Actin gene family of Caenorhabditis elegans

Two genomic sequences that share homology with RpII215, the gene encoding the largest subunit of RNA polymerase H in Drosophila melanogaster, have been isolated from the nematode Caenorhabditis elegans. One of these sequences was physically mapped on chromosome IV within a region deleted by the deficiency mDf4, 25 kilobases (kb) from the left deficiency breakpoint. This position corresponds to ama-i (resistance to a-amanitin), a gene shown previously to encode a subunit of RNA polymerase II. Northern (RNA) blotting and DNA sequencing revealed that ama-i spans 10 kb, is punctuated by 11 introns, and encodes a 5.9-kb mRNA. A cDNA clone was isolated and partially sequenced to confirm the 3' end and several splice junctions. Analysis of the inferred 1,859-residue ama-i product showed considerable identity with the largest subunit of RNAP II from other organisms, including the presence of a zinc finger motif near the amino terminus, and a carboxyl-terminal domain of 42 tandemly reiterated heptamers with the consensus Tyr Ser Pro Thr Ser Pro Ser. The latter domain was found to be encoded by four exons. In addition, the sequence oriented ama-i transcription with respect to the genetic map. The second C. elegans sequence detected with the Drosophila probe, named rpc-1, was found to encode a 4.8-kb transcript and hybridized strongly to the gene encoding the largest subunit of RNA polymerase III from yeast, implicating rpc-i as encoding the analogous peptide in the nematode. By contrast with ama-1, rpc-i was not deleted by mDf4 or larger deficiencies examined, indicating that these genes are no closer than 150 kb. Genes flanking ama-1, including two collagen genes, also have been identified.The nematode Caenorhabditis elegans is being used by an increasing number of laboratories as a model organism for the molecular genetic analysis of metazoan development (33,55). Understanding the control of gene expression in development will require a description of the RNA polymerases and how they interact with regulatory proteins and the DNA template. Much of our knowledge of RNA polymerase has come from studies of the procaryotic enzyme, in which biochemical and genetic methods have elucidated functions of the core polymerase (2%W') and its specificity factors (37, 56). Although eucaryotic nuclear RNA polymerases are structurally complex (48), recent molecular genetic approaches to understanding RNA polymerase II (RNAP II) have also proven useful (4-6, 39, 57). The objective of this work was to determine the structure of the gene encoding the largest subunit of this enzyme in C. elegans and to provide the basis for the molecular analysis of mutants affected in RNAP II function during development.In most eucaryotes, RNAP II binds to a-amanitin, a toxin that inhibits RNA chain elongation (16). Mutations conferring resistance to ot-amanitin have been isolated in a number of cell lines (13,30,49) DNA sequence analysis of the yeast (3, 51), mouse (1), and Drosophila (32) genes has revealed regions of identity between the peptides from o...

Section: Resultssupporting

confidence: 65%

Molecular cloning and sequencing of ama-1, the gene encoding the largest subunit of Caenorhabditis elegans RNA polymerase II.

Bird¹,

Riddle²

1989

“…The N-terminal Met and Cys codons are also found in other mammalian and avian muscle actins (4,12,16,17,21,47,53). The same two codons are also found in all six Drosophila actin genes (13), four nematode actin genes (10), and sea urchin actin genes (6,42). However, the same two codons are not present in mammalian and avian cytoplasmic actins (3,9,29,35).…”

Section: Methodsmentioning

confidence: 86%

Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice.

Kim¹,

Waters²,

Hake³

et al. 1989

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.

“…By quantitating the hybridization of plasmid sequences relative to that of singlecopy genomic sequences and correcting for the number of transformed worms in the population, we could estimate the plasmid copy number per genome in an average transformed worm. Purified worm DNA was spotted onto triplicate nitrocellulose filters and hybridized with excess 32P-labeled pBR322 DNA, a single-copy worm sequence, and a fourfold repeated worm sequence (15). Hybridization was quantitated by scintillation counting and standardized for the hybridization efficiencies of the different probes.…”

Section: Downloaded Frommentioning

confidence: 99%

“…Three edges bf the coverslip were sealed with petrolatum; the fdurth edge was covered with the halocarbon oil (3S Voltalef oil equilibrated with M9 buffer or series 700 Halocarbon oil). Anesthetized nematodes (10 to 15) were transferred to the oil and then aligned with their vulvae in apposition to the edge of the cover slip. The nematodes remained viable and paralyzed at this water/oil interphase for several hours.…”

mentioning

confidence: 99%

Extrachromosomal DNA transformation of Caenorhabditis elegans.

Stinchcomb¹,

Shaw²,

Carr³

et al. 1985

304

251

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 highmolecular-weight arrays. Surprisingly, these high-molecular-weight arrays were Similarly, techniques for DNA transformation of multicellular organisms facilitate the molecular analysis of developmental processes. DNA transformation of multicellular organisms has been achieved by microinjection of fertilized eggs of mice (6,10,18,53), frogs (14), sea urchins (16), and Drosophila melanogaster (39). In D. melanogaster (17,42,45) and in several cases in mice (7,19,50) the reintroduced genes appear to be properly regulated during development. Thus, DNA transformation permits studies of the expression of isolated sequences in specific tissues at specific times. DNA transformation may also be used to identify and isolate other sequences that encode functions vital for determination and development.The nematode Caenorhabditis elegans has been used widely for studies of developmental genetics. Its simple anatomy and life cycle have permitted the characterization of mutants defective in particular tissues, behaviors, and developmental pathways (5). The cell lineage of the entire worm is known (25,48,49) and developmental physiology of the nematode. Here we describe the introduction of foreign DNA into the C. elegans genome by microinjection into the' gonad. The exogenous DNA formed a high-molecular-weight concatamer which was extrachromosomal and heritable. These experiments, demonstrating DNA transformation of C. elegans, provide a foundation for studying transformation and expression of developmentally interesting genes. MATERIALS AND METHODSStrains and media. C. elegans var. Bristol N2 (5) or a strain lacking the major DNase of the worm [nuc-J(e1392)X] (47) were used for the injections. DSB40 (alias Escherichia coli HB101) was used for bacterial transformation experiments. C. elegans w,as grown and manipulated as described by Brenner (5). 'To genetically manipulate the transformants, males were isolated from a population 'of 'transformed hermaphrodites that had been incubated at 30°C for 5 h. Individual males were mated with MT465 [dpy-5(e61)I bli-2(e768)1I; unc-32(e189)III] and MT464 [unc-5(e53)IV; dpy-11(e224)V; lon-2(e678)XJ provided by the C. elegans Genetic Stock Center. Bacteria were grown and manipulated as described by Davis et al. (11). J3acterial strains harboring plasmids used for injection were generously provided by P. Southern (pSV2neo) and R. Jefferson (pCEV70.3-neo). YRp17 has been described previously (46). YRp17-unc was constructed by inserting the 3.8-kilobase-pair (kpb) BglII fragment of the unc-54 ge...