Search PubMed⌕ Search

Biomedical subjects

I M Jones

Publications and source records attributed to I M Jones.

At least 73 records · Page 4Linked to original sources
PubMed1993

Analyses of alternative poly(A) signals for use in baculovirus expression vectors.

Synthetic, duplicated, or heterologous sequences encompassing putative 3' mRNA processing (polyadenylation, poly(A)) sites have been analyzed with regard to their involvement in processing mRNA species synthesized from the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The data indicate that a synthetic sequence based on the 3' processing signal of the rabbit beta-globin gene is used more efficiently than an SV40-based, early gene poly(A) signal, or the AcNPV 1629 ORF poly(A) signal. Evidence is presented that indicates that the hexanucleotide AAUAAA alone is inadequate to effect mRNA processing. This supports the view that like other eukaryotic mRNAs additional signals are involved.

Animals↗
PubMed1993

Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step.

Amino Acid Sequence↗
PubMed1993

Complement activation upon binding of mannan-binding protein to HIV envelope glycoproteins.

OBJECTIVE: Retroviruses can activate the complement system in the absence of antibodies, and the purpose of this study was to examine whether the serum collection, mannan-binding protein (MBP), could mediate such complement activation. DESIGN: Virus envelope proteins gp120 and gp110 from HIV-1 and HIV-2 were incubated in microtitre wells coated with anti-gp120 or anti-gp110 antibodies. After further incubation with serum, complement activation was measured as deposition of complement factor C4 and C3 onto the wells. Deposited C4 and C3 were detected with enzyme-labelled antibodies. Normal human serum depleted of endogenous lectins by affinity chromatography was used as the complement source. Serum from C1q-deficient patients was used in some experiments. Complement activation was then assessed with and without prior addition of MBP to the wells. Complement activation was also correlated with the quantity of endogenous MBP in a number of normal sera. RESULTS: Complement activation by HIV envelope glycoproteins was found to be mediated by the binding of MBP to carbohydrates on natural envelope protein produced in virus-infected cells, as well as on glycosylated recombinant envelope proteins produced in insect cells. Non-glycosylated recombinant envelope proteins produced in Escherichia coli did not induce this type of complement activation. CONCLUSIONS: Activation of the classical complement pathway by retrovirus envelope proteins can be initiated by the binding of MBP to carbohydrate side chains of envelope glycoproteins.

Carrier Proteins↗
PubMed1992

Monoclonal antibodies to a CD4 peptide derivative which includes the region corresponding to an immunoglobulin CDR3: evidence of the involvement of pre-CDR3-related region in HIV-1 and host cell interaction.

A CD4 peptide of amino acid residues 68-130 [CD4(68-130)], which had the capacities to inhibit HIV-1 replication and HIV-1-induced syncytium formation, was used as an immunogen for the preparation of mAb. The mAbs prepared were classified into at least five types (I-V) in terms of their recognition sites by ELISA using various kinds of smaller CD4 peptides. Among them, the type I mAb no. 35 recognizing amino acid residues 72-84, which lies just before the region corresponding to an immunoglobulin third complementarity-determining region (CDR3), showed the strongest effects in reducing both HIV-1 infection and HIV-1-induced syncytium formation, although a large amount of no. 35 mAb was necessary to reduce such HIV-1 activities compared with those of anti-Leu-3a and OKT4A mAbs which recognize CD4 epitopes near a portion corresponding to an immunoglobulin CDR2. Western blot analysis showed that the reactivities of CD4 molecule in CD4-positive cells or sCD4 molecule with types I-V mAbs were stronger than that with anti-Leu-3a mAb. Flow cytometry showed that no. 35 mAb was faintly reactive with native CD4 molecule on cell surface at the concn showing the inhibitory effects on HIV-1 infection and syncytium formation. In addition, a smaller peptide CD4(66-92), one of the good epitope peptides for no. 35 mAb, also showed strong inhibitory effect on HIV-1 infection as well as a weaker inhibitory effect on syncytium formation. These results suggest that, in addition to the CD4 CDR2-related region, the pre-CDR3-related region is also involved in the early events of the interactions between the host cell and HIV-1.

Amino Acid Sequence↗
PubMed1992

Distinct signals in human immunodeficiency virus type 1 Pr55 necessary for RNA binding and particle formation.

The human immunodeficiency virus type 1 (HIV-1) gag gene product Pr55 self-assembles to form virus-like particles when expressed in Spodoptera frugiperda cells using recombinant baculoviruses. The particles resemble immature HIV and are released from the infected cell into the culture medium. Using this system we have progressively truncated the gag open reading frame from the C terminus and examined each deleted gag protein for its particle-producing capability. We show that deletion of Pr6 and deletions that progressively remove the distal region of the Pr7 domain, including one Cys-His box thought to function as an RNA capture signal, do not affect particle formation. However deletion of two Cys-His boxes causes production of slightly larger particles with altered sedimentation properties. Sequence-specific North-Western assays using an RNA probe representative of the HIV-1 packaging signal revealed specific RNA binding by all mutants that maintained both Cys-His boxes. However, deletion of one Cys-His box reduced RNA binding substantially and loss of two Cys-His boxes abolished binding entirely. We conclude that HIV-1 gag particle formation per se does not require viral RNA encapsidation, but that it may act as a cofactor in the condensation of the immature core. Further deletion of gag sequences upstream of the Cys-His boxes led to the abolition of particle-forming ability, and we show that one boundary of the gag sequence necessary for particle formation lies within eight amino acids spanning one of the known protease cleavage sites at the C terminus of Pr24.

Amino Acid Sequence↗
PubMed1992

Human immunodeficiency virus type 1 (HIV-1) superinfection of a cell clone converting it from production of defective to infectious HIV-1 is mediated predominantly by CD4 regions other than the major binding site for HIV-1 glycoproteins.

A cell clone, L-2, which produces non-infectious doughnut-shaped human immunodeficiency virus type 1 (HIV-1) particles, was permissive for HIV-1 superinfection, which resulted in the production of infectious particles. The superinfection showed slow kinetics compared with primary HIV-1 infection of M10 cells, the parent of the L-2 cell clone. Inhibition studies on the superinfection of L-2 cells using several CD4-related reagents showed that the CD4 molecule was an essential component of the receptor for superinfection. Strong inhibitory effects were obtained using CD4 peptides such as CD4(68-130), which includes a portion homologous to the immunoglobulin third complementarity-determining region (CDR3), as well as recombinant soluble CD4. In contrast, a CD4(45-60) peptide, which includes most of the CDR2-related region, was not effective, although the Leu-3a monoclonal antibody (MAb), which recognizes a site near the CDR2-related region, did slightly, but significantly, delay the superinfection kinetics. Comparative flow cytometry of L-2 and M10 cells revealed that the cell surface of L-2 cells despite expressing HIV-1 env protein, reacted slightly with OKT4 or anti-CD4(68-130) MAb, but not with Leu-3a or OKT4A MAb. In contrast, no reaction was detected with any of these anti-CD4 MAbs on the surface of another HIV-1 superinfection-resistant cell clone, MOLT-#8IIIB-14, which expresses HIV-1 env proteins but does not produce infectious HIV-1 particles. These results strongly suggest that expression of the CD4 major receptor site for primary HIV-1 infection is preferentially decreased on the surface of L-2 cells, but that the OKT4 epitope and the nearby region corresponding to immunoglobulin CDR3 remain exposed on the cell surface. Consequently, the CD4 CDR3-related region could play a major role as the receptor for the superinfection reported here.

Binding Sites↗
PubMed1992

Complementation of human immunodeficiency virus glycoprotein mutations in trans.

The external glycoproteins of human immunodeficiency virus type 1 (HIV-1) (gp120) and HIV-2 (gp105) are responsible for binding the cellular receptor CD4. The proteins are functionally identical although their affinity for CD4 varies, with gp120 binding 10- to 20-fold more efficiently than gp105. To investigate the structural requirements for CD4 binding in each molecule we have constructed a number of hybrid glycoproteins in which sequences are exchanged between the two molecules via conserved residues and subsequently tested for their ability to bind to CD4. We found that two constructs in which the V1/V2 or V3 loops of gp105 are exchanged for those of gp120 continue to bind to CD4. Surprisingly, however, all other domain exchange mutants failed to bind to CD4 suggesting that long-range interactions within the molecule are sequence-specific. Mixing mutant molecules in vitro did not rescue CD4 binding. However, co-expression of a number of mutant glycoprotein pairs within the same cell produced complementation of CD4 binding ability; complementing molecules were shown to be heteromeric in structure. Alignment of the molecules within each complementation group allowed the interactive sequences necessary for receptor binding to be determined. These sequences constitute a novel target for the disruption of gp120 function.

Animals↗
PubMed1991

Reduction in CD4 binding affinity associated with removal of a single glycosylation site in the external glycoprotein of HIV-2.

The role of selected glycosylation sites in the ability of the envelope glycoprotein of HIV-2 (gp 105) to bind to CD4 has been investigated. Loss of glycosylation sites at amino acids 410 and 447 did not affect the CD4 binding ability of gp 105 even when removed in pair combination. Loss of a single glycosylation site at amino acid 400, however, was sufficient to cause a reduction of at least 50-fold in the efficiency of receptor binding. These data support the hypothesis that some of the carbohydrate side chains on gp 105 have a profound effect on biological activity.

Base Sequence↗
PubMed1990

Review of the molecular characteristics of gene mutations of the germline and somatic cells of the human.

Molecular analyses of the limited number of de novo germinal mutations identified in humans indicate that an array of alterations in gene structure can be generated. Similar conclusions are derived from the large data set obtained from molecular analyses of alleles that segregate in the human population and cause genetic diseases. The molecular alterations include nucleotide substitutions as well as insertions, deletions and other rearrangements of the DNA. The lesions may be located in the coding or the noncoding regions of genes or may involve the flanking sequences. The insertions and deletions involve fragments ranging from single nucleotides to many kilobases, and involve both unique sequences and repetitive elements. The nature of the lesions observed to date as either de novo mutations or segregating variants suggests there are locus-specific characteristics of the alterations in DNA structure that are recovered as genetic diseases. Differences in mutation spectra among genetic loci appear to reflect both the structure of the target sequences and the relationship between gene structure and gene function. No induced germinal mutations have been identified, thus no data are available that reveal the relationships between mutagenic exposures and the molecular fingerprints of the lesion induced in the human germ cell and transmitted to the subsequent generations. In contrast, the prospects for analyzing the roles of genetic target, exposure history and individual responsiveness to exposure in creating particular molecular lesions in somatic cells are excellent, both for alterations of single nucleotides and for major alterations of gene structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Alleles↗
PubMed1990

HIV-1 envelope protein gp120 expression by secretion in E. coli: assessment of CD4 binding and use in epitope mapping.

A non-glycosylated form of the HIV-1 envelope protein gp120 and four truncated derivatives have been expressed as non-fused secreted products in the periplasmic space of E. coli. We show that the full length molecule, whilst folded and soluble, fails to bind to CD4 consistent with other work that suggests an essential role for carbohydrate in gp120 function. In addition, when used in conjunction with the truncated derivatives, rapid epitope mapping of anti-gp120 monoclonal antibodies is achieved using both Western-blot and ELISA formats.

Antibodies, Monoclonal↗
PubMed1990

Expression of HIV-1 gp120 and human soluble CD4 by recombinant baculoviruses and their interaction in vitro.

The soluble domains of the envelope glycoprotein of HIV-1 (gp120) and human CD4 (sCD4) have been individually expressed in insect cells using recombinant baculoviruses. Each product is secreted from infected cells and accumulates in the surrounding media to levels of 1-2 mg/liter of 2 x 10(9) cells. Both molecules have full biological activity, and conditioned media from infected cells have been used to establish a simple assay for gp120-sCD4 interaction that is highly specific and amenable to mass screening. The crystallization of sCD4 purified from this source is reported.

Animals↗
PubMed1990

Characterization of recombinant gp120 and gp160 from HIV-1: binding to monoclonal antibodies and soluble CD4.

We compared four preparations of recombinant HIV-1 envelope glycoprotein: mammalian (Chinese hamster ovary cells) gp120 (Celltech); baculovirus gp120 from American Biotechnologies Inc. (ABT) and from MicroGeneSys (MGS); and baculovirus gp160 (Institute of Virology, Oxford, UK). Each envelope glycoprotein binds to a neutralizing monoclonal antibody (MAb) directed against the V3 loop, confirming the integrity of this type-specific neutralization epitope. MGS gp120 binds abnormally well to a MAb which recognizes an epitope preferentially exposed on denatured gp120. Consistent with this finding, MGS gp120 binds to soluble CD4 (sCD4) with an affinity 50-100-fold lower than that of Celltech gp120. The affinity of Celltech gp120 from sCD4 is 2.3 nM, indistinguishable from that of gp120 extracted from HIV-1 virions. Baculovirus gp120 (ABT) and gp160 also have a high affinity for sCD4. A significant proportion of anti-gp120 antibodies in HIV-positive human sera recognize epitopes that are dependent on the mammalian glycosylation pattern, and a human HIV-positive serum inhibits the binding of mammalian gp120 to sCD4 five- to 10-fold more potently than it inhibits baculovirus gp120 binding to sCD4.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Expression and purification of p24, the core protein of HIV, using a baculovirus-insect cell expression system.

The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been genetically manipulated to yield a recombinant virus capable of expressing p24, the major core protein of HIV-1, in insect cell culture. The expressed product is a p24 protein flanked by short regions of p17 at the amino terminus and p12 at the carboxy terminus. It has been identified and characterized using monoclonal antibodies on Western blots and by amino-terminal sequence analysis. The presence of p24 in the soluble fraction of infected cells following lysis by detergent or sonication, combined with a high level of expression (in excess of 50 mg/l of culture) facilitates the enrichment of large quantities of recombinant HIV antigen in a simple two-step procedure involving ammonium sulphate fractionation and gel filtration. p24 antigen purified in this way is shown to be an efficient diagnostic reagent.

Amino Acid Sequence↗