bluuebunny/crossref_metadata_2025 huggingface dataset:
MOD5 translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA.
Authors: Gillman E C , Slusher L B , Martin N C , Hopper A K
Published: October 2015

MOD5 is one of several genes that code for enzymes found in mitochondria and another cellular compartment. Like other such genes, it contains two in-frame ATGs that could be used to produce two proteins, differing from each other by an amino-terminal extension. Certain other genes produce heterogeneous mRNAs with some 5' ends falling upstream of the longest open reading frame and some 5' ends falling between the first and second ATGs. In these cases, selection of transcription start sites appears to play a significant role in translation start site selection. MOD5, in contrast, produces mRNAs with 5' ends that all fall upstream of both ATGs. To determine how MOD5 encodes isozymes that are located in different cellular compartments and to determine whether they differ in structure, we constructed MOD5 and MOD5-COXIV fusions with mutations of the first, second, or both ATGs. The effect of these alterations on protein production, tRNA modification, and cellular location was assessed. Both the first and second ATGs are used to produce MOD5 protein in vivo, but only the long form of the protein is imported into mitochondria. Thus, the first 11 amino acids present on the amino-terminal extended protein are necessary for mitochondrial import. Surprisingly, this extension does not promote complete import of the long form of the protein, but rather a functional pool of the extended protein remains in the cytoplasm. The amino-terminal extension is also unusual because it is probably not proteolytically removed upon import and therefore does not constitute part of a mitochondrial presequence.
Genetic evidence that different functional domains of the PET54 gene product facilitate expression of the mitochondrial genes COX1 and COX3 in Saccharomyces cerevisiae.
Authors: Valencik M L , McEwen J E
Published: October 2015

Expression of the yeast mitochondrial genes COX1 and COX3, which encode subunits I and III of cytochrome oxidase, respectively, is controlled by a common nuclear-encoded trans-acting factor. This protein, encoded by the PET54 gene, controls expression of COX1 at the level of RNA splicing and COX3 at the level of mRNA translation. While the steps of COX1 and COX3 gene expression affected by the PET54 gene product are different, it is possible that the PET54 protein is monofunctional and affects expression of each gene by a single mechanism, such as modulation of RNA secondary structure. The goal of this study was to address whether the PET54 protein is monofunctional or multifunctional with respect to its role in COX1 and COX3 gene expression. Ten insertion mutations, which each resulted in the in-frame addition of four amino acids within the PET54 polypeptide, were generated, and the resulting mutants were characterized for respiration phenotype and mitochondrial gene expression. Five of the ten mutants were respiration deficient. Two of these five mutants were defective in expression of COX3 but not in expression of COX1, while two other mutants had the opposite phenotype (primarily defective in expression of COX1). The fifth mutant was equally defective in expression of both genes. These results demonstrate that the two functions of PET54 are genetically separable and support the idea that the PET54 protein is multifunctional.
Stereospecific alignment of the X and Y elements is required for major histocompatibility complex class II DRA promoter function.
Authors: Vilen B J , Cogswell J P , Ting J P
Published: October 2015

The regulatory mechanisms controlling expression of the major histocompatibility complex (MHC) class II genes involve several cis-acting DNA elements, including the X and Y boxes. These two elements are conserved within all murine and human class II genes and are required for accurate and efficient transcription from MHC class II promoters. Interestingly, the distance between the X and Y elements is also evolutionarily conserved at 18 to 20 bp. To investigate the function of the invariant spacing in the human MHC class II gene, HLA-DRA, we constructed a series of spacing mutants which alters the distance between the X and Y elements by integral and half-integral turns of the DNA helix. Transient transfection of the spacing constructs into Raji cells revealed that inserting integral turns of the DNA helix (+20 and +10 bp) did not reduce promoter activity, while inserting or deleting half-integral turns of the DNA helix (+15, +5, and -5 bp) drastically reduced promoter activity. The loss of promoter function in these half-integral turn constructs was due neither to the inability of the X and Y elements to bind proteins nor to improper binding of the X- and Y-box-binding proteins. These data indicate that the X and Y elements must be aligned on the same side of the DNA helix to ensure normal function. This requirement for stereospecific alignment strongly suggests that the X- and Y-box-binding proteins either interact directly or are components of a larger transcription complex which assembles on one face of the DNA double helix.
Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.
Authors: Engler D A , Hauser M R , Cook J S , Niyogi S K
Published: October 2015

The third disulfide loop (amino acids 33 to 42) of human epidermal growth factor (hEGF) encompasses the region of highest amino acid conservation among all of the EGF-like family of molecules. The importance of some of these highly conserved residues for the maintenance of biological activity, especially the aromatic amino acid tyrosine at position 37, has until now been considered essential on the basis of previous studies with the EGF-like molecule transforming growth factor alpha. Variants at the Tyr-37 position of hEGF were constructed by site-directed mutagenesis. The substituting amino acids were phenylalanine, histidine, serine, alanine, aspartic acid, arginine, and glycine. The variants were tested for their ability to competitively displace native [125I]hEGF from its receptor and to stimulate the protein-tyrosine kinase activity of the receptor; the order of efficacy of substituting amino acids was Phe greater than His greater than Ser greater than Ala greater than Asp greater than Arg greater than Gly in both assays. All were effective, with no or only moderate reduction in potency, in stimulating the incorporation of [3H]thymidine into acid-insoluble material of quiescent mouse A31 cells. Only Tyr-37----Ala, Tyr-37----Arg and Tyr-37----Gly were slightly less potent in the cell assay. Thus, neither tyrosine nor another aromatic amino acid at position 37 in hEGF is essential for full biological activity.
Heterodimers of myogenic helix-loop-helix regulatory factors and E12 bind a complex element governing myogenic induction of the avian cardiac alpha-actin promoter.
Authors: French B A , Chow K L , Olson E N , Schwartz R J
Published: October 2015

Recent studies have shown that two genes regulating myogenesis (MyoD and myogenin) are coexpressed with cardiac alpha-actin during early stages of skeletal muscle development. Myogenin and MyoD are members of a family of regulatory proteins which share a helix-loop-helix (HLH) motif required for dimerization and DNA binding. Myogenin and MyoD form heterodimers with the ubiquitous HLH protein E12 which bind cis-acting DNA elements that have an E box (CANNTG) at their core. E boxes are present in the control regions of numerous muscle-specific genes, although their functional importance in regulating many of these genes has not yet been evaluated. In this report we examine the possibility that myogenin (or MyoD) directly transactivates the cardiac alpha-actin promoter. Heterodimers of myogenin and E12 (or MyoD and E12) specifically bound a restriction fragment extending from -200 to -103 relative to the start of cardiac alpha-actin transcription. Methylation interference footprints pinpointed the site of interaction to an E box immediately adjacent to a previously identified CArG box (CArG3). Site-directed mutations to the DNA-binding site revealed that either an intact E box or an intact CArG3 is required for induction of the cardiac alpha-actin promoter in myoblasts and for transactivation by myogenin in cotransfected fibroblasts. However, deletion and substitution experiments indicate that the complex E box/CArG3 element alone does not confer muscle-specific expression to a minimal promoter. These results suggest that direct and indirect pathways involving multiple cis-acting elements mediate the induction of the cardiac alpha-actin promoter by myogenin and MyoD.
An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro.
Authors: Brewer G
Published: October 2015

Transient expression of some proto-oncogenes, cytokines, and transcription factors occurs as a cellular response to growth factors, 12-O-tetradecanoylphorbol-13-acetate, antigen stimulation, or inflammation. Expression of these genes is mediated in part by the rapid turnover of their mRNAs. A + U-rich elements in the 3' untranslated regions of these mRNAs serve as one recognition signal targeting the mRNAs for rapid degradation. I report the identification of a cytosolic factor that both binds to the proto-oncogene c-myc A + U-rich element and specifically destabilizes c-myc mRNA in a cell-free mRNA decay system which reconstitutes mRNA decay processes found in cells. Proteinase K treatment of the factor abolishes its c-myc mRNA degradation activity without affecting its RNA-binding capacity. Thus, RNA substrate binding and degradation appear to be separable functions. These findings should aid in understanding how the cell selectively targets mRNAs for rapid turnover.
Characterization of VSG gene expression site promoters and promoter-associated DNA rearrangement events.
Authors: Gottesdiener K , Chung H M , Brown S D , Lee M G , Van der Ploeg L H
Published: October 2015

The expressed variant cell surface glycoprotein (VSG) gene of Trypanosoma brucei is located at the 3' end of a large, telomeric, polycistronic transcription unit or expression site. We show that the region 45 kb upstream of the VSG gene, in the expression site on a 1.5-Mb chromosome, contains at least two promoters that are arranged in tandem, directing the transcription of the expression site. DNA rearrangement events occur specifically, at inactivation of the expression site, and these events delete the most upstream transcribed region and replace it with a large array of simple-sequence DNA, leaving the downstream promoter intact. Because of the placement of simple-sequence DNA, the remaining downstream promoter now becomes structurally identical to previously described VSG promoters. The downstream promoter is repetitive in the genome, since it is present at several different expression sites. Restriction fragment length polymorphism mapping allows grouping of the expression sites into two families, those with and those without an upstream transcription unit, and the DNA rearrangement events convert the expression sites from one type to the other. Deletion of the upstream transcription unit also leads to the loss of several steady-state RNAs. The findings may indicate a role for promoter-associated DNA rearrangement events, and/or interactions between tandemly arranged promoters, in expression site transcriptional control.
Transcriptional role of a conserved GATA-1 site in the human epsilon-globin gene promoter.
Authors: Gong Q H , Stern J , Dean A
Published: October 2015

The epsilon-globin gene is the first of the human beta-like globin genes to be expressed during development. We have analyzed protein-DNA interactions in the epsilon-globin promoter region by DNase I footprinting and electrophoretic mobility shift experiments using nuclear extracts from K562 human erythroid cells and from nonerythroid HeLa cells. A restricted set of ubiquitous proteins, including Sp1, bound to regions of the promoter including the CACCC and CCAAT sites. Three interactions, at positions -213, -165, and +3 relative to the transcription start site, were erythroid specific and corresponded to binding of GATA-1, a transcription factor highly restricted to the erythroid lineage. Interestingly, the GATA-1 site at -165 has been conserved in the promoters of 10 mammalian embryonic globin genes. Point mutations demonstrate that GATA-1 binding to this site is necessary for interaction with an erythroid-specific enhancer but that in the absence of an enhancer, GATA-1 does not increase transcription.
Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1.
Authors: Tishkoff D X , Johnson A W , Kolodner R D
Published: October 2015

Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is parallel to the RAD50 pathway.
Both the 5' untranslated region and the sequences surrounding the start site contribute to efficient initiation of translation in vitro.
Authors: Falcone D , Andrews D W
Published: October 2015

The role of RNA sequences in the 5' leader region between the cap site and initiating AUG in mediating translation was examined in vitro. Hybrid mRNAs were synthesized in which the cognate leader sequence was replaced with either optimized or compromised leader sequences, and translational efficiency was measured for six different coding regions. Translation was most efficient with a leader containing the 5' untranslated region from Xenopus beta-globin and an optimized initiation sequence. Compared with the cognate leaders, this hybrid was observed to increase translation of the various coding regions as much as 300-fold. The translational efficiencies of the different coding regions also varied substantially. In contrast to earlier suggestions that increased leader efficiency results from higher affinity of the leader for a limiting factor, our experiments suggest that increased translation from the beta-globin hybrid leader sequence results from more rapid initiation of translation.
Mutational studies reveal a complex set of positive and negative control elements within the chicken vitellogenin II promoter.
Authors: Seal S N , Davis D L , Burch J B
Published: October 2015

The endogenous chicken vitellogenin II (VTGII) gene is transcribed exclusively in hepatocytes in response to estrogen. We previously identified two estrogen response elements (EREs) upstream of this gene. We now present an analysis of the VTGII promoter activated by these EREs in response to estrogen. Chimeric VTGII-CAT genes were cotransfected into LMH chicken hepatoma cells along with an estrogen receptor expression vector, and transient CAT expression was assayed after culturing the cells in the absence or presence of estrogen. An analysis of constructs bearing deletions downstream of the more proximal ERE indicated that promoter elements relevant to transcription in LMH cells extend to between -113 and -96. The relative importance of sequences within the VTGII promoter was examined by using 10 contiguous linker scanner mutations spanning the region from -117 to -24. Although most of these mutations compromised VTGII promoter function, one dramatically increased expression in LMH cells and also rendered the VTGII promoter capable of being activated by cis-linked EREs in fibroblasts cotransfected with an estrogen receptor expression vector. Gel retardation and DNase I footprinting assays revealed four factor-binding sites within this promoter. We demonstrate that three of these sites bind C/EBP, SP1, and USF (or related factors), respectively; the fourth site binds a factor that we denote TF-V beta. The biological relevance of these findings is suggested by the fact that three of these binding sites map to sites previously shown to be occupied in vivo in response to estrogen.
A single base pair dominates over the novel identity of an Escherichia coli tyrosine tRNA in Saccharomyces cerevisiae.
Authors: Trézéguet V , Edwards H , Schimmel P
Published: October 2015

The Escherichia coli su+3 tyrosine tRNA was shown recently to be a leucine-specific tRNA in Saccharomyces cerevisiae. This finding raises the possibility that some determinants for tRNA identity in E. coli may be different in S. cerevisiae. To investigate whether the fungal system is sensitive to the major determinant for alanine acceptance in E. coli, a single G3 . U70 base pair was introduced into the acceptor helix of the su+3 tyrosine tRNA. This substitution converts the identity of the E. coli suppressor in S. cerevisiae from leucine to alanine. Thus, as in E. coli, G3 . U70 is a strong determinant for alanine acceptance that can dominate over other features in a tRNA that might be recognized by alternative charging enzymes.
The rat leukocyte antigen MRC OX-44 is a member of a new family of cell surface proteins which appear to be involved in growth regulation.
Authors: Bellacosa A , Lazo P A , Bear S E , Tsichlis P N
Published: October 2015

Moloney murine leukemia virus (MoMuLV)-induced rat T-cell lymphomas express discrete 1.8-, 2.2-, and 4-kb mRNA transcripts hybridizing under conditions of reduced stringency to a probe derived from a region upstream of the first exon of the Tpl-1/Ets-1 gene. Screening a cDNA library from one rat T-cell lymphoma with this genomic probe yielded 15 cDNA clones which were derived from 10 different genes. One of these genes, defined by the cDNA clone pRcT7a, was expressed as a 1.8-kb mRNA transcript in spleen and thymus but not in other normal rat tissues. Expression of the gene defined by the pRcT7a cDNA clone in a series of MoMuLV-induced rat T-cell lymphomas showed a perfect correlation with the expression of the rat leukocyte antigen MRC OX-44. Because of this observation, the pRcT7a clone was sequenced and it was shown to identify a gene coding for a 219-amino-acid protein. The homology between pRcT7a and the Tpl-1 probe used for its detection mapped within the 3' untranslated region of the pRcT7a cDNA clone. The pRcT7a protein, which exhibits four putative transmembrane regions and three putative glycosylation sites, contains a region which is nearly identical in sequence to a peptide derived from the rat leukocyte antigen MRC OX-44. This finding suggested that the pRcT7a cDNA clone defines the gene coding for OX-44. To confirm this finding, a pRcT7a construct in the retrovirus vector pZipNeo was introduced into the OX-44- T-cell lymphoma line 2788. Immunostaining with the MRC OX-44 monoclonal antibody followed by flow cytometry revealed that following gene transfer, the 2788 cells became OX-44+. Sequence comparisons revealed that pRcT7a/MRC OX-44 is a member of a family of genes which includes the melanoma-specific antigen ME491; the human leukocyte antigen CD37; the protein TAPA-1, which is expressed on the surface of human T cells and appears to be involved in growth regulation; the human gastrointestinal tumor antigen CO-029; and the Schistosoma mansoni-associated antigen Sm23.
A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein.
Authors: Sasaki T , Kaibuchi K , Kabcenell A K , Novick P J , Takai Y
Published: October 2015

Evidence is accumulating that smg p25A, a small GTP-binding protein, may be involved in the regulated secretory processes of mammalian cells. The SEC4 protein is known to be required for constitutive secretion in yeast cells. We show here that the mammalian GDP dissociation inhibitor (GDI), which was identified by its action on smg p25A, is active on the yeast SEC4 protein in inhibiting the GDP/GTP exchange reaction and is capable of forming a complex with the GDP-bound form of the SEC4 protein but not with the GTP-bound form. These results together with our previous findings that smg p25A GDI is found in mammalian cells with both regulated and constitutive secretion types suggest that smg p25A GDI plays a role in both regulated and constitutive secretory processes, although smg p25A itself may be involved only in regulated secretory processes. These results also suggest that a GDI for the SEC4 protein is present in yeast cells.
Transformation mapping of the regulatory elements of the ecdysone-inducible P1 gene of Drosophila melanogaster.
Authors: Maschat F , Dubertret M L , Lepesant J A
Published: October 2015

The transcription of the P1 gene is induced by 20-hydroxyecdysone in fat bodies of third-instar larvae. Germ line transformation showed that sequences between -138 to +276 contain elements required for a qualitatively correct developmental and hormonal regulation of P1 transcription. Sequences from -138 to -68 are essential for this expression.
Effects of excess centromeres and excess telomeres on chromosome loss rates.
Authors: Runge K W , Wellinger R J , Zakian V A
Published: October 2015

The linear chromosomes of eukaryotes contain specialized structures to ensure their faithful replication and segregation to daughter cells. Two of these structures, centromeres and telomeres, are limited, respectively, to one and two copies per chromosome. It is possible that the proteins that interact with centromere and telomere DNA sequences are present in limiting amounts and could be competed away from the chromosomal copies of these elements by additional copies introduced on plasmids. We have introduced excess centromeres and telomeres into Saccharomyces cerevisiae and quantitated their effects on the rates of loss of chromosome III and chromosome VII by fluctuation analysis. We show that (i) 600 new telomeres have no effect on chromosome loss; (ii) an average of 25 extra centromere DNA sequences increase the rate of chromosome III loss from 0.4 x 10(-4) events per cell division to 1.3 x 10(-3) events per cell division; (iii) centromere DNA (CEN) sequences on circular vectors destabilize chromosomes more effectively than do CEN sequences on 15-kb linear vectors, and transcribed CEN sequences have no effect on chromosome stability. We discuss the different effects of extra centromere and telomere DNA sequences on chromosome stability in terms of how the cell recognizes these two chromosomal structures.
Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor.
Authors: Martinez E , Dusserre Y , Wahli W , Mermod N
Published: October 2015

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.
Alternative splicing generates isoforms of the met receptor tyrosine kinase which undergo differential processing.
Authors: Rodrigues G A , Naujokas M A , Park M
Published: October 2015

The met proto-oncogene is a member of the family of tyrosine kinase growth factor receptors. We describe the isolation and characterization of a cDNA clone (pOK) for the met receptor from a gastric carcinoma cell line. This clone differs from the published cDNA clone by the absence of 54 bp predicted to encode 18 amino acids in the extracellular domain. The pOK cDNA corresponds to the most abundant met RNA species of 8 kb expressed in human cell lines and tissue, and we show that there are in fact two 8-kb met receptor tyrosine kinase (RTK) isoforms that are generated by alternative splicing. This newly described met isoform when transiently expressed in COS cells encodes a protein of 190 kDa which corresponds in size to the p190 met alpha beta heterodimer expressed in human cell lines. Furthermore, we show that the 190-kDa product of pOK consists of the 140-kDa met beta subunit associated with the 50-kDa met alpha subunit. This finding suggests that both the alpha and beta met chains are encoded by this construct and confirms the hypothesis that a single chain precursor is cleaved to produce both subunits of met. In contrast, the previously characterized met isoform corresponds to a minor met RNA species and encodes a protein of 170 kDa that is not cleaved yet is processed in a manner that allows cell surface expression. Both met RTK isoforms are autophosphorylated in the in vitro kinase assay. These results suggest that different isoforms of the met RTK may have distinct biological activities.
The Steel/W transduction pathway: kit autophosphorylation and its association with a unique subset of cytoplasmic signaling proteins is induced by the Steel factor.
Authors: Rottapel R , Reedijk M , Williams D E , Lyman S D , Anderson D M , Pawson T , Bernstein A
Published: October 2015

The W/c-kit and Steel loci respectively encode a receptor tyrosine kinase (Kit) and its extracellular ligand, Steel factor, which are essential for the development of hematopoietic, melanocyte, and germ cell lineages in the mouse. To determine the biochemical basis of the Steel/W developmental pathway, we have investigated the response of the Kit tyrosine kinase and several potential cytoplasmic targets to stimulation with Steel in mast cells derived from normal and mutant W mice. In normal mast cells, Steel induces Kit to autophosphorylate on tyrosine and bind to phosphatidylinositol 3'-kinase (PI3K) and phospholipase C-gamma 1 but not detectably to Ras GTPase-activating protein. Additionally, we present evidence that Kit tyrosine phosphorylation acts as a switch to promote complex formation with PI3K. In mast cells from mice homozygous for the W42 mutant allele, Kit is not tyrosine phosphorylated and fails to bind PI3K following Steel stimulation. In contrast, in the transformed mast cell line P815, Kit is constitutively phosphorylated and binds to PI3K in the absence of ligand. These results suggest that Kit autophosphorylation and its physical association with a unique subset of cytoplasmic signaling proteins are critical for mammalian development.
A generic intron increases gene expression in transgenic mice.
Authors: Choi T , Huang M , Gorman C , Jaenisch R
Published: October 2015

To investigate the role of splicing in the regulation of gene expression, we have generated transgenic mice carrying the human histone H4 promoter linked to the bacterial gene for chloramphenicol acetyltransferase (CAT), with or without a heterologous intron in the transcription unit. We found that CAT activity is 5- to 300-fold higher when the transgene incorporates a hybrid intron than with an analogous transgene precisely deleted for the intervening sequences. This hybrid intron, consisting of an adenovirus splice donor and an immunoglobulin G splice acceptor, stimulated expression in a broad range of tissues in the animal. Although the presence of the hybrid intron increased the frequency of transgenics with significant CAT activity, it did not affect the integration site-dependent variation commonly seen in transgene expression. To determine whether the enhancement is a general outcome of splicing or is dependent on the particular intron, we also produced equivalent transgenics carrying the widely used simian virus 40 small-t intron. We found that the hybrid intron is significantly more effective in elevating transgene expression. Our results suggest that inclusion of the generic intron in cDNA constructs may be valuable in achieving high levels of expression in transgenic mice.
Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein.
Authors: Minvielle-Sebastia L , Winsor B , Bonneaud N , Lacroute F
Published: October 2015

In Saccharomyces cerevisiae, temperature-sensitive mutations in the genes RNA14 and RNA15 correlate with a reduction of mRNA stability and poly(A) tail length. Although mRNA transcription is not abolished in these mutants, the transcripts are rapidly deadenylated as in a strain carrying an RNA polymerase B(II) temperature-sensitive mutation. This suggests that the primary defect could be in the control of the poly(A) status of the mRNAs and that the fast decay rate may be due to the loss of this control. By complementation of their temperature-sensitive phenotype, we have cloned the wild-type genes. They are essential for cell viability and are unique in the haploid genome. The RNA14 gene, located on chromosome H, is transcribed as three mRNAs, one major and two minor, which are 2.2, 1.5, and 1.1 kb in length. The RNA15 gene gives rise to a single 1.2-kb transcript and maps to chromosome XVI. Sequence analysis indicates that RNA14 encodes a 636-amino-acid protein with a calculated molecular weight of 75,295. No homology was found between RNA14 and RNA15 or between RNA14 and other proteins contained in data banks. The RNA15 DNA sequence predicts a protein of 296 amino acids with a molecular weight of 32,770. Sequence comparison reveals an N-terminal putative RNA-binding domain in the RNA15-encoded protein, followed by a glutamine and asparagine stretch similar to the opa sequences. Both RNA14 and RNA15 wild-type genes, when cloned on a multicopy plasmid, are able to suppress the temperature-sensitive phenotype of strains bearing either the rna14 or the rna15 mutation, suggesting that the encoded proteins could interact with each other.
DNA repair in the c-myc proto-oncogene locus: possible involvement in susceptibility or resistance to plasmacytoma induction in BALB/c mice.
Authors: Beecham E J , Mushinski J F , Shacter E , Potter M , Bohr V A
Published: October 2015

This report describes an unexpected difference in the efficiency of removal of UV-induced DNA damage in the c-myc locus in splenic B lymphoblasts from two inbred strains of mice. In cells from plasmacytoma-resistant DBA/2N mice, 35% of UV-induced damage in the regulatory and 5' flank of c-myc is removed by 12 h. However, in cells from plasmacytoma-susceptible BALB/cAn mice, damage is not removed from this region. In the protein-encoding region and 3' flank of c-myc as well as in two dihydrofolate reductase gene fragments, UV damage is repaired with similar efficiency in B lymphoblasts from both strains of mice. Furthermore, in the protein-encoding portion and 3' flank of c-myc, damage is selectively removed from only the transcribed strand. No repair is detected in the nontranscribed strand. In contrast, DNA repair in the 5' flank of c-myc is not strand specific; in DNA from DBA/2N cells, UV damage is rapidly removed from both the transcribed and nontranscribed strands. In BALB/cAn cells no repair was detected in either strand in the 5'flank, consistent with the results with double-stranded, nick-translated probes to this region of c-myc. In addition to the repair studies, we have detected post-UV-damage formation: in most of the genes studied, we find that additional T4 endonuclease-sensitive sites are formed in the DNA 2 h after irradiation. Our findings provide new insights into the details of gene-specific and strand-specific DNA repair and suggest that there may be close links between DNA repair and B-cell neoplastic development.
A ras effector domain mutant which is temperature sensitive for cellular transformation: interactions with GTPase-activating protein and NF-1.
Authors: DeClue J E , Stone J C , Blanchard R A , Papageorge A G , Martin P , Zhang K , Lowy D R
Published: October 2015

A series of v-rasH effector domain mutants were analyzed for their ability to transform rat 2 cells at either low or high temperatures. Three mutants were found to be significantly temperature sensitive: Ile-36 changed to Leu, Ser-39 changed to Cys (S39C), and Arg-41 changed to Leu. Of these, the codon 39 mutant (S39C) showed the greatest degree of temperature sensitivity. When the same mutation was analyzed in the proto-oncogene form of ras(c-rasH), this gene was also found to be temperature sensitive for transformation. Biochemical analysis of the proteins encoded by v-rasH(S39C) and c-rasH(S39C) demonstrated that the encoded p21ras proteins were stable and bound guanine nucleotides in vivo at permissive and nonpermissive temperatures. On the basis of these findings, it is likely that the temperature-sensitive phenotype results from an inability of the mutant (S39C) p21ras to interact properly with the ras target effector molecule(s) at the nonpermissive temperature. We therefore analyzed the interaction between the c-rasH(S39C) protein and the potential target molecules GTPase-activating protein (GAP) and the GAP-related domain of NF-1, on the basis of stimulation of the mutant p21ras GTPase activity by these molecules in vitro. Assays conducted across a range of temperatures revealed no temperature sensitivity for stimulation of the mutant protein, compared with that of authentic c-rasH protein. We conclude that for this mutant, there is a dissociation between the stimulation of p21ras GTPase activity by GAP and the GAP-related domain NF-1 and their potential target function. Our results are also consistent with the existence of a distinct, as-yet-unidentified effector for mammalian ras proteins.
The juxtamembrane regions of the epidermal growth factor receptor and gp185erbB-2 determine the specificity of signal transduction.
Authors: Segatto O , Lonardo F , Wexler D , Fazioli F , Pierce J H , Bottaro D P , White M F , Di Fiore P P
Published: October 2015

The epidermal growth factor receptor (EGFR) and gp185erbB-2 are closely related tyrosine kinases. Despite extensive sequence and structural homology, these two receptors display quantitative and qualitative differences in their ability to couple with mitogenic signalling pathways. By using chimeric molecules between EGFR and erbB-2, we found that the determinants responsible for the specificity of mitogenic signal transduction are located in the amino-terminal half of the tyrosine kinase domain of either receptor. In the EGFR, mutational analysis within this subdomain revealed that deletion of residues 660 to 667 impaired receptor mitogenic activity without affecting its tyrosine kinase properties. This sequence is therefore likely to contribute to the specificity of substrate recognition by the EGFR kinase.
The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter.
Authors: Bun-Ya M , Nishimura M , Harashima S , Oshima Y
Published: October 2015

The PHO84 gene specifies Pi-transport in Saccharomyces cerevisiae. A DNA fragment bearing the PHO84 gene was cloned by its ability to complement constitutive synthesis of repressible acid phosphatase of pho84 mutant cells. Its nucleotide sequence predicted a protein of 596 amino acids with a sequence homologous to that of a superfamily of sugar transporters. Hydropathy analysis suggested that the secondary structure of the PHO84 protein consists of two blocks of six transmembrane domains separated by 74 amino acid residues. The cloned PH084 DNA restored the Pi transport activity of pho84 mutant cells. The PHO84 transcription was regulated by Pi like those of the PHO5, PHO8, and PHO81 genes. A PHO84-lacZ fusion gene produced beta-galactosidase activity under the regulation of Pi, and the activity was suggested to be bound to a membrane fraction. Gene disruption of PHO84 was not lethal. By comparison of nucleotide sequences and by tetrad analysis with GAL80 as a standard, the PHO84 locus was mapped at a site beside the TUB3 locus on the left arm of chromosome XIII.
Nur77 is differentially modified in PC12 cells upon membrane depolarization and growth factor treatment.
Authors: Hazel T G , Misra R , Davis I J , Greenberg M E , Lau L F
Published: October 2015

The rat pheochromocytoma cell line PC12 can be induced by growth factors to undergo proliferation and neuronal differentiation. These cells also have excitable membranes that can be depolarized by neurotransmitters or elevated levels of extracellular KCl. Treatment of PC12 cells with growth factors or membrane-depolarizing agents rapidly activates the expression of specific genes whose products are thought to mediate the subsequent biological responses. One such gene, nur77, is a member of the steroid and thyroid hormone receptor gene superfamily. We have identified the Nur77 protein and shown that it is synthesized rapidly and transiently in PC12 cells following stimulation, has a short half-life of 30 to 40 min, and is located in both the nucleus and the cytoplasm. Nur77 is posttranslationally modified, primarily by phosphorylation on serine residues. Phosphopeptide analysis reveals that Nur77 is modified differently upon membrane depolarization than after treatment with growth factors. We hypothesize that the activity of Nur77 is regulated by both differential gene expression and posttranslational modification and that these modes of regulation contribute to distinct downstream responses specific to membrane depolarization and growth factor treatment.
Partial characterization of a cellular factor that regulates the double-stranded RNA-dependent eIF-2 alpha kinase in 3T3-F442A fibroblasts.
Authors: Judware R , Petryshyn R
Published: October 2015

The interferon-induced double-stranded RNA-dependent eIF-2 alpha kinase (dsI) has an established role in mediating part of interferon's antiviral effects. Numerous studies have suggested that dsI also has regulatory functions in cells not infected with virus. Our previous results have indicated that the activation of this kinase may be an important regulatory signal in controlling growth arrest of mouse 3T3-F442A fibroblasts prior to their subsequent differentiation to adipocytes. Here, we report that extracts from 3T3-F442A cells cultured under conditions nonpermissive for differentiation exhibit significantly reduced dsI activity and that this reduction is due, at least in part, to the presence of elevated levels of a novel inhibitor of dsI activation (dRF). This inhibitor is also detected in reduced amounts in extracts from cells cultured under conditions which are permissive for differentiation. We have achieved a 1,000-fold purification of dRF activity, and highly purified dRF preparations were found to be greatly enriched for a 15-kDa protein that was greater than 90% pure. Our results indicate that dRF is not a protein phosphatase or protease but a reversible inhibitor of dsI autophosphorylation. In addition, our results imply that dRF is a physiologic regulator of dsI, since dRF activity correlates with the ability of 3T3-F442A cells to undergo adipose conversion.
Involvement of single-stranded tails in homologous recombination of DNA injected into Xenopus laevis oocyte nuclei.
Authors: Maryon E , Carroll D
Published: October 2015

Homologous recombination of DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient when those molecules are linear and have overlapping homologous ends. It was previously shown that a 5'----3' exonuclease activity in oocytes attacks injected linear DNAs and leaves them with single-stranded 3' tails. We tested the hypothesis that such tailed molecules are early intermediates on the pathway to recombination products. Substrates with 3' tails were made in vitro and injected into oocytes, where they recombined rapidly and efficiently. In experiments with mixed substrates, molecules with 3' tails entered recombination intermediates and products more rapidly than did molecules with flush ends. Molecules endowed in vitro with 5' tails also recombined efficiently in oocytes, but their rate was not faster than for flush-ended substrates. In most cases, the 5' tails served as templates for resynthesis of the 3' strands, regenerating duplex ends which then entered the normal recombination pathway. In oocytes from one animal, some of the 5' tails were removed, and this was exacerbated when resynthesis was partially blocked. Analysis by two-dimensional gel electrophoresis of recombination intermediates from 5'-tailed substrates confirmed that they had acquired 3' tails as a result of the action of the 5'----3' exonuclease. These results demonstrate that homologous recombination in oocytes proceeds via a pathway that involves single-stranded 3' tails. Molecular models incorporating this feature are discussed.
An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3' untranslated region of lymphokine mRNA.
Authors: Bohjanen P R , Petryniak B , June C H , Thompson C B , Lindsten T
Published: October 2015

Considerable evidence suggests that the metabolism of lymphokine mRNAs can be selectively regulated within the cytoplasm. However, little is known about the mechanism(s) that cells use to discriminate lymphokine mRNAs from other mRNAs within the cytoplasm. In this study we report a sequence-specific cytoplasmic factor (AU-B) that binds specifically to AUUUA multimers present in the 3' untranslated region of lymphokine mRNAs. AU-B does not bind to monomeric AUUUA motifs nor to other AU-rich sequences present in the 3' untranslated region of c-myc mRNA. AU-B RNA-binding activity is not present in quiescent T cells but is rapidly induced by stimulation of the T-cell receptor/CD3 complex. Induction of AU-B RNA-binding activity requires new RNA and protein synthesis. Stabilization of lymphokine mRNA induced by costimulation with phorbol myristate acetate correlates inversely with binding by AU-B. Together, these data suggest that AU-B is a cytoplasmic regulator of lymphokine mRNA metabolism.
Structural analyses of the 7SK ribonucleoprotein (RNP), the most abundant human small RNP of unknown function.
Authors: Wassarman D A , Steitz J A
Published: October 2015

The human 7SK ribonucleoprotein (RNP) has been analyzed to determine its RNA secondary structure and protein constituents. HeLa cell 7SK RNA alone and within its RNP have been probed by chemical modification and enzymatic cleavage, and sites of modification or cleavage have been mapped by primer extension. The resulting secondary structure suggests that structural determinants necessary for capping (a 5' stem followed by the sequence AUPuUPuC) and nuclear migration (the sequence AUPuUPuC) of 7SK RNA may be similar to those for U6 small nuclear RNA (snRNA). It also supports existence of a 3' stem structure which could serve to self-prime cDNA synthesis during pseudogene formation. Oligonucleotide-directed RNase H digestion indicated regions of 7SK RNA capable of base pairing with other nucleic acids. Antisense 2'-O-methyl RNA oligonucleotides were used to affinity select the 7SK RNP from an in vivo 35S-labeled cell sonic extract and identify eight associated proteins of 83, 48, 45, 43, 42, 21, 18, and 13 kDa. 7SK RNA has extensive sequence complementarity to U4 snRNA, within the U4/U6 base pairing domain, and also to U11 snRNA. The possibility that the 7SK RNP is an unrecognized component of the pre-mRNA processing machinery is discussed.
An endoplasmic reticulum-specific cyclophilin.
Authors: Hasel K W , Glass J R , Godbout M , Sutcliffe J G
Published: October 2015

Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression.
The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors.
Authors: Krauskopf A , Bengal E , Aloni Y
Published: October 2015

We have previously reported that both in vivo and in vitro, RNA polymerase II pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCl concentrations (0.2 to 1.5 M) but not at the standard KCl concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCl, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or HeLa cells. The general transcription factors IIF and IIX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.