Carl D. Bennett

Identification and structure of the gene encoding gpII, a major glycoprotein of varicella-zoster virus

The genome of varicella-zoster virus (VZV) encodes three major families of glycoproteins (gpI, gpII, and gpIII). mRNA from VZV-infected cells was hybrid selected using a library of VZV recombinant plasmids and translated in vitro; polypeptide products were immunoprecipitated by polyclonal monospecific guinea pig antibodies to gpII. The mRNA encoding a 100-kD polypeptide precipitable by anti-gpII antibodies mapped to the HindIII D fragment near the center of the UL region. DNA sequence analysis of this region of the VZV genome revealed a 2.6-kbp open reading frame (ORF) potentially encoding a 98-kDa polypeptide possessing the characteristics of a glycoprotein. The 100-kDa polypeptide was specified by mRNA isolated by hybrid selection using a plasmid containing part of the 2.6-kbp ORF, and immunoprecipitation of this protein by anti-gpII antibodies and by convalescent zoster serum was blocked specifically by purified gpII. We conclude that the 2.6-kbp ORF encodes gpII. The imputed primary amino acid sequence of gpII shows a high degree of homology to that of herpes simplex virus type 1 (HSV-1) gB, a result consistent with the equivalent map locations of the respective genes in the HSV and VZV genomes and with the recently reported serological cross-reactivity of HSV-1 gB and VZV gpII. Unlike the mature gene products of gB, those of gpII have been described as a pair of glycoproteins with approximate molecular weights of 60 kDa in reducing gels, products of a single glycoprotein species with approximate mol mass of 125-140 kDa in nonreducing gels. Amino-terminal sequences of purified gpII were determined and compared to the imputed amino acid sequence. This comparison implies that the primary translational product is cleaved approximately into halves in vivo and suggests that mature gpII is a disulfide-linked heterodimer.

Synthesis of a proposed growth hormone releasing factor.

Abstract The synthesis of Val-His-Leu-Ser-Ala-Glu-Glu-Lys-Glu-Ala, isolated by Schally, et al. from porcine hypothalamus and reported by him to possess growth hormone releasing activity, is described. Similarities between this decapeptide and the amino-terminal sequence of the β-chain of porcine hemoglobin are pointed out. The syntheses of two analogs including the amino-terminal decapeptide of the β-chain of human hemoglobin are also described.

Rat alkaline phosphatase: II. Structural similarities between the osteosarcoma, bone, kidney, and placenta isoenzymes

A mouse monoclonal antibody raised against rat osteosarcoma alkaline phosphatase (AP) was covalently coupled to protein A-Sepharose and used to purify this enzyme from preparations of rat osteosarcoma, calvaria, kidney, and placenta in a single-step procedure. The tissue-specific isoenzymes purified in this manner showed identity in the immunodiffusion reaction with a polyclonal anti-AP antibody, but differed in apparent molecular weight and degree of polydispersity on sodium dodecyl sulfate-polyacrylamide gels. Treatment with N-glycanase abolished these differences, yielding proteins with an apparent molecular weight of 52,000 Da and identical V8 protease digestion patterns. Alkaline phosphatase from these tissues showed no significant difference in amino acid composition and identity in the first 20 N-terminal amino acids. These findings provide structural evidence which supports the hypothesis that the tissue-specific alkaline phosphatase isoenzymes share a common protein sequence subject to different glycosylation pattern.

High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin

A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. coli cheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [Leu-28]Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.

Micro-edman degradation: The use of high pressure liquid chromatography and gas chromatography in the amino terminal sequence determination of 8 nanomoles of dihydrofolate reductase from a mouse sarcoma

Summary A combination of high pressure liquid chromatography and gas chromatography has been used to identify and quantitate all of the phenylthiohydantoin derivatives of the amino acids produced in the sequence determination of the first 25 amino acids of 8 nanomoles of mouse sarcoma dihydrofolate reductase (EC 1.5.1.3). A column of Lichrosorb C-18 bonded-phase packing eluted with a gradient of sodium acetate and acetonitrile was used to separate and quantitate ten of the phenylthiohydantoin amino acids, while gas chromatography was used for the other nine. High pressure liquid chromatography was capable of detecting as little as 50 picomoles of standard phenylthiohydantoin amino acids, and was able to separate phenylthiohydantoin-Leu from phenylthiohydantoin-Ile.

BIOSYNTHESIS OF POSTERIOR PITUITARY HORMONES

Publisher Summary This chapter focuses on the biosynthesis of posterior pituitary hormones. Vasopressin (VP), oxytocin (OT), and their “carrier proteins” the neurophysins are synthesized in large neurons in the hypothalamic supraoptic and paraventricular nuclei. They are transported intraaxonally via the median eminence to nerve endings in the posterior pituitary. There they are stored and released into the systemic circulation. In 1977, a pulse-labeling paradign was used to identify separate precursors for the vasopressin associated neurophysin (Np-VP) and the oxytocin associated neurophysin (Np-OT). Both of these precursors were found to have molecular weights of approximately 20,000 daltons and both reacted with anti-rat neurophysin antibodies. They could be separated frcm one another by means of isoelectric focussing because they had different isoelectric points (PI): 5.4 and 6.1. Only the PI 5.4 precursor is synthesized in Brattleboro rats—animals with hereditary diabetes insipidus that make no vasopressin. Each of the precursors generated a 16-17,000 dalton intermediate that also reacted with the anti-neurophysin antibody.

Rat hepatoma 5123TC albumin mRNA directs the synthesis of pre-proalbumin identical to rat liver pre-proalbumin.

Summary Total polysomal poly(A)-containing RNA from rat hepatoma 5123 C was translated in a wheat germ cell-free system. The product of translation precipitable with rat serum albumin antibody was larger than rat serum albumin with an NH2-terminal extension of 2500 daltons. This extension was identical in length and sequence to that of normal rat liver pre-proalbumin, as determined by positions of leucine and methionine residues. Since hepatoma 5123 C does not secrete albumin but does synthesize it on free-polyribosomes, the results suggest that the pre-piece is not sufficient to cause binding of hepatoma nascent albumin chains to rough endoplasmic reticular membranes and that these membranes are deficient in some other component necessary for binding.