Polyomavirus genome and polyomavirus enhancer-driven gene expression during myogenesis.

The mRNAs for myogenic functions are coordinately transcribed with polyomavirus (Py) early mRNA during in vitro differentiation of mouse C2 myoblast cells. Sequence analysis shows that the A domain of the Py enhancer includes an E1A-like consensus sequence that is also found in the 5' upstream region of two genes expressed during myoblast differentiation: alpha-actin and myosin light chain. Therefore, the coordinate expression of such genes with Py early mRNA may be activated by a common cellular regulatory factor. In the present work, we report that C2 cells surviving Py infection are unable to differentiate and do not express alpha-actin and myosin light-chain mRNAs. Hybrids between such Py-resistant myoblast cells and the parental cells exhibited dominance of the permissibility to Py growth and of the expression of myogenic mRNAs. In C2 cells transiently transfected with a chimeric plasmid (pSVPy12CAT) harboring the bacterial chloramphenicol acetyltransferase (CAT) gene driven by the Py enhancer-promoter region, the CAT gene was expressed irrespective of their stage of differentiation. Moreover, undifferentiated stably transfected cells expressing the CAT gene restricted viral growth. Py-resistant C2 myoblasts transiently transfected with pSVPy12CAT also expressed the CAT gene driven by the Py enhancer. This contradictory finding is similar to results previously obtained by other investigators with cloned genes specific for myogenic functions, and it may be explained by a structural difference between the pSVPy12CAT and the Py genomic organizations in which the viral enhancer operates.     

An inducible eukaryotic host-vector expression system: amplification of genes under the control of the polyoma late promoter in a cell line producing a thermolabile large T antigen

We have taken advantage of the inherent instability of integrated polyoma (Py) DNA sequences in the presence of a functional viral large T antigen (LT) to develop a eukaryotic host-vector system where copy number is controlled by temperature. A mouse cell line WOP32-4, that constitutively expresses a temperature sensitive (ts) LT, was transfected with plasmids containing the Py origin of DNA replication (ori) and either a neomycin-resistance gene (neo) or chloramphenicol acetyl transferase gene (cat) linked to the Py late promoter. Stable transformants were selected at 39 degrees C, the non-permissive temperature for the ts LT function. Upon shift to 33 degrees C, the resident Py sequences present in the WOP32-4 cells cannot excise due to an ori deletion. However, excision of the transfected plasmid molecules and subsequent extrachromosomal replication occur at high rates leading in some cases to the production of 1000-2000 copies per cell (average) of the plasmid. Proportional increases in either neo-specific mRNA or CAT activity were also observed. In situ hybridization for one cell line indicated that about 20% of temperature-shifted cells contained amplified plasmid DNA.     

Escherichia coli heat-labile enterotoxin genes are flanked by repeated deoxyribonucleic acid sequences.

The enterotoxin regions of the heat-labile and heat-stable enterotoxin (LT+ ST+) plasmid, pJY11, originating in a clinically isolated Escherichia coli strain, have been isolated as various-sized deoxyribonucleic acid (DNA) fragments by using cloning vehicles. The structure of the LT+ region and its neighboring DNA regions was studied by utilizing these recombinant plasmids. The LT+ region consisted of at least two genes, toxA and toxB, which could complement each other in trans. The toxA- and toxB-encoded polypeptides (LT subunits A and B, respectively) were identified by their immunological cross-reactivity with Vibrio cholerae enterotoxin subunit A or B. These tox genes and the promoter(s) were localized with respect to the restriction endonuclease cleavage map. The LT+ region was flanked by repeated DNA sequences (designated as beta). Another tox gen(s), encoding ST (designated as toxS), which was also flanked by inverted, repeated DNA sequences (designated as alpha), was located between one of the beta sequences and the LT+ region. These novel DNA structures (beta-alpha-toxS-alpha-toxA-toxB-beta) suggest the possibility that the LT+ region is on a transposon containing an ST transposon within the structure.     

Endothelial cell tumors develop in transgenic mice carrying polyoma virus middle T oncogene

Inoculation of newborn mice with the murine polyoma (Py) virus leads to tumor formation in a wide range of tissues. In order to investigate viral oncogenesis, we generated transgenic mice carrying either the Py large T antigen (LT) gene or the Py middle T antigen (MT) gene linked to Py early region regulatory sequences. While Py LT mice exhibit no phenotype, Py MT mice develop multifocal tumors of the vascular endothelium. These hemangiomas are lethal to the animals and can be passaged in vivo. Transgene RNAs and protein are present in both hemangiomas and the testes of these mice, and the Py middle T protein in both tissues is complexed to a cellular tyrosine kinase. The expression of complexed middle T protein in both tumorigenic endothelial cells and unperturbed testes implies that endothelial cells may be particularly susceptible to the action of the middle T oncogene. These observations indicate that Py middle T disrupts the normal strict controls on vascular growth, and suggest that Py MT transgenic mice will provide a model for studying the control of angiogenesis.     

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.     

Plasmid vectors with multiple cloning sites and cat-reporter gene for promoter cloning and analysis in animal cells

We have constructed two vectors, pGCAT-A and pGCAT-C, designed to facilitate the construction of recombinant plasmids containing the bacterial gene (cat) coding for chloramphenicol acetyltransferase (CAT) under the control of eukaryotic promoter and/or enhancer elements. The cat gene was inserted downstream from a multiple cloning site (MCS) region with eleven unique restriction sites. The MCS region is in opposite orientation in the two vectors. The CAT activity of control extracts from cells transfected with pGCAT-A or pGCAT-C is very low. Insertion of the viral SV40 early promoter into one of the sites of the MCS region of pGCAT-A or pGCAT-C results in a 30- to 400-fold stimulation of the CAT activity.     

Activation of the human epsilon- and beta-globin promoters by SV40 T antigen.

We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.     

The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0

The herpes simplex virus type 1 (HSV-1) alpha proteins ICP4, ICP0, and ICP27 are trans-acting proteins which affect HSV-1 gene expression. To investigate potential interactions between these alpha products and to determine the specificity of action of the alpha proteins in combination with each other compared with their activities individually, we performed a series of transient-expression assays. In these assays we used plasmids containing the alpha genes encoding ICP4, ICP0, and ICP27 either singly or in combination as effectors and HSV-1 genes of different kinetic classes and heterologous genes as targets. The HSV-1 targets consisted of promoter-regulatory domains from alpha (ICP0 and ICP27), beta (thymidine kinase and alkaline exonuclease), beta-gamma (glycoprotein D, glycoprotein B, and VP5), and gamma (glycoprotein C) genes, each fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target genes consisted of the simian virus 40 early promoter with enhancer and the Rous sarcoma virus long terminal repeat promoter and enhancer each fused to the CAT gene. Target promoter activity was measured by the assay of CAT activity in extracts of transfected cells and by Northern (RNA) blot hybridization of CAT mRNA. The results of these experiments showed that ICP4 activated only HSV-1 target genes, whereas ICP0 activated all of the targets and ICP27 had little effect on any of the targets. ICP4 and ICP0 had a synergistic effect when inducing HSV-1 targets, but they did not have this effect on the heterologous targets pSV2-CAT or pRSV-CAT. In fact, lower levels of CAT activity and CAT mRNA were found in the presence of both effectors than with ICP0 alone. Most interestingly, although the effector plasmid containing the ICP27 gene had little effect on its own, two different and marked effects depending on the target were observed when ICP27 was combined with ICP4 or ICP0 or both. A trans-repression of the induction seen with ICP4 and ICP0 was found when ICP27 was present in the transfections with pSV2-CAT, pRSV-CAT, pICP0-CAT, pICP27-CAT, pTK-CAT, pgD-CAT, pgB-CAT, and pgC-CAT. This resulted in CAT activity levels which were similar to or lower than the basal level of expression of the target genes in the absence of effector plasmids. This trans-repression occurred over a wide range of concentrations of input ICP27 plasmid. In contrast to this repressive effect of ICP27, a trans-activation was seen when ICP4, ICP0, and ICP27 plasmids were combined in transfections with pAE-CAT and pVP5-CAT as targets. This trans-activation also occurred over a 10-fold range of input ICP27 plasmid. These results suggest that ICP27 can facilitate both down     

Interaction of nuclear factor EF-1A with the polyomavirus enhancer region.

Enhancer factor 1A (EF-1A) is a mammalian nuclear protein that previously was shown to bind cooperatively to the repeated core enhancer element I sequence in the adenovirus E1A enhancer region. We now have characterized three binding sites for EF-1A in the polyomavirus A2 (Py) enhancer region. Site 1 resides in the Py A enhancer domain, and sites 2 and 3 reside in the Py B enhancer domain. EF-1A binding to Py site 1 is independent of cooperation with other EF-1A sites or the adjacent binding sites for PEA-1 and PEA-2, two murine nuclear factors that bind in the Py A enhancer domain. EF-1A binding to Py sites 2 and 3, in contrast, is cooperative, similar to the situation previously observed with binding sites in the adenovirus E1A enhancer region. In a transient replication assay, EF-1A site 1 functions synergistically with the PEA-1 and PEA-2 sites in the A enhancer domain to enhance Py replication. The functional cooperativity observed with the EF-1A, PEA-1, and PEA-2 sites in vivo does not reflect cooperative DNA binding interactions, as detected in vitro. Py EF-1A site 1 alone is capable of weakly stimulating Py replication. EF-1A site 1 overlaps with the binding sites for the murine nuclear protein PEA-3 and the ets family of oncoproteins.     

Transformation by hamster polyomavirus: identification and functional analysis of the early genes.

A strategy involving polymerase chain reaction amplification of cDNAs was designed to study the expression of the hamster polyomavirus (HaPV) early region in HaPV-transformed rat fibroblasts, productively HaPV-infected cells, and HaPV-induced lymphoma. We identified three mRNAs resulting from alternative splicing of open reading frames leading to coding capacities for three polypeptides with molecular weights similar to those of the murine polyomavirus large T, middle T (MT), and small T (ST) antigens. The corresponding intronless cDNAs direct the in vitro synthesis of polypeptides with the expected electrophoretic mobilities. The biological activities carried by the HaPV early genes were assayed by transfection of appropriate cell systems. The fragment of genomic viral DNA that encodes the three early antigens contains all of the genetic information necessary for immortalization of primary rat embryo fibroblasts and transformation of F111 rat cells. The large T antigen is sufficient for immortalization, although the MT and ST antigens stimulate the growth and modify the phenotype of immortal cell lines. A stringent cooperative effect was observed in the transformation of F111 cells, which requires the simultaneous presence of the MT and ST antigens, as opposed to the transformation by murine polyomavirus, which can be carried out by the MT antigen alone.     

鲤鱼金属硫蛋白基因启动区功能的研究

以氯霉素乙酰化酶作为报讯基因、利用草鱼肾培养细胞瞬时表达系统,对已克隆的鲤鱼金属硫蛋白基因５’－调节区１．６ｋｂ的序列进行了功能分析。从顺式效应和反式效应研究证明：所克隆的鲤鱼ＭＴ基因５‘－调节区具有典型ＭＴ启动子的特性实验发现哺乳动物病毒ＳＶ４０增强子要以加强鱼类ＭＴ启动子的活性,提示在系统进化上鱼类基因不但存在增强子元件,并具有哺乳动物增强子相似的作用方式,而且作用于增强子的反式效应因子也存在     

Acquisition and maintenance of enterotoxin plasmids in wild-type strains of Escherichia coli

Enterotoxigenic strains of Escherichia coli (ETEC) may produce a heat-labile enterotoxin (LT), a heat-stable enterotoxin (ST) or both enterotoxins. Certain serogroups are represented more frequently than others in ETEC isolated from humans. The transfer of three plasmids encoding enterotoxin production (Ent) to 22 non-toxigenic E. coli strains of many different O:H serotypes was studied. The Ent plasmids encoded ST (TP276), or LT (TP277), or ST + LT (TP214), and all carried antibiotic-resistance determinants. Twenty-one recipient strains acquired TP214, 18 acquired TP277 and 14 acquired TP276. Strains of those serotypes to which ETEC in diarrhoeal studies commonly belong neither acquired nor maintained Ent plasmids with a higher frequency than strains of those serotypes to which ETEC rarely belong. The recipient strains, with one exception, all expressed ST, or LT, or ST and LT, when they had acquired the appropriate plasmid; a non-motile strain belonging to O serogroup 88 expressed LT but failed to express ST when it acquired TP214 or TP277.