bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities.
Authors: Werner S , Duan D S , de Vries C , Peters K G , Johnson D E , Williams L T
Published: October 2015

We have cloned a genomic region of the murine fibroblast growth factor (FGF) receptor 1 (FGFR1) gene that includes three alternative exons for the third immunoglobulinlike domain in the extracellular region of the receptor. The mRNA of one of these splice variants encodes a secreted receptor that lacks transmembrane and cytoplasmic sequences as well as a portion of the third immunoglobulinlike domain. Highest levels of mRNA encoding this variant were found in brain, skeletal muscle, and skin. We expressed this form of FGFR1 in CHO cells and showed that the recombinant secreted protein binds acidic FGF. We also discovered a novel alternative exon in the third immunoglobulinlike domain that encodes part of a transmembrane FGFR1 mRNA. This exon is highly homologous to the corresponding region of the keratinocyte growth factor receptor. Transcripts including this exon were present at highest levels in the skin. We cloned an FGFR1 cDNA which includes this exon and expressed this receptor variant in L6 rat skeletal muscle myoblasts. The new receptor variant had a 50-fold-lower affinity for basic FGF than does the published FGFR1 variant, whereas both forms of receptor bound acidic FGF with high affinity. These results show that the third immunoglobulinlike domain plays an important role in determining the binding specificities for different FGFs. Our data provide the first evidence that differential splicing in the extracellular region of a receptor gene generates receptor variants with different ligand-binding specificities.
Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.
Authors: Wang S W , Speck N A
Published: October 2015

The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes.
The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae.
Authors: Deshpande A M , Newlon C S
Published: October 2015

Replication origins have been mapped to positions that coincide, within experimental error (several hundred base pairs), with ARS elements. To determine whether the DNA sequences required for ARS function on plasmids are required for chromosomal origin function, the chromosomal copy of ARS306 was deleted and the chromosomal copy of ARS307 was replaced with mutant derivatives of ARS307 containing single point mutations in domain A within the ARS core consensus sequence. The chromosomal origin function of these derivatives was assayed by two-dimensional agarose gel electrophoresis. Deletion of ARS306 deleted the associated replication origin. The effects on chromosomal origin function of mutations in domain A paralleled their effects on ARS function, as measured by plasmid stability. These results demonstrate that chromosomal origin function is a property of the ARS element itself.
Identification of a 60-kilodalton Rb-binding protein, RBP60, that allows the Rb-E2F complex to bind DNA.
Authors: Ray S K , Arroyo M , Bagchi S , Raychaudhuri P
Published: October 2015

Several reports have indicated that the product of the retinoblastoma gene (Rb) complexes with the transcription factor E2F. We present evidence that the DNA-binding of the Rb-E2F complex involves another cellular factor. Addition of Rb to purified preparations of E2F does not generate an Rb-E2F complex that can bind DNA, and in fact, we see an inhibition of the DNA-binding ability of E2F. On the other hand, addition of Rb to cruder preparations of E2F results in the formation of an Rb-E2F complex (E2Fr) that can bind DNA and produces a distinct complex in gel retardation assays. We have identified and purified a 60-kDa protein that allows the Rb-E2F complex to bind DNA, and we show that this 60-kDa protein exerts its effect by directly interacting with Rb.
The MRF4 activation domain is required to induce muscle-specific gene expression.
Authors: Mak K L , To R Q , Kong Y , Konieczny S F
Published: October 2015

MRF4 is a member of the basic helix-loop-helix muscle regulatory factor family that also includes MyoD, myogenin, and Myf-5. Overexpression of MRF4 or the other muscle regulatory factors in fibroblasts converts the cells to differentiated muscle fibers and transcriptionally activates expression of endogenous and cotransfected muscle genes. Although these factors induce a similar phenotype, they also exhibit some distinct biological activities. For example, MyoD trans activates alpha-actin and troponin I reporter genes to very high levels, whereas MRF4 efficiently activates only alpha-actin expression. Since these proteins have a common basic helix-loop-helix domain, it is likely that portions of the proteins outside of this region impart some specificity to the activity of each muscle regulatory factor. As an initial step in determining the mechanism by which MRF4 and MyoD activate gene transcription, the transcriptional activation domain of MRF4 has been characterized. Experiments utilizing chimeric proteins containing the yeast GAL4 DNA-binding domain and portions of the MRF4 protein indicate that the MRF4 activation domain is located within amino acids 10 to 30. This amino terminus is both necessary and sufficient to elicit a transcriptional response in transfected cells. The MRF4 activation domain and the related amino-terminal MyoD activation domain are capable of substituting for one another in converting fibroblasts to a myogenic phenotype and in activating expression of an alpha-actin reporter gene, although the MRF4 and MyoD activation domains on these chimeric proteins also dictate the specificity of transcriptional activation. The different primary amino acid sequences of these regions leave open the possibility that different coregulator proteins interact with the muscle regulatory factors to elicit their correct transcriptional activity during skeletal muscle development.
The plant amino acid mimosine may inhibit initiation at origins of replication in Chinese hamster cells.
Authors: Mosca P J , Dijkwel P A , Hamlin J L
Published: October 2015

An understanding of replication initiation in mammalian cells has been hampered by the lack of mutations and/or inhibitors that arrest cells just prior to entry into the S period. The plant amino acid mimosine has recently been suggested to inhibit cells at a regulatory step in late G1. We have examined the effects of mimosine on cell cycle traverse in the mimosine [corrected]-resistant CHO cell line CHOC 400. When administered to cultures for 14 h after reversal of a G0 block, the drug appears to arrest the population at the G1/S boundary, and upon its removal cells enter the S phase in a synchronous wave. However, when methotrexate is administered to an actively dividing asynchronous culture, cells are arrested not only at the G1/S interface but also in early and middle S phase. Most interestingly, two-dimensional gel analysis of replication intermediates in the initiation locus of the amplified dihydrofolate reductase domain suggests that mimosine may actually inhibit initiation. Thus, this drug represents a new class of inhibitors that may open a window on regulatory events occurring at individual origins of replication.
A single MEF-2 site is a major positive regulatory element required for transcription of the muscle-specific subunit of the human phosphoglycerate mutase gene in skeletal and cardiac muscle cells.
Authors: Nakatsuji Y , Hidaka K , Tsujino S , Yamamoto Y , Mukai T , Yanagihara T , Kishimoto T , Sakoda S
Published: October 2015

In order to analyze the transcriptional regulation of the muscle-specific subunit of the human phosphoglycerate mutase (PGAM-M) gene, chimeric genes composed of the upstream region of the PGAM-M gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were constructed and transfected into C2C12 skeletal myocytes, primary cultured cardiac muscle cells, and C3H10T1/2 fibroblasts. The expression of chimeric reporter genes was restricted in skeletal and cardiac muscle cells. In C2C12 myotubes and primary cultured cardiac muscle cells, the segment between nucleotides -165 and +41 relative to the transcription initiation site was sufficient to confer maximal CAT activity. This region contains two E boxes and one MEF-2 motif. Deletion and substitution mutation analysis showed that a single MEF-2 motif but not the E boxes had a substantial effect on skeletal and cardiac muscle-specific enhancer activity and that the cardiac muscle-specific negative regulatory region was located between nucleotides -505 and -165. When the PGAM-M gene constructs were cotransfected with MyoD into C3H10T1/2, the profile of CAT activity was similar to that observed in C2C12 myotubes. Gel mobility shift analysis revealed that when the nuclear extracts from skeletal and cardiac muscle cells were used, the PGAM-M MEF-2 site generated the specific band that was inhibited by unlabeled PGAM-M MEF-2 and muscle creatine kinase MEF-2 oligomers but not by a mutant PGAM-M MEF-2 oligomer. These observations define the PGAM-M enhancer as the only cardiac- and skeletal-muscle-specific enhancer characterized thus far that is mainly activated through MEF-2.
Selection of optimal kappa B/Rel DNA-binding motifs: interaction of both subunits of NF-kappa B with DNA is required for transcriptional activation.
Authors: Kunsch C , Ruben S M , Rosen C A
Published: October 2015

Analysis of the p50 and p65 subunits of the NF-kappa B transcription factor complex has revealed that both proteins can interact with related DNA sequences through either homo- or heterodimer formation. In addition, the product of the proto-oncogene c-rel can bind to similar DNA motifs by itself or as a heterodimer with p50 or p65. However, these studies have used a limited number of known kappa B DNA motifs, and the question of the optimal DNA sequences preferred by each homodimer has not been addressed. Using purified recombinant p50, p65, and c-Rel proteins, optimal DNA-binding motifs were selected from a pool of random oligonucleotides. Alignment of the selected sequences allowed us to predict a consensus sequence for binding of the individual homodimeric Rel-related proteins, and DNA-protein binding analysis of the selected DNA sequences revealed sequence specificity of the proteins. Contrary to previous assumptions, we observed that p65 homodimers can interact with a subset of DNA sequences not recognized by p50 homodimers. Differential binding affinities were also obtained with p50- and c-Rel-selected sequences. Using either a p50- or p65-selected kappa B motif, which displayed differential binding with respect to the other protein, little to no binding was observed with the heterodimeric NF-kappa B complex. Similarly, in transfection experiments in which the selective kappa B binding sites were used to drive the expression of a chloramphenicol acetyltransferase reporter construct, the p65- and p50-selected motifs were activated only in the presence of p65 and p50/65 (a chimeric protein with the p50 DNA binding domain and p65 activation domain) expression vectors, respectively, and neither demonstrated a significant response to stimuli that induce NF-kappa B activity. These findings demonstrate that interaction of both subunits of the heterodimeric NF-kappa B complex with DNA is required for DNA binding and transcriptional activation and suggest that transcriptional activation mediated by the individual rel-related proteins will differ dramatically, depending on the specific kappa B motifs present.
Retroviral insertions downstream of the heterogeneous nuclear ribonucleoprotein A1 gene in erythroleukemia cells: evidence that A1 is not essential for cell growth.
Authors: Ben-David Y , Bani M R , Chabot B , De Koven A , Bernstein A
Published: October 2015

A large number of novel cellular proto-oncogenes have been identified and cloned by analysis of common integration sites in retrovirally induced malignancies. In the multistage erythroleukemias induced by the various strains of Friend leukemia virus, the analysis of proviral-integration events has led to the identification of two genes, Fli-1 and Spi-1, both novel members of the ets oncogene family of transcription factors. In this report, we describe the identification of another integration site, designated Fli-2 (Friend leukemia virus integration-2), in an erythroleukemia cell line induced by Friend murine leukemia virus (F-MuLV). Rearrangements at the Fli-2 locus were found in two erythroleukemia cell lines independently induced by F-MuLV and one leukemic cell line derived from the spleen of a mouse infected with the polycythemia strain of Friend leukemia virus. The deduced amino acid sequence of a cDNA corresponding to a transcript originating from genomic DNA adjacent to Fli-2 is identical to that of the human heterogeneous nuclear ribonucleoprotein A1 gene, a member of the gene family of RNA-binding proteins involved in RNA splicing. In one erythroleukemia cell line, A1 expression was undetectable as a result of F-MuLV integration in one allele and loss of the other allele. These results suggest that perturbations in RNA splicing mechanisms may contribute to malignant transformation and provide direct evidence that the A1 protein is not required for cell growth.
Accumulation of U14 small nuclear RNA in Saccharomyces cerevisiae requires box C, box D, and a 5', 3' terminal stem.
Authors: Huang G M , Jarmolowski A , Struck J C , Fournier M J
Published: October 2015

U14 is one of several nucleolar small nuclear RNAs required for normal processing of rRNA. Functional mapping of U14 from Saccharomyces cerevisiae has yielded a number of mutants defective in U14 accumulation or function. In this study, we have further defined three structural elements required for U14 accumulation. The essential elements include the U14-conserved box C and box D sequences and a 5', 3' terminal stem. The box elements are coconserved among several nucleolar small nuclear RNAs and have been implicated in binding of the protein fibrillarin. New mutational results show that the first GA bases of the box C sequence UGAUGA are essential, and two vital bases in box D have also been identified. An intragenic suppressor of a lethal box C mutant has been isolated and shown to contain a new box C-like PyGAUG sequence two bases upstream of normal box C. The importance of the terminal stem was confirmed from new compensatory base changes and the finding that accumulation defects in the box elements can be complemented by extending the terminal stem. The results suggest that the observed defects in accumulation reflect U14 instability and that protein binding to one or more of these elements is required for metabolic stability.
Effects of p56lck deficiency on the growth and cytolytic effector function of an interleukin-2-dependent cytotoxic T-cell line.
Authors: Karnitz L , Sutor S L , Torigoe T , Reed J C , Bell M P , McKean D J , Leibson P J , Abraham R T
Published: October 2015

The growth, differentiation, and functional activities of antigen-stimulated T lymphocytes are regulated by the interaction of the T-cell-derived cytokine, interleukin-2 (IL-2), with the high-affinity IL-2 receptor (IL-2R). IL-2R occupancy initiates a rapid increase in intracellular protein tyrosine phosphorylation, suggesting that a receptor-coupled protein tyrosine kinase (PTK) serves as a proximal signaling element for the IL-2R. Previous studies implicated the src-family kinase, p56lck, as a potential IL-2R-linked signal transducer. In this study, we have characterized a spontaneous variant of the IL-2-dependent cytotoxic T-cell line, CTLL-2, which contains no detectable lck-derived mRNA transcripts, protein, or PTK activity. The p56lck-deficient CTLL-2 cells retained strict dependence on IL-2 for both viability and growth, indicating that p56lck activity was not required for the transduction of IL-2-mediated mitogenic signals. However, the p56lck-deficient cells exhibited a moderate decrease in their rate of IL-2-dependent proliferation. In contrast to this relatively modest proliferative defect, the p56lck-deficient cell line displayed a profound reduction in T-cell antigen receptor-dependent cytolytic effector functions. Both the proliferative and the cytolytic defects observed in the p56lck-deficient cells were completely reversed by transfection of these cells with a wild-type lck expression vector. These results indicate that p56lck expression is not obligatory for IL-2-mediated T-cell growth stimulation; however, this PTK plays a central role in the generation T-cell-mediated cytotoxic responses.
Examination of the DNA-binding ability of estrogen receptor in whole cells: implications for hormone-independent transactivation and the actions of antiestrogens.
Authors: Reese J C , Katzenellenbogen B S
Published: October 2015

We describe an assay employing the competitive binding of estrogen receptor (ER) with basal transcription factors on a constitutive promoter (cytomegalovirus-hormone response element[s]-chloramphenicol acetyltransferase [CMV-(HRE)n-CAT, containing a hormone response element(s) between the TATA box and the start site of transcription]) to examine the DNA-binding ability of the human ER in whole cells. We used this promoter interference assay to examine the DNA binding of ER in cell lines containing high and low levels of endogenous ER, as well as in CHO cells expressing wild-type and mutant ERs from cotransfected expression vectors. The ER is capable of binding to the promoter interference constructs in the absence of added ligand, and estrogen (estradiol) or antiestrogen (trans-hydroxytamoxifen or ICI 164,384) enhances or stabilizes this interaction. The binding of unoccupied ER to reporter gene activation plasmids results in ligand-independent transactivation, presumably due to the TAF-1 function of the receptor. DNA binding of ER in the absence of ligand is observed in cells containing endogenous ER, or expressed ER, and occurs in cells with high or low receptor contents. Although estrogen- and antiestrogen-occupied ER complexes bind to DNA and reduce the template promoter activity, the extent of suppression achieved by ICI-bound ERs is consistently less than that achieved with the other ligands, presumably caused by the fact that ICI rapidly reduces the level of ER in most of the cells examined. However, the ICI-ER complexes that remain are in sufficient quantity to bind to gene activation reporter constructs, and in these cells, ICI still behaves as a pure antagonist of gene transcription and does not activate reporter genes. Hence, obstruction of ER DNA binding or reduction of ER in target cells may contribute to, but cannot fully explain, the pure antagonist character of the antiestrogen ICI 164,384. In addition, DNA binding by the ER alone is clearly not sufficient for ensuring full activation of transcription and argues for an intermediate in the receptor activation of promoters.
Mutations in the WSAWSE and cytosolic domains of the erythropoietin receptor affect signal transduction and ligand binding and internalization.
Authors: Quelle D E , Quelle F W , Wojchowski D M
Published: October 2015

The terminal development of erythroid progenitor cells is promoted in part through the interaction of erythropoietin (EPO) with its cell surface receptor. This receptor and a growing family of related cytokine receptors share homologous extracellular features, including a well-conserved WSXWS motif. To explore the functional significance of this motif in the murine EPO receptor, five WSAWSE mutants were prepared and their signal-transducing, ligand binding, and endocytotic properties were compared. EPO receptors mutated at tryptophan residues (W-232, W-235----G; W-235----G; W-235----F) failed to mediate EPO-induced growth or pp100 phosphorylation, while S-236----T and E-237----K mutants exhibited partial to full activity (50 to 100% of wild-type growth and induced phosphorylation). Ligand affinity was reduced for mutant receptors (two- to fivefold), yet expression at the cell surface for all receptors was nearly equivalent. Also, the ability of mutated receptors to internalize ligand was either markedly reduced or abolished (W-235----F), indicating a role for the WSAWSE region in hormone internalization. Interestingly, receptor forms lacking 97% of the cytosolic domain (no signal-transducing capacity; binding affinity reduced two- to threefold) internalized EPO efficiently. This and all WSAWSE receptor forms studied also mediated specific cross-linking of 125I-EPO to three accessory membrane proteins (M(r)s, 120,000, 105,000, and 93,000). These findings suggest that the WSAWSE domain of the EPO receptor is important for EPO-induced signal transduction and ligand internalization. In contrast, although the cytosolic domain is required for growth signaling, it appears nonessential for efficient endocytosis.
A DNA element that regulates expression of an endogenous retrovirus during F9 cell differentiation is E1A dependent.
Authors: Lamb B T , Satyamoorthy K , Solter D , Basu A , Xu M Q , Weinmann R , Howe C C
Published: October 2015

The retinoic acid-induced differentiation of F9 cells into parietal endoderm-like cells activates transcription of the endogenous mouse retrovirus, the intracisternal A-particle (IAP). To investigate the elements that control IAP gene differentiation-specific expression, we used methylation interference, Southwestern (DNA-protein), and transient-transfection assays and identified the IAP-proximal enhancer (IPE) element that directs differentiation-specific expression. We find that the IPE is inactive in undifferentiated F9 cells and active in differentiated parietal endoderm-like PYS-2 cells. Three proteins of 40, 60, and 68 kDa bind to the sequence GAGTAGAC located between nucleotides -53 and -47 within the IPE. The 40- and 68-kDa proteins from both the undifferentiated and differentiated cells exhibit similar DNA-binding activities. However, the 60-kDa protein from differentiated cells has greater binding activity than that from undifferentiated cells, suggesting a role for this protein in F9 differentiation-specific expression of the IAP gene. The IAP gene is negatively regulated by the adenovirus E1A proteins, and the E1A sequence responsible for repression is located at the N terminus, between amino acids 2 and 67. The DNA sequence that is the target of E1A repression also maps to the IPE element. Colocalization of the differentiation-specific and E1A-sensitive elements to the same protein-binding site within the IPE suggests that the E1A-like activity functions in F9 cells to repress IAP gene expression. Activation of the IAP gene may result when the E1A-like activity is lost or inactivated during F9 cell differentiation, followed by binding of the 60-kDa positive regulatory protein to the enhancer element.
A mammalian factor that binds telomeric TTAGGG repeats in vitro.
Authors: Zhong Z , Shiue L , Kaplan S , de Lange T
Published: October 2015

We have identified a DNA-binding activity with specificity for the TTAGGG repeat arrays found at mammalian telomeres. This factor, called TTAGGG repeat factor (TRF), is present in nuclear extracts of human, mouse, and monkey cells. TRF from HeLa cells was characterized in detail by electrophoretic mobility shift assays. It binds double-stranded TTAGGG repeats in linear and circular DNAs. Single-stranded repeats are not recognized. The optimal site for TRF appears to contain more than six contiguous TTAGGG repeats. Tandem arrays of TAGGG, TTTAGGG, TTTTAGGG, TTGGGG, and TTAGGC repeats do not bind TRF well, indicating that TRF preferentially recognizes the telomeric repeat sequence present at mammalian chromosome ends. The apparent molecular mass of this factor, based on recovery of TRF from sodium dodecyl sulfate-polyacrylamide gels, is approximately 50 kDa. We suggest that TRF binds along the length of mammalian telomeres.
Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A.
Authors: Virshup D M , Russo A A , Kelly T J
Published: October 2015

The catalytic subunit of protein phosphatase 2A (PP2Ac) stimulates the initiation of replication of simian virus 40 DNA in vitro by dephosphorylating T antigen at specific phosphoserine residues (K. H. Scheidtmann, D. M. Virshup, and T. J. Kelly, J. Virol. 65:2098-2101, 1991). To better define the biochemical mechanism responsible for this stimulation, we investigated the effect of PP2Ac on the interaction of T antigen with wild-type and mutant origins of replication. Analysis of the binding of T antigen to the wild-type origin as a function of protein concentration revealed that binding occurs in two relatively discrete steps: the assembly of a T-antigen hexamer on one half-site of the origin, followed by the assembly of the second hexamer on the other half-site. The major effect of PP2Ac was to stimulate binding of the second hexamer, so that the binding reaction became much more cooperative. This observation suggests that dephosphorylation of T antigen by PP2Ac primarily affects interactions between the two hexamers bound to the origin. Pretreatment with PP2Ac increased the ability of the bound T antigen to unwind the origin of replication but had no effect on the intrinsic helicase activity of the protein. Thus, dephosphorylation of PP2Ac appears to increase the efficiency of the initial opening of the origin by T antigen. An insertion mutation at the dyad axis in the simian virus 40 origin, which altered the structural relationship of the two halves of the origin, abolished the effect of the phosphatase on the cooperativity of binding and completely prevented origin unwinding. These findings suggest that the ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.
The osmotic integrity of the yeast cell requires a functional PKC1 gene product.
Authors: Paravicini G , Cooper M , Friedli L , Smith D J , Carpentier J L , Klig L S , Payton M A
Published: October 2015

Seven temperature-sensitive cell lysis (cly) mutant strains of Saccharomyces cerevisiae were isolated which lyse at the restrictive temperature on hypotonic but not on osmotically supported medium. The seven mutants fell into four complementation groups, CLY12 to CLY15. The wild-type CLY15 gene was isolated by complementation of the cly15 temperature-sensitive growth defect. Sequence analysis revealed that the complementing DNA fragment encoded a partial PKC1 gene, which has previously been isolated as an S. cerevisiae homolog of mammalian protein kinase C genes (D. E. Levin, F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner, Cell 62:213-224, 1990). Subsequent genetic analysis showed that CLY15 and PKC1 represent identical loci in the yeast genome. A truncated PKC1 gene encoding only the predicted catalytic domain of Pkc1p was able to complement pkc1 mutant strains. Similar to what has been reported recently (D. E. Levin and E. Bartlett-Heubusch, J. Cell Biol. 116:1221-1229, 1992), we observed that cells deleted for the PKC1 gene are viable when grown on osmotically stabilized medium but are osmotically fragile and lyse rapidly after a shift to hypotonic medium. As shown by light and electron microscopic examinations, the delta pkc1 strain exhibits many cells with a strongly elongated bud or chains of incompletely budded cells when grown on solid medium.
A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.
Authors: Mink S , Härtig E , Jennewein P , Doppler W , Cato A C
Published: October 2015

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites.
Existence of different Fos/Jun complexes during the G0-to-G1 transition and during exponential growth in mouse fibroblasts: differential role of Fos proteins.
Authors: Kovary K , Bravo R
Published: October 2015

We have determined the different Fos/Jun complexes present in Swiss 3T3 cells either following serum stimulation of quiescent cells or during exponential growth by immunoprecipitation analyses. We have shown that while c-Fos is the major Fos protein associated with the Jun proteins (c-Jun, JunB, and JunD) soon after serum stimulation, at later times Fra-1 and Fra-2 are the predominant Fos proteins associated with the different Jun proteins. During exponential growth, the synthesis of Fra-1 and Fra-2 is maintained at a significant level, in contrast to c-Fos and FosB, which are expressed at very low or undetectable levels. Consequently, Fra-1 and Fra-2 are the main Fos proteins complexed with the Jun proteins in asynchronously growing cells. To determine whether the Fos proteins are differentially required during the G0-to-G1 transition and exponential growth for the entrance into S phase, we microinjected affinity-purified antibodies directed against c-Fos, FosB, Fra-1, and Fra-2. We have found that while the activities of c-Fos and FosB are required mostly during the G0-to-G1 transition, Fra-1 and Fra-2 are involved both in the G0-to-G1 transition and in asynchronous growth.
Conformational activation of a basic helix-loop-helix protein (MyoD1) by the C-terminal region of murine HSP90 (HSP84).
Authors: Shaknovich R , Shue G , Kohtz D S
Published: October 2015

A murine cardiac lambda gt11 expression library was screened with an amphipathic helix antibody, and a recombinant representing the C-terminal 194 residues of murine HSP90 (HSP84) was cloned. Both recombinant and native HSP90s were then found to rapidly convert a basic helix-loop-helix protein (MyoD1) from an inactive to an active conformation, as assayed by sequence-specific DNA binding. The conversion process involves a transient interaction between HSP90 and MyoD1 and does not result in the formation of a stable tertiary complex. Conversion does not require ATP and occurs stoichiometrically in a dose-dependent fashion. HSP90 is an abundant, ubiquitous, and highly conserved protein present in most eukaryotic cells. These results provide direct evidence that HSP90 can affect the conformational structure of a DNA-binding protein.
Definition of the transcriptional activation domain of recombinant 43-kilodalton USF.
Authors: Kirschbaum B J , Pognonec P , Roeder R G
Published: October 2015

The cellular transcription factor USF is involved in the regulation of both cellular and viral genes and consists of 43- and 44-kDa polypeptides which independently show site-specific DNA binding. Cloning of the corresponding cDNA revealed that the 43-kDa polypeptide (USF43) is a member of the basic (B)-helix-loop-helix (HLH)-leucine zipper (LZ) family of proteins and provided a means for its functional dissection. Initial structure-function studies revealed that the HLH and LZ regions are both important for USF43 oligomerization and DNA binding. The studies presented here have focused on the determination of domains that contribute to transcriptional activation in vitro and show that (i) both a small region close to the N terminus and a region between residues 93 and 156 contribute strongly to transcriptional activation, (ii) full activation depends on the presence of both domains, (iii) the B-HLH-LZ region has no intrinsic activation potential but DNA binding is absolutely required for transcriptional activation, and (iv) the B-HLH-LZ region can be replaced by the Gal4 DNA binding domain without loss of activation potential.
A Drosophila rotund transcript expressed during spermatogenesis and imaginal disc morphogenesis encodes a protein which is similar to human Rac GTPase-activating (racGAP) proteins.
Authors: Agnel M , Röder L , Vola C , Griffin-Shea R
Published: October 2015

The rotund (rn) locus of Drosophila melanogaster at cytogenetic position 84D3,4 has been isolated and cloned on the basis of the mutant phenotype: an absence of structures in the subdistal regions of the appendages. The shortened appendages are the consequence of a localized cell death in the imaginal discs, precursors of the adult appendages. Physical characterization of the rn locus has demonstrated that it is relatively large, occupying a minimum of 50 kb. There are two major transcripts of 1.7 kb (m1.7) and 5.3 kb (m5.3). We present here the sequence analysis of m1.7 and its putative product, rnprot1.7, and show that rnprot1.7 is similar to the product of the human n-chimaerin gene, which is expressed in brain and testes. Recently, the GAP activity of n-chimaerin was demonstrated and shown to be specific for the Rac subfamily of the Ras oncoproteins. The Rac proteins have been implicated in the regulation of secretory processes. In addition to being expressed in the imaginal discs, the m1.7 racGAP transcript was detected in developmentally specific germ line cells of the testes, the primary spermatocytes.
Sodium butyrate inhibits myogenesis by interfering with the transcriptional activation function of MyoD and myogenin.
Authors: Johnston L A , Tapscott S J , Eisen H
Published: October 2015

Sodium butyrate reversibly inhibits muscle differentiation and blocks the expression of many muscle-specific genes in both proliferating myoblasts and differentiated myotubes. We investigated the role of the basic helix-loop-helix (bHLH) myogenic determinator proteins MyoD and myogenin in this inhibition. Our data suggest that both MyoD and myogenin are not able to function as transcriptional activators in the presence of butyrate, although both apparently retain the ability to bind DNA. Transcription of MyoD itself is extinguished in butyrate-treated myoblasts and myotubes, an effect that may be due to the inability of MyoD to autoactivate its own transcription. We present evidence that the HLH region of MyoD is essential for butyrate inhibition of MyoD. In contrast to MyoD and myogenin, butyrate does not inhibit the ubiquitous basic HLH protein E2-5 from functioning as a transcriptional activator.
p53 is covalently linked to 5.8S rRNA.
Authors: Fontoura B M , Sorokina E A , David E , Carroll R B
Published: October 2015

We report here the isolation and identification of the RNA specifically immunoprecipitated and covalently linked to the tumor suppressor gene product p53. After treatment with proteinase K, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band of p53 yields a single, discrete 157-nucleotide RNA, which was cloned, sequenced, and identified as 5.8S rRNA. 5.8S rRNA was obtained only after proteolysis of the p53 SDS-PAGE band. Free 5.8S rRNA did not comigrate with p53 in SDS-PAGE. This RNA was only immunoprecipitated from cells containing p53. Protein-free RNA obtained by proteolysis of the p53 band hybridized to the single-stranded DNA vector containing the antisense sequence of 5.8S rRNA. The covalence of the p53-5.8S rRNA linkage was demonstrated by the following findings: (i) p53 and the linked 5.8S rRNA comigrated in SDS-PAGE; (ii) only after treatment of the p53-RNA complex with proteinase K did the 5.8S rRNA migrate differently from p53-linked 5.8S rRNA; and (iii) this isolated RNA was found linked to phosphoserine, presumably at the 5' end. Covalent linkage to the single, specific RNA suggests that p53 may be involved in regulating the expression or function of 5.8S rRNA.
Tumorigenicity of the met proto-oncogene and the gene for hepatocyte growth factor.
Authors: Rong S , Bodescot M , Blair D , Dunn J , Nakamura T , Mizuno K , Park M , Chan A , Aaronson S , Vande Woude G F
Published: October 2015

The met proto-oncogene is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). It was previously shown that, like the oncogenic tpr-met, the mouse met proto-oncogene transforms NIH 3T3 cells. We have established NIH 3T3 cells stably expressing both human (Methu) and mouse (Metmu) met proto-oncogene products. The protein products are properly processed and appear on the cell surface. NIH 3T3 cells express endogenous mouse HGF/SF mRNA, suggesting an autocrine activation mechanism for transformation by Metmu. However, the tumor-forming activity of Methu in NIH 3T3 cells is very low compared with that of Metmu, but efficient tumorigenesis occurs when Methu and HGF/SFhu are coexpressed. These results are consistent with an autocrine transformation mechanism and suggest further that the endogenous murine factor inefficiently activates the tumorigenic potential of Methu. The tumorigenicity observed with reciprocal chimeric human and mouse receptors that exchange external ligand-binding domains supports this conclusion. We also show that HGF/SFhu expressed in NIH 3T3 cells produces tumors in nude mice.
TFIIA induces conformational changes in TFIID via interactions with the basic repeat.
Authors: Lee D K , DeJong J , Hashimoto S , Horikoshi M , Roeder R G
Published: October 2015

DNA-binding studies with Saccharomyces cerevisiae TFIID point mutants indicated that TFIIA interacts with the basic repeat region of TFIID and induces structural changes. The latter was shown by the ability of TFIIA to compensate for TFIID point mutants defective for DNA binding. Interaction with TFIIA also rendered TFIID binding temperature independent, thus mimicking the effect of removing the nonconserved N terminus of TFIID. In addition, N-terminal truncation of the TFIID point mutants defective for DNA binding mimicked the ability of TFIIA to restore DNA binding of those mutants. Taken together, these results suggest that TFIIA enhances TFIID binding to DNA by eliminating an otherwise inhibitory effect of the nonconserved N terminus of TFIID. Furthermore, analyses of TFIID contact points on DNA and binding studies with TATA-containing oligonucleotide probes showed that TFIIA decreases the effect of sequences flanking the adenovirus major late TATA element on TFIID binding to DNA, suggesting a possible role of TFIIA in allowing TFIID to recognize a wider variety of promoters.
Characterization of a new tissue-specific transcription factor binding to the simian virus 40 enhancer TC-II (NF-kappa B) element.
Authors: Lattion A L , Espel E , Reichenbach P , Fromental C , Bucher P , Israël A , Baeuerle P , Rice N R , Nabholz M
Published: October 2015

We have biochemically and functionally characterized a new transcription factor, NP-TCII, which is present in nuclei from unstimulated T and B lymphocytes but is not found in nonhematopoietic cells. This factor has a DNA-binding specificity similar to that of NF-kappa B but is unrelated to this or other Rel proteins by functional and biochemical criteria. It can also be distinguished from other previously described lymphocyte-specific DNA-binding proteins.
Interactions between double-stranded RNA regulators and the protein kinase DAI.
Authors: Manche L , Green S R , Schmedt C , Mathews M B
Published: October 2015

The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.
A Raf-1-related p110 polypeptide associates with the CD4-p56lck complex in T cells.
Authors: Prasad K V , Rudd C E
Published: October 2015

The CD4 and CD8 antigens on T cells have been shown to associate with the Src family member p56lck and a GTP-binding protein, p32. The identification of receptor interactions with intracellular mediators is essential in the elucidation of downstream signals mediated by engagement of these receptor complexes. In this study, we report the detection of an additional 110-kDa polypeptide (p110) associated with the CD4-p56lck complex in human peripheral blood T lymphocytes and leukemic T-cell lines. p110 bound preferentially to CD4-p56lck as an assembled complex and poorly, if at all, to the individual components. p110 was recognized directly by an antiserum to the C-terminal region of the serine/threonine kinase Raf-1 and is related to a p110 polypeptide detected in anti-Raf-1 immunoprecipitates. Despite its association with the CD4-p56lck complex, p110 was found to be phosphorylated predominantly on serine residues. Furthermore, phorbol ester treatment of cells resulted in a transient increase in the detection of p110 associated with CD4-p56lck, concomitant with the modulation of CD4-p56lck from the cell surface. This Raf-1-related p110 is therefore likely to play a role in signals generated from the CD4-p56lck complex. p110 may serve as a bridge between the CD4-p56lck complex and the serine/threonine kinase pathways of T-cell activation.
Specific changes of Ras GTPase-activating protein (GAP) and a GAP-associated p62 protein during calcium-induced keratinocyte differentiation.
Authors: Filvaroff E , Calautti E , McCormick F , Dotto G P
Published: October 2015

Induction of tyrosine phosphorylation occurs as an early and specific event in keratinocyte differentiation. A set of tyrosine-phosphorylated substrates which transduce mitogenic signals by tyrosine kinases has previously been identified. We show here that of these substrates, the Ras GTPase-activating protein, GAP, is specifically affected during calcium-induced keratinocyte differentiation. As early as 10 min after calcium addition to cultured primary mouse keratinocytes, GAP associates with tyrosine-phosphorylated proteins and translocates to the membrane. In addition, a GAP-associated protein of approximately 62 kDa (p62) becomes rapidly and heavily tyrosine phosphorylated in both membrane and cytosolic fractions. This protein corresponds to the major tyrosine-phosphorylated protein that is induced in differentiating keratinocytes as early as 5 min after calcium addition. p62 phosphorylation was not observed after exposure of these cells to epidermal growth factor, phorbol ester, or transforming growth factor beta. In contrast, PLC gamma and P13K were tyrosine phosphorylated after epidermal growth factor, but not calcium, stimulation. Thus, changes of Ras GAP and an associated p62 protein occur as early and specific events in keratinocyte differentiation and appear to involve a calcium-induced tyrosine kinase.