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D Hirsh

Publications and source records attributed to D Hirsh.

At least 55 records · Page 3Linked to original sources
PubMed1986

beta-Glucuronidase from Escherichia coli as a gene-fusion marker.

We have developed a gene-fusion system based on the Escherichia coli beta-glucuronidase gene (uidA). The uidA gene has been cloned from E. coli K-12 and its entire nucleotide sequence has been determined. beta-Glucuronidase has been purified to homogeneity and characterized. The enzyme has a subunit molecular weight of 68,200, is very stable, and is easily and sensitively assayed using commercially available substrates. We have constructed gene fusions of the E. coli lacZ promoter and coding region with the coding region of the uidA gene that show beta-glucuronidase activity under lac control. Plasmid vectors have been constructed to facilitate the transfer of the beta-glucuronidase coding region to heterologous control regions, using many different restriction endonuclease cleavage sites. There are several biological systems in which uidA-encoded beta-glucuronidase may be an attractive alternative or complement to previously described gene-fusion markers such as beta-galactosidase or chloramphenicol acetyltransferase.

Base Sequence↗
PubMed1985

Expression of the Caenorhabditis elegans collagen genes col-1 and col-2 is developmentally regulated.

The total collagen gene expression as well as the specific expression of two sequenced Caenorhabditis elegans collagen genes, col-1 and col-2, has been investigated. Northern blots of RNA isolated from animals at different developmental stages were probed under conditions that allow cross-hybridization of all collagen sequences. The majority of hybridization is to transcripts of 1.1-1.4 kilobases (kb) in length, with weak hybridization to some larger transcripts. Different size patterns, within the 1.1-1.4-kb ranges, are seen in RNAs from different developmental stages. Gene-specific probes were produced from the C. elegans collagen genes col-1 and col-2, and each was shown to hybridize to a single size transcript in the 1.1-1.4-kb region. The col-1 transcript was found in all the developmental stages examined, but its abundance varied between stages. The col-2 transcript was detected only in a single developmental stage, during formation of the dauer larvae. The 5' and the 3' ends of the col-1 and col-2 transcripts were determined by S1 nuclease digestion experiments. Both genes have the common "TATA" and "CAAT" box sequences preceding the 5' end of their transcripts and there is strong sequence homology in their 5' untranslated regions. Multiple copies of an eight-nucleotide repeat sequence were found upstream from both col-1 and col-2.

Animals↗
PubMed1985

Use of a psoralen-induced phenocopy to study genes controlling spermatogenesis in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, spermatogenesis represents one of two alternative developmental pathways open to premeiotic germ cells. At least two genes, fem-1 and fem-2, control the initiation of spermatogenesis in XX (hermaphrodite) worms, and the entire spectrum of male differentiation in XO animals. Low-dose irradiation of worms treated with the light-activated DNA crosslinking drug trimethylpsoralen, at levels that do not affect cell division or growth rates, blocks spermatogenesis in C. elegans hermaphrodites and produces an identical phenotype to that of temperature-sensitive mutations in the fem genes. Psoralen treatment does not, however, produce corresponding phenotypes of these mutants in XO animals. The developmental age for phenocopy production is the same as the hermaphrodite temperature-sensitive period of the two mutants. The effects of pulses of restrictive temperature and psoralen treatment on fem-2 mutant hermaphrodites are additive, suggesting that psoralen crosslinking may reduce the level of the fem-2 gene product. Microbeam experiments localize the target for the psoralen effect to the primary germ cells in the first stage larvae, indicating that a critical step occurs in a small number of precursor cells prior to their commitment to spermatogenesis.

Animals↗
PubMed1985

Caenorhabditis elegans DNA that directs segregation in yeast cells.

We have isolated seven DNA fragments from Caenorhabditis elegans that enhance the mitotic segregation of autonomously replicating plasmids in the yeast Saccharomyces cerevisiae. These segregators, designated SEG1-SEG7, behave like isolated yeast chromosomes: they increase the stability and simultaneously lower the copy number of circular plasmids during mitotic growth in yeast. During meiosis, plasmids containing the C. elegans segregators show higher levels of precocious or aberrant disjunction than do plasmids bearing isolated yeast centromeres. Yet one of the segregators improved the meiotic segregation of the parental plasmid. We estimate that there may be as many as 30 segregator sequences in the C. elegans genome, a value that is consistent with the polycentric nature of C. elegans chromosomes. Five of the seven segregators are linked to sequences that are repeated in the worm genome, and four of these five segregators cross-hybridize. Other members of this family of repetitive DNA do not contain segregator function. Segregator sequences may prove useful for probing the structure of centromeres of both C. elegans and S. cerevisiae chromosomes.

Animals↗
PubMed1985

Genetic mapping of Caenorhabditis elegans collagen genes using DNA polymorphisms as phenotypic markers.

In Caenorhabditis elegans collagens comprise a dispersed family of 40-150 genes, the majority of which probably code for collagen proteins found in the animal's cuticle. The conserved (Gly-X-Y)n triple helix coding sequence of collagen genes has facilitated the isolation of a large number of C. elegans collagen genes by recombinant DNA methods. We have begun a study of the chromosomal organization of these genes by screening laboratory strains of C. elegans for DNA polymorphisms in the regions surrounding collagen genes. Polymorphisms near seven genes have been identified and have been used as phenotypic markers in genetic crosses to assign the genes to linkage groups II, III, IV, and X. Four genes are shown by multifactor crosses to map to a 2-3 map unit interval between unc-24 and unc-22 on chromosome IV.

Animals↗
PubMed1985

Extrachromosomal DNA transformation of Caenorhabditis elegans.

DNA was introduced into the germ line of the nematode Caenorhabditis elegans by microinjection. Approximately 10% of the injected worms gave rise to transformed progeny. Upon injection, supercoiled molecules formed a high-molecular-weight array predominantly composed of tandem repeats of the injected sequence. Injected linear molecules formed both tandem and inverted repeats as if they had ligated to each other. No worm DNA sequences were required in the injected plasmid for the formation of these high-molecular-weight arrays. Surprisingly, these high-molecular-weight arrays were extrachromosomal and heritable. On average 50% of the progeny of a transformed hermaphrodite still carried the exogenous sequences. In situ hybridization experiments demonstrated that approximately half of the transformed animals carried foreign DNA in all of their cells; the remainder were mosaic animals in which some cells contained the exogenous sequences while others carried no detectable foreign DNA. The presence of mosaic and nonmosaic nematodes in transformed populations may permit detailed analysis of the expression and function of C. elegans genes.

Animals↗
PubMed1985

Stage-specific patterns of collagen gene expression during development of Caenorhabditis elegans.

Collagens are the major protein components of the Caenorhabditis elegans cuticle and are encoded by a large family of 40 to 150 closely related but nonidentical genes. We have determined temporal patterns of mRNA accumulation for a large number of collagen genes by screening recombinant phages and plasmids containing cloned collagen genes under high stringency conditions with 32P-labeled cDNA preparations specific for eggs or three postembryonic molts. We find that collagen mRNA levels are regulated both temporally and quantitatively during C. elegans development. Most genes studied exhibit one of four patterns of mRNA accumulation which correlate with changes in cuticle morphology and collagen protein composition during development. Our results suggest that, in general, there is a progressive activation of new collagen genes during normal development.

Animals↗
PubMed1984

Dominant mutations affecting muscle structure in Caenorhabditis elegans that map near the actin gene cluster.

By examining F1 progeny of mutagenized Caenorhabditis elegans larvae, we recovered several dominant mutations which affect muscle structure. Five of these new mutations resulted in phenotypes unlike the previously recognized unc-54 and unc-15 dominant alleles. Mapping studies placed all five mutations in the same small region of linkage group V. Polarized light, fluorescence and electron microscopic studies showed that a prominent feature of the disorganized myofilament lattice is the abnormal placement of thin filaments within the body wall muscle cells. Pharyngeal musculature is also affected by three of the mutations when homozygous. Of the five mutations only three are homozygous viable. All three of these have unusually high intragenic reversion rates either spontaneously (approximately 10(-6)) or after ethyl methanesulfonate mutagenesis (2 X 10(-5)), suggesting that reversion occurs through loss of function mutations. No unlinked suppressor mutations were found. The dominance of the mutations, the effect on thin filaments and the reversion properties suggested that these new dominant mutations lie in a gene or genes specifying a structural component of the thin filament. The positioning of a set of three actin sequences in the same region (Files et al., 1983) led us to speculate that these mutations lie in actin genes.

Actins↗
PubMed1984

DNA rearrangements of the actin gene cluster in Caenorhabditis elegans accompany reversion of three muscle mutants.

We present evidence that associates dominant mutations in Caenorhabditis elegans that disrupt muscle structure and motility with a cluster of three actin genes mapped in the same region of linkage group V. We examined spontaneous and mutagen-induced wild-type revertants of these dominant alleles for alterations in the DNA of the actin gene cluster. Four of 73 revertants contain detectable DNA rearrangements within the cluster of actin genes including an insertion, a deletion and gene fusions. We postulate that these rearrangements inactivate or delete at least one gene in the cluster and consequently the original mutations are within the actin gene cluster.

Actins↗
PubMed1984

Specification of male development in Caenorhabditis elegans: the fem genes.

Mutation of the gene fem-2 causes feminization of both sexes: hermaphrodites make no sperm, and males produce oocytes in an intersexual somatic gonad. A double mutant harboring ts alleles of both fem-1 (formerly named isx-1; G. A. Nelson, K. K. Lew, and S. Ward, 1978, Dev. Biol. 66, 386-409) and fem-2 causes transformation of XO animals (normally male) into spermless hermaphrodites at restrictive temperature. The phenotypes, temperature-sensitive periods, and maternal effects observed in mutants of each fem gene are found to be similar. It is suggested that the fem genes are centrally involved in specification of male development in Caenorhabditis elegans--both in the germ line of hermaphrodites and in somatic and germ line tissues of males.

Alleles↗
PubMed1984

Number and organization of collagen genes in Caenorhabditis elegans.

We analyzed the number and organization of collagen genes in the nematode Caenorhabditis elegans. Genomic Southern blot hybridization experiments and recombinant phage library screenings indicated that C. elegans has between 40 and 150 distinct collagen genes. A large number of recombinant phages containing collagen genes were isolated from C. elegans DNA libraries. Physical mapping studies indicated that most phage contained a single small collagen gene less than 3 kilobases in size. A few phage contained multiple collagen hybridizing regions and may contain a larger collagen gene or several tightly linked small collagen genes. No overlaps were observed between phages containing different collagen genes, implying that the genes are dispersed in the C. elegans genome. Consistent with the small size of most collagen genes, we found that the predominant class of collagen mRNA in C. elegans is 1.2 to 1.4 kilobases in length. Genomic Southern blot experiments under stringent hybridization conditions revealed considerable sequence diversity among collagen genes. Our data suggest that most collagen genes are unique or are present in only a few copies.

Animals↗
PubMed1983

Target sequences for the C. elegans transposable element Tc1.

The target sequences for two independent insertions of the transposable element Tc1 from Caenorhabditis elegans show homology. Because both insertions are at palindromic TA/AT sequences, the exact boundaries of Tc1 cannot be distinguished; Tc1 could be 1610 bp and flanked by a 2-bp duplication of the target site or it could be 1612 bp and without target site duplication. The latter possibility implies a novel manner for insertion of a transposable element.

Animals↗
PubMed1983

Sequence of the C. elegans transposable element Tc1.

The complete nucleotide sequence was determined for Tc1, a transposable element in the nematode Caenorhabditis elegans. The 1610-base-pair element terminates in 54-base-pair perfect inverted repeats and is flanked by a 2-base-pair duplication of the target sequence. The Tc1 sequence contains two long open reading frames on the same DNA strand but in different translational reading frames. The positions of transcriptional control sequences suggest that a single transcript is made, which could produce two polypeptides, 273 and 112 amino acids in length. These features, i.e. terminal repeats, target site duplication and open reading frames, make Tc1 similar to transposable elements from other species.

Animals↗
PubMed1983

Actin gene family of Caenorhabditis elegans.

Four actin genes have been isolated from Caenorhabditis elegans that account for all of the major actin hybridization to total genomic DNA. Actin genes I, II and III are clustered within a 12 X 10(3) base region; gene IV is unlinked to the others. All four genes have been sequenced from at least nucleotide -109 to +250. Genes I and III are identical for the first 307 coding nucleotides. Genes I and II differ in 14 positions within the first 250 coding nucleotides; one difference substitutes an aspartic acid for a glutamic acid at codon 5. Genes I and IV differ in 18 positions within the first 259 coding nucleotides without causing any amino acid differences. Genes I, II and III have introns after the first nucleotide of codon 64 and gene IV has an intron between codons 19 and 20. The four nucleotide sequences thus far define two different amino acid sequences. Both of the amino acid sequences resemble vertebrate cytoplasmic actin more than vertebrate muscle actin. A DNA polymorphism between the Bristol and Bergerac strains has been used as a phenotypic marker in genetic crosses to map the cluster of actin genes within a 2% recombination interval on linkage group V between unc-23 and sma-1 in order to begin a molecular genetic analysis of the actin loci.

Actins↗
PubMed1983

Analysis of a transposable element in Caenorhabditis elegans.

A transposable element, designated Tc1, has been characterized in Caenorhabditis elegans. Tc1 is 1.7 kilobases long, has an inverted terminal repeat of less than 100 base pairs, and is repeated as a highly conserved element. The copy number and genomic positions of Tc1 are extremely variable among strains, implying that Tc1 is mobile. However, progeny of interstrain crosses did not show hybrid dysgenic traits that might be due to Tc1 transposition.

Animals↗