Cloning, sequencing and bacterial expression of human glycine tRNA synthetase.

The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da. The protein sequence has approximately 60% identity with B. mori GlyRS and 45% identity with S. cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. The enzyme was expressed in E. coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa. The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS. Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with [14C]-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.     

Identification of immunoreactive antigens of human papillomavirus type 6b by using Escherichia coli-expressed fusion proteins.

Human papillomavirus (HPVs) infect the genital epithelium and are found in proliferative lesions ranging from benign condylomata to invasive carcinomas. The immunological response to these infections is poorly understood because of the lack of purified viral antigens. In this study, bacterially derived fusion proteins expressing segments of all the major open reading frames (ORFs) of HPV type 6b (HPV-6b) have been used in Western blot (immunoblot) assays to detect antibodies directed against HPV-encoded proteins. The most striking reactivities present in sera from patients with genital warts were to the HPV-6b L1 ORF protein and, to a lesser extent, to the HPV-6b L2 ORF protein. Two cases of reactivity to HPV-6b E2 ORF were observed, but no reactivities were seen with other HPV-6b constructs. Two sera reacted with the HPV-16 L2 fusion protein, and two sera reacted with the HPV-16 E4 protein. The antibodies directed against the HPV-6b fusion proteins showed no cross-reactivity with comparable regions of the HPV-16 ORFs. This assay provides a useful approach for further studies of HPV serology.     

The varicella-zoster virus immediate-early protein IE62 is a major component of virus particles.

Varicella-zoster virus (VZV) open reading frame (ORF) 62 potentially encodes a protein with considerable amino acid homology to the herpes simplex virus (HSV) immediate-early regulatory polypeptide ICP4 (or IE3). To identify and characterize its protein product(s) (IE62), we used a rabbit antiserum prepared against a synthetic peptide corresponding to the C-terminal 13 amino acids of the predicted protein. This antiserum reacted with phosphorylated polypeptides of 175 to 180 kDa that were made in VZV-infected cells and in cells infected with a vaccinia virus recombinant expressing IE62, but not in control-infected cells, confirming its specificity and reactivity to the IE62 protein. The antiserum recognized a 175-kDa polypeptide in purified virions that comigrated with a major structural protein. Comparison of this reactivity with that of an antipeptide antiserum directed against the VZV ORF 10 product (homologous to the HSV major structural protein VP16) indicates similar levels of ORF 62 and ORF 10 polypeptides in VZV virions. In contrast, antipeptide antiserum directed against the VZV ORF 29 product, the homolog of the HSV major DNA-binding protein, failed to recognize any protein in our virion preparations. Treatment of virions with detergents that disrupt the virion envelope did not dissociate IE62 from the nucleocapsid-tegument structure of the virion. Differential sensitivity of VZV virion IE62 to trypsin digestion in the presence or absence of Triton X-100 indicates that IE62 is protected from trypsin degradation by the virus envelope; since it is not a nucleocapsid protein, we conclude that it is part of the tegument. Finally, we show that the virion 175-kDa protein either can autophosphorylate or is a major substrate in vitro for virion-associated protein kinase activity.     

Human antibodies react with an epitope of the human papillomavirus type 6b L1 open reading frame which is distinct from the type-common epitope.

Recombinant proteins encoded by the human papillomavirus type 6b (HPV6b) L1 open reading frame react with sera from patients with condylomata acuminata and also react with rabbit antiserum raised against sodium dodecyl sulfate-disrupted bovine papillomavirus type 1 (BPV1) virions. To map the immunoreactive epitopes, a series of procaryotic expression plasmids was made which contained a nested set of 3' to 5' deletions in the HPV6b L1 open reading frame. The deleted plasmids expressed a set of carboxy to amino terminus truncated fusion proteins. Regions containing the immunoreactive epitopes were mapped by determining which of the deleted fusion proteins retained reactivity with sera in Western immunoblot assays. The coding sequence for a human antibody-reactive linear epitope mapped between HPV6b nucleotide coordinates 7045 and 7087, and the rabbit anti-BPV1-reactive epitope coding sequence mapped between coordinates 6377 and 6454. Synthetic peptides derived from the epitope mapping were reacted with sera in enzyme-linked immunosorbent assay. Human sera reacted with synthetic peptide QSQAITCQKPTPEKEKPDPYK (HPV6b L1 amino acids 417 through 437). Rabbit anti-BPV1 and rabbit antisera raised against HPV16 L1 recombinant proteins reacted with the synthetic peptide DGDMVDTGFGAMNFADLQTNKSDVPIDI (HPV6b L1 amino acids 193 through 220). Human sera which reacted with HPV6b L1 fusion proteins cross-reacted with an HPV11 L1 fusion protein but did not react with fusion proteins encoded by HPV1a, HPV16, or HPV18. Rabbit anti-BPV1 reacted with L1 fusion proteins encoded by all of these HPV types. In contrast to the type-common (rabbit anti-BPV1-reactive) epitope, the human antibody-reactive epitope appears to be relatively HPV type specific.     

Cloning and characterization of two immunophilin-like genes, ilpA and fkpA, on a single 3.9-kilobase fragment of Aeromonas hydrophila genomic DNA.

Antiserum to Aeromonas hydrophila A6 cell envelopes was shown in a previous study (C. Y. F. Wong, G. Mayrhofer, M. W. Heuzenroeder, H. M. Atkinson, D. M. Quinn, and R. L. P. Flower, FEMS Immunol. Med. Microbiol. 15:233-241, 1996) to protect mice against lethal infection by this organism. In this study, colony blot analysis of an A. hydrophila genomic library using antiserum to A. hydrophila A6 cell envelopes revealed a cosmid clone expressing a 30-kDa protein which has not been described previously in aeromonads. The nucleotide sequence of a 3.9-kb fragment derived from this cosmid which expressed the 30-kDa protein revealed two potential open reading frames (ORFs) with homology to known immunophilin proteins. ORF1 encoded a 212-amino-acid protein (molecular mass, 22.4 kDa) with 56% identity to the immunophilin SlyD protein of Escherichia coli. ORF1 was subsequently designated ilpA (immunophilin-like protein). ORF3 encoded a potential gene product of 268 amino acids with a typical signal sequence and a predicted molecular size of 28.7 kDa. The inferred amino acid sequence showed 46% identity with the sequence of the FkpA protein of E. coli and 40% identity with the sequence of the macrophage infectivity potentiator (Mip) protein of Legionella pneumophila. ORF3 was designated fkpA (FK506 binding protein) by analogy with the E. coli FkpA protein. Expression of the FkpA protein was confirmed by Western blot (immunoblot) analysis, which detected a 30-kDa protein, with antiserum to the Mip protein of Legionella longbeachae and a specific antiserum to anA. hydrophila 30-kDa membrane protein. PCR and Southern analysis showed that a DNA sequence encoding FkpA was found in all 178 aeromonads of diverse origins tested. A nonpolar insertion mutation in the fkpA gene did not attenuate virulence in a suckling mouse model nor did it affect the expression of hemolysins or DNase. This suggests that either the fkpA gene is not essential in the virulence of A. hydrophila under these conditions or there are other genes in A. hydrophila coding for proteins with similar functions.     

Use of a novel strategy for the preparation and characterization of an antipeptide antibody capable of recognizing members of the annexin family

Annexins are structurally-related proteins which bind phospholipids in a Ca2+-dependent manner. We have used a novel coupling strategy to prepare an antiserum directed against a 17-amino acid synthetic peptide that resembles the sequence of a highly-conserved portion of these proteins. This antipeptide serum specifically recognizes 5 of 6 human annexins on Western blots, despite differences between the protein and peptide sequences of 3 or 4 amino acids. The antiserum does not recognize endonexin II, whose sequence differs from that of the peptide by 6 amino acids. The availability of multiple proteins with known amino acid sequence has allowed analysis of structural requirements for recognition by this antibody. In some situations, use of such an antibody may allow the identification of a protein as a member of a family.     

Palmitoylation of the Intracytoplasmic R Peptide of the Transmembrane Envelope Protein in Moloney Murine Leukemia Virus

Previously it was reported that the 16-amino-acid (aa) C-terminal cytoplasmic tail of Moloney murine leukemia virus (MoMLV) transmembrane protein Pr15E is cleaved off during virus synthesis, yielding the mature, fusion active transmembrane protein p15E and the 16-aa peptide (R peptide or p2E). It remains to be elucidated how the R peptide impairs fusion activity of the uncleaved Pr15E. The R peptide from MoMLV was analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostained with antiserum against the synthetic 16-aa R peptide. The R peptide resolved with an apparent molecular mass of 7 kDa and not the 4 kDa seen with the corresponding synthetic peptide. The 7-kDa R peptide was found to be membrane bound in MoMLV-infected NIH 3T3 cells, showing that cleavage of the 7-kDa R-peptide tail must occur before or during budding of progeny virions, in which only small amounts of the 7-kDa R peptide were found. The 7-kDa R peptide was palmitoylated since it could be labeled with [(3)H]palmitic acid, which explains its membrane association, slower migration on gels, and high sensitivity in immunoblotting. The present results are in contrast to previous findings showing equimolar amounts of R peptide and p15E in virions. The discrepancy, however, can be explained by the presence of nonpalmitoylated R peptide in virions, which were poorly detected by immunoblotting. A mechanistic model is proposed. The uncleaved R peptide can, due to its lipid modification, control the conformation of the ectodomain of the transmembrane protein and thereby govern membrane fusion.     

Identification of retinal insulin receptors using site-specific antibodies to a carboxyl-terminal peptide of the human insulin receptor alpha-subunit. Up-regulation of neuronal insulin receptors in diabetes

Insulin receptor-specific polyclonal antipeptide serum was generated against a synthetic pentadecapeptide (residues 657-670) of the deduced amino acid sequence of human insulin proreceptor cDNA for use in the analysis of insulin receptors in the retina. The affinity-purified antibodies recognized peptide antigen but not keyhole limpet hemocyanin as determined by dot blot analysis and solid phase radioimmunoassay. Addition of either synthetic peptide or the affinity-purified serum had no effect on 125I-insulin binding to placental membranes or to cells in culture. alpha-Subunits of approximately 125 kDa from human placental membranes and liver membranes were labeled by immunoblot analysis with this antiserum. In membranes isolated from human retina and brain, two classes of alpha-subunits of approximately 125 and 115 kDa were detectable. The 115-kDa subunit was neuraminidase resistant whereas the 125-kDa subunit was digested to a band of 115 kDa, indicating that these bands represent peripheral and neuronal receptors, respectively. Analysis of human retinas obtained from type I diabetic donors revealed an increased level of neuronal receptor as compared with normal retinas. These data indicate that human retina expresses neuronal insulin receptor subtypes that are up-regulated in diabetes.     

Sequence of the phosphoprotein gene of pneumonia virus of mice: expression of multiple proteins from two overlapping reading frames.

The gene encoding the phosphoprotein of the pneumovirus pneumonia virus of mice (PVM) has been cloned and sequenced. The gene is 903 nucleotides in length and contains a long open reading frame (ORF) capable of encoding a polypeptide of 295 amino acid residues. A smaller, second, overlapping ORF encoding a polypeptide 137 amino acids in length was also present. The large ORF directed the synthesis of a 39-kDa polypeptide and four additional polypeptides with masses of 37 kDa, 26 kDa, 23 kDa, and 16 kDa in vitro. The smaller polypeptides were generated by internal initiation on in-frame AUG initiation codons to generate carboxy co-terminal products. Western immunoblot analysis indicated that at least two of these proteins and several other related polypeptides are present in infected cells, and the possible origins of these are discussed. Western blot analysis using antiserum raised against a synthetic peptide and specific for the predicted second ORF product identified a polypeptide of 23 kDa in PVM-infected cells. The pattern of PVM P gene expression is unlike that of the closely related respiratory syncytial virus and is reminiscent of that of paramyxoviruses such as Sendai virus. This is the first example of a pneumovirus encoding multiple polypeptide products from a single mRNA in vivo.     

The cauliflower mosaic virus open reading frame VII product can be expressed in Saccharomyces cerevisiae but is not detected in infected plants.

Antiserum was prepared against a synthetic peptide corresponding to the N-terminal 20 amino acids of the protein encoded by cauliflower mosaic virus (CaMV) open reading frame VII (ORF VII). This antiserum was used to detect the expression of CaMV ORF VII either in Saccharomyces cerevisiae transformed by an expression vector containing CaMV ORF VII or in CaMV-infected plants. Only in S. cerevisiae has a 14-kilodalton protein been detected.     

Cloning and sequencing of the gene encoding a 125-kilodalton surface-layer protein from Bacillus sphaericus 2362 and of a related cryptic gene.

Using the vector pGEM-4-blue, a 4,251-base-pair DNA fragment containing the gene for the surface (S)-layer protein of Bacillus sphaericus 2362 was cloned into Escherichia coli. Determination of the nucleotide sequence indicated an open reading frame (ORF) coding for a protein of 1,176 amino acids with a molecular size of 125 kilodaltons (kDa). A protein of this size which reacted with antibody to the 122-kDa S-layer protein of B. sphaericus was detected in cells of E. coli containing the recombinant plasmid. Analysis of the deduced amino acid sequence indicated a highly hydrophobic N-terminal region which had the characteristics of a leader peptide. The first amino acid of the N-terminal sequence of the 122-kDa S-layer protein followed the predicted cleavage site of the leader peptide in the 125-kDa protein. A sequence characteristic of promoters expressed during vegetative growth was found within a 177-base-pair region upstream from the ORF coding for the 125-kDa protein. This putative promoter may account for the expression of this gene during the vegetative growth of B. sphaericus and E. coli. The gene for the 125-kDa protein was followed by an inverted repeat characteristic of terminators. Downstream from this gene (11.2 kilobases) was an ORF coding for a putative 80-kDa protein having a high sequence similarity to the 125-kDa protein. Evidence was presented indicating that this gene is cryptic.     

Antibodies to 100- and 60-kDa surface proteins inhibit substratum attachment and differentiation of rodent skeletal myoblasts

A rabbit polyclonal antiserum was raised against membrane vesicles shed from the surface of fusing L6 rat myoblasts. In immunoblots the antiserum recognized fibronectin, a protein of approximately 100,000 Da (100-kDa), and a protein of approximately 60,000 Da (60 kDa). If added prior to cellular alignment, immunoglobulins from this serum inhibited fusion of both rat (L6) and mouse (C2) myoblasts in a dose-dependent fashion. To determine which component of this serum was responsible for fusion inhibition, antibodies against fibronectin, the 100- and 60-kDa proteins were microaffinity purified and tested, individually, for their effects on myoblast fusion. Antibodies against fibronectin had no effect on fusion. Antibodies against the 100-kDa protein released most cells from the substratum. Antibodies against the 60-kDa protein completely inhibited fusion. Fusion inhibition was accompanied by a corresponding inhibition of expression of two differentiation markers, creatine phosphokinase and the acetylcholine receptor. The 60-kDa protein was found, by immunoblot analysis, in smooth muscle-like cells (BC3H1 cells) and in variant L6 cells that do not differentiate and do not fuse. However, in the differentiation incompetent cells, the 60-kDa antigen appeared to be present in reduced amount. Indirect immunofluorescence of unpermeabilized L6 cells revealed alterations in the distribution of all three antigens during development. Fibronectin first appeared in long fibrillar arrays above the surface of cells that were beginning to align and fuse; fibronectin was not present on myotubes. The 100-kDa protein was seen initially in prominent fibrillar projections at the tips of prefusion myoblasts. During fusion the antigen was observed at sites of cell-cell contact and on extracellular vesicles. The 100-kDa protein appeared to be less abundant on myotubes. The 60-kDa protein first appeared in regions of cell-cell contact on cells that were beginning to align and fuse. As. fusion progressed, the 60-kDa protein was also found in extracellular vesicles. The 60-kDa protein was not observed on myotubes. As a result of this study we have identified two previously undescribed cell surface proteins involved in rodent skeletal myogenesis. The first is an approximately 100-kDa protein involved in early interactions of skeletal myoblasts with their substratum. The second is an approximately 60-kDa protein involved in myoblast differentiation. Both proteins are shed from the myoblast surface during myotube formation.     

An anti-peptide antibody that recognizes the dopamine D2 receptor from bovine striatum.

The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.     

A gene fusion system for generating antibodies against short peptides

A novel method to obtain specific antibodies against short peptides is described, involving synthesis of the corresponding oligodeoxynucleotides followed by cloning into a new set of fusion vectors, pEZZ8 and pEZZ18, based on two synthetic IgG-binding domains (ZZ) of Staphylococcus aureus protein A. The soluble gene fusion product thus obtained, can be collected from the culture medium of Escherichia coli and rapidly recovered in a one-step procedure by IgG affinity chromatography. The system was used to express a fusion protein consisting of the two Z fragments and the C-terminal part [amino acids (aa) 57-70] of human insulin-like growth factor I (IGF-I). This 16-kDa protein was purified by affinity chromatography on IgG Sepharose and antibodies were raised in rabbits. The fusion protein elicited peptide-specific antibodies, as measured by solid-phase radioimmuno assay and Western blotting, reactive with both synthetic C-terminal peptide and the native human IGF-I protein. The results suggests that the gene fusion system can be used for efficient antibody production against short peptides encoded by synthetic oligodeoxynucleotides.     

Cloning, primary structure and properties of a novel human integrin beta subunit.

The originally described integrin beta subunits that define the three subfamilies of integrin heterodimers are beta 1, beta 2 and beta 3. In this paper, we describe the isolation of a cDNA coding for a novel human integrin beta subunit, designated as beta 5. The beta 5 cDNA was isolated from a human thymic epithelial cell library, using oligonucleotide probes that were designed from a region highly conserved among the known beta 1, beta 2 and beta 3 sequences. The beta 5 cDNA codes for 799 (or 796) amino acids, including a 23 amino acid leader sequence. There are 776 (or 773) amino acids in the mature protein, which includes a long extracellular domain of 696 amino acids, a transmembrane domain and an intracellular C-terminal domain of 57 amino acids. The beta 5 sequence resembled the known beta 3, beta 1 and beta 2 sequences by 55, 43 and 38%, respectively, including conservation of 56/56 cysteines. Rabbit antiserum was prepared against a 20 amino acid synthetic peptide predicted from the beta 5 C-terminal sequence. This serum immunoprecipitated a beta 5 protein that was 100,000 Mr (reduced) and 95,000 Mr (nonreduced). Only a single alpha subunit was detected in association with beta 5, and that alpha subunit was immunochemically indistinguishable from the alpha v subunit previously found as part of the vitronectin receptor complex. By immunoprecipitation, beta 5 was most prevalent on carcinoma cell lines, was also present on hepatoma and fibroblast cell lines, and was absent from lymphoblastoid cells and platelets.     

Demonstration of evolutionary differences between conserved antigenic epitopes in the minor nucleocapsid protein of human papillomavirus types 6b, 16 and 18.

We studied human papillomavirus (HPV) minor nucleocapsid protein (L2) by epitope scanning. Conserved antigenic epitopes identified by rabbit antiserum to bovine papillomavirus (BPV) were revealed in HPV-6b (amino acids, aa, 196-205); HPV-16 (aa:s 376-85) and HPV-18 (aa:s 221-230). L2 proteins. The first two epitopes were situated in hydrophilic regions of the proteins. Aligning the aa-sequences that corresponded to the epitopes with the total L2 sequences of BPV and HPV1a revealed consensus motifs between BPV, HPV1a and the reactive HPV type. In the non-reactive types amino acid alterations were noted. Mismatch between HPV1a sequences and the corresponding HPV-6b and HPV-16, HPV-6b and HPV-18, and HPV-16 and HPV-18 sequences suggests that the alterations may have evolved to facilitate immune surveillance of the genital HPV types.     

水稻OsFAD2、OsFAD6的克隆及其家族成员对非生物胁迫的响应

植物中的不饱和脂肪酸由脂肪酸去饱和酶(Fatty acid desaturase, FAD)合成, 它在植物的生长发育以及植物非生物胁迫方面起着重要的作用。文章采用RT-PCR方法, 从水稻日本晴(Oryza sativa L.)中克隆了分别与FAD2、FAD6同源的脂肪酸脱氢酶序列, 命名为OsFAD2和OsFAD6。OsFAD2的ORF为1 167 bp, 推测其编码蛋白含有388个氨基酸, 等电点为8.17, 分子量为52.24 kDa, C端有内质网定位序列; OsFAD6的ORF长度为1 365 bp, 推测编码454个氨基酸的蛋白质序列, 分子量44.35 kDa, 等电点为9.24, 推测N端38个氨基酸为叶绿体导肽。两者都具有膜整合脂肪酸去饱和酶特有的3个组氨酸簇。RT-PCR分析表明, OsFAD2和OsFAD6在水稻所有器官中都表达, 在叶中表达量为最高。在水稻FAD基因家族中, 叶中OsFAD2、OsFAD6 的mRNA对低温不响应, 而OsFAD7和OsFAD8的 mRNA在低温下上升。水稻叶中OsFAD2、OsFAD6、OsFAD3和OsFAD7的mRNA表达具有昼夜节律性, 在光照下表达量低, 而在随后的黑暗中表达量高, OsFAD6和OsFAD7 mRNA表达的昼夜节律性可能与水稻幼苗叶片中NADPH量的改变有关。