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D F Wirth

Publications and source records attributed to D F Wirth.

At least 91 records · Page 5Linked to original sources
PubMed1986

Chromosome location of four genes in Leishmania.

Chromosome-sized DNA molecules from Leishmania isolates (L. mexicana amazonensis, L. mexicana mexicana, L. chagasi, L. major, L. donovani, and L. braziliensis) were separated by orthogonal field alternation gel electrophoresis. The chromosome locations of four genes were mapped. The alpha-tubulin and rRNA genes each mapped to a single chromosome size class. The beta-tubulin and the 5'-spliced-leader-sequence genes were found on more than one chromosome size class and showed variation of hybridization profiles across species.

Animals↗
PubMed1986

Characterization of a ribosomal DNA clone of Brugia malayi.

The structure of ribosomal DNA (rDNA) clone pBmr7 from microfilariae of the human parasite Brugia malayi has been examined in detail by Southern blot analysis and S-1 mapping techniques. The results demonstrate that this clone contains regions homologous to 28S, 18S and 5.8S rDNAs. A noncoding or 'spacer' region lies between the 3' end of 28S rDNA and the 5' end of 18S rDNA. An AccI-Sau3AI fragment of approximately 900 bp from this spacer region cross-hybridizes to genomic DNA fragments of different sizes from Brugia pahangi and Dirofilaria immitis. The differences observed in hybridization suggest that this rDNA fragment can be used to differentiate between various filariid species.

Animals↗
PubMed1986

A DNA probe cloned in Escherichia coli for the identification of Brugia malayi.

This report describes a specific and sensitive DNA probe for the identification of Brugia malayi. A genomic DNA library produced from subperiodic B. malayi microfilariae was screened to detect clones containing DNA sequences which are highly repeated within the parasite genome. Several clones were further analyzed to identify those which hybridize specifically with B. malayi DNA but not with DNA from B. pahangi and Dirofilaria immitis. From these, clone pBm15 was selected because it hybridized with high sensitivity to B. malayi DNA as detected by autoradiography. Clone pBm15 was sensitive enough to detect two infective larvae or five microfilariae or 300 pg of purified B. malayi microfilarial DNA. This study forms the basis for the development of a specific and sensitive DNA probe for the identification of B. malayi in field specimens.

Animals↗
PubMed1986

Transcriptional mapping of Leishmania enrietti tubulin mRNAs.

We have mapped the mRNAs for alpha- and beta-tubulins of Leishmania enriettii promastigotes and amastigotes and have demonstrated that each RNA contains a 35 nucleotide sequence on its 5' end which is not encoded contiguously with the rest of the message. Sequencing of the 5' end of the alpha- and beta-tubulin mRNAs revealed that this 35 nucleotide leader sequence is identical in both messages and that it is homologous to the spliced leader found on the 5' end of mRNAs in the related parasite Trypanosoma brucei. Additionally, we have sequenced regions of the alpha- and beta-tubulin genomic clones upstream from the mRNA encoding regions and have shown that the leader sequence is not encoded within these regions of DNA. Hence, Leishmania tubulin mRNAs may be synthesized by a discontinuous transcription process that links together the transcription products of two separate gene families, as suggested previously by others for the assembly of T. brucei mRNAs. Homologies between the Leishmania alpha- and beta-tubulin genes themselves and between these genes and the T. brucei alpha- and beta-tubulin genes have revealed sequences which may be important in synthesis or processing of Leishmania tubulin mRNAs.

Animals↗
PubMed1986

Identification of visceral Leishmania species with cloned sequences of kinetoplast DNA.

Several efforts have been made in order to develop more precise and sensitive methods in the identification of Leishmania parasites. We report here the identification of cloned subfragments of minicircle kinetoplast DNA (kDNA) isolated from L. donovani, WR352, which show different taxonomic specificities. Analysis of these fragments demonstrates a significant sequence diversity within the kDNA minicircle. For example, one cloned fragment was found to be present in all visceral Leishmania species tested, but was not present in any of the cutaneous Leishmania species. Another cloned fragment was only found in the strain from which it had been derived, and was not present in any of the other strains tested. In similar experiments with the New World visceral leishmanias (L. chagasi, WR518) several different cloned kDNA fragments were found to react with all of isolates of the L. chagasi tested, but not with any cutaneous Leishmania species, either from the Old World or the New World. It is of interest to note that these cloned L. chagasi kDNA fragments reacted with isolates of African visceral Leishmania species but not with isolates from India.

Animals↗
PubMed1986

Identification of Brugia malayi in vectors with a species-specific DNA probe.

We evaluated the potential value of a cloned sequence of genomic DNA of Brugia malayi as a species-specific probe. Clone pBm 15 reacted with all stages of 8 different geographic isolates of B. malayi and cross-hybridized with microfilariae of B. timori. It did not hybridize with Wuchereria bancrofti or with B. pahangi, W. kalimantani, Dirofilaria repens, Breinlia booliati or Cardiofilaria species, animal filariids that can be sympatric with B. malayi. P32-labeled clone pBm 15 correctly identified mosquitoes infected even with 1 infective larva of B. malayi. This specific DNA probe should be an invaluable tool to monitor control programs of Brugian filariasis.

Aedes↗
PubMed1985

Structure of mRNA encoded by tubulin genes in Leishmania enriettii.

Both the alpha- and beta-tubulin genes of Leishmania enriettii are encoded by mRNA of 2.0-2.2 kb in length. We have shown previously that the alpha- and beta-tubulin genes are arranged in separate, tandem repeats of 2 and 4 kb, respectively, and now report the mapping of mature mRNA onto these cloned genes. Here we show that the alpha-tubulin gene contains a very short intergenic region (100-200 bases) whereas the larger, tandemly repeated beta-tubulin gene contains a 1.8-2.0 kb region not found in mature mRNA. Comparison of S-1 mapping and primer extension results indicates that the messenger RNAs for both alpha- and beta-tubulin contain a sequence of about 35 base pairs located at the 5' end that is not encoded contiguously with the rest of the mRNA. This short 5' sequence may be added to the body of the tubulin mRNAs either through splicing of a precursor RNA molecule or by a novel post-transcriptional processing reaction. The alpha- and beta-tubulin genes are arranged identically in the two developmental stages of the parasite life cycle and are present in equal copy number.

Animals↗
PubMed1985

The primary structure of the rRNA insertions of Plasmodium lophurae.

The DNA sequences of the novel insertion in the 17s rRNA gene and the large insertion in the 25s rRNA gene in the cloned rDNA unit of the avian malaria parasite Plasmodium lophurae are presented, together with a partial sequence of the flanking regions, which code for the mature rRNA. The homology of the mature rRNA coding regions with the rRNA sequences of other eukaryotic organisms is extensive, indicating that the plasmodium rRNA is structurally similar to other eukaryotes. Sequence data also reveal that the region 3' to the insertion in the 17s rRNA contains a second small inserted DNA sequence, in contrast to other known small rRNA sequences. The region containing the 25s insertion shares sequence homology and some secondary structure characteristics with the terminal direct repeat of the Drosophila melanogaster transposable element copia. This is the first such sequence described in plasmodia. The direction of transcription of the cloned rDNA unit of P. lophurae has also been determined. As in other organisms, the direction of transcription is found to be 5' 17s-25s 3'.

Animals↗
PubMed1984

Primary structure of the Streptomyces enzyme endo-beta-N-acetylglucosaminidase H.

We report the DNA and primary amino acid sequences of the Streptomyces plicatus enzyme endo-beta-N-acetylglucosaminidase H. Peptide sequence information was derived from enzyme isolated from Streptomyces culture medium using a combination of mass spectrometric methods and conventional techniques, including Edman degradation and carboxypeptidase Y digestion. The DNA sequence was determined by analysis of the Endo-beta-N-acetylglucosaminidase H gene cloned into the Escherichia coli plasmid pBR322 (Robbins, P. W., Wirth , D. F., and Hering , C. (1981) J. Biol. Chem. 256, 10640-10644). The enzyme from Streptomyces medium is 271 (or 269) amino acids in length and has a ragged NH2-terminal sequence beginning primarily with Ala-Pro-Val or Ala-Pro-Ala-Pro-Val. DNA resection experiments as well as the DNA sequence itself suggest that a proenzyme or, more probably, " prepro " enzyme may be the primary product of translation. The long 42 (or 44) residue leader sequence of the preproenzyme shows striking similarities to leader sequences found on proteins secreted by Bacillus species. The leader sequence is partially removed by E. coli and, as reported previously, endo-beta-N-acetylglucosaminidase H made in E. coli appears in both the periplasmic space and in the cell.

Acetylglucosaminidase↗
PubMed1983

The avian malaria Plasmodium lophurae has a small number of heterogeneous ribosomal RNA genes.

The structure and number of the ribosomal RNA (rRNA) genes of the avian malaria parasite Plasmodium lophurae has been examined using Southern blot analysis and recombinant DNA techniques. The ribosomal DNA (rDNA) of P. lophurae has been cloned into the plasmid pBR322, beginning with size-selected populations of Cla I- and Hind III-restricted parasite DNA. The structure of two clones (CL-1 and HA-2) is presented in detail. These two clones together probably represent the entire 17s and 25s coding regions of P. lophurae. Analysis of quantitative genomic Southern blots reveals that there are approximately six rRNA genes per haploid genome, and that the rRNA genes may be divided into four distinct classes by restriction analysis. Examination of the flanking regions of these genes indicates that they are not organized into easily recognizable tandem repeats.

Animals↗
PubMed1983

The cloned rRNA genes of P. lophurae: a novel rDNA structure.

The detailed structure of two ribosomal DNA (rDNA) clones CL-1 and HA-2, from the avian malaria parasite Plasmodium lophurae has been examined using hybridization and electron microscopy. The results demonstrate that the clone CL-1 contains two regions homologous to 25s rRNA of approximately 2200 base pairs (bp) and 450 bp in length, separated by a non homologous region of 240 bp. CL-1 also contains two regions of approximately 1100 bp and 550 bp homologous to 17s rRNA, separated by a non homologous region of 230 bp. The clone HA-2 contains a single region of 670 bp, which is homologous to 25s rRNA. This region is flanked by non homologous stretches of DNA 940 bp and 110 bp in length. As HA-2 is known to be adjacent to CL-1 in the genome (1), these results suggest that the 25s rDNA is interrupted twice, and the 17s rDNA once, by stretches of DNA not found in mature rRNA.

Animals↗
PubMed1983

Isolation and characterization of an alpha-tubulin gene from Leishmania enriettii.

An alpha-tubulin gene of Leishmania enriettii has been identified in genomic Southern blots by hybridization with a heterologous alpha-tubulin gene from Drosophila melanogaster. A clone containing this gene has been isolated from a plasmid library of size-selected L. enriettii DNA. It was identified by hybridization with the D. melanogaster tubulin gene. The cloned DNA fragment was characterized by restriction analysis and partial DNA sequence analysis. The cloned DNA fragment is 2 kb in length, bounded by Pst I sites, and appears to contain the entire coding region of the alpha-tubulin gene.

Animals↗
PubMed1983

Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii.

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.

Animals↗
PubMed1982

Rapid identification of Leishmania species by specific hybridization of kinetoplast DNA in cutaneous lesions.

Kinetoplast DNA (kDNA) was isolated from various species of the protozoic parasite Leishmania and analyzed by nucleic acid hybridization to detect species-related heterogeneity of kDNA. Purified DNA isolated from L. mexicana and L. braziliensis displayed no homology in nucleic acid hybridization studies. These results confirmed that rapid kDNA sequence change and evolution is occurring in New World species of Leishmania and suggested that such isolated kDNA could be used as a specific hybridization probe for the rapid identification of Leishmania species by using whole organisms. This work further demonstrates that such species-specific identification is feasible on isolated Leishmania promastigotes and, more important, directly on tissue touch blots derived from the cutaneous lesion. Thus, specific hybridization of isolated kDNA provides the basis for a rapid, accurate method for the diagnosis of human leishmaniasis directly from infected tissue.

Animals↗
PubMed1981

Expression of the Streptomyces enzyme endoglycosidase H in Escherichia coli.

Endoglycosidase H is one of a large number of enzymes secreted by Streptomyces plicatus and other Streptomyces species. When the structural gene for this enzyme is introduced into Escherichia coli attached to the plasmid pBR-322 or Charon 4 phage, the enzyme is synthesized and is found in the periplasmic space, culture medium, and cells. Attachment of the UV-5 lac promoter to a site in the plasmid adjacent to the Streptomyces insert stimulates enzyme synthesis as much as 100-fold. This result demonstrates that transcription of the Streptomyces gene can be initiated from sequences outside of the Streptomyces insert. Initiation of transcription on a Streptomyces promoter is also a suggested but unproven possibility. In contrast to the situation in Streptomyces, where the enzyme has a molecular weight of 27,000, the enzyme made in E. coli has a molecular weight of approximately 30,000. Possible explanations for this difference in size are lack of cleavage of the Streptomyces secretion "signal sequence" in E. coli or protein "processing" by enzymes secreted into the medium by STreptomyces.

Bacteriophages↗
PubMed1979

Transformed mammalian cells secrete specific proteins and phosphoproteins.

We have examined the proteins secreted into the growth medium by normal and transformed cells. Transformed cell lines from several mammalian species all secrete proteins in the 58,000 dalton molecular weight range. These proteins are all immunologically related and are secreted at low levels or not at all by the parental normal cell lines. Secretion of the 58K proteins occurs with either DNA or RNA virus transformation and with spontaneous transformation. The transformed cells also secrete phosphoproteins in the same size range, but these are immunologically distinct from the 58K proteins mentioned above. The sizes of the phosphoproteins are species-specific and unrelated to the transforming virus. Incubation of conditioned media from transformed cell cultures with gamma-32P-ATP labels phosphoproteins of the same sizes, indicating the presence in the media of both protein kinase and substrate. All three properties (58K protein, phosphoprotein, in vitro phosphorylation) are closely correlated with transformation in cells transformed by temperature-sensitive viruses. The biological implications of these results remain unknown, but the results may be relevant to recent data on the (phospho)proteins and protein kinase encoded by RNA tumor viruses and the molecular basis of the transformed phenotype.

Cell Line↗