The C. elegans vitellogenin genes: short sequence repeats in the promoter regions and homology to the vertebrate genes.

The nematode Caenorhabditis elegans contains a small family of vitellogenin genes which is expressed abundantly, but only in the intestine of the adult hermaphrodite worm. In order to identify possible regulatory elements, we have sequenced the DNA surrounding the 5' ends of five of the six genes. Contained within regions which have largely diverged from one another, two different heptameric sequences are found repeated within the first 200 bp upstream of each of the genes. The first sequence, TGTCAAT, is present as a perfect heptamer at least once upstream of each gene. It is repeated in both orientations four to six times in each 5' flanking region, allowing a one-base mismatch. The second sequence, CTGATAA, is also present as a perfect heptamer in a restricted region upstream of each gene. These two sequence elements may be involved in regulation of the vitellogenin genes. Remarkably, the CTGATAA sequence is present in a similar location in the promoter regions of vertebrate vitellogenin genes. In fact, our data reveal a surprising degree of similarity between the nematode and vertebrate vitellogenins.     

The Caenorhabditis elegans vitellogenin gene family includes a gene encoding a distantly related protein.

While the nematode Caenorhabditis elegans is more primitive than most egg-laying organisms, it's vitellogenins, or yolk protein precursors, appear to be more complex. C. elegans oocytes accumulate two major classes of yolk proteins. The first consists of two polypeptides with an Mr of about 170,000 (yp170A and yp170B) encoded by a family of five closely related genes called vit-1 through vit-5. The second class consists of two smaller proteins with Mr values of 115,000 (yp115) and 88,000 (yp88) which are cut from a single precursor. Here we report the cloning and analysis of a single-copy gene (vit-6) that encodes this precursor. The lengths of the gene and its mRNA are about 5 X 10(3) base pairs. Like vit-1 through vit-5, vit-6 is expressed exclusively in adult hermaphrodites. Comparison of portions of the coding sequence indicates that vit-6 is distantly related to the vit-1 through vit-5 gene family. Thus, even though the two classes of yolk proteins are antigenically and physically distinct, they are encoded by a single highly diverged gene family.     

Vitellogenin motifs conserved in nematodes and vertebrates

Summary Caenorhabditis elegans vitellogenins are encoded by a family of six genes, one of which,vit-5, has been previously sequenced and shown to be surprisingly closely related to the vertebrate vitellogenin genes. Here we report an alignment of the amino acid sequences of vitellogenins from frog and chicken with those from threeC. elegans genes:vit-5 and two newly sequenced genes,vit-2 andvit-6. The four introns ofvit-6 are all in different places from the four introns ofvit-5, but three of these eight positions are identical or close to intron locations in the vertebrate vitellogenin genes. The encoded polypeptides have diverged from one another sufficiently to allow us to draw some conclusions about conserved positions. Many cysteine residues have been conserved, suggesting that vitellogenin structure has been maintained over a long evolutionary distance and is dependent upon disulfide bonds. In addition, a 20-residue segment shows conservation between the vertebrate and the nematode vitellogenins. This sequence may play a highly conserved role in vitellogenesis, such as specific recognition by oocytes. On the whole, however, selection may be acting more strongly on amino acid composition and codon usage than on amino acid sequence, as might be expected for abundant storage proteins: The amino acid compositions ofvit-2, vit-5, andvit-6 products are remarkably similar, despite the fact that the sequence of thevit-2 protein is only 22% and 50% identical to the sequences ofvit-6 andvit-5 proteins, respectively.     

Trichinella spiralis mtDNA: a nematode mitochondrial genome that encodes a putative ATP8 and normally structured tRNAS and has a gene arrangement relatable to those of coelomate metazoans

The complete mitochondrial DNA (mtDNA) of the nematode Trichinella spiralis has been amplified in four overlapping fragments and 16,656 bp of its sequence has been determined. This sequence contains the 37 genes typical of metazoan mtDNAs, including a putative atp8, which is absent from all other nematode mtDNAs examined. The genes are transcribed from both mtDNA strands and have an arrangement relatable to those of coelomate metazoans, but not to those of secernentean nematodes. All protein genes appear to initiate with ATN codons, typical for metazoans. Neither TTG nor GTT start codons, inferred for several genes of other nematodes, were found. The 22 T. spiralis tRNA genes fall into three categories: (i) those with the potential to form conventional "cloverleaf" secondary structures, (ii) those with TPsiC arm + variable arm replacement loops, and (iii) those with DHU-arm replacement loops. Mt-tRNA(R) has a 5'-UCG-3' anticodon, as in most other metazoans, instead of the very unusual 5'-ACG-3' present in the secernentean nematodes. The sequence also contains a large repeat region that is polymorphic in size at the population and/or individual level.     

Loss and gain of domains during evolution of cut superclass homeobox genes

The cut superclass of homeobox genes has been divided into three classes: CUX, ONECUT and SATB. Given the various completed genomes, we have now made a comprehensive survey. We find that there are only two cut domain containing genes in Drosophila, one CUX and one ONECUT type. Caenorhabditis elegans has undergone an expansion of the ONECUT subclass genes and has a gene cluster with three ONECUT class genes, one of which has lost the cut domain. Two of these genes contain a conserved sequence motif, termed OCAM, which also occurs in another gene in C. elegans this motif seems to be nematode specific. A recently uncovered C. elegans CUX gene has sequence conservation in its amino-terminus with vertebrate CUX proteins. Further, the 5' end of this gene containing the conserved region can undergo alternative splicing to give rise to a protein with a different carboxy-terminus lacking the cut- and homeodomain. This protein is conserved in its entirety with vertebrate genes termed CASP--which are also alternative splice products of the CUX genes--and with plant and fungal genes. The highly divergent SATB genes share a conserved amino terminal domain, COMPASS, with the Drosophila defective proventriculus gene and a C. elegans ORF. These two "COMPASS" family genes encode two highly divergent homeodomains, may be homologues of the SATB genes and thus probably belong to the cut superclass, too.     

The spliceosomal snRNAs of Caenorhabditis elegans.

Nematodes are the only group of organisms in which both cis- and trans-splicing of nuclear mRNAs are known to occur. Most Caenorhabditis elegans introns are exceptionally short, often only 50 bases long. The consensus donor and acceptor splice site sequences found in other animals are used for both cis- and trans-splicing. In order to identify the machinery required for these splicing events, we have characterized the C. elegans snRNAs. They are similar in sequence and structure to those characterized in other organisms, and several sequence variations discovered in the nematode snRNAs provide support for previously proposed structure models. The C. elegans snRNAs are encoded by gene families. We report here the sequences of many of these genes. We find a highly conserved sequence, the proximal sequence element (PSE), about 65 bp upstream of all 21 snRNA genes thus far sequenced, including the SL RNA genes, which specify the snRNAs that provide the 5' exons in trans-splicing. The sequence of the C. elegans PSE is distinct from PSE's from other organisms.     

Analysis of the VPE sequences in the Caenorhabditis elegans vit-2 promoter with extrachromosomal tandem array-containing transgenic strains.

The Caenorhabditis elegans vit genes, encoding vitellogenins, are abundantly expressed in the adult hermaphrodite intestine. Two repeated elements, vit promoter element 1 (VPE1 [TGTCAAT]) and VPE2 (CTGATAA), have been identified in the 5' flanking DNA of each of the vit genes of C. elegans and Caenorhabditis briggsae. These elements have previously been shown to be needed for correctly regulated expression of a vit-2/vit-6 fusion gene in low-copy-number, integrated transgenes. Here we extend the analysis of the function of VPE1 and VPE2 by using transgenic lines carrying large, extrachromosomal arrays of the test genes. The results validate the use of such arrays for transgenic analysis of gene regulation in C. elegans, by confirming previous findings showing that the VPE1 at -45 and both VPE2s are sites of activation. Additional experiments now indicate that when the -45 VPE1 is inverted or replaced by a VPE2, nearly total loss of promoter function results, suggesting that the highly conserved -45 VPE1 plays a unique role in vit-2 promoter function. In contrast, single mutations eliminating the three upstream VPE1s are without effect. However, in combination in double and triple mutants, these upstream VPE1 mutations cause drastic reductions in expression levels. The -150 VPE2 can be replaced by a XhoI site (CTCGAG), and the -90 VPE2 can be eliminated, as long as the overlapping VPE1 is left intact, but when these two replacements are combined, activity is lost. Thus, the promoter must have at least one VPE2 and it must have at least two VPE1s, one at -45 and one additional upstream element.     

Amino acid sequence of phosvitin derived from the nucleotide sequence of part of the chicken vitellogenin gene

The amino acid sequence of the egg yolk storage protein phosvitin has been deduced from the nucleotide sequence of part of the chicken vitellogenin gene. Of the phosvitin sequence, 210 amino acids including the N-terminal residue are contained on one large exon, whereas the remaining six amino acids are encoded on the next exon. Phosvitin contains a core region of 99 amino acids, consisting of 80 serines, grouped in runs of maximally 14 residues interspersed by arginines, lysines, and asparagines. The serines of the core region are encoded by AGC and AGT codons exclusively and the arginines by AGA and AGG, which results in a continuous stretch of 99 codons with adenine in the first position. The N-terminal quarter of the phosvitin sequence contains 16 serines grouped in a cluster with alanines and threonines and coded mainly by TCX triplets. The C-terminal part includes 27 serines, preferentially coded by AGC and AGT, 13 histidine residues, and the sequence ...Asn-Gly-Ser... at which the carbohydrate moiety of phosvitin is attached. Heteroduplex formation between cloned DNAs from chicken and Xenopus vitellogenin genes shows that the phosvitin sequence contains a stretch of highly conserved sequence.     

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.     

Characterization of regions of the Caenorhabditis elegans X chromosome containing vitellogenin genes

Caenorhabditis elegans contains a family of vitellogenin genes consisting of five closely related genes (vit-1 to vit-5) coding for 186,000 Mr yolk proteins, and one distantly related gene (vit-6) encoding a 200,000 Mr precursor to two smaller yolk proteins. We demonstrate here that, although vit-1 to vit-5 are not clustered (with the exception of vit-3 and vit-4), they are all on the X chromosome. In contrast, vit-6 is autosomal. The genes are strictly regulated during development: they are activated in the intestine of the hermaphrodite worm, following the last larval molt. In order to determine whether the vit genes are contained within chromosomal domains of similarly regulated genes, we have used the chromosomal "walking" technique to isolate 55,000 to 60,000 base-pairs of DNA surrounding each of the X-linked genes and determined the developmental specificity of nearby genes. In the total of 235,400 base-pairs of cloned DNA, seven genes, in addition to the five vit genes, were found. The average gene spacing is approximately 20,000 base-pairs per gene but is highly variable, ranging from less than 2000 to more than 38,000 base-pairs. The seven newly identified genes, called uvt-1 to uvt-7, specify RNAs varying in size from 500 to 2700 bases. With the exception of uvt-4, all of the genes are developmentally regulated; but the patterns of regulation are quite variable, and all are different from the vitellogenin genes. The vit genes, therefore, are not contained within co-regulated chromosomal domains. We also searched for the presence of repetitive DNA, but only four such sequences were found.     

Molecular cloning of a steroid-regulated 108K heat shock protein gene from hen oviduct.

The natural gene for a steroid inducible 108K heat shock protein has been isolated from a lambda genomic library prepared from hen oviduct tissue. Genomic DNA blots indicate that it exists as a single copy gene in the chick oviduct haploid genome. The 9.9 kilobase gene codes for a messenger RNA of 2733bp (21) and is split into 18 exons as established by sequence comparison of cDNA and genomic clones. The 3' end of the gene contains a repetitive element which shares homology with the CR1 family of repeats. The first exon contains both the untranslated leader and coding regions of the gene. The promoter region is rich in G + C residues (70%) and the dinucleotide CG. This 5' flanking segment contains bases similar both in sequence and location to the Goldberg-Hogness TATA homology and consensus sequence CCAAT. A consensus sequence located upstream of steroid hormone responsive chicken genes is found at -267 and on a reverse orientation at -593. The structure of this gene is of interest since the presence of introns in heat shock genes is rare in any species examined to date. Furthermore, this gene lacks the previously described heat shock promoter consensus sequence (C-GAA-TTC-G) present in other species.     

Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions

The primary sequence of the major heat shock gene of D. melanogaster, that for the 70,000 protein, has been determined. One of the reading frames is devoid of stop codons for over 2000 bp. The region between the first ATG and the first stop codon encodes a protein of molecular weight 70,270. The 5' end of the messenger RNA was localized in the DNA sequence by two independent methods. The 5' flanking sequences of three distinct 70K genes were also determined. Extensive homology in the primary sequences extends about 500 bp upstream from the ATG, which is the presumptive initiation of protein synthesis. Each 70K gene has the putative promoter sequence TATAAATA about 325 bp upstream from this ATG. A heptanucleotide sequence identified as the capping site for other messengers is found 24-30 bp downstream from the ends of the A-T-rich sequence. A 12 bp sequence with dyad symmetry begins 23 bp upstream from the beginning of the above A-T-rich sequence.     

Operon structure and trans-splicing in the nematode Pristionchus pacificus

In the nematode Caenorhabditis elegans, up to 15% of the genes are organized in operons. Polycistronic precursor RNAs are processed by trans-splicing at the 5' ends of genes by adding a specific trans-spliced leader. Ten different spliced leaders are known in C. elegans that differ in sequence and abundance. The SL1 leader is most abundant and is spliced to the 5' ends of monocistronic genes and to upstream genes in operons. Trans-splicing is common among nematodes and was observed in the genera Panagrellus, Ascaris, Haemonchus, Anisakis, and Brugia. However, little is known about operons in nonrhabditid nematodes. Dolichorhabditis CEW1, another rhabditid nematode that is now called Oscheius CEW1, contains operons and SL2 trans-splicing. We have studied the presence of operons and trans-splicing in Pristionchus pacificus, a species of the Diplogastridae that has recently been developed as a satellite organism in evolutionary developmental biology. We provide evidence that P. pacificus contains operons and that downstream genes are trans-spliced to SL2. Surprisingly, the one operon analyzed so far in P. pacificus is not conserved in C. elegans, suggesting unexpected genomic plasticity.