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N Agabian

Publications and source records attributed to N Agabian.

At least 37 records · Page 2Linked to original sources
PubMed1994

A common core structure for U3 small nucleolar RNAs.

U3 nucleolar small RNA (snRNA) is involved in early processing of the primary rRNA transcript. A secondary structure model for the unusually small Trypanosoma brucei U3 snRNA was deduced by chemical modification and enzymatic cleavage of U3 snRNA in deproteinized and ribonucleoprotein (RNP) forms. Comprehensive alignment of U3 snRNAs from vertebrate, plant, fungal and protozoan species clearly delineated conserved and divergent features. The 5' domain of the T. brucei U3 snRNA appears to form one small, flexible 5' stem loop structure followed by a long single-stranded region; this model is a variation on 5' domain structures proposed for other U3 snRNAs which do not conform to a single model. The 3' domain of T. brucei U3 snRNA contains four single-stranded sequences conserved between U3 snRNAs. Of these, structural probing determined that the configurations of GAU region and box B and C sequences are altered by protein interactions in U3 snRNP. Conspicuously, the 3' domains of trypanosomal U3 snRNAs lack stem loops II and III, indicating that these structures are not required for conserved U3 snRNA functions.

Animals↗
PubMed1994

Mitochondrial minicircle DNA supports plasmid replication and maintenance in nuclei of Trypanosoma brucei.

In a search for trypanosome DNA sequences that permit replication and stable maintenance of extrachromosomal elements, a 1-kilobase-pair (kbp) fragment from a mitochondrial kinetoplast DNA (kDNA) minicircle of Trypanosoma brucei was isolated and characterized. The plasmid pTbo-1, carrying the kDNA element, is maintained in T. brucei as a supercoiled concatemer containing approximately seven to nine pTbo-1 monomer units (5.6 kbp each) in a head-to-tail orientation. The concatemer is found in approximately one copy per cell when procyclic trypanosomes are cultured in the presence of 100 micrograms of hygromycin per ml; however, in the absence of continuous hygromycin selection, the plasmid is lost from the population with a t1/2 of approximately 8.7 days (17 cell generations). A second unrelated kDNA minicircle was also able to serve as an autonomously replicating sequence (ARS) element in T. brucei, suggesting that this is a general property of kDNA minicircles. Replication of mitochondrial DNA in the nucleus may be due to either a specific consensus sequence (such as in yeast ARS elements) or nonspecific sequence characteristics (such as the degree of A&T-richness or bent DNA).

Animals↗
PubMed1994

Trypanosoma brucei RNA polymerase II is phosphorylated in the absence of carboxyl-terminal domain heptapeptide repeats.

Formation of an RNA polymerase II transcription initiation complex requires binding of a polymerase that contains a non-phosphorylated largest subunit carboxyl-terminal domain (CTD). Polymerase binding is followed by elongation after phosphorylation of the CTD by a CTD kinase. Phosphorylation sites are within the repeating heptapeptide motifs which characterize the CTD of all eukaryotic RNA polymerase IIs. In contrast to all other eukaryotes studied, the trypanosome genome contains two genetic loci which encode the large subunit of RNA polymerase II; both genes lack CTD heptapeptide repeat structures. We have examined whether Trypanosoma brucei RNA polymerase II, despite its unique CTD domain, is phosphorylated when isolated from elongating transcription complexes. Elongating trypanosome RNA polymerases were photoaffinity labeled during nuclear run-on assays. The identity of the labeled proteins was established by immunoblotting and immunoprecipitation using polymerase-specific antisera. Analysis of the largest subunit of RNA polymerase II revealed the expected 195-kDa species and an additional larger 220-kDa species. The apparent molecular weight of this larger form of RNA polymerase II decreased incrementally as a function of incubation with increasing concentrations of calf intestinal phosphatase. These results show that extensive phosphorylation of the largest subunit of RNA polymerase-II is a conserved feature between trypanosomes and higher eukaryotes despite the absence of a typical CTD domain.

Affinity Labels↗
PubMed1994

Identification of a small RNA that interacts with the 5' splice site of the Trypanosoma brucei spliced leader RNA in vivo.

In vivo psoralen cross-linking of the trypanosome spliced leader (SL) RNA has led to the discovery of a small RNA that we provisionally call the spliced leader-associated (SLA) RNA. The 72 nt SLA RNA is unlike any known small RNA except for a small region that resembles U5 snRNA. The SL/SLA RNA cross-links map to two regions, the predominant interactions occurring between the 5' splice site region of the SL RNA and a CUUUUA sequence in the SLA RNA. The resemblance between these cross-links and interactions of U5 snRNA with cis-spliced pre-mRNAs suggests that the SLA RNA may be the trans-splicing analog of U5 snRNA in trypanosomes.

Animals↗
PubMed1994

Characterization of putative small nuclear RNAs from Giardia lamblia.

Small nuclear RNAs (snRNAs), which have roles in RNA metabolism, have been found in all eukaryotic cells. Candidate snRNAs were identified in the primitive protozoan parasite, Giardia lamblia, by either immunoprecipitation of total RNA with antiserum directed against the 2,2,7-trimethylguanosine cap structure characteristic of snRNAs or by Northern hybridization with oligonucleotide probes complementary to snRNA consensus sequences. Isolated putative snRNAs include eight 2,2,7-trimethylguanosine-capped species: RNAs A through H, and one non-2,2,7-trimethylguanosine-capped species: RNA J. Single copy genes encoding RNA D, RNA H and RNA J were cloned and sequenced, and the 5' end of each RNA was determined by primer extension analysis. Certain characteristics suggest tentative identification of these Giardia RNAs as nucleolar RNAs with possible roles in rRNA processing.

Animals↗
PubMed1994

A fourth secreted aspartyl proteinase gene (SAP4) and a CARE2 repetitive element are located upstream of the SAP1 gene in Candida albicans.

Candida albicans secreted aspartyl proteinases (Sap), products of the SAP genes, which are presumed to act as virulence factors. In the C. albicans strain WO-1, the ability to secrete Sap1 is regulated with switch phenotype, another putative virulence factor. KpnI restriction fragment length polymorphisms differentiate between several distinct SAP1 alleles in laboratory and clinical strains. Both SAP1 alleles from strain WO-1 along with their 5'- and 3'-flanking regions were cloned and sequenced, as were both alleles from another strain, SS. The 5'-flanking regions were remarkably similar in all four of the sequenced alleles over approximately 1,500 nucleotides. S1 analysis revealed that both alleles of WO-1 are transcribed. Characterization of the one allele from strain WO-1 identified a 284-nucleotide insertion flanked by 8-bp direct repeats that shows homology to the CARE2 repetitive element and that is not present in the other alleles. Characterization of the SAP1 alleles also identified a fourth SAP gene (SAP4) that includes an extended leader sequence. SAP4 is positioned upstream, in tandem to SAP1, in all strains tested and may encode another closely related secreted aspartyl proteinase.

Alleles↗
PubMed1994

A nuclear encoded tRNA of Trypanosoma brucei is imported into mitochondria.

The mitochondrial genome of trypanosomes, unlike that of most other eukaryotes, does not appear to encode any tRNAs. Therefore, mitochondrial tRNAs must be either imported into the organelle or created through a novel mitochondrial process, such as RNA editing. Trypanosomal tRNA(Tyr), whose gene contains an 11-nucleotide intron, is present in both the cytosol and the mitochondrion and is encoded by a single-copy nuclear gene. By site-directed mutagenesis, point mutations were introduced into this tRNA gene, and the mutated gene was reintroduced into the trypanosomal nuclear genome by DNA transfection. Expression of the mutant tRNA led to the accumulation of unspliced tRNA(Tyr) (A. Schneider, K. P. McNally, and N. Agabian, J. Biol. Chem. 268:21868-21874, 1993). Cell fractionation revealed that a significant portion of the unspliced mutant tRNA(Tyr) was recovered in the mitochondrial fraction and was resistant to micrococcal nuclease treatment in the intact organelle. Expression of the nuclear integrated, mutated tRNA gene and recovery of its gene product in the mitochondrial fraction directly demonstrated import. In vitro experiments showed that the unspliced mutant tRNA(Tyr), in contrast to the spliced wild-type form, was no longer a substrate for the cognate aminoacyl synthetase. The presence of uncharged tRNA in the mitochondria demonstrated that aminoacylation was not coupled to import.

Animals↗
PubMed1993

Ribosomal protein L25 from Trypanosoma brucei: phylogeny and molecular co-evolution of an rRNA-binding protein and its rRNA binding site.

The gene encoding ribosomal protein L25, a primary rRNA-binding protein, was isolated from the protozoan parasite Trypanosoma brucei. Hybridization studies indicate that multiple copies of the gene are present per T. brucei haploid genome. The C-terminal domain of L25 protein from T. brucei is strikingly similar to L23a protein from rat, L25 proteins from fungal species, and L23 proteins from eubacteria, archaebacteria, and chloroplasts. A phylogenetic analysis of L23/25 proteins and the putative binding sites on their respective LSU-rRNAs (large subunit rRNAs) provides a rare opportunity to study molecular co-evolution between an RNA molecule and the protein that binds to it.

Amino Acid Sequence↗
PubMed1993

Splicing and 3'-processing of the tyrosine tRNA of Trypanosoma brucei.

Trypanosoma brucei belongs to a family of protozoa that is characterized as having diverged early in the evolution of eukaryotes. Many unusual forms of RNA processing have been discovered in these cells, one of which is trans-splicing, and, although a great number of genes have been sequenced, no evidence for cis-splicing has yet been found. This study shows that tRNA(Tyr) of T. brucei contains an 11-nucleotide intron. tRNA of the size predicted for unspliced precursor was detected by Northern analysis using an oligonucleotide probe complementary to the putative intervening region. Direct sequence analysis of mature tRNA(Tyr) showed that the predicted intron region is absent from this form of the molecule. Mutated versions of the gene encoding tRNA(Tyr) were introduced into trypanosomes by DNA transformation and shown to be expressed in vivo. Introduction into the genome of a tRNA(Tyr) gene containing an amber suppressor mutation led to a significant accumulation of unspliced precursor molecules. Analysis of the tRNA(Tyr) species from both the wild type and transformed cells revealed unspliced and spliced processing intermediates. The relative abundance of each of these intermediates suggests that 3'-processing and splicing occur independently and that, in the wild type, splicing tends to precede 3'-processing and CCA addition.

Animals↗
PubMed1993

Trypanosoma vivax: evidence for only one RNA polymerase II largest subunit gene in a trypanosome which undergoes antigenic variation.

Previous studies suggested a correlation between antigenic variation in Kinetoplastida and the presence of two RNA polymerase (pol) II largest subunit genes in these organisms. We have found that Trypanosoma vivax, an African trypanosome which undergoes antigenic variation, is an exception, and has only one pol II largest subunit gene, indicating that probably neither of the two pol II genes found in other African trypanosomes is uniquely required for antigenic variation.

Animals↗
PubMed1993

Three distinct secreted aspartyl proteinases in Candida albicans.

The secreted aspartyl proteinases of Candida albicans (products of the SAP genes) are thought to contribute to virulence through their effects on Candida adherence, invasion, and pathogenicity. From a single strain of C. albicans (WO-1) which expresses a phenotypic switching system, three secreted aspartyl proteinases have been identified as determined by molecular weight and N-terminal sequence. Each of the three identified proteins represents the mature form of one of three distinct proteinase isoenzymes, two of which correspond to the recently cloned SAP1 and SAP2 genes (previously referred to as CAP, PEP, or PRA). A genomic library was screened under low-stringency hybridization conditions with a polymerase chain reaction fragment from SAP1. In addition to clones of SAP1 and SAP2, a clone containing SAP3, a novel third secreted proteinase gene, was identified and sequenced. The three aspartyl proteinase isoenzymes differ in primary sequence and pI, suggesting that they may play different roles in virulence and pathogenesis. All three of these proteinases are expressed in the same strain. However, the pattern of proteinase expression is correlated with the switch phenotype of the cell. Opaque cells of strain WO-1 express Sap1 and Sap3, while white cells of the same strain express Sap2. The differential expression of three Sap proteinases may contribute to virulence in C. albicans.

Amino Acid Sequence↗
PubMed1993

Characterization and partial nucleotide sequence of the DNA fingerprinting probe Ca3 of Candida albicans.

The moderately repetitive Ca3 fragment of Candida albicans has been used as an effective DNA fingerprinting probe in epidemiological studies. EcoRI digestion of Ca3 DNA results in seven fragments of 4.2 kb (A), 2.98 kb (B), 2.85 kb (C), 0.77 kb (D1), 0.77 kb (D2), 0.38 kb (E), and 0.30 kb (F). Five of these EcoRI fragments have been mapped in the 5'-3' order C B D1 A D2. The intact Ca3 probe and the three largest EcoRI fragments, A, B, and C, were individually used to probe Southern blots of EcoRI-digested DNA of a set of test strains, transverse alternating field electrophoresis-separated chromosomes of strain 3153A, and Northern (RNA) blots of test strain 3153A. Fragments, A, B, and C each generate a different Southern blot hybridization pattern with EcoRI-digested whole-cell DNA; Ca3 sequences are present in at least five of seven separable chromosomes and a minichromosome of strain 3153A; fragments A, B, and C are distributed differently on chromosomes; and fragments A, B, and C do not cross-hybridize. Ca3 hybridizes to three major transcripts of 2.8, 2.3, and 1.5 kb. Fragment A hybridizes intensely to the 1.5-kb transcript, while fragments B and C both hybridize intensely to the 2.8- and 2.3-kb transcripts. The B fragment, which contains 2,980 bp and contributes to the major portion of the Ca3 pattern, was sequenced. Both direct and inverted repeat sequence motifs were identified. These results provide us with initial insights into the evolution of the Ca3 pattern and the nature of the probe.

Base Sequence↗
PubMed1993

RNA B is the major nucleolar trimethylguanosine-capped small nuclear RNA associated with fibrillarin and pre-rRNAs in Trypanosoma brucei.

RNA B is one of three abundant trimethylguanosine-capped U small nuclear RNAs (snRNAs) of Trypanosoma brucei which is not strongly identified with other U snRNAs by sequence homology. We show here that RNA B is a highly diverged U3 snRNA homolog likely involved in pre-rRNA processing. Sequence identity between RNA B and U3 snRNAs is limited; only two of four boxes of homology conserved between U3 snRNAs are obvious in RNA B. These are the box A homology, specific for U3 snRNAs, and the box C homology, common to nucleolar snRNAs and required for association with the nucleolar protein, fibrillarin. A 35-kDa T. brucei fibrillarin homolog was identified by using an anti-Physarum fibrillarin monoclonal antibody. RNA B and fibrillarin were localized in nucleolar fractions of the nucleus which contained pre-rRNAs and did not contain nucleoplasmic snRNAs. Fibrillarin and RNA B were precipitated by scleroderma patient serum S4, which reacts with fibrillarins from diverse organisms; RNA B was the only trimethylguanosine-capped RNA precipitated. Furthermore, RNA B sedimented with pre-rRNAs in nondenaturing sucrose gradients, similarly to U3 and other nucleolar snRNAs, suggesting that RNA B is hydrogen bonded to rRNA intermediates and might be involved in their processing.

Animals↗
PubMed1992

The 7SL RNA homologue of Trypanosoma brucei is closely related to mammalian 7SL RNA.

In eukaryotes, protein translocation across the endoplasmic reticulum is mediated by a signal recognition particle, a small ribonucleoprotein (RNP) containing 7SL RNA. We have cloned and sequenced the gene coding for the Trypanosoma brucei 7SL RNA homologue and found that its sequence shows the highest degree of similarity to the human 7SL RNA sequence. In keeping with the prototype secondary structure of eukaryotic 7SL RNA, the trypanosome 7SL RNA secondary structure can be folded into four domains. The 7SL RNP, which sediments at approximately 11S on sucrose density gradients, was partially purified using column chromatography. A particle containing a 76-nucleotide-long RNA co-purified with the 7SL RNP; however, these particles did not co-fractionate by non-denaturing polyacrylamide gel electrophoresis.

Animals↗
PubMed1992

Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo.

The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.

Adenosine↗
PubMed1992

Low risk of invasive amebiasis in cyst carriers. A longitudinal molecular seroepidemiological study.

A seroepidemiological study of a household cohort, using both clinical observational and molecular criteria was conducted in a periurban area endemic for E. histolytica infection. This longitudinal study was undertaken to determine the risk of asymptomatic cyst carriers to develop invasive illness. Zymodeme patterns of strains isolated from these patients were correlated both with the clinical presentation of disease and with the serological response against the M-17 ameba antigen and further compared with that found in 16 proven cases of amebic liver abscess. From a total of 163 housewives screened, 39, (24%) were asymptomatic cyst carriers; 31 of them (index cases) and 114 members of their households remained in the study over an 8 month follow-up period to detect ameba infection and illness. Of the household members at risk, 46 (40%) became infected within 6 weeks. None of the index or secondary cases developed ameba-related symptoms and cyst excretion followed a chronic persistent, intermittent, or transient pattern over the period of the study. Amebas were recovered and zymodemes determined in 19 of 71 (27%) cyst carriers. Ameba shed from each of these 19 carriers exhibited nonpathogenic zymodeme 1, except for one index case where zymodeme 2 was recovered in one sampling, and returned to zymodeme 1 in subsequent samples. Of 48 of 71 cyst carriers studied, antibodies to crude E. histolytica antigen were detected by ELISA in 16 (31%); antibodies to the M-17 fusion protein were found in 8 (16%) by ELISA and in 2 (4%) by Western-Blot (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗