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D J Hayzer

Publications and source records attributed to D J Hayzer.

17 recordsLinked to original sources
PubMed1992

Molecular cloning of the rat vascular smooth muscle thrombin receptor. Evidence for in vitro regulation by basic fibroblast growth factor.

To study thrombin's receptor-mediated effects on vascular cells, we cloned and characterized a cDNA encoding a rat smooth muscle cell thrombin receptor. A rat aortic smooth muscle (RASM) cell cDNA library was screened with a 500-base pair (bp) sequence from the human thrombin receptor, obtained by polymerase chain reaction (PCR) amplification of cDNA synthesized from human erythropoietic leukemia (HEL) cell mRNA with PCR primers based on the published human thrombin receptor sequence. Clone pRTHR17 contains a 3418-bp insert that includes 50 bp of the 5'-untranslated region and the entire coding and 3'-untranslated regions of the RASM cell thrombin receptor. The sequence of pRTHR17 is 85% similar, at the nucleotide level, and 78% similar, at the deduced amino acid level, to the human thrombin receptor. Although the putative thrombin cleavage and binding sites are present, there are significant differences between the rat and human receptors in their amino-terminal sequences. Detectable signals (consisting of a single band of 3.45 kb) are present by Northern analysis of mRNA from RASM cells, and rat lung, kidney, and testes, but not in aorta or other tissues probed. The results of Southern analysis of rat genomic DNA are consistent with the existence of a single copy of the gene encoding this receptor. The steady state thrombin receptor mRNA level is low in cultured growth-arrested RASM cells and not detectable in rat aorta. To determine whether regulation of the RASM cell thrombin receptor occurs under growth-stimulating conditions, growth-arrested RASM cells were treated with basic fibroblast growth factor (bFGF, recently proposed to be a major mitogen controlling vascular smooth muscle cell growth following injury (Lindner, V., and Reidy, M. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3739-3743)). There was a significant increase in thrombin receptor mRNA following the addition of bFGF. These data demonstrate that: 1) mRNA for a thrombin receptor similar to that reported from human megakaryocyte and hamster fibroblast cell lines is present in proliferating primary culture rat smooth muscle cells, 2) the most significant sequence differences are present in the amino-terminal tail of the thrombin receptor, and 3) the mRNA level for this receptor is regulated under growth-stimulating conditions in vitro.

Amino Acid Sequence↗
PubMed1992

A rat beta-interferon-induced mRNA: sequence characterization.

Polymerase chain reaction amplification of a cDNA derived from rat aortic smooth muscle cells, using sequences from conserved regions of the intramembrane domains of adrenergic receptors as primers, yielded the clone, rat8. This clone possesses a high degree of sequence similarity to a series of human interferon (IFN)-inducible genes. The rat8 sequence is 70% similar to that derived from the human alpha-IFN-induced gene, 9-27; there is 66% similarity between the deduced amino acid sequences encoded by the rat and the human genes. The rat homologue hybridizes with many bands in Southern analysis of rat DNA, suggesting that it is a member of a large multigene family.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of a cDNA encoding a novel protein related to the neuronal vesicle protein synaptophysin.

The structure of synaptophysin, an integral membrane protein present in synaptic vesicles, is highly conserved. We report the sequence analysis of a clone, HL-5, isolated from a human erythroleukemia cell cDNA library, that is similar to synaptophysin in DNA and amino acid sequence. The predicted protein product derived from this clone is truncated, and the tissue distribution of HL-5 is different from that of synaptophysin. Thus, HL-5 appears to be a member of a previously undescribed family of synaptophysin-like genes.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of a human endothelin receptor: subtype A.

The polymerase chain reaction, employing degenerate primers specific for the intramembrane domains III and VI of G-coupled receptors, was used to generate partial clones encoding those receptors carried by cultured rat aorta smooth muscle cells. One clone, spanning the intramembrane domains IV-VI of a receptor specific for endothelin-1 (ET-R[A]), was used as a probe to screen a human placental cDNA library. The clone pL4-3, encoding a selective type of human endothelin receptor (ET-R[A]), has an open reading frame encoding a protein 427 amino acids in length, with a relative molecular weight of 48,625 daltons. The sequence analysis suggests the presence of a signal peptide, two potential sites for glycosylation in the N terminal extracellular domain, the seven transmembrane domains typical of G-protein receptors, and several potential sites for phosphorylation in the C terminal cytoplasmic domain. At the amino acid level, the human ET-R(A) shows 91% and 94% identity with the rat and bovine ET-R(A)s, respectively, and 59% similarity with the human ET-R(B). Xenopus laevis oocytes injected with the cloned cDNA express binding sites specific for endothelin-1. Expression of the message in COS 7 cells gave a membrane-bound product to which binding of the [125I]-ET-1 was inhibited by peptide analogues specific for ET-R(A).

Amino Acid Sequence↗
PubMed1990

Alternative splicing of glucokinase mRNA in rat liver.

The sequences of two near full-length cDNAs encoding rat liver glucokinase are reported. One of the cDNAs is essentially identical to the cDNA cloned by Andreone, Printz, Pilkis, Magnuson & Granner. [(1989) J. Biol. Chem. 264, 363-369]. The other cDNA contains a 151 bp insertion and a downstream 52 bp deletion. The inserted block of bases has been shown to originate from an optional cassette exon, termed 2A, between the previously described exons 1 and 2. The conceptual translation product from the variant mRNA is identical to the original glucokinase protein for the first 15 amino acids. Next there is a novel polypeptide sequence of 87 residues, comprising 50 residues encoded by the cassette exon and 37 residues specified by an altered reading frame in exon 2. Due to the 52 bp deletion, 17 amino acids of the reference sequence are then missing, after which the sequence reverts to the original. Northern blot analysis with oligonucleotide probes has shown that alternatively spliced mRNA represents about 5% of total glucokinase mRNA. Alternative splicing of glucokinase mRNA in liver may explain earlier findings of minor isoforms of hepatic glucokinase.

Amino Acid Sequence↗
PubMed1990

cDNA clones encoding immunoglobulin lambda chains from rabbit expressing the phenotype c7.

A cDNA library derived from spleen cells of an unimmunized rabbit expressing the c7 phenotype of Ig lambda chains (c7+, c21-) was screened with V lambda or C lambda probes of a lambda light chain bearing c21 epitopes. The nucleotide sequences of three hybridizing clones were found to be identical within the V lambda, J lambda and C lambda regions. The V lambda region was 97% similar to that of the functional germ-line gene V lambda 2, and the C lambda region was identical to that of gene C lambda 6, recently identified. Gene C lambda 6 exhibited four codon differences when compared with gene C lambda 5, the latter encoding c21 epitopes. The data presented here and in the accompanying report (Jaton, J.-C. et al., Eur. J. Immunol. 1990, 20:2713) support the view that gene C lambda 6 encodes the C region of c7 lambda chains and that c7 and c21 markers designate two distinct isotypic forms of lambda chains. On the basis of comparative Southern blotting analyses and restriction maps of cloned genomic regions containing V lambda and C lambda genes, a scheme is proposed to account for the c7- and c21- phenotypes.

Amino Acid Sequence↗
PubMed1990

Chemical and immunochemical identification of the second major rabbit immunoglobulin lambda chain polypeptide bearing c7 epitopes.

The purification of lambda chain-containing IgG fraction from pooled sera of Basilea rabbits, which were bred and selected for the expression of a high level of lambda chains positive for c7 but negative for c21, was carried out. On the basis of specific binding to anti-c7 antiserum, the c7 lambda chain fraction in serum IgG was shown to account for up to 70% of the total immunoglobulin light chains, the remaining 30%, bearing the expected k2 bas isotype. Peptide mapping of the mixed light chains (lambda + k2 bas) followed by microsequencing of the constant region fragments indicated that the C lambda region originated from the cDNA sequence derived from a c7+,c21- Basilea rabbit (i.e. identical to gene C lambda 6), as described in the preceding report (Hayzer, D. J. et al., Eur. J. Immunol. 1990. 20: 2707). In addition, a synthetic peptide encompassing residues 139-159 of the constant region derived from the predicted sequence of gene C lambda 6 was shown to partially inhibit the c7-anti-c7 binding reaction in a sensitive enzyme-linked immunosorbent assay. Taken together, the chemical and immunochemical data clearly demonstrate that gene C lambda 6 indeed encodes c7 epitopes.

Amino Acid Sequence↗
PubMed1990

Immunoglobulin lambda light chain evolution: Igl and Igl-like sequences form three major groups.

The nucleotide sequences, and the derived protein sequences, of immunoglobulin (Ig) Igl, Igl-like VpreB genes and the protein sequences of Igl-C regions were aligned and compared. A classification of the Igl and Igl-like VpreB sequences into three categories, designated groups I, II, and III, is proposed. Group I contains the human and mouse Igl-like VpreB genes. Group II contains Igl-V genes of the rabbit and the recently described mouse Igl-Vx gene. Group III includes the Igl-V genes, encoding all other known Igl-V region protein sequences, of mouse, rat, human, pig, sheep, and chicken. An evolutionary analysis of the three groups is presented, and suggests that the group III genes are evolving at a faster rate than those of the other groups and that within this group a further subdivision is possible: the V lambda-encoding genes of mouse, rat, and one human subgroup evolve faster than other group III genes. It is suggested that all mammalian species contain Igl-V genes of each group. A similar comparison between the protein sequences encoded by the known Igl-C genes indicates that the duplication of the Igl-J-C gene pairs occurred independently in each species, after mammalian speciation, and that the Igl-V-(J-C)(J-C) gene clusters of the mouse may not have their homologues in other species.

Amino Acid Sequence↗
PubMed1989

Cloning and sequencing of two functional rabbit germ-line immunoglobulin V lambda genes.

A recombinant-phage library of rabbit genomic DNA was screened for immunoglobulin V lambda genes. Two functional genes, V lambda 2 and V lambda 3, which are separated by 1.6 kb were isolated and sequenced. Both are accompanied by signals required for transcription-translation and the recombination with J lambda genes. The two genes, which are 95% similar in their framework regions, may be the parents of several cDNAs encoding V lambda regions. Rabbit V lambda-region diversity is likely to be generated by somatic mutation and V-J junction flexibility. Two cDNA clones encoding lambda-light chains may have arisen by gene conversion, exchanging the complementarity-determining regions, CDR3s, of the V lambda 2 and V lambda 3 genes.

Animals↗
PubMed1989

Inactivation of rabbit immunoglobulin lambda chain variable region genes by the insertion of short interspersed elements of the C family.

Two rabbit germ-line genes encoding immunoglobulin lambda light chain V regions were cloned from a rabbit genomic liver DNA library and characterized. One, V lambda 1, is separated by at least 8 kb from any other V lambda gene. The second, V lamdba 4, forms part of a three-gene cluster with two functional V lambda genes recently reported. Both V lambda 1 and V lambda 4 have structural features rendering them pseudogenes. The coding regions have frame-shift mutations which would yield defective protein products; both genes are also interrupted by the insertions of short, interspersed repetitive elements of the C family. In the V lambda 1 gene, the 369-bp insert is located upstream of the gene between the putative TATA box and the leader exon, whereas in gene V lambda 4, the 360-bp insert interrupts the FR2 at codon 48c. In addition, the sequence of the complement-determining region 3 of gene V lambda 1 is very similar to the mouse DSP2.6 sequence.

Amino Acid Sequence↗
PubMed1988

A rabbit Ig lambda L chain C region gene encoding C21 allotopes.

Southern blot analyses of germ-line DNA obtained from rabbits expressing lambda chains of C7 and/or C21 allotypes were performed with a rabbit C lambda region-specific probe; a 12-kbp EcoRI- and a 2-kbp BamHI-hybridizing fragment were detected only in the DNA from rabbits expressing the C21 allotype. The 12-kbp EcoRI fragment was cloned and shown to contain two C lambda region-encoding genes in the same orientation. Each is preceded by a J lambda gene segment. Nonamer-12-bp spacer-heptamer recombination signal sequences were found 5' of each J lambda segment, and splicing signals were identified at the 3' ends of the J lambda segments and the 5' ends of the corresponding C lambda genes. The C lambda 5 gene, which exhibits a sequence identical with that found in several cDNA clones, is carried by the 2-kbp BamHI fragment missing from the genomic DNA of rabbits which do not express the C21 allotype. The second C lambda gene, C lambda 6, lies 3' of C lambda 5, in a 1.6-kbp BamHI fragment which is present in genomic DNAs of all tested rabbits, irrespective of their phenotype. Its sequence is identical with that found in one cDNA clone and differs from that of C lambda 5 in 17 base positions resulting in four amino acid substitutions. A fragment of a cDNA, with a J-C region sequence identical with that encoded by the J lambda 5-C lambda 5 gene pair, was subcloned into a plasmid expression vector. The resulting polypeptide product could be specifically immunoprecipitated by anti-C21 but not anti-C7 alloantisera, showing that some, if not all, C21 allotopes are encoded by the C lambda 5 gene. In contrast, the C lambda 6 gene product was not precipitable, either by anti-C7 or by anti-C21 alloantisera, although it was readily immunoprecipitated by a goat anti-rabbit lambda chain antiserum.

Amino Acid Sequence↗
PubMed1987

cDNA clones encoding rabbit immunoglobulin lambda chains. Evidence for length variation of the third hypervariable region and for a novel constant region.

Five cDNA clones designated pDH2, pDH8, pDH9, pDH31 and pDH101 encoding rabbit immunoglobulin lambda light chain sequences have been characterized. Comparison of the V lambda sequences suggests that, in addition to an increased divergence in all of the complementarity-determining regions (CDRs), variable-region diversity is amplified by the length heterogeneity of the CDR3, at the V lambda-J lambda junction. An insertion of four codons at positions 48a-d has been noted in three cDNA sequences. This insert, not found in lambda nor kappa light chains of other species, has a variable sequence, suggesting its possible implication in expanding variability of the CDR2. One of the cDNA clones was shown to encode a novel C lambda region which differs by four amino acid substitutions from the C lambda region common to all the other clones. Thus, the rabbit can use two different C lambda genes, which might correlate with the expression of the two known allotypes of lambda chains, C7 and C21. Southern blotting experiments indicate a small number of germ-line V lambda genes and the cDNA nucleotide sequence data reported here suggest that several of these genes can be expressed. The possibility of at least two V-J-C gene clusters is discussed.

Alleles↗
PubMed1987

Nucleotide sequence of a cDNA clone encoding a rabbit immunoglobulin-lambda light chain: the V lambda region differs markedly from that of other species.

A cDNA clone (pDH7) has been isolated which encodes the entire leader peptide and variable (V) region and most of the constant (C) region of a rabbit lambda-light chain. Although similar to amino acid sequences derived from fragments of isolated lambda-chains from several Basilea rabbits, differences in the first framework region (FR1) suggest that at least two germ-line V lambda genes are expressed. There are major differences between rabbit V lambda sequences and light chains of other species: in particular, rabbit lambda-chains have an additional four amino acids in the vicinity of the FR2-CDR2 junction. The same region also has significant homology with the human D2 germ-line mini-gene sequence, especially with a 14-nucleotide sequence previously shown to be homologous to human and rabbit heavy chain CDR2 sequences. Similar homologies in other heavy and light chain sequences suggest that D-gene segments may be derived from VH genes, perhaps by transposition. The framework regions of the rabbit lambda-chain encoded by clone pDH7 show the greatest homologies with those of human kappa- and lambda-sequences (46 to 54% homology), with that of chicken sequence (55%), and least with murine V lambda sequences (40%).

Amino Acid Sequence↗
PubMed1978

Proline biosynthesis by cell-free extracts of Escherichia coli and potential errors arising from the use of a bioradiological assay procedure.

1. The growth of Escherichia coli proline auxotrophs on medium containing L-proline (50 microgram/ml) induces catabolic enzymes. A bioradiological assay system for proline, using proB cells of E. coli, might give erroneous results owing to proline catabolism by the proline auxotrophs on which the assay depends. 2. Differential utilization of proline and 1-pyrroline-5-carboxylate by the proB cells for the synthesis of protein, and failure of the method to distinguish between these two possible products of the proline-biosynthetic enzymes, might also give rise to error. 3. The proline-dependent incorporation of [14C]phenylalanine into the protein of proline-starved proB auxotrophs was to some degree directly influenced by the presence of crude cell extract from E. coli, even though this was not supplied with substrate and cofactors, and could thus not itself synthesize proline. 4. The kinetics of proline biosynthesis by cell-free extracts were linear and biphasic, only the last phase being affected by the concentrations of substrate and extract. This phenomenon is not understood. 5. Proline biosynthesis is inhibited, not only by high concentrations of ATP, but also by aspartate, glycine, alanine and serine, aspartate having the greatest effect. 6. Attempts at complementation in vitro between extracts of proline auxotrophic mutants were not successful, suggesting the possibility that strain X680 (proA) and/or X278 (proB) may be a double mutant. 7. The enzymes of proline biosyntehsis are co-eluted from a column of Bio-Gel A1.5M in a position corresponding to a mol.wt. of 350000. 8. Comparisons between rates of proline biosynthesis in vivo and in vitro were made.

Adenosine Triphosphate↗
PubMed1978

The enzymes of proline biosynthesis in Escherichia coli. Their molecular weights and the problem of enzyme aggregation.

1. By using Bio-Gel A1.5M and Sephadex G-150 columns, crude cell-free extracts of Escherichia coli were fractionated to demonstrate the existence of a proline-biosynthetic aggregate. 2. Sephadex G-150 resolves two glutamyl kinases that are inhibited by proline, with mol.wts. of 125000 and 38000, the reactions of which are Mg2+-dependent. The heavier species is more sensitive to inhibition by proline. 3. Gamma-Glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase (EC 1.5.1.2) have mol.wts. of approx. 125000 and 190000 respectively, the specific activity of the latter being 5 X 10(3)-fold greater than either of the other two biosynthetic enzymes or of the total pathway in vivo. 4. Bio-Gel A1.5M chromatography gave a single glutamyl kinase of mol.wt. 250000, and the possibility of this being a constituent of an enzyme complex is discussed.

Aldehyde Oxidoreductases↗
PubMedDate not supplied

Conjugation of plasminogen activators and fibrin-specific antibodies to improve thrombolytic therapeutic agents.

Here we have reviewed chemical and recombinant approaches to the construction of hybrid molecules that combine a "targeting" antibody and an "effector" enzyme activity. There are advantages and disadvantages to both chemical and recombinant methods, and one goal of this review has been to elucidate these so that the appropriate method can be used by those interested in using hybrid molecules to study questions of basic or therapeutic importance. The system studied in greatest detail has as its goal the targeting of a plasminogen activator to an occlusive intravascular thrombus. We have, therefore, used this system as an example of currently available approaches. Now that these methodologies have been studied and put into use, it is anticipated that this principle will be generalized both to other therapeutic applications, as well as to the design and construction of molecules that will allow more basic questions to be addressed.

Animals↗