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PubMed1986

Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae.

Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the beta-glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl-beta-glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the beta-glucosidase gene originated from C. pelliculosa. beta-Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, beta-glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of beta-glucosidase synthesis in S. cerevisiae carrying the cloned beta-glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the beta-glucosidase gene negatively in S. cerevisiae.

Candida↗
PubMed1987

Transformation of Aspergillus niger using the homologous orotidine-5'-phosphate-decarboxylase gene.

A homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5'-phosphate-decarboxylase gene. A. niger Pyr- mutants have been selected from 5-fluoro-orotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/micrograms DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA+ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.

Aspergillus niger↗
PubMed1986

Differential distribution of lysine and arginine residues in the closely related histones H1 and H5. Analysis of a human H1 gene.

A human H1 histone gene and its flanking sequences were isolated from a human gene library using a fragment of the duck H5 histone gene as a hybridization probe. The primary structure of this human H1 histone (as deduced from the nucleotide sequence of the gene) reveals a close homology of H1 and H5 histones and fits the three-domain organization of all members of the H1 histone family. Within this protein organization, the C-terminal domain of H1 differs from the arginine-rich H5 in its distribution of the basic amino acids: the C-terminal domain of the human H1 shows only one arginine and most of the H5 specific arginine positions show lysine instead.

Amino Acid Sequence↗
PubMed1987

Organization of phosphoribulokinase and ribulose bisphosphate carboxylase/oxygenase genes in Rhodopseudomonas (Rhodobacter) sphaeroides.

A heterologous phosphoribulokinase (PRK) gene probe was used to analyze two recombinant plasmids isolated from a Rhodopseudomonas (Rhodobacter) sphaeroides gene library. These plasmids were previously shown to carry the genes for form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O). Southern blot hybridization analysis indicated that there were two PRK genes linked to the RuBPC/O coding sequences. Restriction mapping showed the arrangement of the duplicate sets of PRK and RuBPC/O to be distinct. Subcloning of the hybridizing PRK sequences downstream of the lac promoter of pUC8 allowed expression of the two PRK enzymes in Escherichia coli. Analysis of the purified proteins by sodium dodecyl sulfate-slab gel electrophoresis revealed polypeptides with molecular weights of 32,000 and 34,000 corresponding to the form I and form II PRKs, respectively. Preliminary experiments on sensitivity to NADH regulation suggested that the two PRK enzymes differ in catalytic properties.

Cloning, Molecular↗
PubMed1992

Isolation of three novel Drosophila melanogaster genes containing repetitive sequences rich in GCN triplets.

Several cDNA and genomic clones were isolated from Drosophila melanogaster gene libraries by hybridization with a region of a mammalian gene that contains a simple repetitive sequence of six GCN repeats. One of the cDNA clones, E6, was completely sequenced and it was shown that it contains a region of 16 GCN repeats; these repeats encode a polyalanine stretch within a long open reading frame. The sequencing of three different genomic clones (A, B, and D) revealed that all the isolated Drosophila clones are similar to one another in a short region containing variable numbers of the GCN repeat. The genomic clone B was found to be the genomic counterpart of the cDNA clone E6. The other genomic clones, A and D, also hybridize with Drosophila cDNA clones at high stringency. These results indicate that the short GCN repetitive sequences, which we have named ala, are found within transcribed regions of the Drosophila genome. These Drosophila genes containing the ala repeat do not show significant sequence similarity to any presently known gene; we have named these novel genes ala-A, ala-B, and ala-D. The cDNA clone from gene ala-B was named ala-E6.

Amino Acid Sequence↗
PubMed1987

Chromosomal gene structure of human myeloperoxidase and regulation of its expression by granulocyte colony-stimulating factor.

By using the full-length cDNA as a probe, the chromosomal gene for human myeloperoxidase was isolated from a human gene library. Comparison of the nucleotide sequence of the chromosomal gene with that of the cDNA has revealed that the human myeloperoxidase gene is composed of 12 exons and 11 introns. S1 mapping analysis of human myeloperoxidase mRNA identified the single transcription initiation site at 180 base pairs upstream of the ATG initiation codon. In the 5'-flanking region of the human myeloperoxidase gene, there are several blocks of sequences which are homologous to the sequences found on the 5'-promoter region of the human c-myc proto-oncogene. The myeloperoxidase gene is expressed in mouse myeloid leukemia WEHI-3B D+ and NFS-60 cells. When these cells were treated with human recombinant granulocyte colony-stimulating factor, the steady-state level of myeloperoxidase mRNA declined to zero within 36 h in WEHI-3B D+ cells, but not in NFS-60 cells.

Amino Acid Sequence↗
PubMed1993

[Cloning and sequencing of attacus ricini nuclear polyhedrosis virus polyhedrin gene].

Attacus ricini is a species of insect which only grows in China. It is expected to construct a new expression system for genetic engineering by using ArNPV as a vector. We have established an ArNPV gene library. The 1.1 kb DNA fragment containing ArNPV Ph gene was subcloned. Sequencing analysis shows that the 735 bp Ph structural gene has the homology of 76% and 81% with those of AcNPV and BmNPV respectively. The Rohrmann box in the ArNPV 5'-end regulation region is very similar to those of various other NPVs, but the 3'-end downstream sequence has almost no homology with those of AcNPV and BmNPV, demonstrating the characteristic property of the structure of ArNPV Ph gene. Other structural features of the Ph gene promoter are also discussed in this paper.

Base Sequence↗
PubMed1992

Derivation of a mathematical expression useful for the construction of complete genomic libraries.

We present and derive a formula that is useful for the design and evaluation of gene cloning experiments in which a complete gene library of the entire genome of an organism is desired. The formula n = ln(1-phi f)/ln(1-f) (in which n is the number of recombinant clones required to ensure a probability, phi, of obtaining at least one of each of all possible gene sequences, and f is the fraction of the genome contained in an average-sized DNA fragment) applies to construction of libraries, in which at least one copy of all the genetic information of a genome is required. The use of this formula for quantitative evaluation of genomic libraries should give greater assurance that a given library would be complete.

Cloning, Molecular↗
PubMed2005

Complete, gene-specific siRNA libraries: production and expression in mammalian cells.

Short interfering RNAs (siRNAs) are widely used to silence the expression of specific genes. Current practice for designing effective siRNAs is to use algorithms based on sequence-efficacy correlations; however, there are many highly effective sequences that these algorithms do not anticipate. To ensure that the best siRNAs are identified, all possible gene-specific siRNA sequences of appropriate lengths should be screened in cell culture. Synthesizing and testing all such sequences individually is costly. A potentially much easier alternative is to prepare a mixture of all these sequences (a gene-specific library), express them in cells, select cells having the desired phenotype, and identify the siRNA contained within the selected cells. Here we describe two new methods for preparing and expressing such libraries. The first uses cloned Dicer or RNase III to digest gene-specific RNA duplexes to siRNAs, which are then converted to the corresponding DNA sequences by attaching RNA primers and performing reverse transcription-PCR. The second method involves partial DNase I digestion of gene-specific DNA, purification of a 20-30-bp fraction, and amplification by attaching DNA adapters followed by PCR. DNA libraries specific for TNF-alpha, DsRed, and part of the hepatitis C virus genome, generated by methods, were inserted into siRNA expression vectors between convergent human U6 and H1 promoters. Randomly selected clones from each library together with vectors expressing the corresponding target genes were cotransfected into 293FT cells and assayed for target gene inhibition. About 10%-20% of siRNAs represented in these libraries show significant inhibition of their target genes. Most of these inhibitory sequences are not predicted by existing algorithms.

Animals↗
PubMed2006

Uncultured Archaea in a hydrothermal microbial assemblage: phylogenetic diversity and characterization of a genome fragment from a euryarchaeote.

The polychaete Alvinella pompejana lives in organic tubes on the walls of active hydrothermal chimneys along the East Pacific Rise. To examine the diversity of the archaeal community associated with the polychaete tubes, we constructed libraries by direct PCR amplification and cloning of 16S rRNA genes. Almost half of the sequences of the 16S rRNA gene libraries clustered with uncultured archaeal groups. In an effort to access genomic information from uncultured archaeal members we further constructed a fosmid library from the same DNA source. One of the clones, Alv-FOS5, was sequenced completely. Its sequence analysis revealed an incomplete rRNA operon and 32 predicted ORFs. Seventeen of these ORFs have been assigned putative functions, including transcription and translation, cellular processes and signalling, transport systems and metabolic pathways. Phylogenetic analyses of the 16S rRNA gene suggested that Alv-FOS5 formed a new lineage related to members of Deep-Sea Hydrothermal Vent Euryarchaeota group II. Phylogenetic analyses of predicted proteins revealed the existence of likely cases of horizontal gene transfer, both between Crenarchaeota and Euryarchaeota and between Archaea and Bacteria. This study is the first step in using genomics to reveal the physiology of an as yet uncultured group of archaea from deep-sea hydrothermal vents.

Archaea↗
PubMed1994

Kinetic analysis of engineered antibody-antigen interactions.

Molecular engineering of antibodies has made it possible to produce specific domains of the antibody molecule and combine them with other protein domains to achieve new properties. Using site directed mutagenesis, amino acid residues can be exchanged within the binding site; and, by analysis of crystal structures, the positions of these amino acids can be determined in three dimensions at atomic resolution. In addition, gene libraries and phage selection technology can be used to generate new antibody fragments directly from a gene pool. Both mutagenesis and selection from libraries offer opportunities to identify antibody-derived molecules with altered and useful antigen recognition properties. The detailed analysis of both kinetic and equilibrium binding affinity are therefore essential to understand the activity of the molecules resulting from antibody engineering and to guide the progress of their further design. This paper reviews recently evolving techniques for the binding analysis of antibodies, their functional domains and antibody chimerae.

Animals↗
PubMed1984

Cloning and organisation of some genes for nitrogen fixation from Azotobacter chroococcum and their expression in Klebsiella pneumoniae.

By DNA hybridisation, restriction fragments of genomic DNA from Azotobacter chroococcum and A. vinelandii bearing sequences homologous to Klebsiella pneumoniae nitrogenase structural genes were detected. These were different in the two species and inconsistent with the arrangement of the homologous sequences as a contiguous cluster of unique genes. The use of a DNA probe specific for nifH showed that in A. chroococcum two nifH-like sequences were present in the genome. From gene libraries for A. chroococcum, several recombinant cosmid clones bearing nif genes were identified and physically mapped. One copy of the nifH-like sequences was closely linked to nifD and K, the order of genes being as for K. pneumoniae. This cluster was sub-cloned into the broad host-range vector pKT230. The resultant plasmid complemented for C2H2-reduction but not growth in N2 several Nif- mutants of A. vinelandii and K. pneumoniae and also abolished growth in N2 in Nif+ parents. The inhibition was ascribed to a short region adjacent to nifH, which probably corresponds to the promoter as its inhibitory affects were alleviated by provision of K. pneumoniae nifA in multiple copies. 3 sizes of transcripts are produced from the region containing nifH and nifD of A. chroococcum in cultures derepressing for nif. A region bearing homology to a fragment of the K. pneumoniae nif cluster bearing nifV was identified 15 Kb away from nifHDK in A. chroococcum however the order of genes is probably similar to that of K. pneumoniae.

Azotobacter↗
PubMed1979

Molecular structure and flanking nucleotide sequences of the natural chicken ovomucoid gene.

Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.

Animals↗
PubMed1983

A frameshift addition causes silencing of the delta-globin gene in an old world monkey, an anubis (Papio doguera).

A delta-globin gene of an anubis, Papio doguera, was cloned, and the nucleotide sequence of a region of about 2,000 bp including the gene was determined. In comparison with that of pSP3, a delta-globin clone from the gene library of a normal Japanese, the anubis delta-globin gene showed one base addition in the second exon at the codon for amino acid 55, generating a termination codon at the new 59th codon. The other differences found in the region analyzed were not considered to affect expression of the delta-globin gene. Therefore, it was concluded that this frameshift addition was at least one of the causes of silencing of the delta-globin gene in the anubis. In addition, from the sequence divergence in the delta-globin gene, the period of this silencing was estimated to be at most 4 million years, and the divergence of anubis occurred at most 30 million years ago.

Animals↗
PubMed1999

Cloning, overexpression, purification, and physicochemical characterization of a cold shock protein homolog from the hyperthermophilic bacterium Thermotoga maritima.

Thermotoga maritima (Tm) expresses a 7 kDa monomeric protein whose 18 N-terminal amino acids show 81% identity to N-terminal sequences of cold shock proteins (Csps) from Bacillus caldolyticus and Bacillus stearothermophilus. There were only trace amounts of the protein in Thermotoga cells grown at 80 degrees C. Therefore, to perform physicochemical experiments, the gene was cloned in Escherichia coli. A DNA probe was produced by PCR from genomic Tm DNA with degenerated primers developed from the known N-terminus of TmCsp and the known C-terminus of CspB from Bacillus subtilis. Southern blot analysis of genomic Tm DNA allowed to produce a partial gene library, which was used as a template for PCRs with gene- and vector-specific primers to identify the complete DNA sequence. As reported for other csp genes, the 5' untranslated region of the mRNA was anomalously long; it contained the putative Shine-Dalgarno sequence. The coding part of the gene contained 198 bp, i.e., 66 amino acids. The sequence showed 61% identity to CspB from B. caldolyticus and high similarity to all other known Csps. Computer-based homology modeling allowed the conclusion that TmCsp represents a beta-barrel similar to CspB from B. subtilis and CspA from E. coli. As indicated by spectroscopic analysis, analytical gel permeation chromatography, and mass spectrometry, overexpression of the recombinant protein yielded authentic TmCsp with a molecular weight of 7,474 Da. This was in agreement with the results of analytical ultracentrifugation confirming the monomeric state of the protein. The temperature-induced equilibrium transition at 87 degrees C exceeds the maximum growth temperature of Tm and represents the maximal Tm-value reported for Csps so far.

Bacterial Proteins↗
PubMed1991

Exopolysaccharide mutants of Rhizobium loti are fully effective on a determinate nodulating host but are ineffective on an indeterminate nodulating host.

By Tn5 mutagenesis of Rhizobium loti PN184 (NZP2037 str-1) and selection for nonfluorescence of colonies on Calcofluor agar, eight independently generated expolysaccharide (EPS) mutants (three smooth and five rough) were isolated. The parent strain, PN184, was found to produce an acidic EPS. This EPS was produced. with reduced O acetylation, by the smooth EPS mutants but not by the rough EPS mutants. Lipopolysaccharide was isolated from all mutants and was identical to that of PN184 as defined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All mutants were resistant to lysis by R. loti bacteriophage phi 2037/1. Cosmids that complemented the mutations in the rough EPS mutants were isolated from a pLAFR1 gene library of NZP2037 by complementation of the nonfluorescent phenotype. The genes identified were shown to be unlinked and located on the chromosome. All mutants were fully effective when inoculated onto Lotus pedunculatus, a determinate nodulating host, but were ineffective, inducing the formation of very small nodules or tumorlike growths, when inoculated onto Leucaena leucocephala, an indeterminate nodulating host. These results, obtained in an isogenic Rhizobium background, support suggestions that acidic EPS is required for effective nodulation of indeterminate nodulating legumes but is not required for effective nodulation of determinate nodulating legumes.

Cosmids↗
PubMedDate not supplied

Rapid isolation of tissue-specific genes from rat kidney.

A systematic effort to isolate kidney-specific genes was performed using recently described PCR-select methodology. Using this technique, a kidney-specific mini-gene library was generated and a number of kidney-specific genes that share significant homology to previously characterized kidney genes from rats and other species were isolated. These included three renal-specific transporters (an ADH water channel, the anion transporters RST and ROAT1), a cell adhesion molecule (K-cadherin) and a kidney-specific protein upregulated in renal carcinoma (DD96). In addition, we isolated two novel genes from a rat kidney. One of the genes shares limited homology to rat profilin-1 while the other did not share any similarity to genes in the Genbank. Northern blot analysis revealed that the mRNA for each of these genes is expressed in a highly kidney-restricted fashion. Our results suggested that tissue-specific genes can be rapidly isolated and characterized using PCR-select techniques and this methodology may be generally applicable to isolate specific genes from a variety of tissues.

Animals↗
PubMed2001

A gene encoding phosphatidylethanolamine N-methyltransferase from Acetobacter aceti and some properties of its disruptant.

Phosphatidylcholine (PC) is a major component of membranes not only in eukaryotes, but also in several bacteria, including Acetobacter. To identify the PC biosynthetic pathway and its role in Acetobacter sp., we have studied Acetobacter aceti IFO3283, which is characterized by high ethanol oxidizing ability and high resistance to acetic acid. The pmt gene of A. aceti, encoding phosphatidylethanolamine N-methyltransferase (Pmt), which catalyzes methylation of phosphatidylethanolamine (PE) to PC, has been cloned and sequenced. One recombinant plasmid that complemented the PC biosynthesis was isolated from a gene library of the genomic DNA of A. aceti. The pmt gene encodes a polypeptide with molecular mass of either 25125, 26216, or 29052 for an about 27-kDa protein. The sequence of this gene showed significant similarity (44.3% identity in the similar sequence region) with the Rhodobacter sphaeroides pmtA gene which is involved in PE N-methylation. When the pmt gene was expressed in E. coli, which lacks PC, the Pmt activity and PC formation were clearly demonstrated. A. aceti strain harboring an interrupted pmt allele, pmt::Km, was constructed. The pmt disruption was confirmed by loss of Pmt and PC, and by Southern blot analyses. The null pmt mutant contained no PC, but tenfold more PE and twofold more phosphatidylglycerol (PG). The pmt disruptant did not show any dramatic effects on growth in basal medium supplemented with ethanol, but the disruption caused slow growth in basal medium supplemented with acetate. These results suggest that the lack of PC in the A. aceti membrane may be compensated by the increases of PE and PG by an unknown mechanism, and PC in A. aceti membrane is related to its acetic acid tolerance.

Acetobacter↗