bluuebunny/crossref_metadata_2025 huggingface dataset:
Cotransformation and gene targeting in mouse embryonic stem cells.
Authors: Reid L H , Shesely E G , Kim H S , Smithies O
Published: October 2015

We have investigated cotransformation in mammalian cells and its potential for identifying cells that have been modified by gene targeting. Selectable genes on separate DNA fragments were simultaneously introduced into cells by coelectroporation. When the introduced fragments were scored for random integration, 75% of the transformed cells integrated both fragments within the genome of the same cell. When one of the cointroduced fragments was scored for integration at a specific locus by gene targeting, only 4% of the targeted cells cointegrated the second fragment. Apparently, cells that have been modified by gene targeting with one DNA fragment rarely incorporate a second DNA fragment. Despite this limitation, we were able to use the cotransformation protocol to identify targeted cells by screening populations of colonies that had been transformed with a cointroduced selectable gene. When hypoxanthine phosphoribosyltransferase (hprt) targeting DNA was coelectroporated with a selectable neomycin phosphotransferase (neo) gene into embryonic stem (ES) cells, hprt-targeted colonies were isolated from the population of neo transformants at a frequency of 1 per 70 G418-resistant colonies. In parallel experiments with the same targeting construct, hprt-targeted cells were found at a frequency of 1 per 5,500 nonselected colonies. Thus, an 80-fold enrichment for targeted cells was observed within the population of colonies transformed with the cointroduced DNA compared with the population of nonselected colonies. This enrichment for targeted cells after cotransformation should be useful in the isolation of colonies that contain targeted but nonselectable gene alterations.
Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor delta gene enhancer.
Authors: Ko L J , Yamamoto M , Leonard M W , George K M , Ting P , Engel J D
Published: October 2015

A family of transcriptional activators has recently been identified in chickens; these transcriptional activators recognize a common consensus motif (WGATAR) through a conserved C4 zinc finger DNA-binding domain. One of the members of this multigene family, cGATA-3, is most abundantly expressed in the T-lymphocyte cell lineage. Analysis of human and murine GATA-3 factors shows a striking degree of amino acid sequence identity and similar patterns of tissue specificity of expression in these three organisms. The murine and human factors are abundantly expressed in a variety of human and murine T-cell lines and can activate transcription through a tissue-specific GATA-binding site identified within the human T-cell receptor delta gene enhancer. We infer that the murine and human GATA-3 proteins play a central and highly conserved role in vertebrate T-cell-specific transcriptional regulation.
Ras GTPase-activating protein physically associates with mitogenically active phospholipids.
Authors: Tsai M H , Roudebush M , Dobrowolski S , Yu C L , Gibbs J B , Stacey D W
Published: October 2015

The physical interaction between GTPase-activating protein (GAP) and lipids has been characterized by two separate analyses. First, bacterially synthesized GAP molecules were found to associate with detergent-mixed micelles containing arachidonic but not with those containing arachidic acid. This association was detected by a faster elution time during molecular exclusion chromatography. Second, GAP molecules within a crude cellular lysate were specifically retained by a column on which certain lipids had been immobilized. The lipids able to retain GAP on such columns were identical to those which were shown previously to be most active in blocking GAP activity. The association between lipids and GAP was dependent upon magnesium ions. Lipids unable to inhibit GAP activity were also unable to physically associate with GAP. The tight association of GAP with these lipids was predicted by and helps to rationalize their ability to inhibit GAP activity.
Phosphorylation of eukaryotic translation initiation factor 4E is increased in Src-transformed cell lines.
Authors: Frederickson R M , Montine K S , Sonenberg N
Published: October 2015

Eukaryotic initiation factor 4F (eIF-4F) is a three-subunit complex that binds the 5' cap structure (m7GpppX, where X is any nucleotide) of eukaryotic mRNAs. This factor facilitates ribosome binding by unwinding the secondary structure in the mRNA 5' noncoding region. The limiting component of the 4F complex is believed to be the 24-kDa cap-binding phosphoprotein, eIF-4E. In this report, we describe the phosphorylation of eIF-4E in response to expression of the tyrosine kinase oncoproteins pp60v-src and pp60c-src527F. The results suggest that eIF-4E functions as a downstream target of the phosphorylation cascade induced by tyrosine-specific protein kinases as well as by effectors of the mitogenic response.
Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae.
Authors: Schnier J , Schwelberger H G , Smit-McBride Z , Kang H A , Hershey J W
Published: October 2015

Translation intitiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond. One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A. To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae. Two genes named TIF51A and TIF51B were cloned and sequenced. The two yeast proteins are closely related, sharing 90% sequence identity, and each is ca. 63% identical to the human protein. The purified protein expressed from the TIF51A gene substitutes for HeLa eIF-5A in the mammalian methionyl-puromycin synthesis assay. Strains lacking the A form of eIF-5A, constructed by disruption of TIF51A with LEU2, grow slowly, whereas strains lacking the B form, in which HIS3 was used to disrupt TIF51B, show no growth rate phenotype. However, strains with both TIF51A and TIF51B disrupted are not viable, indicating that eIF-5a is essential for cell growth in yeast cells. Northern (RNA) blot analysis shows two mRNA species, a larger mRNA (0.9 kb) transcribed from TIF51A and a smaller mRNA (0.8 kb) encoded by TIF51B. Under the aerobic growth conditions of this study, the 0.8-kb TIF51B transcript is not detected in the wild-type strain and is expressed only when TIF51A is disrupted. The TIF51A gene was altered by site-directed mutagenesis at the site of hypusination by changing the Lys codon to that for Arg, thereby producing a stable protein that retains the positive charge but is not modified to the hypusine derivative. The plasmid shuffle technique was used to replace the wild-type gene with the mutant form, resulting in failure of the yeast cells to grow. This result indicates that hypusine very likely is required for the vital in vivo function of eIF-5A and suggests a precise, essential role for the polyamine spermidine in cell metabolism.
In vitro analysis of the tissue plasminogen activator promoter reveals a GC box-binding activity present in murine brain but undetectable in kidney and liver.
Authors: Pecorino L T , Darrow A L , Strickland S
Published: October 2015

Tissue plasminogen activator (t-PA) mRNA levels are high in murine brain, lower in kidney, and undetectable in liver. Differences in t-PA mRNA levels are regulated in part at the transcriptional level. Brain, kidney, and liver nuclear extracts direct regulated transcription from the murine t-PA promoter in a manner that reflects the relative levels of t-PA gene expression in these tissues in vivo. Analysis of mutants has defined two GC box motifs as important elements for regulated transcription in vitro. Upon investigation of protein-DNA binding, we detected an activity in brain extracts which was not detected in kidney or liver extracts. An Sp1-like factor also binds to this region in all three tissue types. DNA interference experiments show that the brain-enriched binding activity and the Sp1-like factor contact the same GC-rich sequences. These studies provide additional evidence that brain-enriched DNA-binding activities can interact with sequences also recognized by ubiquitous transcription factors.
Translational control of discoidin lectin expression in drsA suppressor mutants of Dictyostelium discoideum.
Authors: Alexander S , Leone S , Ostermeyer E
Published: October 2015

Genetic analysis in Dictyostelium discoideum has identified regulatory genes which control the developmental expression of the discoidin lectin multigene family. Among these, the drsA mutation is a dominant second-site suppressor of another mutation, disB, which has the discoidinless phenotype. We now demonstrate a novel mechanism by which the drsA allele exerts its suppressive effect on the disB mutation. Interestingly, drsA does not merely bypass the disB mutation and restore the wild-type pattern of lectin expression. Rather, drsA mutant cells have high levels of discoidin lectin synthesis during growth but do not express lectins during aggregation. In contrast, wild-type cells only express lectin protein during the aggregation period of development. Phenocopies of the drsA mutation show a pattern of discoidin expression similar to that seen in the bona fide mutant. These data suggest that there may be a mechanism of negative feedback, resulting from the high levels of discoidin lectin made during growth, which inhibits further discoidin lectin expression during development. Northern (RNA) analysis of developing drsA mutant cells shows that these cells contain high levels of discoidin mRNA, although no discoidin lectin protein is being translated from these messages. Therefore, expression of the discoidin gene family can be controlled at the level of translation as well as transcription.
The human CCG1 gene, essential for progression of the G1 phase, encodes a 210-kilodalton nuclear DNA-binding protein.
Authors: Sekiguchi T , Nohiro Y , Nakamura Y , Hisamoto N , Nishimoto T
Published: October 2015

The human CCG1 gene complements tsBN462, a temperature-sensitive G1 mutant of the BHK21 cell line. The previously cloned cDNA turned out to be a truncated form of the actual CCG1 cDNA. The newly cloned CCG1 cDNA was 6.0 kb and encoded a protein with a molecular mass of 210 kDa. Using an antibody to a predicted peptide from the CCG1 protein, a protein with a molecular mass of over 200 kDa was identified in human, monkey, and hamster cell lines. In the newly defined C-terminal region, an acidic domain was found. It contained four consensus target sequences for casein kinase II and was phosphorylated by this enzyme in vitro. However, this C-terminal region was not required to complement tsBN462 mutation since the region encoding the C-terminal part was frequently missing in complemented clones derived by DNA-mediated gene transfer. CCG1 contains a sequence similar to the putative DNA-binding domain of HMG1 in addition to the previously detected amino acid sequences common in nuclear proteins, such as a proline cluster and a nuclear translocation signal. Consistent with these predictions, CCG1 was present in nuclei, possessed DNA-binding activity, and was eluted with similar concentrations of salt, 0.3 to 0.4 M NaCl either from isolated nuclei or from a DNA-cellulose column.
Developmental regulation of the alpha-mannosidase gene in Dictyostelium discoideum: control is at the level of transcription and is affected by cell density.
Authors: Schatzle J , Rathi A , Clarke M , Cardelli J A
Published: October 2015

In Dictyostelium discoideum, there is a group of genes that are expressed following starvation and when exponentially growing cells reach high densities. We have examined the expression of one of these genes, alpha-mannosidase. Using an alpha-mannosidase cDNA probe in Northern (RNA) blot analysis, we have shown that the previously observed increase in alpha-mannosidase enzyme-specific activity during development is due to an increase in the levels of alpha-mannosidase mRNA. mRNA levels reach a maximum by 8 h of development and then begin to decline by 14 to 22 h. Using nuclear run-on analysis, we have found that this gene is regulated at the level of transcription. We also examined the effects of cell-cell contacts, cyclic AMP levels, and protein synthesis on expression of this gene and found that they were not critical in regulating its expression. However, cell density did play a major role in the expression of alpha-mannosidase. High cell density or the presence of buffer conditioned by high-density cells was sufficient to induce expression of alpha-mannosidase, indicating that this is one of the prestarvation response genes. Finally, the alpha-mannosidase gene was not expressed in aggregation-negative mutant strain HMW 404.
The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.
Authors: Burd C G , Matunis E L , Dreyfuss G
Published: October 2015

The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).
Excess wild-type p53 blocks initiation and maintenance of simian virus 40 transformation.
Authors: Fukasawa K , Sakoulas G , Pollack R E , Chen S
Published: October 2015

Wild-type (wt) murine p53 has been tested for its ability to block and reverse the transforming effects of simian virus 40 (SV40) large T antigen. Established and precrisis mouse cells overexpressing exogenously introduced wt p53 became resistant to SV40 transformation. The introduction of excess wt p53 into SV40-transformed precrisis cells reverted their transformed phenotype. However, the phenotype of SV40-transformed established cells was not reverted by excess wt p53. We conclude that an antioncogenic action of wt p53 is exerted during SV40 transformation and that in precrisis cells, the antitransforming action of wt p53 can be exerted both at initiation and during the maintenance of transformation.
Poly(ADP-ribose) polymerase inhibitors suppress UV-induced human immunodeficiency virus type 1 gene expression at the posttranscriptional level.
Authors: Yamagoe S , Kohda T , Oishi M
Published: October 2015

Gene expression of human immunodeficiency virus type 1 (HIV-1) is induced not only by trans activation mediated through a gene product (tat) encoded by the virus but also by treatment of virus-carrying cells with DNA-damaging agents such as UV light. Employing an artificially constructed DNA in which the chloramphenicol acetyltransferase gene was placed under the control of the HIV-1 long terminal repeat, we analyzed the induction process in HeLa cells and found that inhibitors of poly(ADP-ribose) polymerase suppressed UV-induced HIV-1 gene expression but not tat-mediated expression. We also found that suppression occurs at the posttranscriptional level. These results indicate that HIV-1 gene expression is activated by at least two different mechanisms, one of which involves poly-ADP ribosylation. A possible new role of poly-ADP ribosylation in the regulation of specific gene expression is also discussed.
Alteration of homeobox gene expression by N-ras transformation of PA-1 human teratocarcinoma cells.
Authors: Buettner R , Yim S O , Hong Y S , Boncinelli E , Tainsky M A
Published: October 2015

We used a series of cell clones from a human teratocarcinoma cell line, PA-1, to study the effect of transformation by an activated N-ras oncogene on the expression of genes involved in retinoic acid (RA)-induced differentiation and growth regulation. Recently, it has been shown that expression of human HOX 2 genes is sequentially activated by RA beginning from Hox 2.9 at the 3' end of the HOX 2 cluster (A. Simeone, D. Acampora, L. Arcioni, P. W. Andrews, E. Boncinelli, and F. Mavilio, Nature [London] 346:763-766, 1990). We now report that six different genes of the cluster HOX 1 are sequentially induced by RA in a similar temporal pattern, beginning with genes at the 3' end of the cluster. However, in N-ras-transformed cell clones, RA-induced expression of these homeobox genes is delayed. Hox 1.4 and Hox 1.3, genes abundantly induced in nontransformed clones after 3 days of RA treatment, are expressed in N-ras-transformed cells only after 10 days of RA treatment. At this time, the cells' growth is arrested at very high density, and no differentiated morphologic characteristics are observed. Constitutive expression of a transfected Hox 1.4 gene under the control of a simian virus 40 promotor leads to differentiated cell morphology similar to that of the RA-induced phenotype and restores the growth-inhibitory effects of RA in N-ras-transformed cells. These observations provide evidence that enhanced proliferation in N-ras-transformed cells compromises teratocarcinoma cell differentiation by a mechanism that transiently suppresses homeobox gene induction and implies a central role for homeobox genes in RA-induced cell differentiation. We conclude that stimulation of a putative growth factor signal pathway, associated with ras-induced proliferation, transiently suppresses the induction of transcription factors functionally involved in cell growth and differentiation.
Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II.
Authors: Manak J R , Prywes R
Published: October 2015

Casein kinase II (CKII) phosphorylates the mammalian transcription factor serum response factor (SRF) on a serine residue(s) located within a region of the protein spanning amino acids 70 to 92, thereby enhancing its DNA-binding activity in vitro. We report here that serine 83 appears to be the residue phosphorylated by CKII but that three other serines in this region can also be involved in phosphorylation and the enhancement of DNA-binding activity. A mutant that contained glutamate residues in place of these serines had only low-level binding activity; however, when the serines were replaced with glutamates and further mutations were made that increased the negative charge of the region, the resulting mutant showed a constitutively high level of binding equal to that achieved by phosphorylation of wild-type SRF. We have investigated the mechanism by which phosphorylation of SRF increases its DNA-binding activity. We have ruled out the possibilities that phosphorylation affects SRF dimerization or relieves inhibition due to masking of the DNA-binding domain by an amino-terminal region of the protein. Rather, using partial proteolysis to probe SRF's structure, we find that the conformation of SRF's DNA-binding domain is altered by phosphorylation.
The myosin light chain enhancer and the skeletal actin promoter share a binding site for factors involved in muscle-specific gene expression.
Authors: Ernst H , Walsh K , Harrison C A , Rosenthal N
Published: October 2015

The myosin light chain (MLC) 1/3 enhancer (MLC enhancer), identified at the 3' end of the skeletal MLC1/3 locus, contains a sequence motif that is homologous to a protein-binding site of the skeletal muscle alpha-actin promoter. Gel shift, competition, and footprint assays demonstrated that a CArG motif in the MLC enhancer binds the proteins MAPF1 and MAPF2, previously identified as factors interacting with the muscle regulatory element of the skeletal alpha-actin promoter. Transient transfection assays with constructs containing the chloramphenicol acetyltransferase reporter gene demonstrated that a 115-bp subfragment of the MLC enhancer is able to exert promoter activity when provided with a silent nonmuscle TATA box. A point mutation at the MAPF1/2-binding site interferes with factor binding and abolishes the promoter activity of the 115-bp fragment. The observation that an oligonucleotide encompassing the MAPF1/2 site of the MLC enhancer alone cannot serve as a promoter element suggests that additional factor-binding sites are necessary for this function. The finding that MAPF1 and MAPF2 recognize similar sequence motifs in two muscle genes, simultaneously activated during muscle differentiation, implies that these factors may have a role in coordinating the activation of contractile protein gene expression during myogenesis.
Human alpha-globin genes demonstrate autonomous developmental regulation in transgenic mice.
Authors: Albitar M , Katsumata M , Liebhaber S A
Published: October 2015

Recent studies have demonstrated that transcriptional activation of the human adult beta-globin transgene in mice by coinsertion of the beta-globin cluster locus control region (beta-LCR) results in loss of its adult restricted pattern of expression. Normal developmental control is reestablished by coinsertion of the fetal gamma-globin transgene in cis to the adult beta-globin gene. To test the generality of this interdependence of two globin genes for their proper developmental control, we generated transgenic mice in which the human adult alpha-globin genes are transcriptionally activated by the beta-LCR either alone or in cis to their corresponding embryonic zeta-globin gene. In both cases, the human globin transgenes were expressed at the appropriate developmental period. In contrast to the beta-globin gene, developmental control of the human adult alpha-globin transgenes appears to be autonomous and maintained even when activated by an adjacent locus control region.
Secretion and transcriptional regulation of transforming growth factor-beta 3 during myogenesis.
Authors: Lafyatis R , Lechleider R , Roberts A B , Sporn M B
Published: October 2015

Transforming growth factor-beta 3 (TGF-beta 3) mRNA is differentially expressed in developing and mature mouse tissues, including high-level expression in developing and adult cardiac tissue. We show now that TGF-beta 3 mRNA is also expressed highly in skeletal muscle as well as in the mouse skeletal myoblast cell line C2C12. We also show that C2C12 cells secrete TGF-beta 3, and that this TGF-beta is able to inhibit C2C12 myoblast fusion after activation. In order to begin to understand how the TGF-beta 3 promoter is regulated in specific tissues during development, we therefore studied the regulation of TGF-beta 3 during myoblast fusion. After fusion of C2C12 cells into myotubes, TGF-beta 3 mRNA levels increased eightfold as a result of increased TGF-beta 3 transcription. TGF-beta 3 transcriptional regulation was studied in myoblasts and myotubes by transfection of chimeric TGF-beta 3/CAT promoter plasmids. Chloramphenicol acetyltransferase (CAT) activity was stimulated in myoblasts by several upstream regions between -301 and -47 of the TGF-beta 3 promoter and by the TGF-beta 3 5' untranslated region. CAT activity directed by the TGF-beta 3 promoter in myotubes was stimulated by a distinct upstream region located between -499 and -221. Therefore, the high level of TGF-beta 3 mRNA expression in muscle cells appears to be dependent on multiple regulatory events during different stages of myogenesis.
A retinoic acid-responsive element in the apolipoprotein AI gene distinguishes between two different retinoic acid response pathways.
Authors: Rottman J N , Widom R L , Nadal-Ginard B , Mahdavi V , Karathanasis S K
Published: October 2015

The gene coding for apolipoprotein AI, a plasma protein involved in the transport of cholesterol and other lipids in the plasma, is expressed predominantly in liver and intestine. Previous work in our laboratory has shown that hepatocyte-specific expression is determined by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located in the region -222 to -110 nucleotides upstream of the apolipoprotein AI gene transcription start site (+1). In this study, it was found that site A is a highly selective retinoic acid-responsive element (RARE) that responds preferentially to the recently identified retinoic acid receptor RXR alpha over the previously characterized retinoic acid receptors RAR alpha and RAR beta. Control experiments indicated that a RARE in the regulatory region of the laminin B1 gene responds preferentially to RAR alpha and RAR beta over RXR alpha, while a previously described palindromic thyroid hormone-responsive element responds similarly to all three of these receptors. Gel retardation experiments showed that the activity of these RAREs is concordant with receptor binding. These results indicate that different RAREs may play a fundamental role in defining distinctive retinoic acid cellular response pathways and suggest that retinoic acid response pathways mediated by RXR alpha play an important role in cholesterol and retinoid transport and metabolism.
Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix.
Authors: Dijkwel P A , Vaughn J P , Hamlin J L
Published: October 2015

Two complementary two-dimensional gel electrophoretic techniques have recently been developed that allow initiation sites to be mapped with relative precision in eukaryotic genomes at least as complex as those of yeast and Drosophila melanogaster. We reported the first application of these mapping methods to a mammalian genome in a study on the amplified dihydrofolate reductase (DHFR) domain of the methotrexate-resistant CHO cell line CHOC 400 (J.P. Vaughn, P.A. Dijkwel, and J.L. Hamlin, Cell 61:1075-1087, 1990). Our results suggested that in this 240-kb domain, initiation of nascent DNA strands occurs at many sites within a 30- to 35-kb zone mapping immediately downstream from the DHFR gene. In the course of these studies, it was necessary to develop methods to stabilize replication intermediates against branch migration and shear. This report describes these stabilization methods in detail and presents a new enrichment protocol that extends the neutral/neutral two-dimensional gel mapping method to single-copy loci in mammalian cells. Preliminary analysis of replication intermediates purified from CHO cells by this method suggests that DNA synthesis may initiate at many sites within a broad zone in the single-copy DHFR locus as well.
Overexpression of RPI1, a novel inhibitor of the yeast Ras-cyclic AMP pathway, down-regulates normal but not mutationally activated ras function.
Authors: Kim J H , Powers S
Published: October 2015

A high-copy-number plasmid genomic library was screened for genes that when overexpressed down-regulate Ras protein activity in Saccharomyces cerevisiae. We report on the structure and characterization of one such gene, RPI1, which potentially encodes a novel 46-kDa negative regulator of the Ras-cyclic AMP pathway. Three lines of evidence suggest that the RPI1 gene product operates upstream to negatively regulate the activity of normal but not mutationally activated Ras proteins: (i) overexpressed RPI1 lowers cyclic AMP levels in wild-type yeast cells but not in yeast cells carrying the RAS2Val-19 mutation, (ii) overexpressed RPI1 suppresses the heat shock sensitivity phenotype induced by overexpression of normal RAS2 but does not suppress the same phenotype induced by RAS2Val-19, and (iii) disruption of RPI1 results in a heat shock sensitivity phenotype which can be suppressed by mutations that lower normal Ras activity. Thus, RPI1 appears to encode an inhibitor of Ras activity that shares a common feature with Ras GTPase-activating proteins in that it fails to down-regulate activated RAS2Val-19 function. We present evidence that the down-regulatory effect of RPI1 requires the presence of one of the two Ras GTPase activators, IRA1 and IRA2.
In vivo assembly of yeast mitochondrial alpha-ketoglutarate dehydrogenase complex.
Authors: Repetto B , Tzagoloff A
Published: October 2015

The assembly of alpha-ketoglutarate dehydrogenase complex (KGDC) has been studied in wild-type Saccharomyces cerevisiae and in respiratory-deficient strains (pet) with mutations in KGD1 and KGD2, the structural genes for alpha-ketoglutarate dehydrogenase (KE1) and dihydrolipoyl transsuccinylase (KE2) components, respectively. Mutants unable to express KE1 or KE2 form partial complexes similar to those reported in earlier studies on the resolution and reconstitution of bacterial and mammalian KGDC. Thus mutants lacking KE1 assemble a high-molecular-weight subcomplex consisting of a KE2 core particle with bound dihydrolipoyl dehydrogenase (E3). Similarly, mitochondrial extracts of mutants lacking KE2 contain dimeric KE1 and E3. These components, however, are not associated with each other. The partial complexes detected in the mutants are capable of reconstituting normal KGDC when supplied with the missing subunit. Complete restoration of overall alpha-ketoglutarate dehydrogenase activity is achieved by mixing appropriate ratios of mitochondrial extracts from mutants deficient in KE1 and KE2. The reconstitution of enzymatic activity correlates with binding of KE1 to the KE2-E3 particle to form a complex with the same sedimentation properties as wild-type KGDC. Overexpression of KE2 relative to KE1 results in a preponderance of incompletely assembled complexes with substoichiometric contents of KE1. Formation of a complex with a full complement of KE1 therefore depends on a balanced output of KE1 and KE2 from their respective genes. Biochemical screens of a pet mutant collection have led to the identification of a new gene required for the expression of enzymatically active KGDC. Mitochondria of the mutant have all of the catalytic subunits of KGDC. Sedimentation analysis of these components indicates that while the mutant has a stable KE2-E3 subcomplex, the interaction of KE1 with KE2 core is much weaker in the mutant than in the wild type. The gene product responsible for this phenotype, therefore, appears to function at a late stage of assembly of KGDC, most likely by posttranslational modification of one of the subunits.
Displacement of Xenopus transcription factor IIIA from a 5S rRNA gene by a transcribing RNA polymerase.
Authors: Campbell F E , Setzer D R
Published: October 2015

In the absence of other components of the RNA polymerase III transcription machinery, transcription factor IIIA (TFIIIA) can be displaced from both strands of its DNA-binding site (the internal control region) on the somatic-type 5S rRNA gene of Xenopus borealis during transcription elongation by bacteriophage T7 RNA polymerase, regardless of which DNA strand is transcribed. Furthermore, substantial displacement is observed after the template has been transcribed only once. Since the complete 5S rRNA transcription complex has previously been shown to remain stably bound to the gene during repeated rounds of transcription by either RNA polymerase III or bacteriophage SP6 RNA polymerase, these results indicate that a factor(s) in addition to TFIIIA is required to create a complex that will remain stably associated with the template during transcription. Thus, transcription complex stability during passage of RNA polymerase cannot be explained solely on the basis of the DNA-binding properties of TFIIIA.
Intrachromosomal rearrangements fusing L-myc and rlf in small-cell lung cancer.
Authors: Mäkelä T P , Kere J , Winqvist R , Alitalo K
Published: October 2015

Chromosomal abnormalities affecting proto-oncogenes are frequently detected in human cancer. Oncogenes of the myc family are activated in several types of tumors as a result of gene amplification or chromosomal translocation. We have recently found the L-myc gene involved in a gene fusion in small-cell lung cancer (SCLC). This results in a chimeric protein with amino-terminal sequences from a novel gene named rif joined to L-myc. Here we present a preliminary structural characterization of the rlf-L-myc fusion gene, which has been found only in cells with an amplified L-myc gene. In addition, we have used somatic cell hybrids to assign the normal rlf locus to the same chromosome (chromosome 1) on which L-myc resides. Finally, we have been able to establish a physical linkage between rif and L-myc with pulsed-field gel electrophoresis. Our results demonstrate that normal rlf and L-myc genes are separated by less than 800 kb of DNA. Thus, the rlf-L-myc gene fusions are due to similar but not identical intrachromosomal rearrangements at 1p32. The presence of independent genetic lesions that cause the formation of identical chimeric rlf-L-myc proteins suggests a role for the fusion protein in the development of these tumors.
Striking similarities between the regulatory mechanisms governing yeast mating-type genes and mammalian major histocompatibility complex genes.
Authors: Weissman J D , Singer D S
Published: October 2015

Expression of a mammalian major histocompatibility complex (MHC) class I gene is in part regulated by a silencer DNA sequence element which binds a complex of silencer factors. This negative regulatory system is shown to be strikingly similar to the yeast alpha 2 mating-type repression system. A moderate DNA sequence homology exists between the MHC class I silencer DNA element and the yeast alpha 2 operator. Mammalian silencer factors specifically bind to the yeast alpha 2 operator DNA and also specifically interact with a yeast alpha 2-binding protein. Furthermore, the alpha 2 operator functions as a silencer element in mammalian cells when placed upstream of a MHC class I promoter.
RNA editing intermediates of cox2 transcripts in maize mitochondria.
Authors: Yang A J , Mulligan R M
Published: October 2015

Eighteen cytidines are changed to uridines in the coding sequence of transcripts for cytochrome c oxidase subunit 2 (cox2) in maize mitochondria. The temporal relationship of editing and splicing was examined in cox2 transcripts by sequence analysis of spliced and unspliced cDNAs. Cloned cDNAs of unspliced cox2 transcripts ranged from clones with no edited nucleotides to completely edited forms, while spliced cDNAs were nearly completely edited. Incompletely edited transcripts in the nascent pool of unspliced transcripts represent intermediates of the editing process. These results indicate that editing proceeds without a strong directional bias and suggest that RNA editing is a posttranscriptional process.
Interleukin 2- and polyomavirus middle T antigen-induced modification of phosphatidylinositol 3-kinase activity in activated T lymphocytes.
Authors: Augustine J A , Sutor S L , Abraham R T
Published: October 2015

Stimulation of activated T lymphocytes with interleukin 2 (IL-2) results in rapid increases in intracellular protein tyrosine phosphorylation. Both the identity of the protein tyrosine kinase (PTK) activated by IL-2 receptor ligation and the identities of the critical target proteins for this PTK remain largely undefined. In this article, we demonstrate that stimulation of activated murine or human T cells with IL-2 for 10 to 30 min induces two- to threefold increases in the level of phosphatidylinositol (PtdIns) 3-kinase activity present in antiphosphotyrosine (p-Tyr) antibody immunoprecipitates from these cells. Furthermore, substantial levels of PtdIns 3-kinase activity were coprecipitated from IL-2-deprived T cells by antibodies to the src-related PTK p59fyn. Cellular stimulation with IL-2 induced a two- to threefold increase in the level of p59fyn-associated PtdIns 3-kinase activity. To examine the effect of a constitutive increase in PtdIns 3-kinase activity on the growth factor responsiveness of activated T cells, murine CTLL-2 cells were transfected with a polyomavirus middle T antigen (MTAg) expression vector. Anti-p-Tyr and anti-p59fyn immunoprecipitates from MTAg-transfected CTLL-2 cells contained three- to sixfold higher levels of PtdIns 3-kinase activity than wild-type cells. Immune complex kinase assays revealed that MTAg expression concomitantly induced a constitutive threefold increase in the PTK activity of p59fyn in these cells. However, stable MTAg expression did not abrogate the dependence of CTLL-2 cells on exogenous IL-2 for continued growth and proliferation.
Synapsis, strand scission, and strand exchange induced by the FLP recombinase: analysis with half-FRT sites.
Authors: Amin A , Roca H , Luetke K , Sadowski P D
Published: October 2015

We have used a previously described cross-linking assay and half-FRT site substrates to examine the requirements for synapsis, strand exchange, and strand scission. The cross-linking assay showed that the minimum functional FRT site needed for synapsis contains two inverted FLP-binding elements surrounding an 8-bp core. This indicates that four FLP molecules interact with four binding elements in a synaptic complex. The analysis using half-sites showed that the enzyme can catalyze efficient strand exchange between a half-site and the intact FRT site. The reaction occurred only if the half-site had at least 2 bp but no more than 4 bp of the adjoining core sequence. The exchange occurred exclusively at the regions of limited core homology between the respective half-site and the FRT site. The absence of strand exchange between an intact site and a half-site bearing regions of core nonhomology indicates that 1 bp of homology is not sufficient for the formation of stable recombinant structures. Qian et al. (X.-H. Qian, R. B. Inman, and M. M. Cox, J. Biol. Chem. 265:21779-21788, 1990) have recently shown that the FLP protein can catalyze the formation of dimeric, trimeric, and tetrameric complexes with half-FRT sites. We show that only half-sites that contained at least 2 bp of adjacent core could form stable dimer products and be cleaved by the enzyme. Stable dimers were formed between a noncleavable half-site and a cleavable half-site, suggesting that only a single cleavage event is needed for the formation of the dimer.
Telomerase is processive.
Authors: Greider C W
Published: October 2015

Telomerase synthesizes tandem repeats of the sequence d(TTGGGG) onto input d(TTGGGG)n primer oligonucleotides (C. W. Greider and E. H. Blackburn, Cell 43:405-413). An intrinsic RNA component of the enzyme provides the template for d(TTGGGG)n repeat synthesis [C. W. Greider and E. H. Blackburn, Nature (London) 337:331-337, 1989; G.-L. Lu, J. D. Bradley, L. D. Attardi, and E. H. Blackburn, Nature (London) 344:126-132, 1990]. In a typical reaction, products greater than 2,000 nucleotides were synthesized in 60 min. Dilution and primer challenge experiments showed that these long products were synthesized processively. The apparent processivity was not due to a higher affinity of the enzyme for long d(TTGGGG) products over the shorter competitors. The degree of processivity was quantitated; telomerase synthesized approximately 520 nucleotides before half of the enzyme had dissociated. After dissociating, telomerase reinitiated d(TTGGGG)n synthesis on new primer oligonucleotides. The products from a telomerase reaction have a characteristic 6-nucleotide banding pattern (C. W. Greider and E. H. Blackburn, Cell 51:887-898, 1987). A strong pause in the reaction occurs after the addition of the first G in the sequence d(TTGGGG). Both the processivity and the banding pattern analysis imply that in the elongation mechanism there must be a translocation step after the 9 nucleotides of internal template RNA have been copied to the extreme 5' end.
5'-flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells.
Authors: Glauber J G , Wandersee N J , Little J A , Ginder G D
Published: October 2015

A stable transfection assay was used to test the mechanism by which embryonic globin gene transcription is stimulated in adult erythroid cells exposed to butyric acid and its analogs. To test the appropriate expression and inducibility of chicken globin genes in murine erythroleukemia (MEL) cells, an adult chicken beta-globin gene construct was stably transfected. The chicken beta-globin gene was found to be coregulated with the endogenous adult mouse alpha-globin gene following induction of erythroid differentiation of the transfected MEL cells by incubation with either 2% dimethyl sulfoxide (DMSO) or 1 mM sodium butyrate (NaB). In contrast, a stably transfected embryonic chicken beta-type globin gene, rho, was downregulated during DMSO-induced MEL cell differentiation. However, incubation with NaB, which induces MEL cell differentiation, or alpha-amino butyrate, which does not induce differentiation of MEL cells, resulted in markedly increased levels of transcription from the stably transfected rho gene. Analysis of histone modification showed that induction of rho gene expression was not correlated with increased bulk histone acetylation. A region of 5'-flanking sequence extending from -569 to -725 bp upstream of the rho gene cap site was found to be required for both downregulation of rho gene expression during DMSO-induced differentiation and upregulation by treatment with NaB or alpha-amino butyrate. These data are support for a novel mechanism by which butyrate compounds can alter cellular gene expression through specific DNA sequences. The results reported here are also evidence that 5'-flanking sequences are involved in the suppression of embryonic globin gene expression in terminally differentiated adult erythroid cells.
Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.
Authors: Fien K , Stillman B
Published: October 2015

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.
The mouse Wnt-1 gene can act via a paracrine mechanism in transformation of mammary epithelial cells.
Authors: Jue S F , Bradley R S , Rudnicki J A , Varmus H E , Brown A M
Published: October 2015

The mouse Wnt-1 gene plays an essential role in fetal brain development and can contribute to tumorigenesis when activated aberrantly in the mammary gland. The gene encodes secretory glycoproteins associated with the extracellular or pericellular matrix, and it has been proposed that Wnt-1, as well as its Drosophila homolog wingless, may function in intercellular signalling. We show here that fibroblasts expressing Wnt-1 protein, although not transformed themselves, are able to elicit morphological transformation of neighboring C57MG mammary epithelial cells in coculture experiments. Heparin inhibits this effect, possibly by displacing Wnt-1 protein from its normal site of action. Our results indicate that the Wnt-1 gene can act via a paracrine mechanism in cell culture and strongly support the notion that in vivo the gene may function in cell-to-cell communication.
Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice.
Authors: Tal M , Thorens B , Surana M , Fleischer N , Lodish H F , Hanahan D , Efrat S
Published: October 2015

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.