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Biomedical subjects

A Laban

Publications and source records attributed to A Laban.

18 recordsLinked to original sources
PubMed1992

Gene expression in Leishmania: analysis of essential 5' DNA sequences.

A major unanswered question in Kinetoplastida parasites is the mechanism of regulating gene expression. Using a transfection system, we have previously shown that the intergenic region of the alpha-tubulin gene of Leishmania enriettii contained sequences required for gene expression. The goal of the work reported here was to determine whether the Leishmania-derived sequences were providing transcriptional control signals or functioning at a post-transcriptional level, most likely in trans-splicing. The chloramphenicol acetyltransferase (cat) gene was used as the reporter gene and was stably introduced into L. enriettii as part of an extrachromosomal element by transfection. We show here that the production of cat mRNA was dramatically dependent on the presence of the intergenic region 5' to the cat gene. The intergenic region could be substituted by a smaller fragment (222 base pairs) that contained the trans-splice acceptor site and an adjacent polypyrimidine tract. This native fragment could be replaced by a synthetic polypyrimidine tract containing an AG site. The native and the synthetic fragments had unidirectional activity. No effect on transcription of the cat gene by the wild-type fragment or the synthetic polypyrimidine was detected. The results indicate that both regions contain signals that affect RNA stability, probably sequences involved in trans-splicing.

Animals↗
PubMed1992

A stereological investigation of rat endocrine pancreas after a long-term low-protein diet.

Stereological analysis of rat endocrine pancreas was carried out after 4 months of low-protein intake. Thirty make Wistar rats, aged 2 months at the beginning of the experiment, were divided into three groups. The control group (NP) received a standard laboratory diet. The experimental group (LP) received a low-protein diet, and the pair-fed animals also received a standard laboratory diet, as the NP rats did, but restricted to the amounts consumed by the LP group. All groups were offered drinking water ad lib. Weibel's multipurpose lattice was used to determine the volume fraction of cells and numerical density of islets in the endocrine pancreas. The results showed that the long-term low-protein diet decreased the volume fraction of B-cells and increased that of A-cells. Numerical density of islets was significantly increased, whereas the absolute number was decreased. The mean number of B-cells per islet was found to be decreased and that of D-cells increased. The mean radius of the islets was decreased, as was the mean surface area.

Animals↗
PubMed1991

Homologous recombination in Leishmania enriettii.

We have used derivatives of the recently developed stable transfection vector pALT-Neo to formally demonstrate that Leishmania enriettii contains the enzymatic machinery necessary for homologous recombination. This observation has implications for gene regulation, gene amplification, genetic diversity, and the maintenance of tandemly repeated gene families in the Leishmania genome as well as in closely related organisms, including Trypanosoma brucei. Two plasmids containing nonoverlapping deletions of the chloramphenicol acetyltransferase (CAT) gene, as well as the neomycin-resistance gene, were cotransfected into L. enriettii. Analysis of the DNA from these cells by Southern blotting and plasmid rescue revealed that a full-length or doubly deleted CAT gene could be reconstructed by homologous crossing-over and/or gene conversion between the two deletion plasmids. Additionally, parasites cotransfected with pALT-Neo and pALT-CAT-S, a plasmid containing two copies of the chimeric alpha-tubulin-CAT gene, resulted in G418-resistant parasites expressing high levels of CAT activity. The structure of the DNA within these cells, as shown by Southern blot analysis and the polymerase chain reaction, is that which would be expected from a homologous exchange event occurring between the two plasmids.

Animals↗
PubMed1990

Stable expression of the bacterial neor gene in Leishmania enriettii.

Molecular genetic studies in parasitic protozoa have been hindered by the lack of methods for the introduction and expression of modified or foreign genes in these organisms. Two recent reports described the transient expression of the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of parasite-specific sequences. We now describe the stable expression of a selectable marker, the gene for neomycin resistance (neor) in Leishmania enriettii. A chimaeric gene containing the neor gene inserted between two alpha-tubulin intergenic sequences was introduced into the cells and drug-resistant L. enriettii were observed which stably expressed the neor gene. One goal of this work was to analyse the sequences necessary for trans-splicing of messenger RNA, as trypanosomatids have a novel process of RNA trans-splicing, described initially in Trypanosome brucei and subsequently in several other trypanosomatids, including L. enriettii. Many trypanosomatid genes are arranged in tandem arrays and the intergenic sequences contain both the splice acceptor site for the addition of the spliced leader sequence and a putative polyadenylation site. Messenger RNA isolated from several different neor L. enrietti lines contained the spliced leader sequence joined to the neor gene at the position of the splice acceptor site in the alpha-tubulin intergenic sequence. The neor mRNA was also polyadenylated. Plasmid DNA is present within the drug-resistant organisms and appears to be extrachromosomal. The development of these methods allows the functional analysis of sequences necessary for trans-splicing.

Animals↗
PubMed1989

Transfection of Leishmania enriettii and expression of chloramphenicol acetyltransferase gene.

We report a transient expression transfection system in Leishmania enriettii. A hybrid gene containing an intergenic region of the alpha-tubulin cluster and the bacterial chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene is expressed after transfection of L. enriettii with the hybrid plasmid. The expression of the CAT gene is dependent on the presence of sequences from the alpha-tubulin gene. The hybrid gene is also active in Leishmania braziliensis and Leishmania major.

Animals↗
PubMed1984

The effect of nonhomologous DNA sequences on interplasmidic recombination.

The effect of nonhomologous DNA sequences at one or both sides of short genetic intervals on recombination within that interval was investigated, using an interplasmidic recombination system in Escherichia coli K-12. The recombining plasmids were derivatives of pBR322 and pACYC184, which share a 1330-nucleotide sequence that includes the tet gene. The genetic interval was defined by the HindIII and BamHI or BamHI and the SalI restriction endonuclease sites of this gene. The substantial differences between recombination frequencies measured within intervals bracketed or bounded on one side by major nonhomologies suggests that, in this system, strand exchange is polar and is blocked by major nonhomologies. This conclusion is substantiated by results of three-factor crosses and by structural analysis of recombination products. Results of two-factor crosses in recA genetic background and structural analysis of recombination products suggest that strand exchange occurs in the absence of a functional recA gene. "Opening" a bracketed BamHI-SalI genetic interval of the tet gene, at the SalI site, by substituting a major insertion with a short deletion, results in an increase in recombination frequency, within this genetic interval, which is greater than expected on the basis of the ratio of the length of homology on the two sides of the SalI site. This observation suggests that a genetic element that may affect rate of recombination initiation, polarity of strand exchange, template specificity in mismatch repair or more than one of these events may be present on the outer side of the SalI site of the tet gene.

Base Sequence↗
PubMed1983

Plasmidic recombination in Escherichia coli K-12: the role of recF gene function.

Interplasmidic and intraplasmidic recombination proficiencies were determined in E. coli bacterial strains carrying rec mutations. Our results defined the role of recF gene function, recB, recC, and sbcB gene products (exonuclease V and exonuclease I) in plasmidic recombination in wild-type E. coli cells and in cells in which the recE recombination pathway is activated. RecF gene function is required for interplasmidic recombination regardless of the recB recC genotype. Intraplasmidic recombination is recF dependent in cells having a functional exonuclease V, but not in recB recC mutants. Exonuclease V activity inhibits both interplasmidic and intraplasmidic recombination via the recE pathway.

Bacterial Proteins↗
PubMedDate not supplied

Herpes simplex virus type 1 thymidine kinase gene expression in Escherichia coli.

The HSV-1 thymidine kinase (TK) gene of Herpes simplex virus was inserted into plasmids pBR322, pMOB45 and pHA10. The recombinant hybrid plasmids were used to transfect a tdk- mutant of Escherichia coli (Ky895) and the synthesis of the viral TK in the bacterial host was studied. Recombinant plasmids containing the entire BamHI-BamHI DNA fragment carrying the viral TK gene and the upstream sequences containing its promoter were able to produce the thymidine kinase, but removal of the BamHI-BglII DNA fragment containing the viral promoter of the TK gene resulted in enhanced expression of the viral gene, originating from the pBR322 tetr gene promoter. When the BglII-BamHI DNA fragment containing the viral TK gene was cloned in a plasmid with temperature-dependent copy number control (pMOB45), expression of the TK gene was enhanced fourfold by the temperature shift. Cloning of the HSV-1 TK gene in plasmid pHA10 showed that the lambda pL promoter allowed transcription of the viral gene but not the synthesis of active TK. The BamHI-BglII DNA fragment containing the viral upstream sequences with the promoter of the TK gene was found to contain transcription-termination sequences that prevented expression of the lambda kil gene of pHA10 in the presence of lambda N function.

DNA, Recombinant↗