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J S Salstrom

Publications and source records attributed to J S Salstrom.

11 recordsLinked to original sources
PubMed1984

Bacterial synthesis of herpes simplex virus types 1 and 2 glycoprotein D antigens.

We have used elements of the E. coli lactose (lac) operon to produce a collection of herpes simplex virus types 1 and 2 glycoprotein D (gD-1 and gD-2) antigens. Our approach employed recombinant DNA techniques to construct plasmids with various segments of the gD-1 and gD-2 coding sequences fused to the lacZ gene. Such hybrid genes were expressed in a regulated manner in E. coli by joining them to the lac promoter-operator region. Efficient translation of these hybrid genes was facilitated by incorporating a coding sequence specifying a short peptide leader (lambda cro) in the plasmid expression vectors resulting in synthesis of chimeric Cro-gD-beta-galactosidase proteins. In addition, insertion of synthetic translation terminators at the junction of gD and lacZ enabled us to produce specific truncated gD polypeptide sequences unfused to beta-galactosidase. The gD antigens produced in E. coli were not glycosylated and were generally recovered as dense insoluble aggregates. Proteins containing portions of gD-1 or gD-2 were analyzed by immunoprecipitation using anti-HSV rabbit serum and a number of monoclonal antibodies recognizing different epitopes of gD-1. Initial animal studies were done with antigens that reacted with neutralizing antisera or monoclonal antibodies. When these bacterially produced proteins were injected into rabbits, antibodies were produced that specifically immunoprecipitated authentic gD polypeptides and neutralized the infectivity of both virus types. These studies suggest that gene fusion techniques can be used to produce immunogenic proteins in large quantity. These polypeptides are not only useful in analyses of gene structure and function, but also can provide novel diagnostic reagents and well-defined pure antigens for vaccine development.

Amino Acid Sequence↗
PubMed1983

An immunologically active chimaeric protein containing herpes simplex virus type 1 glycoprotein D.

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) cause both persistent and latent infections, including recurrent cutaneous disease, lethal neonatal disease, central nervous system disease and other clinical syndromes. Modified live vaccines or conventionally prepared subunit vaccines have generally been unsuccessful in the treatment of HSV-1 and HSV-2 infections from the standpoints of safety and efficacy. It has been established that HSV-1 and HSV-2 infectivity may be neutralized in vitro with antisera directed specifically against each of the four major glycoproteins of the virus (gA/gB, gC, gD and gE) and antisera against glycoprotein gD, of either HSV-1 or HSV-2, are capable of neutralizing both HSV-1 and HSV-2 infectivity in vitro and in vivo. We have previously reported on the identification, DNA sequence and expression at low level in Escherichia coli of the gD gene of HSV-1 strain Patton. Here we describe construction of a hybrid gene encoding a chimaeric protein containing HSV-1 gD, bacteriophage lambda Cro and E. coli beta-galactosidase (gD-beta-gal) protein, which is expressed at high level in E. coli. Moreover, the chimaeric protein elicits antibodies in rabbits that not only immunoprecipitate gD from cells infected with HSV-1 and HSV-2 but also neutralize HSV-1 and HSV-2 infectivity in vitro.

Antigens, Viral↗
PubMed1982

Herpes simplex virus type-1 glycoprotein D gene: nucleotide sequence and expression in Escherichia coli.

The protein coding region of the herpes simplex virus type-1 glycoprotein D (gD) gene was mapped, and the nucleotide sequence was determined. The predicted amino acid sequence of the gD polypeptide was found to contain a number of features in common with other virus glycoproteins. Insertion of this protein coding region into a bacterial expressor plasmid enabled synthesis in Escherichia coli of an immunoreactive gD-related polypeptide. The potential of this system for preparation of a type-common herpes simplex virus vaccine is discussed.

Amino Acid Sequence↗
PubMed1982

Sequence changes in coliphage lambda mutants affecting the nutL antitermination site and termination by tL1 and tL2.

The 17-bp sequence designated nutL is required for the N-mediated antitermination of transcription in the major leftward operon of coliphage lambda. The single-stranded sequence can be folded into a hairpin structure. Ten independently isolated spontaneous lambda nutL- mutants have changes that affect the same nucleotide, located in the loop of the hairpin structure, changing the guanine to adenine, thymine or cytosine. Another mutant (lambda nutL3), selected by a different means, has a deletion of one GC base pair and thus eliminates one C in the stem of the hairpin structure, destabilizing it -11.2 to -2.2 kcal/mol. True reversions of the nutL point mutations restore the guanine. The second-site revertant lambda ninL99 was found to have a deletion of 417 bp between the tL1 terminator and the N gene, removing bases +523 to +939 (counted from SL = +1). This deletion include codons for the six carboxy-terminal amino acids of gene N product, but the fusion allows continuation of translation for 53 additional amino acid residues beyond the truncated N gene before reaching a nonsense codon. The fused N product is active.

Bacteriophage lambda↗
PubMed1980

A biochemical assay for the transcription-antitermination function of the coliphage lambda N gene product.

An in vitro assay has been developed for the antiterminating activity of the N gene product (pN) of bacteriophage lambda, based on the N-dependent stimulation of trp mRNA synthesis from the DNA templates of lambda trp transducing phages. Using this assay system, we show (a) that the stimulation of trp mRNA synthesis by pN requires the cis-dominant nutL site on the lambda chromosome and (b) that pN can participate in vitro in the formation of functional antiterminating transcription complexes.

Bacteriophage lambda↗
PubMed1979

N-independent leftward transcription in coliphage lambda: deletions, insertions and new promoters bypassing termination functions.

Lambda mutants capable of N-independent red-gam gene expression were isolated by selecting Fec+ plaque-forming derivatives of lambda N+ nutL- (Fec-) strains. In addition to true nutL+ reversions, three classes of second-site mutations were identified: (1) ninL deletions that remove a region containing either tL1 or both tL1 and tL2 termination signals, or only a small region (defining the rut site) just upstream from tL1, (2) new constitutive promoters that map just upstream from the tL2 termination site and which are created either by point mutations (hip) or by short insertion sequences (isp), (3) small internal deletions in gene cro. The positions and individual effects of these mutations, some of which only partially abolish termination function, provide evidence for a complex multipartite structure of the termination signals.

Chromosome Mapping↗
PubMed1979

The site controlling the specificity of N action is outside the promoter-operator region: a triple hybrid phage lambda N21 imm434nin5.

A short interval of homology between imm lambda, imm434 and imm21 DNAs was identified near the leftward promoter-operator region. This homology, denoted Hs, was revealed by electron microscopic examination of lambda imm lambda/lambda imm21 and lambda imm434/lambda imm21 heteroduplexes, and permitted us to construct a special lambda hybrid (lambda hyB) which contains the N region of phage 21 and the adjacent imm region from phage 434. This triple hybrid, labmda N21 imm434nin5, was analysed by genetic, transcriptional and electronic micrographic techniques. Its leftward and rightward promoter-operator regions are of phage 434 specificity and are controlled by the 434 repressor. Surprisingly, the N21 gene of lambda hyB was found to be defective, perhaps to preserve the viability of the hybrid. Its leftward N-recognition system (nutL) is of phage 21 specificity since it responds only to the N21 function in complementation tests, as measured by antitermination of leftward transcription initiated at the pL promotor in the imm434 region. We conclude, therefore, that the pLoL region of 434 contains no information for the specificity of N antitermination. Both lambda imm21 and lambda hyB were found to be missing the tL1 terminator function (see also Salstrom and Szybalski, 1978b). In these phages, the tL2 terminator was found to be only 60% effective under N21 conditions, and therefore expression of their red-gam genes is sufficient to endow the lambda hyB and lambda N21- imm21nin5 phages with the Fec+ phenotype.

Coliphages↗