Worm Breeder's Gazette 9(2): 45
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.
We have previously described our increasingly fine structure mapping of Bergerac Tc1 elements in the lin-14 gene region (Newsletter Vol.8 No.l, No.3). This mapping generated via a lin-14 intragenic recombination event a hybrid Bristol:Bergerac lin-14 gene containing the wild type Bergerac portion to the left and the dominant lin-14 allele (n536) in the Bristol portion to the right, lin- 14( Bergerac:Bristol n536). The recombination point which generated this hybrid gene was mapped by detecting two EcoRI fragment length polymorphisms flanking this recombination point: a Bergerac-specific 1. 5 kb EcoRI fragment and a Bristol-specific 1.4 kb EcoRI fragment were both found in the lin-14 intragenic recombinant strain. These data show that this recombination event took place between the two RFLPs which are located 25 kb apart on one of the cosmids we isolated from this region, EEG4. Therefore cosmid EEG4 must contain the site of the lin-14 intragenic recombination event and must contain at least part of the lin-14 gene. We used cosmid EEG4 as a probe to Southern blots of DNAs isolated from a total of 19 strains containing independent lin-14 alleles and 15 non-lin-14 strains. We have detected allele specific polymorphisms associated with both of the existing dominant lin-14 mutations and two of 17 lin-14 recessive mutations. Three of these four detected alleles were induced by gamma-ray and one by EMS. The two recessive alleles map to adjacent EcoRI fragments and are probably deletions. The two dominant alleles map about 15 kb to the right of the recessive alleles and to the same 6.0 kb HindIII fragment but probably do not overlap. They are deletions of 600 bp (n536) and 300 bp (n355). These polymorphisms associated with dominant alleles are also detected in all recessive revertants of lin-14 dominant mutations, showing that the revertants still contain a cryptic dominant allele. Using cosmid EEG4 as a probe, no changes in the lin-14 restriction map were detected in any of 15 non-lin-14 strains examined. The cosmid EEG4 was used as a probe to RNA isolated from staged N2 and unsynchronized cultures of a variety of lin-14 mutant strains. Two mRNA species were detected using this probe, one at about 7 kb and one at 3.5 kb. The 3.5 kb mRNA decreased in size to about 3.0 kb in all strains containing the lin-14 dominant allele n536 and to about 2. 0 kb in all strains containing the lin-14 dominant allele n355. The 7 kb mRNA is unchanged in these strains. In addition, in all strains containing the dominant alleles the level of the 3.5 kb related mRNA is increased about 2 to 4 fold relative to N2 (normalizing to actin-1 mRNA), regardless of whether a recessive lin-14 allele is present in cis (6 out of 14 alleles have been tested). The fact that the level of the 3.5 kb lin-14 mRNA is increased, for example, in both lin-14( n536) strains showing a dominant retarded phenotype and in lin-14( n538n536) strains showing a recessive precocious phenotype argues that this increase in mRNA is not a result of either retarded or precocious development but under a control independent of the execution of early or late cell lineages in the animal. We have not analyzed all mutants yet, but so far none of the recessive lin-14 mutations significantly decreases the amount of mRNA. Northern analysis with individual DNA probes from the region of the recessive mutations and from region of the dominant mutations showed that the 3.5 kb related mRNA is transcribed from both of these genomic regions which are about 12 kb apart. Most likely, there is an intron(s) in this interval. In staged wild-type worms, the amount of 3.5 kb lin-14 mRNA relative to actin-1 mRNA is relatively high in eggs and L1 and decreases until it is down about 20-fold in the L4. The mRNA level increases again in adults presumably due to oogenesis. The level of this mRNA is 0.005% to 0.01% of the total mRNA in unsynchronized N2. The 7 kb mRNA detected with cosmid EEG4 is present at the same level relative to actin-1 at all stages of N2 development. We find that the direction of transcription of the 3.5 kb related mRNA is oriented left to right on the genetic map. Therefore the recessive mutations are located toward the 5' end of the gene and the dominant mutations at the 3' end. We have not yet explained how a 3' deletion in an mRNA can cause an increase in the amount of that same mRNA and inappropriate activity of the gene product, although two attractive but speculative models come to mind. In one model, there is a sequence located at the 3' end of the lin-14 mRNA which is involved in the degradation of the mRNA as larval development progresses; deletion of that sequence results in abnormal stability of the mRNA and associated temporally inappropriate lin-14 gene activity. In the other model, there is a COOH terminal domain of the lin-14 gene product which is involved in either direct or indirect negative feedback control of lin-14 transcription; deletion of that domain results in loss of this negative feedback control and excessive late larval lin-14 gene activity. These models predict very different DNA sequences in the region of the mRNA deleted in these dominant mutants. These data corroborate the predictions of the genetic and developmental analyses of lin-14 which showed that the lin-14 gene product functions during early postembryonic development. Presumably the lin-14 mRNA is synthesized in the egg to allow the L1 function of the gene product. In addition the genetic analysis of the dominant mutations suggested that in normal development the level of lin-14 gene activity decreases as the larvae develops, and that the dominant mutations inappropriately express a wild-type lin- 14 gene activity at late times in development. Our finding that the level of lin-14 mRNA follows this pattern suggests that of lin-14 gene activity in both wild-type and dominant mutant animals is controlled at least in part by changing lin-14 gene transcription or mRNA stability.