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PubMed1995

A new gene with sequence and structural similarity to the gene encoding human lysyl oxidase.

We have isolated a number of recombinant clones from a human skin fibroblast cDNA library that contain extensive sequence homology to several coding domains within the human lysyl oxidase mRNA. Using one of these lysyl oxidase-like cDNAs, we obtained several overlapping genomic DNA recombinants. Restriction mapping and DNA sequence analysis revealed that the complete sequence of the lysyl oxidase-like mRNA was encoded by seven exons distributed throughout 25 kilobases of genomic DNA. Exons 2-6 encoded the region of greatest homology to lysyl oxidase. The size of these five exons, moreover, was exactly the same as the size of the corresponding exons within the lysyl oxidase gene. Northern blot analysis also revealed the concomitant appearance of lysyl oxidase and lysyl oxidase-like mRNA in several human tissues. It appears therefore that the genes encoding lysyl oxidase and a lysyl oxidase-like protein share a common evolutionary origin and may also be functionally related.

Amino Acid Sequence↗
PubMed1993

Intragenic recombination and a chimeric outer membrane protein in the relapsing fever agent Borrelia hermsii.

The spirochete Borrelia hermsii, a relapsing fever agent, evades the host's immune response through multiphasic antigenic variation. Antigen switching results from sequential expression of genes for serotype-specific outer membrane proteins known as variable major proteins (Vmp's); of the 25 serotypes that have been identified for the HS1 strain, serotypes 7 and 21 have been studied in greatest detail. In the present study, an atypical variant was predominant in the relapse from a serotype 21 infection in mice; relapse cells were bound by monoclonal antibodies specific for Vmp21 as well as antibodies specific for Vmp7. In Western blots (immunoblots), the variant had a single Vmp that was reactive with monoclonal antibodies representing both serotypes. The gene encoding this Vmp, vmp7/21, was cloned and characterized by restriction mapping and sequence analysis to determine the likely recombination event. Whereas the 5' end of vmp7/21 was identical to that of vmp21, its 3' end and flanking sequences were identical to the 3' end of vmp7. Unlike other vmp genes examined thus far, the vmp7/21 gene existed only in an expressed form; a silent, storage form of the gene was not detected. We conclude that the vmp7/21 gene was created by an intragenic recombination between the formerly expressed vmp21 gene and a silent vmp7 gene. This finding suggests that the lack of cross-reactivity between variants, which is usually observed, results from immunoselection against variants possessing chimeric Vmp's rather than from a switching mechanism that excludes partial gene replacements.

Antigens, Bacterial↗
PubMed1995

Sequence of a transposon identified as Tn1000 (gamma delta).

We report the complete sequence of a transposon found in a cosmid clone of a human DNA sequence. The transposon is identified as the Escherichia coli transposon Tn1000 (also known as gamma delta) on the basis of the identity of the restriction map of the new sequence with that previously recorded for Tn1000 and homology between parts of the new sequence and that of published fragments of Tn1000 sequence. The transposon, which comprises 5,981 nucleotides including two 35 bp inverted terminal repeat sequences (ITRs), contains three open reading frames. The sequence of the resolvase coding region (tnpR) is identical to that published by others. A second reading frame can be identified as the tnpA gene, coding for the transposase, on the grounds of its strong homology with the corresponding gene from transposon Tn3. The third reading frame has the potential to code for a protein of unknown function containing 698 amino acids.

Amino Acid Sequence↗
PubMed1995

Characterization of a repetitive DNA element in a brown algal virus.

We describe a family of repetitive sequences found in viruses infecting the brown alga Feldmannia sp. Previously we have demonstrated that the dsDNA genomes of viruses infecting one Feldmannia sp. isolate exist as two size classes of 160 and 179 kb. Repetitive sequences within these genomes were first demonstrated based on the anomalous hybridization among five BamHI fragments from digested virus DNA. Sequence analysis of one of those fragments, B2.4, revealed the presence of 173-bp direct repeats. The restriction maps of the cross-hybridizing BamHI fragments in the two FsV (Feldmannia sp. virus) genome size classes show that these repeats are not widely dispersed, rather they are confined to a small region of each virus genome. We estimate the number of these repeats in the 179-kb genome to be about 109 and in the 169-kb genome to be about 41. In the 179-kb genome, the repeats are contained within a 22-kb region, about 12% of that virus genome, and in the 160-kb genome the repeats are contained within a 10-kb region, about 6% of the genome. The difference in repeat numbers can account for 62% of the size difference between the two size classes of the FsV genome.

Base Sequence↗
PubMed1993

Transport properties of a C. albicans amino-acid permease whose putative gene was cloned and expressed in S. cerevisiae.

Using a gene bank of C. albicans, the lysine-permease deficiency in a strain of S. cerevisiae was complemented, and the restriction map of the corresponding C. albicans DNA fragment was constructed. Its expression in S. cerevisiae showed that the permease of C. albicans actively transports arginine (KT = 18 mumol/l, Jmax = 26 nmol/min per mg dry weight), lysine (KT = 12 mumol/l, Jmax = 18 nmol/min per mg dry weight), histidine (KT = 37 mumol/l, Jmax = 9.7 nmol/min per mg dry weight), as well as their toxic analogues canavanine and thialysine, with high affinity. The intracellular concentration of basic amino acids transported into S. cerevisiae by the C. albicans permease reaches more than a thousand-times-higher value compared to the external concentration in the medium. Accumulated amino acids do not leave the cells. The uptake is strongly reduced by the protonophores and inhibitors of plasma membrane H(+)-ATPase.

Amino Acid Transport Systems↗
PubMed1990

Biotinylated probes for epidemiological studies of drug resistance in Salmonella krefeld.

A gene probe for ampicillin resistance and one for sulphonamide resistance were prepared to study the origin and the relation of multiple drug resistances in Salmonella krefeld. The resistance genes were cloned into the pACYC184 vector of Escherichia coli from a common plasmid of S. krefeld that encoded for resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, sulphonamide and tetracycline resistance. Restriction map analysis and deletion analysis of a recombinant plasmid (pACSS1) showed that the gene determining ampicillin resistance was located on a 1.34 and 1.12 kb PstI fragment, and that the gene for sulphonamide resistance was located on a 0.85 kb PstI fragment. These fragments were used as probes. Their specificity was tested by colony hybridization with various bacterial species, including sensitive and resistance S. krefeld isolates. Further study indicated that the ampicillin resistance gene probe reacted with the gene for TEM-1 beta-lactamase and that the gene probe for sulphonamide resistance reacted with the gene for type II dihydropteroate synthase. The two probes were sufficiently specific to allow study of the epidemiology of resistance in S. krefeld and other enteric bacteria.

Ampicillin Resistance↗
PubMed1988

Recombinational resolution in primate cells of two homologous human DNA segments with a gradient of sequence divergence.

Human alpha-thalassemia-2 genotype -alpha 4.2 is the result of meiotic recombination between two 1.3 kb long, homologous DNA segments, X(alpha 2) and X(alpha 1), located in the adult alpha globin locus. The two segments can also undergo intramolecular recombination on extrachromosomal vectors transfected into mitotically dividing primate cells (COS 7). The existence of a gradient of sequence divergence between X(alpha 2) and X(alpha 1) makes them an interesting system to study the relationship between efficiencies of homologous DNA recombination and the extent of dispersed and localized base mismatches. By partial restriction mapping and DNA sequencing of plasmids recombined in COS 7 cells and rescued from bacteria HB 101, we have determined the distribution of recombinational resolution sites along the two X blocks. Most, if not all, of the homologous recombination events between the two X blocks appear to be single crossing-over without efficient gene correction or repair of base mismatches. The distribution of the sites of recombinational resolution is inversely correlated with that of the gradient of sequence divergence, with only approximately 7% of the X recombinants resolved within the 3' third of the X blocks where two diverged Alu family repeats reside. The Alu sequence within which one of the X recombinants resolved is homologous to a previously characterized alpha thalassemia deletion point.

Animals↗
PubMed1991

Characterization of a maize cDNA that complements an enolase-deficient mutant of Escherichia coli.

A cDNA encoding maize enolase (2-phospho-D-glycerate hydrolase) was purified by functional genetic complementation using an enolase deficient mutant of Escherichia coli, DF261. This cDNA, pZM245, was characterized by restriction mapping and DNA sequence analysis. The cDNA contained an open reading frame encoding a protein of 446 amino acids with a high degree of similarity to enolase sequences from other organisms (72% identity to yeast enolase and 82% identity to human enolase). The pZM245 contains a correctly positioned consensus prokaryotic translation initiation sequence. The specific activity of enolase in maize increases to about twice its initial level after 48 hours of anaerobiosis. Northern-blot analysis showed a five-fold anaerobic induction in enolase mRNA, while heat shock or cold shock increased enolase mRNA levels only slightly. Southern-blot analysis of maize genomic DNA indicated that there is one copy of the pZM245 hybridizing sequence per haploid genome in maize.

Amino Acid Sequence↗
PubMed1991

A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP-specific glutamate dehydrogenase(s) in Chlorella sorokiniana.

Chlorella sorokiniana possesses ammonium-inducible, chloroplastic, NADP-specific glutamate dehydrogenase (NADP-GDH) homo- or heterohexamers composed of alpha- and/or beta-subunits which were previously shown to derive from precursor protein(s) of identical size. From the present studies, data are consistent with these two subunits being encoded by a single nuclear gene. The NADP-GDH gene is greater than 7 kb in length due to the presence of at least 21 introns, an unusually large number for a eukaryotic microorganism. The exons, identified by comparison with sequences of NADP-GDH cDNA clones, include a region which is highly conserved among NADP-GDH genes. This region in the C. sorokiniana gene is 77% and 73% identical to the corresponding regions in the Escherichia coli and Neurospora crassa NADP-GDH genes, respectively. Seventeen independent NADP-GDH cDNA clones were analyzed by restriction mapping and partial sequencing, and no differences were detected among them. The longest cDNA was fused in frame with lacZ in a Bluescript vector and was expressed in E. coli as NADP-GDH antigen. During a 240 min induction period, under conditions in which both types of subunits were synthesized, only a single (2.2 kb) NADP-GDH mRNA band was detected on northern blots using cDNA probes from the highly conserved and 3'-untranslated regions. Collectively, these results are consistent with a single mRNA encoding a precursor-protein which is differentially processed to yield either an alpha- or beta-subunit.

Amino Acid Sequence↗
PubMed1990

Use of non-radioactive DNA probes for detection of Campylobacter jejuni and Campylobacter coli in stool specimens.

DNA probes specific for C. jejuni (pDT1720 containing a 1475 base pair fragment) and for C. jejuni and C. coli (pDT1719 containing a 1845 base pair fragment) were isolated from a bacteriophage lambda gt11 genomic library of C. jejuni, using antiserum prepared against a 46 kDa major outer membrane protein of C. jejuni. The two probe-fragments had different restriction maps and were only moderately related by DNA hybridization analysis. A non-radioactive labelling kit which consisted of alkaline phosphatase conjugated anti-digoxigenin antiserum and 5-bromo-4-chloro-3-indoyl phosphate with nitroblue tetrazolium as the colour substrate, which gives a purple colour for positive hybridization, was used to test 140 stool specimens, 70 of which were culture positive and 70 of which wer culture negative for Campylobacter spp. The pDT1720 fragment (C. jejuni probe) could detect a minimum of 1 x 10(5) C. jejuni cells on filters, whereas the pDT1719 fragment (C. coli probe) was 100-fold less sensitive. The C. jejuni probe demonstrated a sensitivity of 93% with culture positive stool samples, however, 15% of culture negative samples were also recorded as positive using this non-radioactive DNA probe.

Blotting, Southern↗
PubMed1991

Antigenic and genomic identity between simian herpesvirus aotus type 2 and bovine herpesvirus type 4.

Herpesvirus aotus type 2 (HVA-2) was isolated from a culture of kidney cells from a healthy owl monkey (Aotus trivirgatus). Bovine herpesvirus type 4 (BHV-4) is frequently isolated from diseased and even healthy cattle and occasionally from sheep, wild ruminants and cats. The two viruses are related antigenically, as was revealed by an indirect fluorescent antibody test using polyclonal antisera from experimentally infected rabbits or monoclonal antibodies raised against six BHV-4 proteins, three of which were glycosylated. The genome structures of the two viruses consist of a unique central sequence flanked at both ends by G + C-rich tandem repeats. Restriction maps (produced using EcoRI, BamHI and HindIII) of these two viruses were nearly identical but the unique sequence of the HVA-2 genome possessed two additional BamHI sites. Four genomic regions of variable size were detected, two located in the unique part, one in the repetitive part and one in the left junction between the unique and the repeated part of the genome; these slight variations were similar to those observed between various BHV-4 isolates. These results suggest that HVA-2 and BHV-4 belong to the same virus species; HVA-2 could be either a BHV-4 contaminant of owl monkey kidney cell cultures or an isolate from an owl monkey accidentally infected with BHV-4.

Animals↗
PubMed1992

Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines.

Nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra were generated with N-methyl-N-nitro-N'-nitrosoguanidine to identify and characterize pathogenicity genes of the bacterium. A total of 16 nonpathogenic mutants were isolated from 2,000 colonies. One mutant, NP1, was chosen for further study. NP1 did not multiply in soybean cotyledons. A genomic library of strain 8ra was constructed in the cosmid pLAFR3, and the cosmids were tested for complementation in NP1. One cosmid clone, pIH1, which contained a 31-kb insert, complemented mutant NP1. A restriction map of pIH1 was constructed, and deletion analyses identified a 10-kb HindIII fragment that restored pathogenicity to NP1. Southern hybridization analysis indicated that DNA sequences in the 10-kb HindIII fragment are conserved among other X. campestris pathovars tested. Three regions responsible for restoring pathogenicity have been identified by Tn3-HoHo1 mutagenesis. A 2.7-kb ClaI fragment was sequenced, and two possible open reading frames (ORF1 and ORF2) were found. Results indicated that ORF2 but not ORF1 may be expressed in Escherichia coli and in X. campestris pv. glycines. The carboxy terminus of the potential polypeptide encoded by ORF2 has an amino acid sequence similar to that of the gamma subunit of oxaloacetate decarboxylase, which is involved in sodium ion transport in Klebsiella pneumoniae.

Amino Acid Sequence↗
PubMed1992

Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris.

Nonpathogenic mutants of Xanthomonas campestris pv. campestris, generated from transposon mutagenesis, accumulated extracellular polygalacturonate lyase, alpha-amylase, and endoglucanase in the periplasm. The transposon Tn5 was introduced by a mobilizable, suicidal plasmid, pSUP2021 or pEYDG1. Genomic banks of wild-type X. campestris pv. campestris, constructed on the broad-host-range, mobilizable cosmid pLAFR1 or pLAFR3, were conjugated with one of the mutants, designated XC1708. Recombinant plasmids isolated by their ability to complement XC1708 can be classified into two categories. One, represented by pLASC3, can complement some mutants, whereas the other, represented by a single plasmid, pLAHH2, can complement all of the other mutants. Restriction mapping showed that the two recombinant plasmids shared an EcoRI fragment of 8.9 kb. Results from subcloning, deletion mapping, and mini-Mu insertional mutation of the 8.9-kb EcoRI fragment suggested that a 4.2-kb fragment was sufficient to complement the mutant XC1708. Sequence analysis of this 4.2-kb fragment revealed three consecutive open reading frames (ORFs), ORF1, ORF2, and ORF3. Hybridization experiments showed that Tn5 in the genome of XC1708 and other mutants complemented by pLASC3 was located in ORF3, which could code for a protein of 83.5 kDa. A signal peptidase II processing site was identified at the N terminus of the predicted amino acid sequence. Sequence homology of 51% was observed between the amino acid sequences predicted from ORF3 and the pulD gene of Klebsiella species.

Amino Acid Sequence↗
PubMedDate not supplied

[Multiple genes of delta-endotoxins from Bacillus thuringiensis subspecies galleriae].

Cloning and expression in E. coli delta-endotoxin genes cryIAb7, cryIG and, cryIX from Bacillus thuringiensis ssp galleriae has been performed. Restriction mapping and partial sequencing have shown the identity of the 3'-terminal parts of cryIG and cryIX, although their 5'-terminal halves corresponding to the toxin are unique. Sequencing 5'-flanking region of cryIX revealed no translation initiation site, which indicates a nonfunctional state of the gene. The clusterized locating of the cryIG and cryIG genes has been shown. The absence of a promoter-like structure in the 5'-flanking region of cryIG suggests transcription of the gene as a bicistronic mRNA with cryIX. The extremely high homology of the cryIG and cryIX 3'-terminal parts (2 kB long) suggests a recombination act in the gene origin.

Amino Acid Sequence↗
PubMed2006

Characterization of a novel type of MLSB resistance plasmid from Staphylococcus saprophyticus carrying a constitutively expressed erm(C) gene.

An erm(C)-carrying plasmid of unusual size and restriction map, designated pSES22, was identified in a Staphylococcus saprophyticus strain and sequenced completely. Constitutive expression of the erm(C) gene from pSES22 is based on a novel 22-bp tandem duplication in the erm(C) translational attenuator. Comparative analysis of the deduced Erm(C) amino acid sequence revealed that Erm(C) from pSES22 - together with an Erm(C) methylase from S. hyicus - represented a separate branch in the homology tree of Erm(C) methylases. Structural comparisons showed that plasmid pSES22 differed distinctly from all other completely sequenced erm(C)-carrying resistance plasmids. However, pSES22 was similar to several members of a diverse group of small plasmids, all of which carried closely related plasmid backbones consisting of the genes repU and pre/mob, but differed in their resistance genes.

Amino Acid Sequence↗
PubMed1997

Genetic localization of the type I trimethoprim resistance gene and its dissemination in urinary tract isolates in Taiwan.

In a total of 425 urinary isolates of E. coli, Enterobacter spp., and Klebsiella spp. selected, there were 169 (45.4%) isolates harbouring type I dihydrofolate reductase (DHFR) gene among 374 trimethoprim-resistant isolates. In these 169 isolates, only 17.2% hybridized with the Tn7 probe. According to another probe specific for the integrase gene of integron, 87.6% showed a positive reaction. Further analysis by restriction mapping proved that the type I DHFR gene was inserted into a integron-like structure. These results indicate that the type I DHFR gene that was initially observed in association with transposable element Tn7 is becoming associated with an integrase function similar to integrons in most instances. Further analysis of the distribution of Tn21-like integrase gene in clinical isolates indicated that the prevalence rates were 86.4%, 84.8%, and 76.7% respectively in E. coli, Enterobacter spp., and Klebsiella spp.. Furthermore, the integrase gene found in our clinical isolates proved to be mediated by a plasmid, demonstrated by Southern hybridization. Thus, the trimethoprim-resistant gene that developed under selective pressure from the double drug trimethoprim and sulphonamide was transmitted by insertion into integron-like structure and then mediated by plasmid transfer for dissemination.

Animals↗
PubMed1991

Organisation and expression of small subunit ribosomal RNA genes encoded by a 35-kilobase circular DNA in Plasmodium falciparum.

A restriction map of the 35-kb circular DNA molecule of Plasmodium falciparum showed that a region of about 6 kb, encoding both a large and a small subunit ribosomal RNA gene, has been duplicated in inverted orientation. The complete sequence of one small subunit rRNA gene is presented as well as an analysis of transcripts from erythrocytic stage parasites. Comparative sequence analysis of the rRNA gene and the proposed secondary structure of the rRNA suggest that it is of organellar origin. Intriguingly, while some characteristics of the small subunit rRNA gene are similar to mitochondrial sequences, others are more like those of plastids. The origin of the circular DNA molecule and evolutionary implications of its genetic content are discussed.

Animals↗
PubMed1990

Amplification of a long sequence that includes a processed pseudogene for elongation factor 2 in the mouse.

Quantitative Southern blotting analysis has demonstrated that mouse cells contain about 70 copies per haploid genome of a DNA sequence related to the gene for elongation factor 2. The restriction maps of seven cosmids that each carry one copy of the EF2-related sequence (MER) and nucleotide sequences of MERs were highly conserved among the cosmids. Data obtained by such analyses suggest that MERs were produced by the integration of one copy of MER derived from poly(A)+ mRNA for EF2 into a specific site in the mouse genome, with subsequent amplification of MER together with its large flanking sequences during the evolution of the mouse. Furthermore, it appears that the size of each repeating unit is more than 60 kb. Analysis by pulse-field gel electrophoresis suggested that multiple copies of a repeating unit of more than 400 kb (or two units) are clustered at a specific site (or each specific site) in the mouse genome.

Animals↗