Michael R. Klass

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.

The location of the major protein in Caenorhabditis elegans sperm and spermatocytes.

Abstract Using an affinity-purified antibody to the major sperm protein (MSP) in Caenorhabditis elegans sperm we have shown by immunofluorescence that the MSP is localized in the fibrous bodies of spermatocytes and early spermatids, in the cytoplasm of late spermatids, and in the pseudopods of spermatozoa. The MSP can also form crystalline inclusions in mutant and wild-type sperm. The function of this protein is still unknown, but its ability to form filaments and its localization in the pseudopod, together with the lack of actin in these sperm suggest that the MSP may be required for amoeboid motility.

Expression of chimeric genes in Caenorhabditis elegans

We have shown the expression of transformed genes in the nematode Caenorhabditis elegans using a new gene fusion system. Vectors consisting of the flanking regions of a collagen gene (col-1) or a major sperm protein gene of C. elegans fused to the Escherichia coli uidA gene, encoding beta-glucuronidase, were microinjected into worms and found to be propagated as high-copy extrachromosomal tandem arrays. We have detected beta-glucuronidase activity in transformed lines, and have shown that the activity is dependent upon the correct reading frame of the construction and on the presence of the worm sequences. The enzyme activity was shown to be encoded by the chimeric beta-glucuronidase gene by co-segregation analysis and by inactivation with specific antisera. Expression is at a very low level, and seems to be constitutive. We have used histochemical techniques to visualize the enzyme activity in embryos.

Age-correlated changes in the DNA template in the nematode Caenorhabditis elegans

Analysis of DNA from the nematode Caenorhabditis elegans demonstrated a number of significant age-correlated changes. The number of single-strand breaks as assayed by an in vitro assay procedure using Escherichia coli DNA polymerase I increased significantly with age. There was also an exponential increase in the amount of 5-methylcytosine in C. elegans DNA as the worm matured and aged. Furthermore, DNA isolated from older worms exhibited reduced transcriptional capacity when assayed in a HeLa cell in vitro transcription system. Finally, a biological assay to determine age-correlated changes in the DNA of aging sperm demonstrated a significant reduction in the capacity of the sperm to support zygotic development as the age of the male increased. These findings demonstrated significant age-correlated alterations and modifications occurring in the DNA template of the nematode, and their implications to the aging process are discussed.

Cell-specific transcriptional regulation of the major sperm protein in Caenorhabditis elegans

Abstract The major protein found in nematode sperm exhibits a distinct pattern of developmental regulation. In the nematode Caenorhabditis elegans , the synthesis of the major sperm protein (15K) begins with the onset of spermatogenesis in both the male and hermaphrodite. Both spermatogenesis and 15K synthesis continue for the life of the male while in the protandrous hermaphrodite the major sperm protein is synthesized only during the fourth larval stage. Inhibitor studies using actinomycin D and α-amanitin as well as Northern blot analysis have shown that the primary regulatory mechanism of this gene is at the transcriptional level. Recombinant molecules have been selected bearing the 15K genomic sequence by a positive hybridization translation assay. Using one of these cloned fragments as a probe for in situ hybridization, 15K transcripts have been localized to a specific region of the male gonad. These studies indicate that the gene for the major sperm protein is regulated by a cell-specific transcriptional control mechanism coincidental with the onset of sexual differentiation in the nematode.

Conservation in the 5′ flanking sequences of transcribed members of the Caenorhabditis elegans major sperm protein gene family☆

The major sperm proteins (MSPs) are encoded in the Caenorhabditis genome by a multigene family with more than 50 genes dispersed in small clusters at three chromosomal loci. In spite of their dispersed locations, all of the MSP genes appear to be expressed at the same time exclusively in the testis, indicating co-ordinate temporal and spatial regulation of these dispersed genes. Many of the MSP genes must be transcribed, because RNA hybridization with gene-specific probes showed that individual genes each contribute less than 3% to the total poly(A)+ RNA, and 13 out of 14 sequenced cDNAs came from different genes. Primer extension assays from MSP mRNA showed that most of the MSP mRNAs must be initiated at position -35 from the translation start codon. Extensive similarity was found in the first 100 nucleotides of genomic sequence flanking the start codons of ten MSP genes from different chromosomal locations. All MSP genes contained a consensus ribosome binding site, a consensus TATA homology 27 nucleotides distal to the site of mRNA initiation, and ten highly conserved nucleotides adjacent to the site of initiation. All the MSP genes contained the sequence AGATCT located approximately 65 nucleotides upstream from the transcriptional start, but little or no similarity was found more distal to this. Some of these conserved sequences may be cis-acting control elements that ensure the cell and temporal specificity of transcription of these co-ordinately regulated genes.

Cell-Specific Gene Expression in the Nematode

Publisher Summary The detailed molecular analysis of genes has provided important information on the mechanisms of gene expression. The free-living soil nematode Caenorhabditis elegans is an excellent organism for the multidisciplinary study of gene regulation during development. C. elegans is a genetically manipulable, multicellular eukaryote that differentiates specific tissue types. One of the additional advantage of the nematode as an experimental system is its simple and extensively characterized anatomy. This chapter describes a number of genes in C.elegans whose tissue-specific regulation is currently being investigated. It also presents the culture methods, development, and genetics of the organism. C.elegans can be easily cultured in the laboratory and is routinely grown on agar plates and fed Escherichia coli. Many different approaches have been used for the study of the cell-specific expression of genes of C.elegans . Most analyses use a specific antibody to demonstrate the tissue-specific location and temporal regulation of the protein. Another general approach that has been used is to construct a cDNA clone bank followed by differential screening to identify genes expressed at specific developmental stages or in certain tissues. Such a procedure is used to identify genes expressed during spermatogenesis in the nematode.