Tc1 Insertion Research Articles

The sqt-1 gene of C. elegans encodes a collagen critical for organismal morphogenesis

Different mutations in the sqt-1 gene of C. elegans can lengthen, shorten, or helically twist the entire animal. We have cloned the sqt-1 gene and have shown that it encodes a collagen. sqt-1 was localized to a 35 kb region of DNA by physical mapping of chromosomal deficiencies. A transposon (Tc1)-induced mutation of sqt-1 was generated and utilized to identify the sqt-1 gene within this 35 kb region. Sequence analysis of the sqt-1 gene shows that it encodes a 32 kd collagen polypeptide that is similar in size and structure to other members of the C. elegans collagen family. The Tc1 insertion mutant has no detectable sqt-1 transcripts, yet it is morphologically normal, indicating that the null phenotype of sqt-1 is wild type. These results demonstrate that collagen mutations can have dramatic effects on organismal morphology.

Molecular cloning and transcript analysis of fem-3, a sex-determination gene in Caenorhabditis elegans.

The fem-3 gene is required for specification of the male fate in the nematode Caenorhabditis elegans: XO males need fem-3 for male differentiation in both soma and germ line; XX hermaphrodites need it for the production of sperm. We have cloned fem-3 by transposon tagging. Among eight spontaneous fem-3 mutations generated in a strain in which the transposable element Tc1 is mobile, six contain Tc1 insertions in a 2-kb region of the genome. From this region, we have identified three fem-3 transcripts. Two, 1.7 kb and 1.62 kb, are presented in embryos, and two, 1.62 kb and 1.55 kb, are present in L4s and adults. The fem-3 transcripts are not XO specific; however, in XX adult hermaphrodites, they appear to be limited to the germ line--a tissue involved in male development (both for spermatogenesis and for the maternal contribution of fem-3 to the embryo). The amount of fem-3 RNA in XO embryos is approximately sixfold greater than in XX embryos. The significance of this difference in specifying male development in XO but not in XX embryos is discussed.

Insertion and excision of Caenorhabditis elegans transposable element Tc1.

The transposable element Tc1 is responsible for most spontaneous mutations that occur in Caenorhabditis elegans variety Bergerac. We investigated the genetic and molecular properties of Tc1 transposition and excision. We show that Tc1 insertion into the unc-54 myosin heavy-chain gene was strongly site specific. The DNA sequences of independent Tc1 insertion sites were similar to each other, and we present a consensus sequence for Tc1 insertion that describes these similarities. We show that Tc1 excision was usually imprecise. Tc1 excision was imprecise in both germ line and somatic cells. Imprecise excision generated novel unc-54 alleles that had amino acid substitutions, amino acid insertions, and, in certain cases, probably altered mRNA splicing. The DNA sequences remaining after Tc1 somatic excision were the same as those remaining after germ line excision, but the frequency of somatic excision was at least 1,000-fold higher than that of germ line excision. The genetic properties of Tc1 excision, combined with the DNA sequences of the resulting unc-54 alleles, demonstrated that excision was dependent on Tc1 transposition functions in both germ line and somatic cells. Somatic excision was not regulated in the same strain-specific manner as germ-line excision was. In a genetic background where Tc1 transposition and excision in the germ line was not detectable, Tc1 excision in the soma still occurred at high frequency.

Insertion and excision of Caenorhabditis elegans transposable element Tc1.

The transposable element Tc1 is responsible for most spontaneous mutations that occur in Caenorhabditis elegans variety Bergerac. We investigated the genetic and molecular properties of Tc1 transposition and excision. We show that Tc1 insertion into the unc-54 myosin heavy-chain gene was strongly site specific. The DNA sequences of independent Tc1 insertion sites were similar to each other, and we present a consensus sequence for Tc1 insertion that describes these similarities. We show that Tc1 excision was usually imprecise. Tc1 excision was imprecise in both germ line and somatic cells. Imprecise excision generated novel unc-54 alleles that had amino acid substitutions, amino acid insertions, and, in certain cases, probably altered mRNA splicing. The DNA sequences remaining after Tc1 somatic excision were the same as those remaining after germ line excision, but the frequency of somatic excision was at least 1,000-fold higher than that of germ line excision. The genetic properties of Tc1 excision, combined with the DNA sequences of the resulting unc-54 alleles, demonstrated that excision was dependent on Tc1 transposition functions in both germ line and somatic cells. Somatic excision was not regulated in the same strain-specific manner as germ-line excision was. In a genetic background where Tc1 transposition and excision in the germ line was not detectable, Tc1 excision in the soma still occurred at high frequency.

Transposable element Tc1 of Caenorhabditis elegans recognizes specific target sequences for integration.

The frequency of movement of Tc1, a 1.6-kilobase transposable element in the nematode Caenorhabditis elegans, is under genetic control, and Tc1 insertion sites are widely but nonrandomly distributed. The usually high frequency of insertions at multiple sites in the gene unc-22 suggested that this gene might be particularly rich in preferred target sites. To discover the features of Tc1 target sites, we have sequenced the sites of seven independent Tc1 transpositions into unc-22 and three other sites. Our comparison of these and two other sites from the literature indicates that in all cases Tc1 integrates at the dinucleotide T-A when it is flanked both 5' and 3' by particular preferred nucleotides. Our analysis revealed the following consensus target for Tc1 integration: G-A-K-A-T-A-T-G-T, in which K = G or T. This target site sequence specificity has implications both for the mechanism of Tc1 transposition and the use of Tc1 in cloning genes by transposon-tagging.

Identification and intracellular localization of the unc-22 gene product of Caenorhabditis elegans.

The unc-22 gene is one of a set of genes identified using classical genetics that affect muscle structure and function in the free-living nematode Caenorhabditis elegans. Since cloning the unc-22 gene by transposon tagging, we have used conventional techniques combined with a set of Tc1 transposon insertion alleles to characterize the gene and its products. The gene extends over more than 20 kb of genomic sequence and produces a transcript of approximately 14 kb. A polyclonal antibody raised against an Escherichia coli beta-galactosidase-unc-22 fusion protein recognizes a polypeptide in nematode extracts that is between 500,000 and 600,000 daltons and labels the muscle A-band in indirect immunofluorescent microscopy. The Tc1-induced alleles have been used at every stage to verify these conclusions. The Tc1 insertions are spread over much of the region that contributes to the mature transcript; in most alleles, Tc1 sequences are incorporated into a composite unc-22-Tc1 transcript. The large protein is either absent or severely reduced in amounts in the mutants. In one case, a truncated polypeptide was also identified. The location of the protein in the A-band, along with earlier genetic data, suggests that the unc-22 product may interact with myosin to regulate its function.

Lin-12, a nematode homeotic gene, is homologous to a set of mammalian proteins that includes epidermal growth factor.

The lin-12 gene of the nematode Caenorhabditis elegans controls certain binary decisions during development. The lin-12 locus was cloned by means of Tc1 transposon tagging: spontaneous lin-12 null alleles were isolated in a genetic background permissive for Tc1 transposition, and seven independently isolated mutations were found to be associated with Tc1 insertion events. All of these Tc1-induced mutations mapped to a single 2.9 kb restriction fragment within a 50 kb region examined. The DNA sequence of this fragment revealed that, although it does not contain the entire gene, it does include three complete exons. These three exons together encode 11 peptide units that are homologous to one another. The repeated peptide motif is also homologous to a set of mammalian proteins that includes epidermal growth factor.

Cloning within the unc-43 to unc-31 interval (linkage group IV) of the Caenorhabditis elegans genome using Tc1 linkage selection.

The region around the twitcher gene, unc-22, flanked by unc-43 on the left and by unc-31 on the right, has been intensively studied in our laboratory over the period of the last 8 years. In this paper we describe the identification and isolation of probes specific for several restriction fragment length differences (RFLDs) which lie within this region. Many RFLDs in Caenorhabditis elegans are caused by the insertion of a transposable element, Tc1. The method we used involved the isolation of Tc1-containing genomic fragments. These were recovered from a lambda gt 10 library of DNA from a specially constructed genetic strain containing the unc-43 to unc-31 interval from the BO strain and the rest of the genome from N2. Because the BO strain is rich in Tc1 insertion sites and the N2 strain has few, the majority of Tc1-bearing genomic fragments in the constructed strain were derived from the unc-22 region. Of nine such Tc1-bearing genomic fragments isolated, six were found which mapped within the region of interest. The 350 kilobases of genomic sequences isolated as a result of these studies are being used to study the molecular organization of this region. The method described here for Tc1 linkage selection is one that is rapid, general, and may be targeted to any genetically characterized region of the C. elegans genome.