The nucleotide sequence of a nematode vitellogenin gene.

The nematode, Caenorhabditis elegans, contains a family of six genes that code for vitellogenins. Here we report the complete nucleotide sequence of one of these genes, vit-5. The gene specifies a mRNA of 4869 nucleotides, including untranslated regions of 9 bases at the 5' end and 51 bases at the 3' end. Vit-5 contains four short introns totalling 218 bp. The predicted vitellogenin, yp170A, has a molecular weight of 186,430. At its N terminus it is clearly related to the vitellogenins of vertebrates. However, the vit-5-encoded protein does not contain a serine-rich sequence related to the vertebrate vitellin, phosvitin. In fact, the amino acid composition of the nematode protein is very similar to that of the vertebrate protein without phosvitin. Vit-5 has a highly asymmetric codon choice dictionary. The favored codons are different from those favored in other organisms, but are characteristic of highly expressed C. elegans genes. The strong selection against rare codons is not as great near the 5' end of the gene; rare codons are 15 times more frequent within the first 54 bp than in the next 4.8 kb.     

Domain organization and intron positions inCaenorhabditis elegans collagen genes: The 54-bp module hypothesis revisited

Summary The amino acid (aa) sequences of the polypeptides encoded by five collagen genes of the nematodeCaenorhabditis elegans, col-6, col-7 (partial),col-8, col-14, andcol-19, were determined. These collagen polypeptides, as well as those encoded by the previously sequencedC. elegans collagen genescol-1 andcol-2, share a common organization into five domains: an amino-terminal leader, a short (30–33 aa) (Gly-X-Y) _n domain, a non(Gly-X-Y) spacer, a long (127–132 aa) (Gly-X-Y) _n domain, and a short carboyl-terminal domain. The domain organizations and intron positions of these polypeptides were compared with those of the polypeptides encoded byDrosophila andStrongylocentrotus type IV, and vertebrate types I, II, III, IV, and IX collagen genes; theC. elegans collagen polypeptides are most similar to the vertebrate type IX collagents. It is suggested that the collagen gene family comprises two divergent subfamilies, one of which includes the vertebrate interstitial collagen genes, and the other of which includes the invertebrate collagen genes and the vertebrate type IV and type IX collagen genes. Only the vertebrate interstitial collagen genes display clear evidence of evolution via the tandem duplication of a 54-bp exon.     

The plastid aldolase gene fromChlamydomonas reinhardtii: Intron/exon organization, evolution, and promoter structure

Genomic clones encoding the plastidic fructose- 1,6-bisphosphate aldolase ofChlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of theC. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5 border sequences of introns in the aldolase gene ofC. reinhardtii exhibit the conserved plant consensus sequence. The 3 acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in theC. reinhardtii genome.     

Potential regulatory elements of nematode vitellogenin genes revealed by interspecies sequence comparison

Summary The nematode,Caenorhabditis elegans, has a six-member gene family encoding vitellogenins, the yolk protein precursors. These genes are expressed exclusively in the intestine of the adult hermaphrodite. Here we report the cloning of all five members of the homologous gene family from anotherCaenorhabditis species,Caenorhabditis briggsae. Nucleotide sequence analysis of these genes reveals they are about 85% identical to theC. elegans genes in the coding regions. Oveerall similarity is much reduced in noncoding and flanking regions. However, two repeated heptamers, previously identified in the upstream regions of theC. elegans genes, are largely conserved in both location and sequence inC. briggsae. Conservation of certain of these heptamers suggests that proteins bound at these positions may be especially important to promoter function and/or regulation. Comparative sequence analysis also suggests the possibility that the first 70 bases of the vitellogenin mRNAs can be folded into stable secondary structures. Almost all base differences between the two species occur in sequences predicted to be unpaired, suggesting that the ability to form intrastrand base pairs has been selected duringCaenorhabditis evolution.     

Sequence organization of the chloroplast ribosomal spacer ofSpinacia oleracea including the 3′ end of the 16S rRNA and the 5′ end of the 23S rRNA

The 2201-bp spacer between the chloroplast ribosomal 16S and 23S genes ofSpinacia oleracea was sequenced. It contains the genes of the tRNA^Ile (GAU) and tRNA^Ala (UGC) which are both interrupted by introns of respectively 728 and 816 bp. These introns belong to the class II according to the classfication of Michel and Dujon [17]. Comparison of the rDNA spacer sequence of maize, tobacco and spinach indicates that no conserved polypeptide is encoded within the introns of the two tRNA genes and that the two main insertions/deletions between the three plants are located within two loops of the class II introns secondary structure, which is therefore conserved. Based on the sequence complementarity observed between the upstream and downstream parts, of the 16S and 23S rRNA genes, RNase III-like secondary structures involved in the processing of the rRNA precursor are proposed.     

Isolation and characterization of the gene from Pseudomonas syringae pv. phaseolicola encoding the phaseolotoxin-insensitive ornithine carbamoyltransferase

Summary The gene coding for the phaseolotoxin-insensitive ornithine carbamoyltransferase (OCTase) fromPseudomonas syringae pv.phaseolicola has been cloned and sequenced. The gene has a deduced coding capacity for a polypeptide with a calculated M, of 36520 daltons. Comparison of the amino acid sequence of the OCTase enzymes encoded by theP. aeruginosa argF and theEscherichia coli argI andargF genes with the deduced sequence of the newly identified gene shows that 79 amino acid residues are strictly conserved in all four polypeptides; among these 7 out of 9 residues are involved in enzyme function. Of three amino acid regions that have been implicated in substrate binding or catalysis, two are strictly conserved, and the third involved in carbamoylphosphate binding differs. This correlates well with published data showing that phaseolotoxin competes for the carbamoylphosphate binding site in the phaseolotoxin-sensitive OCTases. We propose that the gene be namedargK.     

Rudimentary phosvitin domain in a minor chicken vitellogenin gene

We have determined the nucleotide sequence and the derived amino acid sequence of the phosphoprotein-encoding region of the chicken vitellogenin III gene. The sequence of this minor vitellogenin could be aligned with exon 22 up to exon 27 of the previously sequenced major vitellogenin II gene (van het Schip et al., 1987). The exon 23 and 25 sequences are rich in serine codons (26% and 41%, respectively), and this region encodes at least one of the small egg yolk phosphoproteins. The major egg yolk phosphoprotein, phosvitin, is encoded by the analogous region in vitellogenin II. Comparison of the vitellogenin II and vitellogenin III sequences shows a great reduction in the size of the putative exon 23 of the latter (321 base pairs as opposed to 690). The number of serine codons is also drastically reduced from 124 in exon 23 of the vitellogenin II gene to 28 in vitellogenin III. The grouping of synonymous serine codons, as has hitherto been observed in sequenced vitellogenin phosphoproteins, has been maintained in vitellogenin III. A putative asparagine-linked N-glycosylation site which was conserved in the chicken vitellogenin II and the Xenopus laevis vitellogenin A2 gene, at the beginning of exon 23, is also present in vitellogenin III. The two chicken vitellogenins show a low conservation in the phosphoprotein-encoding region (average 33%, at the protein level) compared to that in the peripheral sequences (58% identity), which indicates that it is a rapidly evolving domain of the vertebrate vitellogenin gene.     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

Active MHC class Ib genes in rat are pseudogenes in the mouse

The most telomeric class I region of the MHC in rat and mouse is the M region, which contains about 20 class I genes or gene fragments. The central part carries three class I genes—M4, M5, and M6—which are orthologous between the two species. M4 and M6 are pseudogenes in the mouse but transcribed, intact genes in the rat. To analyze the pseudogene status for the mouse genes in more detail, we have sequenced the respective exons in multiple representative haplotypes. The stop codons are conserved in all mouse strains analyzed, and, consistent with the pseudogene status, all strains show additional insertions and deletions, taking the genes further away from functionality. Thus, M4 and M6 indeed have a split status. They are silent in the mouse but intact in the closely related rodent, the rat.GenBank accession numbers: AF057065 to AF057072 (exon 3 of H2-M4 of reported mouse strains), AF057976 to AF057985 (exon 3 of RT1.M4 of reported rat strains), AF058923 and AF058924 (exon 2 of RT1.M4 of strains PVG and BN), AY286080 to AY286092 (exon 4 of H2-M6 of reported mouse stains), and AY303772 (full-length genomic sequence of RT1.M6-1^l)     

Caenorhabditis Globin genes: Rapid intronic divergence contrasts with conservation of silent exonic sites

Globin genes from theCaenorhabditis speciesbriggsae andremanei were identified and compared with a previously describedC. elegans globin gene. The encoded globins share between 86% and 93% amino acid identity, with most of the changes in or just before the putative B helix.C. remanei was found to have two globin alleles,Crg1-1 andCrgl-2. The coding sequence for each is interrupted by a single intron in the same position. The exons of the two genes are only 1 % divergent at the nucleotide level and encode identical polypeptides. In contrast, intron sequence divergence is 16% and numerous insertions and deletions have significantly altered the size and content of both introns. Genetic crosses show thatCrg1-1 andCrgl-2 segregate as alleles. Homozygous lines for each allele were constructed and northern analysis confirmed the expression of both alleles. These data reveal an unusual situation wherein two alleles encoding identical proteins have diverged much more rapidly in their introns than the silent sites of their coding sequences, suggesting multiple gene conversion events. Correspondence to: D. Goldberg     

An orphan gyrB in the Mycobacterium smegmatis genome uncovered by comparative genomics

DNA gyrase is an essential topoisomerase found in all bacteria. It is encoded bygyrB andgyrA genes. These genes are organized differently in different bacteria. Direct comparison ofMycobacterium tuberculosis andMycobacterium smegmatis genomes reveals presence of an additionalgyrB inM. smegmatis flanked by novel genes. Analysis of the amino acid sequence of GyrB from different organisms suggests that the orphan GyrB inM. smegmatis may have an important cellular role.     

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I inNicotiana sylvestris

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear genepsaD. We isolated apsaD genomic clone fromNicotiana sylvestris, by screening a genomic library with apsaD cDNA which we previously cloned fromN. sylvestris (Yamamotoet al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains apsaD gene, which does not correspond to thepsaD cDNA, so we designated these genespsaDb andpsaDa, respectively. ThepsaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for theN. sylvestris PSI-D protein encoded bypsaDb begins at the 49th residue. The products ofpsaDa andpsaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies ofpsaD are present in theN. sylvestris genome. Ribonuclease protection assays and immunoblot analysis inN. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio ofpsaDb topsaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms controlpsaDa andpsaDb expression at both the mRNA and protein levels during leaf development.     

The myrosinase (thioglucoside glucohydrolase) gene family in Brassicaceae

The glucosinolate hydrolyzing enzymes myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) are encoded by a multigene family consisting of two subgroups. The first two nuclear genes representing each of these two subgroups of the new gene family, Myr1.Bn1 and Myr2.Bn1, from Brassica napus have been cloned and sequenced. Based on conserved regions in cDNA of three species, PCR (polymerase chain reaction) primers were made, and used to amplify and characterize the structure of the myrosinase genes in seven species of Brassiceae. Southern hybridization analysis of PCR products and genomic DNA indicates that myrosinase is encoded by at least 14 genes in B. napus, with similar numbers in the other species of Brassicaceae investigated. The Myr1 gene cloned from B. napus has a 19 amino acid signal peptide and consists of 11 exons of sizes ranging from 54 to 256 bp and 10 introns of sizes from 75 to 229 bp. The Myr2 gene has a 20 amino acid signal peptide and consists of 12 exons ranging in size from 35 to 262 bp and 11 introns of sizes from 81 to 131 bp. The exons from the two genes have 83% homology at the amino acid level. The intron-exon splice sites are of GT..AG consensus type. The signal peptides and presence of sites for N-linked glycosylation, suggest transport and glycosylation through the ER-Golgi complex. The differences between the two genes are discussed on the basis of their predicted expression at different developmental stages in the plant. Both genes show homology to a conserved motif representing the glycosyl hydrolase family of enzymes.     

AgTHR4, a new selection marker for transformation of the filamentous fungusAshbya gossypii, maps in a four-gene cluster that is conserved betweenA. gossypii andSaccharomyces cerevisiae

Single-read sequence analysis of the termini of eight randomly picked clones ofAshbya gossypii genomic DNA revealed seven sequences with homology toSaccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of theS. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putativeAgTHR4 gene ofA. gossypii. It comprises 512 codons, two less than theS. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of theA. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying theAgTHR4 gene and variousS. cerevisiae ARS elements. Using these plasmids only very weak complementation of aS. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to theAgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved inA. gossypii andS. cerevisiae.     

Osmia cornifrons vitellogenin: cDNA cloning,structural analysis and developmental expression

Osmia cornifrons plays a major role in the pollination of orchards, but basic information on vitellogenin and oocyte development is limited. To better understand vitellogenin in hymenopteran insects, we cloned a cDNA encoding vitellogenin from the hornfaced bee O. cornifrons. Osmia cornifrons vitellogenin cDNA contains 5477 bp with an open reading frame of 1783 amino acid residues, and has a predicted molecular mass of approximately 200.21 kDa and a pI of 6.55. Osmia cornifrons vitellogenin possesses four consensus (RXXR/S) cleavage sites and has conserved DGXR and GL/ICG motifs in the C‐terminus. The deduced amino acid sequence of the O. cornifrons vitellogenin cDNA showed a 66% identity with Megachile rotundata, 53% to Apis mellifera, 51% to Bombus ignitus and 42%–30% with other hymenopteran insect vitellogenins. Phylogenetic analysis showed that O. cornifrons vitellogenin clustered with vitellogenins from Megachilidae, Apidae, Vespidae and Formicidae species but not with those from Pteromalidae, Aphelinidae or Ichneumonidae species. The expression profile of O. cornifrons vitellogenin mRNA during development revealed that O. cornifrons vitellogenin was first detected in the pupal stage and was continuously detected during the adult stage. Interestingly, O. cornifrons vitellogenin mRNA expression was low in mid‐diapause, then gradually increased beginning on day 3 of the newly emerged adult stage, and subsequently declined. These results suggest that the expression level of O. cornifrons vitellogenin mRNA is stage‐specific.