Biomedical subjects

L E Post

Publications and source records attributed to L E Post.

At least 37 records · Page 2Linked to original sources

PubMed1987

Replication and virulence of pseudorabies virus mutants lacking glycoprotein gX.

Pseudorabies virus (PRV) glycoprotein gX accumulates in the medium of infected cells. In an attempt to study the function of gX, two viruses were constructed that lacked a functional gX gene. One virus, PRV delta GX1, was derived by insertion of the herpes simplex virus thymidine kinase gene into the gX-coding region. The other virus, PRV delta GXTK-, was derived by subsequent deletion of the inserted herpes simplex virus thymidine kinase gene. Both viruses replicated in cell cultures but produced no gX. Furthermore, PRV delta GX1 was capable of killing mice with a 50% lethal dose of less than 100 PFU.

Animals↗

PubMed1987

Evaluation of pseudorabies virus glycoprotein gp50 as a vaccine for Aujeszky's disease in mice and swine: expression by vaccinia virus and Chinese hamster ovary cells.

Pseudorabies virus (PRV) is an alphaherpesvirus which causes an economically important disease of swine. One of the PRV glycoproteins, gp50, was previously identified as the sequence homolog of herpes simplex virus glycoprotein gD (E.A. Petrovskis, J.G. Timmins, M.A. Armentrout, C.C. Marchioli, R.J. Yancey, Jr., and L.E. Post, J. Virol. 59:216-223, 1986). gp50 was evaluated as a PRV subunit vaccine candidate. gp50 protected mice from PRV-induced mortality either when delivered via infection with a recombinant vaccinia virus or when administered as a subunit vaccine produced in a eucaryotic cell line, Chinese hamster ovary (CHO) cells. In addition, gp50 synthesized in CHO cells protected pigs from lethal infection with PRV. This result demonstrates that a single viral glycoprotein could induce a protective immune response in the natural host of a herpesvirus infection.

Animals↗

PubMed1987

A vaccine strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein X genes.

A pseudorabies virus (PRV) mutant with deletions in genes for glycoprotein X (gX) and thymidine kinase, designated delta GX delta TK, was constructed and evaluated as a vaccine for protecting swine against PRV-induced mortality. Doses greater than or equal to 10(3) plaque-forming units (PFU) of this strain given to mice provided protection from challenge exposure with virulent PRV. Sera tested from mice inoculated with delta GX delta TK had high titers of neutralizing antibody to PRV, but reactivity in the same sera was not significantly different from that in sera from noninoculated mice (controls) when sera from both groups were evaluated by use of an ELISA with gX antigen produced in Escherichia coli. Compared with noninoculated pigs (controls), those given delta GX delta TK (greater than or equal to 10(2) PFU) were protected completely from lethal challenge exposure, without experiencing adverse effects on weight gain and with reduction of shedding of virulent challenge virus. Serotest results indicated that, although inoculated pigs responded with strong neutralizing antibody titers, the response of delta GX delta TK-inoculated pigs to gX, as determined by ELISA before challenge exposure, was not significantly greater than the ELISA values obtained from control pigs. The ELISA values from a group of pigs inoculated with a commercially available vaccine were significantly (P less than 0.05) higher than those of control pigs. The experimental vaccine, delta GX delta TK, was avirulent for mice, swine, and sheep, but was mildly virulent for calves (mortality, 1 of 12) and more virulent for dogs (mortality, 3 of 6) and cats (mortality, 2 of 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Isolation and characterization of native human renin derived from Chinese hamster ovary cells.

Transfection of Chinese hamster ovary (CHO) cells with a plasmid containing the cDNA for human preprorenin has provided cell lines that secrete 15 mg of native prorenin per liter of culture medium. Tryptic activation of the prorenin occurs by selective cleavage of the Arg66-Leu67 bond (numbering as in preprorenin). The renin product, purified in a single step and in high yield by affinity chromatography, is fully stable for as long as 8 months when stored in solution at 4 degrees C and pH 6.5. Purity of the renin was judged to be greater than 95% by gel electrophoresis, compositional and N-terminal sequence analyses, and specific enzyme activity. An important aspect of the present work is the development of a direct assay for renin which permits accurate and reproducible evaluation of enzyme units and kinetic parameters. Application of methods described herein, combined with appropriate scale-up fermentation capabilities, provides the means for generating gram quantities of human renin and its zymogen.

Animals↗

PubMed1986

The processing of pseudorabies virus glycoprotein gX in infected cells and in an uninfected cell line.

Pseudorabies virus (PRV) produces a glycoprotein, gX, that accumulates in the medium of infected cells. The gX gene was expressed in Chinese hamster ovary cells (CHOgX cells) using the cytomegalovirus Towne major immediate early promoter. Like PRV-infected cells, CHOgX cells produced gX and exported it into the medium. Tunicamycin reduced the molecular weight of the gX in the medium to 89 kDa, compared with 99 kDa for gX made in the absence of drug. In the presence of tunicamycin gX produced by both PRV-infected cells and CHOgX cells was still glycosylated, as indicated by incorporation of [14C]glucosamine. The most likely form of this glycosylation is O-linked. In a pulse-chase experiment, gX first appeared in a 90-kDa form, then a 115-kDa form. This 115-kDa form is probably cleaved to give the 99-kDa form of gX that is released into the medium. The 115-kDa form was much more persistent in the PRV-infected Vero cells than in the CHOgX cells. In both cell types, gX was labeled by [35S]sulfate in the presence and absence of tunicamycin.

Animals↗

PubMed1986

DNA sequence of the gene for pseudorabies virus gp50, a glycoprotein without N-linked glycosylation.

The DNA sequence was determined for a region of the pseudorabies virus (PRV) genome to which a mutation defining resistance to a monoclonal antibody has been mapped (M. W. Wathen and L. M. K. Wathen, J. Virol., 51:57-62, 1984). This sequence was found to contain an open reading frame that did not include an amino acid sequence directing N-linked glycosylation. This open reading frame was expressed in uninfected Chinese hamster ovary cells to produce the PRV glycoprotein gp50. When PRV-infected Vero cells were incubated in the presence of tunicamycin, the gp50 that was produced had an identical molecular weight to that produced in the absence of drug. When infected cells were incubated in the presence of monensin, the molecular weight of gp50 was reduced from 60,000 to 45,000, but was not sensitive to endo-beta-N-acetylglucosaminidase H. These observations led to the conclusion that gp50 does not contain N-linked carbohydrate, as predicted from the DNA sequence. A region of the amino acid sequence and the positions of the cysteine residues of PRV gp50 are homologous to glycoprotein D of herpes simplex virus.

Amino Acid Sequence↗

PubMed1986

Use of lambda gt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins.

A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambda gt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambda gt11 vector, the cloned proteins were expressed in Escherichia coli as beta-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of [14C]glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.

Bacteriophage lambda↗

PubMed1986

Deletions in vaccine strains of pseudorabies virus and their effect on synthesis of glycoprotein gp63.

The pseudorabies virus vaccine strains Norden and Bartha each have been reported to have deletions in the small unique component of the genome (B. Lomniczi, M. L. Blankenship, and T. Ben-Porat, J. Virol. 49:970-979, 1984). The deletion in Norden was shown to delete the entire coding region for gI but not any of the coding sequences for gp63. However, gp63 in Norden-infected cells was only 36 kilodaltons, and a 44-kilodalton form of gp63 was released into the medium. In Bartha, the deletion removed the coding region for all but 89 amino acids of gp63, and no gp63 was detected in either Bartha-infected cells or medium.

Base Sequence↗

PubMed1985

A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins.

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.

Acetone↗

PubMed1985

Mapping and sequence of the gene for the pseudorabies virus glycoprotein which accumulates in the medium of infected cells.

RNA from pseudorabies virus (PRV)-infected cells was translated in a reticulocyte lysate with and without the addition of dog pancreas microsomes. Upon addition of the microsomes to the translation reaction, an additional prominent protein product was observed that was not present when microsomes were omitted. The gene coding for this processed protein and its lower-molecular-weight precursor was mapped within the small unique region of the genome by hybridization of mRNA to cloned fragments of PRV DNA and translation of the selected mRNAs. A fragment of the coding region of this gene was inserted into an open reading frame cloning vector to express part of this gene as a hybrid protein in Escherichia coli. This hybrid protein was injected into mice to raise an antiserum which was found to precipitate the glycoprotein which accumulates in the medium of PRV-infected cells. This allows us to conclude that the gene for the "excreted" glycoprotein (gX) maps to the small unique region of the genome, and that the precursor of this glycoprotein is readily processed by dog pancreas microsomes. The region of the PRV genome which codes for this glycoprotein was sequenced and found to include an open reading frame coding for 498 amino acids, flanked by sequences which contain features common to eucaryotic promoters and polyadenylation signals. The predicted protein sequence includes a hydrophobic sequence at the N-terminus which could be a signal sequence, and a hydrophobic sequence followed by a hydrophilic sequence at the C-terminus.

Amino Acid Sequence↗

PubMed1982

Chicken ovalbumin gene fused to a herpes simplex virus alpha promoter and linked to a thymidine kinase gene is regulated like a viral gene.

We are describing a system for the introduction, selection, and expression of eucaryotic genes in higher eucaryotic cells. The carrier consisted of the herpes simplex virus 1 (HSV-1) tk gene covalently linked to an HSV-1 alpha promoter directed away from the tk gene. In this study we fused to the alpha promoter the 5' transcribed noncoding sequences and the coding sequences of the chicken oviduct ovalbumin gene. Cells converted to the TK+ phenotype with this chimeric fragment produced an ovalbumin precursor which was processed and secreted into the extracellular fluid. The ovalbumin gene utilized the HSV-1 alpha promoter and was regulated as a viral gene inasmuch as inversion of the genomic DNA relative to the alpha promoter resulted in no ovalbumin synthesis, and production of ovalbumin was enhanced after superinfection with HSV-1. Synthesis of ovalbumin was not detected when cDNA was linked to the HSV-1 alpha promoter. The carrier system described in this study is suitable for introduction, selection, and expression of eucaryotic genes whose natural promoter is either weak or requires the presence of regulatory elements which may be absent from undifferentiated cells in culture.

Animals↗

PubMed1981

A generalized technique for deletion of specific genes in large genomes: alpha gene 22 of herpes simplex virus 1 is not essential for growth.

We describe a general method for inactivation and deletion of genes at specific sites in large DNA genomes. In the first step of the procedure, the herpes simplex virus thymidine kinase is inserted into the genome at a specific site. In the second step, the thymidine kinase gene is desired sequences flanking the insertion site are deleted. Both steps involve recombination of the genomes with cloned chimeric fragments and utilize the available selection for or against thymidine kinase to select the desired genomes. We have applied the procedure to inactivate and to delete portions of an alpha gene of herpes simplex virus 1 specifying protein 22. The recombinant virus carrying the thymidine kinase inserted into the gene 22 and viruses exhibiting 0.1 kb and 0.7 kb deletions in the gene 22 specify new alpha polypeptides with molecular weights approximately 30% of the wild-type gene 22 product and grown normally in Vero cell cultures.

Animals↗

PubMed1981

Regulation of alpha genes of herpes simplex virus: expression of chimeric genes produced by fusion of thymidine kinase with alpha gene promoters.

We report a system for investigating promoters of eucaryotic cell and virus genes based on analyses of the regulation of herpes simplex virus 1 (HSV-1) thymidine kinases whose structural gene sequences have been fused to the promoter of the gene under study. In infected cells, the polypeptides specified by HSV-1 form at least three groups, alpha, beta and gamma, whose synthesis is coordinately regulated and sequentially ordered at the transcriptional level. To identify the DNA sequence responsible for the regulation of transcription of alpha genes, we fused the sequence encoding the 5' end of an alpha gene to the structural gene sequence of the thymidine kinase, a beta gene. The resultant recombinant DNA was inserted into the viral genome and was also used to convert Ltk- cells to tk+ phenotype. In cells infected with recombinant virus, the thymidine kinase gene was regulated and expressed as an alpha gene-that is, it was transcribed and processed in the absence of prior infected cell protein synthesis. Moreover, mRNA selected by hybridization to sequences encoding the thymidine kinase contains at its 5' terminus sequences homologous to the donor sequence encoding the t'terminus of the alpha mRNA. In converted tk+ cells, the fused thymidine kinase gene, like the wild-type gene, is stimulated by superinfection with the tk- virus. However, the stimulation is many times greater and is due to non-alpha-gene products, whereas in cells converted by the wild-type gene, the stimulation is by alpha gene products. We conclude that the alpha genes are identified for transcription by sequences at or near those encoding the 5' terminus of the mRNA, and transposition of these sequences to a beta gene is all that is required to convert it to an alpha gene. Transcription of alpha genes appears to be regulated by non-alpha-gene products, which could be contained within the structure of the virion. In converted Ltk+ cells, the thymidine kinase gene uses its own promoter.

Base Sequence↗

PubMed1980

DNA sequence of the promoter region for the alpha ribosomal protein operon in Escherichia coli.

Previous studies showed that the gene for RNA polymerase subunit alpha at 72 min on the Escherichia coli chromosome is co-transcribed with genes for ribosomal proteins (r-proteins) S11, S4, and L17, and probably S13. DNA sequence analysis of a deletion mutant has now established that the S13 gene is a part of the alpha operon and the gene order is promoter (P alpha), rpsM (S13), rpsK (S11), rpsD(S4), rpoA(alpha), and rplQ(L17). The DNA sequence extending 650 bases before S13 gene was determined. In vitro transcription experiments establish the probable location of the alpha promoter (P alpha) within this sequence. The start site is 94 nucleotides upstream from the initiation codon (GUG) of the S13 gene. This promoter has features previously noted as common to E. coli promoters. However, comparison with four other sequenced promoters of r-protein operons shows no unique common features that might account for the common regulation of synthesis of r-proteins. This lack of sequence similarity in promoters of r-protein operons may be because r-protein synthesis is regulated at least partially at a post-transcriptional level.

Amino Acid Sequence↗

PubMed1980

DNA sequences from the str operon of Escherichia coli.

The str operon at 72 min on the Escherichia coli chromosome contains genes for ribosomal proteins (r-proteins) S12 (str or rpsL) and S7 (rpsG) and elongation factors G (fus) and Tu (tufA). The sequence of the entire S12 gene, the S12-S7 intercistronic region, and the beginning of the S7 gene is reported. Also, the sequence of the end of the S7 gene, the S7-G intercistronic region, and the beginning of the elongation fractor G gene is reported. The S12-S7 intercistronic region is 96 base pairs long, in contrast to other intercistronic regions in r-protein operons which have been found to vary from 3 to 66 base pairs. The S7-G intercistronic region is only 27 bases long, supporting the previous conclusion that r-protein and elongation factor genes are co-transcribed. A comparison of translation initiation sites of the S12 and S7 genes, and other examples of co-transcribed r-protein genes, reveals no obvious features that could account for equimolar synthesis of all r-proteins. The codon usage in the S12 and S7 genes follows the pattern observed in other r-protein genes; that is, there is a highly preferential usage of codons recognized by the most abundant of isoaccepting tRNA species. This pattern could reflect the cell's need for efficient translation or minimal errors, or both, in r-protein synthesis.

Amino Acid Sequence↗

PubMed1980

Molecular engineering of the herpes simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversions.

We have developed a technique for the insertion of any DNA fragment into the herpes simplex virus (HSV) genome at specific sites. This technique was used to resolve a specific problem concerning the isomerization of the HSV genome. Briefly, HSV DNA consists of four isomers differing in the orientation of two covalently linked components, L and S, relative to each other. Each component consists of unique sequences flanked by inverted repeats. To determine whether the isomerization of HSV DNA is the result of generalized recombinatin between homologous reiterated sequences in the inverted repeats or the result of site-specific recombination, we constructed plasmids in which DNA fragments derived from various regions of the viral genome were inserted in both orientations into the thymidine kinase gene, rendering it nonfunctional. The HSV DNA sequences in the plasmids were then recombined into the viral genome, and viral recombinants were selected for their thymidine kinase-deficient phenotype. The insertion of these fragments by homologous recombination was highly efficient in that all the viral clones isolated contained the inserted fragment at the expected location. The only fragments that promoted additional inversions of the viral genome were those spanning the junction between the L and S components. Furthermore, analysis of isomers formed by these recombinants indicates that the inversions occur only when sequences in the inserted fragment are in inverted orientation in relation to homologous sequences at the termini or at the authentic junction.

Base Sequence↗

PubMed1980

Feedback regulation of ribosomal protein gene expression in Escherichia coli: structural homology of ribosomal RNA and ribosomal protein MRNA.

Certain ribosomal proteins (r proteins) in Escherichia coli, such as S4 and S7, function as feedback repressors in the regulation of r-protein synthesis. These proteins inhibit the translation of their own mRNA. The repressor r proteins so far identified are also known to bind specifically to rRNA at an initial stage in ribosome assembly. We have found structural homology between the S7 binding region on 16S rRNA and a region of the mRNA where S7 acts as a translational repressor. Similarly, there is structural homology between one of the reported S4 binding regions on 16S rRNA and the mRNA target site for S4. The observed homology supports the concept that regulation by repressor r proteins is based on competition between rRNA and mRNA for these proteins and that the same structural features and of the r proteins are used in their interactions with both rRNA and mRNA.

Bacterial Proteins↗

PubMed1980

Cloning of reiterated and nonreiterated herpes simplex virus 1 sequences as BamHI fragments.

Over 95% of the herpes simplex virus type 1 (strain F) DNA sequences have been cloned as BamHI fragments in the pBR322 plasmid. With one exception, all of the cloned fragments have the same electrophoretic mobilities and restriction enzyme cleavage sites as do the authentic fragments derived from the BamHI digests of the viral genome. The exception is the BamHI B fragment mapping at the right end of L component in the prototype arrangement of the DNA. Thus, a small deletion mapping near the left end of the fragment was present in two independently derived plasmids. Included in the collection of plasmids are several clones containing DNA sequences that span the junction between the L and S components of the virus DNA. Several plasmids containing the junction fragment were found to be sufficiently stable to permit the preparation of large amounts of the DNA fragment for fine-structure mapping of the restriction enzyme cleavage sites. Preliminary studies on one cloned fragment (BamHI G) have shown that it is biologically active in marker rescue of a temperature-sensitive mutation and in transfer of a plaque morphology marker.

Cloning, Molecular↗