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Modern Genetic Analysis Chapter 11. Applications of Recombinant DNA Technology
Solved Problem

2.

A yeast plasmid carrying the yeast leu2+ gene is used to transform nonrevertible haploid leu2 yeast cells. Several leu+-transformed colonies appear on a medium lacking leucine. Thus, leu2+ DNA presumably has entered the recipient cells, but now we have to decide what has happened to it inside these cells. Crosses of transformants to leu2 testers reveal that there are three types of transformants, A, B, and C, representing three different fates of the leu2+ gene in the transformation. The results are:

What three different fates of the leu2+ DNA do these results suggest? Be sure to explain all the results according to your hypotheses. Use diagrams if possible.

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Solution

If the yeast plasmid does not integrate, then it replicates independently of the chromosomes. In meiosis, the daughter plasmids would be distributed to the daughter cells, resulting in 100 percent transmission. This percentage was observed in transformant type C.

If one copy of the plasmid is inserted, in a cross with a leu2 line, the resulting offspring would have a ratio of 1 leu+:1 leu. This ratio is seen in type A and type B.

When the resulting leu+ cells are crossed with standard leu2 lines, the data from type A cells suggest that the inserted gene is segregating independently of the standard leu2+ gene so the leu2+ transgene has inserted ectopically into another chromosome.

When this is crossed with a standard wild-type strain,

then the following segregation results:

The data from type B cells suggest that the inserted gene has replaced the standard leu2+ allele at its normal locus.

When crossed with a standard wild type, all the progeny will be leu+.

See question

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