Biomedical subjects

G A Buck

Publications and source records attributed to G A Buck.

54 records · Page 3Linked to original sources

PubMed1990

In vitro capping in Trypanosoma cruzi identifies and shows specificity for the spliced leader RNA and U-RNAs.

Messenger RNA maturation in trypanosomes requires a trans-splicing event in which a capped 39 nucleotide leader sequence, the spliced leader (SL), from the 5' terminus of a small RNA (SL-RNA) is joined to the 5' termini of protein coding gene transcripts. We have developed nuclear extracts from Trypanosoma cruzi that label three small endogenous RNAs in the presence of [alpha-32P]GTP. Herein, we have characterized this labelling as 5' capping and shown that the capping activity exhibits an unusual ATP dependence. Moreover, partial sequence analysis identified the three cap-labelled RNAs as the T. cruzi SL-RNA, and two U-RNAs previously uncharacterized in T. cruzi, U2 and Ux. Finally, the capping reaction in the T. cruzi extracts showed apparent specificity for these RNAs--other endogenous or exogenous transcripts were not capped. The apparent specificity of this in vitro capping activity closely reflects the in vivo requirements; i.e., only the SL- and U-RNAs need to be capped since mature mRNAs are capped via trans-splicing. These observations are consistent with the hypothesis that one of the functions of trans-splicing is to supply 5' caps to mature trypanosome mRNAs.

Adenosine Triphosphate↗

PubMed1989

Characterization of the spliced leader genes and transcripts in Trypanosoma cruzi.

Trypanosome mRNA is processed to maturity in a novel trans-splicing reaction during which a 35-nucleotide (nt) spliced leader (SL) is joined to the 5' ends of most structural gene transcripts. We have examined this process in Trypanosoma cruzi, the causative agent of Chagas' disease in Central and South America. In this communication, we characterize the genes encoding the SL (SL gene) in five different strains of T. cruzi by hybridization analysis and show that the genome of each of these strains contains numerous tandemly repeated copies of the SL gene. We demonstrate that the SL genes show remarkable intrastrain homogeneity, but significant interstrain heterogeneity. We have cloned and sequenced one of the SL repeats from T. cruzi strain CL and used synthetic oligodeoxyribonucleotides designed to hybridize to SL gene transcripts in Northern analyses of T. cruzi RNA to identify an approx. 110-nt putative SL primary transcript (SL-RNA). The 5' end of the SL-RNA was mapped to the first nt of the SL by primer extension analyses. The sequence of the 110-nt SL-RNA was used to generate a predicted secondary structure, and this structure compared favorably to the predicted secondary structures of SL transcripts of other trypanosomatids.

Animals↗

PubMed1989

In vitro 3' end processing and poly(A) tailing of RNA in Trypanosoma cruzi.

Pre-mRNA in kinetoplastids is processed to maturity following unique pathways requiring a transplicing event that links a common 39 nucleotide leader to the 5' termini of the mature mRNAs. The mechanisms of this reaction and other steps of mRNA processing; i.e., 5' capping and 3' cleavage and polyadenylation, have not been resolved. Herein, we describe a 3' polyadenylation activity in cell-free extracts prepared from nuclei isolated from Trypanosoma cruzi, the kinetoplastid agent of Chagas' Disease. Synthetic RNA transcripts incubated in these extracts in the presence of ATP are 3' polyadenylated. This polyadenylation activity is sensitive to heat or pre-treatment of the extract with Micrococcal nuclease, suggesting that an RNA-protein complex is required. As these are characteristics of polyadenylation activities in other eukaryotes, we believe that this activity may participate in the in vivo trypanosome mRNA polyadenylation system. Several other modification activities specific for RNA 3' termini, including terminal nucleotide transferases, a tRNA CCA maturation activity, and a 3' exonuclease were also identified in these T. cruzi nuclear extracts.

Animals↗

PubMed1989

An electrophoretic karyotype and assignment of ribosomal genes to resolved chromosomes of Pneumocystis carinii.

Pulsed-field gel electrophoresis was used to generate a molecular karyotype of chromosomes from the opportunistic AIDS pathogen, Pneumocystis carinii. P. carinii cysts and trophozoites were isolated from immunosuppressed rats, lysed in situ in agarose blocks, and subjected to orthogonal-field gel electrophoresis (OFAGE) and contour-clamped homogeneous-field gel electrophoresis (CHEF). OFAGE and CHEF gels resolved, respectively, 16 or 20 chromosome bands ranging in size from 0.32-1.5 megabase pairs. Summation of the estimated sizes of these chromosomes suggested a total genome complexity for P. carinii of 8-16 megabase pairs. Homologous probes for the genes encoding the 18S, 5.8S, and 5S ribosomal RNAs were hybridized to filter blots of the pulsed-field gels to map these genes to specific P. carinii chromosomes.

Animals↗

PubMed1985

DNA relationships among some tox-bearing corynebacteriophages.

The DNA genomes of a number of tox-bearing, temperate corynebacteriophages isolated from strains of Corynebacterium diphtheriae and Corynebacterium ulcerans were compared. With one exception, these phages displayed similarities in their restriction enzyme digest profiles and extensive homology with prototypic beta converting phage. The exception, phage delta, had a unique restriction profile and exhibited homology with beta over a limited portion of its genome. DNAs of phages from each host contained cohesive ends and integrated as prophage by a mechanism analogous to that employed by coliphage lambda. It is proposed that these tox-bearing phages belong to a common family, the beta family. The role of the beta family in the movement of the tox gene between strains of C. diphtheriae and C. ulcerans is discussed.

Bacteriophages↗

PubMed1984

Re-expression of an inactivated variable surface glycoprotein gene in Trypanosoma equiperdum.

Variable surface glycoprotein (VSG) genes in African trypanosomes are often activated by the duplicative transposition of a silent basic copy (BC) gene into an unlinked telomerically located expression site, producing an active expression-linked copy (ELC) of that gene. However, some BC genes that are already linked to a telomere are activated without apparent duplication or transposition. We have recently shown that an active VSG ELC can be inactivated in situ, apparently without rearrangement. To explain these observations it has been suggested that VSG genes that are associated with chromosome telomeres are activated by chromosome end exchanges that occur at a considerable distance upstream from the genes themselves and place them cis to a unique VSG expression element. In an attempt to test this model we derived five VSG-1 expressing variants from BoTat-2, a VSG-2 expressing variant of Trypanosoma equiperdum which carries an inactive residual VSG-1 ELC (R-ELC) as well as the active VSG-2 ELC near unlinked chromosome telomeres. We examined the fates of the VSG-2 ELC and the VSG-1 R-ELC in these variants. All five had maintained the VSG-1 R-ELC; three in a reactivated form and two in an inactive state. The latter two variants carried new, active VSG-1 ELCs: one in the site that had previously contained the VSG-2 ELC and one in a previously unidentified site. The VSG-2 ELC was lost in all five of the variants. The results are not consistent with the simple chromosome end exchange model, which predicts that the VSG-2 ELC would be inactivated but not deleted when the VSG-1 R-ELC was reactivated.

Animals↗

PubMed1983

Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage beta.

A 1,942-base-pair DNA segment encoding the structural gene for diphtheria toxin was sequenced, and the primary structure of the toxin was deduced. Restriction enzyme fragments corresponding to nontoxic or hypotoxic peptides of the toxin were isolated from corynebacteriophage beta and cloned into Escherichia coli on plasmid pBR322, and the sequence was determined. The mature toxin molecule deduced from the sequence has 535 amino acid residues and a molecular weight of 58,342. The deduced sequence for the fragment A moiety was the same as that determined at the protein level, except for a single serine residue, which had been mispositioned in the earlier study. Several differences were noted with respect to the partial sequence data available on the fragment B moiety, some or all of which may reflect genetic variations among populations of corynephages carrying the toxin gene. The DNA sequence predicts a 25-residue leader peptide preceding the mature protein, which is presumably involved in secretion of the toxin from lysogenized Corynebacterium diphtheriae. We infer that initiation of translation probably occurs at a GTG codon (codon -25). Cloned restriction fragments containing sequences for the amino-terminal region of toxin, together with 5' flanking regions, were expressed in E. coli. Toxin-related peptides were synthesized and secreted into the periplasmic space. These results provide a basis for applying recombinant DNA methods to the study of diphtheria toxin and for producing novel, genetically altered forms of the toxin suited to the construction of new classes of immunotoxins.

Amino Acid Sequence↗

PubMed1982

Immunological cross-reactivity in the absence of DNA homology between Pseudomonas toxin A and diphtheria toxin.

The immunodominant determinant of Pseudomonas toxin A was shown to cross-react with a normally inaccessible determinant in fragment A of diphtheria toxin. Trypsin-treated diphtheria toxin and fragment A of diphtheria toxin inhibited binding of toxin A antibody to whole toxin A, whereas whole diphtheria toxin did not inhibit this reaction. However, even at the lowest stringency no hybridization was detected between diphtheria tox probe and Pseudomonas aeruginosa DNA.

ADP Ribose Transferases↗

PubMed1981

Physical mapping of beta-converting and gamma-nonconverting corynebacteriophage genomes.

Deoxyribonucleic acids (DNAs) from wild-type and mutant strains of beta-converting and gamma-nonconverting corynebacteriophages were isolated and physically characterized. The data obtained from DNA heteroduplexes, restriction enzyme banding profiles, and restriction maps reinforce the conclusion that beta and gamma phages are very closely related. The major physical differences seen in the DNA heteroduplexes are a small substitution bubble and one or two insertions which are present on the gamma phage genome. The insertions account for the differences in the genome sizes of beta and gamma phages, and with the substitution they are responsible for most of the differences in the restriction endonuclease profiles and maps of the corynebactriophage genomes, two special sites and the DNA fragments carrying them were identified. These were the cohesive (cos) sites and the specific attachment (attP) site of the vegetative phage genome. The behavior of these sites indicated that the transition of phage DNA from the vegetative to the prophage state involves the circularization of vegetative DNA through the cos sites and its integration into the bacterial chromosome via the attP site. The mechanism of corynebacteriophage integration was similar to that employed by Escherichia coli phage gamma. From the data assembled the physical and genetic maps of beta and gamma phage were oriented with respect to one another. The extensive similarity in their maps provides additional confirmation of a close evolutionary relationship.

Attachment Sites, Microbiological↗

PubMed1981

Genetic elements novel for Corynebacterium diphtheriae: specialized transducing elements and transposons.

It was shown in an accompanying paper (Buck and Groman, J. Bacteriol. 148: 131-142, 1981) that gamma-tsr-1 phage stocks produced by heat induction of lysogens are a mixture of two phages which differ in the content of their deoxyribonucleic acid (DNA). This difference is evidenced by the appearance of "heterogeneous" (HET) fragments in restriction enzyme digests of gamma-tsr-1 phage DNA. It was estimated that 20 to 80% of the phage in these lysates produced HET fragments. The appearance of HET fragments correlated with the appearance of a DNA insertion (DI-1) in the gamma phage genome as revealed in heteroduplexes of DNA from gamma-tsr-1 and beta corynebacteriophages. The HET fragments were seen in DNA from heat-induced lysates, but not in DNA from phage stocks produced by lytic infection. By DNA-DNA hybridization analysis it was shown that a fraction of gamma-tsr-1 phages from heat-induced lysates carried an insertion of bacterial DNA in the vegetative phage attachment site (attP), and that this insertion was responsible for the formation of HET fragments. Since the phage produced by this event carried a complete phage genome plus a small segment of bacterial DNA, they were called transducing elements. On the basis of these facts it was concluded that heat-induced gamma-tsr-1 prophage was excised at an abnormal site at a very high frequency. Abnormal excision was highly specific, and the change in excision specificity occurred simultaneously with the spontaneous mutation of the phage to heat inducibility. From this and other data it was postulated that a mutation in the immune repressor was reponsible for an alteration in the specificity of the normal excision process. This distinguishes the mechanism of formation of gamma-tsr-1 transducing elements from that employed by other phages. A second DNA insertion (DI-2) in the tox (diphtheria toxin) gene of gamma-tsr-1 and gamma-tsr-2 was also identified as an insertion of bacterial DNA. The DI-2 insertion had a stem-and-loop structure similar to that seen in heteroduplexes visualizing transposons or insertion elements. It seems likely that gamma wild-type phage, which is mutant for tox, was originally tox(+), but that transposition of bacterial DNA into the gene inactivated it.

Attachment Sites, Microbiological↗

PubMed1981

Identification of deoxyribonucleic acid restriction fragments of beta-converting corynebacteriophages that carry the gene for diphtheria toxin.

Deoxyribonucleic acid fragments bearing the gene for diphtheria toxin have been identified in restriction enzyme digests of deoxyribonucleic acids from beta-converting and gamma-nonconverting corynebacteriophages. A combination of physical and genetic evidence has established that the Bam HI band C fragment of beta phage deoxyribonucleic acid, which carries the specific phage attachment site (Buck and Groman, J. Bacteriol. 148:131-142, 1981), also carries most, and probably all, of the gene for diphtheria toxin. A detailed restriction map of this tox-bearing Bam HI fragment has been developed, and the locations and orientation of the tox gene and the attP site within this fragment have been established.

Attachment Sites, Microbiological↗

PubMedDate not supplied

Stability of expression-linked surface antigen gene in Trypanosoma equiperdum.

African trypanosomes evade clearance in immune-competent hosts by periodically replacing their major surface glycoprotein with an antigenically different glycoprotein. Expression of many of these variant surface glycoproteins (VSGs) is associated with the duplication and transposition of silent basic copy genes (BCs) into unlinked genomic expression sites. The new expression-linked VSG gene copies (ELCs) are oriented with their 3' ends proximal to chromosome telomeres. Other VSG genes are activated without the production of an ELC. The 3' ends of these VSG genes are near chromosome telomeres both when they are active and when they are inactive. Recently, we have shown that activation of the VSG-1 gene in the BoTaR (Bordeaux trypanozoon antigen repertoire) serodeme of Trypanosoma equiperdum involves the duplication and transposition of a telomeric BC gene into one of at least three unlinked telomeric sites. Here we show that the VSG-1 ELC is inactivated but not eliminated in some antigenic variants derived from a VSG-1 expressor. In addition, a subsequent variant that again expresses VSG-1 has not reactivated the residual VSG-1 ELC (R-ELC), but instead contains a new, active VSG-1 ELC in an unlinked telomeric site. These results show that the simple presence of an ELC in a potential expression site is not sufficient for its expression.

Animals↗

PubMedDate not supplied

Molecular karyotyping of multiple yeast species isolated from nine patients with AIDS during prolonged fluconazole therapy.

Variations in molecular karyotype and fluconazole susceptibility of serial yeast isolates from the oral cavities of nine patients with AIDS receiving fluconazole for single or multiple episodes of oropharyngeal candidiasis were monitored. Multiple yeast species were isolated from the initial oral specimens in six patients. Molecular karyotyping identified at least eight different DNA subtypes of C. albicans, at least eight of T. glabrata and only one DNA subtype each of C. krusei, C. tropicalis and C. parapsilosis. Among isolates of T. glabrata, fluconazole MICs in each patient were consistently within one or two dilutions, regardless of strain variations. Similarly, among five patients monitored during one course of therapy, the MICs of fluconazole of C. albicans isolates of either the same or different DNA subtypes remained within two dilutions. However, increases in MICs of fluconazole of C. albicans were observed in four patients who received two or more courses of fluconazole, three of whom had the same DNA subtype and one of whom changed from one DNA subtype to another.

Acquired Immunodeficiency Syndrome↗

PubMedDate not supplied

An arrayed bacteriophage P1 genomic library of Pneumocystis carinii.

We have constructed an arrayed, large insert, multiple coverage genomic library of Pneumocystis carinii DNA using the bacteriophage P1 cloning system. The library consists of approximately 4800 independent clones with an average insert size of approximately 55 kbp individually arrayed in 50 microtiter plates, and is readily screened on ten or fewer microtiter plate-sized filters using a high density colony replicating device. Screening of the library for unique P. carinii sequences detected an average of 4-5 positive clones for each, consistent with a several-fold coverage of the approximately 10-mbp P. carinii genome. Restriction and hybridization analyses demonstrated that the P1 clones in this library are quite stable and contain few, if any, chimeric inserts. Thus, this arrayed, large insert library of P. carinii genomic DNA will be a valuable tool in the future genetic dissection of this important pathogen.

Animals↗

PubMedDate not supplied

The HSP70 gene family in Pneumocystis carinii: molecular and phylogenetic characterization of cytoplasmic members.

Pneumocystis carinii, a major opportunistic lung pathogen of AIDS patients, is found in a number of mammals and is proposed to be a member of the fungi. In this work, several members of the highly conserved HSP70 multigene family were characterized from rat-derived P. carinii. Previously, we reported characterization of the ER resident HSP70 homolog known as BiP from prototype (P.c. carinii) and variant (P. c. rattus) strains of the organism. We report here, from P. c. carinii, characterization of Pcsa1, an HSP70 homolog that encodes a cognate/stress-induced HSP70 homolog of the SSA subfamily in Saccharomyces cerevisiae. We also identify, from both rat strains and from a human isolate of P. carinii (P.c. hominis), a third set of HSP70 homologs that apparently encode a ribosome-associated cytoplasmic HSP70 homologous to the S. cerevisiae SSB subfamily. Our data indicate that Pcsal mRNA, like Pcbip mRNA, bears an intron in the 5' untranslated region, is induced by heat shock, and suggest that this gene undergoes alternative transcription and splicing. The SSB homologs display significant sequence heterogeneity between P. carinii source strains, supporting the genetic divergence and likely speciation of P. carinii isolates within and between host species. Phylogenetic analysis with the PcSA1 protein supports inclusion of P. carinii among the higher fungi.

Alternative Splicing↗

PubMedDate not supplied

Polymorphisms at the topoisomerase II gene locus provide more evidence for the partition of Trypanosoma cruzi into two major groups.

We have dissected the topoisomerase II gene of members of the two recently characterized subgroups of Trypanosoma cruzi to obtain further evidence to support this dichotomy of isolates in this important parasite. Pulsed field gel electrophoresis showed a striking heterogeneity in the molecular karyotypes of the strains analyzed. Southern analysis of these chromosome gels also showed heterogeneity in the size and number of chromosomes containing the topoisomerase II gene. Analysis of DNA restriction fragment length polymorphisms of the topoisomerase II gene also showed two principal patterns consistent with the two previously characterized groups. Finally, the sequences of portions of the topoisomerase II genes from members of the T. cruzi groups showed two distinct patterns, again consistent with the previous grouping of this parasite. Thus, this work clearly supports previous observations suggesting an ancient divergence of known T. cruzi isolates into two main branches.

Animals↗

PubMedDate not supplied

Transfusion-transmitted virus (TTV) in Brazil. Preliminary report.

TTV is a recently discovered DNA virus, isolated from a patient with post-transfusion hepatitis of unknown etiology by Japanese researchers. In the present study, we evaluated the presence of TTV among chronic liver diseases patients in São Paulo and Pará states, representing two geographically distinct Brazilian regions. TTV DNA was found in 21/105 (20%) and 9/20 (45%) cases from São Paulo and Pará States, respectively. DNA sequence data confirmed the presence of TTV genotypes 1a and 2a, as well as other genotypes not yet described. In conclusion, TTV is present in chronic liver diseases cases from Southeast and North Brazil. However, further studies involving healthy populations are necessary before establishing any causal relationship among TTV and human hepatitis.

Brazil↗

PubMedDate not supplied

Colony polymerase chain reaction of stably transfected trypanosoma cruzi grown on solid medium.

Tools for the genetic manipulation of Trypanosoma cruzi are largely unavailable, although several vectors for transfection of epimastigotes and expression of foreign or recombinant genes have been developed. We have previously constructed several plasmid vectors in which recombinant genes are expressed in T. cruzi using the rRNA promoter. In this report, we demonstrate that one of these vectors can simultaneously mediate expression of neomycin phosphotransferase and green fluorescent protein when used to stably transfect cultured epimastigotes. These stably transfected epimastigotes can be selected and cloned as unique colonies on solid medium. We describe a simple colony PCR approach to the screening of these T. cruzi colonies for relevant genes. Thus, the methodologies outlined herein provide important new tools for the genetic dissection of this important parasite.

Animals↗