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Results for “Genetic characterization”

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PubMed2001

Clinical and genetic characterization of an autosomal dominant nephropathy.

Autosomal dominant familial nephropathies with adult onset, no macroscopic cysts, and progressive deterioration include medullary cystic disease (ADMCKD) as well as other less specific entities. We studied a kindred of Jewish ancestry in which 15 members (both male and female) have suffered from chronic renal failure. The first evidence of renal involvement was observed between 18 and 38 years. It included hypertension followed by progressive renal insufficiency. No polyuria, anemia, gout, hematuria, nor proteinuria were seen. An average of 4.5 years elapsed from diagnosis to end-stage renal disease. Renal pathology at early stages of the disease showed extensive tubulointerstitial fibrosis and global glomerulosclerosis. Linkage analysis was performed at the two known loci of ADMCKD, on Chromosomes 1 and 16. Linkage to the chromosome 16 locus was excluded. However, linkage to the chromosome 1q21 locus of ADMCKD was established with a maximum two-point LOD score of 3.82 to D1S394. The disease interval could be narrowed to about 9 cM/7.4 Mb between D1S1156 and D1S2635. Multiple-point linkage analysis revealed a maximum LOD of 4.21, with a broad peak from markers D1S2858 and D1S2624. This report establishes linkage between a familial nephropathy characterized by hypertension and progressive renal failure to the locus described for ADMCKD, a disease classically associated with macroscopic corticomedullary cysts, salt-losing tubulointerstitial nephropathy, and anemia. This finding broadens the clinical spectrum of ADMCKD positioned on chromosome 1q21 locus.

Adult↗

PubMed2003

Genetic characterization of rebounding human immunodeficiency virus type 1 in plasma during multiple interruptions of highly active antiretroviral therapy.

Various strategies of interrupting highly active antiretroviral therapy (HAART) are being investigated for the treatment of human immunodeficiency virus (HIV) infection. Interruptions of greater than 2 weeks frequently result in rebound of plasma HIV RNA. In order to discern changes in the viral population that might occur during cycles of treatment interruption, we evaluated the homology of HIV-1 envelope gene sequences over time in 12 patients who received four to seven cycles of 4 weeks off HAART followed by 8 weeks on HAART by using the heteroduplex tracking assay and novel statistical tools. HIV populations in 9 of 12 patients diverged from those found in the first cycle in at least one subsequent cycle. The substantial genetic changes noted in HIV env did not correlate with increased or decreased log changes in levels of plasma HIV RNA (P > 0.5). Thus, genetic changes in HIV env itself did not contribute in a systematic way to changes in levels of plasma viremia from cycle to cycle of treatment interruption. In addition, the data suggest that there may be multiple compartments contributing to the rebound of plasma viremia and to viral diversity from cycle to cycle of intermittent therapy.

Antiretroviral Therapy, Highly Active↗

PubMed2005

Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand.

The H5N1 avian influenza virus outbreak among zoo tigers in mid-October 2004, with 45 animals dead, indicated that the avian influenza virus could cause lethal infection in a large mammalian species apart from humans. In this outbreak investigation, six H5N1 isolates were identified and two isolates (A/Tiger/Thailand/CU-T3/04 and A/Tiger/Thailand/CU-T7/04) were selected for whole genome analysis. Phylogenetic analysis of the 8 gene segments showed that the viruses clustered within the lineage of H5N1 avian isolates from Thailand and Vietnam. The hemagglutinin (HA) gene of the viruses displayed polybasic amino acids at the cleavage site, identical to those of the 2004 H5N1 isolates, which by definition are highly pathogenic avian influenza (HPAI). In addition, sequence analyses revealed that the viruses isolated from tigers harbored few genetic changes compared with the viruses having infected chicken, humans, tigers and a leopard isolated from the early 2004 H5N1 outbreaks. Sequence analyses also showed that the tiger H5N1 isolated in October 2004 was more closely related to the chicken H5N1 isolated in July than that from January. Interestingly, all the 6 tiger H5N1 isolates contained a lysine substitution at position 627 of the PB2 protein similar to the human, but distinct from the original avian isolates.

Amino Acid Sequence↗

PubMed2004

Genetic characterization of the Drosophila homologue of coronin.

We report cloning and characterization of coro, which codes for the Drosophila homologue of the F-actin binding protein coronin. Viable alleles of coro produce a variety of phenotypes in leg, wing and eye development, which are similar to the phenotypes observed as a result of mutations in genes associated with the actin cytoskeleton and/or membrane trafficking. Homozygous lethal mutations in coro results in the disruption of the actin cytoskeleton in wing imaginal discs. Formation of both basolateral septate junctions and apical adherens junctions are also adversely affected in epithelial cells. Both viable and lethal alleles of coro show genetic interactions with syntaxin1A, a gene required for membrane trafficking. They also show enhanced response to over-expression of Decapentaplegic (Dpp) and its receptor Thick vein. Tracing of Dpp morphogen using a Dpp::GFP fusion construct suggested defects in the endocytic pathway, which resulted in uniform distribution of Dpp along the AP axis rather than a gradient from the AP boundary. Our results provide a genetic link between endocytosis/exocytosis events involving F actin-coated vesicles and the establishment of morphogen gradient.

5' Untranslated Regions↗

PubMed1993

Antigenic and genetic characterization of Borrelia species isolated from Ixodes persulcatus in Hokkaido, Japan.

Ten characteristic strains of spirochetes (HT2, HT7, HT10, HT15, HT17, HT19, HT20, HT22, HT32, and HT59) isolated from Ixodes persulcatus adult ticks in Hokkaido, Japan, were selected and analyzed by polyacrylamide gel electrophoresis, with monoclonal and polyclonal antibodies, and by pulsed-field gel electrophoresis. The protein profiles of the borrelial isolates were variable and markedly different from that of the type strain Borrelia burgdorferi B31. The 41-kDa flagellin protein was present in all isolates, but the outer surface protein A (OspA) was absent in four isolates (HT15, HT2, HT20, and HT32). The molecular weights of the OspA proteins in six isolates were found to differ from one isolate to another. No two isolates examined had the same plasmid profile. These findings show the antigenic and genetic heterogeneity of the Japanese isolates, and some isolates are strikingly different from North American, European, and Asian strains.

Animals↗

PubMed2000

Genetic characterization of DNA region containing the trh and ure genes of Vibrio parahaemolyticus.

We have demonstrated that possession of the gene for thermostable direct hemolysin-related hemolysin (trh) coincides with the presence of the urease gene among clinical Vibrio parahaemolyticus strains and that the location of the two genes are in close proximity on the chromosome. Here, we cloned and sequenced the 15,754-bp DNA region containing the trh gene and the gene cluster for urease production from the chromosome of clinical V. parahaemolyticus (TH3996). We found 16 open reading frames (ORFs) and a lower G+C content (41%) compared with the total genome of this bacterium (46 to 47%). The ure cluster consisted of eight genes, namely, ureDABCEFG and ureR. ureR was located 5.2 kb upstream of the other seven genes in the opposite direction. The genetic organization and sequences of the ure genes resembled those found in Proteus mirabilis. Between ureR and the other ure genes, there were five ORFs, which are homologous with the nickel transport operon (nik) of Escherichia coli. We disrupted each of the ureR, ureC, and nikD genes in TH3996 by homologous recombination and analyzed the phenotype of the mutants. In the presence of urea these mutant strains had dramatically less urease activity than the strain they were derived from. Disruption of ureR, nikD, or ureC, however, had no effect on TRH production. The DNA region containing the trh, nik, and ure genes was found in only trh-positive strains and not in Kanagawa phenomenon-positive and environmental V. parahaemolyticus strains. At the end of the region, an insertion sequence-like element existed. These results suggest that the DNA region was introduced into V. parahaemolyticus in the past through a mechanism mediated by insertion sequences. This is the first reported case that the genes for an ATP-binding cassette-type nickel transport system, which may play a role in nickel transport through bacterial cytoplasmic membrane, are located adjacent to the ure cluster on the genome of an organism.

ATP-Binding Cassette Transporters↗

PubMed1999

Genetic characterization of the silkworm Bombyx mori by simple sequence repeat (SSR)-anchored PCR.

Thirteen diverse strains of the silkworm Bombyx mori were analysed using the simple sequence repeat anchored polymerase chain reaction (SSR-anchored PCR) or Inter-SSR-PCR (ISSR-PCR). A set of four 5'-anchored and two 3'-anchored repeat primers amplified a total of 239 bands out of which 184 (77%) were polymorphic. The 5'-anchored primers revealed more distinct polymorphic markers than the 3'-anchored primers and the ISSR-PCR method showed greater variability than RAPDs. The strain-specific pattern was shown to be inherited and segregated in a Mendelian fashion. A dendrogram constructed using the UPGMA method revealed two distinct groups, one comprising nondiapausing and one comprising diapausing strains. These results suggest that the ISSR-PCR method is potentially useful for genetic fingerprinting of silkworm genotypes and as a mapping tool in the silkworm.

Animals↗

PubMed1975

Genetic characterization of the metK locus in Escherichia coli K-12.

Three independently isolated metK mutants have been shown to have leisions lying between speB and glc near 57 min on the Escherichia coli chromosome. Two deletions result in a lack of the metC gene product but neither extends into the metK glc region. The three metK mutations are recessive to the wild-type allele carried on the KLF16 episome.

Arginine↗

PubMed1985

Isolation and genetic characterization of human KB cell lines resistant to multiple drugs.

Human KB cell lines resistant to high levels of colchicine were isolated by several successive single-step selections. Most of these selection steps resulted in cross-resistance to vincristine, vinblastine, adriamycin, actinomycin D, and puromycin; however, at the highest levels of colchicine resistance, increased cross-resistance to other drugs was not observed. There was no major change in protein synthesis or alteration in protein phosphorylation or [14C]glucosamine labeling patterns accompanying the development of multiple drug resistance as measured by analysis of metabolically labeled proteins on SDS gels. Cell-cell hybridization experiments showed that the colchicine-resistant and multiple drug-resistant phenotypes were incompletely dominant. In addition, colchicine resistance was found to segregate independently from resistance to other drugs in one somatic cell hybrid, suggesting that complex genetic loci are involved in the development of the multiple drug-resistant phenotype. These mutants should be useful for the study of the clinically important problem of multiple drug resistance in human cancer.

Antineoplastic Agents↗

PubMed1989

Expression of two insulin-like growth factor-binding proteins in a human endometrial cancer cell line: structural, immunological, and genetic characterization.

Insulin-like growth factors (IGFs) bind with high affinity to specific proteins in human serum, cerebrospinal fluid (CSF), and amniotic fluid. In serum, IGFs are bound to a complex with an apparent mol wt of 150K in which an acidstable binding protein of 53K, termed BP-53, is found. In amniotic fluid, a different binding protein with an apparent mol wt of 25K, termed hBP-25, has been identified. This binding protein is secreted by endometrial cells and has been shown to block the binding of IGFs to their membrane receptors and inhibit the mitogenic action of the IGFs on human choriocarcinoma cells. It has been proposed that hBP-25 modulates the action of IGFs on endometrial tissue, especially during pregnancy. We have identified two binding proteins produced by an endometrial adenocarcinoma, HEC 1A, neither of which is related structurally or genetically to hBP-25. One is present in two glycosylated forms with apparent mol wt of 37K and 40K, both of which are immunoprecipitated with an antiserum made to BP-53. Both forms are reduced to a core protein of 30K upon digestion with endoglycosidase-F. Furthermore, BP-53-specific RNA was detected in HEC 1A cells. A second binding protein with an apparent mol wt of 32K was also detected and did not increase in size upon treatment with endo glycosidase F. These two binding proteins were partially purified by a combination of wheat germ agglutinin and IGF affinity chromatography, and polyclonal antibodies were generated. The polyclonal antiserum specifically recognizes a 32K protein in human CSF, suggesting that the HEC 1A cells produce the same binding protein that is predominant in CSF. The findings suggest that IGF action in the endometrium may be modulated by more than a single binding protein, and that at least three structurally distinct human IGF binding proteins exist.

Blotting, Northern↗

PubMed2000

Biochemical and genetic characterization of coagulin, a new antilisterial bacteriocin in the pediocin family of bacteriocins, produced by Bacillus coagulans I(4).

A plasmid-linked antimicrobial peptide, named coagulin, produced by Bacillus coagulans I(4) has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42-50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I(4). Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different Pediococcus acidilactici strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI(4) DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from P. acidilactici and pI(4) when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus.

Amino Acid Sequence↗

PubMed1992

Arabidopsis mutants deficient in polyunsaturated fatty acid synthesis. Biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase.

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by reduced levels of polyunsaturated 18-carbon fatty acids and an increased proportion of oleate as a consequence of a single recessive nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids demonstrated that all the major phospholipids of the extrachloroplast membranes are affected by the mutation, whereas the chlorplast lipids show fatty acid compositions only slightly different from those of wild type plants. These results are consistent with the parallel operation of two pathways of lipid synthesis in plant leaf cells (the prokaryotic pathway in the chloroplast and the eukaryotic pathway in the endoplasmic reticulum) and with genetic evidence (Browse, J., Kunst, L., Anderson, S., Hugly, S., and Somerville, C.R. (1989) Plant Physiol 90, 522-529) that an independent 18:1/16:1 desaturase operates on chloroplast membrane lipids. Direct enzyme assays confirmed that the mutant plants are deficient in the activity of a microsomal oleoyl-phosphatidycholine desaturase and demonstrated that this desaturase is the major enzyme responsible for the synthesis of polyunsaturated phospholipids. Despite this deficiency in 18:1-desaturase activity, mutant plants contained relatively high levels of 18:3 in their leaf phospholipids. This finding is interpreted as additional evidence that considerable two-way exchange of lipid occurs between the chloroplast and endoplasmic reticulum and that this exchange allows the chloroplast desaturases to provide lipids containing 18:3 to the extrachloroplast compartment, thus partially alleviating the deficiency in 18:1 desaturase activity.

Chloroplasts↗

PubMed1997

Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme.

The Lactobacillus helveticus L-(+)-lactate dehydrogenase (L-LDH) gene (ldhL) was isolated from a lambda library. The nucleotide sequence of the ldhL gene was determined and shown to have the capacity to encode a protein of 323 amino acids (35.3 kDa). The deduced sequence of the 35-kDa protein revealed a relatively high degree of identity with other lactobacillar L-LDHs. The highest identity (80.2%) was observed with the Lactobacillus casei L-LDH. The sizes and 5' end analyses of ldhL transcripts showed that the ldhL gene is a monocistronic transcriptional unit. The expression of ldhL, studied as a function of growth, revealed a high expression level at the logarithmic phase of growth. The ldhL gene is preceded by two putative -10 regions, but no corresponding -35 regions could be identified. By primer extension analysis, the ldhL transcripts were confirmed to be derived from the -10 region closest to the initiation codon. However, upstream of these regions additional putative -10/-35 regions could be found. The L-LDH was overexpressed in Escherichia coli and purified to homogeneity by two chromatographic steps. The purified L-LDH was shown to be a nonaliosteric enzyme, and amino acid residues involved in allosteric regulation were not conserved in L. helveticus L-LDH. However, a slight enhancement of enzyme activity was observed in the presence of fructose 1,6-diphosphate, particularly at neutral pH. A detailed enzymatic characterization of L-LDH was performed. The optimal reaction velocity was at pH 5.0, where the kinetic parameters K(m), and Kcat for pyruvate were 0.25 mM and 643 S-1, respectively.

Amino Acid Sequence↗

PubMed1991

Kinase activity and genetic characterization of a growth related antigen of Drosophila.

The Drosophila developmental antigen recognized by the monoclonal antibody F7D6 is expressed in dividing embryonic and imaginal cells but is lost from all differentiating tissues except electrogenic cells of the nervous system and spontaneously contracting muscles. The 63 kDa antigen is associated with the inner surface of plasma membranes and is expressed in several classes of tumorous mutants of Drosophila. The monoclonal antibody was used for immunoprecipitating the antigen for biochemical characterization and for screening expression vector cDNA libraries. Here we report that this oncodevelopmental antigen is a phosphoprotein and a serine-threonine specific protein kinase. A 1.6 kb cDNA isolated by immunological screening of an ovarian library hybridized to a single band on polytene chromosomes, localizing the gene to 72F on the left arm of the third chromosome. Immunofluorescence assays of deficiency stocks in the region confirmed the location of the gene and identity of the cDNA clone, and mapped the gene between the left breakpoints of Df(3L) st100.62 and Df(3L) stj7, i.e., between 72F3-7 and 73A1-2. The biochemical and genetic properties indicate that this is a novel growth-related kinase of Drosophila.

Animals↗

PubMed1988

Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli.

In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage lambda poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA+ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.

Bacteriophage lambda↗

PubMed2004

Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia.

Porcine circovirus type 2 (PCV2) from the Circoviridae family has recently been associated with two serious diseases of swine, post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). During 2002, several outbreaks of clinical disease in pigs with weights ranging from 10 to 70 kg occurred on four farms in different locations in Croatia. The signs were consistent with PMWS and PDNS. Apart from progressive weight loss, pneumonia and/or diarrhoea, multifocal erythematous skin lesions and dermal necrosis were also observed. The PCR results obtained from PCV2 specific oligonucleotide primers confirmed a PCV2 infection. In addition, archive samples that were classical swine fever virus positive and derived from domestic pigs during an outbreak in 1997 were included in this study and one out of the three isolates was found to be positive for PCV2. For a better epizootiological understanding, genetic typing of representative isolates was carried out and compared with available isolates reported in the GenBank databases.

Animals↗

PubMed2002

Genetic characterization of wild-type measles viruses circulating in suburban Khartoum, 1997-2000.

Measles remains endemic in many East African countries, where it is often associated with high morbidity and mortality. We collected clinical specimens from Sudanese measles patients between July 1997 and July 2000. Sequencing of the 3' 456 nucleotides of the nucleoprotein gene from 33 measles virus (MV) isolates and 8 RNA samples extracted from clinical specimens demonstrated the presence of a single endemic MV strain with little sequence variation over time (overall nucleotide divergence of 0 to 1.3%). This was confirmed by sequencing of the complete H gene of two isolates from 1997 and two from 2000, in which the overall divergence ranged between 0 and 0.5%. Comparison with MV reference strains demonstrated that the viruses belonged to clade B, genotype B3, and were most closely related to a set of viruses recently isolated in Nigeria. Our study demonstrates a remarkable genetic stability of an endemically circulating MV strain.

Genetic Variation↗

PubMed1984

Genetic characterization of Salmonella typhimurium LT2 ara mutations.

Seventeen independently isolated L-arabinose utilization-deficient mutants of Salmonella typhimurium LT2 were characterized. Four complementation groups (araA, araB, araC, and araD) were identified and were equivalent to the same genes in the ara system in Escherichia coli. The order of the four genes was determined to be araD-araA-araB-araC-leu. Two transcription units were found: the araBAD operon was transcribed counterclockwise, and the araC gene was transcribed clockwise.

Arabinose↗