Alcohol dehydrogenase gene of Drosophila melanogaster: relationship of intervening sequences to functional domains in the protein

The gene that codes for Drosophila alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase EC 1.1.1.1) was identified in a bacteriophage lambda library of genomic Drosophila DNA by using ADH cDNA cloned DNA as a probe. The DNA sequence of the protein encoding region was shown to be in agreement with the amino acid sequence of the ADH. Two intervening DNA sequences (introns) were identified within the protein encoding region: one was 65 nucleotides and located between the codons for amino acid residues 32 and 33, and one was 70 nucleotides and located between the codons for amino acid residues 167 and 168. Both contained the 5' G-T and 3' A-G dinucleotides characteristic of intron boundaries of eukaryotic genes. On the basis of secondary structure predictions, the first 140 amino acid residues of Drosophila ADH are in an alternating beta-sheet/alpha-helix arrangement which is characteristic of the coenzyme binding domain of dehydrogenases. The smaller of the two introns interrupts the domain predicted to bind the adenine portion of the coenzyme.     

Internal structure of a mitochondrial intron of Aspergillus nidulans.

The intron of the mitochondrial apocytochrome b gene, cobA, of Aspergillus nidulans has been subjected to sequence analysis. It contains an open reading frame of 957 base pairs contiguous with the preceding exon. Regions of the translated open reading frames of cobA and the third intron of the cob gene in yeast show high amino acid homology. Comparison of the cobA intron with this and other yeast introns indicates that cobA codes for a maturase protein that splices out the intron encoding it and possibly other mitochondrial introns. Two very similar decamer peptides are found in the protein sequences of the cobA intron, four mitochondrial yeast introns, and the yeast mitochondrial sequence reading frame 1 (RF-1) and may be diagnostic of one class of maturase-coding introns. Four short DNA sequences, two of which are in the region defined by box9 and box2 mutations in the cob gene of yeast, are conserved in cobA and certain yeast introns. Comparison with three yeast introns strongly suggests that the first 200 base pairs of the open reading frame of the cobA intron do not code for any amino acids present in the putative maturase protein but are required for splicing or the control of splicing, or both.     

Molecular cloning, characterization, and overexpression of ERG7, the Saccharomyces cerevisiae gene encoding lanosterol synthase.

We report the cloning, characterization, and overexpression of Saccharomyces cerevisiae ERG7, which encodes lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme responsible for the complex cyclization/rearrangement step in sterol biosynthesis. Oligonucleotide primers were designed corresponding to protein sequences conserved between Candida albicans ERG7 and the related Arabidopsis thaliana cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A PCR product was amplified from yeast genomic DNA using these primers and was used to probe yeast libraries by hybridization. Partial-length clones homologous to the two known epoxysqualene mutases were isolated, but a full-length sequence was found neither in cDNA nor genomic libraries, whether in phage or plasmids. Two overlapping clones were assembled to make a functional reconstruction of the gene, which contains a 2196-bp open reading frame capable of encoding an 83-kDa protein. The reconstruction complemented the erg7 mutation when driven from either its native promoter or the strong ADH1 promoter.     

Isolation and properties of a nitrile hydratase from the soil fungus Myrothecium verrucaria that is highly specific for the fertilizer cyanamide and cloning of its gene.

A protein was purified from crude extracts of the soil fungus Myrothecium verrucaria by gel filtration and hydrophobic chromatography to homogeneity; this protein catalyzed the stoichiometric hydration of the fertilizer cyanamide to urea with high substrate specificity. This cyanamide hydratase (urea hydro-lyase; EC 4.2.1.69) contained zinc and consisted of six identical subunits with Mr = 27,700. It was partially sequenced. The protein was detectable only when the fungus was grown on cyanamide as the sole nitrogen source. Genomic DNA from the fungus was cloned, and the gene encoding the enzyme was mapped with an oligonucleotide probe derived from the amino acid sequence within a 25,800-base-pair DNA region. The subunit of the enzyme is encoded by a 795-base-pair DNA sequence containing a 63-base-pair intron. A cDNA clone containing the intronless gene with an open reading frame encoding a sequence of 244 amino acids expressed the enzyme in active form in Escherichia coli with excellent yield.     

Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains.

The sequence of nitrate reductase (EC 1.6.6.1) mRNA from the plant Arabidopsis thaliana has been determined. A 3.0-kilobase-long cDNA was isolated from a lambda gt10 cDNA library of Arabidopsis leaf poly(A)+ RNA. The cDNA hybridized to a 3.2-kilobase mRNA whose level increased 15-fold in response to treatment of the plant with nitrate. An open reading frame encoding a 917 amino acid protein was found in the sequence. This protein is very similar to tobacco nitrate reductase, being greater than 80% identical within a section of 450 amino acids. By comparing the Arabidopsis protein sequence with other protein sequences, three functional domains were deduced: (i) a molybdenum-pterin-binding domain that is similar to the molybdenum-pterin-binding domain of rat liver sulfite oxidase, (ii) a heme-binding domain that is similar to proteins in the cytochrome b5 superfamily, and (iii) an FAD-binding domain that is similar to NADH-cytochrome b5 reductase.     

Recognition and cleavage site of the intron-encoded omega transposase.

The optional group I intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae contains a 235-codon-long open reading frame the translation product of which (the omega transposase) catalyzes the formation of a double-strand break within the intron-minus (omega-) copies of the same gene. Purified omega transposase generates in vitro a 4-base-pair staggered cut with 3' hydroxyl overhangs at the exact position where the intron eventually inserts in the gene. Using randomly mutagenized synthetic oligonucleotides, single-base mutants were produced at 21 positions around the cleavage site. Experiments with these oligonucleotides show that the recognition site extends over an 18-base pair-long sequence within which minimal sequence degeneracy is tolerated. The intron-encoded omega transposase is, therefore, one of the most specific restriction endonucleases known to date.     

Arabidopsis thaliana gamma-glutamylcysteine synthetase is structurally unrelated to mammalian, yeast, and Escherichia coli homologs.

A mutant of Escherichia coli, JTG10, deficient in gamma-glutamylcysteine synthetase (gamma-ECS; EC 6.3.2.2) is unable to synthesize glutathione (GSH) and is sensitive to 8-hydroxyquinoline. This phenotype was exploited for the isolation of Arabidopsis thaliana gamma-ECS cDNAs by expression cloning, and clones were selected through functional complementation by growth on 8-hydroxyquinoline. High levels of gamma-ECS activity were detectable in extracts derived from cultures of JTG10 expressing the Arabidopsis gamma-ECS open reading frame, although these complemented mutants accumulated GSH to only 10% of the wild-type level. The derived amino acid sequence constitutes a polypeptide of 59.9 kDa and shows only 44-48% similarity with previously published sequences of rat kidney, human liver, yeast, and E. coli gamma-ECS. When the gamma-ECS cDNA was used as a probe, Southern blot analysis of Arabidopsis genomic DNA revealed that it is present as a low copy number gene. Furthermore, the Arabidopsis gamma-ECS cDNA probe failed to hybridize to maize and tobacco genomic DNA at low stringency, suggesting that heterogeneity in gamma-ECS structure exists between plant species. The activity of recombinant Arabidopsis gamma-ECS was inhibited by buthionine sulfoximine and GSH, indicating that, while differences in the primary and secondary structure of gamma-ECS from different sources exist, the enzymes may have similar active site structures.     

Presence of a member of the Tc1-like transposon family from nematodes and Drosophila within the vasotocin gene of a primitive vertebrate, the Pacific hagfish Eptatretus stouti.

Molecular cloning of the vasotocin gene of a cyclostome, the Pacific hagfish Eptatretus stouti, reveals, in contrast to other known members of the vertebrate vasopressin/oxytocin hormone gene family, an unusual exon-intron organization. Although the location of three exons and two introns is conserved, an additional intron is present 5' of the coding region of the hagfish gene. The third intron, which is greater than 14 kilobase pairs in size, contains on the opposite DNA strand to that encoding vasotocin an open reading frame exhibiting striking similarity to the putative transposase of Tc1-like nonretroviral mobile genetic DNA elements, so far reported only from nematodes and Drosophila. The hagfish element, called Tes1, is flanked by inverted terminal repeats representing an example of the existence of a typical inverted terminal-repeat transposon within vertebrates. The presence of Tc1-like elements in nematodes, Drosophila, and cyclostomes indicates that these genetic elements have a much broader phylogenetic distribution than hitherto expected.     

Isolation and characterization of the gene encoding 2,3-oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae.

The ERG7 gene encoding oxidosqualene-lanosterol cyclase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] from Saccharomyces cerevisiae has been cloned by genetic complementation of a cyclase-deficient erg7 strain. The DNA sequence of this gene has been determined and found to contain an open reading frame of 2196 nt (including stop codon) that encodes a predicted protein of 731 amino acids. The predicted molecular mass of the S. cerevisiae cyclase, 83.4 kDa, is similar to the predicted molecular masses of the oxidosqualene-lanosterol cyclase from Candida albicans and the oxidosqualene-cycloartenol cyclase from Arabidopsis thaliana, as well as to the molecular masses assigned to vertebrate oxidosqualene-lanosterol cyclases; however, it is substantially larger than the molecular mass assigned to purified S. cerevisiae cyclase. At the level of DNA and predicted amino acid sequences, the S. cerevisiae and C. albicans cyclases share 56% and 63% identity, respectively. Tryptophan and tyrosine residues are unusually abundant in the predicted amino acid sequences of (oxido)-squalene cyclases, leading to a hypothesis that electron-rich aromatic side chains from these residues are essential features of cyclase active sites.     

A gene encoding the tryptophan synthase beta subunit of Arabidopsis thaliana.

The DNA sequence of a tryptophan synthase gene and the flanking 5' and 3' regions has been determined for Arabidopsis thaliana. The sequence encodes only the beta subunit domain, indicating that alpha and beta subunits are specified by separate genes. The gene contains four introns and encodes 470 amino acid residues. The plant amino acid sequence is highly conserved with respect to corresponding microbial sequences. The NH2-terminal amino acid sequence is characteristic of chloroplast transit peptides. Identity of the sequences of the genomic exons and a cDNA clone and the presence of cellular RNA corresponding in size and 5' sequence to the gene indicate that the gene is expressed. Analysis of Arabidopsis genomic DNA suggests the presence of a second gene for the beta subunit.     

Bipartite mRNA for chicken alpha-fibrinogen potentially encodes an amino acid sequence homologous to beta- and gamma-fibrinogens.

Overlapping cDNAs derived from the chicken alpha-fibrinogen mRNA have been sequenced, beginning from within the coding region for the signal peptide of this subunit and terminating within the poly(A) extension. The predicted size of chicken alpha-fibrinogen is 54,187 daltons, which is the smallest of any alpha chain reported; the oligopeptide repeats that characterize the central regions of the other alpha subunits were conspicuously absent. A further unexpected finding was the presence on the mRNA of a separate, long open reading frame (752 nucleotides), beginning 312 nucleotides downstream from the alpha-fibrinogen coding sequence and containing intron-like features near its 5' end. The protein sequence predicted from this second open reading frame lacks an initiating methionine but is homologous to the C-terminal regions of all known beta- and gamma-fibrinogens as well as the C termini of two nonfibrinogen proteins: cytotactin (tenascin), an extracellular matrix protein, and pT49, a putative protein specific to cytotoxic T cells. The intron-like features of the second open reading frame immediately precede the region of common homology, and the beginnings of the corresponding homologous segments in the beta- and gamma-fibrinogen sequences are marked by aligned intron positions. Based on these findings, it is proposed that fibrinogen gene evolution included a fusion of two distinct ancestral genes.     

Human renal carcinoma expresses two messages encoding a parathyroid hormone-like peptide: evidence for the alternative splicing of a single-copy gene.

A peptide secreted by tumors associated with the clinical syndrome of humoral hypercalcemia of malignancy was recently purified from human renal carcinoma cell line 786-0. The N-terminal amino acid sequence of this peptide has considerable similarity with those of parathyroid hormone (PTH) and of peptides isolated from human breast and lung carcinoma (cell line BEN). In this study we obtained the nucleotide sequence of a 1595-base cDNA complementary to mRNA encoding the PTH-like peptide produced by 786-0 cells. The cDNA contains an open reading frame encoding a leader sequence of 36 amino acids and a 139-residue peptide, in which 8 of the first 13 residues are identical to the N terminus of PTH. Through the first 828 bases the sequence of this cDNA is identical with one recently isolated from a BEN cell cDNA library; however, beginning with base 829 the sequences diverge, shortening the open reading frame by 2 amino acids. Differential RNA blot analysis revealed that 786-0 cells express two major PTH-like peptide mRNAs with different 3' untranslated sequences, one of which hybridizes with the presently described sequence and the other one with that reported for the BEN cell PTH-like peptide cDNA. Primer-extension analysis of 786-0 poly(A)+ RNA together with Southern blot analysis of human DNA confirmed the presence of a single-copy gene coding for multiple mRNAs through alternate splicing. In addition, the 3' untranslated sequence of the cDNA described here has significant similarity to the c-myc protooncogene.     

Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y.

Escherichia coli replication factor Y (protein n') functions in the assembly of a mobile multiprotein replication-priming complex called the primosome. Although the role of factor Y in primosome assembly during replication in vitro of bacteriophage phi X174 and plasmid pBR322 DNA is clear, its role in E. coli chromosomal replication is not. To address this issue, the gene for factor Y has been cloned molecularly and its DNA sequence has been determined. The cloned fragment of DNA contained an open reading frame capable of encoding a polypeptide of 81.7 kDa. This open reading frame contains amino acid sequences identical to 13 N-terminal amino acids of purified factor Y, as well as to a 10-amino acid internal sequence (from a cyanogen bromide fragment) as determined by gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame using a bacteriophage T7 transient expression system resulted in the accumulation of a polypeptide with an apparent molecular mass of 78 kDa that comigrated with bona fide factor Y during SDS/polyacrylamide gel electrophoresis. Soluble extracts made from cells overexpressing the product of the putative factor Y open reading frame showed a 2000-fold increase in factor Y activity during bacteriophage phi X174 complementary-strand DNA synthesis in vitro when compared to control extracts. The gene encoding factor Y, which maps to 88.5 min on the E. coli chromosome, has been designated primosome A (priA).     

Primary structure of the Escherichia coli ribonucleoside diphosphate reductase operon.

The nucleotide sequence of the Escherichia coli K-12 DNA comprising the operon for the structural genes of the subunits of ribonucleotide diphosphate reductase has been determined. The DNA sequenced maps at 48.5 minutes on the E. coli chromosome and includes a total length of 8557 nucleotides. An open reading frame between nucleotides 3506 and 5834, encoding a 776-amino acid polypeptide chain with a molecular weight of 87,532, has been identified as the nrdA gene. An open reading frame between nucleotides 6012 and 7139, encoding a 375-amino acid polypeptide with a molecular weight of 43,466, has been identified as the nrdB gene. The sequences reveal not only the primary structures for both subunits, but also some interesting aspects of potential regulatory sites.