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J E Coligan

Publications and source records attributed to J E Coligan.

At least 181 records · Page 10Linked to original sources
PubMed1986

Alternative protein products with different carboxyl termini from a single class I gene, H-2Kb.

In addition to mRNA encoding the canonical form of the murine class I antigen H-2Kb (348 amino acid residues), mRNA that would encode a shortened form of H-2Kb (missing 9 amino acids from the C-terminus) has been identified in C57BL/6 spleen cells by RNase-protection studies. The alternative transcripts of H-2Kb arise through the use of different AG acceptor splice sites for exon VIII. The existence of a shortened H-2Kb protein was demonstrated by sequential immunoprecipitation. Lysates of spleen cells that had been labeled with [35S]methionine and [3H]histidine were precleared with rabbit anti-peptide serum reactive with the C-terminus of the canonical H-2Kb. The shortened form of H-2Kb was immunoprecipitated from this lysate with H-2Kb alloantiserum. Both forms of H-2Kb were isolated by NaDodSO4/PAGE. Tryptic peptide mapping confirmed that these molecules differed only at their C-terminus. The shortened form of H-2Kb is also found in a B-cell line (R8) but not in three cloned T-cell lines or in a T-cell lymphoma (EL4), suggesting that regulatory events are involved in producing the two forms of H-2Kb. Putative lariat branch points involved in these alternative splicing events for the 3' coding region of H-2 class I pre-mRNAs are proposed.

Animals↗
PubMed1986

Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin.

The human laminin receptor was purified and molecularly cloned to investigate its biosynthetic regulation. Laminin receptor from normal and neoplastic tissue was preparatively affinity purified to homogeneity based on the high affinity of the receptor for laminin. The apparent molecular weight of the receptor from different carcinoma sources and from normal placental tissue is in the range of 68-72 kDa. Isoelectric focusing and two-dimensional gel electrophoresis indicated that the receptor protein consists of one major polypeptide chain with a pI value of 6.4 +/- 0.2. Laminin receptor cDNA clones were isolated after screening a human endothelial lambda gt11 cDNA library with a monoclonal antibody directed against a domain of the laminin receptor involved in ligand binding. Definitive identification of the cDNA clones was based on comparison of cDNA sequence with the amino acid sequence of a cyanogen bromide-generated octapeptide of purified placental laminin receptor. The laminin receptor mRNA is approximately 1700 bases long. The level of laminin receptor mRNA in a variety of human carcinoma-derived cell lines correlated with the number of laminin receptors on the cell surfaces of those cells. This suggests that the amount of laminin receptor mRNA may be a rate-limiting control step in the biosynthesis of the laminin receptor, and hence in the regulation of cellular attachment to basement membranes via laminin.

Amino Acid Sequence↗
PubMed1986

Tumorigenicity of hamster and mouse cells transformed by adenovirus types 2 and 5 is not influenced by the level of class I major histocompatibility antigens expressed on the cells.

Inbred hamster and mouse cells transformed by the nononcogenic adenovirus (Ad) serotypes, Ad2 and Ad5, are nontumorigenic in syngeneic adult animals, while cells from these species transformed by the highly oncogenic Ad12 are tumorigenic in such rodents. By immunoprecipitation and flow cytometry, cells from four of six Ad2- and Ad5-transformed hamster and mouse lines expressed high levels of cell-surface class I major histocompatibility complex (MHC) antigens, while cells from two of these six lines expressed low levels of cell-surface class I MHC antigens. The levels of class I MHC proteins expressed by cells from these latter two lines were comparable to the levels of cell-surface class I MHC proteins expressed by cells from Ad12-transformed hamster and mouse lines. Moreover, an Ad2-transformed line that had become highly oncogenic after in vivo adaptation showed the same high level of MHC expression as the nononcogenic parent. The amounts of class I mRNA, analyzed by RNA blotting, were, in general, consistent with the levels of class I antigens expressed on the surfaces of these cells. These results indicate that there is no correlation between the tumorigenicity in immunocompetent syngeneic adult rodents of Ad2- and Ad5-transformed hamster and mouse cells and the level of class I MHC antigens expressed on the surfaces of these cells. Thus, the expression of different levels of class I MHC proteins does not seem to explain the differences in the oncogenicity between nononcogenic and highly oncogenic human Ad serotypes.

Adenoviruses, Human↗
PubMed1986

Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein.

The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5.

Amino Acid Sequence↗
PubMed1986

Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus.

Automated N-terminal microsequencing of immune affinity-purified acquired immunodeficiency syndrome retrovirus polypeptides from infected cells was used to locate the N termini of 64-, 51-, and 34-kilodalton (kDa) polypeptides within the pol open reading frame (ORF) of the proviral DNA. The 64- and 51-kDa proteins had identical N termini (Pro-Ile-Ser-Pro-IIe-Glu-Thr-Val-) positioned 156 residues from the beginning of the pol ORF. The N terminus of the 34-kDa pol gene product, Phe-Leu-Asp-Gly-Ile-Asp-Lys-, mapped 716 residues into the pol ORF. These polypeptides were absent in an RT-negative, CD4-negative, persistently infected cell line (8E5) carrying a single defective copy of a constitutively expressed, integrated proviral DNA.

Amino Acid Sequence↗
PubMed1986

Characteristics of the expression of the murine soluble class I molecule (Q10).

Q10 is a class I molecule previously proven to be secreted rather than membrane bound. To measure the amount of Q10 in various mouse sera, a quantitative Western blot assay was developed. Q10 was the only class I molecule detectable in mouse sera. It occurs as a high m.w. complex of 200,000 to 300,000. The amount of Q10 in serum varies among different mouse strains and is controlled by a region telomeric to H-2S. Mice of the f haplotype do not express Q10, but all other mice examined (20 strains) with inbred or wild-derived H-2 haplotypes do. The H-2 haplotypes rank according to their levels of Q10 as follows: z, s greater than k, b greater than d, q greater than f; and the actual values range from to 60 micrograms/ml to undetectable levels in serum. In some strains the levels are higher in males than in females. The levels increase with age and decrease during pregnancy but not during lactation. There is a dramatic decrease after the injection of irritants or syngeneic tumor transplantation, but allostimulation has no effect on Q10 levels. The possible significance of this soluble class I molecule is discussed in the light of our findings.

Animals↗
PubMed1985

A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients.

A newly identified protein from HTLV-III/LAV, the virus implicated as the etiologic agent of the acquired immune deficiency syndrome, was studied. This protein, which has a molecular weight of 27,000 (p27), was shown by amino acid sequencing to have a coding origin 3' to the env gene on the HTLV-III genome. The presence of antibodies to p27 in virus-exposed individuals indicated that this gene is functional in the natural host.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Respiratory syncytial virus envelope glycoprotein (G) has a novel structure.

Amino acid sequence of human respiratory syncytial virus envelope glycoprotein (G) was deduced from the DNA sequence of a recombinant plasmid and confirmed by limited amino acid microsequencing of purified 90K G protein. The calculated molecular mass of the protein encoded by the only long open reading frame of 298 amino acids was 32,588 daltons and was somewhat smaller than the 36K polypeptide translated in vitro from mRNA selected by this plasmid. Inspection of the sequence revealed a single hydrophobic domain of 23 amino acids capable of membrane insertion at 41 residues from the N-terminus. There was no N-terminal signal sequence and the hydrophilic N-terminal 20 residues probably represent the cytoplasmic tail of the protein. The N-terminally oriented membrane insertion was somewhat analogous to paramyxovirus hemagglutinin-neuraminidase (HN) and influenza neuraminidase (NA). The protein was moderately hydrophilic and rich in hydroxy-amino acids. It was both N- and O-glycosylated with the latter contributing significantly to the net molecular mass 90K.

Amino Acid Sequence↗
PubMed1985

Variable major proteins of Borrelia hermsii. Epitope mapping and partial sequence analysis of CNBr peptides.

The variable major proteins (VMP) of serotypes 7 and 21 of the relapsing fever agent Borrelia hermsii were isolated by detergent extraction and high performance liquid chromatography. Cyanogen bromide (CNBr) digestion of the isolated VMP yielded two peptides of apparent molecular weights 20,000 (20 K) and 16 K from VMP7, and three peptides of 14.5, 14, and 7 K mol wt from VMP21. Serotype-specific monoclonal antibodies bound in Western blots to one of each of the two or three CNBr fragments from the homologous VMP. A single monoclonal antibody bound to the whole cells, the isolated VMP, and a CNBr fragment of both serotype 7 and serotype 21. (This crossreactive antibody did not, however, bind to any of four other serotypes examined.) Regional conservation of structure between VMP7 and VMP21 was also shown by amino acid sequence analysis of the N-termini of the five CNBr fragments. One pair of aligned fragments from VMP7 and VMP21 had 80% amino acid homology in sequence; a second pair had 40% homology. The partial amino acid homologies between two VMP suggest that these proteins are products of members of a polygene family.

Amino Acid Sequence↗
PubMed1985

Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III.

Antibodies from the serum of patients with the acquired immune deficiency syndrome (AIDS) or with the AIDS-related complex and from the serum of seropositive healthy homosexuals, recognize two major glycoproteins in cells infected with human T-cell lymphotropic virus type III (HTLV III). These glycoproteins, gp160 and gp120, are encoded by the 2.5-kilobase open reading frame located in the 3' end of the HTLV-III genome, as determined by amino terminus sequence analysis of the radiolabeled forms of these proteins. It is hypothesized that gp160 and gp120 represent the major species of virus-encoded envelope gene products for HTLV-III.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1.

Amino Acid Sequence↗
PubMed1985

Analysis of the structure of the human T cell surface antigen T8 by limited proteolysis and chemical cleavage.

The structure of the human T lymphocyte surface antigen T8 (Leu 2) has been explored utilizing limited proteolysis on viable cells and cellular lysates. The positions of the cleavage sites of trypsin and papain were placed relative to the single CNBr cleavage point. Additional data allowed the location of the amino and carboxyl termini relative to the enzymatic and chemical cleavage sites. This information, together with earlier evidence concerning the position of a membrane binding site, allowed the construction of a model illustrating the vectorial orientation of the molecule on the cell. Within this model, the approximate positions of disulfide linkages were indicated based on the results of nonreduced/reduced two-dimensional sodium sulfide-polyacrylamide gel electrophoresis. Carbohydrate moieties were localized using cleavage with trifluoromethanesulfonic acid, a reagent which cleaves both N-linked and O-linked oligosaccharides. Finally, the implications of the proteolysis experiments in relation to the function of T8 were discussed.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1985

Biosynthesis of rat insulin-like growth factor II. II. Localization of mature rat insulin-like growth factor II (7484 daltons) to the amino terminus of the approximately 20-kilodalton biosynthetic precursor by radiosequence analysis.

In previous studies, we have identified possible biosynthetic precursors of rat insulin-like growth factor II (rIGF-II) using specific immunoprecipitation, approximately 22-kDa prepro-rIGF-II and 20-kDa pro-rIGF-II. We now provide chemical evidence that amino acid sequences corresponding to mature 7484-dalton rIGF-II are present at the NH2 terminus of the putative approximately 20-kDa pro-rIGF-II. BRL-3A cultures have been labeled individually with several radioactive amino acid precursors, the cells have been lysed, and the lysates have been immunoprecipitated with antiserum to rIGF-II. Following electrophoresis of the immunoprecipitated proteins, labeled approximately 20-kDa pro-rIGF-II was eluted from the gels and subjected to automated radiosequence analysis. Discrete peaks of radioactivity were observed in 12 of the first 30 cycles of Edman degradation. The deduced partial amino acid sequence was identical at each position with that of mature 7484-dalton rIGF-II. These results directly demonstrate that mature rIGF-II sequences are present in the approximately 20-kDa protein, as required if the approximately 20-kDa protein were pro-rIGF-II. In addition, they localize the 7484-dalton rIGF-II to the NH2 terminus of the precursor molecule. A second NH2-terminal sequence differing only in the absence of the NH2-terminal residue, alanine, also was present in an approximately equal amount. Similar NH2-terminal heterogeneity has been reported for 7484-dalton rIGF-II and most likely reflects ambiguity in the cleavage sites for the signal peptidase.

Amino Acid Sequence↗
PubMed1985

Secretion of a soluble class I molecule encoded by the Q10 gene of the C57BL/10 mouse.

The DNA sequence of the Q10 genes appears to be highly conserved amongst strains of mice and has only been found to be transcribed in the liver. An examination of the nucleotide sequence of the exon that normally encodes the transmembrane domain of class I molecules suggested that the Q10 gene encodes a secreted protein. We have established this by showing that L cells transformed with an expression vector containing the Q10 gene secrete a class I molecule which was identified with an antiserum raised against a peptide predicted by the Q10 transmembrane exon. Both the L cell-derived Q10 molecule and a class I protein immunoprecipitated from serum with this anti-peptide antiserum have mol. wts. of approximately 38 000; the Q10 molecule secreted by L cells is heterogeneous in mol. wt. This heterogeneity was drastically reduced after endoglycosidase F treatment, suggesting that Q10 molecules secreted into the serum by the liver may be glycosylated differently from those secreted by L cells. Endoglycosidase F treatment of both the L cell and serum forms of the soluble molecule yielded two products with mol. wts. of approximately 32 000 and 35 000; this is consistent with the observation that the predicted Q10 protein sequence has two potential glycosylation sites. In contrast to previous published results, the Q10 molecule reacted with rabbit anti-H-2 antisera which is consistent with its greater than 80% homology to the classical transplantation antigens.

Animals↗
PubMed1985

Human cytotoxic T-lymphocyte recognition of an HLA-A3 gene product expressed on murine L cells: the only human gene product required on the target cells for lysis is the class I heavy chain.

To dissect the molecular basis for T-cell recognition of class I major histocompatibility complex antigens, we have examined the ability of human cytotoxic T lymphocytes (CTL) to recognize murine L cells transformed with a human class I gene. Three transformed L-cell lines were generated that expressed the human HLA-A3 gene from donor E1 at levels comparable to those of the endogenous L-cell H-2Kk molecules. CTL were generated in secondary and tertiary mixed lymphocyte culture against the HLA-A3 subtype of donor E1 by culturing irradiated E1 peripheral blood lymphocytes with the peripheral blood lymphocytes of responder donor M3 (M3 shares all defined class I antigens with E1 but expresses a different HLA-A3 subtype). Each of the HLA-A3-transformed L cells was lysed by M3 anti-E1 CTL in a short-term 51Cr release assay and this recognition was blocked by a monoclonal anti-HLA-A3 antibody. Antibodies specific for the human T8 and LFA-1 molecules on the CTL effectors (but absent from the transformed targets) also blocked lysis of each of the HLA-A3 transformed L-cell targets. Antibodies to other T-cell molecules failed to block lysis. The present results demonstrate that human CTL can recognize human class I molecules on targets that do not express any other human gene product and further suggest that effector T-cell molecules T8 and LFA-1 are functionally involved in this recognition process.

Animals↗
PubMed1985

DNA sequence of the mouse H-2Dd transplantation antigen gene.

The inbred BALB/c mouse has three transplantation antigens, H2-Kd, H2-Ld, and H2-Dd. We present the complete nucleotide sequence of the H2-Dd gene as well as 777 residues of previously unpublished H-2Dd protein sequence. These data complete the sequences of all the BALB/c transplantation antigen genes and permit detailed comparison with each other and with their counterparts from the inbred C57BL/10 mouse. Transplantation antigens may differ from one another by as much as 5%-15% of their amino acid sequence for the external domains. These extensive differences may arise by gene conversion. The H-2D region of the BALB/c mouse encodes the H2-Dd and the H2-Ld genes. Serologic data suggest that at least two additional transplantation antigen molecules, H2-Rd and H2-Md, are encoded in the H-2D region of the major compatibility complex. Paradoxically, gene cloning studies have only identified the H2-Dd and the H2-Ld genes in the H-2D region. A complete DNA sequence of the H2-Dd gene shows that a variety of alternative splice sites exist throughout the gene, which may lead to additional gene products and may explain the multiplicity of H-2D-encoded polypeptides.

Amino Acid Sequence↗
PubMed1985

Partial amino acid sequence and molecular cloning of the encoding gene for the major outer membrane protein of Chlamydia trachomatis.

The first 25 N-terminal amino acids of the major outer membrane protein of Chlamydia trachomatis serovar L2 were determined. The amino acid sequence was used to construct an oligonucleotide probe specific for the major outer membrane protein gene. Using this oligonucleotide as a hybridization major outer membrane protein gene. Using this oligonucleotide as a hybridization probe, we discovered one recombinant clone that produced a 15-kilodalton polypeptide which reacted with a monoclonal antibody directed against the major outer membrane protein type-specific epitope. In a separate set of experiments, we uncovered another recombinant clone that produced a 51-kilodalton polypeptide which was reactive with an anti-major outer membrane protein subspecies-specific monoclonal antibody. The expression of these recombinant DNA plasmids in Escherichia coli is discussed.

Amino Acid Sequence↗