Biomedical subjects

V Bellofatto

Publications and source records attributed to V Bellofatto.

24 records · Page 2Linked to original sources

PubMed1988

Characterization of RNA transcripts from the alpha tubulin gene cluster of Leptomonas seymouri.

A tandem cluster of alpha tubulin genes was identified in the trypanosomatid protozoan Leptomonas seymouri. One repeat unit and the first gene of the cluster, with its upstream flanking region, were cloned and analyzed for their transcriptional and coding capacities. The 12-15 copies per cell of the 4 kb repeat encode a stable 2 kb transcript, which contains a mini-exon at its 5' end and two closely spaced polyadenylation sites. Transcription of the alpha tubulin gene cluster in isolated nuclei was unidirectional. Intergenic and coding regions were transcribed at the same rate, and nascent intergenic and coding region transcripts were quantitatively linked. These results are consistent with the possibility that the primary transcripts are polycistronic, or that there is a single small intercistronic gap.

Amino Acid Sequence↗

PubMed1987

Biochemical changes associated with alpha-difluoromethylornithine uptake and resistance in Trypanosoma brucei.

Procyclic Trypanosoma brucei grown in semi-defined media are sensitive to alpha-difluoromethylornithine (DFMO) (EC50 100 microM), an inhibitor of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis. Organisms resistant to 5 mM DFMO (EC50 greater than 20 mM) were obtained by passage in incremental amounts of drug. Resistant and wild-type cells accumulated DFMO by passive diffusion with a consequent decrease in polyamine levels, indicating inhibition of ODC in both cell types. The resistant phenotype was stable in the absence of DFMO, in which state there was no increase in ODC abundance or activity. By kinetic analysis, the ODC of resistant cells appeared normal. In wild-type and resistant cells, [3H]DFMO equally and uniquely affinity-labelled a 50 kDa polypeptide corresponding to the ODC subunit. Levels of ODC and tubulin mRNAs were elevated 4-fold in resistant cells grown in the presence of DFMO, although there was no indication of gene amplification. The intracellular concentration of dihydrotrypanothione (N1,N8-bis(glutathionyl)-spermidine), a redox intermediate unique to kinetoplastids, was unchanged in resistant cells growing in DFMO but was halved in wild-type cells exposed to DFMO for 48 h. The exceptionally elevated levels of ornithine found in DFMO-treated resistant cells most likely play a crucial role in cell survival by maintaining intracellular concentrations of dihydrotrypanothione by competing with DFMO for ODC.

Animals↗

PubMed1986

Transcription initiation in vitro and in vivo at a highly conserved promoter within a 16 S ribosomal RNA gene.

Transcription initiation has been shown to occur in vitro at several sites within a cloned Caulobacter crescentus ribosomal RNA gene cluster that lacks the major promoter region 5' to the 16 S rRNA gene. The predominant transcription start site in vitro was located near the 3' end of the 16 S rRNA gene. Transcription initiation from this region was also detected in vivo, when the cloned rRNA gene cluster was present on a multi-copy plasmid. The transcription start sites in vitro and in vivo were shown to be identical by S1 nuclease mapping and were found to be located approximately 300 nucleotides upstream from the 3' end of the 16 S rRNA gene. The transcript synthesized in vitro was shown to be cleaved by C. crescentus RNase III and to release the transfer RNA genes from the downstream 16 S/23 S intergenic spacer region. Analysis of the nucleotide sequence near the internal 16 S rRNA transcription start site revealed the presence of a consensus promoter sequence followed by the beginning of an open reading frame approximately 90 nucleotides downstream. Examination of the 16 S rRNA genes from other bacterial species and chloroplasts and 18 S rRNA genes from Xenopus and yeast revealed that the nucleotide sequence of this internal 16 S rRNA promoter region was highly conserved. Although the length of these 16 S and 18 S rRNA genes is slightly variable, the distance of the conserved promoter sequence from the 3' end of these genes has been conserved.

Base Sequence↗

PubMed1985

Organization and nucleotide sequence analysis of an rRNA and tRNA gene cluster from Caulobacter crescentus.

rRNA genes of Caulobacter crescentus CB13 were isolated and shown to be present in two gene clusters in the genome. The organization of each rRNA gene cluster was found to be 5'-16S-tRNA spacer-23S-5S-3'. The DNA sequence of 40% of the 16S rRNA gene, the entire 16S/23S intergenic spacer region, and portions of the 23S rRNA gene were determined. Analysis of the nucleotide sequence in the 16S-23S intergenic spacer region revealed the presence of tRNAIle and tRNAAla genes. Large invert repeat sequences were found surrounding the 16S rRNA gene. These inverted repeat sequences are analogous to the RNase III-processing sites in the E. coli rRNA precursor. Small invert repeat sequences were also found flanking the individual tRNA genes. RNA polymerase-binding studies with restriction fragments of the rRNA gene cluster revealed three regions which bound enzyme, and these regions were shown to contain transcription initiation sites. One of these sites was located within the 16S gene near its 3' end, and the other two were found at the 5' end of the 23S gene.

Base Sequence↗

PubMed1984

Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus.

A promoter probe, Tn5-VB32, was constructed and placed in a P group R plasmid containing bacteriophage Mu sequences, allowing transfer of the transposon to bacteria such as Caulobacter, Rhizobium, and Agrobacterium without retention of the plasmid. The probe carries an altered Tn5 transposon that allows detection of chromosomal promoter regions by virtue of acquired kanamycin resistance. A fragment of DNA containing the neomycin phosphotransferase II (NPT II) gene from Tn5, lacking its promoter region but retaining its translation initiation signal, was inserted into a Tn5 derivative that lacked the entire NPT II gene and a large portion of the IS50L sequence while retaining its ability to transpose. This Tn5 derivative also contained the intact tetracycline resistance-encoding region of the transposon Tn10. Transposition of the Tn5-VB32 promoter probe into the Caulobacter crescentus chromosome generated auxotrophic and motility mutants and Southern blot analysis of DNA from these mutants showed Tn5-VB32 sequences in random-sized chromosomal restriction fragments. Transcriptional regulation by exogenous cysteine of NPT II gene expression was demonstrated in a cysteine auxotroph generated by Tn5-VB32 insertional inactivation. NPT II synthesis, measured by agar plate assays of kanamycin resistance and by immunoprecipitation of the NPT II protein, was repressed in the presence of cysteine and derepressed in its absence. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. Insertions within temporally regulated genes, such as those involved in flagellar biosynthesis and chemotaxis functions, can now be used directly to monitor transcriptional regulation from Caulobacter promoter sequences.

Bacteria↗

PubMed1983

Purification and characterization of an RNA processing enzyme from Caulobacter crescentus.

An RNA processing enzyme has been isolated from Caulobacter crescentus which is specific for double-stranded RNA, has an absolute requirement for monovalent cations, and can be eluted from a poly I:C agarose affinity column in pure form. This enzyme, like RNase III isolated from Escherichia coli, processes precursor ribosomal RNAs and polycistronic phage mRNAs and has a monomeric Mr of approximately 20,000. The two enzymes differ, however, in the recognition of specific cleavage sites and yield different digestion products when either coliphage T7 or C. crescentus phage phi Cdl early mRNA is used as substrate. Two lines of evidence are presented which show that an RNase III activity functions as a processing enzyme in C. crescentus. (a) In an in vitro reaction, C. crescentus phage phi Cdl major early mRNA synthesized in vitro by host RNA polymerase was processed by RNase III to yield RNA species which co-migrated with phage RNA synthesized in vivo in phi Cdl-infected cells, and (b) an in vitro transcript of a C. crescentus DNA clone containing the entire 16 S gene and part of the 23 S gene was processed by C. crescentus RNase III to yield an RNA product which co-migrated with 16 S RNA. The RNase III activity isolated from C. crescentus cell extracts has potential use in the analysis of specific RNA species because it was found to be more stringent in the recognition of cleavage sites than the E. coli enzyme.

Cloning, Molecular↗