bluuebunny/crossref_metadata_2025 huggingface dataset:
The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras.
Authors: Jaiswal R K , Moodie S A , Wolfman A , Landreth G E
Published: October 2015

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.
The dual-specificity CLK kinase induces neuronal differentiation of PC12 cells.
Authors: Myers M P , Murphy M B , Landreth G
Published: October 2015

CLK is a dual-specificity protein kinase capable of phosphorylating serine, threonine, and tyrosine residues. We have investigated the action of CLK by establishing stable PC12 cell lines capable of inducibly expressing CLK. Expression of CLK in stably transfected PC12 cells mimicked a number of nerve growth factor (NGF)-dependent events, including the morphological differentiation of these cells and the elaboration of neurites. Moreover, CLK expression enhanced the rate of NGF-mediated neurite outgrowth of these cells, indicating that CLK expression and NGF treatment activate similar signal transduction pathways. CLK expression, unlike NGF, was not able to promote PC12 cell survival in serum-free media, demonstrating that CLK only partially recapitulated the actions of NGF on these cells and that the biochemical pathways necessary for morphological differentiation can be stimulated without also stimulating those necessary for survival. Induction of CLK expression also resulted in the selective activation of protein kinases that are components of growth factor-stimulated signal transduction cascades, including ERK1, ERK2, pp90RSK, and S6PKII. Induction of CLK expression, however, did not stimulate pp70S6K or Fos kinase, two NGF-sensitive protein kinases. These data indicate that CLK action mediates the morphological differentiation of these cells through its capacity to independently stimulate signal transduction pathways normally employed by NGF.
RNA-protein interactions in the nuclei of Xenopus oocytes: complex formation and processing activity on the regulatory intron of ribosomal protein gene L1.
Authors: Santoro B , De Gregorio E , Caffarelli E , Bozzoni I
Published: October 2015

The gene encoding ribosomal protein L1 in Xenopus laevis is known to be posttranscriptionally regulated; the third intron can be processed from the pre-mRNA in two alternative ways, resulting either in the production of L1 mRNA or in the release of a small nucleolar RNA (U16). The formation of splicing complexes was studied in vivo by oocyte microinjection. We show that spliceosome assembly is impaired on the L1 third intron and that the low efficiency of the process is due to the presence of suboptimal consensus sequences. An analysis of heterogeneous nuclear ribonucleoprotein (hnRNP) distribution was also performed, revealing a distinct site for hnRNP C binding proximal to the 5' end of the L1 third intron. Cleavage, leading to the production of the small nucleolar RNA U16, occurs in the same position, and we show that conditions under which hnRNP C binding is reduced result in an increase of the processing activity of the intron.
The requirement for yeast superoxide dismutase is bypassed through mutations in BSD2, a novel metal homeostasis gene.
Authors: Liu X F , Culotta V C
Published: October 2015

Oxygen toxicity in Saccharomyces cerevisiae strains lacking superoxide dismutase can be suppressed through mutations in either the BSD1 or BSD2 gene. In this report, we demonstrate that the BSD2 gene normally functions in the homeostasis of heavy metal ions. A mutation in BSD2 not only reverses the aerobic defects of yeast strains lacking superoxide dismutase but also is associated with an increased sensitivity to copper and cadmium toxicity and an elevation in copper ion accumulation. The BSD2 gene was cloned by functional complementation and is predicted to encode a novel 37.5-kDa protein with three potential transmembrane domains. The mutant bsd2-1 allele was isolated and found to contain a single C-to-T transition changing a centrally located proline to a serine. This substitution results in total inactivation of BSD2, since the bsd2-1 mutation is identical to a bsd2 delta gene deletion in phenotype. BSD2 is expressed in yeast cells as a 1.5-kb mRNA. Although the gene functions in copper detoxification, BSD2 is not induced by copper ions, as is the case with S. cerevisiae metallothioneins. A probable role for copper ions in the bsd2 reversal of oxidative damage is discussed.
Transcriptional regulation of the phosphoenolpyruvate carboxykinase gene by cooperation between hepatic nuclear factors.
Authors: Yanuka-Kashles O , Cohen H , Trus M , Aran A , Benvenisty N , Reshef L
Published: October 2015

To study the transcriptional regulation of the liver gluconeogenic phenotype, the underdifferentiated mouse Hepa-1c1c7 (Hepa) hepatoma cell line was used. These cells mimicked the fetal liver by appreciably expressing the alpha-fetoprotein and albumin genes but not the phosphoenolpyruvate carboxykinase (PEPCK) gene. Unlike the fetal liver, however, Hepa cells failed to express the early-expressed factors hepatocyte nuclear factor 1 alpha (HNF-1 alpha) and HNF-4 and the late-expressed factor C/EBP alpha, thereby providing a suitable system for examining possible cooperation between these factors in the transcriptional regulation of the PEPCK gene. Transient transfection assays of a chimeric PEPCK-chloramphenicol acetyltransferase construct showed a residual PEPCK promoter activity in the Hepa cell line, which was slightly stimulated by cotransfection with a single transcription factor from either the C/EBP family or HNF-1 alpha but not at all affected by cotransfection of HNF-4. In contrast, cotransfection of the PEPCK construct with members from the C/EBP family plus HNF-1 alpha resulted in a synergistic stimulation of the PEPCK promoter activity. This synergistic effect depended on the presence in the PEPCK promoter region of the HNF-1 recognition sequence and on the presence of two C/EBP recognition sequences. The results demonstrate a requirement for coexistence and cooperation between early and late liver-enriched transcription factors in the transcriptional regulation of the PEPCK gene. In addition, the results suggest redundancy between members of the C/EBP family of transcription factors in the regulation of PEPCK gene expression.
Reciprocal regulation of the Epstein-Barr virus BamHI-F promoter by EBNA-1 and an E2F transcription factor.
Authors: Sung N S , Wilson J , Davenport M , Sista N D , Pagano J S
Published: October 2015

The Epstein-Barr virus BamHI-F promoter (Fp) is one of three used to transcribe the EBNA latency proteins, in particular, EBNA-1, the only viral gene product needed for episomal replication. Fp is distinguished by possession of the only EBNA-1 binding sites (the Q locus) in the Epstein-Barr virus genome outside oriP. Activity of Fp is negatively autoregulated by interaction of EBNA-1 at two sites in the Q locus, which is situated downstream of the RNA start site. We demonstrate in transient assays that this EBNA-1-mediated repression of Fp can be overcome by an E2F transcription factor which interacts with the DNA at a site centered between the two EBNA-1 binding sites within the Q locus. An E2F-1 fusion protein protects the sequence 5'-GGATGGCGGGTAATA-3' from DNase I digestion, and a DNA probe containing this sequence binds an E2F-specific protein complex from cell extracts, although this region is only loosely homologous with known consensus binding sites for E2F transcription factors. In mobility shift assays, E2F can displace the binding of EBNA-1 from the Q locus but not from oriP, where the E2F binding site is not present. E2F also activates expression of Fp in epithelial cells. These findings identify a potentially new binding site for members of the E2F family of transcription factors and suggest that such a factor is important for expression of EBNA-1 in lymphoid and epithelial cells by displacing EBNA-1 from the Q locus. In addition, the possibility that Fp activity is under cell cycle control is raised. Since the supply of functional E2F varies during the cell cycle and since in these assays overexpression of E2F can overcome repression of Fp by EBNA-1, control of transcription of EBNA-1 mRNA by cell cycle regulatory factors may help to bring about ordered replication of episomes.
The REC2 gene encodes the homologous pairing protein of Ustilago maydis.
Authors: Kmiec E B , Cole A , Holloman W K
Published: October 2015

Amino acid sequence analysis has established that the homologous pairing protein of Ustilago maydis, known previously in the literature as rec1, is encoded by REC2, a gene essential for recombinational repair and meiosis with regional homology to Escherichia coli RecA. The 70-kDa rec1 protein is most likely a proteolytic degradation product of REC2, which has a predicted mass of 84 kDa but which runs anomalously during sodium dodecyl sulfate-gel electrophoresis with an apparent mass of 110 kDa. To facilitate purification of the protein product, the REC2 gene was overexpressed from a vector that fused a hexahistidine leader sequence onto the amino terminus, enabling isolation of the REC2 protein on an immobilized metal affinity column. The purified protein exhibits ATP-dependent DNA renaturation and DNA-dependent ATPase activities, which were reactions characteristic of the protein as purified from cell extracts of U. maydis. Homologous pairing activity was established in an assay that measures recognition via non-Watson-Crick bonds between identical DNA strands. A size threshold of about 50 bp was found to govern pairing between linear duplex molecules and homologous single-stranded circles. Joint molecule formation with duplex DNA well under the size threshold was efficiently catalyzed when one strand of the duplex was composed of RNA. Linear duplex molecules with hairpin caps also formed joint molecules when as few as three RNA residues were present.
Immortalization-susceptible elements and their binding factors mediate rejuvenation of regulation of the type I collagenase gene in simian virus 40 large T antigen-transformed immortal human fibroblasts.
Authors: Imai S , Fujino T , Nishibayashi S , Manabe T , Takano T
Published: October 2015

Dramatic changes occur in expression of the type I collagenase gene during the process of immortalization in simian virus 40 large T antigen-transformed human fibroblasts (S. Imai and T. Takano, Biochem. Biophys. Res. Commun. 189:148-153, 1992). From transient transfection assays, it was determined that these changes involved the functions of two immortalization-susceptible cis-acting elements, ISE1 and ISE2, located in a 100-bp region about 1.7 kb upstream. The profiles of binding of an activator, Proserpine, to the enhancer ISE1 were similar in the extracts of young, senescent preimmortalized and immortalized cells. ISE2 contained both negative and positive regulatory elements located adjacent to each other. The positive regulatory element consisted of a tandem array of putative Ets family- and AP-1-binding sites. An activator, Pluto, interacted with this positive regulatory element and had an AP-1-related component as a complex. The binding activity of Pluto was predominantly detected only in the extract from senescent preimmortalized cells. In contrast, a repressor, Orpheus, which bound to the ATG-rich negative regulatory element of ISE2, was prominently detected in extracts from both young preimmortalized and immortalized cells and appeared to suppress transcription in an orientation-dependent manner. Thus, the interplay of Pluto and Orpheus was suggested to be crucial for regulation of the collagenase gene accompanying in vitro aging and immortalization. Proserpine seemed to interact with Pluto to mediate strong expression of the collagenase gene in cellular senescence. On the basis of these results, we propose a model for regulation of the collagenase gene during in vitro aging and immortalization.
A monomeric derivative of the cellular transcription factor CREB functions as a constitutive activator.
Authors: Krajewski W , Lee K A
Published: October 2015

The mammalian transcriptional activator CREB binds as a dimer to a broad spectrum of inducible promoters. CREB activity is modulated by several signalling agents (protein kinase A [PKA], Ca2+, and transforming growth factor beta) and via functional interactions with cell-specific transcription factors. In addition, CREB can activate transcription constitutively and repress the activity of several other transcriptional activators. The mechanisms that allow CREB to act in such a malleable manner and the role that CREB dimerization might play in this are poorly understood. To probe the latter issue, we have created monomeric forms of CREB by fusing CREB to the DNA-binding domain of a protein (B-cell specific activator protein [BSAP]) that binds to DNA as a monomer. Remarkably, monomeric CREB acts as a potent, constitutive activator under conditions in which native CREB is inducible by PKA. Thus, CREB contains constitutive activation regions that are unable to function in native CREB. Two glutamine-rich domains that are important for native, PKA-inducible CREB activity are required for the constitutive activity of monomeric CREB. In contrast, two elements within the kinase-inducible domain of CREB are dispensable for constitutive activity. We discuss our results in relation to inducible and constitutive CREB activity and the potential modes of action of other activators that directly interact with CREB.
Regulation of RAG-2 protein expression in avian thymocytes.
Authors: Ferguson S E , Accavitti M A , Wang D D , Chen C L , Thompson C B
Published: October 2015

The recombinase-activating genes, RAG-1 and RAG-2, have been shown to be necessary to initiate the process of V(D)J recombination during the ontogeny of lymphocytes. While much is known about the end products of this rearrangement process, little is known about the function or regulation of the components of the recombinase system. To this end, we have generated a monoclonal antibody to the chicken RAG-2 protein. Chicken thymocytes were found to express high levels of RAG-2, part of which is phosphorylated. Within thymocytes, RAG-2 is expressed primarily within the nucleus. RAG-2 protein levels are high in the CD4- CD8- and CD4+ CD8+ immature thymocytes but absent at the single-positive CD4+ CD8- or CD4- CD8+ stage of thymocyte development. Mitogenic stimulation of thymocytes with phorbol myristate acetate and ionomycin results in down-regulation of RAG-2 expression. Consistent with these data, in vivo levels of RAG-2 are markedly lower in proliferating thymocytes than in smaller, G0/G1 cells. Down-regulation of RAG-2 expression appears to occur before cells enter S phase, suggesting that RAG-2 function may be limited to noncycling cells.
A homeodomain protein related to caudal regulates intestine-specific gene transcription.
Authors: Suh E , Chen L , Taylor J , Traber P G
Published: October 2015

The continually renewing epithelium of the intestinal tract arises from the visceral endoderm by a series of complex developmental transitions. The mechanisms that establish and maintain the processes of cellular renewal, cell lineage allocation, and tissue restriction and spatial assignment of gene expression in this epithelium are unknown. An understanding of the regulation of intestine-specific gene regulation may provide information on the molecular mechanisms that direct these processes. In this regard, we show that intestine-specific transcription of sucrase-isomaltase, a gene that is expressed exclusively in differentiated enterocytes, is dependent on binding of a tissue-specific homeodomain protein (mouse Cdx-2) to an evolutionarily conserved promoter element in the sucrase-isomaltase gene. This protein is a member of the caudal family of homeodomain genes which appear to function in early developmental events in Drosophila melanogaster, during gastrulation in many species, and in intestinal endoderm. Unique for this homeodomain gene family, we show that mouse Cdx-2 binds as a dimer to its regulatory element and that dimerization in vitro is dependent on redox potential. These characteristics of the interaction of Cdx-2 with its regulatory element provide for a number of potential mechanisms for transcriptional regulation. Taken together, these findings suggest that members of the Cdx gene family play a fundamental role both in the establishment of the intestinal phenotype during development and in maintenance of this phenotype via transcriptional activation of differentiated intestinal genes.
Human T-cell leukemia virus type I Tax activation of NF-kappa B/Rel involves phosphorylation and degradation of I kappa B alpha and RelA (p65)-mediated induction of the c-rel gene.
Authors: Sun S C , Elwood J , Béraud C , Greene W C
Published: October 2015

The tax gene product of human T-cell leukemia virus type I (HTLV-I) is a potent transcriptional activator that both stimulates viral gene expression and activates an array of cellular genes involved in T-cell growth. Tax acts indirectly by inducing or modifying the action of various host transcription factors, including members of the NF-kappa B/Rel family of enhancer-binding proteins. In resting T cells, many of these NF-kappa B/Rel factors are sequestered in the cytoplasm by various ankyrin-rich inhibitory proteins, including I kappa B alpha. HTLV-I Tax expression leads to the constitutive nuclear expression of biologically active NF-kappa B and c-Rel complexes; however, the biochemical mechanism(s) underlying this response remains poorly understood. In this study, we demonstrate that Tax-stimulated nuclear expression of NF-kappa B in both HTLV-I-infected and Tax-transfected human T cells is associated with the phosphorylation and rapid proteolytic degradation of I kappa B alpha. In contrast to prior in vitro studies, at least a fraction of the phosphorylated form of I kappa B alpha remains physically associated with the NF-kappa B complex in vivo but is subject to rapid degradation, thereby promoting the nuclear translocation of the active NF-kappa B complex. We further demonstrate that Tax induction of nuclear c-Rel expression is activated by the RelA (p65) subunit of NF-kappa B, which activates transcription of the c-rel gene through an intrinsic kappa B enhancer element. In normal cells, the subsequent accumulation of nuclear c-Rel acts to inhibit its own continued production, indicating the presence of an autoregulatory loop. However, the pathologic action HTLV-I Tax leads to the deregulated and sustained nuclear expression of both NF-kappa B and c-Rel, a response that may contribute to HTLV-I-induced T-cell transformation.
Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning.
Authors: Kawai J , Hirose K , Fushiki S , Hirotsune S , Ozawa N , Hara A , Hayashizaki Y , Watanabe S
Published: October 2015

Restriction landmark genomic scanning (RLGS) is a novel method which enables us to simultaneously visualize a large number of loci as two-dimensional gel spots. By this method, the status of DNA methylation can efficiently be determined by monitoring the appearance or disappearance of spots by using a methylation-sensitive restriction enzyme. In the present study, using RLGS with NotI, we examined, in comparison with a brain RLGS profile, the status of DNA methylation of more than 900 loci among three types of mouse cell lines: the embryonal carcinoma cell line P19, the stable mesenchymal cell line 10T1/2, and our established neuroepithelial (EM) cell lines. We found that the relative numbers of RLGS spots which appeared were less than 3.3% of those surveyed in all cell lines examined. However, 5 to 14% of spots disappeared, the numbers increasing with an increase in the length of the culture period, and many spots were commonly lost in 10T1/2 and in three EM cell lines. Thus, for these cell lines, many more spots disappeared than appeared. However, the numbers of spots disappearing and appearing were well balanced, and the ratio in P19 cells was almost equal to that in liver cells in vivo. These RLGS experimental observations suggested that permanent cell lines such as 10T1/2 are hypermethylated and that our newly established EM cell lines are also becoming heavily methylated at common loci. On the other hand, methylation and demethylation seem to be balanced in P19 cells in a manner similar to that in in vivo liver tissue.
The yeast TEM1 gene, which encodes a GTP-binding protein, is involved in termination of M phase.
Authors: Shirayama M , Matsui Y , Toh-E A
Published: October 2015

LTE1 belongs to the CDC25 family that encodes a guanine nucleotide exchange factor for GTP-binding proteins of the ras family. Previously we have shown that LTE1 is essential for termination of M phase at low temperatures. We have identified TEM1 as a gene that, when present on a multicopy plasmid, suppresses the cold-sensitive phenotype of lte1. Sequence analysis of TEM1 and GTP-binding analysis of the gene product revealed that TEM1 encodes a novel low-molecular-weight GTP-binding protein. The defect of TEM1 was lethal, and the tem1-defective cells were arrested at telophase with high H1-kinase activity under restrictive conditions, indicating that TEM1 is required to exit from M phase. The defect of TEM1 was suppressed by a high dose of CDC15, which encodes a protein kinase homologous to mitogen-activated protein kinase kinase kinases. The genetic interaction among LTE1, TEM1, and CDC15 indicates that they cooperatively play an essential role for termination of M phase.
The SR protein B52/SRp55 is essential for Drosophila development.
Authors: Ring H Z , Lis J T
Published: October 2015

B52, also called SRp55, is a 52-kDa member of the Drosophila SR protein family of general splicing factors. Escherichia coli-produced B52 is capable of both activating splicing and affecting the alternative splice site choice in human in vitro splicing reactions. Here we report the isolation of a B52 null mutant generated by remobilizing a P element residing near the B52 gene. The resulting deletion, B52(28), is confined to the B52 gene and its neighbor the Hrb87F gene. Second-instar larvae homozygous for the deletion are deficient in both B52 mRNA and protein. The B52 null mutant is lethal at the first- and second-instar larval stages. Germ line transformation of Drosophila flies with B52 genomic DNA rescues this lethality. Thus, B52 is an essential gene and has a critical role in Drosophila development. Larvae deficient in B52 are still capable of splicing the five endogenous pre-mRNAs tested here, including both constitutively and alternatively spliced genes. Therefore, B52 is not required for all splicing in vivo. This is the first in vivo deficiency analysis of a member of the SR protein family.
Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple-stranded coiled-coil structure.
Authors: Zuo J , Baler R , Dahl G , Voellmy R
Published: October 2015

Heat stress regulation of human heat shock genes is mediated by human heat shock transcription factor hHSF1, which contains three 4-3 hydrophobic repeats (LZ1 to LZ3). In unstressed human cells (37 degrees C), hHSF1 appears to be in an inactive, monomeric state that may be maintained through intramolecular interactions stabilized by transient interaction with hsp70. Heat stress (39 to 42 degrees C) disrupts these interactions, and hHSF1 homotrimerizes and acquires heat shock element DNA-binding ability. hHSF1 expressed in Xenopus oocytes also assumes a monomeric, non-DNA-binding state and is converted to a trimeric, DNA-binding form upon exposure of the oocytes to heat shock (35 to 37 degrees C in this organism). Because endogenous HSF DNA-binding activity is low and anti-hHSF1 antibody does not recognize Xenopus HSF, we employed this system for mapping regions in hHSF1 that are required for the maintenance of the monomeric state. The results of mutagenesis analyses strongly suggest that the inactive hHSF1 monomer is stabilized by hydrophobic interactions involving all three leucine zippers which may form a triple-stranded coiled coil. Trimerization may enable the DNA-binding function of hHSF1 by facilitating cooperative binding of monomeric DNA-binding domains to the heat shock element motif. This view is supported by observations that several different LexA DNA-binding domain-hHSF1 chimeras bind to a LexA-binding site in a heat-regulated fashion, that single amino acid replacements disrupting the integrity of hydrophobic repeats render these chimeras constitutively trimeric and DNA binding, and that LexA itself binds stably to DNA only as a dimer but not as a monomer in our assays.
Breakpoint junctions of chromosome 9p deletions in two human glioma cell lines.
Authors: Pomykala H M , Bohlander S K , Broeker P L , Olopade O I , Díaz M O
Published: October 2015

Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p21. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5' of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One- and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangement is discussed.
In organello footprint analysis of human mitochondrial DNA: human mitochondrial transcription factor A interactions at the origin of replication.
Authors: Ghivizzani S C , Madsen C S , Nelen M R , Ammini C V , Hauswirth W W
Published: October 2015

Using in organello footprint analysis, we demonstrate that within human placental mitochondria there is a high level of protein-DNA binding at regularly phased intervals throughout a 500-bp region encompassing the D-loop DNA origins and two promoter regions. Comparison with in vitro DNase I protection studies indicates that this protein-DNA interaction is due to non-sequence-specific binding by human mitochondrial transcription factor A (h-mtTFA). Since h-mtTFA can bend and wrap DNA, like its yeast counterpart ABF2, a primary function of h-mtTFA appears to be specific packaging of the mitochondrial DNA control region in vivo. Intervals of protein binding coincide with the spacing of the RNA start sites and prominent D-loop DNA 5' ends, suggesting a role for phased h-mtTFA binding in defining transcription and H-strand DNA replication origins. Significant protein-DNA interaction was also observed within the human homolog of conserved sequence block 1, both in organello and in vitro, using purified h-mtTFA.
Identification and analysis of a Saccharomyces cerevisiae copper homeostasis gene encoding a homeodomain protein.
Authors: Knight S A , Tamai K T , Kosman D J , Thiele D J
Published: October 2015

Yeast metallothionein, encoded by the CUP1 gene, and its copper-dependent transcriptional activator ACE1 play a key role in mediating copper resistance in Saccharomyces cerevisiae. Using an ethyl methanesulfonate mutant of a yeast strain in which CUP1 and ACE1 were deleted, we isolated a gene, designated CUP9, which permits yeast cells to grow at high concentrations of environmental copper, most notably when lactate is the sole carbon source. Disruption of CUP9, which is located on chromosome XVI, caused a loss of copper resistance in strains which possessed CUP1 and ACE1, as well as in the cup1 ace1 deletion strain. Measurement of intracellular copper levels of the wild-type and cup9-1 mutant demonstrated that total intracellular copper concentrations were unaffected by CUP9. CUP9 mRNA levels were, however, down regulated by copper when yeast cells were grown with glucose but not with lactate or glycerol-ethanol as the sole carbon source. This down regulation was independent of the copper metalloregulatory transcription factor ACE1. The DNA sequence of CUP9 predicts an open reading frame of 306 amino acids in which a 55-amino-acid sequence showed 47% identity with the homeobox domain of the human proto-oncogene PBX1, suggesting that CUP9 is a DNA-binding protein which regulates the expression of important copper homeostatic genes.
Oligonucleotides complementary to the Oxytricha nova telomerase RNA delineate the template domain and uncover a novel mode of primer utilization.
Authors: Melek M , Davis B T , Shippen D E
Published: October 2015

The telomerase reverse transcriptase uses an essential RNA subunit as a template to direct telomeric DNA synthesis. The 190-nucleotide Oxytricha nova telomerase RNA was identified by using an oligonucleotide probe complementary to the predicted CCCCAAAA template. This RNA displays extensive sequence similarity to the Euplotes crassus telomerase RNA and carries the same 5' CAAAACCCCAAAACC 3' telomeric domain. Antisense oligonucleotides were used to map the boundaries of the functional template and to investigate the mechanism of primer recognition and elongation. On the basis of their ability to inhibit or to prime telomerase, oligonucleotides were classified into three categories. Category 1 oligonucleotides, which extended 5' of residue 42 in the RNA, abolished elongation of (T4G4)3 and (G4T4)3 primers in vitro. In contrast, oligonucleotides terminating between residues 42 and 50 (categories 2 and 3), served as efficient telomerase primers. We conclude that the O. nova template comprises residues 42 to 50 in the 190-nucleotide RNA, a different set of nucleotides than are used by the E. crassus enzyme. Category 2 primer reactions amassed short products, and their abundance could be decreased by altering the 5' sequence of the primer, consistent with the two-primer-binding-site model for telomerase. Category 3 primers generated a bimodal distribution of short and long products, each having a unique elongation profile. The long-product profile is inconsistent with sequence-specific primer alignment. Rather, each primer was extended by the same register of TTTTGGGG repeats, suggesting shuttling to a default position within the template. The parallels between telomerase and RNA polymerase elongation mechanisms are discussed.
Inhibition of platelet-derived growth factor- and epidermal growth factor-mediated mitogenesis and signaling in 3T3 cells expressing delta Raf-1:ER, an estradiol-regulated form of Raf-1.
Authors: Samuels M L , McMahon M
Published: October 2015

We have recently described the properties of delta Raf-1:ER, a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the human estrogen receptor. In this study, we demonstrate that activation of delta Raf-1:ER in quiescent 3T3 cells (C2 cells), while sufficient to promote morphological oncogenic transformation, was insufficient to promote the entry of cells into DNA synthesis. Indeed, activation of delta Raf-1:ER potently inhibited the mitogenic response of cells to platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) treatment. Addition of beta-estradiol to quiescent C2 cells led to rapid, sustained activation of delta Raf-1:ER and MEK but only two- to threefold activation of p42 mitogen-activating protein (MAP) kinase activity. Addition of PDGF or EGF to quiescent C2 cells in which delta Raf-1:ER was inactive led to rapid activation of Raf-1, MEK, and p42 MAP kinase activities, and entry of the cells into DNA synthesis. In contrast, when delta Raf-1:ER was activated in quiescent C2 cells prior to factor addition, there was a significant inhibition of certain aspects of the signaling response to subsequent treatment with PDGF or EGF. The expression and activation of PDGF receptors and the phosphorylation of p70S6K in response to PDGF treatment were unaffected by prior activation of delta Raf-1:ER. In contrast, PDGF-mediated activation of Raf-1 and p42 MAP kinases was significantly inhibited compared with that of controls. Interestingly, the mitogenic and signaling responses of quiescent C2 cells to stimulation with fetal bovine serum or phorbol myristate acetate were unaffected by prior activation of delta Raf-1:ER. It seems likely that at least two mechanisms contribute to the effects of delta Raf-1:ER in these cells. First, activation of delta Raf-1:ER appeared to uncouple the activation of Raf-1 from the activation of the PDGF receptor at the cell surface. This may be due to the fact that mSOS1 is constitutively phosphorylated as a consequence of the activation of delta Raf-1:ER. Second, quiescent C2 cells expressing activated delta Raf-1:ER appear to contain an inhibitor of the MAP kinase pathway that, because of its apparent sensitivity to sodium orthovanadate, may be a phosphotyrosine phosphatase. It is likely that the inhibitory effects of delta Raf-1:ER observed in these cells are a manifestation of the activation of some of the feedback inhibition pathways that normally modulate a cell's response to growth factors. 3T3 cells expressing delta Raf-1:ER will be a useful tool in unraveling the role of Raf-1 kinase activity in the regulation of such pathways.
Further analysis of cytoplasmic polyadenylation in Xenopus embryos and identification of embryonic cytoplasmic polyadenylation element-binding proteins.
Authors: Simon R , Richter J D
Published: October 2015

Early development in Xenopus laevis is programmed in part by maternally inherited mRNAs that are synthesized and stored in the growing oocyte. During oocyte maturation, several of these messages are translationally activated by poly(A) elongation, which in turn is regulated by two cis elements in the 3' untranslated region, the hexanucleotide AAUAAA and a cytoplasmic polyadenylation element (CPE) consisting of UUUUUAU or similar sequence. In the early embryo, a different set of maternal mRNAs is translationally activated. We have shown previously that one of these, C12, requires a CPE consisting of at least 12 uridine residues, in addition to the hexanucleotide, for its cytoplasmic polyadenylation and subsequent translation (R. Simon, J.-P. Tassan, and J.D. Richter, Genes Dev. 6:2580-2591, 1992). To assess whether this embryonic CPE functions in other maternal mRNAs, we have chosen Cl1 RNA, which is known to be polyadenylated during early embryogenesis (J. Paris, B. Osborne, A. Couturier, R. LeGuellec, and M. Philippe, Gene 72:169-176, 1988). Wild-type as well as mutated versions of Cl1 RNA were injected into fertilized eggs and were analyzed for cytoplasmic polyadenylation at times up to the gastrula stage. This RNA also required a poly(U) CPE for cytoplasmic polyadenylation in embryos, but in this case the CPE consisted of 18 uridine residues. In addition, the timing and extent of cytoplasmic poly(A) elongation during early embryogenesis were dependent upon the distance between the CPE and the hexanucleotide. Further, as was the case with Cl2 RNA, Cl1 RNA contains a large masking element that prevents premature cytoplasmic polyadenylation during oocyte maturation. To examine the factors that may be involved in the cytoplasmic polyadenylation of both C12 and C11 RNAs, we performed UV cross-linking experiments in egg extracts. Two proteins with sizes of ~36 and ~45 kDa interacted specifically with the CPEs of both RNAs, although they bound preferentially to the C12 CPE. The role that these proteins might play in cytoplasmic polyadenylation is discussed.
G1 cyclin degradation: the PEST motif of yeast Cln2 is necessary, but not sufficient, for rapid protein turnover.
Authors: Salama S R , Hendricks K B , Thorner J
Published: October 2015

The 545-residue Cln2 protein, like the other G1 cyclins of Saccharomyces cerevisiae, is a very unstable protein. This instability is thought to play a critical role in regulating cell cycle progression. The carboxyl-terminal domains of Cln2 and the other G1 cyclins contain sequences rich in Pro, Glu (and Asp), Ser, and Thr (so-called PEST motifs) that have been postulated to make up the signals that are responsible for the rapid degradation of these and other unstable proteins. To test this hypothesis, the carboxyl-terminal 178 residues of Cln2 were fused to the C terminus of a reporter enzyme, a truncated form of human thymidine kinase (hTK delta 40). The resulting chimeric protein (hTK delta 40-Cln2) retained thymidine kinase activity but was markedly less stable than hTK, hTK delta 40, or an hTK-beta-galactosidase fusion protein, as judged by enzyme assay, immunoblotting with anti-hTK antibodies, pulse-chase analysis of the radiolabeled polypeptides, and ability to support the growth of a thymidylate auxotroph (cdc21 mutant) on thymidine-containing medium. Thus, the presence of the Cln2 PEST domain was sufficient to destabilize a heterologous protein. Furthermore, the half-life of hTK delta 40-Cln2 was similar to that of authentic Cln2, and the rate of degradation of neither protein was detectably enhanced by treatments known to cause G1 arrest, including exposure of MATa haploids to alpha-factor mating pheromone and shifting cdc28ts and cdc34ts mutants to the restrictive temperature. These results suggest that the major signals responsible for Cln2 instability are confined to its C-terminal third. Because hTK delta 40-Cln2 and Cln2 were expressed from heterologous promoters yet their half-lives both in asynchronous cultures and when arrested at various cell cycle stages were always similar, the Cln2 PEST domain contains a signal for rapid protein turnover that is constitutively active and operative throughout the cell cycle. Removal of the 37 codons that encode the most prominent PEST-like segment from either hTK delta 40-Cln2 or Cln2 decreased the turnover rate of the resulting proteins, as expected; however, an hTK delta 40 chimera containing only this 37-residue segment was not detectably destabilized, suggesting that this PEST sequence, when removed from its normal context, is not a self-contained determinant of protein instability.
Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene.
Authors: Yasumoto K , Yokoyama K , Shibata K , Tomita Y , Shibahara S
Published: October 2015

Tyrosinase is a rate-limiting enzyme in melanin biosynthesis and is specifically expressed in differentiated melanocytes. We have identified the enhancer element in the 5'-flanking region of the human tyrosinase gene that is responsible for its pigment cell-specific transcription and have termed it tyrosinase distal element (TDE) (positions -1861 to -1842). Transient expression assays showed that TDE confers efficient expression of a firefly luciferase reporter gene linked to the tyrosinase gene promoter in MeWo pigmented melanoma cells but not in HeLa cells, which do not express tyrosinase. TDE was specifically bound by nuclear proteins of MeWo and HeLa cells, the binding properties of which were indistinguishable in gel mobility shift assays. TDE contains the CATGTG motif in its center, and mutation analysis indicates that the CA dinucleotides of this motif are crucial for protein binding and pigment cell-specific enhancer function. The CATGTG motif is consistent with the consensus sequence recognized by a large family of transcription factors with a basic helix-loop-helix structure, which prompted us to examine the possible involvement of a ubiquitous transcription factor, USF, and a novel factor, microphthalmia-associated transcription factor (MITF), recently cloned as the human homolog of the mouse microphthalmia (mi) gene product. The mi phenotype is associated with a mutant mi locus and characterized by small eyes and loss of melanin pigments. Both USF and MITF are predicted to contain a basic helix-loop-helix structure and a leucine zipper structure. We provide evidence that USF binds to TDE, whereas we were unable to detect the DNA-binding activity of MITF. Transient coexpression assays showed that MITF specifically transactivates the promoter activity of the tyrosinase gene through the CATGTG motif of TDE but not the promoter of the ubiquitously expressed heme oxygenase gene, while USF is able to activate both promoters. These results indicate that MITF is a cell-type-specific factor that is capable of activating transcription of the tyrosinase gene.
Ligand-dependent occupancy of the retinoic acid receptor beta 2 promoter in vivo.
Authors: Dey A , Minucci S , Ozato K
Published: October 2015

Retinoic acid (RA) activates transcription of the RA receptor beta 2 (RAR beta 2) gene in embryonal carcinoma (EC) cells. This activation involves binding of the RAR/retinoid X receptor (RAR/RXR) heterodimer to the RA-responsive element (beta RARE). Dimethyl sulfate-based genomic footprinting was performed to examine occupancy of this promoter in P19 EC cells. No footprint was detected at the beta RARE prior to RA treatment, but a footprint was detected within the first hour of RA treatment. Concomitantly, other elements in the promoter, the cyclic AMP-responsive element and tetradecanoyl phorbol acetate-like-responsive element became footprinted. Footprints at these elements were induced by RA without requiring new protein synthesis and remained for the entire duration of RA treatment but rapidly reversed upon withdrawal of RA. A delayed protection observed at the initiator site was also reversed upon RA withdrawal. The RA-inducible footprint was not due to induction of factors that bind to these element, since in vitro assays showed that these factors are present in P19 cell extracts before RA treatment. Significantly, no RA-induced footprint was observed at any of these elements in P19 cells expressing a dominant negative RXR beta, in which RXR heterodimers are unable to bind to the beta RARE. Results indicate that binding of a liganded heterodimer receptor to the beta RARE is the initial event that allows other elements to gain access to the factors. In accordance, reporter analyses showed that a mutation in the beta RARE, but not those in other elements, abrogates RA activation of the promoter. It is likely that the RAR beta 2 promoter opens in a hierarchically ordered manner, signalled by the occupancy of liganded heterodimers.
MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating.
Authors: Iida H , Nakamura H , Ono T , Okumura M S , Anraku Y
Published: October 2015

By establishing a unique screening method, we have isolated yeast mutants that die only after differentiating into cells with a mating projection, and some of them are also defective in Ca2+ signaling. The mutants were classified into five complementation groups, one of which we studied extensively. This mutation defines a new gene, designated MID1, which encodes an N-glycosylated, integral plasma membrane protein with 548 amino acid residues. The mid1-1 mutant has low Ca2+ uptake activity, loses viability after receiving mating pheromones, and escapes death when incubated with high concentrations of CaCl2. The MID1 gene is nonessential for vegetative growth. The efficiency of mating between MATa mid1-1 and MAT alpha mid1-1 cells is low. These results demonstrate that MID1 is required for Ca2+ influx and mating.
The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase.
Authors: Roberts B T , Farr K A , Hoyt M A
Published: October 2015

Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement, we previously screened for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. Two of the identified genes, BUB2 and BUB3, have been cloned and described (M. A. Hoyt, L. Totis, and B. T. Roberts, Cell 66:507-517, 1991). Here we present the characterization of the BUB1 gene and its product. Genetic evidence was obtained suggesting that Bub1 and Bub3 are mutually dependent for function, and immunoprecipitation experiments demonstrated a physical association between the two. Sequence analysis of BUB1 revealed a domain with similarity to protein kinases. In vitro experiments confirmed that Bub1 possesses kinase activity; Bub1 was able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1 was found to localize to the cell nucleus.
Efficiency of insertion versus replacement vector targeting varies at different chromosomal loci.
Authors: Hasty P , Crist M , Grompe M , Bradley A
Published: October 2015

We have analyzed the targeting frequencies and recombination products generated with isogenic vectors at the fah and fgr loci in embryonic stem cells. A single vector which could be linearized at different sites to generate either a replacement or an insertion vector was constructed for each locus. A replacement event predominated when the vectors were linearized at the edge of the homologous sequences, while an insertion event predominated when the vectors were linearized within the homologous sequences. However, the ratio of the targeting frequencies exhibited by the different vector configurations differed for the two loci. When the fgr vector was linearized as an insertion vector, the ratio of targeted to random integrations was four- to eightfold greater than when the vector was linearized as a replacement vector. By contrast, the ratio of targeted to random integrations at the fah locus did not vary with the linearization site of the vector. The different relationships between the targeting frequency and the vector configuration at the fgr and fah loci may indicate a DNA sequence or chromatin structure preference for different targeting pathways.