bluuebunny/crossref_metadata_2025 huggingface dataset:
Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change.
Authors: Lieberman P M , Schmidt M C , Kao C C , Berk A J
Published: October 2015

Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.
Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with mitogenesis.
Authors: Miura O , D'Andrea A , Kabat D , Ihle J N
Published: October 2015

A role for tyrosine phosphorylation in the signal-transducing mechanisms of several hematopoietic growth factors has been hypothesized. To extend these observations, we have examined the effects of erythropoietin (Epo) on tyrosine phosphorylation in an Epo-responsive cell that was obtained by transfecting the murine erythropoietin receptor (EpoR) into an interleukin-3 (IL-3)-dependent cell line. By two-dimensional analysis of phosphotyrosine-containing proteins isolated with a monoclonal antibody (1G2) against phosphotyrosine, Epo and IL-3 were found to rapidly induce tyrosine phosphorylation of comparable substrates of 92, 70, and 56 kDa. In addition, Epo uniquely induced phosphorylation of a 72-kDa substrate while IL-3 uniquely induced phosphorylation of a 140-kDa substrate. Immunoprecipitation and mixing experiments indicated that the 72-kDa substrate may represent a small fraction of the EpoR. To explore the significance of tyrosine phosphorylation, we generated two mutants of the EpoR that lacked 108 or 146 amino acids at their carboxyl termini. In addition we constructed an internally deleted mutant that lacked 20 amino acids in a region of sequence homology with the IL-2 receptor beta chain. Although all mutants were expressed at comparable levels and had comparable binding affinities for Epo, only the mutant lacking 108 amino acids at the carboxyl terminus retained significant mitogenic activity or the ability to induce tyrosine phosphorylation.
Highly conserved residues in the bZIP domain of yeast GCN4 are not essential for DNA binding.
Authors: Pu W T , Struhl K
Published: October 2015

Yeast GCN4 and the Jun oncoprotein are transcriptional activators that bind DNA via a bZIP domain consisting of a leucine zipper dimerization element and an adjacent basic region that directly contacts DNA. Two highly conserved alanines (Ala-238 and Ala-239 in GCN4) and an invariant asparagine (Asn-235) in the basic region have been proposed to play important roles in DNA sequence recognition by bZIP proteins. Surprisingly, these conserved residues can be functionally replaced in GCN4 and in a derivative containing the Jun basic region (Jun-GCN4). The ability of an amino acid to functionally substitute for Asn-235 does not correlate with its preference for assuming the N-cap position of an alpha helix. This finding argues against the proposal of the scissors grip model that the invariant asparagine forms an N cap that permits the basic region to bend sharply and wrap around the DNA. In contrast to a prediction of the induced fork model, the pattern of functional substitutions of the conserved alanines together with the results of uracil interference experiments suggests that Ala-238 and Ala-239 do not make base-specific DNA contacts. Finally, the Jun-GCN4 chimeric proteins appear much more active in vivo than expected from their DNA-binding properties in vitro. The mechanistic and evolutionary implications of these results are discussed.
Thyroid-specific enhancer-binding protein (T/EBP): cDNA cloning, functional characterization, and structural identity with thyroid transcription factor TTF-1.
Authors: Mizuno K , Gonzalez F J , Kimura S
Published: October 2015

A cDNA clone encoding a thyroid-specific enhancer-binding protein (T/EBP) was isolated from a rat thyroid-derived FRTL-5 cell lambda gt 11 expression library, using a double-stranded oligonucleotide probe. This oligonucleotide was previously demonstrated to have the strongest binding affinity among three cis-acting DNA elements within the thyroid-specific enhancer region located 5.5 kbp upstream of the human thyroid peroxidase gene transcription start site. Nucleotide and deduced amino acid sequences of the cDNA revealed that T/EBP is identical to the previously reported thyroid-specific transcription factor 1 (TTF-1), which binds to the promoter of the rat thyroglobulin gene and controls its thyroid-specific expression. Expression of the T/EBP cDNA under control of the human cytomegalovirus major immediate-early gene promoter conferred thyroid-specific enhancer activity of as high as 26-fold to nonpermissive human hepatoma HepG2 cells when cotransfected with a vector containing 6.3 kbp of upstream sequence of the human thyroid peroxidase gene connected to a luciferase reporter gene. T/EBP was further expressed in HepG2 cells by using the vaccinia virus expression system. The expressed protein was partially purified by using sequence-specific affinity column chromatography and was further shown, by gel mobility shift experiments, to specifically bind to the enhancer-derived double-stranded oligonucleotide. These results clearly indicate that the binding of T/EBP (TTF-1) to the specific cis-acting enhancer element is largely responsible for thyroid-specific enhancer activity.
Regulation of the yeast CYT1 gene encoding cytochrome c1 by HAP1 and HAP2/3/4.
Authors: Schneider J C , Guarente L
Published: October 2015

Mitochondrial biogenesis requires the coordinate induction of hundreds of genes that reside in the nucleus. We describe here a study of the regulation of the nuclear-encoded cytochrome c1 of the b-c1 complex. Unlike cytochrome c, which is encoded by two genes, CYC1 and CYC7, c1 is encoded by a single gene, CYT1. The regulatory region of the CYT1 promoter contains binding sites for the HAP1 and HAP2/3/4 transactivators that regulate CYC1. The binding of HAP1 to the CYT1 element was studied in detail and found to differ in two important respects from binding to the CYC1 element. First, while CYC1 contains two sites that bind HAP1 cooperatively, CYT1 has a single high-affinity site. Second, while the CYT1 site and the stronger HAP1-binding site of CYC1 share a large block of homology, the HAP1 footprints at these sites are offset by several nucleotides. We discuss how these differences in HAP1 binding might relate to the difference in the biology of cytochrome c and cytochrome c1.
Induction of NF-kappa B DNA-binding activity during the G0-to-G1 transition in mouse fibroblasts.
Authors: Baldwin A S , Azizkhan J C , Jensen D E , Beg A A , Coodly L R
Published: October 2015

A DNA-binding factor with properties of NF-kappa B and another similar activity are rapidly induced when growth-arrested BALB/c 3T3 cells are stimulated with serum growth factors. Induction of these DNA-binding activities is not inhibited by pretreatment of quiescent cells with the protein synthesis inhibitor cycloheximide. Interestingly, the major NF-kappa B-like activity is not detected in nuclear extracts of proliferating cells, and thus its expression appears to be limited to the G0-to-G1 transition in 3T3 cells. These DNA-binding activities bind many of the expected NF-kappa B target sequences, including elements in the class I major histocompatibility complex and human immunodeficiency virus enhancers, as well as a recently identified NF-kappa B binding site upstream of the c-myc gene. Furthermore, both the class I major histocompatibility complex and c-myc NF-kappa B binding sites confer inducibility on a minimal promoter in 3T3 cells stimulated with serum growth factors. The results demonstrate that NF-kappa B-like activities are immediate-early response proteins in 3T3 cells and suggest a role for these factors in the G0-to-G1 transition.
Light represses transcription of asparagine synthetase genes in photosynthetic and nonphotosynthetic organs of plants.
Authors: Tsai F Y , Coruzzi G
Published: October 2015

Asparagine synthetase (AS) mRNA in Pisum sativum accumulates preferentially in plants grown in the dark. Nuclear run-on experiments demonstrate that expression of both the AS1 and AS2 genes is negatively regulated by light at the level of transcription. A decrease in the transcriptional rate of the AS1 gene can be detected as early as 20 min after exposure to light. Time course experiments reveal that the levels of AS mRNA fluctuate dramatically during a "normal" light/dark cycle. This is due to a direct effect of light and not to changes associated with circadian rhythm. A novel finding is that the light-repressed expression of the AS1 gene is as dramatic in nonphotosynthetic organs such as roots as it is in leaves. Experiments demonstrate that the small amount of light which passes through the soil is sufficient to repress AS1 expression in roots, indicating that light has a direct effect on AS1 gene expression in roots. The negative regulation of AS gene expression by light was shown to be a general phenomenon in plants which also occurs in nonlegumes such as Nicotiana plumbaginifolia and Nicotiana tabacum. Thus, the AS genes can serve as a model with which to dissect the molecular basis for light-regulated transcriptional repression in plants.
axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase.
Authors: O'Bryan J P , Frye R A , Cogswell P C , Neubauer A , Kitch B , Prokop C , Espinosa R , Le Beau M M , Earp H S , Liu E T
Published: October 2015

Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.
C/EBP-like proteins binding to the functional box-alpha and box-beta of the second enhancer of hepatitis B virus.
Authors: Yuh C H , Ting L P
Published: October 2015

The second enhancer (enhancer II) of hepatitis B virus is functionally liver specific. Located within an open reading frame of the virus and immediately upstream of the initiation sites of viral major transcripts, enhancer II furnishes a unique model for use in investigating the structure and function of an enhancer. In this study, two functional constituents, a 23-bp box-alpha and a 12-bp box-beta, are identified as being both necessary and sufficient for enhancer II function. Examination of the box-alpha and box-beta sequences reveals a weak homology to the extended consensus for a C/EBP binding site. Gel shift and footprinting analyses indicate that multiple proteins bind to these sequences and thus are candidate transcription factors that mediate the enhancer function. One heat-resistant protein, protein a, and one heat-sensitive protein, protein b, bind to box-alpha. Protein a, which binds to box-alpha in a way indistinguishable from that seen with a recombinant C/EBP, appears not to be identical to C/EBP in that the binding of protein a requires a minimal sequence larger than the canonical C/EBP sites. Two box-beta-binding proteins, c and d, show greater affinity for the C/EBP consensus than for box-beta. However, both proteins c and d are relatively heat sensitive and display a distinct sequence preference from the recombinant C/EBP protein. Since the function of enhancer II is strictly dependent on a bipartite architecture, this system provides a unique model for studies of how the interactions of its binding proteins lead to the enhancer function.
Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein.
Authors: Brott B K , Decker S , O'Brien M C , Jove R
Published: October 2015

GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases.
GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats.
Authors: Flick J S , Johnston M
Published: October 2015

Growth of the yeast Saccharomyces cerevisiae on glucose leads to repression of transcription of many genes required for alternative carbohydrate metabolism. The GRR1 gene appears to be of central importance to the glucose repression mechanism, because mutations in GRR1 result in a pleiotropic loss of glucose repression (R. Bailey and A. Woodword, Mol. Gen. Genet. 193:507-512, 1984). We have isolated the GRR1 gene and determined that null mutants are viable and display a number of growth defects in addition to the loss of glucose repression. Surprisingly, grr1 mutations convert SUC2, normally a glucose-repressed gene, into a glucose-induced gene. GRR1 encodes a protein of 1,151 amino acids that is expressed constitutively at low levels in yeast cells. GRR1 protein contains 12 tandem repeats of a sequence similar to leucine-rich motifs found in other proteins that may mediate protein-protein interactions. Indeed, cell fractionation studies are consistent with this view, suggesting that GRR1 protein is tightly associated with a particulate protein fraction in yeast extracts. The combined genetic and molecular data are consistent with the idea that GRR1 protein is a primary response element in the glucose repression pathway and is required for the generation or interpretation of the signal that induces glucose repression.
A hydrophobic protein sequence can override a nuclear localization signal independently of protein context.
Authors: van Zee K , Appel F , Fanning E
Published: October 2015

Simian virus 40 T antigen is specifically targeted to the nucleus by the signal Pro-Lys-Lys-128-Lys-Arg-Lys-Val. We have previously described the isolation of a simian virus 40 T-antigen mutant, 676FS, which retains a wild-type nuclear localization signal but fails to accumulate properly in the nucleus and interferes with the nuclear localization of heterologous proteins. Here we report that the hydrophobic carboxy-terminal sequence novel to 676FS T antigen overrides the nuclear localization signal if fused to other proteins, thereby anchoring the proteins in the cytoplasm. We discuss possible mechanisms by which missorting of such a fusion protein could interfere with the nuclear transport of heterologous proteins.
Specificity of a retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter: consequences of both retinoic acid and thyroid hormone receptor binding.
Authors: Lucas P C , Forman B M , Samuels H H , Granner D K
Published: October 2015

The ability of a retinoic acid (RA) response element (RARE) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter to mediate effects of either RA or thyroid hormone (T3) on gene expression was studied. Fusion gene constructs consisting of PEPCK promoter sequences ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were used for this analysis. While T3 induced CAT expression to a small degree (about twofold) when such constructs were transiently transfected into H4IIE rat hepatoma cells, along with an expression vector encoding the alpha subtype of the T3 receptor (TR), this effect was mediated by promoter sequences distinct from the PEPCK RARE. Although TRs were capable of binding the PEPCK RARE in the form of putative monomers, dimers, and heterodimers with RA receptors (RARs), this element failed to mediate any positive effect of T3 on gene expression. In contrast, the PEPCK RARE mediated six- to eightfold induction of CAT expression by RA. When TRs were coexpressed along with RARs in transfected H4IIE cells, this RA induction was substantially blunted in a T3-independent manner. This inhibitory effect may be due to the binding of nonfunctional TRs or TR-RAR heterodimers to the PEPCK RARE. A model is proposed to explain the previously observed in vivo effects of T3 on PEPCK gene expression.
In vivo analysis of the Saccharomyces cerevisiae centromere CDEIII sequence: requirements for mitotic chromosome segregation.
Authors: Jehn B , Niedenthal R , Hegemann J H
Published: October 2015

In the yeast Saccharomyces cerevisiae, the complete information needed in cis to specify a fully functional mitotic and meiotic centromere is contained within 120 bp arranged in the three conserved centromeric (CEN) DNA elements CDEI, -II, and -III. The 25-bp CDEIII is most important for faithful chromosome segregation. We have constructed single- and double-base substitutions in all highly conserved residues and one nonconserved residue of this element and analyzed the mitotic in vivo function of the mutated CEN DNAs, using an artificial chromosome. The effects of the mutations on chromosome segregation vary between wild-type-like activity (chromosome loss rate of 4.8 x 10(-4)) and a complete loss of CEN function. Data obtained by saturation mutagenesis of the palindromic core sequence suggest asymmetric involvement of the palindromic half-sites in mitotic CEN function. The poor CEN activity of certain single mutations could be improved by introducing an additional single mutation. These second-site suppressors can be found at conserved and nonconserved positions in CDEIII. Our suppression data are discussed in the context of natural CDEIII sequence variations found in the CEN sequences of different yeast chromosomes.
Overexpression of transforming growth factor-beta in transgenic mice carrying the human T-cell lymphotropic virus type I tax gene.
Authors: Kim S J , Winokur T S , Lee H D , Danielpour D , Kim K Y , Geiser A G , Chen L S , Sporn M B , Roberts A B , Jay G
Published: October 2015

Human T-cell lymphotropic virus type I (HTLV-I) has been associated with an adult form of T-cell leukemia as well as tropical spastic paraparesis, a neurodegenerative disease. Adult T-cell leukemia patients express high levels of the type 1 isoform of transforming growth factor-beta (TGF-beta 1), which is mediated by the effects of the HTLV-I Tax transactivator protein on the TGF-beta 1 promoter. To understand further the regulation of TGF-beta 1 expression by Tax, we examined its expression in transgenic mice carrying the HTLV-I tax gene. We show that tumors from these mice and other tissues, such as submaxillary glands and skeletal muscle, which express high levels of tax mRNA selectively express high levels of TGF-beta 1 mRNA and protein. Moreover, TGF-beta 1 significantly stimulated the incorporation of tritiated thymidine into one of three cell lines derived from neurofibromas of tax-transgenic mice, which suggests that the excessive production of TGF-beta 1 may play a role in tumorigenesis and that these mice may serve as a useful model for studying the biological effects of TGF-beta in vivo.
LyF-1, a transcriptional regulator that interacts with a novel class of promoters for lymphocyte-specific genes.
Authors: Lo K , Landau N R , Smale S T
Published: October 2015

We have studied transcriptional control of the murine terminal deoxynucleotidyltransferase (TdT) gene, which is activated specifically in immature B and T lymphocytes. This analysis has led to the identification and purification of a 50-kDa sequence-specific DNA-binding protein, LyF-1, that interacts with the approximate consensus sequence PyPyTGGGAGPu and is enriched in cells at most stages of B- and T-cell differentiation. LyF-1 binds tightly to an element in the TdT promoter that we show is required for transcription in lymphocytes. LyF-1 also interacts with an element in the immunoglobulin mu enhancer, called microB, that was recently shown to be important for lymphocyte-specific enhancer activity. Moreover, LyF-1 binds to the promoters for the lymphocyte-specific genes lambda 5, VpreB, and lck, all of which we speculate have additional features in common with the TdT promoter. Thus, LyF-1 may be a general transcriptional activator for genes whose expression is restricted to the B- and/or T-lymphocyte lineages.
Inhibition of chromatin assembly in Xenopus oocytes correlates with derepression of the mouse mammary tumor virus promoter.
Authors: Perlmann T , Wrange O
Published: October 2015

The mouse mammary tumor virus (MMTV) promoter is positively regulated by glucocorticoid hormone via binding of glucocorticoid receptor to a specific response element. Upon addition of hormone, a nucleosome containing the glucocorticoid response element is removed or structurally altered, suggesting that the nucleosome interferes with transcription. Accordingly, inhibition of chromatin assembly should relieve the repression and result in an increased constitutive activity. We have tested this hypothesis by injecting nonspecific competitor DNA into Xenopus laevis oocytes to titrate endogenous histones. The coinjection of competitor DNA altered chromatin structure: nucleosomal ladders produced by micrococcal nuclease were disrupted, and the unique helical setting of the MMTV promoter in a nucleosome was lost, as shown by in situ DNase I footprinting. Basal MMTV transcription was drastically increased by competitor DNA, whereas a coinjected, constitutively active adenovirus 2 major late promoter was not stimulated. These results show that the uninduced MMTV promoter is under negative control and provide direct support for the theory that the nucleosomal organization maintains the repression of this promoter in its uninduced state.
Yeast glycolytic mRNAs are differentially regulated.
Authors: Moore P A , Sagliocco F A , Wood R M , Brown A J
Published: October 2015

The regulation of glycolytic genes in response to carbon source in the yeast Saccharomyces cerevisiae has been studied. When the relative levels of each glycolytic mRNA were compared during exponential growth on glucose or lactate, the various glycolytic mRNAs were found to be induced to differing extents by glucose. No significant differences in the stabilities of the PFK2, PGK1, PYK1, or PDC1 mRNAs during growth on glucose or lactate were observed. PYK::lacZ and PGK::lacZ fusions were integrated independently into the yeast genome at the ura3 locus. The manner in which these fusions were differentially regulated in response to carbon source was similar to that of their respective wild-type loci. Therefore, the regulation of glycolytic mRNA levels is mediated at the transcriptional level. When the mRNAs are ordered with respect to the glycolytic pathway, two peaks of maximal induction are observed at phosphofructokinase and pyruvate kinase. These enzymes (i) catalyze the two essentially irreversible steps on the pathway, (ii) are the two glycolytic enzymes that are circumvented during gluconeogenesis and hence are specific to glycolysis, and (iii) are encoded by mRNAs that we have shown previously to be coregulated at the translational level in S. cerevisiae (P. A. Moore, A. J. Bettany, and A. J. P. Brown, NATO ASI Ser. Ser. H Cell Biol. 49:421-432, 1990). This differential regulation of glycolytic mRNA levels might therefore have a significant influence upon glycolytic flux in S. cerevisiae.
Exon recognition and nucleocytoplasmic partitioning determine AMPD1 alternative transcript production.
Authors: Mineo I , Holmes E W
Published: October 2015

Two mature transcripts are produced from the rat AMP deaminase 1 (AMPD1) gene, one that retains exon 2 and one from which exon 2 has been removed. The ratio of these two transcripts is controlled by stage-specific and tissue-specific signals (I. Mineo, P. R. H. Clarke, R. L. Sabina, and E. W. Holmes, Mol. Cell. Biol. 10:5271-5278, 1990; R. L. Sabina, N. Ogasawara, and E. W. Holmes, Mol. Cell. Biol. 9:2244-2246, 1989). By using transfection studies with native, mutant, and chimeric minigene constructs, two steps in RNA processing that determine the ratio of these two transcripts have been identified. The first step is recognition of this exon in the primary transcript. The primary transcript is subject to alternative splicing in which exon 2 is either recognized and thereby included in the mature mRNA or is ignored and retained in a composite intron containing intron 1-exon 2-intron 2. The following properties of the primary transcript influence exon recognition. (i) Exon 2 is intrinsically difficult to recognize, possibly because of its small size (only 12 bases) and/or a suboptimal 5' donor site at the exon 2-intron 2 boundary. (ii) Intron 2 plays a permissive role in recognition of exon 2 because it is removed at a relatively slow rate, presumably because of the suboptimal polypyrimidine tract in the putative 3' branch site. The second step in RNA processing that influences the ratio of mature transcripts produced from the AMPD1 gene occurs subsequent to the ligation of exon 2 to exon 1. An RNA intermediate, composed of exon 1-exon 2-intron 2-exon 3, is produced in the first processing step, but it is variably retained in the nucleus. Retention of this intermediate in the nucleus is associated with accumulation of the mature mRNA containing exon 2, while cytoplasmic escape of this intermediate is reactions, exon recognition and nucleocytoplasmic partitioning, determine the relative abundance of alternative mRNAs derived from the AMPD1 gene.
Heteroduplex formation and mismatch repair of the "stuck" mutation during mating-type switching in Saccharomyces cerevisiae.
Authors: Ray B L , White C I , Haber J E
Published: October 2015

We sequenced two alleles of the MATa locus of Saccharomyces cerevisiae that reduce homothallic switching and confer viability to HO rad52 strains. Both the MATa-stk (J. E. Haber, W. T. Savage, S. M. Raposa, B. Weiffenbach, and L. B. Rowe, Proc. Natl. Acad. Sci. USA 77:2824-2828, 1980) and MATa-survivor (R. E. Malone and D. Hyman, Curr. Genet. 7:439-447, 1983) alleles result from a T----A base change at position Z11 of the MAT locus. These strains also contain identical base substitutions at HMRa, so that the mutation is reintroduced when MAT alpha switches to MATa. Mating-type switching in a MATa-stk strain relative to a MATa Z11T strain is reduced at least 50-fold but can be increased by expression of HO from a galactose-inducible promoter. We confirmed by Southern analysis that the Z11A mutation reduced the efficiency of double-strand break formation compared with the Z11T variant; the reduction was more severe in MAT alpha than in MATa. In MAT alpha, the Z11A mutation also creates a mat alpha 1 (sterile) mutation that distinguishes switches of MATa-stk to either MAT alpha or mat alpha 1-stk. Pedigree analysis of cells induced to switch in G1 showed that MATa-stk switched frequently (23% of the time) to produce one mat alpha 1-stk and one MAT alpha progeny. This postswitching segregation suggests that Z11 was often present in heteroduplex DNA that was not mismatch repaired. When mismatch repair was prevented by deletion of the PMS1 gene, there was an increase in the proportion of mat alpha 1-stk/MAT alpha sectors (59%) and in pairs of switched cells that both retained the stk mutation (27%). We conclude that at least one strand of DNA only 4 bp from the HO cut site is not degraded in most of the gene conversion events that accompany MAT switching.
Cell-specific regulation of oncogene-responsive sequences of the c-fos promoter.
Authors: Gutman A , Wasylyk C , Wasylyk B
Published: October 2015

We have identified oncogene-responsive sequences in the human c-fos promoter that mediate induction of transcription by several nonnuclear oncoproteins and the tumor promoter TPA. These sequences are regulated in a cell-specific manner. (i) In NIH 3T3 cells, the CArG box of the c-fos promoter is sufficient to mediate activation by oncogenes. (ii) In contrast, in HeLa cells, additional flanking sequences are also required, including the outer arm of the serum response element and the FAP site. We also show that the serum response factor, which binds to the CArG box, activates transcription in vivo in NIH 3T3 cells but not in HeLa cells. Finally, we present evidence that the intracellular level of the c-Fos protein could be a major determinant of cell-specific regulation of these oncogene-responsive elements of the c-fos promoter.
Conserved mechanism of tRNA splicing in eukaryotes.
Authors: Zillmann M , Gorovsky M A , Phizicky E M
Published: October 2015

The ligation steps of tRNA splicing in yeast and vertebrate cells have been thought to proceed by fundamentally different mechanisms. Ligation in yeast cells occurs by incorporation of an exogenous phosphate from ATP into the splice junction, with concomitant formation of a 2' phosphate at the 5' junction nucleotide. This phosphate is removed in a subsequent step which, in vitro, is catalyzed by an NAD-dependent dephosphorylating activity. In contrast, tRNA ligation in vertebrates has been reported to occur without incorporation of exogenous phosphate or formation of a 2' phosphate. We demonstrate in this study the existence of a yeast tRNA ligase-like activity in HeLa cells. Furthermore, in extracts from these cells, the entire yeastlike tRNA splicing machinery is intact, including that for cleavage, ligation, and removal of the 2' phosphate in an NAD-dependent fashion to give mature tRNA. These results argue that the mechanism of tRNA splicing is conserved among eukaryotes.
Androgen responsiveness of the murine beta-glucuronidase gene is associated with nuclease hypersensitivity, protein binding, and haplotype-specific sequence diversity within intron 9.
Authors: Lund S D , Gallagher P M , Wang B , Porter S C , Ganschow R E
Published: October 2015

The tissue specificity and genetic variability of the murine beta-glucuronidase (GUS) response to androgen provide useful markers for identifying elements which underlie this responsiveness. While GUS is expressed constitutively in all examined cell types, kidney epithelial cells uniquely exhibit a manyfold yet slow rise in GUS mRNA and enzyme levels when stimulated by androgens. Three major phenotypes of this androgen response have been described among inbred strains of mice: (i) a strong response in strains of the Gusa haplotype, (ii) a reduced response in strains of the Gusb and Gush haplotypes, and (iii) no response, as observed in Gusor mice. These response variants define a cis-active element(s) which is tightly linked to the GUS structural gene. Nuclease hypersensitivity scans of kidney chromatin within and surrounding the structural gene revealed an androgen-inducible hypersensitive site in intron 9 of the gene in Gusa but not in Gusor mice. When a radiolabeled fragment of Gusa DNA containing this hypersensitive site was incubated with kidney nuclear extracts and then subjected to gel electrophoresis, two shifted bands were observed whose levels were dramatically higher in extracts of androgen-treated than in those of untreated Gusa mice. The shifted bands reflect binding of a kidney-specific factor(s) to a 57-bp region of complex dyad symmetry in Gusa and Gusor mice which is partially deleted in Gusb and Gush mice. This binding site is located approximately 130 bp downstream of a glucocorticoid response element sequence motif which is totally deleted in [Gus]or mice. Taken together, our results suggest that the androgen responsiveness of GUS in murine kidney epithelial cells is controlled by elements within the proximal end of intron 9 of the GUS structural gene.
Structural characterization of SIL, a gene frequently disrupted in T-cell acute lymphoblastic leukemia.
Authors: Aplan P D , Lombardi D P , Kirsch I R
Published: October 2015

The SIL (SCL interrupting locus) gene was initially discovered at the site of a genomic rearrangement in a T-cell acute lymphoblastic leukemia cell line. This rearrangement, which occurs in a remarkably site-specific fashion, is present in the leukemic cells of 16 to 26% of patients with T-cell acute lymphoblastic leukemia. We have now cloned a normal SIL cDNA from a cell line which does not carry the rearrangement. The SIL cDNA has a long open reading frame of 1,287 amino acids, with a predicted molecular size of 143 kDa. The predicted protein is not homologous with any previously described protein; however, a potential eukaryotic topoisomerase I active site was identified. Cross-species hybridization using a SIL cDNA probe indicated that the SIL gene was conserved in mammals. A survey of human and murine cell lines and tissues demonstrated SIL mRNA to be ubiquitously expressed, at low levels, in hematopoietic cell lines and tissues. With the exception of 11.5-day-old mouse embryos, SIL mRNA was not detected in nonhematopoietic tissues. The genomic structure of SIL was also analyzed. The gene consists of 18 exons distributed over 70 kb, with the 5' portion of the gene demonstrating alternate exon utilization.
Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.
Authors: Dumont M E , Cardillo T S , Hayes M K , Sherman F
Published: October 2015

Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.
Sequences and factors required for the F9 embryonal carcinoma stem cell E1a-like activity.
Authors: Murray E J , Stott D , Rigby P W
Published: October 2015

F9 embryonal carcinoma (EC) stem cells contain an E1a-like activity that is absent from differentiated derivatives. We have previously characterized proteins present in F9 EC cell extracts that bind to the E1a-dependent E2A promoter and have shown that two of them, TF68 and DRTF1, are required for efficient transcription in vitro (N. B. La Thangue, B. Thimmapaya, and P. W. J. Rigby, Nucleic Acids Res. 18:2929-2938, 1990). We now show that the E1a-like activity is detectable in transient transfection assays. Deletion mutations show that a distal sequence element, which includes the ATF/CREB consensus, is required for expression in both cell types, although it does not mediate the down-regulation of promoter activity that accompanies differentiation. A series of point mutations generated by in vitro mutagenesis confirm this and show that sequences around -60 are necessary for efficient expression in stem cells but not in differentiated derivatives. These sequences bind DRTF1, the activity of which is strongly down-regulated during differentiation. Surprisingly, mutations in a previously uncharacterized region of the promoter restore activity to a promoter carrying the -60 mutation and lead to the formation of a new DNA-protein complex.
Identification of a conserved lipopolysaccharide-plus-interleukin-4-responsive element located at the promoter of germ line epsilon transcripts.
Authors: Rothman P , Li S C , Gorham B , Glimcher L , Alt F , Boothby M
Published: October 2015

Treatment of splenic B lymphocytes and certain B-lineage cell lines with the mitogen lipopolysaccharide (LPS) and the lymphokine interleukin-4 (IL-4) induces expression of germ line immunoglobulin C epsilon transcripts and class switching to the C epsilon gene. We show that LPS-plus-IL-4 induction of germ line epsilon transcripts (termed I epsilon transcripts) occurs at the transcriptional level in an Abelson murine leukemia virus-transformed pre-B-cell line. A 1.1-kb region of DNA surrounding the I epsilon promoter endows inducible transcription to a heterologous reporter gene stably transfected into these cells; such inducible expression depends on combined treatment with LPS and IL-4. Analyses of constructs transiently introduced into a B-cell lymphoma line demonstrated that LPS-plus-IL-4-inducible expression can be conferred by a 179-bp segment of DNA spanning the I epsilon transcriptional initiation site. Mutational analyses demonstrated that this expression depended on DNA sequences within a conserved region directly upstream from the I epsilon transcriptional initiation region. One nuclear protein that is constitutively expressed in normal B cells binds to the downstream end of the conserved sequence; its binding specificity correlates with the functional effect of several mutations. Two additional proteins, which are induced by IL-4 treatment of splenic B cells, bind to the transcription initiation sites of I epsilon. These proteins are indistinguishable in binding assays from proteins previously shown to bind an enhancer region of the class II major histocompatibility complex gene A alpha.
U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.
Authors: Yean S L , Lin R J
Published: October 2015

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.
The length of homology required for gene targeting in embryonic stem cells.
Authors: Hasty P , Rivera-Pérez J , Bradley A
Published: October 2015

Homologous recombination has been used to introduce site-specific mutations into murine embryonic stem (ES) cells with both insertion and replacement vectors. In this study, we compared the frequency of gene targeting with various lengths of homology and found a dramatic increase in targeting with an increase in homology from 1.3 to 6.8 kb. We examined in detail the relationship between the length of homology and the gene-targeting frequency for replacement vectors and found that a critical length of homology is needed for targeting. Adding greater lengths of homology to this critical length has less of an effect on the targeting frequency. We also analyzed the lengths of homology necessary on both arms of the vector for gene replacement events and found that 472 bp of homology is used as efficiently as 1.2 kb in the formation and resolution of crossover junctions.
The intron-containing hsp82 gene of the dimorphic pathogenic fungus Histoplasma capsulatum is properly spliced in severe heat shock conditions.
Authors: Minchiotti G , Gargano S , Maresca B
Published: October 2015

We have isolated and characterized a heat-inducible gene, hsp82, from the dimorphic pathogenic fungus Histoplasma capsulatum, which is a filamentous mold at 25 degrees C and a unicellular yeast at 37 degrees C. This gene, which has a high degree of homology with other members of the hsp82 gene family, is split into three exons and two introns of 122 and 86 nucleotides, respectively. Contrary to what has been demonstrated in Drosophila melanogaster, Saccharomyces cerevisiae, and other organisms, hsp82 mRNA in H. capsulatum is properly spliced during the severe heat conditions of 37 to 40 degrees C in the temperature-sensitive Downs strain. Splicing accuracy was also observed at 42 degrees C in the temperature-tolerant G222B strain, which showed no evidence of accumulation of primary transcripts. Furthermore, the intron containing the beta-tubulin gene is also properly spliced at the upper temperature range, suggesting that the lack of a block in splicing may be a general phenomenon in this organism.
Nonhomologous recombination at sites within the mouse JH-C delta locus accompanies C mu deletion and switch to immunoglobulin D secretion.
Authors: Owens J D , Finkelman F D , Mountz J D , Mushinski J F
Published: October 2015

Plasma cells secrete immunoglobulins other than immunoglobulin M (IgM) after a deletion and recombination in which a portion of the immunoglobulin heavy-chain locus (IgH), from the 5'-flanking region of the mu constant-region gene (C mu) to the 5'-flanking region of the secreted heavy-chain constant-region gene (CH), is deleted. The recombination step is believed to be targeted via switch regions, stretches of repetitive DNA which lie in the 5' flank of all CH genes except delta. Although serum levels of IgD are very low, particularly in the mouse, IgD-secreting plasmacytomas of BALB/c and C57BL/6 mice are known. In an earlier study of two BALB/c IgD-secreting hybridomas, we reported that both had deleted the C mu gene, and we concluded that this deletion was common in the normal generation of IgD-secreting cells. To learn how such switch recombinations occur in the absence of a switch region upstream of the C delta 1 exon, we isolated seven more BALB/c and two C57BL/6 IgD-secreting hybridomas. We determined the DNA sequences of the switch recombination junctions in eight of these hybridomas as well as that of the C57BL/6 hybridoma B1-8. delta 1 and of the BALB/c, IgD-secreting plasmacytoma TEPC 1033. All of the lines had deleted the C mu gene, and three had deleted the C delta 1 exon in the switch recombination event. The delta switch recombination junction sequences were similar to those of published productive switch recombinations occurring 5' to other heavy-chain genes, suggesting that nonhomologous, illegitimate recombination is utilized whenever the heavy-chain switch region is involved in recombination.
Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16.
Authors: Moritz M , Pulaski B A , Woolford J L
Published: October 2015

Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomyces cerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heat-sensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is inhibited at their restrictive temperatures. However, many of the cells remain viable if returned to their permissive temperatures. All of the mutants are deficient in 60S ribosomal subunits and therefore accumulate translational preinitiation complexes. Three of the mutants exhibit a shortage of mature 25S rRNA, and one accumulates rRNA precursors. The accumulation of rRNA precursors suggests that ribosome assembly may be slowed in this mutant. These phenotypes lead us to propose that mutants containing the rpl16b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rpl16b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rpl16b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.