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Biomedical subjects

A F Cowman

Publications and source records attributed to A F Cowman.

At least 109 records · Page 6Linked to original sources
PubMed1988

Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum.

We describe the isolation and the sequence of the gene for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS; EC 1.5.1.3 and EC 2.1.1.45, respectively) from two pyrimethamine-resistant clones of Plasmodium falciparum, HB3 and 7G8. We have also derived the sequence of the DHFR portion of the gene, by amplification using polymerase chain reaction, for the pyrimethamine-sensitive clone 3D7 and the pyrimethamine-resistant strains V-1, K-1, Csl-2, and Palo-alto. The deduced protein sequence of the resistant DHFR portion of the enzyme from HB3 contained a single amino acid difference from the pyrimethamine-sensitive clone 3D7. It is highly likely that this difference is involved in the mechanism of drug resistance in HB3. The sequence of the DHFR gene from other pyrimethamine-resistant strains contains the same amino acid difference from the sensitive clone 3D7. However, they all differ at one other site that may influence pyrimethamine resistance. The DHFR-TS gene is present as a single copy on chromosome 4 in all pyrimethamine-sensitive and pyrimethamine-resistant isolates tested. Therefore, the molecular basis of pyrimethamine resistance in the parasites tested is not amplification of the DHFR-TS gene.

Amino Acid Sequence↗
PubMed1987

Babesia bovis: molecular and biological characteristics of cloned parasite lines.

An in vivo limiting dilution technique was used to produce several Babesia bovis cloned lines with which to study the basis of virulence and immunogenicity in this parasite. DNA hybridization using a cloned DNA fragment from the BabR locus demonstrated that the cloned lines were a more restricted genetic population than the parent strain. Biosynthetic labeling and immunoprecipitation studies indicated that the cloned lines differed from each other and from the parentals in the expression of a small number of polypeptides and antigens. Animal trials with three of the lines demonstrated that the parental line contains both virulent and avirulent parasite populations, at least three of which are not tick transmissible, and that while the lines do provide significant protection against heterologous challenge, they may not give as effective protection as the parental line. These experiments demonstrated the existence of subpopulations with distinctive molecular and biological properties, providing evidence that the attenuation process is based on the selection of preexisting parasite subpopulations combined with the ability of these parasites to vary genetically.

Animals↗
PubMed1987

Changes in repeat number, sequence, and reading frame in S-antigen genes of Plasmodium falciparum.

The S antigens from different isolates of Plasmodium falciparum exhibit extensive size, charge, and serological diversity. We show here that the S-antigen genes behave as multiple alleles of a single locus. The size heterogeneity results from different numbers, lengths, and/or sequences of tandem repeat units encoded within the S-antigen genes. Two genes studied here encode antigenically different S antigens but nevertheless have closely related tandem repeat sequences. We show that antigenic differences can arise because repeats are translated in different reading frames.

Alleles↗
PubMed1986

An opsin gene expressed in only one photoreceptor cell type of the Drosophila eye.

We have isolated an opsin gene from D. melanogaster that is expressed specifically in photoreceptor cell 8 of the Drosophila compound eye. This opsin is 381 amino acid residues long and is 67% homologous to the ninaE opsin, which is expressed in photoreceptor cells 1-6. The gene is divided into four exons; only one of the intron positions is conserved with that of the ninaE gene.

Amino Acid Sequence↗
PubMed1985

Conserved sequences flank variable tandem repeats in two S-antigen genes of Plasmodium falciparum.

We describe the isolation of two chromosomal DNA fragments from Plasmodium falciparum. These fragments encode the antigenically distinct S antigens of two different P. falciparum isolates, namely FC27 from Papua New Guinea and NF7 from Ghana. The complete nucleotide sequences of both fragments are presented. The fragments are homologous over most of their lengths, including the entire regions flanking the protein coding sequences. Whereas the N- and C-terminal portions of sequences encoding the S antigens are homologous, major portions of the coding sequences are not. The nonhomologous regions are comprised of tandemly repeated sequences, of 33 bp in FC27 and predominantly of 24 bp in NF7. The 33 bp tandem repeats encoded by the FC27 S-antigen gene could not be detected in the NF7 genome. Conversely, the 24 bp tandem repeats encoded by the NF7 S-antigen gene could not be detected in the FC27 genome. The pattern of sequence variation within the repeats of both genes suggests a mechanism for the generation of S-antigen diversity.

Amino Acid Sequence↗
PubMed1985

Isolation and structure of a rhodopsin gene from D. melanogaster.

Using a novel method for detecting cross-homologous nucleic acid sequences we have isolated the gene coding for the major rhodopsin of Drosophila melanogaster and mapped it to chromosomal region 92B8-11. Comparison of cDNA and genomic DNA sequences indicates that the gene is divided into five exons. The amino acid sequence deduced from the nucleotide sequence is 373 residues long, and the polypeptide chain contains seven hydrophobic segments that appear to correspond to the seven transmembrane segments characteristic of other rhodopsins. Three regions of Drosophila rhodopsin are highly conserved with the corresponding domains of bovine rhodopsin, suggesting an important role for these polypeptide regions.

Amino Acid Sequence↗
PubMed1984

The expression of Plasmodium falciparum bloodstage antigens in Escherichia coli.

A library of cDNA clones expressing proteins of the asexual blood stages of a Papua New Guinean isolate of Plasmodium falciparum (isolate FCQ27/PNG (FC27] was constructed in the bacteriophage vector lambda gt11-Amp3. In an in situ colony immunoassay, human serum was used to identify colonies producing natural immunogens. Sera from donors of defined clinical status, or reactive to a defined subset of natural immunogens were used to identify clones of particular interest (for example, clones reacting with convalescent but not with acute serum or clones expressing the isolate specific S-antigen of FC27). Antisera raised by immunizing mice and rabbits with cloned antigens were used to characterize the P. falciparum proteins corresponding to the antigen-positive clones. Nucleotide sequence analysis of an antigen found on the surface of cells infected with ring stage parasites revealed an unusual sequence coding for eight, four and three amino acid repeats rich in acidic amino acids. The discussion centres on the use of cloned antigens as tools for the analysis of the host-protective immune response and selection of candidate vaccine molecules.

Animals↗
PubMed1984

Genes of the protozoan parasite Babesia bovis that rearrange to produce RNA species with different sequences.

We describe a highly polymorphic gene family from the protozoan parasite Babesia bovis that encodes a complex pattern of transcripts. Gene rearrangements at this locus (designated BabR ) have generated RNA species that differ in sequence. The naturally occurring virulent field isolate Kv expresses two different sized transcripts that are identical in sequence at their 5' ends but different at the 3' ends. An avirulent derivative of this isolate, Ka, expresses not only these two transcripts but a third, of intermediate size. Analysis of cloned genomic fragments from Ka identified genes encoding all three transcripts. Two fragments contained tandemly repeated copies of the BabR genes. The gene encoding the intermediate sized transcript has undergone a rearrangement, substituting a 5' region different to that shared by the other transcripts. These results suggest that sequence diversity may play an important role in the biological function of the BabR locus.

Amino Acid Sequence↗
PubMed1984

Accurate transcription of a cloned gene from Babesia bovis in Saccharomyces cerevisiae.

Two cloned chromosomal segments of the hemoprotozoan Babesa bovis bearing genes from the polymorphic locus designated BabR were inserted into plasmid vector YCp19, cloned in Escherichia coli and then introduced into Saccharomyces cerevisiae. Both genes were transcribed producing poly(A+) RNAs. In at least one case, the transcript was very similar if not identical to that produced in B. bovis. It is therefore possible that S. cerevisiae can recognize at least some hemoprotozoan promoter and poly(A+)-addition signals.

Animals↗
PubMed1984

DNA polymorphisms and subpopulations in Babesia bovis.

Independent isolates of Babesia bovis differ by only a limited number of polypeptides, some of which may be important as host protective antigens. Avirulent derivatives of these parasites also differ from their virulent counterparts in only a few polypeptides. To identify genes encoding such polypeptides we have isolated cDNA clones corresponding to poly(A)+ RNAs that are expressed only in certain isolates. For this purpose a cDNA clone library was constructed from poly(A)+ RNA of the K-avirulent isolate (KA). These clones were screened by colony hybridization using [32P]cDNA complementary to poly(A)+ RNA from KA and from virulent isolates, in order to identify clones that selectively hybridize to one cDNA probe. Hybridization of DNA from three clones, designated pK4, pK5 and pK6 to poly(A)+ RNA from various isolates revealed different and complex patterns. The gene represented by clone pK5 appeared to be transcribed predominantly in avirulent parasites. Analysis of genomic DNA by the Southern procedure enabled each isolate to be distinguished and suggested that most isolates are comprised of a heterogeneous mixture of subpopulations. Analysis of genomic DNA from parasites obtained after passage of KA through the tick vector (Boophilus microplus) suggested that a subpopulation was being selected that more closely resembled KV than KA.

Animals↗
PubMed1984

Differential antibody screening of cloned Plasmodium falciparum sequences expressed in Escherichia coli: procedure for isolation of defined antigens and analysis of human antisera.

We describe a procedure that uses polyspecific human sera for screening Escherichia coli colonies expressing cloned Plasmodium falciparum cDNA sequences in order to detect colonies that react differentially with different sera. This procedure can be used for two distinct purposes. First, it enables the isolation of clones encoding specified antigenic sequences present in the complex mixture, without purification of either antigens or antibodies by conventional procedures. This requires that the antigen can be expressed in E. coli and that antisera are available that differ substantially in their reactivities to the component of interest. To develop the procedure, we used two polyspecific sera that shared many anti-P. falciparum specificities but differed in that only one was reactive to the isolate-specific S antigen of P. falciparum strain FCQ27/PNG (called FC27). Differential screening with the two sera identified 30 cDNA clones, and colony hybridization confirmed that 25 of these express S-antigen sequences. Second, the procedure identifies defined antibody specificities within polyspecific human sera by virtue of their ability to react with any given cDNA clone. The procedure has been used here to identify antibody specificities that increase dramatically in titer between the acute and convalescent phases of malaria in certain individuals and, hence, to isolate clones encoding the corresponding antigens.

Animals↗
PubMed1984

The ring-infected erythrocyte surface antigen (RESA) polypeptide of Plasmodium falciparum contains two separate blocks of tandem repeats encoding antigenic epitopes that are naturally immunogenic in man.

We showed previously that the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum contains a repetitive amino acid sequence. We have investigated here the sequence and antigenic relationships between RESA from FC27, a Papua New Guinea isolate, and from NF7, a Ghanaian isolate. The complete nucleotide sequences of eight different cDNA clones demonstrate that RESA from the two strains are closely homologous over the region that can be compared. A series of related eight, four and three amino acid repeats is located at the 3' end, forming the C terminus of RESA in FC27. RESA contains a second block of repeats based on an 11 amino acid sequence and separated from the 3' block by 381 amino acids in NF7. Antibodies from Papua New Guineans react with RESA from the African isolate, and vice-versa. Antigenic determinants that are naturally immunogenic in man are present in the 5' repeats of RESA, as well as in the 3' repeats, and antibodies that cross-react with both blocks of repeats were detected by reacting affinity-purified human antibodies with cloned subfragments of the cDNA clones, expressed in Escherichia coli.

Africa↗
PubMed1984

Plasmodium falciparum complementary DNA clones expressed in Escherichia coli encode many distinct antigens.

A library of cDNA clones expressing antigens of the asexual blood-stages of Plasmodium falciparum (isolate FCQ27/PNG) was constructed in the bacteriophage vector gamma gt11-Amp3. Clones expressing P. falciparum antigens (as polypeptides fused to beta-galactosidase) were selected by their reactivity in an in situ colony immunoassay with affinity-purified malaria antibodies. A detailed analysis of 78 antigen-positive clones selected from approximately 10,000 recombinant clones has shown them to correspond to many different parasite antigens. cDNA hybridization studies on this array of 78 antigen-positive clones have so far identified 18 families of sibling clones with 22 clones as yet unassigned, the majority of which may represent additional unique sequences. Only about 20% of the clones synthesized abundant amounts of the malaria antigen/beta-galactosidase fused polypeptide but each multi-member family except one was represented by at least one clone producing a fused polypeptide in abundance. Antisera have been raised against cloned malaria antigens by immunizing mice and rabbits with bacterial lysates and purified fused polypeptides, respectively. These antisera have been used to characterize the antigens in P. falciparum that correspond to the various antigen-positive clones. The variety of distinct antigens recognized by these antisera confirms that the clone library contains coding sequences for many different antigens.

Animals↗
PubMed1983

Production of RNA for secreted immunoglobulin mu chains does not require transcriptional termination 5' to the microM exons.

The mouse immunoglobulin mu gene encodes both secreted and surface-bound mu heavy chains produced by cells of the B lymphoid series. Transcripts of the mu gene are processed into mu mRNA species which differ at their 3' termini, bearing either 'microsecond' or 'microM' segments, distinguishing secreted and cell-membrane-bound mu polypeptides. During maturation of surface IgM-bearing B cells to IgM-secreting plasma cells, both the total amount of mu mRNA and the ratio of microsecond- to microM-terminated mRNA increase greatly. Two possible mechanisms for the developmental regulation of 3' RNA processing cannot yet be distinguished. One mechanism would yield the microsecond terminus by specific cleavage of a common presursor transcript encompassing both microsecond and the microM exons (Fig. 1), the other by regulated termination of transcription upstream from the microM exons. While the first mechanism would produce, as a by-product, RNA fragments containing microM exons, the second would not. We report here the detection of such microM fragments in cells producing predominantly microsecond-terminated RNA species.

Base Sequence↗
PubMed1983

Expression of Plasmodium falciparum blood-stage antigens in Escherichia coli: detection with antibodies from immune humans.

Many proteins produced by blood stages of the malaria parasite Plasmodium falciparum are natural immunogens in man. As an approach to determining which of these are relevant to protective immunity we have constructed an expression library of P. falciparum cDNA sequences, cloned in Escherichia coli. The cDNA sequences were inserted into the beta-galactosidase gene of an ampicillin-resistant derivative of the temperature-sensitive lysogenic bacteriophage lambda gt11. About 5% of the resulting clones expressed P. falciparum sequences as polypeptides fused to beta-galactosidase. We have identified many clones that express P. falciparum antigens by immunological screening in situ with antibodies from immune human sera that inhibit P. falciparum growth in vitro. The antigen-positive clones contain P. falciparum cDNA sequences, as determined by hybridization. Some express polypeptides that are larger than beta-galactosidase and react both with antibodies to beta-galactosidase and with antibodies from humans immune to P. falciparum. The cloned P. falciparum antigens should facilitate new approaches to the identification of potential vaccine molecules.

Animals↗
PubMed1982

mRNA for surface immunoglobulin gamma chains encodes a highly conserved transmembrane sequence and a 28-residue intracellular domain.

To probe the structure of the gamma heavy chain of membrane IgG and the mRNA and gene segments that encode it, we have analyzed cDNA clones derived from a gamma 1 membrane RNA of B lymphoma 2PK-3. The nucleotide sequence of the clones indicated that membrane gamma 1 chains bear a COOH-terminal 71-residue segment that is absent from secretory gamma 1 chains. This terminus includes a 26-residue hydrophobic transmembrane region homologous to that of membrane mu chains and, significantly, a 28-residue intracellular domain found only on gamma chains. The extra domain suggests that receptor IgG, on memory B cells, may generate a different signal on binding antigen than does receptor IgM, on virgin B cells. The gamma 1 membrane terminus is encoded by two gene segments 1.5 and 2.4 kilobase pairs downstream from the C gamma 1 gene, and homologous segments occur 3' to the C gamma 2a and C gamma 3 genes. Small amounts of membrane gamma mRNAs persist in plasmacytomas secreting IgG1, IgG2a, or IgG2b, suggesting that competition between alternative RNA processing pathways governs the synthesis of membrane and secretory gamma chain mRNAs.

Animals↗