Identification of a putative hypothalamic mRNA coding for somatostatin and of its product in cell-free translation

Poly(A)-containing RNA from rodent hypothalamic tissue has been used to direct the synthesis of polypeptides in cell-free systems derived from wheat germ extract and rabbit reticulocyte lysate in the presence of [³⁵S]-L-cysteine. Immunoprecipitation of translation products with antiserum to somatostatin followed by sodium dodecylsulfate gel electrophoresis demon-strated the existence of a 15,000 dalton polypeptide species which was displaceable by synthetic somatostatin. In addition, hybridization of fractionated hypothalamic poly(A)-RNA, blotted against DBM-paper, with a probe containing a synthetic gene for somatostatin resulted in specific hybridization of a 550 nucleotide RNA species to the probe. These results suggest that the primary translation product for hypothalamic somatostatin is a 15,000 dalton polypeptide species.     

Characterization of a beta-actin mRNA zipcode-binding protein.

Localization of beta-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' untranslated region of the mRNA, including a 54-nucleotide element which has been termed the "zipcode" (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize beta-actin mRNA.     

Glycoprotein H-related complexes of human cytomegalovirus: identification of a third protein in the gCIII complex.

Previous studies have described three disulfide-bonded glycoprotein complexes within the envelope of human cytomegalovirus (HCMV). These have been designated gCI, gCII, and gCIII. Although gCI has been identified as homodimeric glycoprotein B (gB, gpUL55), the compositions of gCII and gCIII remain incompletely defined. Earlier studies suggested that gCIII was composed of glycoprotein H (gH, gpUL75) complexed with a second glycoprotein, the gL homolog of HCMV. We characterized the gCIII complex of HCMV using recombinant vaccinia virus-expressed gH and gL. Our results indicated that authentic gCIII was not reconstituted by coexpression of gH and gL. The presence of a third, structurally and antigenically unique glycoprotein with an estimated molecular mass of 125,000 Da in virion-derived gCIII complexes suggested that at least three proteins were necessary for formation of this envelope glycoprotein complex. This third glycoprotein, gp125, contained both simple and complex N-linked carbohydrates and had an estimated deglycosylated mass of 64,000 Da. Furthermore, we demonstrated that mature gH existed as both a covalently complexed and noncovalently associated component of the gCIII complex within the envelope of infectious extracellular virions. These findings provide further evidence for the structural complexity of the envelope of HCMV and emphasize the uncertainties associated with the previous assignment of specific functions to envelope proteins of HCMV.     

Characterization of the human herpesvirus 6 U69 gene product and identification of its nuclear localization signal

To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminal deletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescent protein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into the nucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. The nuclear transport of the GST-GFP fusion protein containing the N-terminal NLS of U69 was inhibited by wheat germ agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into the nucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that the nuclear transport of the U69 protein was mediated by importin alpha/beta in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-alpha subtype, NPI2/importin-alpha7 elicited more efficient transport activity than did Rch1/importin-alpha1 or Qip1/importin-alpha3. These results suggest a relationship between the localization of NPI2/importin-alpha7 and the cell tropism of HHV-6.     

Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides.

At least 10 distinct early virus-induced polypeptides were synthesized within 0 to 6 h after infection of permissive cells with cytomegalovirus. These virus-induced polypeptides were synthesized before and independently of viral DNA replication. A majority of these early virus-induced polypeptides were also synthesized in nonpermissive cells, which do not permit viral DNA replication. The virus-induced polypeptides synthesized before viral DNA replication were hypothesized to be nonstructural proteins coded for by the cytomegalovirus genome. Their synthesis was found to be a sequential process, since three proteins preceded the synthesis of the others. Synthesis of all early cytomegalovirus-induced proteins was a transient process; the proteins reached their highest molar ratios before the onset of viral DNA replication. Late viral proteins were synthesized at the time of the onset of viral DNA replication, which was approximately 15 h after infection. Their synthesis was continuous and increased in molar ratios with the accumulation of newly synthesized viral DNA in the cells. The presence of the amino acid analog canavanine or azetadine during the early stage of infection suppressed viral DNA replication. The amount of viral DNA synthesis was directly correlated to the relative amount of late viral protein synthesis. Because synthesis of late viral proteins depended upon viral DNA replication, the proteins were not detected in permissive cells treated with an inhibitor of viral DNA synthesis or in nonpermissive cells that are restrictive for cytomegalovirus DNA replication.     

Characterization of mRNA for a proline-rich protein of cotton fiber.

Cotton (Gossypium hirsutum L.) mRNA (H6) is expressed predominantly in fiber cells and is present during early primary cell wall formation. However, H6 protein is found to accumulate during later stages, when active secondary cell wall formation occurs, indicating possible regulation at the translational level and function in the secondary cell wall assembly. The nucleotide-derived amino acid sequence of pCK-H6 is proline rich (35 mol %) with a calculated molecular mass of 21 kD. Cotton protein H6 contains a repetitive pentameric motif (17) of alanine (serine)-threonine (serine)-proline-proline-proline. Its amino acid composition and solubility indicate that it may belong to the group of arabinogalactan proteins. Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (55 kD) and size-exclusion chromatography (77-83 kD) overestimated the size of in vitro synthesized H6 protein compared to the molecular mass derived from the nucleotide sequence (21 kD). The conformation of the molecule determined by its unusual primary structure may be the cause of its anomalous mobility. The presence of a proline-rich, arabinogalactan-type protein in cotton fiber raises the interesting possibility that it may be an integral part of the plasmalemma taking part in the development and architecture of the secondary wall of cotton fiber.