PubMed Health⌕ Search

Biomedical subjects

S M Beverley

Publications and source records attributed to S M Beverley.

At least 19 recordsLinked to original sources
PubMed1992

A member of the aldoketo reductase family confers methotrexate resistance in Leishmania.

Methotrexate (MTX)-resistant mutants of the parasitic protozoan Leishmania have been used as models for the mechanism and genetic basis of drug resistance in trypanosomatids and other cells. Three resistance mechanisms to MTX, a dihydrofolate reductase inhibitor, have been described in Leishmania: decreased uptake and accumulation of MTX via the folate/MTX transporter, amplification and overexpression of the dihydrofolate reductase-thymidylate synthase gene, and extrachromosomal amplification of H region DNA. We have now identified hmtxr as the H region gene conferring MTX resistance using a transfection-based approach. Data base searches show that the predicted HMTXr protein is related to members of the polyol dehydrogenase/carbonyl reductase family of aldoketo reductases, whose substrates include polyols, quinones, steroids, prostaglandins, fatty acids, and pterins. We therefore propose that HMTXr is also an oxidoreductase and suggest several biochemical mechanisms of resistance in Leishmania that could be exploited in the design of parasite-specific inhibitors.

Amino Acid Sequence↗
PubMed1992

Leishmania tarentolae taxonomic relatedness inferred from phylogenetic analysis of the small subunit ribosomal RNA gene.

The sequence of the Leishmania tarentolae SSU rRNA (small subunit or 18S rRNA) gene was completely determined from 2 different strains and used to determine phylogenetic relationships between this organism and other trypanosomatids. Extensive structural similarities were observed between L. tarentolae and mammalian leishmanias the SSU rRNA. Phylogenetic reconstructions, using distance matrix or parsimony methods, showed large evolutionary distances between trypanosomes, either African and American, and L. tarentolae. Further analysis using intergenic rDNA spacer (IGS) sequences as probes in dot blot experiments confirmed the results obtained with the SSU rDNA comparisons. The data presented here clearly indicate that L. tarentolae is closely related to the mammalian parasite Leishmania donovani and highly divergent from trypanosomes.

Animals↗
PubMed1992

Stable amplification of a linear extrachromosomal DNA in mycophenolic acid-resistant Leishmania donovani.

Pulsed field gel electrophoretic analysis of chromosomes of MPA100 cells, a strain of Leishmania donovani that possesses an approx. 15-fold amplified IMP dehydrogenase (IMPDH) gene copy number, revealed a new 280-kb extrachromosomal DNA, IMPDH-280, that was not present in wild type parental cells. Southern blots of these pulsed field gels revealed that the vast majority of the amplified impdh genes were localized on IMPDH-280. In addition to the 700-kb wild type chromosome, the impdh probe also recognized a 740-kb chromosome in the MPA100 genome. The pulse time-dependent relative mobility of IMPDH-280 in pulsed field gels, the failure of limited gamma-irradiation to generate a new discrete DNA fragment, and the susceptibility of IMPDH-280 to lambda-exonuclease digestion, demonstrated that IMPDH-280 was a linear molecule. IMPDH-280 was also recognized by a telomere probe but not by fragments derived from amplified DNAs found in other drug-resistant Leishmania. IMPDH-280 and the drug resistance phenotype remained stable when MPA100 cells were propagated in the absence of drug for 2 years. The appearance of IMPDH-280 in MPA100 cells represents one of the first examples of an amplification of a linear extrachromosomal DNA element mediating drug resistance in Leishmania and the first instance of a linear DNA amplification that is stable in the absence of selective pressure.

Animals↗
PubMed1992

Loss of the GP46/M-2 surface membrane glycoprotein gene family in the Leishmania braziliensis complex.

Immunization with the GP46/M-2 membrane glycoprotein of Leishmania amazonensis has been shown to induce a protective immune response against infection. We have surveyed a variety of trypanosomatid species and genera for the presence and expression of this gene family, information that will be relevant to future vaccine studies against leishmaniasis. Molecular karyotype analysis revealed the presence of GP46/M-2 genes in all members of the Leishmania mexicana complex, Leishmania major, Leishmania donovani, Leishmania tarentolae, and Crithidia fasciculata. In contrast, DNAs from species of the Leishmania braziliensis complex (L. braziliensis, Leishmania guyanensis, and Leishmania panamensis) failed to hybridize to GP46/M-2 probes. Western blot analyses with several polyclonal antisera against the GP46/M-2 protein revealed protein expression in L. major and L. donovani, but not L. panamensis or L. braziliensis. Phylogenetic analysis suggests that a loss of the GP46A gene family occurred following separation of the L. braziliensis complex, prior to speciation events within this complex. These data indicate that GP46/M-2 membrane glycoprotein may not be critical to parasite survival, but may play an ancillary role during the developmental cycle.

Animals↗
PubMed1992

Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani.

We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.

Animals↗
PubMed1991

Heavy metal resistance: a new role for P-glycoproteins in Leishmania.

P-glycoproteins are responsible for multidrug resistance in tumor cell lines and are thought to have a physiologic role in exporting cellular metabolites. We now report that a P-glycoprotein gene in the H region of the trypanosomatid protozoan Leishmania confers resistance to heavy metals when present in multiple copies. The Leishmania H region is frequently amplified in drug-resistant lines and is associated with metal resistance. Leishmania expression vectors were used to introduce multiple copies of segments of the Leishmania major H region into wild-type L. major promastigotes. Only constructs bearing a segment of L. major DNA containing the P-glycoprotein lmpgpA conferred arsenite resistance. Deletional analysis of the arsenite-resistant construct mapped resistance to the lmpgpA protein coding region. Lines expressing lmpgpA showed resistance to arsenite and trivalent antimonials, but not to pentavalent antimonials, zinc, cadmium, or the typical multidrug-resistant P-glycoprotein substrates vinblastine and puromycin. Transfection of the Leishmania tarentolae P-glycoprotein homologue ltpgpA resulted in a similar resistance profile. Thus, these pgpAs represent a functionally distinct group of P-glycoproteins which exhibit a substrate specificity similar to prokaryotic heavy metal pumps. Additionally, several arguments suggest that pgpAs may play a role in the susceptibility of Leishmania to clinically utilized antimonials.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Double targeted gene replacement for creating null mutants.

We have used double gene targeting to create homozygous gene replacements in the protozoan parasite Leishmania major, an asexual diploid. This method uses two independent selectable markers in successive rounds of gene targeting to replace both alleles of an endogenous gene. We developed an improved hygromycin B-resistance cassette encoding hygromycin phosphotransferase (HYG) for use as a selectable marker for Leishmania. HYG-containing vectors functioned equivalently to those containing the neomycin phosphotransferase (NEO) cassette previously used for extrachromosomal transformation or gene targeting. Drug resistances conferred by the NEO and HYG markers were independent, allowing simultaneous selection for both markers. A HYG targeting vector was utilized to replace the single dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene remaining in a line heterozygous for a NEO replacement at the dhfr-ts locus (+/neo), with a targeting efficiency comparable to that seen with wild-type recipients. The resultant dhfr-ts- line (hyg/neo) was auxotrophic for thymidine. The double targeted replacement method will enable functional genetic testing in a variety of asexual diploids, including cultured mammalian cells and fungi such as Candida albicans. Additionally, it may be possible to use Leishmania bearing conditionally auxotrophic gene replacements as safe, improved live vaccines for leishmaniasis.

Alleles↗
PubMed1991

Simultaneous transient expression assays of the trypanosomatid parasite Leishmania using beta-galactosidase and beta-glucuronidase as reporter enzymes.

We describe a transient transfection protocol for cultured Leishmania major promastigotes, utilizing Escherichia coli genes encoding beta-galactosidase and beta-glucuronidase inserted into an expression vector derived from the dihydrofolate reductase-thymidylate synthase locus. Less than 0.1 pg of either reporter enzyme can be detected with a simple fluorimetric assay, and transfection of 10 micrograms of either reporter construct yields activities at least 100-fold over background. Simultaneous introduction of both constructs showed that the activity of each reporter gene was unaffected by the presence of the other, allowing one reporter construct to serve as a control for experimental variability in test gene constructs containing the second reporter gene. These results show that it is feasible to apply transient expression assays to the identification of cis-acting elements of genes encoding nonabundant mRNAs in the genus Leishmania.

Animals↗
PubMed1991

Stable DNA transfection of a wide range of trypanosomatids.

We have shown that the Leishmania major transfection vector pR-NEO (or derivatives thereof) can be introduced and stably maintained in four species complexes of pathogenic Leishmania (L. tropica, L. mexicana, L. donovani, L. braziliensis), and the genera Endotrypanum and Crithidia; transfection of Trypanosoma cruzi or Trypanosoma brucei was not successful. Quantitative plating assays showed that the transfection efficiencies were high in L. major and Leishmania amazonensis (5x10(-5)/cell) and about 10-fold less for Leishmania panamaensis and Crithidia. Leishmania donovani transfected with pR-NEO retained the ability to infect hamsters, and amastigotes recovered after 2 months yielded G418-resistant promastigotes which retained high levels of extrachromosomal pR-NEO DNA. In promastigotes, the transfected DNA existed as extrachromosomal circles, and expressed the predicted 2.4-kb hybrid NEO/DHFR-TS mRNA bearing the trans-spliced miniexon. Large quantitative differences were observed only in Crithidia: relative to transfected Leishmania species, the copy number of pR-NEO was elevated 20-fold, while the levels of the NEO/DHRFR-TS mRNA or Escherichia coli beta-galactosidase (synthesized from the expression vector pX-beta GAL) were reduced 80 and more than 1000-fold, respectively. Thus, genetic signals derived from L. major DNA that mediate RNA expression or stability are recognized by the heterologous Leishmania species but less efficiently by Crithidia. These studies suggest that pR-NEO derived vectors may be applied to the study of genes expressed throughout the life cycle in a wide range of pathogenic trypanosomatids.

Animals↗
PubMed1990

Nuclease mapping and DNA sequence analysis of transcripts from the dihydrofolate reductase-thymidylate synthase (R) region of Leishmania major.

Trypanosomatid protozoan parasites utilize a number of nonstandard mechanisms in expressing their genes. To probe these phenomena in a genetically accessible system, we have mapped termini of eight transcripts arising from the amplified R region including the DHFR-TS gene of methotrexate-resistant Leishmania major. Poly(A)+ RNAs transcribed from the DHFR-TS-coding strand exhibit features similar to those observed around other trypanosomatid protein-coding genes. These include close spacing, the presence of a transpliced miniexon on the 5' termini, heterogeneity at both 5' and 3' ends, and in some cases S1 nuclease protection of intertranscript regions. Other than the splice acceptor site, no consensus sequence elements associated with either 5' or 3' ends were detected, although polydinucleotide tracts tended to be near inter-transcript regions. Two poly(A)+ RNAs transcribed from the opposite strand of the upstream flanking regions lacked the miniexon. Sequencing of DNA encoding the overlapping 1.7 kb opposite strand transcripts (one bearing and one lacking the miniexon, both found on polysomes) revealed no reading frames likely to encode proteins, suggesting that at least some of these RNAs could be nonfunctional by-products of RNA processing.

Animals↗
PubMed1990

Gene replacement in parasitic protozoa.

Trypanosomatid protozoa frequently cause severe diseases in humans. Many molecules likely to have a role during the infectious cycle have been identified, yet proof of their function is often lacking. We describe studies in Leishmania major of homologous gene targeting, a powerful method for testing gene function in other organisms. Following introduction of a construct containing dihydrofolate reductase-thymidylate synthase (dhfr-ts) flanking sequences fused to neomycin phosphotransferase, 45% of the colonies contained the planned homologous replacement; this frequency rose to nearly 100% in transfections using low amounts of DNA. Integrative transfection in Leishmania thus resembles that of Saccharomyces cerevisiae in giving predominantly homologous events. To facilitate studies of folate metabolism and chemotherapy the sole dhfr-ts copy in a heterozygous deletion line was replaced, yielding lines that were functionally DHFR-TS-. Although most genes are diploid in trypanosomatids, methods exploiting the high frequency of homologous recombination should permit complete replacement of any parasite gene.

Animals↗
PubMed1990

Evolution of nuclear DNA and the occurrence of sequences related to new small chromosomal DNAs in the trypanosomatid genus Endotrypanum.

Comparisons of nuclear DNA restriction fragment patterns were used to examine the evolutionary relatedness among 17 strains previously identified as Endotrypanum, a trypanosomatid parasite of sloths. Fragments were obtained with 6 restriction enzymes and analyzed by Southern blotting with hybridization probes from three loci. An estimate of the percent nucleotide sequence divergence among strains, delta, was calculated and used to construct molecular evolutionary trees. The 17 isolates fell into four distinct groups, one of which (group D) showed no more relationship to groups A-C than it did to other genera (Leishmania, Crithidia, Leptomonas, Trypanosoma), being too distant to be resolved with this method. These and other data suggest that group D may not actually be Endotrypanum. Molecular karyotype analysis revealed considerable variation among the chromosomes of these strains. One strain (LV88, group B) contained a linear 70-kb chromosome not evident in other isolates. Hybridization probes specific for this chromosome (LV88-70) were developed and revealed that related sequences were present at high levels in group B isolates and low levels in group A isolates, although a complex hybridization pattern was evident. Sequences related to LV88-70 were not present in groups C and D, nor in Leishmania major, showing that this DNA has a disjunct distribution which curiously parallels that of virus-like particles present in these isolates.

Animals↗
PubMed1990

Recurrent de novo appearance of small linear DNAs in Leishmania major and relationship to extra-chromosomal DNAs in other species.

We have detected several new chromosome-sized DNAs in lines derived from the LT252 isolate of Leishmania major. These DNAs appeared de novo in two clonal lines undergoing methotrexate (MTX) selection (clone 7-R50, clone 15-R50), in a stably MTX-resistant population reverting from MTX pressure (R1000-11-P55rev), and spontaneously during routine serial passage of the wild-type LT252 line in vitro (LT252+). No association of these new DNAs with drug resistance was detected. The new chromosomes were present in multiple copies, stably maintained, linear, and hybridized to a telomere-specific probe, and ranged in size from 180 to 220 kb. Southern blot hybridization revealed that all four new DNAs were related, and the family was designated the 715 class of small linear DNAs (SLDs). A 715-class SLD hybridization probe also identified small chromosomes described previously in Leishmania donovani (LD-1, HU-3 minichromosome) and Leishmania braziliensis (LD-1); LD-1 is known to be related to a smaller circular DNA, CD-1. A cloned probe derived from CD-1 (from K. Stuart and C. Tripp) identified two of the four L. major 715 class of SLDs in addition to the LD-1 DNAs of L. donovani and L. braziliensis, however the 715-class SLD probe did not identify CD-1 itself. L. major and L. donovani possess a homologous 1.5 Mb chromosome containing both the CD-1 and 715 sequences within a 40-kb region, whose size remained unaltered following appearance of the SLDs. These data suggest that SLDs and CD-1 may arise from an evolutionarily conserved chromosomal reservoir.

Animals↗
PubMed1990

Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes.

Trypanosomatid protozoan parasites cause several important tropical diseases and have been a fertile ground for the discovery of molecular paradigms such as trans-splicing and RNA editing. Transfection-based methods for the study of these organisms have recently been developed, and we have now designed an expression vector, pX, which contains only 2.3 kilobases of Leishmania DNA and can be stably transfected with high efficiency. Genes encoding Escherichia coli beta-galactosidase or a Leishmania amazonensis protective membrane glycoprotein (GP46A/M-2) were inserted into the pX expression site and transfected into Leishmania major, where they directed the synthesis of high levels of mRNAs formed by 5' and 3' processing events occurring predominantly at the sites used by the normal transcripts. Colony assays and immunoblot analysis showed that both proteins were produced; enzymatically active beta-galactosidase comprised approximately 1% of total protein. Sizes of the GP46A protein synthesized in transfected L. major or L. amazonensis were similar and differed from the predominant L. amazonensis GP46, suggesting that the GP46A gene may encode a variant GP46 family member. Because these vectors function efficiently in pathogenic species of Leishmania, pX will facilitate the genetic analyses of parasite proteins crucial for infectivity as well as the identification of cis-acting elements mediating transcription and replication.

Animals↗
PubMed1990

Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression.

To delineate segments of the genome of the human protozoan parasite Leishmania major necessary for replication and expression, we developed a vector (pR-NEO) which can be reproducibly introduced into L. major. This DNA was derived from a 30-kilobase extrachromosomal amplified DNA bearing the dihydrofolate reductase-thymidylate synthase gene, with the coding region for neomycin phosphotransferase substituted for that of dihydrofolate reductase-thymidylate synthase and a bacterial origin of replication and selectable marker added. G418-resistant lines were obtained at high efficiency by electroporation of pR-NEO (approaching 10(-4) per cell), while constructs bearing an inverted neo gene or lacking Leishmania sequences did not confer resistance. pR-NEO replicated in L. major and gave rise to correctly processed transcripts bearing the trans-spliced miniexon. Molecular karyotype analysis showed that in some lines pR-NEO DNA exists exclusively as an extrachromosomal circle, a finding supported by the rescue of intact pR-NEO after transformation of Escherichia coli. These data genetically localize all elements required in cis for DNA replication, transcription, and trans splicing to the Leishmania DNA contained within pR-NEO DNA and signal the advent of stable transfection methodology for addressing molecular phenomena in trypanosomatid parasites.

Animals↗