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D J Kemp

Publications and source records attributed to D J Kemp.

At least 145 records · Page 8Linked to original sources
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

A search for messenger RNA molecules bearing immunoglobulin VH nucleotide sequences in T cells.

Expression of VH-coded mRNA molecules in T cells, antigen-specific T cell lines, or T cell hybridomas was not detected using four different VH DNA probes under conditions that permitted cross-hybridization between distantly related VH genes. In contrast, VH gene expression was readily detected in two B cell lymphomas and in splenic B cells. Less than one molecule per cell of RNA, exactly complementary to the DNA probes used, would have been detected in these T cell populations. The results thus seriously question the proposition that T cells use the B cell VH repertoire to code for antigen receptors.

Animals↗
PubMed1981

Direct immunoassay for detecting Escherichia coli colonies that contain polypeptides encoded by cloned DNA segments.

We describe a simple in situ immunoassay for screening Escherichia coli colonies, to detect those that express polypeptide antigens encoded by cloned DNA segments. In the colony immunoassay that we have developed, only one antibody molecule need bind to the polypeptide molecule. Hence short fragments of coding sequences should be detectable, and problems imposed by the conformation of polypeptides "fused" to protein fragments encoded by the vector should be minimized. The method should allow the use of monoclonal antibodies, which bind to only one determinant. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated filter papers. Antigens covalently bound to the CNBr paper were detected by reaction with antiserum, followed by 125I-labeled protein A from Staphylococcus aureus and autoradiography. We have used the colony immunoassay to determine what proportion of ptrpED5-1 clones containing mouse mu chain cDNA segments express mu polypeptides. Surprisingly, about 50% of the clones were positive. Because only one in six clones would be expected to contain the insert in the correct orientation and reading frame to encode a fused polypeptide, it is likely that polypeptides that initiate within the insert can be detected. A similarly high proportion of pBR322 clones containing mu cDNA were positive, and about 25% of pBR322 clones containing mouse gamma 2a chain cDNA were positive to anti-gamma 2a serum. EAch of the positive mu clones reacted with affinity-purified anti-mu antibodies but not with normal serum, and clones containing irrelevant sequences were negative. The mu cDNA segment was inserted in the correct orientation in all six clones studied, and discrete mu polypeptides corresponding in size both to fused polypeptides and internally initiated polypeptides were detected by electrophoresis of protein extracts.

Cloning, Molecular↗
PubMed1981

Organization of genes and spacers within the mouse immunoglobulin VH locus.

The germline organization of mouse immunoglobulin VH genes has been investigated using cloned VH sequences. Hybridization studies with VH probes from plasmacytomas HPC76 (H76), S107, HOPC1 (H1), and lymphoma ABLS-8 (A8) demonstrated that the VH locus contains at least three distinct VH gene families. Comparison with other results suggests a total of about 10 such families, and of the order of 160 germline VH genes. Nucleotide sequencing revealed that the H76 family includes anti-inulin VH sequences, and the S107 family is known to encode antiphosphorylcholine sequences. The three VH gene families studied were mapped in the order H76-S107-A8/H1-CH by determining which VH genes had been deleted from several plasmacytomas by VH rearrangement events. Nine genomic clones from athe H76 family, and one each from the S107 and A8/H1 families, were characterized; collectively they span 103 kilobases (kb). Two clones from the H76 family and one from the S107 family each bore a pair of VH genes separated by approximately 14 kb, suggesting that related VH genes in these families are clustered with a typical spacing of approximately 14 kb. No other VH genes were detected within the spacers, arguing against intermingling of different families. Within the VH76 family cluster, however, two closely homologous VH genes were shown not to be adjacent. While spacer sequences were strongly conserved in the A8/H1 family, the H76 family had minimal conservation and that was restricted to regions immediately surrounding the genes. Hence conservation of spacer sequences cannot be essential for VH gene function, nor for maintenance of a VN family. Spacers in both the H76 and S108 family contained small repeat elements, some of which behaved like mobile DNA sequences.

Animals↗
PubMed1980

The immunoglobulin mu constant region gene is expressed in mouse thymocytes.

It has been a matter of controversy whether the functional capacity of T cells to discriminate between antigens is mediated via immunoglobulin, an immunoglobulin-like molecule, or by the product(s) of unrelated genes. The progenitors of immunoglobulin-secreting cells, B cells, express membrane-bound immunoglobulin as the antigen-specific receptor on their surface. For T cells, although products of immunoglobulin heavy chain variable region genes are implicated as receptor components, there has been no compelling immunochemical evidence for participation of either immunoglobulin light chains or heavy chain constant regions (see refs 2-6 for the disparate views). Recently, using cloned immunoglobulin DNA sequences as hybridization probes, we have demonstrated that the immunoglobulin Cmu gene, but not the Cmu gene, is expressed as polyadenylated RNA in some T cell tumour (T lymphoma) cell lines. Individual T lymphoma lines yielded up to three discrete mu RNA species of different sizes (1.9, 2.2 and 3.0 kilobases), each species being different in size from the major mu RNA species present in B lymphoma cells (2.4 and 2.7 kilobases). We show here that cells from the normal mouse thymus contain mu RNA species, indistinguishable in size from those in T lymphoma cells, but contain little if any kappa RNA.

Animals↗
PubMed1980

Intervening sequences divide the gene for the constant region of mouse immunoglobulin mu chains into segments, each encoding a domain.

To elucidate the structure of the gene for the constant region of immunoglobulin mu chains, we have cloned a 9.9-kilobase-pair fragment of mouse DNA bearing a gene for the constant region of the mu chain (C mu gene) from an IgM-secreting mouse plasmacytoma. The sequence around this gene has apparently undergone somatic rearrangement; the gene occurs in an EcoRI restriction endonuclease fragment of a different size from that in embryo or liver DNA and no C mu-bearing fragment of embryo size remains in the plasmacytoma. The cloned sequence lacks a variable region gene; hence, if this C mu gene is active, its position within the clone indicates that the gene for the variable region of a heavy chain (VH gene) must be more than 3.7 kilobase pairs away. The C mu gene is divided by three intervening sequences into four coding segments, each of which encodes one of the domains (homology units) of the polypeptide. The nucleotide sequence coding for amino acids near the V-C junction is not present within the C mu clone or clones bearing homologous embryonic VH genes. This suggests that an immunoglobulin heavy chain, in common with light chains, is encoded not only by a V and C gene, but also by an independent joining region (JH) gene.

Animals↗
PubMed1980

Transcripts of the immunoglobulin C mu gene vary in structure and splicing during lymphoid development.

Two classes of transcripts of the mouse immunoglobulin mu constant region (C mu) gene can be distinguished. Whereas B cells contain mu mRNAs of 2.4 and 2.7 kilobases (kb), many T lymphoma, Abelson pre-B lymphoma, and myeloid tumor cell lines contain polyadenylylated RNA species bearing C mu sequences (C mu RNAs) of 1.9, 2.1, 2.3, and 3.0 kb. Production of C mu RNAs, unlike mu mRNAs, does not require recombination with the joining region (JH) locus. To define the structure of C mu RNAs, RNA from representative cell lines was fractionated by gel electrophoresis, transferred to diazobenzyloxymethyl paper, and tested for hybridization with 23 DNA fragments that collectively span a chromosomal mu gene, cloned from a plasmacytoma. All the C mu RNAs bear sequences derived from each of the four C mu domains, but none contain the intervening sequences separating domains; thus each represents a spliced RNA species. The 1.9-kb C mu RNA contains the 3' sequence characteristic of secreted mu chain, whereas the longer species bear that of membrane-bound mu chin. Hence C mu RNAs and mu mRNAs are equivalent throughout the C mu and 3' terminal regions. They differ markedly, however, in their 5' regions, because the 3.0-and 2.3-kb C mu RNAs bear sequences from within the conventional JH-C mu intervening sequence. Because these sequences are several kb from C mu, this region must contain at least one hitherto unsuspected splice site. C mu RNAs may not express immunological diversity, because no evidence was found that they bear variable regions. T and pre-B lymphoid and myeloid cells contain equivalent C mu RNA species, which coexist with mu mRNAs in some pre-B and B lymphoid lines. C mu RNA expression appears to reflect an activated state of the C mu gene common to cells at early stages of T, B, and myeloid development.

Animals↗
PubMed1980

Expression of the immunoglobulin C mu gene in mouse T and B lymphoid and myeloid cell lines.

We have used cloned nucleotide sequences as probes to search for immunoglobulin RNA in T and B lymphoid and nonlymphoid tumor cell lines. Polyadenylated RNA from the cells was fractionated electrophoretically by size and fixed to diazobenzyloxymethyl-paper, and then immunoglobulin RNA species were revealed by hybridization with 32P-labeled cloned sequences for mu heavy chain and kappa light chain. kappa mRNA was detected only in B lymphoid lines known to synthesize kappa chains. No mu RNA was detectable in erythroleukemia, mastocytoma, or sarcoma cells. RNA bearing mu constant region (C mu) sequences, however, was detected in four of nine T lymphoma and four of five myeloid tumor cell lines tested, as well as in B lymphoma and "pre-B" Abelson lymphoma cells. In contrast to the single mu RNA species of 2.6 kilobases (kb) in a plasmacytoma, individual T lymphoma and myeloid lines yielded up to three discrete mu RNA species of apparent size 1.9, 2.2, and 3.0 kb, each different from the two mu RNA species in a B lymphoma line (2.4 and 2.7 kb) or the single species in "pre-B" lymphoma cells (2.9 kb). Both chromosomal complements of the C mu gene in STRij-4 T cells were found to be rearranged from their embryonic (germ line) context. The results suggest that the C mu gene functions not only in T cells, wherein the ability to recognize antigen is long established, but also in myeloid cells.

Animals↗
PubMed1980

Somatic rearrangements forming active immunoglobulin mu genes in B and T lymphoid cell lines.

We have cloned an active gene for an immunoglobulin mu heavy (H) chain, bearing the variable (VH), joining (JH), and constant (C mu) sequences expressed in the IgM-secreting mouse plasmacytoma HPC-76. The mu gene was formed by somatic recombination between a VH gene and one of several JH genes, which are located about 7.7 kilobase pairs from the C mu gene in embryo DNA. The JH-C mu intervening sequence has suffered a deletion of about 2.7 kilobase pairs in HPC-76. Because the delection encompasses sequences required to switch an expressed VH-JH gene from C mu to another CH gene, it may represent a mechanism for "freezing" a lymphocyte clone at the stage of IgM expression. For the second (inactive) C mu allele in HPC-76, the entire joining and switch regions have been deleted; functional inactivation of one allele may thus represent one mechanism by which a lymphocyte clone restricts expression to a single allele (allelic exclusion). Probes generated from the cloned mu gene allowed examination of the JH locus in B, Abelson "pre-B," and T lymphoma cell lines and a myeloid line, all of which cotain RNA species bearing C mu sequences. The B and pre-B lines exhibited recombination within both alleles of the JH locus, suggesting that both alleles may be expressed in some cells. The absence of the JH gene 5' to the recombination sites favors a deletion mechanism for VH-JH joining. Recombination within the JH locus was also detected in two out of four T lymphoma lines, but not in the myeloid line. This indicates that the mechanism by which B cells generate immunoglobulin diversity is operational in some T cells. Lines that synthesize mu RNA without JH rearrangement may have activated the C mu gene directly or have undergone recombination at a more distant locus.

Animals↗
PubMed1979

Cloned pairs of variable region genes for immunoglobulin heavy chains isolated from a clone library of the entire mouse genome.

To investigate the organization of immunoglobulin genes, we have constructed a clone library containing 10(6) randomly generated fragments of mouse embryo DNA, corresponding to eight equivalents of the genome. The cloning method involved methylation of embryo DNA at EcoRI recognition sites, partial digestion by EcoRI* endonclease activity, and direct ligation of the resulting large fragments to the lambda phage vector Charon 4A. The library was searched for sequences homologous to a cloned complementary DNA copy of a mu heavy chain mRNA. Nine clones bearing variable heavy chain (VH) sequences were isolated, representing at least eight distinct VH genes. Thus, multiple related VH genes are available in the genome to contribute to immunoglobulin diversity. Each of the two clones carries a pair of VH genes, one pair separated by 15 +/- 1 kilobase pairs of mouse DNA and the other by 14 +/- 2 kilobase pairs. This indicates that related VH genes are clustered and may occur in a tandem array having a repeating unit of 14--16 kilobase pairs. The large spacer sequences between VH genes cannot, however, be highly conserved.

Animals↗
PubMed1978

Messenger RNA for the insect storage protein calliphorin: in vitro translation and chromosomal hybridization analyses of a 20 S poly(A)-RNA fraction.

A major poly(A)-containing RNA fraction of the approximate size expected of a monocistronic mRNA for the storage protein calliphorin has been isolated from the larval fat bodies of Calliphora vicina during early instar 3. This 20 S RNA fraction programs the synthesis by cell-free wheat embryo extracts of polypeptides of 86,000 daltons identified by tryptic peptide fingerprinting as precursors of the authentic calliphorin subunits of 83,000 daltons. Complementary DNA synthesized by AMV reverse transcriptase using the same 20 S RNA as template hybridized in situ to a single segment of one or two bands in the salivary polytene chromosomes of C. vicina.

Adipose Tissue↗