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Biomedical subjects

G A Buck

Publications and source records attributed to G A Buck.

At least 37 records · Page 2Linked to original sources
PubMed1997

Trypanosoma cruzi strains partition into two groups based on the structure and function of the spliced leader RNA and rRNA gene promoters.

We have previously identified a major proximal sequence element (PSE) responsible for transcription of the spliced leader (SL) gene from Trypanosoma cruzi strain CL, and showed that the sequence encompassing this PSE exhibits approximately 30% divergence between two major groups of T. cruzi isolates, but strong conservation within the groups. In this report, we show that the SL RNA gene promoter from the CL strain (group I) is efficiently expressed only in T. cruzi isolates from group I. Similarly, the sequence of the approximately 643 bp promoter region of the T. cruzi rRNA is strongly conserved within, but diverged approximately 20% between, the two groups. Reporter constructs driven by the rRNA promoter sequences from group I strains are strongly expressed after electroporation into other group I strains, but not expressed in group II strains. In contrast, constructs bearing rRNA promoter sequences from group II strains are active in strains from both groups. Phylogenetic analyses performed with both the rRNA and the SL RNA gene promoter sequences yielded similar trees, and these trees strongly reinforce the partitioning of known T. cruzi into two major groups that parallel the observed functional specificity of the promoters. Given the well-documented species specific pattern of both rRNA promoters and PSEs in higher eukaryotes, these results suggest an ancient evolutionary divergence among organisms currently classified as T. cruzi.

Animals↗
PubMed1997

Profound biotinidase deficiency caused by a point mutation that creates a downstream cryptic 3' splice acceptor site within an exon of the human biotinidase gene.

Biotinidase recycles the vitamin biotin from biocytin upon the degradation of the biotin-dependent carboxylases. We have identified a novel point mutation within the biotinidase gene that encodes the signal peptide in two unrelated individuals with profound biotinidase deficiency. Sequence analysis of genomic DNA from these individuals revealed a G to A transition (G100-->A) located 57 bases downstream of the authentic splice acceptor site in exon B. Although this mutation predicts a G34S substitution, it also generates a 3' splice acceptor site. Sequence of the PCR-amplified cDNA from the homozygous child revealed that all the product was shorter than that of normal individuals and was the result of aberrant splicing. The aberrantly spliced transcript lacked 57 bases, including a second in-frame ATG, that encode most of the putative signal peptide and results in an in-frame deletion of 19 amino acids. The mutation results in failure to secrete the aberrant protein into the blood. This is the first reported example in which a point mutation creates a cryptic 3' splice acceptor site motif that is used preferentially over the upstream authentic splice site. The preferential usage of the downstream splice site is not consistent with the 5'-3' scanning model, but is consistent with the exon definition model of RNA splicing.

Amidohydrolases↗
PubMed1997

Mutations in the human biotinidase gene that cause profound biotinidase deficiency in symptomatic children: molecular, biochemical, and clinical analysis.

Biotinidase deficiency is an autosomal recessively inherited disorder that results in the inability to recycle the vitamin biotin. The disorder can cause neurologic and cutaneous abnormalities that can be treated effectively with pharmacologic doses of biotin. We identified 21 mutations that cause profound biotinidase deficiency in 37 symptomatic children (30 different probands and 7 siblings), as well as provide relevant biochemical and clinical information for each child. The two most common mutations (G98:d7i3 and R538C) were found in 31 of 60 alleles (52%), whereas the remainder of the alleles are accounted for by the 19 other unique mutations. Serum samples were available from 18 children, of these 11 had no detectable cross-reacting material (CRM) to antibody prepared against normal human serum biotinidase, three had reduced quantities of CRM and four had normal quantities of CRM in serum. All of these mutations result in complete absence of biotinyl-transferase activity in serum. Two polymorphisms were also identified in normal individuals. It is apparent that a child who inherits any of these mutations, either in the homozygous state or in combination, can develop the clinical features of the disorder if untreated. There are, however, no clear genotype/phenotype correlations that would allow for the prediction of the type, severity, or age of onset of symptoms.

Acyltransferases↗
PubMed1996

Trypanosomatidae: a spliced-leader-derived probe specific for the genus Phytomonas.

We probed DNA from all trypanosomatid genera by slot blot hybridization with an oligonucleotide (SL3') complementary to a sequence of the Phytomonas spliced-leader or mini-exon RNA. The 19-nucleotide probe target site was previously shown to be highly conserved among a limited number of Phytomonas isolates, but diverges in other kinetoplastid genera. Our examination of 84 isolates of various genera of trypanosomatids showed hybridization of this probe exclusively with isolates from plants or insects which could, by morphological, biochemical, and molecular criteria, be considered to belong to the genus Phytomonas. In contrast, no hybridization was observed with flagellates of the genera Blastocrithidia, Crithidia, Endotrypanum, Herpetomonas, Leptomonas, Leishmania, and Trypanosoma. The method detected DNA quantities as low as 50 ng using either radioactive or nonradioactive probes, and was effective with as few as 10(4) intact flagellates. Together, these results suggest that this probe will serve as a convenient marker for taxonomic and epidemiological studies requiring reliable identification of Phytomonas spp. in plants or in putative insect vectors.

Animals↗
PubMed1996

Deletion/insertion mutation that causes biotinidase deficiency may result from the formation of a quasipalindromic structure.

Biotinidase is responsible for recycling the vitamin biotin from biocytin that is formed after the proteolytic degradation of the biotin-dependent carboxylases. We have identified a deletion/insertion mutation within exon D of the human biotinidase gene in a child with biotinidase deficiency. The mutation causes a frame shift and premature termination which are predicted to result in a truncated protein. We propose that the mutation occurred during DNA replication by either of two mechanisms. Both mechanisms involve formation of a quasipalindromic hairpin loop in the template and dissociation of DNA polymerase alpha. This mutation supports the formation of palindromic structures as a possible cause of deletions in eukaryotes, and supports the proposal, derived from in vitro studies, that polymerase alpha may preferentially arrest or dissociate at specific template sequences.

Amidohydrolases↗
PubMed1996

Identification, characterization, and expression of the BiP endoplasmic reticulum resident chaperonins in Pneumocystis carinii.

We have isolated, characterized, and examined the expression of the genes encoding BiP endoplasmic reticulum (ER) resident chaperonins responsible for transport, maturation, and proper folding of membrane and secreted proteins from two divergent strains of Pneumocystis carinii. The BiP genes, Pcbip and Prbip, from the P. c. carinii (prototype) strain and the P. c. rattus (variant) strain, respectively, are single-copy genes that reside on chromosomes of approximately 330 and approximately 350 kbp. Both genes encode approximately 72.5-kDa proteins that are most homologous to BiP genes from other organisms and exhibit the amino-terminal signal peptides and carboxyl-terminal ER retention sequences that are hallmarks of BiP proteins. We established short-term P. carinii cultures to examine expression and induction of Pcbip in response to heat shock, glucose starvation, inhibition of protein transport or N-linked glycosylation, and other conditions known to affect proper transport, glycosylation, and maturation of membrane and secreted proteins. These studies indicated that Pcbip mRNA is constitutively expressed but induced under conditions known to induce BiP expression in other organisms. In contrast to mammalian BiP genes but like other fungal BiP genes, P. carinii BiP mRNA levels are induced by heat shock. Finally, the Prbip and Pcbip coding sequences surprisingly exhibit only approximately 83% DNA and approximately 90% amino acid sequence identity and show only limited conservation in noncoding flanking and intron sequences. Analyses of the P. carinii BiP gene sequences support inclusion of P. carinii among the fungi but suggest a large divergence and possible speciation among P. carinii strains infecting a given host.

Amino Acid Sequence↗
PubMed1996

Multi-facility survey of oligonucleotide synthesis and an examination of the performance of unpurified primers in automated DNA sequencing.

The purity of 208 crude synthetic 25- and 50-base oligonucleotides synthesized in 71 DNA core facilities was assessed by capillary electrophoresis (CE), and the average coupling efficiency of each synthesis was determined. The median average coupling efficiencies of the 25-mers and 50-mers were 98.9% and 98.7%, respectively, and 85% of the samples exceeded the minimum industry standard of 98% average coupling efficiency. The overall yields estimated by on-line trityl monitors showed poor agreement with the empirically determined yield, and accuracy of the monitors decreased as synthesis efficiency decreased. The performance of the unpurified 25-base oligonucleotides, ranging in purity from 14% to 94%, as primers for automated DNA sequencing was evaluated. Over 85% of these oligonucleotides exhibited an unedited sequencing accuracy of > 97.5% over the 400-base test sequence. Surprisingly, sequencing performance was not strictly related to primer purity, though a marked loss of performance was observed for primers < or = 70% pure (< or = 98.5% coupling efficiency). Thus, the vast majority of the oligonucleotides synthesized by the 71 core facilities participating in this study were of high quality and performed well as sequencing primers without post-synthesis purification or desalting. Finally, our results suggest that an increase in the standard minimum performance specifications of DNA synthesis instruments and reagents from > or = 98% to > or = 98.5% average coupling efficiency, or the development of rapid, inexpensive and efficient methods to detect syntheses below the 98.5% threshold, could obviate post synthesis purification of sequencing primers.

DNA Primers↗
PubMed1995

Transient expression mediated by the Trypanosoma cruzi rRNA promoter.

Plasmid constructs containing a putative Trypanosoma cruzi rRNA promoter and transcription start point upstream from the bacterial chloramphenicol acetyltransferase (CAT) reporter gene were transfected into cultured T. cruzi epimastigotes to verify the presence of a promoter activity. Constructs bearing the putative promoter and a 3' trans-splicing acceptor site in the proper orientation yielded approx. two orders of magnitude greater CAT expression than that previously observed with the T. cruzi spliced leader (SL) gene promoter. In contrast, similar constructs lacking the known 3' splice site yielded reduced but readily measurable expression suggesting that sequences near the promoter may function as cryptic 3' splice sites. A repeated sequence upstream from the putative basal rRNA promoter in a position analogous to rRNA gene enhancer elements in other eukaryotes did not enhance expression from the T. cruzi rRNA promoter. Finally, these constructs were functional in some but not all T. cruzi isolates, and were inactive in other kinetoplastid species, suggesting that the T. cruzi rRNA promoter may have a limited host range.

Animals↗
PubMed1995

Mutational hotspot in the human biotinidase gene causes profound biotinidase deficiency.

Biotinidase deficiency is an autosomal recessive inherited disorder that is characterized by neurological and cutaneous symptoms. Biotinidase-deficient children cannot recycle endogenous biotin, an essential water-soluble B vitamin. Biotin is covalently attached to epsilon-amino groups of lysyl residues of four carboxylases. These carboxylases are subsequently degraded to biocytin (biotin-epsilon-lysine). Biotinidase cleaves biocytin to biotin and lysine, thereby completing the biotin cycle. The symptoms of biotinidase deficiency can be resolved or prevented by treatment with biotin. Therefore, it is important that biotinidase deficiency is diagnosed early so that permanent neurological damage can be prevented. Many states and countries currently perform newborn screening for biotinidase deficiency. We have recently isolated and characterized the cDNA for normal human biotinidase and localized the gene to chromosome 3p25 (ref. 9). We have now identified the first mutation that causes profound biotinidase deficiency. It occurs in a distinct region of the gene that encodes the putative signal peptide. Fifty percent of symptomatic children studied have a 7-bp deletion coupled with a 3-bp insertion in at least one of their alleles of the biotinidase gene. This mutation appears to be a common cause of biotinidase deficiency in symptomatic children.

Alleles↗
PubMed1995

Accuracy of automated DNA sequencing: a multi-laboratory comparison of sequencing results.

A double-stranded (ds)DNA template of "unknown" sequence was distributed to approximately 80 core DNA sequencing laboratories by the Association of Biomolecular Resource Facilities (ABRF) for automated DNA sequence analysis. Forty-four different facilities responded with 83 usable sequence submissions. These sequences were grouped by both sequencing protocol (dye-primer or dye-terminator) and whether manually edited or not. The sequences were aligned with the known sequence, and the number of correct base calls, insertions, deletions, no-calls and miscalls were determined for each group. The dye-primer sequencing protocol provided the longest and most accurate sequence. The edited dye-primer data were > 95% accurate out to 400-450 bp, while the edited dye-terminator data could call only 300-350 bases at this accuracy. However, 75% of the laboratories in this sampling preferred the dye-terminator protocol, presumably because of its versatility and convenience. Laboratories that manually edited the automatically called data were able to obtain an additional 100 bases of good sequence when the dye-primer protocol was used. Surprisingly though, editing of dye-terminator results did not increase the amount of good sequence, although the dye-terminator protocol had a superior base-calling ability within the first 100 bases of called sequence.

Autoanalysis↗
PubMed1994

Human serum biotinidase. cDNA cloning, sequence, and characterization.

Biotinidase (EC 3.5.1.12) catalyzes the hydrolysis of biocytin, the product of biotin-dependent carboxylase degradation, to biotin and lysine. Biotinidase deficiency is an inherited metabolic disorder of biotin recycling that is characterized by neurological and cutaneous abnormalities, and can be successfully treated with biotin supplementation. Sequences of tryptic peptides of the purified human serum enzyme were used to design oligonucleotide primers for polymerase chain reaction amplification from human hepatic total RNA to generate putative biotinidase cDNA fragments. Sequence analysis of a cDNA isolated from a human liver library by plaque hybridization with the largest cDNA probe revealed an open reading frame of 1629 bases encoding a protein of 543 amino acid residues, including 41 amino acids of a potential signal peptide. Comparison of the open reading frame with the known biotinidase tryptic peptides and recognition of the expressed protein encoded by this cDNA by monoclonal antibodies prepared against purified biotinidase demonstrated the identity of this cDNA. Southern analyses suggested that biotinidase is a single copy gene and revealed that human cDNA probes hybridized to genomic DNA from mammals, but not from chicken or yeast. Northern analysis indicated the presence of biotinidase mRNA in human heart, brain, placenta, liver, lung, skeletal muscle, kidney, and pancreas.

Amidohydrolases↗
PubMed1994

A survey of nucleic acid services in core laboratories.

Core facility services related to DNA synthesis and sequencing were surveyed by the Association of Biomolecular Resource Facilities. Responses from 85 facilities offering DNA synthesis and 37 facilities offering DNA sequencing were obtained. Data on instrumentation, volume, number of users, cost, methodology and a number of other criteria were obtained. The volume of work performed by these centralized core facilities was quite substantial (combined synthesis output of 4 million bases per year and a combined sequencing output of 35 million bases per year). The large number of users supported by these facilities and the high sample throughput make these core resource facilities good indicators of technological trends.

Costs and Cost Analysis↗
PubMed1993

Optimization of coupled PCR amplification and cycle sequencing of cloned and genomic DNA.

We describe optimization of a coupled amplification and cycle sequencing (CAS) method for rapid characterization of cloned or genomic DNA. Our modification of this method, termed coupled PCR amplification and cycle sequencing (CPACS), utilizes commercially available reagents, does not require template purification and produces high-quality sequence ladders from nanogram quantities of complex genomic DNA. The reactions have been streamlined to permit automation. Finally, we show that the technique can be applied more efficiently in conjunction with the AutoTrans 350 Direct Transfer Electrophoresis System and 33P-labeled sequencing primers.

Autoanalysis↗
PubMed1992

Characterization of the rRNA-encoding genes and transcripts, and a group-I self-splicing intron in Pneumocystis carinii.

Although Pneumocystis carinii is the most common opportunistic pathogen infecting individuals with AIDS, very little is known of the basic biology of the organism. We have examined the ribosomal RNA (rRNA) and the DNA encoding it (rDNA) in P. carinii in an attempt to clarify its taxonomic position and to begin to study its genetic processes. Electrophoretic analysis showed that the sizes of the P. carinii rRNAs are quite similar to the sizes of the corresponding rRNAs from Saccharomyces cerevisiae. Direct sequence analysis of approx. 60% of the 18S small subunit-rRNA (Ss-rRNA) confirmed that its sequence is similar to that of yeast-like fungi and that a putative group-I intron previously observed in the 18S rDNA is, in fact, excised from the mature rRNA. PCR analysis of the intron in P. carinii genomic DNA showed that each of the multiple rDNA genes bears the group-I intron and in vitro transcripts of the intron autocatalytically excise from the rRNA primary transcript in the presence of GTP. Finally, analogues of GTP inhibit the self-splicing reaction, indicating that the guanosine-binding site of the intron closely resembles that of other well-characterized group-I introns. Since no group-I introns have been found in higher eukaryotes, this self-splicing process represents a viable target for chemotherapy of P. carinii pneumonia (PCP).

Antifungal Agents↗
PubMed1991

RNA-protein complexes mediate in vitro capping of the spliced-leader primary transcript and U-RNAs in Trypanosoma cruzi.

A 39-nucleotide spliced leader (SL) is joined to the 5' ends of trypanosome mRNAs in a bimolecular or trans-splicing process. The SL in Trypanosoma cruzi is transcribed as an approximately 110-nucleotide RNA (SL-RNA or SL primary transcript) bearing the 39-nucleotide SL at the 5' end. The SL-RNA is 5' capped by a guanylyltransferase activity prior to trans-splicing and trypanosome mRNAs thus obtain their mature caps from the SL by trans-splicing. We have previously characterized a guanylyltransferase activity from T. cruzi nuclear extracts and shown that this capping activity has an unusual ATP dependence and an apparent specificity for the SL-RNA and U-RNAs. Herein, we show that the capping activity sediments as a 12-15S particle during velocity sedimentation in glycerol gradients and fractionates as a greater than 150-kDa particle during large-pore gel filtration chromatography. Moreover, the endogenous substrate RNAs--the SL-RNA and U-RNAs--consistently copurify with the capping activity, suggesting that the activity and the substrates form a ribonucleoprotein particle. The capping activity and substrate RNAs are not dissociated in isopycnic Cs2SO4 gradients and band at a density expected for an RNA-protein complex, confirming the existence of ribonucleoprotein particles bearing both the activity and its substrate RNAs. Finally, we partially purified these ribonucleoprotein particles and showed that the capping activity remains ATP dependent and highly specific for the SL-RNA and the U-RNAs. These observations are consistent with the hypothesis that one of the functions of trans-splicing is for mRNA capping.

Animals↗
PubMed1991

Expression of an exogenous gene in Trypanosoma cruzi epimastigotes.

We have constructed a plasmid vector system into which a segment of the Trypanosoma cruzi gene encoding the spliced leader (SL) has been inserted to drive expression of a downstream gene encoding bacterial chloramphenicol acetyltransferase (CAT). We used this construct to establish conditions that permit reproducible transfection of cultured T. cruzi epimastigotes where transfection was mediated by electroporation and measured by assaying expression of CAT. CAT activity was detected only in T. cruzi lysates from cells transfected with constructs containing a properly oriented SL gene; constructs in which the gene was inserted in reverse orientation did not express CAT. The optimal electric field strength, cell density and DNA concentration for efficient transfection were established. This and similar systems will permit genetic dissection of this parasite.

Animals↗