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Biomedical subjects

G A Buck

Publications and source records attributed to G A Buck.

At least 19 recordsLinked to original sources
PubMed2003

Association of erosive hand osteoarthritis with a single nucleotide polymorphism on the gene encoding interleukin-1 beta.

OBJECTIVE: Certain forms of primary osteoarthritis (OA), particularly those affecting hand joints, have a genetic component. Recent studies have shown suggestive evidence that hand and knee OA are linked with the interleukin-1 (IL-1) region on human chromosome 2q. This study was undertaken to assess the association of primary OA of the hand (hand OA) with IL-1 region markers. METHODS: Sixty-eight US Caucasoid cases and 51 US Caucasoid controls aged 60 years or older were recruited from the Mid-Atlantic region of the United States. Hand OA was classified by American College of Rheumatology (ACR) Clinical Criteria, and cases were subjected to radiographic examination for subgrouping. Genotyping was done for seven previously described single nucleotide polymorphisms (SNPs) of genes for IL-1alpha (encoded by IL1A), IL-1beta (IL1B), and the IL-1 receptor antagonist (IL1RN), as well as an IL1RN variable number of tandem repeat (VNTR) marker. Six microsatellite markers on other chromosomes (null loci) were also typed. RESULTS: The IL1B 5810 G>A SNP genotypes marker were not in Hardy-Weinberg equilibrium (p<0.05 in both non-erosive and erosive hand OA subgroups). Statistically significant association with the IL1B 5810 AA genotype was found in the erosive hand OA subgroup (relative risk 3.8, p=0.007). This IL1B 5810 AA genotype association was also significant between erosive and non-erosive hand OA subjects (relative risk 4.01, p=0.008). As expected, significant linkage disequilibrium was present between IL1B 5810 SNP and IL1A (-)889 SNP, other IL1B SNPs, and the nearest IL1RN SNP examined. The IL1B 5810A allele occurs most frequently on haplotypes with the SNP alleles IL1B 1423C, IL1B 1903T, IL1B 5887C, and IL1A (-)889C. Genotypes at null loci failed to show evidence suggesting population stratification that might account for spurious association. CONCLUSION: Statistical evidence shows association between erosive hand OA and a genomic region containing the IL1B 5810 SNP in a US Caucasoid population. This supports a potential role for IL-1 in the pathogenesis of a severe phenotype of hand OA.

Aged↗
PubMed2002

Genetic relatedness among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a synapomorphic DNA fragment for species-specific diagnosis.

In this study we employed randomly amplified polymorphic DNA patterns to assess the genetic relatedness among 14 Brazilian Trypanosoma evansi stocks from domestic and wild hosts, which are known to differ in biological characteristics. These akinetoplastic stocks were compared with one another, to three Old World (Ethiopia, China and Philippines) dyskinetoplastic stocks of T. evansi, and also with Trypanosoma equiperdum, Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Randomly amplified polymorphic DNA analysis showed limited heterogeneity in T. evansi stocks from different hosts and geographical regions of the world, or in other species of the subgenus Trypanozoon. However, minor variations generated random amplification of polymorphic DNA analysis disclosed a pattern consisting of a unique synapomorphic DNA fragment (termed Te664) for the T. evansi cluster that was not detected in any other trypanosome species investigated. Pulsed field gel electrophoresis analysis demonstrated that the Te664 fragment is a repetitive sequence, dispersed in intermediate and minichromosomes of T. evansi. Based on this sequence, we developed a conventional PCR assay for the detection of T. evansi using crude preparations of blood collected either on glass slides or on filter paper as template DNA. Our results showed that this assay may be useful as a diagnostic tool for field-epidemiological studies of T. evansi.

Animals↗
PubMed2001

Identification of a spliced leader RNA binding protein from Trypanosoma cruzi.

Nuclear mRNAs in trypanosomatids are generated by trans-splicing. Although trans-splicing resembles cis-splicing in many ways and most of the U RNA participants have been characterized, relatively few involved proteins have been identified. Herein, we employed a yeast three-hybrid system to identify a protein, XB1, which binds to the Trypanosoma cruzi SL RNA. XB1 is a approximately 45 kDa protein which is homologous to the essential pre-mRNA-splicing factor PRP31p from Saccharomyces cerevisiae. Gel shift assays and UV cross-linking experiments with recombinant XB1 confirmed that this T. cruzi protein binds the SL RNA in vitro. The binding site of XB1 on the SL RNA was mapped to stem-loop II by deletion of the SL RNA 'bait' in the three-hybrid system. Finally, UV cross-linking SL RNA with S100 extract indicated native XB1 protein and SL RNA interaction in T. cruzi extract.

Amino Acid Sequence↗
PubMed2001

Trypanosoma cruzi: exogenously regulated gene expression.

A regulated expression vector would provide a strong tool for the dissection of gene function in Trypanosoma cruzi. Herein, we establish a system in which genes in T. cruzi expression vectors can be exogenously regulated by tetracycline. We first generated strains of T. cruzi that stably express the repressor of the bacterial tetracycline resistance gene and T7 RNA polymerase. Based on these strains, we developed two T. cruzi expression systems regulated by tetracycline--the first by use of a regulated rRNA promoter and the second by use of a regulated T7 promoter. In the former, we constructed an expression vector in which tetracycline resistance gene operators flank the transcription start point of the T. cruzi rRNA gene promoter. Reporter gene activity from this modified promoter was regulated up to 20-fold in the presence of different concentrations of tetracycline. In the T7 system, tetracycline resistance gene operators flank the transcription start point of the T7 promoter. Reporter gene activity from this modified promoter was regulated up to 150-fold in the presence of different concentrations of tetracycline. Expression in these systems was repressed when tetracycline was removed even after full induction for extended periods in the presence of tetracycline. We are now using these two systems to test protein function in T. cruzi.

Animals↗
PubMed2001

Trypanosoma vivax: characterization of the spliced-leader gene of a Brazilian stock and species-specific detection by PCR amplification of an intergenic spacer sequence.

The sequence of the spliced-leader gene repeat of a Brazilian Trypanosoma vivax stock from cattle showed high similarity to sequences of West African T. vivax in both intron and intergenic sequences. This is the first evidence based on DNA sequences of close-relatedness between Brazilian and West African T. vivax stocks. A T. vivax-specific diagnostic PCR assay based on spliced-leader gene intergenic sequences was able to amplify DNA from T. vivax stocks from South America (Brazil, Bolivia, and Colombia) and West Africa. Species-specificity of this method was confirmed by results obtained by testing 15 other trypanosomes, including other species and subspecies that can also infect cattle. The PCR assay developed presented high sensitivity, detecting the DNA content of only one parasite and also revealing T. vivax infection in asymptomatic animals without detectable parasitemia by microhematocrit or in Giemsa-stained blood smears. Use of crude preparations from field-blood samples collected on both filter paper and glass slides as DNA template suggested that this method could be useful for the diagnosis of T. vivax in large epidemiological studies.

Animals↗
PubMed2000

Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parvum.

Human cryptosporidiosis is attributed to two major Cryptosporidium parvum genotypes of which type 1 appears to be the predominant. Most laboratory investigations however are performed using genotype 2 isolates, the only type which readily infects laboratory animals. So far type 1 has only been identified in humans and primates. A type 1 isolate, obtained from an individual with HIV and cryptosporidiosis, was successfully adapted to propagate in gnotobiotic piglets. Genotypic characterization of oocyst DNA from this isolate using multiple restriction fragment length polymorphisms, a genotype-specific PCR marker, and direct sequence analysis of two polymorphic loci confirmed that this isolate, designated NEMC1, is indeed type 1. No changes in the genetic profile were identified during multiple passages in piglets. In contrast, the time period between infection and onset of fecal oocyst shedding, an indicator of adaptation, decreased with increasing number of passages. Consistent with other type 1 isolates, NEMC1 failed to infect mice. A preliminary survey of the NEMC1 genome covering approximately 2% of the genome and encompassing 200 kb of unique sequence showed an average similarity of approximately 95% between type 1 and 2 sequences. Twenty-four percent of the NEMC1 sequences were homologous to previously determined genotype 2 C. parvum sequences. To our knowledge, this is the first successful serial propagation of genotype 1 in animals, which should facilitate characterization of the unique features of this human pathogen.

Animals↗
PubMed2000

PPB1, a putative spliced leader RNA gene transcription factor in Trypanosoma cruzi.

In trypanosomatids, the spliced leader RNA, or SL RNA, donates its 5' 39 nucleotides to mature nuclear mRNAs in a process termed trans-splicing. We have previously characterized the SL RNA gene from Trypanosoma cruzi and identified its transcription promoter, including a 14 nt proximal sequence element, or PSE, that binds a putative transcription factor and activates transcription of the gene. Herein, we describe establishment of a yeast one-hybrid system using the 14 nt PSE as bait, and use this system to select T. cruzi cDNAs encoding a putative transcription factor that activates transcription of the SL RNA gene. The cDNA was selected from a normalized library and encodes an approximately 45 kDa putative PSE promoter-binding protein, PPB1. PPB1 in vitro translated or overexpressed in and isolated from transformed E. coli, showed PSE-specific binding activity by electrophoretic mobility shift assays. Finally, overexpression of PPB1 in T. cruzi led to increased expression of the SL RNA gene as well as reporter genes in episomal constructs under the control of the SL RNA gene promoter. These observations suggest that PPB1 is a transcription factor that plays an important role in SL RNA gene expression.

Amino Acid Sequence↗
PubMed2000

Simultaneous stable expression of neomycin phosphotransferase and green fluorescence protein genes in Trypanosoma cruzi.

The ribosomal RNA (rRNA) gene promoter was used to construct plasmid vectors that simultaneously express multiple exogenous genes in Trypanosoma cruzi. Vector pBSPANEO expresses neomycin phosphotransferase, and pPAGFPAN expresses both green fluorescent protein and neomycin phosphotransferase from a single promoter. Both vectors require the presence of the rRNA promoter for stable transfection; epimastigotes transfected with pPAGFPAN strongly fluoresced due to green fluorescent protein expression. Intact plasmids were rescued from the T. cruzi-transfected population after >8 mo of culture, indicating stable replication of these vectors. Vectors were integrated into the rRNA locus by homologous recombination and into other loci, presumably by illegitimate recombination. Parasites bearing tandem concatamers of plasmids were also found among the transfectants. Transfectants expressing green fluorescent protein showed a bright green fluorescence distributed throughout the cell. Fluorescence was also detected in amastigotes after infection of mammalian cells with transfected parasites, indicating that the rRNA promoter can drive efficient expression of these reporter genes in multiple life-cycle stages of the parasite. Expression of the heterologous genes was detected after passage in mice or in the insect vector. These vectors will be useful for the genetic dissection of T. cruzi biology and pathogenesis.

Animals↗
PubMed1999

PCR amplification of the spliced leader gene for the diagnosis of trypanosomatid parasites of plants and insects in methanol-fixed smears.

A PCR-based method was adapted for the amplification of DNA from methanol-fixed smears of insects and plants parasitized by trypanosomatids. The PCR target was the multicopy spliced leader (SL) gene. Amplicons were hybridized with an oligonucleotide probe (SL3') specific for Phytomonas. The method has the advantage of dispensing with the cultivation of parasites, many of which are very fastidious or non-cultivable. The technique was applied to archival glass slides and to newly collected material. It proved to specific for Phytomonas spp., enabling their detection in plants and insects. Sequence comparison of the amplicons obtained revealed the existence of different strains/species of Phytomonas circulating among diseased palsms and fruit.

Animals↗
PubMed1999

Trypanosomatidae: Phytomonas detection in plants and phytophagous insects by PCR amplification of a genus-specific sequence of the spliced leader gene.

In this paper we describe a method for the detection of Phytomonas spp. from plants and phytophagous insects using the PCR technique by targeting a genus-specific sequence of the spliced leader (SL) gene. PCR amplification of DNA from 48 plant and insect isolates previously classified as Phytomonas by morphological, biochemical, and molecular criteria resulted in all cases in a 100-bp fragment that hybridized with the Phytomonas-specific spliced leader-derived probe SL3'. Moreover, this Phytomonas-specific PCR could also detect Phytomonas spp. in crude preparations of naturally infected plants and insects. This method shows no reaction with any other trypanosomatid genera or with plant and insect host DNA, revealing it to be able to detect Phytomonas spp. from fruit, latex, or phloem of various host plants as well as from salivary glands and digestive tubes of several species of insect hosts. Results demonstrated that SLPCR is a simple, fast, specific, and sensitive method that can be applied to the diagnosis of Phytomonas among cultured trypanosomatids and directly in plants and putative vector insects. Therefore, the method was shown to be a very specific and sensitive tool for diagnosis of Phytomonas without the need for isolation, culture, and DNA extraction of flagellates, a feature that is very convenient for practical and epidemiological purposes.

Animals↗
PubMed1999

High prevalence of GB virus C in Brazil and molecular evidence for intrafamilial transmission.

The prevalence of GB virus C (GBV-C) in candidate Brazilian blood donors with normal and elevated alanine aminotransferase levels was found to be 5.2% (5 of 95) and 6.5% (5 of 76), respectively. Among Brazilian patients, GBV-C was found in 9.5% (13 of 137) of cases of hepatitis not caused by hepatitis A virus (HAV), HBV, HCV, HDV, or HEV (non-A-E hepatitis) and in 18.2% (8 of 44) of individuals infected with HCV. Molecular characterization of GBV-C by partial sequencing of the NS3 region showed clustering between members of a single family, implying intrafamilial transmission. In conclusion, these results together suggest that contagion mechanisms which facilitate intrafamilial transmission of GBV-C may partially explain the high prevalence of viremic carriers worldwide.

Base Sequence↗
PubMed1999

Design strategies and performance of custom DNA sequencing primers.

This study surveyed strategies of sequencing primer selection and evaluated primer performance in automated DNA sequencing. We asked participants to relate their preferred primer design strategies to identify primer characteristics that are considered most important in sequencing primer design. The participants preferred primers of 18-24 nucleotides (nt), 39%-58% G + C, a melting temperature (Tm) of 53 degrees-65 degrees C with a 1-2 nt 3' GC clamp, hairpin stems of less than 2-3 bp, homopolymeric runs of less than 4-5 nt, primer dimers of less than 3-4 bp and secondary priming sites of less than 3-4 bp. We provided a 300-bp test sequence and asked participants to submit sequences of 1-3 optimal sequencing primers. Submitted primers ranged from 17-24 nt and largely conformed to the preferred parameters. Submitted primers were distributed across the test sequence, although some sites were disfavored. Surprisingly, approximately 45% of the primers were selected "manually", more than by any software package. Each of 69 submitted and 95 control primers, distributed at 3-bp intervals across the test sequence, were synthesized, purified and tested using a Model 377 PRISM DNA Sequencer with dichlororhodamine dye terminator reagents (dRhodamine dye terminators). Approximately half of the control primers were also tested using rhodamine dye terminator reagents ("old" rhodamine dye terminators). The results indicated that primer physico-chemical characteristics thought to have a strong impact on sequencing performance had surprisingly little effect. Thus, primers with high or low percent G + C or Tm, strong secondary priming scores or long 3' homopolymeric stretches yielded excellent sequences with the dRhodamine dye terminator reagents, although these characteristics had a stronger effect when the old rhodamine reagents were used. The old rhodamine reagents gave sequences with a similar average read length, but the number of errors and ambiguities or "N's" was consistently higher. Moreover, the effects of the primer physico-chemical characteristics were also more evident with the old rhodamine dyes. We conclude that under optimal sequencing conditions with highly pure template and primer, many of the commonly applied primer design parameters are dispensable, particularly when using one of the new generation of sequencing reagents such as the dichlororhodamine dye terminators.

Autoanalysis↗
PubMed1998

Double mutation (A171T and D444H) is a common cause of profound biotinidase deficiency in children ascertained by newborn screening the the United States. Mutations in brief no. 128. Online.

Biotinidase deficiency is inherited as an antosomal recessive trait that, unless treated with pharmacologic doses of biotin, can result in neurologic and cutaneous symptoms. We have identified two new mutations in exon D of the biotinidase gene of children with profound biotinidase deficiency ascertained by newborn screening. Transition 511G->A near the 5' end of exon D results in a substitution of threonine for alanine 171 (A171T) and transversion 1330G->C occurs close to the 3' end of exon D causing a substitution of histidine for aspartic acid 444 (D444H). The D444H mutation was detected in four individuals from our normal population whose mean serum biotinidase activity is 5.25 nmol/min/ml, which is significantly lower than the mean normal activity (7.1 nmol/min/ml). We calculated that this mutation causes a 52% loss of activity in the aberrant enzyme. Twenty-three individuals with the D444H mutation were found by allele specific oligonucleotide analysis of DNA from 296 randomly-selected, anonymous dried-blood spots. We estimate the frequency of this allele in the general population to be 0.039. In contrast, no individuals in 376 have the A171T mutation. Fourteen children (eleven probands and three siblings) out of the 31 enzyme-deficient children have both the A171T and D444H mutations. Both mutations are inherited from a single parent as a double mutation allele. The nine families in which this allele was identified are of mostly European ancestry, although the mutation cannot be attributed to a specific nationality or ethnic group. The serum of a child who is homozygous for the double mutation allele has very little CRM and the aberrant enzyme has very low biotinylhydrolase activity and no botinyl-transferase activity. This double mutation allele (A171T and D444H) is a common cause of profound biotinidase deficience in children ascertained by newborn screening in the United States.

Acyltransferases↗
PubMed1998

Structure of the human biotinidase gene.

Biotinidase cleaves biotin from biocytin, thereby recycling the vitamin. We have determined the structure of the human biotinidase gene. A genomic clone, containing three exons that code for the mature enzyme, was obtained by screening a human genomic bacteriophage library with the biotinidase cDNA by plaque hybridization. To obtain a clone containing the most 5' exon of the biotinidase cDNA, a human PAC library by PCR was screened. The human biotinidase gene is organized into four exons and spans at least 23 kb. The 5'-flanking region of exon 1 contains a CCAAT element, three initiator sequences, an octamer sequence, three methylation consensus sites, two GC boxes, and one HNF-5 site, but has no TATA element. The region from nt -600 to +400 has features of a CpG island and resembles a housekeeping gene promoter. The structure and sequence of this gene are useful for identifying and characterizing mutations that cause biotinidase deficiency.

Amidohydrolases↗
PubMed1997

The transcription promoter of the spliced leader gene from Trypanosoma cruzi.

A putative promoter element responsible for transcription of the spliced leader (SL) gene of Trypanosoma cruzi was identified by overlapping deletion and linker scanning analyses of the upstream flanking sequences using the bacterial chloramphenicol acetyltransferase (CAT) gene as a reporter in transient transfections of cultured epimastigotes. Deletion or substitution of a proximal sequence element (PSE) between positions -53 and -40 relative to the transcription start point eliminated CAT gene expression. Comparison of SL genes from several strains of T. cruzi revealed two alternative sequence patterns for the putative SL PSE, both composed of a short run of purines followed by a run of pyrimidines. Moreover, an examination of these sequences supports the subdivision of T. cruzi isolates into two divergent groups. Double-stranded oligonucleotides containing the sequence of the PSE exhibited specific gel mobility shifts after incubation with T. cruzi nuclear extracts, suggesting that a transcription factor binds this site. Finally, experiments designed to increase the level of CAT expression from the SL promoter suggest that it is not a strong promoter in cultured T. cruzi epimastigotes.

Animals↗
PubMed1997

Mutation (Q456H) is the most common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States.

Biotinidase deficiency is an autosomal recessive disorder that can result in neurologic and cutaneous symptoms if not treated with biotin supplementation. We have identified the most common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States. 1368A-->C results in a substitution of histidine for glutamine 456 (Q456H) in exon D of the biotinidase gene. This mutation was found in at least one allele in 14 unrelated children from 27 different families or 15 of 54 alleles studied (28%). This mutation was not identified in 41 normal adults using SSCA, nor was it found in 296 normal newborns using allele-specific oligonucleotide analysis, suggesting that this change is not a polymorphism. In addition, biochemical data from a child homozygous for Q456H suggest that the aberrant enzyme has very low biotinyl-hydrolase activity, lacks biotinyl-transferase activity, and is not recognized by antibody prepared to purified, normal human biotinidase. The ethnic backgrounds of the parents contributing the Q456H allele are varied but are generally northern European.

Acyltransferases↗
PubMed1997

Arg538 to Cys mutation in a CpG dinucleotide of the human biotinidase gene is the second most common cause of profound biotinidase deficiency in symptomatic children.

Biotinidase deficiency is an autosomal recessively inherited disorder in the recycling of the vitamin biotin. The most common mutation that causes profound biotinidase deficiency in symptomatic individuals is a deletion/insertion (G98:d7i3) that occurs in exon B of the biotinidase gene. We now report the second most common mutation, a C-to-T substitution (position 1612) in a CpG dinucleotide in exon D of the biotinidase gene. This mutation results in the substitution of a cysteine for arginine538 (designated R538C) and was found in 10 of 30 symptomatic children with profound biotinidase deficiency, 5 of whom also have the G98:d7i3 mutation. This mutation was not found in DNA samples from 32 individuals with normal biotinidase activity, but was found in one individual with enzyme activity in the heterozygous range. This mutation was not detected in 371 randomly selected, normal individuals using allele-specific oligonucleotide hybridization analysis. Aberrant biotinidase protein was not detectable in extracts of fibroblasts from a child who is homozygous for the R538C mutation, but was present in less than normal concentration in identical extracts treated with beta-mercaptoethanol. Because there is no detectable biotinidase protein in sera of children who are homozygous for the R538C mutation and in combination with the deletion/insertion mutation, the R538C mutation likely results in inappropriate intra- or intermolecular disulfide bond formation, more rapid degradation of the aberrant enzyme, and failure to secrete the residual aberrant enzyme from the cells into blood.

Acyltransferases↗