A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae ceUls. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect ceUls.

We used an 8987-EST collection to construct a cDNA microarray system with various genomics information (full-length cDNA, expression profile, high accuracy genome sequence, phenotype, genetic map, and physical map) in rice. This array was used as a probe to hybridize target RNAs prepared from normally grown callus of rice and from callus treated for 6 hr or 3 days with the hormones abscisic acid (ABA) or gibberellin (GA). We identified 509 clones, including many clones that had never been annotated as ABA-or GA-responsive. These genes included not only ABA- or GA-responsive genes but also genes responsive to other physiological conditions such as pathogen infection, heat shock, and metal ion stress. Comparison of ABA- and GA-responsive genes revealed antagonistic regulation for these genes by both hormones except for one defense-related gene, thionin. The gene for thionin was up-regulated by both hormone treatments for 3 days. The upstream regions of all the genes that were regulated by both hormones had cis-elements for ABA and GA response. We performed a clustering analysis of genes regulated by both hormones and various expression profiles that showed three notable clusters (seed tissues, low temperature and sugar starvation, and thionin-gene related). A comparison of the cis-elements for hormone response genes between rice and Arabidopsis thaliana, we identified cis-elements for dehydration-stress response or for expression of amylase gene as Arabidopsis gene-specific or rice gene-specific, respectively.

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ABSTRACTmRNA from human HL-60 cells was used to prepare a cDNA library, from which two full-length clones that encompass the complete c-myc coding region were isolated. One clone, pM1-11, contains all three exons of human c-myc. The second clone, pM4-10, represents a relatively rare transcript that initiated in the farst intron and includes the coding exons 2 and 3. The cDNA insert in pMl-11 was used to express the human c-myc protein in both prokaryotic and eukaryotic cells. Insertion of the coding sequences in exons 2 and 3 into the appropriate expression vectors yielded detectable c-myc protein in Escherichia coli lacking the Lon protease and in Saccharomyces cerevisiae upon induction. The protein produced in E. coli has an apparent size of 60 kDa and appears to be unmodified, as it is identical in size to the protein synthesized in an in vitro system. In contrast, yeast cells synthesize two myc proteins, of 60 kDa and 62 kDa. The difference in apparent molecular mass between the two proteins appears to be due, in part, to phosphorylation. Subcellular fractionation of yeast cells showed that the c-myc phosphoprotein is located predominantly in the nuclear fraction.Activation of the cellular myc gene has been associated with neoplasia in a variety of species. In transformed cells, the c-myc gene can be altered by several mechanisms that result in abnormal or elevated expression of c-myc RNA (1, 2) and, presumably, in increased levels of c-myc protein. Analysis of genomic clones of the human c-myc gene shows that it contains three exons (3). Exon 1 represents a long 5' noncoding leader region, whereas all of the coding sequences are located in exons 2 and 3. The coding sequences are highly conserved among species and with v-myc, the viral oncogene homolog harbored by avian myelocytomatosis virus (4, 5). Immunoprecipitation studies show that myc-specific antisera recognize proteins of =60 kDa in human cells (6-8). The highly conserved nature of the c-myc protein in many vertebrate species suggests that c-myc must supply a crucial function in eukaryotic cells. Such a function has not been elucidated, although studies on v-myc and c-myc indicate that this protein is found predominantly in the nucleus and binds to double-stranded DNA (9-13).Studies on the structure and function of the c-myc gene product have been hampered by the difficulty in obtaining sufficient amounts of protein for biochemical analyses from human cells that express elevated levels of c-myc RNA (unpublished observations). In this report, we describe the isolation and characterization of cDNA clones containing the complete coding region of human c-myc. The cDNA clones were used to express the c-myc gene in heterologous cells under regulated conditions. The c-myc gene products synthesized in Escherichia coli and in Saccharomyces cerevisiae were compared and found to exhibit host-cell-specific modifications. MATERIALS AND METHODSCell Strains. E. coli strain KRR123 was derived from strain RR1 (14) by phage P1 transduction of the lon9 allele from str...

Hybridomas producing human monoclonal antibodies (mAb) against varicella-zoster virus (VZV) were generated by fusing human splenic lymphocytes with mouse myeloma cells. Before cell fusion, lymphocytes were stimulated in vitro with viral antigens and pokeweed mitogen. This combination synergistically increased the generation of VZV-specific hybridomas. Five established hybridomas have been stably producing mAb for at least 9 months. These mAb, designated V1, V2, V6, V8 and V9, were of the IgG1, lambda isotype. They bound to all 6 tested VZV strains but not to other herpes viruses, with the exception that V1 bound to herpes simplex virus (HSV) as well as VZV. Immunoprecipitation analysis showed that V1, V6 and V9 recognized glycoprotein gpII, whereas V2 and V8 recognized gpI. In addition, V1 reacted with the gB glycoprotein of HSV. All these mAb neutralized viral infectivity. The neutralizations by V2 and V8 were more effective and more complement dependent than those by V1, V6 and V9. Immunofluorescence tests revealed that all these mAb bound to the surface membrane of VZV-infected cells. These results suggest that cell fusion between in vitro stimulated lymphocytes and mouse myeloma cells is a reliable method for the generation of hybridomas capable of stable production of human mAb. The human mAb thus developed may provide a new means of passive immunization of humans against VZV infection.