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PubMed1999

[Cloning and expression of a serine proteinase gene from Thermoactinomyces sp. in Bacillus subtilis cells].

A gene coding for thermostable serine protease from Thermoactinomyces sp. K50 is cloned and expressed in Bacillus subtilis cells. Restriction map of cloned DNA fragment is determined. Thermostability and temperature optimum of proteolytic activity of the cloned gene product are lower than those of the natural proteinase of Thermoactinomyces sp. K50. Serine protease, a product of cloned gene, is highly sensitive to proteolysis and its degradation can be prevented by Ca2+ ions.

Bacillus subtilis↗
PubMed2001

[A new thermostable proteinase from Thermoactinomyces SP.27a. Cloning and gene expression].

A library of Thermoactinomyces sp. 27a, producer of thermostable proteases of different groups, has been created. Gene coding for thermostable neutral proteinase was cloned and expressed in Bac. subtilis cells. Restriction map for cloned DNA fragment was created and physicochemical parameters of recombinant proteinase were characterized. The thermostability and optimum of proteolytic activity of the enzyme was lower than in the natural Thermoactinomyces sp. strain, which can be due to heterologous expression of the gene coding for thermostable protein in the mesophilic host.

Cloning, Molecular↗
PubMed1989

Identification of partial mutational sites in human mitochondrial DNA in the Chinese and its significance.

A simple method for the identification of mutational sites in human mitochondrial DNA (mtDNA) was described. It was based on the human Cambridge sequence as a relative standard sequence and a single base pair substitution in mtDNA as a unique mutational form. The partial mutational sites can be determined using this method which was characterized by combining the restriction mapping with the analysis for the table of human mtDNA potential mutational sites with rapidity and simplicity. In the meanwhile, six mtDNA mutational sites found in Chinese population were identified by means of this method.

China↗
PubMed1994

Characterization of the CNRE-1 family of repetitive DNA elements in Cryptococcus neoformans.

The pathogenic yeast Cryptococcus neoformans contains 10-20 dispersed repetitive elements that hybridize to clone CNRE-1.0. Screening of a genomic library with probes derived from CNRE-1.0 identified five phages with restriction maps that overlapped CNRE-1.0 and three additional phages that belonged to two distinct groups. Sequencing of internal 3.5-kb SstI fragments from two CNRE-1-like elements revealed 95% homology, as well as a conserved open reading frame. A PCR-RFLP assay was developed that can distinguish different subfamilies of CNRE-1-like elements.

Base Sequence↗
PubMed1994

Detection and cloning of human papillomavirus DNA associated with recurrent respiratory papillomatosis in Thailand.

Recurrent respiratory papillomatosis (RRP) is highly prevalent in Thailand. In this study, we examined the presence of human papillomavirus (HPV) DNA in 25 RRP patients in Thailand by means of dot blot analysis and/or polymerase chain reaction. Eighty-four percent (21/25) of cases and 4% (1/25) of cases were positive for HPV-11 DNA and HPV-6 DNA, respectively. Three cases (3/25) were negative for all of the examined HPV types. No cases were positive for HPV-16 or 18. Furthermore, we isolated the recombinant HPV-11 DNA clone from a genomic library constructed with the DNA of RRP tissue. The restriction map of the cloned HPV DNA was identical with the map of known HPV-11 DNA. These results suggest at least that no specific HPV type or subtype is likely to be associated with RRP in Thailand.

Base Sequence↗
PubMed1988

RSF1010 and a conjugative plasmid contain sulII, one of two known genes for plasmid-borne sulfonamide resistance dihydropteroate synthase.

The nucleotide sequence of the type II sulfonamide resistance dihydropteroate synthase (sulII) gene was determined. The molecular weight determined by maxicells was 30,000, and the predicted molecular weight for the polypeptide was 28,469. Comparison with the sulI gene encoded by Tn21 showed 57% DNA similarity. The sulII-encoded polypeptide has 138 of 271 amino acids in common with the polypeptide encoded by sulI. The sulII gene is located on various IncQ (broad-host-range) plasmids and other small nonconjugative resistance plasmids. Detailed restriction maps were constructed to compare the different plasmids in which sulII is found. The large conjugative plasmid pGS05 and the IncQ plasmid RSF1010 contained identical nucleotide sequences for the sulII gene. This type of sulfonamide resistance is very frequently found among gram-negative bacteria because of its efficient spread to various plasmids.

Amino Acid Sequence↗
PubMed1991

A low-copy repeat located in subtelomeric regions of 14 different human chromosomal termini.

Two cosmid clones, cG1A3 and cG1A6, containing large blocks of a telomeric repeat sequence, (TTAGGG)n, were isolated independently. Restriction mapping and fingerprinting studies suggested that cosmid cG1A6 is entirely contained within cG1A3. Competitive in situ hybridization localized cG1A3 to 14 different human telomeres and one internal site at 2q14----q21, indicating the presence of a subtelomeric low-copy repeat within this cosmid.

Base Sequence↗
PubMed1990

A new streptomycin-resistance plasmid from Staphylococcus hyicus and its structural relationship to other staphylococcal resistance plasmids.

A small plasmid of 4.4 kb encoding resistance to streptomycin (Smr) was detected in a multiresistant Staphylococcus hyicus culture from a piglet with exudative epidermitis. The plasmid-encoded properties were determined by interspecies protoplast transformation experiments. This plasmid was further characterised by restriction endonuclease analysis and a preliminary restriction map was constructed. The plasmid from S. hyicus that conferred streptomycin resistance was designated as pSAI-1. It showed some structural homology with the streptomycin-chloramphenicol resistance plasmid pSK68 from S. aureus of human origin. The MIC of streptomycin in resistance mediated by pSAI-1 was about 10 times higher than the MICs in resistance mediated by Smr plasmids from human S. aureus strains.

Animals↗
PubMed1988

The allosuppressor gene SAL4 encodes a protein important for maintaining translational fidelity in Saccharomyces cerevisiae.

Allosuppressor (sal) mutations enhance the efficiency of the yeast ochre suppressor SUQ5 and define five unlinked loci, SAL1-SAL5. A number of sal4 mutants were isolated and found to have pleiotropic, allele;specific phenotypes, including hypersensitivity in vivo to paromomycin and other antibiotics that stimulate translational errors in yeast. To examine further the nature of the SAL4 gene product, the wild type SAL4 gene was isolated by complementation of a conditional lethal allele sal4-2, and demonstrated to be a single copy gene encoding a single 1.6 kb transcript. Restriction mapping and DNA hybridisation analysis were used to demonstrate that the SAL4 gene is identical to the previously identified omnipotent suppressor gene SUP45 (SUP1). Our results implicate the SAL4 gene product as playing a major role in maintaining translational accuracy in yeast.

Anti-Bacterial Agents↗
PubMed1999

Genomic organisation of the spinocerebellar ataxia type 7 (SCA7) gene responsible for autosomal dominant cerebellar ataxia with retinal degeneration.

Autosomal dominant cerebellar ataxia with retinal degeneration (ADCA type II) is a progressive neurodegenerative disorder caused by a CAG expansion in the spinocerebellar ataxia 7 (SCA7) gene. Here, we describe the genomic organisation of the human SCA7 gene. The exon-intron boundaries were identified by sequencing plasmid subclones of a P1 artificial chromosome (PAC) clone containing the entire SCA7 gene. We found 13 exons, ranging in size from 69 to 979 bp, with all exon-intron boundaries following the GT-AG rule. The ATG initiation codon at position 554 of the cDNA occurs in exon 3 at position 12 and the coding region extends to the first five codons of exon 13, with the CAG repeat being located in exon 3 starting at codon 30. The intron sizes were determined by long-distance polymerase chain reaction with primers from neighbouring exons and by restriction mapping of the SCA7 PAC clone. The introns varied in size from 233 bp to about 40 kb, resulting in an overall size estimate for the SCA7 gene of 140 kb. Sequence analysis of intron 7 (491 bp) revealed a polymorphic GT/AC repeat, a useful intragenic marker for SCA7 in segregation studies.

Base Sequence↗
PubMed1989

In vivo cloning of Pseudomonas aeruginosa genes with mini-D3112 transposable bacteriophage.

The transposition properties of the Pseudomonas aeruginosa mutator bacteriophage D3112 were exploited to develop an in vivo cloning system. Mini-D replicon derivatives of D3112 were constructed by incorporating broad host range plasmid replicons between short terminal D3112 sequences. These elements were made with small replication regions from the RK2, Sa, and pVS1 plasmids and selectable genes for tetracycline, carbenicillin, kanamycin, and gentamicin resistance. Some of the mini-D replicons also contain the RK2 oriT origin-of-transfer sequence, which allows them to be mobilized by conjugation to many different species of gram-negative bacteria. These elements were used to clone DNA by preparing lysates from P. aeruginosa cells harboring an inducible D3112 cts prophage and a mini-D replicon plasmid. These lysates were used to infect sensitive P. aeruginosa recipients and select recombinant plasmids as drug-resistant transductant colonies. These transductants form a gene library from which particular clones can be selected, such as by their ability to complement specific mutations. This system was used to clone nine different genes from the PAO chromosome. The ability of this system to precisely identify a gene was demonstrated by isolating clones of the argF+ and cys-59+ genes. Restriction maps of clones of these genes, which have different amounts of flanking DNA, located the positions of these genes. The sizes of the chromosomal DNA segments from 10 individual clones examined ranged from 6 to 21 kilobases (kb), with an average of about 10 kb. This is consistent with the approximately 40-kb DNA-packaging size of the D3112 phage.

Bacteriophages↗
PubMed1992

Molecular cloning and expression of the xylanase gene from Chainia in Escherichia coli.

A complete genomic library of Chainia was constructed in coliphage lambda vector gt10 and was screened for the xylanase gene using an 18-mer mixed oligonucleotide probe corresponding to a six-amino acid sequence of low molecular mass Chainia xylanase. Inserts from 11 putative clones, showing hybridization with the oligonucleotide probe at medium stringency, were subcloned in pUC8 and screened for xylanase gene expression using anti-xylanase antibodies. The restriction map of the insert (1.4 kb) from one of the four immunopositive clones (PVX8) showing detectable xylanase activity was constructed. The xylanase activity of PVX8 was not induced by IPTG or xylan. Reorientation of the insert by directional cloning into pUC9 had no effect on the xylanase activity suggesting that an indigenous promoter from Chainia is responsible for the xylanase activity.

Actinomycetales↗
PubMed1997

A tandem array of 5S ribosomal RNA genes in Pythium irregulare.

The 5S ribosomal RNA genes of the oomycete Pythium irregulare exist in tandem arrays unlinked to the rDNA repeat unit. A clone with a 9.2-kb insert containing an array of 5S genes was identified in a lambda genomic library and was characterized by restriction mapping and partial sequencing. The array consisted of 9 apparently identical 5S genes and their spacers in tandem, followed by a diverged 5S-like sequence that is likely to be a pseudogene. This gene arrangement, although almost universal in plants and animals, is rare in fungi and protists.

Base Sequence↗
PubMed1990

Site-directed, recombination-mediated mutagenesis of a complex gene locus.

We have generated a site-specific 17 bp insertion within a 38 kb chick globin gene cluster by employing the recombination abilities of Saccharomyces cerevisiae. This gene cluster contains four beta-type globin genes which share a high degree of sequence homology. In this procedure, a small fragment of beta A-globin DNA containing a 17 bp insertion is subcloned into a URA3-based yeast integrating vector (YIp). This mutated globin subclone is introduced into cells that carry the 38 kb globin cluster clone on a single-copy, circular vector derived from a yeast artificial chromosome (YAC). Insertion of the 17 bp oligomer is achieved by targeted integration of the Ylp subclone. The recombinant contains the normal beta A-globin gene, the mutant gene and Ylp vector sequences between the two copies. Excision of the vector sequences and one copy of the duplicated globin sequences by homologous recombination is required for cell survival when exposed to the selective agent 5-fluoroorotic acid, which is toxic to ura+ yeast cells. Depending upon the point of the cross-over, a ura- yeast cell bearing either a wild-type globin gene or a 17 bp insertion mutation will result. By restriction mapping and in vitro transcription analysis, the beta A-globin gene containing the 17 bp insert has no nonspecific mutations generated during the recombination and selection procedures. Specific mutations of regulatory regions, including protein-DNA binding sites, can be accurately targeted within extensive DNA clones by this method.

Animals↗
PubMed1991

Phenotypic heterogeneity in a panel of infectious molecular human immunodeficiency virus type 1 clones derived from a single individual.

Previously, we and others have demonstrated a relation between the clinical course of human immunodeficiency virus type 1 (HIV-1) infection and biological properties of HIV-1 variants such as replication rate, syncytium-inducing (SI) capacity, and cytotropism. For the molecular analysis of the biological variability in these properties, we generated a panel of phenotypically distinct yet genetically highly homologous infectious molecular clones. These clones were derived from HIV-1 isolates, mostly recovered by direct clonal isolation, from a single individual in whom a transition from non-SI to SI isolates had been identified over time. Of 17 molecular clones tested, 8 were infectious. The clones exhibited differences in SI capacity and T-cell line tropism. Their phenotypes corresponded to those of their parental isolates, formally demonstrating that biological variability of HIV-1 isolates can be attributed to single molecular clones. With these clones we demonstrated that SI capacity and tropism for the H9 T-cell line, almost invariably coupled in primary HIV-1 isolates, are discernible properties. Also different requirements appeared to exist for H9 and Sup T1 cell line tropism. We obtained evidence that T-cell line tropism is not caused by differences in level of HIV-1 expression but most probably is restricted at the level of virus entry. Restriction mapping of four clones with divergent phenotypes revealed a high degree of nucleotide sequence homology (over 96.3%), indicating the usefulness of these clones for the tracking of genetic variability critical for differences in biological phenotype.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Characterization of a new alpha-thalassemia-1 deletion in a Spanish family.

A new type of alpha-thalassemia-1 was characterized in one Spanish patient with Hb H disease and in her mother. The restriction map of this deletion suggests that the deletion of 22 kb has occurred on a chromosome carrying a zeta-globin triplication. The resulting chromosome lacks the alpha 2- and alpha 1-globin genes, the psi alpha 2- and psi alpha 1-globin genes, and one of the three zeta-globin genes, while the other two zeta-globin genes and the theta 1-globin gene have been retained.

Adult↗
PubMed2006

Unusual gene order and organization of the sea urchin hox cluster.

While the highly consistent gene order and axial colinear patterns of expression seem to be a feature of vertebrate hox gene clusters, this pattern may be less well conserved across the rest of the bilaterians. We report the first deuterostome instance of an intact hox cluster with a unique gene order where the paralog groups are not expressed in a sequential manner. The finished sequence from BAC clones from the genome of the sea urchin, Strongylocentrotus purpuratus, reveals a gene order wherein the anterior genes (Hox1, Hox2 and Hox3) lie nearest the posterior genes in the cluster such that the most 3' gene is Hox5. (The gene order is 5'-Hox1, 2, 3, 11/13c, 11/13b, 11/13a, 9/10, 8, 7, 6, 5-3'.) The finished sequence result is corroborated by restriction mapping evidence and BAC-end scaffold analyses. Comparisons with a putative ancestral deuterostome Hox gene cluster suggest that the rearrangements leading to the sea urchin gene order were many and complex.

Animals↗