Cloning and sequencing the HinfI restriction and modification genes

The HinfI restriction and modification genes were cloned on a 3.9-kb PstI fragment inserted into the PstI site of plasmid pBR322. Both genes are confined to an internal 2.3-kb BclI-AvaI subfragment. This subfragment was sequenced. Two large open reading frames (ORF's) are present. ORF1 codes for the methylase [predicted 359 amino acids (aa)] and ORF2 codes for the endonuclease (predicted 262 or 272 aa).     

Cloning and sequencing of hydroxylase genes involved in taxol biosynthesis

Two full-length cDNAs (TCH1 and TCH2) were obtained from a cDNA library of Taxus chinensis mainly by the single specific-primer PCR (SSP-PCR) method. Compared with other reported enzymes from Taxus species, the deduced amino acid sequences of TCH1 and TCH2 exhibit significant homologies to hydroxylases that are involved in taxol biosynthesis. These findings imply that the two new genes are closely related to the biosynthesis of taxol/taxoids. Data Accession No: AF545833 and AY374652.     

Cloning, expression and sequencing of Helicobacter felis urease genes

Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid cione led to the construction of plLL205 (9.5 kb) which conferred a urease activity of 1.2±0.5 μmole urea min^-1 mg^-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on plLL205 which were necessary for the expression of an urease-positive phenotype in E. coii clones. To localize the putative structural genes of H. felis on plLL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant cione did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had caicuiated moiecuiar masses of 26 074 and 61 663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.     

Cloning and sequencing of Pseudomonas genes encoding vanillate demethylase.

A 2,598-base-pair (bp) SalI-HincII DNA fragment has been cloned which codes for vanillate demethylase, the enzyme responsible for the demethylation of vanillate (3-methoxy-4-hydroxybenzoate) to protocatechuate (3,4-dihydroxybenzoate). Complementation and insertional inactivation experiments have shown that this fragment carries two genes (vanA and vanB) which are predominantly cotranscribed from a promoter upstream of vanA. Nucleotide sequencing of the SalI-HincII fragment confirmed the genetic data: two open reading frames of 987 and 942 bp were present in the transcribed orientation. These had a very high G + C content in the third base of each codon, which is characteristic of Pseudomonas chromosomal genes. Expression of the genes in Escherichia coli with the T7 RNA polymerase-promoter system gave rise to two polypeptides of 36 and 33 kilodaltons which could be identified by deletion analysis as the products of vanA and vanB, respectively. A search of the protein sequence data bank indicated that the vanB gene product was related to the ferredoxin family.     

Cloning and sequencing of mutantpsbB genes of the cyanobacteriumSynechocystis PCC 6803

Ten strains from a collection of mutants ofSynechocystis 6803 defective in Photosystem II (PS II) function were transformed with chromosomal DNA of wild-type and mutant cells. Cross hybridization data allowed to identify four groups of PS II-mutants. Highly efficient transformation was observed between different mutant groups, but not within the groups. Restoration of photosynthetic activity of the mutant cells was also achieved by transformation with different parts of a 5.6 kbBam HI fragment of wild typeSynechocystis DNA containing thepsbB gene. Each group of mutants was transformed to photoautotrophic growth by specific subfragments of thepsbB gene. DNA fragments of four selected mutant strains hybridizing with thepsbB gene were isolated and sequenced. The mutations were identified as a single nucleotide insertion or substitution leading to stop codon formation in two of the mutants, as a deletion of 12 nucleotides, or as a nucleotide substitution resulting in an amino acid substitution in the other two mutants. Deletion of 12 nucleotides in mutant strain PMB1 and stop codon formation in strain NF16 affect membrane-spanning regions of the gene product, the CP 47 protein.     

Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus

Molecular Genetics and Genomics - Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H2 at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H2ase) was...     

Cloning and sequencing of genes encoding malonate decarboxylase in Acinetobacter calcoaceticus

Malonate decarboxylase from Acinetobacter calcoaceticus was isolated and characterized (Kim, Y.S., Byun, H.S., J. Biol. Chem. 269 (1994) 29636–29641), and its subunits were reanalyzed recently to be α, β, γ, and δ. The genes for the subunits, MdcA (548 a.a.), B (295 a.a.), C (238 a.a.), and D (102 a.a.), of the enzyme have been cloned by using oligonucleotide primers deduced from amino acid sequences of peptides isolated from the purified enzyme, and sequenced to be clustered in an operon in the order of A-D-B-C. The operon was found to encode more genes than mdcABCD. The Escherichia coli, transformed with the vector containing the insert mdcADBC and about 1.7 kb of an upstream region, expressed the four subunits of the enzyme but the proteins did not show enzyme activity. It indicates that, like the enzymes from Malonomonas rubra and Klebsiella pneumoniae, more genes are needed for the formation of the functional malonate decarboxylase.     

Cloning and sequencing the HinfI restriction and modification genes

The HinfI restriction and modification genes were cloned on a 3.9-kb PstI fragment inserted into the PstI site of plasmid pBR322. Both genes are confined to an internal 2.3-kb BclI-AvaI subfragment. This subfragment was sequenced. Two large open reading frames (ORF's) are present. ORF1 codes for the methylase [predicted 359 amino acids (aa)] and ORF2 codes for the endonuclease (predicted 262 or 272 aa).     

Cloning of the genes encoding urease from Proteus vulgaris and sequencing of the structural genes

A fragment of chromosomal DNA from proteus vulgaris encoding urease was cloned and expressed in Escherichia coli. A 3 kbp region was sequenced and revealed three open reading frames with homology to jack bean (Canavalia ensiformis) urease. The smallest protein (11 kDa) was homologous to the N-terminus of the plant enzyme and the largest polypeptide (61 kDa) corresponded to the C-terminus. The large protein contained conserved regions and a cysteine residue which is known to be catalytically important in the plant enzyme. A protein of 12 kDa showed homology to residues 132 to 237 of jack bean urease.     

Cloning and sequencing of endochitinase genes from gliocladium virens and trichoderma species

Nucleotide sequences containing the genetic determinants of the endochitinase of Gliocladiurn virences as well as those of Trichoderma harzianum, T. hamatum, T. viride and T. pseudokoningii were determined. The ORFs of 1287 or 1293 nucleotides were interrupted by three introns. The deduced amino acid sequences of endochitinase of G. virens were highly homologous to those of Trichoderma species (82–97% identity), although the culture filtrates from G. virens showed a much stronger inhibitory effect on growth of Helicobasidium mompa than those from Trichoderma species. Phylogenic analysis showed that these sequences could be divided into three clusters regardless of genus or species.     

Cloning and sequencing of Pseudomonas genes determining sodium dodecyl sulfate biodegradation.

The nucleotide sequences of two genes involved in sodium dodecyl sulfate (SDS) degradation, by Pseudomonas, have been determined. One of these, sdsA, codes for an alkyl sulfatase (58,957 Da) and has similarity (31.8% identity over a 201-amino acid stretch) to the N terminus of a predicted protein of unknown function from Mycobacterium tuberculosis. The other gene, sdsB, codes for a positive activator protein (33,600 Da) that has extensive similarity with the lysR family of helix-turn-helix DNA-binding activator proteins.     

Cloning and sequencing of cDNAs for hypothetical genes from chromosome 2 of Arabidopsis

About 25% of the genes in the fully sequenced and annotated Arabidopsis genome have structures that are predicted solely by computer algorithms with no support from either nucleic acid or protein homologs from other species or expressed sequence matches from Arabidopsis. These are referred to as "hypothetical genes." On chromosome 2, sequenced by The Institute for Genomic Research, there are approximately 800 hypothetical genes among a total of approximately 4,100 genes. To test their expression under various growth conditions and in specific tissues, we used six cDNA populations prepared from cold-treated, heat-treated, and pathogen (Xanthomonas campestris pv campestris)-infected plants, callus, roots, and young seedlings. To date, 169 hypothetical genes were tested, and 138 of them are found to be expressed in one or more of the six cDNA populations. By sequencing multiple clones from each 5'- and 3'-rapid amplification of cDNA ends (RACE) product and assembling the sequences, we generated full-length sequences for 16 of these genes. For 14 genes, there was one full-length assembly that precisely supported the intron-exon boundaries of their gene predictions, adding only 5'- and 3'-untranslated region sequences. However, for three of these genes, the other assemblies represent additional exons and alternatively spliced or unspliced introns. For the remaining two genes, the cDNA sequences reveal major differences with predicted gene structures. In addition, a total of six genes displayed more than one polyadenylation site. These data will be used to update gene models in The Institute for Genomic Research annotation database ATH1.     

Cloning and sequencing of the genes of 2-hydoxyglutaryl-CoA dehydratase from Acidaminococcus fermentans

Two genomic libraries from Acidaminococcus fermentans DNA constructed with the lambda vectors gt11 and EMBL 3 were screened with antisera raised against 2-hydroxyglutaryl-CoA dehydratase. Two clones giving the strongest reaction in the immunoassay were analyzed further, one was a lambda gt11 clone with an insert of 2050 bp and one was a lambda EMBL-3 clone with an insert of approximately 11,000 bp. Escherichia coli cells infected with the lambda gt11 clone expressed the alpha subunit of the dehydratase (Mr, 53,870), whereas with the lambda EMBL-3 clone, the alpha and beta subunits (Mr, 41,857) were detected on Western blots. Restriction fragments of both clones were subcloned in pUC 8 and sequenced by the chain termination method. Thus the complete sequence of the genes of both subunits, hgdA (alpha) and hgdB (beta) were obtained. The genes have the following order: A-B, with an intergenic region of only 2 bp. The deduced amino acid sequences for the alpha and beta subunits were confirmed by four peptides sequenced by protein chemical methods. Both chains are extremely rich in cysteine (13 in alpha, including a CNC and two CC clusters, and nine in beta) but no similarities to other known protein sequences were found.     

Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus

Summary Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H₂ at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H₂ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H₂ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H₂ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H₂ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H₂ases.     

Cloning and sequencing of the xylose isomerase and xylulose kinase genes of Escherichia coli

A 4.2-kilobase-pair fragment of the Escherichia coli chromosome which contains the genes for xylose isomerase and xylulose kinase was cloned into plasmid pBR322. The hybrid plasmid (designated pECX14) complements strains deficient in either or both of the two enzymes. Deletion derivatives of pECX14 were used to localize the two genes on the cloned fragment. The entire nucleotide sequence of the cloned fragment was determined. Open reading frames which, if translated, would encode proteins of molecular weights 54,000 and 52,000 were found. These were identified as the isomerase and kinase structural genes, respectively.     

Cloning,sequencing and expression of the nhaA and nhaR genes from Salmonella entiritidis

Na⁺/H⁺ antiporter activity is wide-spread and plays essential physiological roles. We found that several Enterobacteriaceae share conserved sequences with nhaA, the gene coding for an E. coli antiporter. A nhaA strain which is sensitive to Na⁺ and Li⁺, was used to clone by complementation a DNA fragment from Salmonella enteritidis which confers resistance to the ions. The cloned fragment increased Na⁺/H⁺ antiport activity in membranes isolated from strains carrying the respective hybrid plasmid. DNA sequence analysis of the insert revealed two open reading frames. Both encode putative polypeptides which are closely homologous to the nhaA and nhaR gene products from Escherichia coli. The antiporter activity displays properties very similar to that of the E. coli NhaA, namely, it is activiated by alkaline pH and recognizes Li⁺ with high affinity.Abbreviations _H ⁺ Proton electrochemical potential - pH transmembrane pH gradient - _Na ⁺ Sodium electrochemical potential - SDS Sodium dodecyl sulfate - CIP Calf intestine alkaline phosphates - ORF open reading frame     

Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction

A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5'-oligodeoxyribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from gamma and mu heavy chains and from kappa light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5'-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from gamma and mu isotypes and the universal light-chain primer was used to amplify three separate kappa light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG1/kappa, IgG2b/kappa and IgM/kappa isotypes.     

Cloning, sequencing, and overexpression of genes for ribosomal proteins from Bacillus stearothermophilus

Although a low resolution model for the arrangement of the proteins of the small and large ribosomal subunits is known, a detailed mechanistic understanding of the function of the ribosome awaits a high resolution structure of its components. While crystals have been obtained of several ribosomal proteins from Bacillus stearothermophilus, determination of atomic resolution structures of these proteins is impeded by the difficulty of obtaining large amounts of native proteins for crystallographic or NMR studies. We describe here the cloning and overexpression in Escherichia coli of the genes for ribosomal proteins S5, L6, L9, and L18 from B. stearothermophilus. S5 is extremely toxic to E. coli when overexpressed, and we have taken advantage of a new tightly regulated expression system to obtain high yields (more than 100 mg of pure protein/liter of culture) of this protein. The B. stearothermophilus S5 produced in E. coli crystallizes, and the crystals are identical to those obtained from the native protein. The crystals diffract to 2-A resolution.