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N Agabian

Publications and source records attributed to N Agabian.

At least 91 records · Page 5Linked to original sources
PubMed1984

The IsTat 1.3 VSG multigene family in Trypanosoma brucei: retention of the expression linked copy through multiple antigenic switches.

In the IsTaR 1 serodeme of T. brucei the 3 variant surface glycoprotein (VSG) gene family contains about 10 members, one of which has a telomeric location on a minichromosome. The expression linked copy (ELC) of the 3 VSG gene which occurs in an antigenic variant expressing the 3 VSG, also has a telomeric location but unlike the minichromosomal 3 VSG gene has restriction sites upstream from the 5' barren region. This ELC is retained on the same telomere in a subsequent variant that expresses a telomeric 7 VSG ELC and in relapse variants and procyclic forms derived from variant antigenic types (VATs) 3 and 7. The 7 ELC has a restriction map upstream from the 5' barren region that differs from, but is similar to, that of the 3 ELC. These data indicate that the 3 and 7 ELCs are on different telomeres when expressed.

Animals↗
PubMed1984

Antigenic variation in African trypanosomes by gene replacement or activation of alternate telomeres.

We have analyzed antigenic variants with a known lineage and show that there are several telomeres on which variant surface glycoprotein (VSG) genes can be expressed. These telomeres have similar restriction maps 5' to the barren region. In addition, the same VSG gene was expressed on different telomeres. Some antigenic switches in the lineage were accomplished by duplicative replacement of one VSG gene with another. Other switches occurred without duplication by transcriptional activation of an alternate telomeric VSG gene. We call the latter process telomeric activation and propose that these two processes can occur independently. We further propose that antigenic switching by telomeric activation is mediated by the regulatory system that controls which telomere is transcriptionally active, while the duplicative mechanism does not.

Animals↗
PubMed1984

Identification of a small RNA containing the trypanosome spliced leader: a donor of shared 5' sequences of trypanosomatid mRNAs?

The 35 nucleotide spliced leader (SL) sequence is found on the 5' end of numerous trypanosome mRNAs, yet the tandemly organized reiteration units encoding this leader are not detectably linked to any of these structural genes. Here we report the presence of a class of discrete small SL RNA molecules that are derived from the genomic SL reiteration units of Trypanosoma brucei, Trypanosoma cruzi, and Leptomonas collosoma. These small SL RNAs are 135, 105, and 95 nucleotides, respectively, and contain a 5'-terminal SL or SL-like sequence. S1 nuclease analyses demonstrate that these small SL RNAs are transcribed from continuous sequence within the respective SL reiteration units. With the exception of the SL sequence and a concensus donor splice site immediately following it, these small RNAs are not well conserved. We suggest that the small SL RNAs may function as a donor of the SL sequence in an intermolecular process that places the SL at the 5' terminus of many trypanosomatid mRNAs.

Animals↗
PubMed1984

Trypanosome mRNAs share a common 5' spliced leader sequence.

A 5'-terminal leader sequence of 35 nucleotides was found to be present on multiple trypanosome RNAs. Based on its representation in cDNA libraries, we estimate that many, if not all, trypanosome mRNAs contain this leader. This same leader was originally identified on mRNAs encoding the molecules responsible for antigenic variation, variant surface glycoproteins. Studies of selected cDNAs containing this leader sequence revealed that leader-containing transcripts can be stage-specific, stage-regulated, or constitutive. They can be abundant or rare, and transcribed from single or multigene families. No linkage between the genomic leader sequences and the structural gene exons was observed. Possible mechanisms by which the leader sequences are added to trypanosome mRNAs are discussed.

Amino Acid Sequence↗
PubMed1984

Expression of a Trypanosoma brucei brucei variant antigen in Escherichia coli.

A cDNA library derived from antigenically homogeneous bloodstream stage Trypanosoma brucei brucei was screened with an antiserum directed against the variant surface antigen (VSA) using an enzyme-linked filter immunoassay. Several recombinant clones were detected and the clone giving the most intense reaction was further analyzed. It contained a VSA-specific cDNA insert and synthesized a protein of the expected molecular weight bearing VSA determinants. The nucleotide sequence of the insert was determined and shown to have the unusual codon bias characteristic of T. brucei VSAs, frequently employing codons specifying tRNAs rare in Escherichia coli. These results indicate that a codon bias very different from that of E. coli does not preclude the expression of a cloned sequence to detectable levels in this heterologous host.

Amino Acid Sequence↗
PubMed1984

Variant antigen genes of Trypanosoma brucei: genomic alteration of a spliced leader orphon and retention of expression-linked copies during differentiation.

Variant surface glycoprotein (VSG) gene expression in Trypanosoma brucei involves not only the sequential activation of individual VSG genes during mammalian bloodstream stage antigenic variation but also the regulation of gene expression during cyclic transmission through alternate mammalian and insect hosts. In the bloodstream stage, transcriptional activation of many VSG genes is correlated with the appearance of an additional copy of the gene in a novel genomic location, the expression-linked copy. The parasite loses the ability to synthesize VSG during differentiation from mammalian bloodstream to insect procyclic stage. Five different bloodstream populations were individually converted to procyclic forms. In each case, the procyclic cells retained the expression-linked copy of the bloodstream parent, and it remained in the same immediate genomic context. Transcripts homologous to the VSG structural gene exon were found in bloodstream stage RNA but not in procyclic RNA. Nevertheless, transcripts containing sequences homologous to the VSG mRNA spliced leader were abundant in both procyclic and bloodstream stage cells. When the genomic organization of sequences homologous to the VSG leader was examined, a specific alteration correlated with procylic differentiation was found. These data are discussed in light of biological studies on antigenic variation.

Animals↗
PubMed1984

Cloning of the major protein of the Caulobacter crescentus periodic surface layer: detection and characterization of the cloned peptide by protein expression assays.

A precisely ordered crystalline array is found on the surface of the bacterium Caulobacter crescentus CB15. Using an immunological assay, we identified recombinant bacteriophage clones expressing the predominant protein of this structure from a lambda 1059 library of C. crescentus CB15 DNA. A single 4.4-kilobase HindIII fragment encoded a polypeptide whose antigenic determinants, molecular weight, and peculiar solubilization properties were identical with those of the authentic predominant polypeptide (130K) of the surface array. The 130K protein was produced as a discrete product as a result of gene transcription initiated from a lambda promoter; several experiments suggested that the Caulobacter promoter for this gene is not efficiently recognized by the Escherichia coli transcription machinery. Genomic Southern analysis revealed a single copy of the 130K protein gene per genome. The 130K protein gene was hybridized with DNA of two closely related laboratory strains of C. crescentus which have lost their ability to produce a surface array. One of these strains, CB2, possesses an homologous copy of the 130K gene, whereas DNA from the other strain, CB13B1a, showed a lesser degree of hybridization to the 130K gene probe; genomic fragments which did hybridize were of different sizes in CB13 as compared with those of CB15. These findings are discussed in relation to studies of the surface array function and its role in cellular morphogenesis in this stalk-forming bacterium.

Bacterial Proteins↗
PubMed1984

The IsTaR 1 serodeme of Trypanosoma brucei: development of a new serodeme.

An extensive serodeme of sequentially-isolated antigenic variants of African trypanosomes has been produced from both syringe-passaged and cyclically-transmitted Trypanosoma brucei of the IsTaR 1 clone derived from EATRO 164. The majority of the antigenic variants were isolated from chronically-infected deer mice (Peromyscus leucopus). The pattern of parasitemias during the course of infections initiated with syringe-passaged trypanosomes differed from those initiated with cyclically-transmitted trypanosomes. Trypanosome populations from syringe-passaged (192) and cyclically-transmitted (31) clones were each amplified by growth in lethally-irradiated mice and cryopreserved for retrospective analysis. Five clones derived from a single deer mouse during the first 44 days of infection, and 2 clones derived from an acutely-infected rat were established from these amplified populations. Homogeneous populations were grown in lethally-irradiated rats and mice for antigenic analysis purification of variant-specific glycoprotein. Six of the 7 clones were distinct variants by immunological criteria using antisera derived from whole cells or purified surface glycoproteins. Two clones, one derived from the acutely-infected rat, and the other from the first parasitemia in a chronic infection that was initiated with the former clone, were immunologically identical. Production of these clones established a well-defined serodeme that will allow detailed analysis of antigenic variation.

Animals↗
PubMed1983

The nucleotide sequence of the Mr = 28,500 flagellin gene of Caulobacter crescentus.

The DNA sequences which encode the Mr = 28,500 flagellin polypeptide of Caulobacter crescentus CB15 have been determined. The size of the protein, deduced from its DNA sequence (276 amino acids), is in agreement with its apparent molecular weight as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The distribution of arginine residues within the protein sequence encoded by the gene correlates with their relative location as predicted by peptide alignment analysis (Gill, P.R., and Agabian, N. (1982) J. Bacteriol. 150, 925-933). DNA sequences 5' and 3' to the coding sequence were also determined. In the 5' region, DNA sequences homologous to consensus sequences associated with RNA polymerase recognition and transcription initiation sites in Escherichia coli (Pribnow box) are found. These are centered around 60, 90, and 120 base pairs upstream from the ATG codon at the beginning of the structural gene. Sequences 3' to the coding region were identified which might signal transcription termination. A typical E. coli 16 S ribosomal binding site (Shine-Dalgarno sequence) is located just 5' to the coding sequence, and for most of the amino acids there is a strong codon usage preference. Although this protein is exported from the cell (Gill, P.R., and Agabian, N. (1982) J. Bacteriol. 150, 925-933), the encoded NH2-terminal amino acid sequence is not different from the mature product.

Amino Acid Sequence↗
PubMed1983

Caulobacter flagellin mRNA segregates asymmetrically at cell division.

Molecular processes which promote the spatial localization of subcellular components are fundamental to cell development and differentiation. At various stages in development unequal segregation of molecular information must occur to result in the differentiated characteristics which distinguish cell progeny. Biological attributes of the dimorphic bacterium, Caulobacter crescentus, provide an experimental system permitting examination of the generation of asymmetry at the molecular level. When a Caulobacter cell divides, two different daughter cells are produced--a motile swarmer cell with a polar flagellum and a non-motile cell with a static appendage referred to as a stalk. The two cell types are distinct with respect to surface morphology, developmental potential, protein composition and biosynthetic capabilities. One of the more conspicuous manifestations of asymmetric expression of macromolecules in this system, the flagellum, has been studied extensively. We have cloned the flagellin genes of Caulobacter and report here the use of these sequences as probes to demonstrate that (1) the level of flagellin mRNA is regulated during the cell cycle in a pattern coincident with flagellum polypeptide synthesis and (2) flagellin mRNA synthesized before cell division is segregated with progeny swarmer cells. This provides molecular evidence of specific partitioning of an mRNA at the time of cell division.

Bacteria↗
PubMed1983

Genomic organization of variant surface glycoprotein genes in Trypanosoma brucei procyclic culture forms.

The production of the variant surface glycoprotein coat of bloodstream form African trypanosomes ceases after conversion to the procyclic form. In the bloodstream stage alternate expression of different variant surface glycoprotein genes is responsible for the antigenic variation that occurs during relapse infections in the mammalian host. We have examined procyclic stage populations, derived from different bloodstream variant antigen types, for the two types of genomic alterations associated with variant surface glycoprotein genes in the bloodstream stage. Transcriptional activation of some variant antigen genes is accompanied by the generation of a new copy of the gene, the expression-linked copy. We find that the expression-linked copy is maintained after conversion to procyclic form, indicating that the presence of an expression-linked copy is not sufficient for the expression of a surface coat. Sequences 3' to other variant surface glycoprotein genes show expression-independent variation in bloodstream stage trypanosomes. The same genes showed variation between procyclic populations of different origin, and between procyclics and their bloodstream parent. These data are discussed in light of observations on the sequence of variant antigen expression after cyclic transmission.

Animals↗
PubMed1983

Tubulin genes are tandemly linked and clustered in the genome of trypanosoma brucei.

We have isolated cDNA and genomic clones containing alpha- and beta-tubulin genes from Trypanosoma brucei. Each clone has been mapped, and the identity of the tubulin genes has been established by cross-hybridization with cloned chicken tubulin genes and by hybridization-selection and translation of trypanosome tubulin mRNA. In contrast with the dispersed organization of tubulin genes in other organisms, trypanosome alpha- and beta-tubulin genes are physically linked and clustered in tandem repeats of approximately 13-17 copies per haploid genome of alternating alpha- and beta-tubulin sequences.

Animals↗
PubMed1983

Sequences homologous to the variant antigen mRNA spliced leader are located in tandem repeats and variable orphons in trypanosoma brucei.

We have examined the organization of genomic sequences homologous to the spliced leader of Trypanosoma brucei variant surface glycoprotein (VSG) mRNA, using a synthetic oligodeoxynucleotide probe. These sequences are highly reiterated in the trypanosome genome and most are located in 1.4 kb units arranged in a direct tandem repeat. However, some of the 1.4 kb sequences are dispersed from the cluster(s) of tandem repeats and are flanked by non-repeat DNA. The number and arrangement of these leader sequence orphons varies among different T. brucei stocks. Within the IsTat serodeme, the arrangement of three of four spliced leader orphons observed with Eco RV digestion was stable during a chronic infection and cyclic transmission through the insect vector. The fourth Eco RV orphon, however, undergoes rearrangement during antigenic variation and life-cycle differentiation.

Animals↗
PubMed1983

Molecular characterization of initial variants from the IsTat I serodeme of Trypanosoma brucei.

Variant surface glycoproteins (VSGs) were isolated from variant antigen types (VATs) of the IsTat 1 serodeme. Molecular weight and isoelectric focusing analysis demonstrate that seven early VSGs possess properties generally attributed to VSGs isolated from other trypanosome serodemes. Six of the seven VSGs characterized are distinct from one another, while two (D and 1) appear identical. The presence of VSG specific mRNA in corresponding VATs was demonstrated by in vitro translation of RNA from each of the VATs, followed by immunoprecipitation with homologous and heterologous antisera. Hybridization of VSG cDNA clones with RNA from each VAT confirm that VSG mRNA is present only in homologous VATs and verifies the transcriptional control of these VSG genes. The two VATs D and 1 express indistinguishable VSGs by a variety of biochemical criteria, as well as by reactivity with 24 monoclonal antibodies. The VSG mRNAs in VATs D and 1 also appear identical. However, this identity is not reflected at the genomic level. Data is presented which establishes that DNA rearrangements can occur around both expressed and non-expressed VSG genes without qualitatively affecting VSG gene expression.

Animals↗
PubMed1983

Genomic organization of Trypanosoma brucei variant antigen gene families in sequential parasitemias.

cDNA libraries were made from mRNA purified from each of seven sequentially isolated variant antigen types (VATs) of the IsTat 1 serodeme. Plasmids containing variant surface glycoprotein (VSG) sequences corresponding to each of the isolates were used in Southern analyses to examine the genomic organization of VSG nucleotide sequences. In most cases, cells expressing a given VSG were shown to have an extra copy of the corresponding VSG gene. In one case an expression-linked copy (ELC) was not detectable. VSG gene rearrangements not obviously correlated with the expression of homologous sequences were detected in four of six VSG gene families. Thus, even cDNAs which detected an ELC revealed additional genomic reorganization in regions flanking VSG sequences. The cells used to initiate the chronic infection expressed the same VSG as those isolated from the first parasitemia. The extent of genomic rearrangement observed between these two sequentially derived populations was comparable to that observed between any of the other serially derived VATs. Thus, within a short period of time and in the absence of detectable antigenic variation, the amount of genetic flux in sequences associated with VSG genes can be substantial.

Animals↗
PubMed1982

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

Immunoprecipitation of Caulobacter crescentus polyribosomes with antiflagellin antibody provided RNA for the synthesis of cDNA probes that were used to identify three specific EcoRI restriction fragments (6.8, 10, and 22 kilobases) in genomic digests of Caulobacter DNA. The RNA was present only in polyribosomes isolated from a time interval in the Caulobacter cell cycle that was coincident with flagellin polypeptide synthesis. The structural gene for Mr 27,500 flagellin polypeptide was assigned to a region of the 10-kilobase EcoRI restriction fragment by DNA sequence analysis. Analysis of mutants defective in motility further established a correlation between the Mr 27,500 flagellin gene and the flaE gene locus [Johnson, R. C. & Ely, B. (1979) J. Bacteriol. 137, 627-634]. The other EcoRI fragments that hybridize with the immunoprecipitated polyribosome-derived cDNA probe are also temporally regulated and have features that suggest they encode other polypeptides associated with the flagellum. Modifications were required to adapt the procedure of immunoprecipitation of polyribosomes for use with Caulobacter and should be applicable to the production of specific structural gene probes from other prokaryotic systems.

Bacteria↗
PubMed1982

Cell surface patterning and morphogenesis: biogenesis of a periodic surface array during Caulobacter development.

Shape changes, extended processes, and other surface elaborations are associated with cellular differentiation, and the cell membranes involved with these developmental changes often are reshaped without a major alteration in biochemical composition. Caulobacter crescentus produces a hexagonally-packed periodic surface layer that covers the entire cell and further, mimics some of the membrane-mediated changes of higher organisms by forming a membranous stalk during its distinctive life cycle. Growth of the surface layer was examined during the cell cycle by treating synchronously growing cells with surface layer antibody, continuing growth, and then labeling for electron microscopy with a protein A-colloidal gold conjugate. Three regions of distinctive surface array biogenesis were resolved. The periodic surface layer on the main cell body was enlarged by insertion of new material at numerous uniformly distributed points. In contrast, the surface layer on the stalk appeared as entirely new synthesis. In examining growth of the stalk in subsequent generations, we noted that growth of stalk surface persisted at the stalk-cell body junction. The region of cell division also showed a pattern of entirely new surface layer production at late stages in division, similar to the stalk. The immunocytological method also facilitated a careful examination of stalk initiation and growth. Although initiation was under precise temporal and spatial regulation, the rate of stalk elongation was variable from cell to cell and apparently no longer under cell cycle control. The similarity of surface layer biogenesis on the stalk and the site of cell division may be a significant reflection of other events occurring at the cell pole. A model suggested by this and other studies that can account for the temporal pattern of polar morphogenesis is discussed, as is the potential relationship between the geometrically ordered surface array and the formation or maintenance of the stalk.

Antigens, Bacterial↗