Worm Breeder's Gazette 13(2): 92 (February 1, 1994)
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.
The generation of polarity during metazoan development involves the formation of asymmetric cells and structures and the orientation of these cells and structures with respect to the rest of the organism. While some progress has been made in understanding how asymmetry arises, how asymmetric cells and structures are oriented has been less explored. To this end we have been studying the gene lin-44 ,mutations in which cause the polarities of the asymmetric cell divisions of the B, F, U, and T cell lineages to be reversed. Animals homozygous for either of two reduction-of-function lin-44 alleles or heterozygous for either allele over the deficiency hDf7 (which spans the lin-44 locus) display polarity reversals. We also have observed polarity reversals of cell divisions in the T cell lineage of deficiency homozygotes. These results indicate that the normal function of the lin-44 gene is to define the polarities of specific cell divisions (Herman and Horvitz, 1994, Development, in press).
To learn how lin-44 controls cell polarity at the molecular level, we have cloned the lin-44 locus. It was previously reported that the endpoints of hDf7 have been mapped to cosmids on the unc-73 contig and that microinjections of a YAC clone, Y48F5 ,that spans this interval rescues the lin-44 phenotype, but that cosmids in the interval do not rescue (Herman and Horvitz, 1991 Meeting Abstracts, p.158). It thus appeared that lin-44 lies in one of two regions in this interval not covered by cosmid clones. Recently, we reported the isolation of derivatives of Y48F5 specific to each "cosmid gap" using homologous recombination in the YAC-containing yeast. A derivative specific to the gap between C24G7 and K05E6 (GAPl) rescues the lin-44 phenotype in microinjection experiments (Herman et al., 1993 Meeting Abstracts, p. 191).
In order to narrow the rescuing region further, we used end sequences from the cosmids adjacent to GAPl to probe genomic lambda libraries (see Figure). The G1 Rprobe was used to isolate SP#11 from a C. elegans genomic lambda library (provided by H. Browning and S. Strome, Indiana University). We constructed a genomic lambda library from the yeast strain containing GAPl. From this library, the G1 Lprobe was used to isolate SP#12 .Southern blot analysis demonstrate that these genomic lambda clones overlap. Microinjection of SP#11 or SP#12 into lin-44 mutant hermaphrodites rescues the lin-44 mutant phenotype. This delimited the rescuing region to about 8 kb. We have microinjected subcloned fragments of SP#11 to test for rescue of the lin-44 mutant phenotype. So far, the smallest rescuing subclone is 5.7 kb (see Figure) and we are currently testing smaller subclones. Using p11 SS c7 .5(see Figure) as a probe, we have isolated several potential lin-44 cDNA clones. We have also generated a transgenic array to use for mosaic analysis of lin-44 by coinjecting p11 SS c7 .5( lin-44 (+)),with cosmid C33C3 ( ncl-1 (+)),and R1 p16 ( unc-36 (+);provided by L. Lobel, MIT into lin-44 ; ncl-1 unc-36 hermaphrodites. We have identified four transgenic lines suitable for mosaic analysis.