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1.
Cell Motil Cytoskeleton ; 63(1): 14-28, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16317718

ABSTRACT

Profilins are actin binding proteins, which also interact with polyphosphoinositides and proline-rich ligands. On the basis of the genome sequence, three diverse profilin homologues (PFN) are predicted to exist in Caenorhabditis elegans. We show that all three isoforms PFN-1, PFN-2, and PFN-3 are expressed in vivo and biochemical studies indicate they bind actin and influence actin dynamics in a similar manner. In addition, they bind poly(L-proline) and phosphatidylinositol 4,5-bisphosphate micelles. PFN-1 is essential whereas PFN-2 and PFN-3 are nonessential. Immunostainings revealed different expression patterns for the profilin isoforms. In embryos, PFN-1 localizes in the cytoplasm and to the cell-cell contacts at the early stages, and in the nerve ring during later stages. During late embryogenesis, expression of PFN-3 was specifically detected in body wall muscle cells. In adult worms, PFN-1 is expressed in the neurons, the vulva, and the somatic gonad, PFN-2 in the intestinal wall, the spermatheca, and the pharynx, and PFN-3 localizes in a striking dot-like fashion in body wall muscle. Thus the model organism Caenorhabditis elegans expresses three profilin isoforms and is the first invertebrate animal with tissue-specific profilin expression.


Subject(s)
Caenorhabditis elegans/metabolism , Profilins/metabolism , Actin Cytoskeleton/drug effects , Actins/chemistry , Animals , Caenorhabditis elegans/embryology , Cell Movement , Cell Survival , Embryo, Nonmammalian/metabolism , In Vitro Techniques , Models, Biological , Molecular Sequence Data , Organ Specificity , Peptides/chemistry , Profilins/genetics , Profilins/pharmacology , Profilins/physiology , Proline/chemistry , Protein Binding , Protein Isoforms/metabolism , Rabbits
2.
Mol Genet Genomics ; 270(2): 121-31, 2003 Nov.
Article in English | MEDLINE | ID: mdl-12905072

ABSTRACT

The let-767 gene encodes a protein that is similar to mammalian steroid enzymes that are responsible for the reduction of 17-beta hydroxysteroid hormones. Caenorhabditis elegans is incapable of the de novo synthesis of cholesterol. Therefore, this free-living nematode must extract cholesterol from its environment and modify it to form steroid hormones that are necessary for its survival. C. elegans is unable to survive in the absence of supplemental cholesterol, and is therefore sensitive to cholesterol limitation. We show that a mutation in let-767 results in hypersensitivity to cholesterol limitation, supporting the hypothesis that LET-767 acts on a sterol derivative. Furthermore, let-767 mutants exhibit defects in embryogenesis, female reproduction and molting. Although ecdysone is the major molting hormone in insects, there is as yet no evidence for ecdysone synthesis in C. elegans, suggesting that a different hormone is required for molting in C. elegans. Our results suggest that LET-767 modifies a sterol hormone that is required both for embryogenesis and for later stages of development.


Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Sterols/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/physiology , Caenorhabditis elegans Proteins/genetics , Cholesterol/metabolism , Cloning, Molecular , DNA, Helminth/genetics , Female , Genes, Helminth , Green Fluorescent Proteins , Luminescent Proteins/genetics , Male , Molecular Sequence Data , Mutation , Phenotype , Recombinant Fusion Proteins/genetics , Reproduction/genetics , Reproduction/physiology , Sequence Homology, Amino Acid
4.
Genome Res ; 11(8): 1346-52, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11483575

ABSTRACT

Gene expression in a developmentally arrested, long-lived dauer population of Caenorhabditis elegans was compared with a nondauer (mixed-stage) population by using serial analysis of gene expression (SAGE). Dauer (152,314) and nondauer (148,324) SAGE tags identified 11,130 of the predicted 19,100 C. elegans genes. Genes implicated previously in longevity were expressed abundantly in the dauer library, and new genes potentially important in dauer biology were discovered. Two thousand six hundred eighteen genes were detected only in the nondauer population, whereas 2016 genes were detected only in the dauer, showing that dauer larvae show a surprisingly complex gene expression profile. Evidence for differentially expressed gene transcript isoforms was obtained for 162 genes. H1 histones were differentially expressed, raising the possibility of alternative chromatin packaging. The most abundant tag from dauer larvae (20-fold more abundant than in the nondauer profile) corresponds to a new, unpredicted gene we have named tts-1 (transcribed telomere-like sequence), which may interact with telomeres or telomere-associated proteins. Abundant antisense mitochondrial transcripts (2% of all tags), suggest the existence of an antisense-mediated regulatory mechanism in C. elegans mitochondria. In addition to providing a robust tool for gene expression studies, the SAGE approach already has provided the advantage of new gene/transcript discovery in a metazoan.


Subject(s)
Caenorhabditis elegans/growth & development , Caenorhabditis elegans/genetics , Gene Expression Regulation, Developmental/physiology , Genes, Helminth/physiology , Animals , Gene Expression Profiling/methods , Longevity/genetics , RNA, Messenger/analysis
5.
Cell ; 106(1): 23-34, 2001 Jul 13.
Article in English | MEDLINE | ID: mdl-11461699

ABSTRACT

RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Here we show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation.


Subject(s)
Caenorhabditis elegans/genetics , Gene Expression Regulation, Developmental , Phylogeny , RNA, Helminth/genetics , Animals , Animals, Genetically Modified , Caenorhabditis elegans/embryology , Caenorhabditis elegans/growth & development , DNA Primers , Drosophila/genetics , Embryo, Nonmammalian/physiology , Female , Gene Silencing , Genes, Helminth , Genes, Reporter , Genomic Imprinting , Heterozygote , Larva , Luciferases/genetics , Polymerase Chain Reaction
6.
J Cell Biol ; 154(2): 403-14, 2001 Jul 23.
Article in English | MEDLINE | ID: mdl-11470827

ABSTRACT

Tissue functions and mechanical coupling of cells must be integrated throughout development. A striking example of this coupling is the interactions of body wall muscle and hypodermal cells in Caenorhabditis elegans. These tissues are intimately associated in development and their interactions generate structures that provide a continuous mechanical link to transmit muscle forces across the hypodermis to the cuticle. Previously, we established that mup-4 is essential in embryonic epithelial (hypodermal) morphogenesis and maintenance of muscle position. Here, we report that mup-4 encodes a novel transmembrane protein that is required for attachments between the apical epithelial surface and the cuticular matrix. Its extracellular domain includes epidermal growth factor-like repeats, a von Willebrand factor A domain, and two sea urchin enterokinase modules. Its intracellular domain is homologous to filaggrin, an intermediate filament (IF)-associated protein that regulates IF compaction and that has not previously been reported as part of a junctional complex. MUP-4 colocalizes with epithelial hemidesmosomes overlying body wall muscles, beginning at the time of embryonic cuticle maturation, as well as with other sites of mechanical coupling. These findings support that MUP-4 is a junctional protein that functions in IF tethering, cell-matrix adherence, and mechanical coupling of tissues.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/metabolism , Cell Adhesion Molecules/metabolism , Epithelial Cells/metabolism , Animals , Animals, Genetically Modified , Caenorhabditis elegans/embryology , Caenorhabditis elegans/growth & development , Cell Adhesion/physiology , Cell Adhesion Molecules/genetics , Cloning, Molecular , Embryo, Nonmammalian/metabolism , Epithelial Cells/cytology , Gene Expression/drug effects , Green Fluorescent Proteins , Helminth Proteins/genetics , Helminth Proteins/metabolism , Hemidesmosomes/metabolism , Larva/metabolism , Larva/ultrastructure , Luminescent Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Muscles/metabolism , Muscles/ultrastructure , Mutation , Organ Specificity , Physical Chromosome Mapping , RNA, Double-Stranded/pharmacology , Sequence Analysis, DNA , Sequence Homology, Amino Acid
7.
J Biol Chem ; 276(6): 3999-4011, 2001 Feb 09.
Article in English | MEDLINE | ID: mdl-11073957

ABSTRACT

Voltage-gated calcium channels represent a heterogenous family of calcium-selective channels that can be distinguished by their molecular, electrophysiological, and pharmacological characteristics. We report here the molecular cloning and functional expression of three members of the low voltage-activated calcium channel family from rat brain (alpha(1G), alpha(1H), and alpha(1I)). Northern blot and reverse transcriptase-polymerase chain reaction analyses show alpha(1G), alpha(1H), and alpha(1I) to be expressed throughout the newborn and juvenile rat brain. In contrast, while alpha(1G) and alpha(1H) mRNA are expressed in all regions in adult rat brain, alpha(1I) mRNA expression is restricted to the striatum. Expression of alpha(1G), alpha(1H), and alpha(1I) subunits in HEK293 cells resulted in calcium currents with typical T-type channel characteristics: low voltage activation, negative steady-state inactivation, strongly voltage-dependent activation and inactivation, and slow deactivation. In addition, the direct electrophysiological comparison of alpha(1G), alpha(1H), and alpha(1I) under identical recording conditions also identified unique characteristics including activation and inactivation kinetics and permeability to divalent cations. Simulation of alpha(1G), alpha(1H), and alpha(1I) T-type channels in a thalamic neuron model cell produced unique firing patterns (burst versus tonic) typical of different brain nuclei and suggests that the three channel types make distinct contributions to neuronal physiology.


Subject(s)
Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/physiology , Alternative Splicing , Amino Acid Sequence , Animals , Barium/metabolism , Base Sequence , Brain/metabolism , Calcium/metabolism , Calcium Channels, T-Type/chemistry , Calcium Channels, T-Type/metabolism , Cell Line , Cloning, Molecular , DNA, Complementary , Expressed Sequence Tags , Humans , Ion Channel Gating , Kinetics , Molecular Sequence Data , Permeability , RNA, Messenger/genetics , Rats , Sequence Homology, Amino Acid
9.
Nat Cell Biol ; 2(7): 415-22, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10878806

ABSTRACT

Epithelial cells are polarized, with apical and basal compartments demarcated by tight and adherens junctions. Proper establishment of these subapical junctions is critical for normal development and histogenesis. We report the characterization of the gene let-413 which has a critical role in assembling adherens junctions in Caenorhabditis elegans. In let-413 mutants, adherens junctions are abnormal and mislocalized to more basolateral positions, epithelial cell polarity is affected and the actin cytoskeleton is disorganized. The LET-413 protein contains one PDZ domain and 16 leucine-rich repeats with high homology to proteins known to interact with small GTPases. Strikingly, LET-413 localizes to the basolateral membrane. We suggest that LET-413 acts as an adaptor protein involved in polarizing protein trafficking in epithelial cells.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/cytology , Caenorhabditis elegans/embryology , Cell Polarity , Epithelial Cells/cytology , Helminth Proteins/metabolism , Intercellular Junctions/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Cell Adhesion , Cell Membrane/chemistry , Cell Membrane/metabolism , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Epithelium/abnormalities , Epithelium/metabolism , Epithelium/ultrastructure , Genes, Helminth/genetics , Helminth Proteins/chemistry , Helminth Proteins/genetics , Intercellular Junctions/chemistry , Intercellular Junctions/ultrastructure , Microscopy, Electron , Molecular Sequence Data , Mutation/genetics , Phenotype , Protein Structure, Tertiary , Sequence Alignment
10.
J Cell Biol ; 145(3): 491-502, 1999 May 03.
Article in English | MEDLINE | ID: mdl-10225951

ABSTRACT

The Caenorhabditis elegans unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin that are implicated in myofibril assembly. Here, we show that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. We found that all homozygous viable unc-60 mutations resided in the unc-60B coding region, indicating that UNC-60B is responsible for the Unc-60 phenotype. Wild-type UNC-60B had F-actin binding, partial actin depolymerizing, and weak F-actin severing activities in vitro. However, mutations in UNC-60B caused various alterations in these activities. Three missense mutations resulted in weaker F-actin binding and actin depolymerizing activities and complete loss of severing activity. The r398 mutation truncated three residues from the COOH terminus and resulted in the loss of severing activity and greater actin depolymerizing activity. The s1307 mutation in a putative actin-binding helix caused greater activity in actin-depolymerizing and severing. Using a specific antibody for UNC-60B, we found varying protein levels of UNC-60B in mutant animals, and that UNC-60B was expressed in embryonic muscles. Regardless of these various molecular phenotypes, actin was not properly assembled into embryonic myofibrils in all unc-60 mutants to similar extents. We conclude that precise control of actin filament dynamics by UNC-60B is required for proper integration of actin into myofibrils.


Subject(s)
Actins/metabolism , Caenorhabditis elegans/genetics , DNA-Binding Proteins/metabolism , Microfilament Proteins/metabolism , Transcription Factors/metabolism , Actin Depolymerizing Factors , Animals , Caenorhabditis elegans/chemistry , Caenorhabditis elegans/metabolism , Cytoskeleton/chemistry , Cytoskeleton/metabolism , DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Microfilament Proteins/genetics , Muscles/chemistry , Muscles/metabolism , Mutagenesis/physiology , Myofibrils/chemistry , Myofibrils/metabolism , Myosins/metabolism , POU Domain Factors , Phenotype , Polymers , Transcription Factors/genetics
11.
Gene ; 230(2): 137-44, 1999 Apr 16.
Article in English | MEDLINE | ID: mdl-10216251

ABSTRACT

In the nematode Caenorhabditis elegans, the maternal effect lethal gene mel-32 encodes a serine hydroxymethyltransferase isoform. Since interspecies DNA comparison is a valuable tool for identifying sequences that have been conserved because of their functional importance or role in regulating gene activity, mel-32(SHMT) genomic DNA from C. elegans was used to screen a genomic library from the closely related nematode Caenorhabditis briggsae. The C. briggsae genomic clone identified fully rescues the Mel-32 phenotype in C. elegans, indicating functional and regulatory conservation. Computer analysis reveals that CbMEL-32(SHMT) is 92% identical (97% similar) to CeMEL-32(SHMT) at the amino acid level over the entire length of the protein (484 amino acids), whereas the coding DNA is 82.5% identical (over 1455 nucleotides). Several highly conserved non-coding regions upstream and downstream of the mel-32(SHMT) gene reveal potential regulatory sites that may bind trans-acting protein factors.


Subject(s)
Caenorhabditis/enzymology , Glycine Hydroxymethyltransferase/genetics , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis/genetics , Conserved Sequence , Exons , Glycine Hydroxymethyltransferase/chemistry , Helminth Proteins/genetics , Introns , Molecular Sequence Data , Mutation , Regulatory Sequences, Nucleic Acid , Sequence Homology, Amino Acid
12.
Genome Res ; 9(4): 348-59, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10207157

ABSTRACT

Comparative genomic analysis was used to investigate the gene structure of the bli-4 locus from two related Caenorhabditis species, C. elegans and C. briggsae. In C. elegans, bli-4 is a complex gene encoding a member of the kex2/subtilisin-like family of proprotein convertases. Genomic sequence comparisons coupled with RT-PCR analysis identified five additional coding exons that had not been identified previously using standard recombinant DNA techniques. The C. briggsae gene was able to rescue both viable blistered and developmentally arrested mutants of C. elegans bli-4, demonstrating functional conservation. In addition, deletion analysis of conserved sequences outside of coding regions, combined with phenotypic rescue experiments, identified regulatory elements that alter the expression of the bli-4 gene. These results demonstrate the utility of genomic sequence comparisons of homologous genes in related species as an effective tool with which to dissect the functional information of complex genes.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis/genetics , Helminth Proteins/genetics , Subtilisins/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , Caenorhabditis elegans/genetics , Conserved Sequence , Isoenzymes/genetics , Molecular Sequence Data , Mutation , Regulatory Sequences, Nucleic Acid , Sequence Homology, Nucleic Acid , Subtilisins/metabolism , Transcription Factors/genetics , Transcription, Genetic
13.
Annu Rev Phytopathol ; 37: 247-65, 1999.
Article in English | MEDLINE | ID: mdl-11701823

ABSTRACT

The completion of the entire genome sequence of the free-living nematode, Caenorhabditis elegans is a tremendous milestone in modern biology. Not only will scientists be poring over data mined from this resource, but techniques and methodologies developed along the way have changed the way we can approach biological questions. The completion of the C. elegans genomic sequence will be of particular importance to scientists working on parasitic nematodes. In many cases, these nematode species present intractable challenges to those interested in their biology and genetics. The data already compared from parasites to the C. elegans database reveals a wealth of opportunities for parasite biologists. It is likely that many of the same genes will be present in parasites and that these genes will have similar functions. Additional information regarding differences between free-living and parasitic species will provide insight into the evolution and nature of parasitism. Finally, genetic and genomic approaches to the study of parasitic nematodes now have a clearly marked path to follow.

14.
Mol Gen Genet ; 260(2-3): 280-8, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9862482

ABSTRACT

The central gene cluster of chromosome III was one of the first regions to be sequenced by the Caenorhabditis elegans genome project. We have performed an essential gene analysis on the left part of this cluster, in the region around dpy-17III balanced by the duplication sDp3. We isolated 151 essential gene mutations and characterized them with regard to their arrest stages. To facilitate positioning of these mutations, we generated six new deficiencies that, together with preexisting chromosomal rearrangements, subdivide the region into 14 zones. The 151 mutations were mapped into these zones. They define 112 genes, of which 110 were previously unidentified. Thirteen of the zones have been anchored to the physical sequence by polymerase chain reaction deficiency mapping. Of the 112 essential genes mapped, 105 are within these 13 zones. They span 4.2 Mb of nucleotide sequence. From the nucleotide sequence data, 920 genes are predicted. From a Poisson distribution of our mutations, we predict that 234 of the genes will be essential genes. Thus, the 105 genes constitute 45% of the estimated number of essential genes in the physically defined zones and between 2 and 5% of all essential genes in C. elegans.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/genetics , Chromosome Mapping , Genes, Lethal , Helminth Proteins/genetics , Mutation , Animals , Caenorhabditis elegans/drug effects , Caenorhabditis elegans/radiation effects , Ethyl Methanesulfonate/pharmacology , Gamma Rays , Genetic Complementation Test , Male , Non-Fibrillar Collagens , Phenotype , Physical Chromosome Mapping , Polymerase Chain Reaction , Ultraviolet Rays
15.
J Biol Chem ; 273(11): 6066-73, 1998 Mar 13.
Article in English | MEDLINE | ID: mdl-9497323

ABSTRACT

The mel-32 gene in the free living soil nematode Caenorhabditis elegans encodes a serine hydroxymethyltransferase (SHMT) isoform. Seventeen ethylmethanesulfonate (EMS)-induced mutant alleles of mel-32(SHMT) have been generated, each of which causes a recessive maternal effect lethal phenotype. Animals homozygous for the SHMT mutations have no observable mutant phenotype, but their offspring display an embryonic lethal phenotype. The Mel-32 phenotype has been rescued with a transgenic array containing only mel-32(SHMT) genomic DNA. Heteroduplex analysis of the 17 alleles allowed 14 of the mutations to be positioned to small regions. Subsequent sequence analysis has shown that 16 of the alleles alter highly conserved amino acids, while one allele introduces a stop codon that truncates two thirds of the predicted protein. mel-32(SHMT) has a 55-60% identity at the amino acid level with both isoforms of SHMT found in yeast and humans and a 50% identity with the Escherichia coli isoform. The C. elegans mel-32 mutation represents the first case where SHMT has been shown to be an essential gene.


Subject(s)
Caenorhabditis elegans/enzymology , Genes, Helminth , Genes, Lethal , Glycine Hydroxymethyltransferase/genetics , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/genetics , Cloning, Molecular , Female , Helminth Proteins/genetics , Heterozygote , Homozygote , Isoenzymes/genetics , Male , Molecular Sequence Data , Mutagenesis , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sex Characteristics
16.
Development ; 124(20): 3919-28, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9374390

ABSTRACT

Mutations in the C. elegans gene egl-38 result in a discrete set of defects in developmental pattern formation. In the developing egg-laying system of egl-38 mutant hermaphrodites, the identity of four uterine cells is disrupted and they adopt the fate of their neighbor cells. Likewise, the identity of two rectal epithelial cells in the male tail is disrupted and one of these cells adopts the fate of its neighbor cell. Genetic analysis suggests that the egl-38 functions in the tail and the egg-laying system are partially separable, as different egl-38 mutations can preferentially disrupt the different functions. We have cloned egl-38 and shown that it is a member of the PAX family of genes, which encode transcription factors implicated in a variety of developmental patterning events. The predicted EGL-38 protein is most similar to the mammalian class of proteins that includes PAX2, PAX5 and PAX8. The sequence of egl-38 mutant DNA indicates that the tissue-preferential defects of egl-38 mutations result from substitutions in the DNA-binding paired domain of the EGL-38 protein. egl-38 thus provides the first molecular genetic insight into two specific patterning events that occur during C. elegans development and also provides the opportunity to investigate the in vivo functions of this class of PAX proteins with single cell resolution.


Subject(s)
Caenorhabditis elegans/embryology , Caenorhabditis elegans/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Nuclear Proteins/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , Base Sequence , Male , Molecular Sequence Data , PAX2 Transcription Factor , PAX5 Transcription Factor , Sequence Alignment
17.
Genome Res ; 7(10): 974-85, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9331368

ABSTRACT

We have generated a library of transgenic Caenorhabditis elegans strains that carry sequenced cosmids from the genome of the nematode. Each strain carries an extrachromosomal array containing a single cosmid, sequenced by the C. elegans Genome Sequencing Consortium, and a dominate Rol-6 marker. More than 500 transgenic strains representing 250 cosmids have been constructed. Collectively, these strains contain approximately 8 Mb of sequence data, or approximately 8% of the C. elegans genome. The transgenic strains are being used to rescue mutant phenotypes, resulting in a high-resolution map alignment of the genetic, physical, and DNA sequence maps of the nematode. We have chosen the region of chromosome III deleted by sDf127 and not covered by the duplication sDp8(III;I) as a starting point for a systematic correlation of mutant phenotypes with nucleotide sequence. In this defined region, we have identified 10 new essential genes whose mutant phenotypes range from developmental arrest at early larva, to maternal effect lethal. To date, 8 of these 10 essential genes have been rescued. In this region, these rescues represent approximately 10% of the genes predicted by GENEFINDER and considerably enhance the map alignment. Furthermore, this alignment facilitates future efforts to physically position and clone other genes in the region. [Updated information about the Transgenic Library is available via the Internet at http://darwin.mbb.sfu.ca/imbb/dbaillie/cos mid.html.]


Subject(s)
Animals, Genetically Modified , Caenorhabditis elegans/genetics , Cosmids , Gene Library , Animals , Chromosome Mapping/methods , Genes, Helminth , Genes, Lethal , Genetic Markers , Genome , Multigene Family , Mutation
18.
EMBO J ; 15(13): 3229-37, 1996 Jul 01.
Article in English | MEDLINE | ID: mdl-8670823

ABSTRACT

The ubc-2 gene in Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme (E2) homologous to yeast UBC4 and UBC5. UBC4 and UBC5 are individually dispensable class I E2 enzymes involved in the degradation of short-lived and abnormal proteins. Transgenic analysis using ubc-2-lacZ fusions and in situ immunofluorescence indicate that ubc-2 is abundantly expressed in most tissues of embryos and early larvae, but becomes specific to the nervous system in L4 larvae and adults. This suggests that the functions of this type of E2 are developmentally regulated in C.elegans. This hypothesis is supported by antisense analysis, which shows that blocking the expression of ubc-2 has a more severe effect in early developmental stages than in later stages. Through complementation of previously identified essential genes in the vicinity of ubc-2, we demonstrate that ubc-2 corresponds to let-70, a gene essential for C.elegans larval development. One let-70(ubc-2) allele contains a His75-->Tyr substitution, while another has an altered splice donor site.


Subject(s)
Caenorhabditis elegans/enzymology , Gene Expression Regulation, Developmental , Ligases/metabolism , Alleles , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/embryology , Cloning, Molecular , DNA, Helminth , Genes, Lethal , Lac Operon , Larva/metabolism , Ligases/genetics , Male , Molecular Sequence Data , Mutation , Nervous System/metabolism , RNA, Antisense/genetics , Transgenes , Ubiquitin-Conjugating Enzymes
19.
Mol Gen Genet ; 248(6): 719-26, 1995 Oct 25.
Article in English | MEDLINE | ID: mdl-7476875

ABSTRACT

A mutation in the let-653 gene of Caenorhabditis elegans results in larval death. The lethal arrest is concurrent with the appearance of a vacuole anterior to the lower pharyngeal bulb. The position of the vacuole is consistent with a dysfunction of the secretory/excretory apparatus. Germline transformation rescue experiments were able to position the let-653 gene to two overlapping cosmid subclones. Sequence data generated from both cDNA and genomic DNA subclones indicated that let-653 encodes a mucin-like protein. Our characterization suggests that a mucin-like protein is essential for effective functioning of the secretory/excretory apparatus within C. elegans.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/genetics , Genes, Helminth , Helminth Proteins/genetics , Mucins/genetics , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/cytology , Caenorhabditis elegans/physiology , Cloning, Molecular , Gene Expression/genetics , Helminth Proteins/chemistry , Helminth Proteins/classification , Helminth Proteins/physiology , Larva/cytology , Larva/genetics , Larva/growth & development , Molecular Sequence Data , Mucins/chemistry , Mucins/classification , Mucins/physiology , Mutation/genetics , Phenotype , RNA Splicing/genetics , Restriction Mapping , Sequence Analysis , Transformation, Genetic/genetics
20.
Mol Gen Genet ; 247(3): 367-78, 1995 May 10.
Article in English | MEDLINE | ID: mdl-7770042

ABSTRACT

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.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/genetics , Genes, Helminth , Helminth Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/metabolism , Chromosome Mapping , Cloning, Molecular , Genomic Library , Genotype , Helminth Proteins/biosynthesis , Kinetics , Molecular Sequence Data , Mutagenesis , Phenotype , Recombinant Proteins/biosynthesis , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Species Specificity , Temperature , Time Factors
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