bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of Candida tropicalis acyl coenzyme A oxidase isozyme function by sequential gene disruption.
Authors: Picataggio S , Deanda K , Mielenz J
Published: October 2015

A recently developed transformation system has been used to facilitate the sequential disruption of the Candida tropicalis chromosomal POX4 and POX5 genes, encoding distinct isozymes of the acyl coenzyme A (acyl-CoA) oxidase which catalyzes the first reaction in the beta-oxidation pathway. The URA3-based transformation system was repeatedly regenerated by restoring the uracil requirement to transformed strains, either through selection for spontaneous mutations or by directed deletion within the URA 3 coding sequence, to permit sequential gene disruptions within a single strain of C. tropicalis. These gene disruptions revealed the diploid nature of this alkane- and fatty acid-utilizing yeast by showing that it contains two copies of each gene. A comparison of mutants in which both POX4 or both POX5 genes were disrupted revealed that the two isozymes were differentially regulated and displayed unique substrate profiles and kinetic properties. POX4 was constitutively expressed during growth on glucose and was strongly induced by either dodecane or methyl laurate and to a greater extent than POX5, which was induced primarily by dodecane. The POX4-encoded isozyme demonstrated a broad substrate spectrum in comparison with the narrow-spectrum, long-chain oxidase encoded by POX5. The absence of detectable acyl-CoA oxidase activity in the strain in which all POX4 and POX5 genes had been disrupted confirmed that all functional acyl-CoA oxidase genes had been inactivated. This strain cannot utilize alkanes or fatty acids for growth, indicating that the beta-oxidation pathway has been functionally blocked.
Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.
Authors: Kanaan M N , Marzluf G A
Published: October 2015

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.
High expression of a 3'----5' exonuclease activity is specific to B lymphocytes.
Authors: Kenter A L , Tredup J
Published: October 2015

V(D)J joining, the immunoglobulin heavy-chain (IgH) class switch, and somatic hypermutation directed at variable regions are unique genetic recombination or mutation events which occur during B-cell differentiation. The enzymatic process directing and controlling these events remains obscure. An assay for exonucleolytic activity has been devised, and an exonuclease activity expressed at high levels in normal B lymphocytes has been detected. The high expression of this enzyme is specific to B lymphocytes and may be developmentally regulated. We have partially purified a B-cell-associated nuclease by column chromatography. Using this preparation, we have begun a rigorous analysis of its activity. This activity is a nonprocessive, 3'----5' exonuclease with a requirement for divalent cations. Our studies demonstrate that EDTA, poly(dI-dC), and glycerol are all inhibitory to B-cell-associated exonucleolytic activity. The exonuclease displays sequence preference but no sequence specificity when tested on a variety of native DNA substrates. This nuclease is distinct from other exonuclease activities previously described.
The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology.
Authors: DiPersio C M , Jackson D A , Zaret K S
Published: October 2015

The extracellular matrix (ECM) promotes tissue morphogenesis, cell migration, and the differentiation of a variety of cell types. However, the mechanisms by which ECM causes differentiated gene expression have been unknown. In this report, we show that culturing the hepatocyte-derived cell line H2.35 on an ECM gel changes cell morphology and selectively stimulates the transcription of a subset of liver-specific genes, including serum albumin. Transcriptional activation by ECM also occurs with transfected plasmids bearing the transcriptional enhancer of the albumin gene. ECM substrates of different composition activated the albumin enhancer only when the ECM promoted a cuboidal, differentiated cell morphology. Enhancer activation by the ECM was mediated by two liver transcription factors, HNF3 alpha and eH-TF, which appear to be regulated differently by matrix. Specifically, we found that a collagen gel substratum caused a selective increase in the factor HNF3 alpha at the levels of mRNA accumulation and DNA-binding activity in nuclear extracts, both in H2.35 cells and in the hepatoma cell line HepG2. We conclude that the ECM can stimulate cell differentiation by selectively activating transcriptional regulatory factors and that such regulation occurs coordinately with ECM-promoted changes in cell shape.
Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae.
Authors: Coschigano P W , Miller S M , Magasanik B
Published: October 2015

We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources. Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized. Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.
Assessment of the transcriptional activation potential of the HMG chromosomal proteins.
Authors: Landsman D , Bustin M
Published: October 2015

Chromosomal proteins HMG-14, HMG-17, and HMG-1 are among the most abundant, ubiquitous, and evolutionarily conserved nonhistone proteins. Analysis of their structure reveals features which are similar to those of certain transcription factors. The distribution of charged amino acid residues along the polypeptide chains is asymmetric: positive charges are clustered toward the N-terminal region, while negative charges are clustered toward the C-terminal region. The residues in the C-terminal region have the potential to form alpha helices with negatively charged surfaces. The abilities of HMG-14, -17, and -1 to function as transcriptional activators were studied in Saccharomyces cerevisiae cells expressing LexA-HMG fusion proteins (human HMG-14 and -17 and rat HMG-1) which bind to reporter molecules containing the beta-galactosidase gene downstream from a lexA operator. Fusion constructs expressing deletion mutants of HMG-14, -17, and -1 were also tested. Analysis of binding to the lexA operator with in vitro-synthesized fusion proteins shows that there are more sites for HMG-14, -17, and -1 binding than for LexA binding and that only the fusion constructs which contain the C-terminal, acidic domains of HMG-17 bind the lexA operator specifically. None of the LexA-HMG fusion protein constructs elevate the level of beta-galactosidase activity in transfected yeast cells. Thus, although HMG-14, -17, and -1 are structurally similar to acidic transcriptional activators, these chromosomal proteins do not function as activators in this test system.
Restriction of neuroblastoma to the prostate gland in transgenic mice.
Authors: Skalnik D G , Dorfman D M , Williams D A , Orkin S H
Published: October 2015

Male transgenic mice that carry a construct containing 5'-flanking sequences of the gp91-phox gene linked to the early region of the simian virus 40 (SV40) genome reproducibly develop tumors arising from the prostate gland. As gp91-phox is expressed exclusively in terminally differentiating hematopoietic cells of the myelomonocytic lineage, the induction of tumors arising from the prostate gland was unexpected. These lesions appear to be due to a novel transcription signal that was generated during the construction of the transgene. Surprisingly, the histopathological and biochemical properties of the tumor are diagnostic of neuroblastoma rather than of adenocarcinoma of the prostate gland. Tumors produce SV40 T antigen and isoforms of neural cell adhesion molecule characteristic of neuronal cells, and they occur in a testosterone-independent manner. Microscopic examination of prostate glands from young transgenic mice reveals the presence of small lesions arising outside of the prostate gland epithelium, which is consistent with the diagnosis of neuroblastoma and further distinguishes this tumor from prostatic adenocarcinoma. Prostate gland tumors occur in all male animals of susceptible lines carrying the gp91-phox promoter/SV40 early-region transgene. However, variability in the time at which gross tumors appear and the presence of cells expressing T antigen prior to tumorigenesis suggest that somatic events in addition to T-antigen production are required for the development of a malignancy. The extraordinary restriction of the site of tumorigenesis in these animals indicates the presence in the prostate gland of a novel, tissue-specific neuroectodermal cell of origin. These transgenic animals provide a model system for the study of neuroectodermal malignancies.
HMG1-related DNA-binding protein isolated with V-(D)-J recombination signal probes.
Authors: Shirakata M , Hüppi K , Usuda S , Okazaki K , Yoshida K , Sakano H
Published: October 2015

In order to isolate cDNA clones for DNA-binding components of the V-(D)-J recombinase, phage libraries from a pre-B-cell line were screened with a radiolabeled probe containing recombination signal sequences (RSS). Among prospective clones, cDNA T160 was analyzed further. It produced a protein of 80.6 kDa which bound to DNA containing RSS but not to DNA in which the RSS had been mutated. A search of a data base revealed that the T160 protein has significant sequence homology (56%) to the nonhistone chromosomal protein HMG1 within the C-terminal region of 80 amino acids. DNA-binding analysis with truncated proteins showed that the HMG homology region is responsible for DNA binding. Using restriction fragment length polymorphisms, the T160 gene was mapped at the proximal end of mouse chromosome 2. Evidence was obtained for genetic linkage between the T160 gene and the recombination activator genes RAG-1 and RAG-2.
Cellular role of yeast Apn1 apurinic endonuclease/3'-diesterase: repair of oxidative and alkylation DNA damage and control of spontaneous mutation.
Authors: Ramotar D , Popoff S C , Gralla E B , Demple B
Published: October 2015

The APN1 gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts. The Apn1 protein is a homolog of Escherichia coli endonuclease IV, which functions in the repair of some oxidative and alkylation damages in that organism. We show here that yeast strains lacking Apn1 (generated by targeted gene disruption or deletion-replacement) are hypersensitive to both oxidative (hydrogen peroxide and t-butylhydroperoxide) and alkylating (methyl- and ethylmethane sulfonate) agents that damage DNA. These cellular hypersensitivities are correlated with the accumulation of unrepaired damages in the chromosomal DNA of apn1 mutant yeast cells. Hydrogen peroxide-treated APN1+ but not apn1 mutant cells regenerate high-molecular-weight DNA efficiently after the treatment. The DNA strand breaks that accumulate in the Apn1-deficient mutant contain lesions that block the action of DNA polymerase but can be removed in vitro by purified Apn1. An analogous result with DNA from methylmethane sulfonate-treated cells corresponded to the accumulation of unrepaired DNA apurinic sites in the apn1 mutant cells. The rate of spontaneous mutation in apn1 mutant S. cerevisiae was 6- to 12-fold higher than that measured for wild-type yeast cells. This increase indicates that under normal growth conditions, the production of DNA damages that are targets for Apn1 is substantial and that such lesions can be mutagenic when left unrepaired.
Identification of a specific exon sequence that is a major determinant in the selection between a natural and a cryptic 5' splice site.
Authors: Domenjoud L , Gallinaro H , Kister L , Meyer S , Jacob M
Published: October 2015

The first intron of the early region 3 from adenovirus type 2 contains a cryptic 5' splice site, Dcr1, 74 nucleotides downstream from the natural site D1. The cryptic site can be activated when the natural site is inactivated by mutagenesis. To investigate the basis for selection between a natural and a cryptic 5' splice site, we searched for cis-acting elements responsible for the exclusive selection of the natural site. We show that both the relative intrinsic strength of the sites and the sequence context affect the selection. A 120-nucleotide segment located at the 3' end of exon 1 enhances splicing at the proximal site D1; in its absence the two sites are used according to their strength. Thus, three cis-acting elements are involved in the silencing of the cryptic site: the sequence of D1, the sequence of Dcr1, and an upstream exonic sequence. We show that the exonic element folds, in solution, into a 113-nucleotide-long stem-loop structure. We propose that this potential stem-loop structure which is located 6 nucleotides upstream of the exon 1-intron junction is responsible for the preferential use of the natural 5' splice site.
trans activation of nerve growth factor in transgenic mice containing the human T-cell lymphotropic virus type I tax gene.
Authors: Green J E
Published: October 2015

Three lines of transgenic mice containing the human T-cell lymphotropic virus type I (HTLV-I) tax gene develop neurofibromas composed of perineural fibroblasts (S. H. Hinrichs, M. Nerenberg, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1340-1343, 1987; M. Nerenberg, S. H. Hinrichs, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1324-1327, 1987). Tumors and tumor cell lines derived from these mice produce neurite outgrowth from PC-12 cells and nerve growth factor (NGF), as determined by RNA (Northern) blot analysis and enzyme-linked immunosorbent assays. In vitro cotransfection studies demonstrate that Tax is able to trans activate the NGF promoter in NIH 3T3 fibroblast cells. The major cis-acting tax-responsive element in the NGF promoter (AGGGTGTGACGA) has 92% homology with a tax-responsive element contained within the 21-bp repeats of the HTLV-I long terminal repeat. The receptor for NGF is also expressed in the transgenic tumor cells, suggesting that Tax may activate an autocrine mechanism through the upregulation of NGF.
The gene encoding the transcription factor SCIP has features of an expressed retroposon.
Authors: Kuhn R , Monuki E S , Lemke G
Published: October 2015

SCIP is a POU domain transcription factor expressed by glial progenitor cells in the peripheral and central nervous systems (dividing Schwann cells and O-2A cells, respectively), where it appears to act as a repressor of myelin-specific genes. We have isolated genomic clones encoding the rat SCIP gene. Comparison of the structure of these clones with genomic Southern blots and SCIP cDNAs demonstrates that SCIP is encoded in a single-copy, intronless gene that has the general features of an expressed retroposon. This gene contributes to an extended CpG island. It is transcribed to produce a 3.1-kb mRNA that encodes a 451-amino-acid protein with a predicted molecular mass of 45 kDa. Immunopurified SCIP antibodies specifically recognize a nuclear protein of this size in cultured proliferating Schwann cells, and gel shift analyses demonstrate that this protein is the predominant octamer-binding protein in these cells.
Inducibility of the DNA repair gene encoding O6-methylguanine-DNA methyltransferase in mammalian cells by DNA-damaging treatments.
Authors: Fritz G , Tano K , Mitra S , Kaina B
Published: October 2015

The inducibility of the mammalian O6-methylguanine-DNA methyltransferase (MGMT) gene encoding the MGMT protein (EC 2.1.1.63) responsible for removal of the procarcinogenic and promutagenic lesion O6-alkylguanine from DNA was examined by an analysis of transcription of the MGMT gene following exposure of repair-competent (Mex+) and repair-deficient (Mex-) cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). While human and rodent Mex- cells (CHO-9, V79, HeLa MR) showed no detectable MGMT mRNA despite the presence of the gene in their genome, the amount of it in several Mex+ lines (NIH 3T3, HeLa S3, HepG2) paralleled their MGMT activity. However, none of these cell lines showed an increase in the MGMT mRNA level after treatment with various concentrations of MNNG. In contrast, MNNG-treated rat hepatoma cells, H4IIE and FTO-2B, both Mex+, had three- to fivefold more MGMT mRNA than the corresponding untreated controls as measured 12 to 72 h after alkylation. N-Methyl-N-nitrosourea, methyl methanesulfonate, N-hydroxyethyl-N-chloroethylnitrosourea, UV light, and X rays caused a similar accumulation of MGMT mRNA in rat hepatoma cells. Studies with inhibitors of RNA and protein synthesis indicate that the induced increase in the amount of MGMT mRNA was due to enhanced transcription of the gene. Furthermore, they revealed the turnover of the MGMT mRNA to be relatively low (half-life, greater than 7 h). Mutagen-induced increase of transcription of MGMT mRNA in H4IIE cells was accompanied by elevation of MGMT repair activity and resulted in reduction of mutation frequency after a challenge dose of MNNG. Although induction of MGMT mRNA transcription has been observed in two rodent hepatoma cell lines so far, this appears to be the first demonstration of inducibility of a mammalian gene encoding a clearly define DNA repair function. The transcription activation of the MGMT gene protects cells from the mutagenic effects of methylating agents.
Tissue factor mRNA in THP-1 monocytic cells is regulated at both transcriptional and posttranscriptional levels in response to lipopolysaccharide.
Authors: Brand K , Fowler B J , Edgington T S , Mackman N
Published: October 2015

Tissue factor (TF) is transiently expressed in human monocytes exposed to the inflammatory agonist bacterial lipopolysaccharide (LPS). Since TF is the major cellular initiator of the coagulation protease cascades, it is inferred that its expression within the vasculature is strictly regulated. In this study, we investigated mechanisms which control TF mRNA expression in the human monocytic cell line THP-1. LPS induced a rapid and transient accumulation of the mature 2.2-kb TF mRNA, which was maximal at 2 h. After stimulation, the rate of transcription of the TF gene was increased (3.3 +/- 1.3)fold. In addition, we observed a significant change in TF mRNA stability: at 1 h after LPS stimulation, TF mRNA was stable during a 60-min period and had a half-life of greater than 120 min, whereas at 2 h, the half-life had declined to 25 +/- 5 min. Furthermore, a larger (3.4-kb) TF RNA species was induced in these cells; the size of this species and data from selective hybridizations with intron-specific probes are consistent with the presence of an unspliced copy of intron 1. These results demonstrate that the LPS-induced accumulation of TF mRNA levels in these monocytic cells is accomplished by both transcriptional and posttranscriptional control mechanisms.
Induction of neurite outgrowth by v-src mimics critical aspects of nerve growth factor-induced differentiation.
Authors: Thomas S M , Hayes M , D'Arcangelo G , Armstrong R C , Meyer B E , Zilberstein A , Brugge J S , Halegoua S
Published: October 2015

PC12 cells treated with nerve growth factor (NGF) or infected with Rous sarcoma virus differentiate into sympathetic, neuronlike cells. To compare the differentiation programs induced by NGF and v-src, we have established a PC12 cell line expressing a temperature-sensitive v-src protein. The v-src-expressing PC12 cell line was shown to elaborate neuritic processes in a temperature-inducible manner, indicating that the differentiation process was dependent on the activity of the v-src protein. Further characterization of this cell line, in comparison with NGF-treated PC12 cells, indicated that the events associated with neurite outgrowth induced by these two agents shared features but could be distinguished by others. Both NGF- and v-src-induced neurite outgrowths were reversible. In addition, NGF and v-src could prime PC12 cells for NGF-induced neurite outgrowth, and representative early and late NGF-responsive genes were also induced by v-src. However, unlike NGF-induced neurite growth, v-src-induced neurite outgrowth was not blocked at high cell density. A comparison of phosphotyrosine containing-protein profiles showed that v-src and NGF each increase tyrosine phosphorylation of multiple cellular proteins. There was overlap in substrates; however, both NGF-specific and v-src-specific tyrosine phosphorylations were observed. One protein which was found to be phosphorylated in both the NGF- and v-src-induced PC12 cells was phospholipase C-gamma 1. Taken together, these results suggest that v-src's ability to function as an inducing agent may be a consequence of its ability to mimic critical aspects of the NGF differentiation program and raise the possibility that Src-like tyrosine kinases are involved in mediating some of the events triggered by NGF.
Lipopolysaccharide-induced NF-kappa B activation in mouse 70Z/3 pre-B lymphocytes is inhibited by mevinolin and 5'-methylthioadenosine: roles of protein isoprenylation and carboxyl methylation reactions.
Authors: Law R E , Stimmel J B , Damore M A , Carter C , Clarke S , Wall R
Published: October 2015

We show that both the lipopolysaccharide (LPS)-induced activation of NF-kappa DNA binding and kappa gene expression are blocked by treating murine pre-B lymphocyte 70Z/3 cells with 5'-methylthioadenosine (MTA), an inhibitor of several S-adenosylmethionine-dependent methylation reactions. We further show that the LPS-induced incorporation of radioactivity from [methyl-3H]methionine into methyl ester-like linkages on a group of membrane polypeptides is also inhibited by MTA treatment, suggesting the involvement of protein methylation reactions in the LPS signal transduction pathway. We also find that NF-kappa B and kappa gene activation in LPS-treated 70Z/3 cells is blocked by mevinolin, an inhibitor that prevents protein isoprenylation. Interestingly, mevinolin-treated cells also exhibited a marked reduction in the methylation of membrane proteins. Neither MTA nor mevinolin significantly inhibited NF-kappa B activation by phorbol myristate acetate, suggesting that these agents act early in signal transduction. These results provide the first evidence that carboxyl methylated and/or isoprenylated proteins play an essential role in the LPS-signaling pathway.
Test of the double-strand-break repair model of recombination in Xenopus laevis oocytes.
Authors: Jeong-Yu S J , Carroll D
Published: October 2015

A direct test was made of predictions of the double-strand-break repair (DSBR) model of recombination in Xenopus laevis oocytes. The DNA substrate injected into oocytes had two directly repeated copies of a 1.25-kb sequence and was cleaved within one of them. Different products were expected to result from concerted, conservative events, as predicted by the DSBR model, and from nonconservative events. Only very low levels of recombination products, both conservative and nonconservative, were observed. When individual, apparent DSBR products were cloned and characterized, it emerged that the majority of them had arisen by nonconservative recombination through short, terminal homologies and not from the gene conversion events predicted for DSBR. Two cloned products among 44 tested corresponded to the predications of the DSBR model, but these could also have been generated by other processes. The most efficient recombination events in oocytes are nonconservative and are based on long, terminal homologous overlaps; when these are not available, short, imperfect overlaps support a lower level of nonconservative recombination; genuine, conservative DSBR events occur rarely, if at all.
The cellular oncogene c-myb can interact synergistically with the Epstein-Barr virus BZLF1 transactivator in lymphoid cells.
Authors: Kenney S C , Holley-Guthrie E , Quinlivan E B , Gutsch D , Zhang Q , Bender T , Giot J F , Sergeant A
Published: October 2015

Regulation of replicative functions in the Epstein-Barr virus (EBV) genome is mediated through activation of a virally encoded transcription factor, Z (BZLF1). We have shown that the Z gene product, which binds to AP-1 sites as a homodimer and has sequence similarity to c-Fos, can efficiently activate the EBV early promoter, BMRF1, in certain cell types (i.e., HeLa cells) but not others (i.e., Jurkat cells). Here we demonstrate that the c-myb proto-oncogene product, which is itself a DNA-binding protein and transcriptional transactivator, can interact synergistically with Z in activating the BMRF1 promoter in Jurkat cells (a T-cell line) or Raji cells (an EBV-positive B-cell), whereas the c-myb gene product by itself has little effect. The simian virus 40 early promoter is also synergistically activated by the Z/c-myb combination. Synergistic transactivation of the BMRF1 promoter by the Z/c-myb combination appears to involve direct binding by the Z protein but not the c-myb protein. A 30-bp sequence in the BMRF1 promoter which contains a Z binding site (a consensus AP-1 site) is sufficient to transfer high-level lymphoid-specific responsiveness to the Z/c-myb combination to a heterologous promoter. That the c-myb oncogene product can interact synergistically with an EBV-encoded member of the leucine zipper protein family suggests c-myb is likely to engage in similar interactions with cellularly encoded transcription factors.
Transgenes expressing the Wnt-1 and int-2 proto-oncogenes cooperate during mammary carcinogenesis in doubly transgenic mice.
Authors: Kwan H , Pecenka V , Tsukamoto A , Parslow T G , Guzman R , Lin T P , Muller W J , Lee F S , Leder P , Varmus H E
Published: October 2015

The Wnt-1 and int-2 proto-oncogenes are transcriptionally activated by mouse mammary tumor virus insertion mutations in virus-induced tumors and encode secretory glycoproteins. To determine whether these two genes can cooperate during carcinogenesis, we have crossed two previously characterized lines of transgenic mice to obtain bitransgenic animals carrying both Wnt-1 and int-2 transgenes under the control of the mouse mammary tumor virus long terminal repeat. Mammary carcinomas appear earlier and with higher frequency in the bitransgenic animals, especially the males, than in either parental line. Nearly all bitransgenic males develop mammary neoplasms within 8 months of birth, whereas only 15% of Wnt-1 transgenic males and none of the int-2 transgenic males have tumors. In virgin bitransgenic females, tumors occur approximately 2 months earlier than in their Wnt-1 transgenic siblings; int-2 transgenic females rarely exhibit tumors. Preneoplastic glands from the bitransgenic animals of either sex demonstrate pronounced epithelial hyperplasia similar to that seen in Wnt-1 transgenic virgin females and males, and both transgenes are expressed in the hyperplastic glands and mammary tumors. RNA from the int-2 transgene is more abundant in mammary glands from bitransgenic animals than from int-2 transgenic animals; the increase is associated with high levels of RNA specific for keratin genes 14 and 18, suggesting that Wnt-1-induced epithelial hyperplasia is responsible for the observed increase in expression of the int-2 transgene.
Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog.
Authors: Lee K S , Levin D E
Published: October 2015

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth and division. To identify additional components of the pathway in which PKC1 functions, we isolated extragenic suppressors of a pkc1 deletion mutant. All of the suppressor mutations were dominant for suppressor function and defined a single locus, which was designated BCK1 (for bypass of C kinase). A molecular clone of one suppressor allele, BCK1-20, was isolated on a centromere-containing plasmid through its ability to rescue a conditional pkc1 mutant. The BCK1 gene possesses a 4.4-kb uninterrupted open reading frame predicted to encode a 163-kDa protein kinase. The BCK1 gene product is not closely related to any known protein kinase, sharing only 45% amino acid identity with its closest known relative (the STE11-encoded protein kinase) through a region restricted to its putative C-terminal catalytic domain. Deletion of BCK1 resulted in a temperature-sensitive cell lysis defect, which was suppressed by osmotic stabilizing agents. Because pkc1 mutants also display a cell lysis defect, we suggest that PKC1 and BCK1 may normally function within the same pathway. Suppressor alleles of BCK1 differed from the wild-type gene in a region surrounding a potential PKC phosphorylation site immediately upstream of the predicted catalytic domain. This region may serve as a hinge between domains whose interaction is regulated by PKC1.
Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes.
Authors: Gupta S K , Gallego C , Lowndes J M , Pleiman C M , Sable C , Eisfelder B J , Johnson G L
Published: October 2015

Expression of GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides and overexpression of the wild-type alpha i2 polypeptide in Rat 1a, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat 1a cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient alpha i2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat 1a cells induced by expression of alpha i2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat 1a cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient alpha i2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat 1a cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells.
Oncogenes result in genomic alterations that activate a transcriptionally silent, dominantly selectable reporter gene (neo).
Authors: Drews R E , Chan V T , Schnipper L E
Published: October 2015

Although oncogenes and tumor suppressor genes have been implicated in carcinogenesis and tumor progression, their relationship to the development of genomic instability has not been elucidated. To examine this role, we transfected oncogenes (polyomavirus middle [Py] and large T [MT and LT]) and adenovirus serotype 5 E1A) into two NIH 3T3-derived cell lines, EN/NIH 2-4 and EN/NIH 2-20. Both cell lines contain two stable integrants of a variant of the retrovirus vector pZipNeoSV(x)1 that has been modified by deletion of the enhancer elements from the long terminal repeats. DNA rearrangements activating the silent neomycin phosphotransferase gene (neo) present in these integrants were identified by selection of cells in the antibiotic G418. Whereas control-transfected EN/NIH cell lines do not yield G418-resistant subclones (GRSs), a fraction of oncogene-transfected EN/NIH 2-4 (8 of 19 Py MT, 5 of 17 Py LT, and 11 of 19 E1A) and 2-20 (7 of 15 Py MT) cell lines gave rise to GRSs at differing frequencies (0.33 x 10(-6) to 46 x 10(-6) for line 2-4 versus 0.11 x 10(-6) to 1.3 x 10(-6) for line 2-20) independent of cell generation time. In contrast, a distinctly smaller fraction of mutant Py MT-transfected EN/NIH cell lines (1 of 10 MT23, 1 of 10 MT1015, and 0 of 10 MT59b) resulted in GRSs. Southern analysis of DNA from selected oncogene-transfected GRSs demonstrated genomic rearrangements of neo-containing cellular DNA that varied in type (amplification and/or novel fragments) and frequency depending on the specific oncogene and EN/NIH cell line used in transfection. Furthermore, only one of the two neo-containing genomic loci present in both EN/NIH cell lines appeared to be involved in these genomic events. In addition to effects related to the genomic locus, these observations support a role for oncogenes in the development of genetic changes associated with tumor progression.
GLC3 and GHA1 of Saccharomyces cerevisiae are allelic and encode the glycogen branching enzyme.
Authors: Rowen D W , Meinke M , LaPorte D C
Published: October 2015

In the yeast Saccharomyces cerevisiae, glycogen serves as a major storage carbohydrate. In a previous study, mutants with altered glycogen metabolism were isolated on the basis of the altered iodine-staining properties of colonies. We found that when glycogen produced by strains carrying the glc-1p (previously called gha1-1) mutation is stained with iodine, the absorption spectrum resembles that of starch rather than that of glycogen, suggesting that this mutation might reduce the level of branching in the glycogen particles. Indeed, glycogen branching activity was undetectable in extracts from a glc3-1p strain but was elevated in strains which expressed GLC3 from a high-copy-number plasmid. These observations suggest that GLC3 encodes the glycogen branching enzyme. In contrast to glc3-1p, the glc3-4 mutation greatly reduces the ability of yeast to accumulate glycogen. These mutations appear to be allelic despite the striking difference in the phenotypes which they produce. The GLC3 clone complemented both glc3-1p and glc3-4. Deletions and transposon insertions in this clone had parallel effects on its ability to complement glc3-1p and glc3-4. Finally, a fragment of the cloned gene was able to direct the repair of both glc3-1p and glc3-4. Disruption of GLC3 yielded the glycogen-deficient phenotype, indicating that glycogen deficiency is the null phenotype. The glc3-1p allele appears to encode a partially functional product, since it is dominant over glc3-4 but recessive to GLC3. These observations suggest that the ability to introduce branches into glycogen greatly increases the ability of the cell to accumulate that polysaccharide. Northern (RNA) blot analysis identified a single mRNA of 2,300 nucleotides that increased in abundance ca. 20-fold as the culture approached stationary phase. It thus appears that the expression of GLC3 is regulated, probably at the level of transcription.
Evidence for the involvement of protein kinase activity in transforming growth factor-beta signal transduction.
Authors: Ohtsuki M , Massagué J
Published: October 2015

Transforming growth factor-beta 1 (TGF-beta 1) rapidly increases the expression of junB transcription factor and plasminogen activator inhibitor-1 (PAI-1) and prevents the cell cycle-dependent phosphorylation of the RB retinoblastoma susceptibility gene product during late G1 phase in Mv1Lu lung epithelial cells. These responses are shown in this report to be blocked by the potent serine/threonine protein kinase inhibitor, H7, added with TGF-beta 1. Added alone, H7 does not alter the basal junB or PAI-1 mRNA levels, the deposition of PAI-1 into the extracellular matrix, or the phosphorylation of RB in late G1 phase, suggesting that this inhibitor does not have a general nonspecific effect on the cell. The analogs H8 and H9, which are preferential inhibitors of cyclic nucleotide-dependent protein kinases, are fivefold less potent than H7 as inhibitors of the TGF-beta response. The PAI-1 response to TGF-beta 1 is not affected by the simultaneous addition of staurosporine, which is a protein kinase C inhibitor, or by the prolonged preincubation of cells with phorbol 12-myristate 13-acetate, which down-regulates protein kinase C. The results suggest the possibility that H7 and its analogs block various early TGF-beta responses by inhibiting a protein serine/threonine kinase(s).
The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription.
Authors: Schwarz J J , Chakraborty T , Martin J , Zhou J M , Olson E N
Published: October 2015

Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types. Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes. Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively. Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription. Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators. These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding. However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain. The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif. Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program. These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain. Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.
The general transcription factor RAP30 binds to RNA polymerase II and prevents it from binding nonspecifically to DNA.
Authors: Killeen M T , Greenblatt J F
Published: October 2015

RAP30/74 is a human general transcription factor that binds to RNA polymerase II and is required for initiation of transcription in vitro regardless of whether the promoter has a recognizable TATA box (Z. F. Burton, M. Killeen, M. Sopta, L. G. Ortolan, and J. F. Greenblatt, Mol. Cell. Biol. 8:1602-1613, 1988). Part of the amino acid sequence of RAP30, the small subunit of RAP30/74, has limited homology with part of Escherichia coli sigma 70 (M. Sopta, Z. F. Burton, and J. Greenblatt, Nature (London) 341:410-414, 1989). To determine which sigmalike activities of RAP30/74 could be attributed to RAP30, we purified human RAP30 and a RAP30-glutathione-S-transferase fusion protein that had been produced in E. coli. Bacterially produced RAP30 bound to RNA polymerase II in the absence of RAP74. Both partially purified natural RAP30/74 and recombinant RAP30 prevented RNA polymerase II from binding nonspecifically to DNA. In addition, nonspecific transcription by RNA polymerase II was greatly inhibited by RAP30-glutathione-S-transferase. DNA-bound RNA polymerase II could be removed from DNA by partially purified RAP30/74 but not by bacterially expressed RAP30. Thus, the ability of RAP30/74 to recruit RNA polymerase II to a promoter-bound preinitiation complex may be an indirect consequence of its ability to suppress nonspecific binding of RNA polymerase II to DNA.
Phospholipase C-mediated hydrolysis of phosphatidylcholine is a target of transforming growth factor beta 1 inhibitory signals.
Authors: Diaz-Meco M T , Dominguez I , Sanz L , Municio M M , Berra E , Cornet M E , Garcia de Herreros A , Johansen T , Moscat J
Published: October 2015

Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor beta (TGF-beta). It has been established that the mechanism whereby TGF-beta 1 inhibits cell growth does not interfere with the triggering of early mitogenic signal transduction mechanisms. Phospholipase C-catalyzed hydrolysis of phosphatidylcholine (PC) is a relatively late step in the cascade activated by growth factors. Therefore, conceivably activation of phospholipase C-catalyzed hydrolysis of PC could be the target of TGF-beta 1 action. In the study reported here, we demonstrate that TGF-beta 1 inhibits the coupling of ras p21 to the activation of PC hydrolysis, which appears to be critical for the antiproliferative effects of TGF-beta 1.
Two conserved essential motifs of the murine immunoglobulin lambda enhancers bind B-cell-specific factors.
Authors: Rudin C M , Storb U
Published: October 2015

Two highly homologous enhancers associated with the two murine immunoglobulin lambda constant-region clusters were recently identified. In order to better understand the molecular basis for the developmental stage- and cell-type-restricted expression of lambda genes, we have undertaken an analysis of the putative regulatory domains of these enhancers. By using a combination of DNase I footprinting, electrophoretic mobility shift assay, and site-specific mutations, four candidate protein binding sites have been identified at analogous positions in both enhancers. A mutation of any of these sites decreases enhancer activity. Two of the sites, lambda A and lambda B, are essential for enhancer function, and both of these sites appear to bind both B-cell-specific and general factors. Nevertheless, isolated lambda A and lambda B sites show no evidence of inherent transactivating potential, alone or together, even when present in up to three copies. We suggest that the generation of transactivating signals from these enhancers may require the complex interaction of multiple B-cell-specific and nonspecific DNA-binding factors.
Molecular characterization of the lam locus and sequences involved in regulation by the AmdR protein of Aspergillus nidulans.
Authors: Richardson I B , Katz M E , Hynes M J
Published: October 2015

The lam locus of Aspergillus nidulans consists of two divergently transcribed genes, lamA and lamB, involved in the utilization of lactams such as 2-pyrrolidinone. Both genes are under the control of the positive regulatory gene amdR and are subject to carbon and nitrogen metabolite repression. The lamB gene and the region between the two genes have been sequenced, and the start points of transcription have been determined. Within the lam locus are two sequences with homology to elements, required for AmdR regulation, found in the 5' regions of the coregulated genes amdS and gatA. In vitro and in vivo assays were used to investigate the lam and gatA regulatory elements. One of the three gatA elements and one of the two lam elements were shown to bind AmdR protein in vivo and activate transcription. With a gel shift mobility assay, in vitro binding of AmdR protein to the functional gatA element was detected. Both the functional gatA and lam boxes contain within them a CAAT sequence. In vitro binding analysis indicates that a CCAAT-specific factor(s) binds at these sequences, adjacent to or overlapping the AmdR protein-binding site.
Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus.
Authors: Flanagan J R , Becker K G , Ennist D L , Gleason S L , Driggers P H , Levi B Z , Appella E , Ozato K
Published: October 2015

The long terminal repeat of Moloney murine leukemia virus (MuLV) contains the upstream conserved region (UCR). The UCR core sequence, CGCCATTTT, binds a ubiquitous nuclear factor and mediates negative regulation of MuLV promoter activity. We have isolated murine cDNA clones encoding a protein, referred to as UCRBP, that binds specifically to the UCR core sequence. Gel mobility shift assays demonstrate that the UCRBP fusion protein expressed in bacteria binds the UCR core with specificity identical to that of the UCR-binding factor in the nucleus of murine and human cells. Analysis of full-length UCRBP cDNA reveals that it has a putative zinc finger domain composed of four C2H2 zinc fingers of the GLI subgroup and an N-terminal region containing alternating charges, including a stretch of 12 histidine residues. The 2.4-kb UCRBP message is expressed in all cell lines examined (teratocarcinoma, B- and T-cell, macrophage, fibroblast, and myocyte), consistent with the ubiquitous expression of the UCR-binding factor. Transient transfection of an expressible UCRBP cDNA into fibroblasts results in down-regulation of MuLV promoter activity, in agreement with previous functional analysis of the UCR. Recently three groups have independently isolated human and mouse UCRBP. These studies show that UCRBP binds to various target motifs that are distinct from the UCR motif: the adeno-associated virus P5 promoter and elements in the immunoglobulin light- and heavy-chain genes, as well as elements in ribosomal protein genes. These results indicate that UCRBP has unusually diverse DNA-binding specificity and as such is likely to regulate expression of many different genes.