The glyceraldehyde-3-phosphate dehydrogenase gene family in the nematode, Caenorhabditis elegans: isolation and characterization of one of the genes.

The isolation and genomic sequence of one of possibly four glyceraldehyde-3-phosphate dehydrogenase genes in the nematode, Caenorhabditis elegans is presented. The complete nucleotide sequence of the coding as well as the noncoding flanking regions of this gene has been determined. The deduced amino-acid sequence agrees with the sequence of typical glyceraldehyde-3-phosphate dehydrogenase enzymes and its molecular weight of 36,235 agrees with its size determined previously (Yarbrough, P. and Hecht, R. (1984) J. Biol. Chem. 259, 14711-14720). That this isolated gene encodes a nematode glyceraldehyde-3-phosphate dehydrogenase is additionally confirmed by demonstrating its immunoreactivity to an anti-nematode glyceraldehyde-3-phosphate dehydrogenase antibody after its expression as a fusion protein with dihydrofolate reductase. Codon utilization follows a pattern typical of other expressed nematode genes. The gene is split by two introns that are highly conserved in comparison to other introns observed in C. elegans. The placement of one of these introns is conserved with respect to the chicken glyceraldehyde-3-phosphate dehydrogenase gene. Within the 5' flanking sequence homology to actin and the homology 2 block of the major myosin gene (unc-54) is noted. It is of interest that the 3' flanking region contains a CAAAT box, followed by a TATAAT box, before an open reading frame of a closely linked gene that also contains a small AT-rich intron with the nematode consensus splice junction.

Use of bacterial DHFR-II fusion proteins to elicit specific antibodies.

Plasmids containing the coding region of the type II dihydrofolate reductase (DHFR) specified by R388 have been used to alter the amino acid (aa) sequence at the C-terminus of this protein. These plasmids have a unique cloning site in the C-terminal portion of the 78-aa coding region. Insertions of DNA fragments into this site produced plasmids that code for proteins with 6- to 80-aa extensions. The vectors were constructed to terminate translation in all three phases beyond the position of insertion of foreign DNA. Random DNA fragments from the major sperm protein (MSP) gene of Caenorhabditis elegans produced by DNase I cleavage were inserted into these vectors. Cell extracts from colonies containing MSP sequences were examined by gel electrophoresis and immunoblotting. One of the hybrid DHFR-MSP proteins was isolated and antibody was prepared to it. This antibody preparation reacted with MSP in immunoblots of purified MSP and whole cell extracts of the worm. A rapid purification procedure for the DHFR is presented.

Requirement for protein synthesis in the regulation of a circadian rhythm by serotonin.

Serotonin (5-HT, 5-hydroxytryptamine) regulates the phase of a circadian pacemaker located within the eye of Aplysia. We are attempting to define the cellular and biochemical events involved in the regulatory pathway through which serotonin acts. Previously, we have shown that an activation of adenylate cyclase and an increase in cAMP are events in the 5-HT phase-shifting pathway. In this paper, we examine the role of protein synthesis in mediating the effect of 5-HT and cAMP on the phase of the circadian rhythm. Exposure of eyes to anisomycin, an inhibitor of protein synthesis, completely blocked the advance shift in phase produced by 5-HT. Although anisomycin by itself can produce phase shifts, it did not affect the rhythm at the phases where the blocking experiments were performed. The specificity of action of anisomycin was investigated in two ways. First, deacetylanisomycin, an analogue of anisomycin that is inactive in inhibiting protein synthesis, did not affect the shift in phase produced by 5-HT. Second, anisomycin did not inhibit two other effects of 5-HT on the eye that also appear to be mediated by cAMP: an inhibition of spontaneous optic nerve activity and an increase in the photosensitivity of the eye. The step in the 5-HT phase-shifting pathway that is sensitive to anisomycin appears to occur after the cAMP step because anisomycin also inhibits the ability of 8-benzylthio-cAMP to shift the phase of the rhythm. We have also examined whether 5-HT directly regulates the synthesis of any proteins in the eye. Using two-dimensional gel electrophoresis, we have found that 5-HT appears to increase the synthesis of a protein with an apparent molecular weight of 67,000. Our results indicate that protein synthesis is necessary for 5-HT to shift the phase of the rhythm and that 5-HT appears to regulate the expression of at least one protein in the eye.

Isolation and characterization of a sperm-specific gene family in the nematode Caenorhabditis elegans.

The major sperm protein (MSP) of the nematode Caenorhabditis elegans is a low-molecular-weight (15,000) basic protein implicated in the pseudopodial movement of mature spermatozoa. Its synthesis occurs in a specific region of the gonad and is regulated at the level of transcription (M. Klass and D. Hirsh, Dev. Biol. 84:299-312, 1981; S. Ward and M. Klass, Dev. Biol. 92:203-208, 1982; Klass et al., Dev. Biol. 93:152-164, 1982). A developmentally regulated gene family has been identified that codes for this MSP. Whole genomic blots, as well as analysis of genomic clone banks, indicate that there are between 15 and 25 copies of the MSP gene in the nematode genome. Southern blot analysis also indicates that there is no rearrangement or amplification within the MSP gene family during development. No evidence was found of methylation at various restriction sites surrounding the MSP gene family, and similarly, no correlation between methylation and expression was observed. Three distinct members of this MSP gene family have been cloned, and their nucleotide sequences have been determined. Differential screening of a cDNA clone bank made from polyadenylated mRNA from adult males yielded 45 male-specific clones, 32 of which were clones of MSP genes. One of these cDNA clones was found to contain the entire nucleotide sequence for the MSP, including part of the 5' leader and all of the 3' trailing sequence. Genomic clones bearing copies of the MSP genes have been isolated. At least one of the members of this gene family is a pseudogene. Another member of the MSP gene family that has been cloned from genomic DNA contains the entire uninterrupted structural sequence for the MSP in addition to a 5' flanking sequence containing a promoter-like region with the classic TATA box, a sequence resembling the CAAT box, and a putative ribosome-binding sequence. The 3' trailing sequences of the genomic and the cDNA clones contain an AATAAA polyadenylation site.

A method for the isolation of longevity mutants in the nematode Caenorhabditis elegans and initial results.

The free-living nematode Caenorhabditis elegans is used as a genetically manipulable experimental system for the study of aging. Utilizing a temperature-sensitive sterile strain with a normal life span, a method is described for the isolation of mutant strains with significantly increased life spans. Eight mutant strains were isolated each having increased life spans. Two mutant strains were spontaneous dauer formers, accounting for their increased longevity. Another was chemotaxis-defective, causing reduced food intake which could account for its increased life span. Five mutants suffered from varying degrees of paralysis affecting their rate of pharyngeal pumping and food ingestion. The high correlation of the decreased rate of food ingestion of these mutants with their increased longevity is interpreted as indicating that the increased longevity is most likely due to reduced caloric intake. These results appear to indicate that specific life span genes are extremely rare or, alternatively, life span is controlled in a polygenic fashion.

Sperm isolation and biochemical analysis of the major sperm protein from Caenorhabditis elegans.

In order to facilitate the biochemical analysis of spermatogenesis in the nematode Caenorhabditis elegans methods have been developed for obtaining large quantities of males and for the isolation of sperm. Males are isolated by a passive filtration method from strains producing high proportions of males and sperm are isolated by physical pressure followed by filtration and differential centrifugation. Biochemical analyses show that sperm contain a major protein component that represents 17% of the total sperm protein. This protein has a molecular weight of 15,600, an isoelectric pH of 8.6, and exists as a dimer. It is shown by immunocytochemical techniques to be a specific product of spermatogenesis. It is localized in the proximal arm of the male gonad and in the sperm of both the male and hermaphrodite but it is not detected in other tissues of the nematode. It is not a nuclear binding protein. Pulse-labeling studies show that this major sperm protein is first synthesized in the proximal arm of the male gonad beginning at 39-42 hr after hatching at 20 degrees C. Poly(A) mRNA coding for this protein is first detected in a translatable form just before synthesis of this sperm protein suggesting transcriptional control.

Analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans.

In order to test for the occurrence of rearrangements in DNA during development and to assess the rate of DNA divergence during evolution, we have compared restriction fragments derived from DNA from four sources: sperm cells and somatic tissues of one strain of the nematode Caenorhabditis elegans, somatic tissues of a second strain of the same species, and whole animals of a closely related species. Restriction fragments were detected by hybridizing radioactive cloned fragments to restriction digests that had been fractionated by size on agarose gels and transferred to nitrocellulose sheets. In this way, approximately 50 BamHI restriction fragments were visualized and compared. Fragments from sperm and somatic DNAs were found to be identical; 15% differed in size between the two strains. Little cross homology was found between the two species. We conclude that, if rearrangements occur in C. elegans DNA during development, they must affect fewer than a few percent of the restriction fragments or restriction sites. The difference found between the two strains and the two species is surprisingly great.

Aging in the nematode Caenorhabditis elegans: major biological and environmental factors influencing life span.

The free-living nematode Caenorhabditis elegans is an excellent experimental system for the study of aging. The present study identifies some of the major biological and environmental factors influencing life span as a prelude to more detailed genetic and biochemical analyses. Life span can be altered during any part of the life cycle by a change in either temperature or food concentration. Parental age and parental life span both have relatively small effects on progeny life span. The nematode accumulates fluorescent pigment resembling lipofuscin, and becomes less sensitive to ultra-violet radiation as it ages.

In situ studies on the effect of acid extraction on the DNA template activity of mature avian erythrocytes.

Exogenous E. coli RNA polymerase was used to determine the in situ DNA template activity of ethanol/acetone fixed avian erythrocytes. No RNA polymerase-catalyzed incorporation of 3H-UTP was detected in mature avian erythrocytes while simultaneously fixed avian lymphocytes did exhibit incorporation of 3H-UTP. Nuclei of mature erythrocytes which were subjected to treatments known to remove histones showed dramatic increases in RNA polymerase-catalyzed incorporation of 3H-UTP. The chromatin of treated cells was presumed to be more accessible to RNA polymerase as determined by the increase in RNA polymerase-catalyzed incorporation of 3H-UTP. Incubation of acid-treated nuclei in poly-L-lysine prior to incubation with RNA polymerase failed to inhibit the incorporation of 3H-UTP. Possible mechanisms for the inactivation of avian erythrocyte nuclei are discussed.