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At least 163 records · Page 9Linked to original sources
PubMed2003

Trimorphic DNA variation in the receptor-like protein kinase gene in the F18L15-130 region of the wild plant Arabidopsis thaliana.

DNA variation in the F18L15-130 region, which contains a receptor-like protein kinase gene, was analyzed for the wild plants Arabidopsis thaliana and Arabis gemmifera. In A. thaliana, at least three divergent sequence types were observed (trimorphism), instead of two distinct sequence types (dimorphism) detected in most of other nuclear gene regions studied for this plant species. Intragenic recombinations among the divergent sequence types generated four recombinant sequence types, although pattern of intragenic recombinations was complex. The estimated nucleotide variation in A. thaliana was the highest (pi = 0.0226 and theta = 0.0228) among genes investigated in this plant so far. The high level of nucleotide variation is due to divergence among the three distinct sequence types, each of which has a low level of nucleotide variation. Tests of Tajima, and Fu and Li did not detected deviation from neutrality, except for replacement sites, for which significantly negative Fu and Li's test value was detected. These results suggest that the receptor-like protein kinase gene in this region is functional. Possible causes for the trimorphic pattern of DNA polymorphism was discussed.

Arabidopsis↗
PubMed1995

Identification of a gene, pilV, required for type 4 fimbrial biogenesis in Pseudomonas aeruginosa, whose product possesses a pre-pilin-like leader sequence.

Type 4 fimbriae are important colonization factors in Pseudomonas aeruginosa and other pathogens that mediate attachment to epithelial cells of the host. They are also responsible for a form of translocation termed 'twitching motility' and are implicated in the susceptibility to fimbrial-specific bacteriophage. Analysis of a transposon mutant which lacks functional fimbriae has identified a new gene which is required for fimbrial biogenesis. This gene, termed pilV, is located on chromosomal SpeI fragment E, 2 kb downstream of the previously characterized pilSR genes involved in transcriptional activation of the fimbrial subunit gene. The pilV gene encodes a 20 kDa membrane-located protein with considerable amino-terminal homology to the type 4 consensus pre-pilin leader sequence, suggesting that it is processed by a leader peptidase. Site-directed mutagenesis has shown that PilV requires such cleavage to be functional. PilV also exhibits close similarity to a group of proteins involved in extracellular protein secretion from a number of Gram-negative bacteria, suggesting that the biogenesis of type 4 fimbriae may have a similar basis.

Amino Acid Sequence↗
PubMed2024

Analysis of molecular epidemiological characteristics and antimicrobial susceptibility of vancomycin-resistant and linezolid-resistant Enterococcus in China.

BACKGROUND: This study investigates the distribution and characteristics of linezolid and vancomycin susceptibilities among Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) and explores the underlying resistance mechanisms. METHODS: A total of 2842 Enterococcus clinical isolates from patients were retrospectively collected, and their clinical data were further analyzed. The minimum inhibitory concentrations (MICs) of vancomycin and linezolid were validated by broth dilution method. The resistance genes optrA, cfr, vanA, vanB and vanM were investigated using polymerase chain reaction (PCR). Housekeeping genes and resistance genes were obtianed through whole-genome sequencing (WGS). RESULTS: Of the 2842 Enterococcus isolates, 88.5% (2516) originated from urine, with E. faecium accounted for 60.1% of these. The vanA gene was identified in 27/28 vancomycin resistant Enterococcus (VRE) isolates, 4 of which carried both vanA and vanM genes. The remaining strain was vanM positive. The optrA gene was identified in all E. faecalis isolates among linezolid resistant Enterococcus (LRE). E. faecium showed a higher multiple antibiotic resistance index (MAR index) compared to E. faecalis. The multi-locus sequence typing (MLST) showed the sequence type of E. faecium mainly belongs to clonal complex (CC) 17, nearly E. faecalis isolates analyzed were differentiated into 7 characteristics of sequence types (STs), among which ST16 of CC16 were the major lineage. CONCLUSION: Urine was the primary source of VRE and LRE isolates in this study. E. faecium showed higher levels of resistance compared to E. faecalis. OptrA gene was detected in 91.6% of LRE, which could explain linezolid resistance, and van genes were detected in all vancomycin resistant Enterococcus strains, while vanA was a key resistance mechanism in VRE identified in this study.

Linezolid↗
PubMed1995

Evolution of the Sindbis virus subgenomic mRNA promoter in cultured cells.

Transcription of the subgenomic mRNA of alphaviruses initiates at an internal site, called the promoter, which is highly conserved. To determine the functional significance of this conservation, we used an approach that randomizes positions -13 to -9 of the promoter to generate a library containing all possible sequences within this region, including the wild-type sequence. Viruses in the mixed population with more-efficient promoters were selected for during passaging in mammalian (BHK-21) cells. Results from early passage populations indicate that a large number of different promoters are functionally active. Analysis of eight individual viruses found that although each contained a promoter with different degrees of sequence identity to the wild-type sequence, all eight viruses produced progeny. This suggests that the mechanism for transcription allows for a diversity of sequences to serve as promoters. Further passaging of the viral library led to a population consensus sequence that increasingly resembled the wild-type sequence, despite the fact that these promoters are not constrained by the need to encode the carboxyl terminus of the nsP4 protein. Thus, conservation of the region of the promoter from -13 to -9 is in large part due to selection for promoter function, and the wild-type sequence and sequences closely similar to it seem to be optimal for promoter function in BHK-21 cells.

Animals↗
PubMed1999

Type II restriction endonucleases from Helicobacter pylori include an enzyme with a novel recognition sequence.

Type II restriction endonuclease activities of Helicobacter pylori strain Roberts and of the type strain H. pylori NCTC 11637 were detected and analysed by conventional techniques. The endonucleases were partially purified, their optima for activity and their recognition and cleavage sites were determined. H. pylori (Roberts) contained at least two enzymes: HpyBI was an isoschizomer of RsaI (GT/AC) and HpyBII was of a novel specificity (GTN/NAC). H. pylori NCTC 11637 was found to contain an isoschizomer of EcoRV (HpyCI: GAT/ATC) and at least one other enzyme which was too unstable to characterise.

Deoxyribonucleases, Type II Site-Specific↗
PubMed2002

New HLA-A*11 allele, A*1112, identified by sequence-based typing.

In this report, we describe the identification of HLA-A*1112, a novel HLA-A*11 allele found in two Italian families. The new allele was detected during routine HLA typing by a polymerase chain reaction sequence-specific primer and was confirmed by high-resolution sequencing-based typing. The nucleotide sequences of HLA-A*1112 exons 2 and 3 are identical to HLA-A*11011 except for a single nucleotide substitution in codon 90 (GAC-->GCC).

Alleles↗
PubMed1995

Listeria monocytogenes exists in at least three evolutionary lines: evidence from flagellin, invasive associated protein and listeriolysin O genes.

Regions of the genes encoding flagellin (flaA), the invasive associated protein (iap), listeriolysin O (hly) and 23S rRNA were sequenced for a range of Listeria monocytogenes isolates of different origin and serotypes. Several nucleotide sequence variations were found in the flaA, iap and hly genes. No differences were found for the rRNA genes, but our approach does not exclude the existence of differences between single copies of these genes. Based on the sequence differences, the L. monocytogenes strains can be divided into three distinct sequence types. Further, the presence of only a small number of sequence differences within each group indicates a strong degree of conservation within the groups. There was a complete correspondence among the groups of strains formed according to the analysis of the flaA, iap and hly genes, and the grouping correlates with serotype, pulsed field gel electrophoretic and multilocus enzyme electrophoretic data. Analysis of the region encoding the threonine-asparagine repeat units in the iap gene revealed some striking features. Sequence type 1 strains were found to have 16-17 repeats, sequence type 2 strains had 16-20 repeats whereas the two sequence type 3 strains analysed had only 11 repeats. Furthermore, within a 19 bp segment there was a 37% difference between the sequences of type 1 and 2 strains and that segment was absent in type 3 strains. Within the threonine-asparagine repeat region the nucleotide differences gave rise to four amino acid changes; however, all were changes among the three amino acids present in the repeat structure indicating a strong selective pressure on the composition of this region.

Animals↗
PubMed1996

Tetrasomy 18p de novo: identification by FISH with conventional and microdissection probes and analysis of parental origin and formation by short sequence repeat typing.

We report a de novo supernumerary isochromosome 18p in a child with tetrasomy 18p, analyzed by a straightforward combination of cytogenetic and molecular cytogenetic methods. The diagnostic procedure consisted of standard banding techniques and fluorescence in situ hybridization (FISH) with centromere and library DNA probes for chromosome 18, and 18p-specific FISH probes prepared by chromosome dissesction and in vitro amplification. The maternal origin as well as the most probable cell stages of formation of the supernumerary isochromosome were determined by typing of short sequence repeats (SSRs). The pattern of allelic distribution suggests a nondisjunction during meiosis followed by a centromeric misdivision in an early postzygotic mitosis as the most probable mode of isochromosome 18p formation. The combination of the applied methods represents a powerful tool to investigate the nature and the origin of de novo marker chromosomes.

Alleles↗
PubMed2000

Identification of a new DRB3*02 allelic variant (DRB3*0209) by high-resolution sequence-based typing.

The HLA-DRB3/B4/B5 sequence-based typing method developed in this study in combination with PCR-SSP, enabled us to identify a new DRB3*02 allele, that was named as DRB3*0209 (GenBank accession number AF148518). This name has been officially assigned by the WHO Nomenclature Committee in May 1999. The new allele differs from DRB3*0207 by one substitution in codon 51 from AGG to ACG and another in codon 60 from TAC to TCC, resulting in aminoacid changes from Arg-->Thr (codon 51) and from Tyr-->Ser (codon 60). The DRB3*0209 allele was discovered in two related North Italian families. The fact that it was present in an hemizygous situation in three members of the paternal family and in one member of the secondary related family enabled us to isolate and sequence the new DRB3 allele without cloning, to identify its association with the DRB1 locus, and to generate an Epstein-Barr virus (EBV)-transformed cell line, now present in our ECBR (European Collection for Biomedical Research) Cell Line Bank.

Alleles↗
PubMed1998

Cyclic nucleotide regulation of type-1 plasminogen activator-inhibitor mRNA stability in rat hepatoma cells. Identification of cis-acting sequences.

Type-1 plasminogen activator-inhibitor (PAI-1) is a major physiologic inhibitor of plasminogen activation. Incubation of HTC rat hepatoma cells with the cyclic nucleotide analogue, 8-bromo-cAMP, causes a dramatic increase in tissue-type plasminogen activator activity secondary to a 90% decrease in PAI-1 mRNA. Although 8-bromo-cAMP causes a modest decrease in PAI-1 transcription, regulation is primarily the result of a 3-fold increase in the rate of PAI-1 mRNA degradation. To determine the cis-acting sequences required for cyclic nucleotide regulation, we have stably transfected HTC cells with chimeric genes containing sequences from the rat PAI-1 cDNA and the mouse beta-globin gene and examined the effect of cyclic nucleotides on the decay rate of these transcripts. The mRNA transcribed from the beta-globin gene is stable and not cyclic nucleotide-regulated, whereas the transcript from a construct containing the beta-globin coding region and the PAI-1 3'-untranslated region (UTR) is destabilized in the presence of 8-bromo-cAMP, suggesting that this response is mediated by sequences in the PAI-1 3'-UTR. Analyses by deletion of sequences from this chimeric construct indicate that, whereas more than one region of the PAI-1 3'-UTR can confer cyclic nucleotide responsiveness, the 3'-most 134-nucleotide sequence alone is sufficient to do so. Insertion of PAI-1 sequences within the beta-globin 3'-UTR confirms that the 3'-most 134 nucleotides of PAI-1 mRNA can confer cyclic nucleotide regulation of stability on a heterologous transcript, suggesting that this sequence may play a major role in hormonal regulation of PAI-1 mRNA stability.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Sequencing of HLA class I genes based on the conserved diversity of the noncoding regions: sequencing-based typing of the HLA-A gene.

We present a sequencing-based typing strategy for the HLA-A locus that is generally applicable to all HLA class I genes. Sequencing-based typing is the method of choice for matching in unrelated bone marrow transplantation on the allelic level. We determined the noncoding sequences of all serological antigens and most of their subtypes and discovered a remarkably conserved diversity characterized by polymorphic sequence motifs. In this study we took advantage of this diversity we uncovered in the 5' flanking region, 5' untranslated region and in the introns 1, 2 and 3, which was related to serological families. We established 12 primer mixes for setting up a PCR-based template preparation. Our strategy is based on the separate amplification of haplotypes and therefore defines the cis/trans linkage of polymorphic sequence motifs. This allowed individual sequencing of the haplotypes in all samples heterozygous for the broad antigens as well as the complete analysis of the polymorphic exons 2 and 3. All templates included the 2nd intron which was used as a priming site for the gene-specific 5' and 3' universal sequencing primers regardless of the amplified haplotypes. The independent sequencing of the haplotypes allows the application of the dye terminator cycle sequencing technique, which is less time-consuming and less-laborious than dye primer chemistry. The lack of heterozygous positions essentially facilitates on the one hand the data analysis and on the other hand the detection of new alleles. Sequencing is only required in one direction due to the absence of peak shift problems. The results will remain unambiguous regardless of a growing HLA sequence data bank since this sequencing technique defines the cis/trans linkage of sequence motifs in more than 95% of the cases.

Base Sequence↗
PubMed1991

HLA class II specificities and haplotypes in South Africa detected using polymerase chain reaction and sequence-specific oligonucleotide typing.

DNA sequencing of HLA class II alleles has revealed a degree of polymorphism much greater than was expected on the basis of the standard serological typing methods. Amplification of the polymorphic second exon of the class II genes using the polymerase chain reaction, followed by hybridization with sequence-specific oligonucleotide probes, allows the unambiguous identification of alleles which could not be detected previously. Using the protocols of the Eleventh International Histocompatibility Workshop, we have applied this procedure for the typing of several individuals and their families with suspected alleles that had been observed using serology, cellular typing and restriction fragment length polymorphism (RFLPs). These included an allele related to DRw8 and DRw14, which has only been observed in the mixed ancestral South African population. In addition, unusual combinations of class II genes forming unique haplotypic associations were seen.

Alleles↗
PubMed1999

Identification of the metabolically active members of a bacterial community in a polychlorinated biphenyl-polluted moorland soil.

The presumptive metabolically active members of a bacterial community in a moorland soil in Germany, highly polluted with polychlorinated biphenyls (PCBs), were identified by sequencing of cloned reverse transcription-polymerase chain reaction (RT-PCR) amplification products of 16S rRNA generated from total RNA extracts. Analysis of the 16S rRNA clone library revealed a considerable diversity of metabolically active bacteria in the soil, despite the acidic pH and high concentrations of PCBs. Cloned sequence types clustered within the Proteobacteria (34% alpha-, 33% beta- and 7% gamma-subclasses), the Holophaga-Acidobacterium phylum (14%), the Actinobacteria (6.5%) and the Planctomycetales (2%). Three cloned sequence types were not affiliated to any described phylogenetic group. An unusual feature of this soil was the abundance of sequence types within the beta-subclass of the Proteobacteria, most of which were similar to the 16S rRNA gene sequences of species from only two genera, Burkholderia and Variovorax. Three other numerous 16S rRNA sequence types were similar to the sequences of Sphingomonas species, members of the Rhodopila globiformis group and Acidobacterium capsulatum. Some of the sequence types retrieved were similar to the 16S rRNA sequences of bacterial isolates able to degrade a variety of organic pollutants, including PCBs. As the PCB contamination is the major source of measurable carbon in this soil, some of the 16S rRNA sequence types detected and presumed to represent the metabolically active members of the community indicate the organisms likely to be involved, directly or indirectly, in the utilization of the PCBs as carbon and energy sources.

Bacteria↗
PubMed1997

A generic sequencing based typing approach for the identification of HLA-A diversity.

Sequencing Based Typing (SBT) is a generic approach for the identification of HLA-A polymorphism. This approach includes the high resolution typing of the HLA-A broad reacting groups, HLA-A subtypes and will identify new alleles directly. The SBT approach described here uses a locus specific amplification of DNA from exon 1 to exon 5. The resulting 2,022 bp PCR product serves as a template for the subsequent sequencing reactions. Amplification is followed by direct sequencing of exons 2, 3 and 4 in both orientations with fluorescently labeled primers to define all polymorphic positions leading to a high resolution typing result. In this study the sequence of exons 2 and 3 of a panel of 49 cell lines was determined. In addition, the exon 4 region of 35 cell lines was also sequenced to evaluate the exon 4 polymorphism. The HLA-A type of most of the cells could be identified by sequencing only exons 2 and 3. However, the sequence of exon 4 was required to discriminate A*0201 from A*0209 and A*0207 from A*0215N. In this panel, an identical new "HLA-A*0103" was identified in two Caucasian samples.

Alleles↗
PubMed2025

Isolation and Molecular Characterization of Three Staphylococcus pseudintermedius Strains from Dogs and Humans in Egypt.

Staphylococcus pseudintermedius is an opportunistic pathogen that is largely associated with canine hosts but is becoming more widely recognized as a zoonotic pathogen. Understanding its genetic and phenotypic properties, such as virulence factors and antimicrobial resistance (AMR) profiles, is critical for infection control and vaccine development. In this study, we isolated and molecularly characterized three S. pseudintermedius isolates from dogs (hereafter referred to as S. pseudintermedius D8) and humans (hereafter referred to as S. pseudintermedius H10 and S. pseudintermedius H11) in Egypt. All three isolates showed 100% sequence identity with the nuc gene of the S. pseudintermedius SP_11304-3A reference genome. Multilocus sequence typing (MLST) revealed novel sequence types (STs) in the three isolates. The AMR determinants varied substantially among the isolates. While the mecA gene was absent, blaZ was detected in the canine isolate, indicating beta-lactamase-mediated penicillin resistance. Additionally, tetK and tetM genes were found conferring tetracycline resistance in different isolates. Resistance genes for aminoglycosides, chloramphenicol, fusidic acid, macrolides, streptothricin, and trimethoprim were also identified. All isolates were positive for key virulence genes, including immune evasion (AdsA), coagulase (coa), immunoglobulin-binding protein (sbi/spsK), exfoliative toxin (speta), enterotoxins (se-int and siet), fibrinogen binding protein gene (fnbB), and two-component pore-forming leukocidin genes (lukF and lukS). The S. pseudintermedius H11 isolate uniquely harbored the neuraminidase gene (nanB), while none of the isolates contained the gene coding for immunoglobulin G binding protein (spsQ). These findings highlight the differences in virulence and antimicrobial resistance genes among these S. pseudintermedius isolates, underlining the need for global surveillance and molecular characterization of this pathogen.

Dogs↗
PubMed2002

Identification of two new HLA-A alleles, A*2419 and A*3011, by sequence-based typing.

In this report, we describe two new HLA-A alleles, A*2419 and A* 3011, that were initially recognized by an aberrant serological pattern. Sequence-based typing revealed sequence differences with other known HLA-A alleles. Allele A*2419 showed 4 nucleotide differences with A*2404, resulting in 4 amino acid differences at codons 70, 76, 77 and 90. Compared with other A*24 alleles, A*2419 lacks the Bw4 motif, as do A*2404 and A*2428. The A*3011 allele showed 2 mismatches with A*3001, resulting in one amino acid difference at codon 80.

Alleles↗
PubMed1995

Elongation activity of poliovirus RNA polymerase derived from Sabin type 1 sequence is not temperature sensitive.

Determinants of attenuation in the Sabin type 1 strain of poliovirus are located in the 5' noncoding region, the capsid coding region and the viral RNA-dependent RNA polymerase (3Dpol) coding region. These mutations also contribute to a temperature sensitive pheno-type of virus replication. We have cloned and expressed the Sabin 1 virus 3Dpol) protein which contains three amino acid differences from the wild-type (Mahoney) sequence, as well as a wild- type polymerase containing only a single Sabin amino acid substitution at nt 6203. These enzymes have been examined and compared for temperature sensitive polymerase activity. Wild-type and mutated polymerases demonstrated identical specific activities at 30, 35 and 39 degrees C. All three showed the same kinetics of heat inactivation after pre-incubation at elevated temperatures. Thus the contribution of Sabin 3Dpol sequences to the inability of the virus to grow at elevated temperatures must lie in a function or activity of the enzyme other than RNA polymerization. A likely reaction is the initiation step of RNA chain synthesis.

Cloning, Molecular↗