Dynamic nuclear localization of the baculovirus proteins IE2 and PE38 during the infection cycle: the promyelocytic leukemia protein colocalizes with IE2

The early gene products IE2 and PE38 of Autographa californica multicapsid nuclear polyhedrosis virus localize to distinct nuclear domains after transient expression. Here, the nuclear localization pattern and the putative association with cellular proteins have been determined during virus infection to shed light on the functional significance of the nuclear domains. IE2 was always localized to distinct nuclear structures while PE38 was partly present in nuclear dots. Confocal imaging indicated colocalization of PE38 and IE2 to common domains, prominently at 2 h p.i. The nuclear dot localization of PE38 in infected cells was different from that in transfected cells. Hence, we have performed cotransfection experiments that suggested that a viral factor influences the nuclear distribution. Since the promyelocytic leukemia protein (PML) that localizes to distinct nuclear multiprotein complexes termed ND10/PODs in mammalian cells functions as a target for some immediate early viral proteins, we have investigated whether baculovirus proteins act similarly. Transiently expressed IE2 and PE38 were found to be associated with endogenous PML in the mammalian cell line BHK21. Infection with a recombinant virus that expresses the human pml gene in insect cells reveals IE2 and PML to be colocalized during the early phase of infection followed by a redistribution of both proteins. Taken together our results provide first evidence that the early baculovirus protein IE2 associates at least with one component of mammalian PODs during virus infection, suggesting that POD-like structures can be formed in insect cells.     

人β—簇珠蛋白基因LCR调控元件中HS3与反式调控因子相互作用的研究

Upstream of the human epsilon-globin gene is the Locus Control Region (LCR) of the human beta-globin cluster, which consists of four DNase-I hypersensitive sites(HS1-HS4). It has been reported in transgenic experiments that HS3 preferentially regulates epsilon-globin gene expression. In order to elucidate the regulatory function of HS3 in the expression of globin gene, nuclear extracts from mouse hematopoietic tissues at several developmental stages were prepared and the binding of the nuclear factors to HS3 was analysed by using electrophoresis mobility shift assay(EMSA). Our results showed that the binding patterns of HS3 with nuclear extracts of mouse hematopoietic tissues at day 13 and day 18 of gestation were completely different; furthermore, by Southwestern Blot, the distinction between both stages was also demonstrated. It has been known that GATA and CACCC binding motifs are contained within HS3 core region. Using competitive gel-retardation assay, we found that no shift bands could be competed by using CACCC motif as a competitor. However one shift band at day 13 and day 18 of gestation could be competed respectively by using GATA motif as a competitor. We suggested that the shift bands, which could not be competed by both motifs, might be novel and stage-specific factors. In addition, by using Western Blot, we demonstrated that the two shift bands at day 13 and day 18 of gestation, competed by GATA motif, were GATA-2 and GATA-1 respectively: GATA-1 was expressed in mouse hematopoietic tissues at day 18 of gestation and not expressed at day 13 of gestation; however, GATA-2 was only expressed in mouse hematopoietic tissues at day 13 of gestation. According to these results, we speculated that HS3 might play an important role in regulation of stage-specific expression of globin genes through interaction between stage-specific nuclear factors and HS3.     

GATA DNA-binding protein expressed in mouse I-10 Leydig testicular tumor cells

A nuclear extract of the mouse I-10 Leydig tumor cell line was analyzed by gel mobility shift assay with a combination of antibodies for various mammalian GATA proteins. Antibodies for GATA-4 caused a super-shift of the DNA-protein complex, which is formed through GATA-4 binding to an oligonucleotide with a typical GATA motif, while ones for GATA-1, GATA-2, GATA-3, and GATA-6 did not. These results indicated that I-10 cells express GATA-4 protein. Western blotting analysis of cellular proteins also demonstrated the presence of GATA-4 protein, the size of which corresponds to that of the rat orthologous protein transiently expressed in Cos-1 cells. A significant level of GATA-4 expression in I-10 cells would be advantageous for studying the roles of this protein, especially in view of gonadal function. We further examined the binding site preference of GATA-4 expressed in I-10 cells. GATA-4 showed broad sequence specificity similar to GATA-6, the order of binding core site preference being GATA > GATT > GATC, and adenine was favored on both sides of the core for strong binding. Thus the conserved zinc finger domain of GATA proteins is suggested to contribute to the binding sequence preference. GATA-4 expressed in I-10 cells was not susceptible to proteolysis coupled with cAMP signaling.     

羟基脲诱导前后HEL细胞内GATA转录因子与人β—类珠蛋白基因调控元件 …

HEL cells, a human erythroleukemia cell line, expressing mainly the gamma-globin genes, small amount of epsilon-globin gene, but not beta-globin gene. Our previous studies demonstrated that beta-globin gene could be expressed in HEL cells induced by hydroxyurea. However, the molecular mechenism is still unknown. Here the binding patterns of GATA factors (GATA-1 and GATA-2) to the regulatory elements of human beta-globin gene were examined with the nuclear extracts from hydroxyurea-induced and uninduced HEL cells. Our results showed in EMSA assay that GATA factors could bind to the core sequence of HS2(-10681 to -10971 bp), the 3' flanking sequence of HS2 core(-10323 to -10680 bp) and the promoter of human beta-globin gene(+20 to -112 bp). However, the binding patterns between hydroxyurea-induced and uninduced HEL cells were different. Furthermore, by using Western-blot analysis, our data showed that the amount of GATA-2 was decreased in hydroxyurea-induced HEL cells. In contrast to GATA-2, the amount of GATA-1 was increased in hydroxyurea-induced HEL cells. These results showed that the different members of GATA family might play different roles during the differentiation of erythrocytes. GATA-1 may stimulate the differentiation of HEL cells, while GATA-2 can probably inhibit the differentiation of HEL cells.     

The promoter of the late p10 gene in the insect nuclear polyhedrosis virus Autographa californica: activation by viral gene products and sensitivity to DNA methylation.

In lepidopteran insect cells infected with the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), two major late viral gene products are expressed: the polyhedrin, a 28 000 mol. wt. protein which makes up the mass of the nuclear inclusion bodies, and a 10 000 mol. wt. protein (p10) whose function is unknown. The nucleotide sequences of these strong promoters conform to those of other eukaryotic promoters and are rich in AT base pairs. We used the pSVO-CAT construct containing the prokaryotic gene chloramphenicol acetyl transferase (CAT) to study the function of the p10 gene promoter in insect and mammalian cells. Upon transfection of the pAcp10-CAT construct, which contained 402 bp of the p10 gene of AcNPV DNA in the HindIII site of pSVO-CAT, CAT activity was determined. The p10 gene promoter was inactive in human HeLa cells and in uninfected Spodoptera frugiperda insect cells. The same promoter was active, however, in AcNPV-infected S. frugiperda cells and exhibited optimal activity when cells were transfected 18 h after infection with the insect virus. This finding demonstrated directly that the p10 gene promoter required other viral gene products for its activity in insect cells. The nature of these products was unknown. The p10 gene promoter sequence contained one 5'-CCGG-3' site 40 bp upstream from the cap site of the gene and two such sites 178 and 192 bp downstream from the ATG initiation codon of the gene. Since Drosophila DNA or S. frugiperda DNA contained no 5-methylcytosine or extremely small amounts of it, we were interested in determining the effect of site-specific methylations on the p10 gene insect virus promoter. Methylation at the 5'-CCGG-3' sites led to a block of this promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteins encoded in the 81.2- to 85.0-map-unit fragment of Autographa californica nuclear polyhedrosis virus DNA can be translated in vitro and in Spodoptera frugiperda cells.

We have previously demonstrated that five open reading frames exist in the nucleotide sequence of the 81.2- to 85.0-map-unit (m.u.) segment of plaque isolate E of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. The corresponding polypeptides are 9.8, 12.1, 36.6, 25.0, and 48.2 kDa in size (C. Oellig, B. Happ, T. Müller, and W. Doerfler, J. Virol. 63:1494, 1989), and we have investigated whether these proteins can be translated in infected cells. On subfragments of this viral DNA segment, mRNAs were selected from AcNPV-infected Spodoptera frugiperda insect cells at different times postinfection (p.i.). The in vitro translation of these RNAs in a rabbit reticulocyte-derived cell-free translation system yielded polypeptides of approximately 10 to 11, 12 to 14, 28, 36 to 38, and 48 to 50-kDa which were commensurate in size with the theoretically expected values. mRNAs for the 28- and 48- to 50-kDa proteins were identified by their translation products at 6 h p.i., and mRNAs for the 10- to 11-, 12- to 14-, and 36- to 38-kDa proteins were identified by their translation products at 12 h p.i. We constructed an AcNPV recombinant which carried in its polyhedrin gene the 3.9-kbp EcoRI-HindIII (81.8 to 84.8 m.u.) subfragment of the EcoRI J segment. Nucleotide sequence determinations revealed that the intact polyhedrin promoter lay adjacent to the additional 81.8- to 84.8-m.u. fragment in this recombinant. In S. frugiperda cells, which were infected with the recombinant AcNPV, a protein of 36 to 38 kDa was detected at 44 h p.i. in larger amounts than after infection with the nonrecombinant virus. However, there was no evidence for larger amounts of RNA derived from the 81.8- to 84.8-m.u. fragment in recombinant-infected cells. Recombinant-infected cells lacked the polyhedrin polypeptide. The synthesis of the 36- to 38-kDa polypeptide in recombinant- or AcNPV-E-infected S. frugiperda cells could be demonstrated by immunoprecipitation experiments. Peculiarly, this polypeptide was present in the cytoplasm as a 64-kDa glycoprotein. These data corroborate the notion that at least some of the open reading frames encoded in the 81.2- to 85.0-m.u. segment of AcNPV can be expressed in S. frugiperda cells.     

与人体β—珠蛋白基因5‘旁侧正调控区（PCR）相结合的调控因子（简报）

Using gel mobility shift assay, three protein factors (Pa, Pb, Pc) binding to the positive control region (PCR) in the 5' flanking sequence of human beta-globin gene were detected in the nuclear extracts from mouse fetal liver at d 18 or d 19 of gestation. Competitive experiment showed that Pb and Pc could bind to GATA-1 motif, therefore could be the members of GATA family. While Pa was a new and developmental stage specific trans-acting factor, we suggested that the factor Pa was related to the activation of beta-globin gene.