Molecular cloning and characterization of the dpy-20 gene of Caenorhabditis elegans.
We describe the molecular analysis of the dpy-20 gene in Caenorhabditis elegans. Isolation of genomic sequences was facilitated by the availability of a mutation that resulted from insertion of a Tc1 transposable element into the dpy-20 gene. The Tc1 insertion site in the m474::Tc1 allele was identified and was found to lie within the coding region of dpy-20. Three revertants (two wild-type and one partial revertant) resulted from the excision of this Tc1 element. Genomic dpy-20 clones' were isolated from a library of wild-type DNA and were found to lie just to the left of the unc-22 locus on the physical map, compatible with the position of dpy-20 on the genetic map. Cosmid DNA containing the dpy-20 gene was successfully used to rescue the mutant phenotype of animals homozygous for another dpy-20 allele, e1282ts. Sequence analysis of the putative dpy-20 homologue in Caenorhabditis briggsae was performed to confirm identification of the coding regions of the C. elegans gene and to identify conserved regulatory regions. Sequence analysis of dpy-20 revealed that it was not similar to other genes encoding known cuticle components such as collagen or cuticulin. The dpy-20 gene product, therefore, identifies a previously unknown type of protein that may be directly or indirectly involved in cuticle function. Northern blot analysis showed that dpy-20 is expressed predominantly in the second larval stage and that the mRNA is not at all abundant. Data from temperature shift studies using the temperature-sensitive allele e1282ts showed that the sensitive period also occurs at approximately the second larval stage. Therefore, expression of dpy-20 mRNA and function of the DPY-20 protein are closely linked temporally.
Clark
D V
DV
Department of Biological Sciences, Simon Fraser University, Burnaby, B.C., Canada.
Suleman
D S
DS
Beckenbach
K A
KA
Gilchrist
E J
EJ
Baillie
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017238:Genes, Helminth; D015698:Genomic Library; D005838:Genotype; D015801:Helminth Proteins / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013696:Temperature; D013997:Time Factors
D029742:Caenorhabditis elegans Proteins; D015801:Helminth Proteins; D011994:Recombinant Proteins; C093944:dpy-20 protein, C elegans