells that have a functioning lac-operon will survive and produce progeny. ..BUT what if we substitute GLUCOSE instead of lactose? ..The lac-operons are no longer needed. ..If they become lost, well -, that's just a little extra DNA that doesn't need to be made. ..Soon, if those glucose-grown cells are plated on MacConkey agar, which contains lactose, one expects to be able to see colonies of those cells which have lost the ability to use lactose. ..(There are other goodies in MacConkey agar upon which the lac- cells feed and grow.) ..If we can do this, then we shall have shown that we have been able to partition or segregate the lac+ alleles from the lac- alleles.


MATERIALS AND METHODS

Bacterial strains:

Sterile L-broth ("L" is for Sal Luria, Nobel laureate): 0.3% glucose, 0.7% tryptone, speck of thiamine (vitamin B1), tap water (for minerals).

Several MacConkey agar plates.

..... Day One: Use one MacConkey agar plate to paint stripes of each strain side-by-side. ..Make sure the bacteria grow up with the color stripes they are supposed to: red, pale, red, respectively.

..... Inoculate the L-broth with very few of the F-lac cells. ..If you use a wire inoculation loop, if you can see any smudge of bacteria - any at all! - on the loop, you have too many!

..... Day Two: Make serial dilutions of the L-broth culture and plate them on the remaining MacConkey agar plates.

..... Day Three: Inspect the plates. ..You should see isolated colonies on many of the plates. ..But do you see only red colonies? ..Or are there a few or many pale colored ones? ..(Those pale ones are those which lost their normal allele for the lac-operon.)



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