Specific protein binding to the simian virus 40 enhancer in vitro.

HeLa cell nuclear extracts and wild-type or mutated simian virus 40 enhancer DNA were used in DNase I footprinting experiments to study the interaction of putative trans-acting factors with the multiple enhancer motifs. We show that these nuclear extracts contain proteins that bind to these motifs. Because point mutations which are detrimental to the activity of a particular enhancer motif in vivo specifically prevent protection of that motif against DNase I digestion in vivo, we suggest that the bound proteins correspond to trans-acting factors involved in enhancement of transcription. Using mutants in which the two domains A and B of the simian virus 40 enhancer are either separated by insertion of DNA fragments or inverted with respect to their natural orientation, we also demonstrate that the trans-acting factors bind independently to the two domains.

Enhancement of pancreatic tumor metastasis in transgenic immunodeficient mice.

Metastatic pancreatic cancer presents a bleak prognosis. Typically, human tumor development has been modelled in animals by generating transgenic mice carrying an oncogene, and metastasis studied by engrafting human tumor cells into immunodeficient mice. We derived mouse lines that spontaneously develop metastatic pancreatic cancer by crossing a transgenic line that develops primary pancreatic adenocarcinomas with lines that are deficient for different lymphocyte components of the immune system. We obtained transgenics carrying the SCID mutation resulting in loss of B and T cell function, those carrying the beige mutation resulting in impaired NK cell and macrophage activity, and those carrying both mutations. Although human graft studies indicated that the SCID mutation permits metastasis of different types of tumor cells, in our mice its effect on metastasis of the pancreatic tumor was minimal. In contrast, the beige mutation resulted in metastasis in almost 90% of the animals. The SCID and beige mutations synergistically resulted in faster growing tumors. Both primary tumors and metastases contained undifferentiated and differentiated cell types. The tissue distribution of metastases was similar to that recorded from human patients with pancreatic cancer, suggesting that mechanisms underlying metastasis in these mice could be similar to those involved in human disease.

Tumor cell splice variants of the transcription factor TEF-1 induced by SV40 T-antigen transformation.

The large tumor antigen (TAg) of simian virus 40 is able to transform cells through interactions with cellular proteins, notably p53 and Rb. Among the other proteins that form complexes with TAg is TEF-1, a transcription factor utilized by the viral enhancer to activate expression of the early gene which encodes TAg. We show that fibroblasts contain several alternately spliced TEF-1 mRNAs, the most abundant of which encodes a protein with an additional four amino acid exon compared to the database entry for Hela cell TEF-1. Transformation by TAg induces alternate splicing, producing a more abundant form lacking this exon and matching the published sequence. Splicing variants lacking this exon were detected in mouse pancreatic tumors and in cell lines derived from human pancreatic cancers, in contrast to a single isoform with the exon in normal mouse pancreas. A total of eight splice variants were identified, with the loss of the four amino acid exon typical of transformed cells. These and other data presented suggest that TAg 're-models' host cell transcription factors that are used early in viral infection, and thereby mimics an event that naturally occurs during transformation. The data indicate that TEF-1 alterations may be a hallmark feature of tumorigenesis.

Differential modulation of yeast actin, tubulin, and YPT1 mRNA levels by cycloheximide.

We have examined the effects of cycloheximide (Chx) on transcription of genes encoding the yeast beta-actin (ACT), beta-tubulin (TUB) and yeast protein 1 (YPT1). As in mammalian cells, Chx caused an increase in levels of ACT, but not TUB, transcripts. The YPT1 gene was also activated. Induction of Chx was further studied by placing the promoter regions of the ACT and YPT1 genes in front of a globin (GLB)-encoding reporter gene (GLB) on yeast plasmids. Induction of GLB mRNA synthesis by Chx was not seen with either promoter; the YPT1 promoter was, however, strongly inducible by Chx if glucose was present. The YPT1 coding sequence conferred Chx inducibility on both the YPT1 and ACT promoters, suggesting that it may contain a transcription regulatory element.

A novel strategy for regulated expression of a cytotoxic gene.

The tetracycline (Tet) transactivator system is a powerful promoter system to control gene expression. However, expression of a cytotoxic gene in this system has been limited due to the lethal effect caused by low levels of basal expression of the toxic gene. In this report, we describe a novel strategy to express a toxic gene using the Tet system. The barstar gene is placed downstream of a minimal promoter and the barnase gene downstream of the tetracycline responsive element minimal promoter. When barnase is expressed at a basal level, its toxicity in human cell culture is offset by the similar basal level expression of barstar. However, when the barnase expression is induced with the transactivator protein, its overproduction leads to cell death. Therefore, this strategy allows cytotoxicity to be effectively regulated by tetracycline.

Stage-dependent redistribution of the V-ATPase during bovine implantation.

The 16-kD subunit of the vacuolar H(+)-ATPase (V-ATPase), or ductin, is essential for the activity of this proton pump and has roles in intercellular communication and control of cell growth and differentiation. The V-ATPase is important for acidification-dependent degradation of tissue matrices through which some cell types move, and for pH regulation across some epithelial cell layers. Placentation involves intricate signaling, cell proliferation, and controlled invasion. We examined the distribution of three subunits of the V-ATPase in bovine trophoblast and endometrium at the time of implantation to determine the relationship of ductin expression to that of two other subunits, A (approximately 73 kD) and B (approximately 58 kD). Epithelial expression of all three subunits was observed, and in nonpregnant animals this expression was apical. As pregnancy proceeded, expression of all subunits became pericellular in luminal but not glandular epithelium, suggesting a redistribution of V-ATPase activity. The trophoblast expressed all three subunits during initial contact with the epithelium. In the stroma, ductin expression was reduced after implantation, and we discuss the possibility that ductin plays a role in the shifting communication between stromal and epithelial cells induced by embryo attachment. (J Histochem Cytochem 47:1247-1254, 1999)

Evaluation of three lines of immunodeficient mice for the study of spontaneous metastatic tumors.

Various immunodeficient animals have been used as transplantation recipients for studying the growth of human tumors. We have been assessing the value of immunodeficiencies for the study of naturally arising tumors, using a model system of transgenic mice that spontaneously develop cancer of the pancreas as a result of elastase promoter-driven expression of the large tumor antigen gene of simian virus 40. We previously reported the establishment of transgenic mice that carried the SCID and/or beige mutations, eliminating B- and T-cell function and reducing lytic NK cell activity, respectively. In SCID beige animals, metastasis rates and target organs for metastases were similar to those observed in humans with pancreatic cancer. We describe here analysis of subsequent more highly inbred generations of these mice. The data show that inbreeding has almost negated the value of these immunodeficiencies for enhancing disease progression, and we observe high rates of metastasis even in immunocompetent animals. The data suggest that SCID and beige immunodeficiencies may not always have the same value for the modeling of spontaneous tumors as they do for the study of xenografts.

Molecular analysis of bovine actin gene and pseudogene sequences: expression of nonmuscle and striated muscle isoforms in adult tissues.

Most studies on the tissue distribution of actin isoform transcripts have been done in small mammals such as rat and mouse. We have begun a characterization of the actin gene family in a large mammal, the bovine. The alpha skeletal gene was isolated, and an isoform-specific probe to the 3' untranslated region of the transcript identified. This probe, in combination with isoform specific probes for alpha cardiac, beta nonmuscle, and gamma nonmuscle actins, was used to examine expression of nonmuscle and striated muscle actin gene transcription in different tissues. In contrast to other species so far examined, striated muscle isoforms were more strictly tissue specific, with virtually no alpha cardiac isoform transcripts detected in skeletal muscle and almost no alpha skeletal transcripts in cardiac tissue. The distribution of the beta and gamma nonmuscle actins was also unique in bovine compared to other species. A partial beta-actin pseudogene, and the chromosomal DNA flanking one end of it, were also cloned and sequenced. This chromosomal site was found to be homologous to a viral integration site previously identified in simian virus 40 (SV40)-transformed rat cells, suggesting that this region of the chromosome may be a preferred target for insertion events.

The nucleotide sequence, structure, and preliminary studies on the transcriptional regulation of the bovine alpha skeletal actin gene.

The promoters of mammalian striated muscle actin gene contain binding sites for a number of transcription factors. Examples are the CArG boxes, which bind a protein identical to or related to serum response factor (SRF), E boxes, which bind myogenic determination factors such as MyoD and myogenin, and -CCGCCC- motifs, which bind the transcription factor Sp1. To date, the only mammalian sequences isolated and analyzed are from rodent and human. We have now isolated and sequenced the bovine gene encoding alpha skeletal actin, including almost 3 kb of 5'-flanking region. When compared to the human and rodent genes (the only ones previously cloned and for which 5'-flanking sequences to only approximately -750 are known), there was the expected conservation in the coding region. A comparison of the promoter regions indicated that the bovine gene has three CArG boxes in the 5'-flanking region in positions identical to those in other species. The bovine proximal promoter is unique from those of human and rodent in that it has only one E box in the vicinity of the TATA box, near -350, whereas the other mammals have three. Far upstream sequences reveal clusters of E boxes near -2,500 and -1,500. A minimal promoter element, to -297, which has no E boxes, is sufficient to activate transcription in myotubes derived from rat L6 and mouse C2C12 myoblasts.

Studies on the use of plant extracts in assessing the effects of plant metabolism on the mutagenicity and toxicity of pesticides.

We have carried out studies on the effects of plant metabolism on the mutagenicity of agricultural chemicals. Our approach is to use a cell-free plant extract, as a source of metabolic enzymes, in a standard Ames test. Using a number of test compounds, we observe that plant metabolism can alter the mutagenicity of several pesticides, and can in some instances give rise to metabolites apparently unique from those which are formed in animal cells. A number of parameters of the assay have been examined, and we find that the assay temperature and preincubation of the pesticide with the extract can significantly alter the outcome of the test. We also have devised a method of controlling for the effects that natural extracts can have on the spontaneous reversion rate of the Salmonella tester strains, in an effort to distinguish slight mutagenic responses from the effects of nutrients (e.g. histidine for his- bacteria) in the assay.

A putative ancestral actin gene present in a thermophilic eukaryote: novel combination of intron positions.

The gene encoding actin in the thermophilic fungus Thermomyces lanuginosus has been isolated and sequenced. It contains five introns, with three being at positions already known to be intron sites in actin genes from other eukaryotes. These three positions have not been found to occur simultaneously in any other organisms to date, suggesting that the actin gene in this fungus may more closely resemble an ancestral form of this highly conserved eukaryotic gene. The 5' flanking region of the gene contains a TATA-like sequence and two CCAAT motifs in positions almost identical to those in the yeast actin gene. Other features of the gene sequence, and possible adaptations to thermophily, are discussed.

Transactivation of both early and late simian virus 40 promoters by large tumor antigen does not require nuclear localization of the protein.

The early gene product of simian virus 40, large tumor antigen (T antigen), is required for the onset of viral replication. This protein has also been reported to transactivate viral late gene expression, independently of replication. In this study I have used a vector that permits simultaneously a precise quantitation of simian virus 40 early and late promoter activity with a single nuclease S1 mapping probe. The results show that T antigen can activate the early promoter as well as the late promoter and that only on replicating templates does a shift occur in the ratio of late-to-early transcription. This simultaneous transactivation of early and late promoters occurs in human (HeLa) and monkey (CV-1) cells but does not occur in mouse embryonal carcinoma cells. It is seen with either wild-type T antigen or with a T antigen protein that carries a mutation in the nuclear localization signal. The mutant protein cannot bring about an early-to-late shift, consistent with its inability to support viral replication.

Regulation of SV40 early gene expression.

The early promoter of the simian virus 40 (SV40) has been used as a model eukaryotic promoter for the study of DNA sequence elements and cellular factors that are involved in transcriptional control and initiation. Site-directed mutagenesis and cell-free transcription systems have enabled the dissection of the functional domains within the 21 bp upstream sequence element and the 72 bp enhancer, and a number of protein factors that bind to various "motifs" within these domains have been identified. This article summarizes recent observations that have led to the conclusion that the SV40 promoter, and particularly, the enhancer region, is composed of multiple sequence elements. Some of these elements are present in cellular genes, and may exhibit tissue-specificity in their action. Furthermore, the proteins that are being identified (e.g., Sp1) may have binding sites within these elements that are sufficiently specific to ensure that only certain sets of genes will be selectively expressed.

Three helical domains form a protein binding site in the 5S RNA-protein complex from eukaryotic ribosomes.

A ribosomal protein binding site in the eukaryotic 5S rRNA has been delineated by examining the effect of sequence variation and nucleotide modification on the RNA's ability to exchange into the EDTA-released, yeast ribosomal 5S RNA-protein complex. 5S RNAs of divergent sequence from a variety of eukaryotic origins could be readily exchanged into the yeast complex but RNA from bacterial origins was rejected. Nucleotide modifications in any of three analogous helical regions in eukaryotic 5S RNAs of differing origin reduced the ability of this RNA molecule to form homologous or heterologous RNA-protein complexes. Because sequence comparisons did not indicate common nucleotide sequences in the interacting helical regions, a model is suggested in which the eukaryotic 5S RNA binding protein does not simply recognize specific nucleotide sequences but interacts with three strategically oriented helical domains or functional groups within these domains. Two of the domains bear a limited sequence homology with each other and contain an unpaired nucleotide or "bulge" similar to that recently reported for one of the 5S RNA binding proteins in Escherichia coli (Peattie, D.A., Douthwaite, S., Garrett, R.A. and Noller, H.F. (1981) Proc. Natl. Acad. Sci. 78, 7331-7335). The results further indicate that the single ribosomal protein of eukaryotic 5S RNA-protein complexes interacts with the same region of the 5S rRNA molecule as do the multiple protein components in complexes of prokaryotic origin.

Suppression of tumor-related glycosylation of cell surface receptors by the 16-kDa membrane subunit of vacuolar H+-ATPase.

The glycosylation of integrins and other cell surface receptors is altered in many transformed cells. Notably, an increase in the number of beta1,6-branched N-linked oligosaccharides correlates strongly with invasive growth of cells. An ectopic expression of the Golgi enzyme N-acetylglucosaminyltransferase V (GlcNAc-TV), which forms beta1,6 linkages, promotes metastasis of a number of cell types. It is shown here that the 16-kDa transmembrane subunit (16K) of vacuolar H(+)-ATPase suppresses beta1,6 branching of beta(1) integrin and the epidermal growth factor receptor. Overexpression of 16K inhibits cell adhesion and invasion. 16K contains four hydrophobic membrane-spanning alpha-helices, and its ability to influence glycosylation is localized primarily within the second and fourth membrane-spanning alpha-helices. 16K also interacts directly with the transmembrane domain of beta(1) integrin, but its effects on glycosylation were independent of its binding to beta(1) integrin. These data link cell surface tumor-related glycosylation to a component of the enzyme responsible for acidification of the exocytic pathway.

Role of the SV40 enhancer in the early to late shift in viral transcription.

Simian virus 40 large tumor antigen is a multifunctional protein, with two of its roles being the promotion of viral DNA replication and replication-independent activation of viral transcription. Replication leads to a shift in transcription from the early-early to the late and late-early cap sites, through mechanisms poorly understood. The viral transcription enhancer contains sequences important for both early and late transcription, and we therefore have carried out experiments to evaluate its role in these events. We find that the ability of replication to lead to a shift diminishes when early-early transcription is made increasingly stronger by multimerizing the enhancer, and suggest that replication might lead to the shift by interfering with the ability of the enhancer to direct initiation to those sites. The natural situation in the virus of having two copies of this element might represent a compromise between maximizing both T antigen expression early in infection and late gene expression after replication begins. We also show that replication-independent transcription activation by T antigen is bidirectional and involves at least in part elements to which the factor TEF-1 binds.

Use of interplasmid recombination to generate stable selectable markers for yeast transformation: application to studies of actin gene control.

A plasmid recombination system has been developed that relies upon interplasmid exchanges for yeast cell viability. Two types of plasmids, one carrying the LEU2 allele inserted within yeast actin gene sequences and the other carrying 2-microns plasmid DNA and an intact actin gene, were constructed. Neither plasmid alone yielded transformants in the haploid Leu- strain AH22, but when cotransformed, a number of colonies were obtained. Southern blot analysis revealed that transformants arose because of recombination events within the homologous actin sequences that transferred the LEU2 gene to the actin gene on the 2-microns plasmid. The recombinant plasmids could be recovered, and sequence analysis of one recombination site revealed that the exchange event was faithful at the nucleotide level. The resulting recombinant plasmids carried a defective actin gene and presumably arose because of a double-crossover event. Deletion mutations that prevented actin gene expression on one donor plasmid enabled the recovery at a high frequency of transformants resulting primarily from single-crossover events between the two plasmids. This was presumably because such events no longer generated an intact actin gene on a multicopy plasmid. Infrequently a transformant from a plasmid with an intact gene was recovered, but in these cases the plasmid was not present in multiple copies. These cells exhibited a slower growth rate, and Northern blot analysis revealed an elevated level of actin mRNA.

Fibronectin receptors in preimplantation development: cloning, expression, and localization of the alpha 5 and beta 1 integrin subunits in bovine trophoblast.

The fibronectin receptor, alpha 5 beta 1, may be involved in many aspects of early development, including migration of endodermal and mesodermal cells during formation of the placenta, trophoblastic outgrowth in culture, and development of an invasive phenotype by fetal cytotrophoblasts. In contrast to the human blastocyst, the bovine blastocyst elongates in the uterine lumen for several days until it begins attachment, and the fetal trophoblast limits its invasion to the maternal epithelium. Fibronectin receptor expression was characterized in bovine embryos before and after their attachment to the uterus. Initially, the polymerase chain reaction (PCR) was conducted with degenerate oligonucleotide primers to isolate bovine cDNAs for the alpha 5 and beta 1 subunits. Bovine-specific primers were then constructed to assay for alpha 5 and beta 1 mRNA expression in embryo RNA during the morula through the attachment stages using reverse-transcriptase PCR. Northern blot analysis was used to quantify mRNA levels from Days 15 to 21. Integrin and fibronectin protein expression was assessed by immunohistochemical examination of embryo sections. Both alpha 5 and beta 1 subunit mRNAs were expressed throughout the stages examined. Expression of both subunit proteins was found in the endoderm at Day 14 but not at Day 18 or later. Fibronectin reactivity was not present at any of the stages examined. Between Days 18 and 21, beta 1-reactivity appeared on the lateral surfaces of the trophoblast cells. Day 24 trophoblast binucleate cells showed intense staining with the beta 1 antibody, suggesting that a beta 1-integrin is involved in binucleate cell migration.

Structural studies of 5 S ribosomal RNAs from a thermophilic fungus, Thermomyces lanuginosus. A comparison of generalized models for eukaryotic 5 S RNAs.

The cytoplasmic ribosomes of the thermophilic fungus Thermomyces lanuginosus contain two types of 5 S RNA. The nucleotide sequence for approximately 80% of the molecules is (pp)pA-C-A-U-G-C-G-A-C-C-A-U-A-G-G-G-U-G-U-G-G-A-A-A-A-C-A-G-G-G-C-U-U-C-C-C-G-U-C-C-G-C-U-C-A-G-C-C-G-U-A-C-U-U-A-A-G-C-C-A-C-A-C-G-C-C-G-G-C-U-G-G-U-U-A-G-U-A-G-U-U-G-G-G-U-G-G-G-U-G-A-C-C-A-C-C-A-G-C-G-A-A-U-C-C-C-A-G-C-U-G-U-U-G-C-A-U-G-UOH. The remainder contains two nucleotide substitutions, C19 and G60, which preserve base complementarity. The secondary structure was probed using partial T1, pancreatic, and S1 nuclease digestion under a variety of ionic and temperature conditions and fragments were analyzed by rapid gel sequencing techniques. The results support the Y-shaped secondary structure model originally proposed by Nishikawa, K., and Takemura, S. (1974) FEBS Lett. 40, 106-109, for eukaryotic 5 S RNAs. When the thermal denaturation profile was compared with that of the yeast 5 S RNA, the thermophilic RNA exhibited not only a higher Tm but also an unusual decline in absorbency at moderate temperatures. This suggests that a functionally important structure may be maintained only at higher temperatures.