PubMed Health⌕ Search

Biomedical subjects

N Agabian

Publications and source records attributed to N Agabian.

At least 19 recordsLinked to original sources
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↗
PubMed1992

The identification and tracking of Candida albicans isolates from oral lesions in HIV-seropositive individuals.

Restriction fragment polymorphism analysis was used to investigate the identity and genotypic relatedness of Candida albicans strains isolated from human immunodeficiency virus (HIV)-infected patients with or without oral candidiasis and from some of their sexual partners. Use of the species-specific DNA probe Ca3 revealed that most subjects carried a single distinct C. albicans strain throughout the course of the study, during both symptomatic and asymptomatic periods. Sexual partners were more likely to carry the same or similar C. albicans isolates than unrelated subjects, raising the possibility of transmission via intimate contact. One patient appeared to acquire his partner's isolate, which then became predominant in both partners in subsequent isolations. These findings indicate that recurrent oral candidiasis is usually caused by a single persistent strain unique to each patient, but that in some cases transmission via intimate contact may occur between sexual partners.

AIDS-Related Opportunistic Infections↗
PubMed1991

mRNA processing in the Trypanosomatidae.

Members of the Trypanosomatidae, which include the African trypanosomes, the American trypanosomes and the leishmanias, cause disease in vast proportions in man and his livestock and are a major detrimental factor to the social and economic well-being of the third world. Current research using the techniques of molecular biology has revealed two unusual types of mRNA processing in these protozoans; these are the addition of a shared leader sequence to the 5' ends of nuclear mRNAs by a mechanism of trans splicing, and the insertion and deletion of specific uridine residues in mitochondrial transcripts by RNA editing. The presence of these two mRNA processing pathways in the Trypanosomatidae has profound consequences for the organization and expression of their genetic information.

Amino Acid Sequence↗
PubMed1991

In vivo UV cross-linking of U snRNAs that participate in trypanosome trans-splicing.

The maturation of mRNAs in Trypanosoma brucei involves a trans-splicing reaction whereby the 5' 39 nucleotides of a small RNA, called the spliced leader (SL) RNA, are joined with a pre-mRNA transcript. The trans-splicing reaction appears mechanistically similar to cis-splicing of nuclear pre-mRNAs, and homologs of the U2, U4, and U6 snRNAs are required for the process. In the work presented here, potential RNA-RNA interactions between the SL RNA and the U snRNAs of trypanosomes were examined by UV light induction of RNA-RNA cross-links in vivo. We detected cross-linkage between U2 and U6 RNAs and, as might be expected, between the trypanosome U4 and U6 RNAs. The latter contain extensive sequence complementarity and are thought to exist predominantly in a single RNP. We also detected an SL RNA species following in vivo UV treatment, which may represent either an intramolecular cross-link in the SL RNA or a cross-link formed between the SL RNA and an as yet unidentified small RNA. Mapping of the cross-link position between U2 and U6 RNAs is consistent with base-pairing between the 5' domain of U2 and the 3' end of U6 RNA. These results reveal the existence, in vivo, of cognate RNA-RNA interactions in the RNA homologs that participate in trans-splicing in trypanosomes and cis-splicing in other eukaryotes.

Animals↗
PubMed1990

Characterization of an immuno-dominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolytica.

A 125-kD surface antigen of Entamoeba histolytica is recognized by 73% of immune sera from patients with amoebic liver abscesses. Using pooled human immune sera a cDNA clone (lambda cM17) encoding this antigen (M17) has been isolated from a lambda gt11 expression library of the virulent stain E. histolytica HM1:IMSS. Monospecific antibodies, purified by binding to phage lysate of lambda cM17, and mAb FA7 reacted exclusively with the 125-kD antigen by Western blot analysis. Surface binding and cap formation are observed with patient sera, purified monospecific antiserum, and mAb FA7. Corresponding genomic clones (pBSgM17-1/2/3) were isolated by hybridization with the cDNA clone. These contained an open-reading frame of 3345 bp, which is in good agreement with the mRNA size of approximately 3.0 kb as revealed by Northern hybridization with lambda cM17. The inferred amino acid sequence predicts a 125,513 dalton protein that contains 17 potential N-linked glycosylation sites and is unusually rich in tyrosine and asparagine residues. A distinctly hydrophobic NH2-terminal region may serve as membrane anchor or signal sequence. In contrast to conservation of an immunodominant epitope recognized in pathogenic and nonpathogenic strains by monoclonal FA7 and human immune sera, amplification and sequence analysis of a 1,4000-bp fragment of this gene from a fresh nonpathogenic isolate by use of the PCR demonstrate regions of significant sequence divergence in this antigen. A 1% sequence variability among different isolates of the pathogenic strain HM1:IMSS and a 12-13% variability between pathogenic and nonpathogenic strains are revealed by comparison to published partial amino acid sequences (Tannich, E., R.D. Horstmann, J. Knobloch, and H.H. Arnold. 1989. Proc. Natl. Acad. Sci. USA. 86:5118). Some restriction enzymes were found that allowed PCR diagnosis of nonpathogenic and pathogenic isolates with the exclusion of E. histolytica-like Laredo, suggesting that a detailed study of nonpathogenic and pathogenic isolates in relation to the M17 antigen sequence will provide a basis of differentiating isolates.

Amebiasis↗
PubMed1990

Isolation of distinct small ribonucleoprotein particles containing the spliced leader and U2 RNAs of Trypanosoma brucei.

Messenger RNA maturation in trypanosomes involves an RNA trans-splicing reaction in which a 39 nucleotide 5'-spliced leader (SL), derived from an independently transcribed 139 nucleotide SL RNA, is joined to pre-mRNAs. Trans-splicing intermediates are structurally consistent with a mechanism of SL addition which is similar to that of cis-splicing of nuclear pre-mRNAs; homologous components (e.g. the U small nuclear RNAs) exist in both cis- and trans-splicing systems, suggesting that these also participate in the two types of splicing reactions. In this study, ribonucleoprotein (RNP) complexes containing the trypanosome SL and U2 RNAs were purified and characterized. Although present at low levels in cellular extracts, the SL and U2 RNPs are the two most abundant of the several non-ribosomal small RNP complexes in these cells. The purification scheme utilizes ion-exchange chromatography, equilibrium density centrifugation, and gel filtration chromatography and reveals that the SL RNP shares biophysical properties with U RNPs of trypanosomes and other eukaryotes; its sedimentation coefficient in sucrose gradients is approximately 10 S, and it is resistant to dissociation during Cs2SO4 equilibrium density centrifugation. Complete separation of the SL and U2 RNPs was achieved by non-denaturing polyacrylamide gel electrophoresis. Proteins purifying with the SL and U2 RNPs were identified by 125I-labeling of tyrosine residues. Four SL RNP proteins with approximate molecular masses of 36, 32, 30, and 27 kDa and one U2 RNP protein of 31 kDa were identified, suggesting that different polypeptides are associated with these two RNAs. These particles are not immunoprecipitated by anti-Sm sera which recognizes U snRNP proteins of other eukaryotes including humans plants and yeast.

Animals↗
PubMed1990

A Trypanosoma brucei small RNP particle containing the 5S rRNA.

In mammalian cells, approximately 50% of the 5S rRNA is found in ribosomes, and the remainder in a small particle, the 5S rRNA/ribosomal protein L5 complex, which is thought to be a precursor in ribosome assembly. Trypanosoma brucei, an African trypanosome, is one of the most primitive eukaryotic organisms which have been studied, and it likewise possesses a 5S rRNA species, a small proportion of which is found in an apparent ribonucleoprotein-(RNP) complex. Like the mammalian RNP particle, the T. brucei particle has a sedimentation coefficient of about 7S in sucrose gradients; unlike its mammalian counterpart, the complex is not disrupted by high salt and can be fractionated in cesium sulfate density gradients at a density characteristic of RNP complexes (1.45 g ml-1). Our studies demonstrate the the T. brucei 7S RNP contains 5S rRNA in association with a 36-kDa rRNA binding protein which not only shares molecular size, but also immunological determinants, with the yeast ribosomal protein YL3, and its mammalian homologue, L5. These results indicate that the RNP complex formed between the 5S rRNA and the 36-kDa ribosomal protein is conserved throughout great evolutionary distances between eukaryotic species.

Animals↗
PubMed1990

Bloodstream and metacyclic variant surface glycoprotein gene expression sites of Trypanosoma brucei gambiense.

Trypanosoma brucei gambiense is the causative agent of chronic human sleeping sickness. Previous studies have indicated that T. b. gambiense isolates expressed the antigens U1 or L2 in both the metacyclic and early bloodstream form of the parasite life cycle. These studies suggested that L2 and U1 were likely to be metacyclic variant surface glycoproteins (mVSG). The basic copies of the genes encoding the VSGs L2 and U1 are present in single copy in non-expressing isolates of T. b. gambiense. Furthermore, they have been found to be maintained stably in a large number of stocks isolated from a wide geographic area over a 30-year period. The genomic DNA comprising the upstream 5' flanking regions of the U1 and L2 putative mVSG gene expression sites have been cloned from bloodstream forms of T. b. gambiense. The L2 expression site clone, containing 12.5 kb of sequences 5' to the VSG gene, was found to lack the 72/76-bp repeat unit generally found in the 'barren' region upstream of bloodstream form expression sites. The U1 expression site clone, containing 13.5 kb of the 5' flanking region, appeared to have the repeats, which were localized to 2 kb of DNA immediately 5' to the U1 mVSG gene. Neither the U1 nor the L2 clone was found to have ESAG2 or ESAG3 gene sequences, but both were found to have ESAG1 genes. The ESAG1 genes from the putative metacyclic expression sites and from the U1 and L2 bloodstream form expression sites (in the form of cDNA clones) were sequenced and compared to all other published ESAG1 sequences.

Amino Acid Sequence↗
PubMed1990

A new U2 RNA secondary structure provided by phylogenetic analysis of trypanosomatid U2 RNAs.

A new model of U2 RNA secondary structure has been developed by comparing U2 RNA sequences from distantly related trypanosomatids, which process their RNAs by trans-splicing, and U2 RNAs from cis-splicing organisms. The trypanosomatid U2 RNA retains structural similarity in important functional domains of cis-splicing U2 RNAs yet differs from previous consensus models in that only two helices, rather than three, can form in the stem-loop II region. This alteration eliminates the capacity for pseudoknot formation and produces a single-stranded region 3' to stem-loop II, which may be accessible for snRNP protein binding. Trypanosomatid U2 RNAs differ in the putative branchpoint recognition sequences, which completely diverge from the conserved GUAGUA consensus of cis-splicing organisms.

Animals↗
PubMed1989

Molecular characterization of the Trypanosoma brucei RNA polymerase I and III largest subunit genes.

We have sequenced a 7.1-kilobase fragment of the Trypanosoma brucei RNA polymerase (pol) I largest subunit gene. The 6.9-kilobase transcript from this gene is of roughly equal abundance in bloodstream- and procyclic-form trypanosomes and has a 39-nucleotide trans-spliced leader 290 nucleotides upsteam of the putative initiation codon. The 1781-amino acid trypanosome pol I polypeptide is considerably less similar to its yeast homolog than the trypanosome pol II and pol III polypeptides are to their counterparts in yeast. The yeast and trypanosome pol I polypeptides are distinguished from pol II and pol III largest subunits by two inserts, 100-200 amino acids in length, which are found in the nonconserved portions of the polypeptides. We also report our sequence of the trypanosome pol III largest subunit gene, which agrees with that reported by Köck et al. (Köck, J., Evers, R., and Cornelissen, A.W.C.A. (1988) Nucleic Acids Res. 16, 8753-8772) except for 13 nucleotide differences. Four spliced leader addition sites between 60 and 99 nucleotides upstream of the AUG initiation codon were observed for the 5.4-kilobase transcript from this gene. Four short stretches of amino acid homology between the yeast and trypanosome pol III largest subunit polypeptides were identified which appear to be specific to this class of RNA polymerase; only one short pol I-specific sequence was identified in our comparison of the yeast and trypanosome pol I largest subunit polypeptides.

Amino Acid Sequence↗
PubMed1989

Two variant surface glycoprotein genes distinguish between different substrains of Trypanosoma brucei gambiense.

Trypanosoma brucei gambiense differs from other T. brucei subspecies in the stability and conservation of its bloodstream form antigenic repertoire. Two variant surface glycoprotein (VSG) cDNA clones corresponding to the antigens U1 and L2 were isolated from T. b. gambiense bacteriophage lambda gt11 expression libraries and characterized. A third VSG cDNA clone, P1, was also examined. The L2 and U1 VSG genes are present in a large number of T. b. gambiense stocks isolated over a thirty-year period from different geographical areas of Africa, suggesting that they are stably maintained in the T. b. gambiense genome. These probes may be useful in epidemiological studies of Gambian sleeping sickness to differentiate between T. b. gambiense isolates.

Animals↗
PubMed1989

In trypanosomes the homolog of the largest subunit of RNA polymerase II is encoded by two genes and has a highly unusual C-terminal domain structure.

We have isolated the genes encoding the largest subunit of all three classes of RNA polymerase from Trypanosoma brucei. While the pol II largest subunit is encoded by a single gene in all organisms examined to date, trypanosomes contain two copies of the gene. Both genes are expressed in the procyclic and bloodstream stages of the trypanosome life cycle. The two pol II genes differ from one another in their coding sequences by 21 silent substitutions and 4 amino acid substitutions. In the core part of the large subunit, the predicted polypeptides are similar to other eukaryotic RNA polymerases. Both trypanosome pol II polypeptides, like those of other eukaryotes, also have a unique C-terminal extension. However, this domain in the trypanosome polypeptides, unlike those of other eukaryotes, is not a tandemly repeated heptapeptide sequence.

Amino Acid Sequence↗
PubMed1989

A transcriptional analysis of the Trypanosoma brucei hsp83 gene cluster.

Ten to twelve copies of the 83-kDa heat-shock protein gene (hsp83) from Trypanosoma brucei are arranged in a head-to-tail tandem array of 2.8-kb repeat units, which are transcribed to give 2.6-kb mature mRNAs. We have cloned and sequenced one of the repeat units. The gene encodes a putative protein of 81 kDa which is highly homologous to Hsp83 of Drosophila melanogaster (75%), Hsp90 of Saccharomyces cerevisiae (72%) and the C62.5 protein of Escherichia coli (61%). The 5' end of the mature mRNA was mapped by primer extension sequence analysis and shown to contain the spliced leader. The mapping of the 3' poly(A) addition sites by S1 analysis indicated that there is 218 nt of intergenic sequence linking the boundaries encoding the mature mRNA. Within this sequence are a number of elements conserved with the trypanosome hsp70 intergenic region, including a 14-nt sequence that also has homology to the Drosophila heat-shock consensus element.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum.

We have used a combination of nucleotide and N-terminal-amino-acid-sequence analyses to determine the primary structure of the 37-kilodalton (kDa) endoflagellar outer layer, or sheath, protein. Initially, a lambda gt11 clone (designated lambda A34) expressing a portion of the 37-kDa protein was selected from a Treponema pallidum genomic library with a murine monoclonal antibody (H9-2) directed against an epitope of the 37-kDa protein. The insert from lambda A34 provided a probe with which a chimeric plasmid (pR14) encoding all but the nine N-terminal amino acids of the entire protein was selected from a T. pallidum(pBR322) genomic library. The nine N-terminal amino acids determined by amino acid sequencing were combined with the DNA sequence encoded by pR14 to determine the primary structure of the entire 37-kDa protein; the combined sequence made up a polypeptide with a calculated molecular mass of 36,948 Da. Approximately one-third of the deduced sequence was confirmed by N-terminal amino acid analysis of tryptic peptides from the purified 37-kDa protein. Repeated attempts to clone upstream portions of the gene (flaA) by using a variety of strategies were unsuccessful, suggesting that unregulated expression of the intact sheath protein or of its most amino-terminal portions is toxic in Escherichia coli. These studies should provide the basis for further molecular investigations of the endoflagellar apparatus and of treponemal motility.

Amino Acid Sequence↗