Worm Breeder's Gazette 12(2): 38 (January 1, 1992)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Using recombination in yeast to narrow the lin-31 locus from 190 kb to 8.5 kb

Leilani M. Miller, Maria E. Gallegos, Stuart K. Kim

Figure 1

Department of Developmental Biology, Stanford University Medical Center, Stanford, CA 94305

We are initiating molecular studies to show how lin-31 acts to alternatively specify three different vulval cell fates, and to determine how upstream vulval determination genes regulate lin-31 activity (see also Morisseau and Kim, WBG, this issue). In our efforts to clone lin-31 ,we used a yeast recombination strategy that allowed us to rapidly narrow lin-31 rescuing activity within a large YAC clone.

The cloning of lin-31 was initiated by identifying a candidate lin-31 Tc1 insertion by transposon tagging (S. Kim and H. R. Horvitz). Although this insertion site appeared to lie within the lin-31 locus (tight genetic linkage, insertions in multiple lin-31 alleles), we now know that this site is about 20 kb away from lin-31 (see below).

Cosmid rescue experiments showed that lin-31 rescuing activity is not located to the right of the Tc1 insertion. We could not use similar experiments for the region to the left of the Tc1 insertion, due to the presence of a 10-30 kb cosmid gap located nearby. Instead, we showed that injection of Y14H12 (a 190 kb YAC that spans the cosmid gap) can rescue the lin-31 mutant phenotype. We then used recombination in yeast to generate a nested series of Y14H12 deletions. To do this, we cloned random fragments from F39D1 (the first cosmid to the left of the cosmid gap) into the yeast recombination vector pRB328 ,which contains the HIS selectable marker and vector sequences homologous to the end of the YAC (amp, ori). We then transformed the linearized library of random clones into a yeast strain carrying Y14H12 and selected for His+ colonies. These colonies represent cases in which the linear ends of the recombination vector have copied sequences from Y14H12 by gap repair (see Figure). The resulting recombination products contain inserts of worm DNA whose endpoints are defined by the random fragment from the cosmid F39D1 and the right end of the YAC Y14H12 .The sizes of the YAC derivatives were determined by Southern blot analysis using the starting cosmid ( F39D1 )as a probe. This recombination technique is useful because a nested series of YAC deletion derivatives can be rapidly created. In our case, it allowed us to engineer a yeast clone that contained a cosmid gap. Also, circular YAC derivatives can be efficiently isolated from yeast by alkaline lysis.

Microinjection experiments showed that a 100 kb circular YAC derivative, but not a 94 kb circular YAC derivative, contains lin-31 rescuing activity. This result suggested that a region contained within F39D1 is required for lin-31 rescuing activity, so we next injected F39D1 itself. Surprisingly, we found that F39D1 also has lin-31 rescuing activity, indicating that the Tc1 insertion sites are not in the lin-31 gene because they are not contained in the rescuing cosmid. Subsequent experiments further delimited the rescuing activity to 8.5 kb. Four lin-31 alleles (3 mutator, 1 EMS) are polymorphic in this 8.5 kb region. In addition, at least three related classes of cDNAs have been isolated and Northern blot analysis using one of the cDNAs as a probe has identified four transcripts in the region. Sequencing of the region is in progress.

[See Figure 1]

Figure 1