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V Bellofatto

Publications and source records attributed to V Bellofatto.

At least 19 recordsLinked to original sources
PubMed2001

The Leptomonas seymouri spliced leader RNA promoter requires a novel transcription factor.

The spliced leader RNA gene promoter in Leptomonas seymouri requires three promoter elements for efficient and accurate transcription of the spliced leader RNA. The upstream most element appears to have a functional homolog in Leishmania species and in the African trypanosomes. The protein factor, promoter binding protein-1, interacts with the upstream element and appears to function as a basal transcription factor. Promoter binding protein-1 has three subunits; 36, 41 and 57 kDa. Using microsequencing techniques, we have obtained peptide sequence from each subunit. These data have enabled us to recently identify the Leptomonas gene that encodes the 41 kDa subunit. The 41 kDa subunit, comprised of 381 amino acids, is a founding member of a new class of transcription factors since extensive database searches revealed no homology to any known protein. This subunit, encoded by a single copy gene, has a potential nuclear localisation signal at amino acid positions 71-76. There are also multiple dileucine repeats with unknown function. Anti-41 kDa protein polyclonal antibodies are being employed to test the function of the 41 kDa subunit in PBP-1 activities.

Amino Acid Sequence↗
PubMed2001

Trypanosome spliced leader RNA genes contain the first identified RNA polymerase II gene promoter in these organisms.

Typical general transcription factors, such as TATA binding protein and TFII B, have not yet been identified in any member of the Trypanosomatidae family of parasitic protozoa. Interestingly, mRNA coding genes do not appear to have discrete transcriptional start sites, although in most cases they require an RNA polymerase that has the biochemical properties of eukaryotic RNA polymerase II. A discrete transcription initiation site may not be necessary for mRNA synthesis since the sequences upstream of each transcribed coding region are trimmed from the nascent transcript when a short m(7)G-capped RNA is added during mRNA maturation. This short 39 nt m(7)G-capped RNA, the spliced leader (SL) sequence, is expressed as an approximately 100 nt long RNA from a set of reiterated, though independently transcribed, genes in the trypanosome genome. Punctuation of the 5' end of mRNAs by a m(7)G cap-containing spliced leader is a developing theme in the lower eukaryotic world; organisms as diverse as EUGLENA: and nematode worms, including Caenorhabditis elegans, utilize SL RNA in their mRNA maturation programs. Towards understanding the coordination of SL RNA and mRNA expression in trypanosomes, we have begun by characterizing SL RNA gene expression in the model trypanosome Leptomonas seymouri. Using a homologous in vitro transcription system, we demonstrate in this study that the SL RNA is transcribed by RNA polymerase II. During SL RNA transcription, accurate initiation is determined by an initiator element with a loose consensus of CYAC/AYR(+1). This element, as well as two additional basal promoter elements, is divergent in sequence from the basal transcription elements seen in other eukaryotic gene promoters. We show here that the in vitro transcription extract contains a binding activity that is specific for the initiator element and thus may participate in recruiting RNA polymerase II to the SL RNA gene promoter.

Amino Acid Sequence↗
PubMed1999

Transcription initiation at the TATA-less spliced leader RNA gene promoter requires at least two DNA-binding proteins and a tripartite architecture that includes an initiator element.

Eukaryotic transcriptional regulatory signals, defined as core and activator promoter elements, have yet to be identified in the earliest diverging group of eukaryotes, the primitive protozoans, which include the Trypanosomatidae family of parasites. The divergence within this family is highlighted by the apparent absence of the "universal" transcription factor TATA-binding protein. To understand gene expression in these protists, we have investigated spliced leader RNA gene transcription. The RNA product of this gene provides an m(7)G cap and a 39-nucleotide leader sequence to all cellular mRNAs via a trans-splicing reaction. Regulation of spliced leader RNA synthesis is controlled by a tripartite promoter located exclusively upstream from the transcription start site. Proteins PBP-1 and PBP-2 bind to two of the three promoter elements in the trypanosomatid Leptomonas seymouri. They represent the first trypanosome transcription factors with typical double-stranded DNA binding site recognition. These proteins ensure efficient transcription. However, accurate initiation is determined an initiator element with a a loose consensus of CYAC/AYR (+1), which differs from that found in metazoan initiator elements as well as from that identified in one of the earliest diverging protozoans, Trichomonas vaginalis. Trypanosomes may utilize initiator element-protein interactions, and not TATA sequence-TATA-binding protein interactions, to direct proper transcription initiation by RNA polymerase II.

Animals↗
PubMed1999

In vivo transcriptional analysis of the spliced leader RNA gene in the trypanosomatid Leptomonas seymouri.

Gene expression in all organisms requires the direct and indirect interaction of multiple proteins with specific DNA sequence elements. Using the monogenetic trypanosomatid, Leptomonas seymouri, we investigated the cis- and trans-acting components that determine expression of a central trypanosomatid RNA, the spliced leader (SL) RNA. Using base substitution mutagenesis and DNA transfection assays, we determined that the SL RNA gene promoter lies exclusively up-stream from the transcription initiation site. Accordingly, the SL RNA gene can be used as a gene cassette to express short heterologous RNAs of interest. We utilized two pharmacological agents, alpha-amanitin and tagetitoxin, and the detergent sarkosyl to assess components of the trans-acting machinery involved in transcription. The SL RNA inhibition pattern was distinct from that of alpha-tubulin, tRNA or ribosomal RNA. Taken together, these data suggest that the upstream SL RNA gene promoter serves to nucleate a transcriptional complex that is distinct, in either its initiation and/or elongation abilities, from other genes. A comparison of trypanosomatid SL RNA gene promoter structures with that found in the nematode Ascaris lumbricoides underscores a taxonomic difference in promoter architectures which may reflect differential requirements for the SL RNA in these organisms.

Animals↗
PubMed1997

Characterization of two protein activities that interact at the promoter of the trypanosomatid spliced leader RNA.

All trypanosome mRNAs have a spliced leader (SL). The SL RNA gene in Leptomonas seymouri is a member of the small nuclear RNA gene family. However, the SL RNA is required in stoichiometric amounts for trans-splicing during mRNA formation. Expression of the SL RNA gene requires sequence elements at bp -60 to -70 and bp -30 to -40 upstream from the transcription initiation site. Using conventional and affinity chromatography, we have identified and characterized an approximately 122-kDa protein, promoter-binding protein (PBP) -1, that binds to double-strand DNA. The PBP-1-binding site is within the bp -60 to -70 element determined by DNase I footprinting. Therefore, PBP-1 is the first characterized double-strand DNA binding activity that interacts with a trypanosome gene promoter. A second protein, PBP-2, interacts with the PBP-1:DNA complex and its DNase I footprint extends to include the second promoter element (bp -30 to -40). An alteration of the spacing between the two promoter elements or mutation of the second element decreases PBP-2/PBP-1:DNA stability. Taken together, these data suggest that PBP-1 and PBP-2 are components of a transcription initiation complex that assembles within the SL RNA gene promoter.

Animals↗
PubMed1997

In vitro transcription of the Leptomonas seymouri SL RNA and U2 snRNA genes using homologous cell extracts.

A cell-free transcription system for the spliced leader (SL) RNA gene of the trypanosomatid Leptomonas seymouri has been developed. Accurately initiated transcription was achieved using cell extracts and a template in which the transcribed region of the SL RNA was replaced with a guanosine-less sequence (G-less cassette). The extract was also able to direct accurate initiation of RNA from an L. seymouri tagged U2 snRNA gene, which may be expressed via a transcriptional apparatus shared by the SL RNA gene. In vivo transcription analysis was used previously to define essential sequence components of the SL RNA gene promoter (Hartree D, Bellofatto V. Mol Biochem Parasitol 1995:71:27-39). A substitution mutation in the upstream promoter element (bp - 50 to - 70) markedly reduced transcription in vitro as did deletion of this and the middle promoter element (bp - 30 to - 40). Thus, the in vitro transcription system correctly responds to promoter mutations and is useful for investigating SL RNA and snRNA gene expression.

Animals↗
PubMed1996

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.

Animals↗
PubMed1995

Structure of the Leptomonas seymouri trans-spliceosomal U2 snRNA-encoding gene; potential U2-U6 snRNA interactions conform to the cis-splicing counterpart.

We have characterized the U2 small nuclear RNA (snRNA)-encoding gene from the monogenetic trypanosomatid, Leptomonas seymouri (Ls), to begin to identify the RNA-RNA interactions that direct trans-splicing in kinetoplastid protozoa. The U2 gene, which is single copy in this organism, was isolated and sequenced. Although the Ls U2 snRNA contains many of the sequence and secondary structure elements that are conserved among the U2 snRNAs of cis-splicing organisms, it lacks the stem-loop III region and the intron branch point-recognition region, as do other trypanosomatid U2 snRNAs. A transcriptional promoter element within the Trypanosoma brucei U2 gene [Fantoni et al., Mol. Cell. Biol. 14 (1994) 2021-2028] is conserved in the homologous Ls gene. A crucial step in cis-splicing reactions involves specific base-pairing interactions between the U2 and U6 snRNAs. We show here that in trypanosomatids, where no cis-splicing occurs, these same interactions are possible. This highlights key similarities between the two RNA processing events.

Animals↗
PubMed1995

Essential components of the mini-exon gene promoter in the trypanosomatid Leptomonas seymouri.

In members of the Trypanosomatidae family of parasitic protozoa, the mini-exon (MX) genes encode the mini-exon donor RNA (medRNA) that contributes a small, 39-nt exon to all pre-mRNAs during mRNA maturation. Previously we have shown that a single copy of a MX gene can be expressed continuously from a stable episome transfected into the monogenetic trypanosomatid Leptomonas seymouri. We now identify components of the MX gene promoter. A series of 10-bp block substitution mutations in a tagged MX gene were transfected into Leptomonas on an episomal vector. Expression of tagged and endogenous medRNA was assessed in stably transformed clonal cell populations. Results show that less than half of the 757-bp MX gene is necessary for medRNA transcription and that the key components of the MX gene promoter lie within the proximal 70-bp sequence upstream from the transcription initiation site. Transcription requires several sequence-specific blocks within this 70-bp region. Leptomonas cell extracts contain protein(s) that appear to interact with a subset of these sequences in gel mobility shift assays. All trypanosomatid MX genes contain an AT-rich region at the +10 to +20 position within the transcribed region of the MX gene. Mutagenesis of this region within an episomal copy of the MX gene did not block tagged medRNA synthesis but did cause a 10-fold increase in the steady-state amount of endogenous medRNA.

Animals↗
PubMed1993

Differential response to RNA trans-splicing signals within the phosphoglycerate kinase gene cluster in Trypanosoma brucei.

In trypanosomatids, nuclear pre-mRNA splicing is exclusively a trans-splicing reaction in which a capped, 39 nt exon, the mini-exon, is positioned 5' to an open reading frame. Differential RNA splicing might reflect specific mini-exon and 3' splice site interactions. To test this hypothesis, we compared the efficiency of mini-exon addition to three natural 3' splice acceptor sites (SASs) located within a single pre-mRNA transcript. In Trypanosoma brucei, the phosphoglycerate kinase A, B and C genes (PGK A, B and C) are co-expressed as three consecutive sequences on a polycistronic pre-mRNA. This pre-mRNA gives rise to unequal amounts of PGK A, B and C mRNAs. When the SAS from each gene was placed upstream of the luciferase open reading frame and the resultant constructs transiently transfected into T. brucei procyclic cells, luciferase activity levels indicated differential SAS utilization. Enzyme activity was low when the SAS from the A gene was present. Levels were indistinguishable when the B and C SASs were compared. After replacing luciferase with chloramphenicol acetyl transferase in the test constructs, enzyme activities were shown to directly correlate with mRNA amounts. Thus, poor splicing efficiency accounts for the differential expression of the PGK A mRNA during PGK pre-mRNA maturation. This reaction appears to reflect the polypyrimidine pattern within the 3' splice acceptor site.

Animals↗
PubMed1993

Leptomonas seymouri as a model system for the analysis of gene expression in trypanosomatids.

Leptomonas seymouri, a monogenetic trypanosomatid originally isolated from Dysdercus suturellus (Hemiptera), was used to develop a reverse genetic system for trypanosomatid flagellates. In many eukaryotic cell types, reverse genetics has proven to be a powerful tool for defining structure/function relationships within genes. The mini-exon genes of trypanosomatids encode key components of all cellular mRNAs. This component is a 5' "leader" RNA that is spliced onto all mRNA precursors during mRNA formation within the cell nucleus. The data presented here indicate that structure/function relationships within the mini-exon gene can be probed using the molecular genetic system developed and characterized for L. seymouri.

Animals↗
PubMed1991

Stable transformation of Leptomonas seymouri by circular extrachromosomal elements.

To define the cis-acting sequences necessary for gene expression and DNA replication in trypanosomatids, we have developed a selectable vector that can be grown in Escherichia coli and maintained stably in the insect trypanosomatid Leptomonas seymouri. The vector is relatively small (6 kilobase pairs) and contains a portion of the L. seymouri alpha-tubulin gene positioned in-frame with a truncated neomycin phosphotransferase gene that confers resistance to the aminoglycoside G418. This construct is maintained in cells as a high-copy-number circular extrachromosomal element containing several head-to-tail copies of the transforming plasmid. In L. seymouri, alpha-tubulin-neomycin phosphotransferase fusion RNAs are polyadenylylated and possess a trans-spliced mini-exon. Additional DNA sequences can be inserted into the vector, propagated, and expressed in transformed cells.

Animals↗
PubMed1990

The new trypanosomatid genetics.

The recent development of transfection systems for trypanosomatids has removed a major obstacle to research and provides an important tool for the biochemist, immunologist and molecular biologist. Obtaining expression of a foreign gene in a trypanosomatid has been difficult. In this review, Vivian Bellofatto describes the problems and pitfalls of the process and final successes achieved.

Journal Article↗
PubMed1990

Transcription of the procyclic acidic repetitive protein genes of Trypanosoma brucei.

The procyclic acidic repetitive protein (parp) genes of Trypanosoma brucei encode a small family of abundant surface proteins whose expression is restricted to the procyclic form of the parasite. They are found at two unlinked loci, parpA and parpB; transcription of both loci is developmentally regulated. The region of homology upstream of the A and B parp genes is only 640 base pairs long and may contain sequences responsible for transcriptional initiation and regulation. Transcription upstream of this putative promoter region is not developmentally regulated and is much less active than that of the parp genes; the polymerase responsible is inhibited by alpha-amanitin, whereas that transcribing the parp genes is not. Transcription of the parp genes is strongly stimulated by low levels of UV irradiation. The putative parp promoter, when placed upstream of the chloramphenicol acetyltransferase gene, is sufficient to cause production of chloramphenicol acetyltransferase in a T. brucei DNA transformation assay. Taken together, these results suggest that a promoter for an alpha-amanitin-resistant RNA polymerase lies less than 600 nucleotides upstream of the parp genes.

Animals↗
PubMed1989

Expression of a bacterial gene in a trypanosomatid protozoan.

A simple and reproducible assay for DNA-mediated transfection in the trypanosomatid protozoan Leptomonas seymouri has been developed. The assay is based on expression of the Escherichia coli chloramphenicol acetyl transferase (CAT) gene flanked by Leptomonas DNA fragments that are likely to contain necessary elements for gene expression in trypanosomes. After electroporation of cells in the presence of plasmid DNA, CAT activity was detected in crude cell lysates. No activity was detected when the orientation of the L. seymouri mini-exon sequence (placed upstream of the CAT gene) was reversed, or in additional control experiments. This system provides a method for defining transcriptional control elements in trypanosomes.

Animals↗
PubMed1988

Discontinuous transcription in Leptomonas seymouri: presence of intact and interrupted mini-exon gene families.

Mature mRNAs of trypanosomatid protozoa result from the joining of at least two exons, which are initially transcribed as separate RNAs. In all trypanosomatids examined to date, the first exon (mini-exon) is encoded by approximately 200 tandemly reiterated genes. In characterizing the mini-exon genes of Leptomonas seymouri, we identified two predominant size classes of repetitive sequences that hybridized strongly to the L. seymouri mini-exon sequence. These two sequences are arranged as interspersed clusters. DNA sequence analysis of a clone representing the smaller size class demonstrated that these sequences have the capacity to encode a mini-exon donor (med)RNA corresponding to the 86 nt component seen in Northern blots of L. seymouri RNA. The larger size class comprises a family of related sequences, some of which contain DNA inserted into the mini-exon portion of the medRNA gene. The specific insert identified here (LINS 1) is exclusively associated with medRNA sequences, and is present in approximately 20% of the larger size class of L. seymouri medRNA genes. Disregarding the insertion, the sequences of the smaller bona fide mini-exon genes and the gene copy containing the insert were almost identical. The insert sequence is transcribed in the same direction as medRNA to yield at least four small non-polyadenylated RNAs, which appeared not to be linked to medRNA sequences.

Animals↗