bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneous nuclear ribonucleoprotein complexes and proteins in Drosophila melanogaster.
Authors: Raychaudhuri G , Haynes S R , Beyer A L
Published: October 2015

Pre-mRNAs cotranscriptionally associate with a small group of proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. We have previously identified two genes in Drosophila melanogaster, Hrb98DE and Hrb87F (i.e., genes at 98DE and 87F encoding putative hnRNA binding proteins), which encode five protein species homologous to the mammalian A-B hnRNP proteins. The studies presented herein show that antibodies against the RNP domains of Hrb98DE reacted with 10 to 15 distinct spots of 38 to 40 kDa in the basic region of two-dimensional gels. These nuclear proteins bound single-stranded nucleic acids and were extracted from Drosophila tissue culture cells as 40 to 80S hnRNP complexes in association with 300 to 800 nucleotide fragments of RNA. The peak of poly(A)+ RNA sequences was coincident with the peak of HRB proteins in sucrose gradients, strongly suggesting that the HRB complexes identified are Drosophila hnRNP complexes. The repertoire of HRB proteins did not change significantly during embryogenesis and was similar to that observed in Drosophila tissue culture cells. Analyses with peptide-specific antisera demonstrated that the major proteins in the hnRNP complex were encoded by the two genes previously identified. Although the Drosophila HRB proteins are only approximately 60% identical throughout the RNP domains to the mammalian A-B hnRNP proteins, features of the basic pre-mRNA packaging mechanism appear to be highly conserved between D. melanogaster and mammals.
Inhibitory effect of myristylation on transrepression by FBR (Gag-Fos) protein.
Authors: Kamata N , Holt J T
Published: October 2015

The myristylated v-fos product, FBR murine sarcoma virus (Gag-Fos) protein, exhibits a lower level of transrepression of the serum response element (SRE) than does c-fos protein (Fos). Mutation of the N-terminal myristylation site in FBR protein restored SRE transrepression. Replacement of N-terminal viral Gag sequences with the Fos N terminus also restored this activity, providing additional evidence that myristylation inhibits transrepression by FBR protein. However, the myristylated Gag domain did not inhibit SRE transrepression when fused to Fos, indicating that myristylation of a fos protein is not by itself sufficient to prevent SRE transrepression and that C-terminal mutation is necessary to inhibit transrepression by N myristylation. Comparison of transfection results with Fos C-terminal deletion mutants and the Fos/FBR chimeric mutant revealed that the FBR C terminus retained the potential for transrepression despite deletion of the normal Fos C terminus, whereas similar Fos deletion mutants did not. These results indicate that both N- and C-terminal mutations are required to inhibit transrepression by FBR protein and that multiple structural mutations accompanied by posttranslational protein modification alter gene regulation by FBR protein.
Role of multifunctional autonomously replicating sequence binding factor 1 in the initiation of DNA replication and transcriptional control in Saccharomyces cerevisiae.
Authors: Rhode P R , Elsasser S , Campbell J L
Published: October 2015

Autonomously replicating sequence (ARS) binding factor 1 (ABF1) is an abundant DNA-binding protein that specifically recognizes the motif RTCRYN5ACG at many sites in the yeast genome, including promoter elements, mating-type silencers, and ARSs. Mutational analysis of these sites suggests that ABF1 is involved in constitutive and carbon source-regulated transcriptional activation, transcriptional silencing, and ARS activity. To better assess the role of ABF1 in DNA replication and transcriptional control, temperature-sensitive lethal mutations in the ABF1 gene were isolated. Several of the abf1(Ts) strains show rapid growth arrest at the nonpermissive temperature. At the semipermissive temperature, these strains show an ARS-specific defect in the mitotic stability of ARS-CEN plasmids, such that the abf1 mutants show defects in ARS function identical to those of mutants bearing the mutations in the cis-acting ABF1 binding sites analyzed previously by numerous investigators. Flow cytometric analysis and in vivo DNA labeling experiments on an alpha-factor synchronized abf1(Ts) strain showed that at the nonpermissive temperature, these cells fail to progress efficiently from G1 through S phase and synthesize DNA at 25% of the level seen in the isogenic ABF1 strain. RNA synthesis is also reduced in the abf1(Ts) strains. In addition, transcriptional activation by an ABF1 binding site upstream activation sequence is completely defective in an abf1(Ts) strain at the semipermissive temperature. These phenotypes provide evidence that the same protein, ABF1, functions in the initiation of DNA replication and transcriptional activation.
Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association.
Authors: Werner M , Hermann-Le Denmat S , Treich I , Sentenac A , Thuriaux P
Published: October 2015

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.
A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei.
Authors: Paindavoine P , Rolin S , Van Assel S , Geuskens M , Jauniaux J C , Dinsart C , Huet G , Pays E
Published: October 2015

The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S. Alexandre, P. Paindavoine, P. Tebabi, A. Pays, S. Halleux, M. Steinert, and E. Pays, Mol. Biochem. Parasitol. 43:279-288, 1990). One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene. Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms. These genes differ primarily in a region presumed to encode a large extracellular domain. We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase. The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated. ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T. brucei (S. Rolin, S. Halleux, J. Van Sande, J. E. Dumont, E. Pays, and M. Steinert. Exp. Parasitol. 71:350-352, 1990). Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells.
Molecular and genetic analysis of the yeast early meiotic recombination genes REC102 and REC107/MER2.
Authors: Cool M , Malone R E
Published: October 2015

By selecting for mutations which could rescue the meiotic lethality of a rad52 spo13 strain, we isolated several new Rec genes required relatively early in the meiotic recombination process. This paper presents data to confirm that two of them, REC102 and REC107, are general, meiosis-specific recombination genes that have no detectable role during mitosis. Sequence analysis and genetic complementation indicate that REC107 is identical to the MER2 gene. No sequences related to REC102 have been found in the GenBank or EMBL collections. REC102 is expressed only in meiosis, prior to the reductional division, at about the time that genetic recombination occurs. Examination of the REC102 sequence indicates the presence of several sequences which may play a role in the regulation of its expression; however, the URS1 sequence commonly found in genes expressed early in meiosis is not present.
Domains of the rat rDNA promoter must be aligned stereospecifically.
Authors: Xie W Q , Rothblum L I
Published: October 2015

Efficient transcription from the rat rDNA promoter results from an undefined interaction between the core (CPE) and upstream (UPE) promoter elements or the protein complexes which form on them. These interactions were demonstrated by the behavior of promoters that contained either linker-scanning or deletion mutations of the UPE in combination with point mutations of the CPE (bidomain mutants). In vivo transcription experiments using point mutations within the CPE (G----A mutation at either -16 or -7) demonstrated that the CPE may in fact consist of two domains. Whereas both of these mutants were rescued by the addition of UBF to in vitro transcription reactions, the CPE mutant -7A/G was inactive in vivo. Experiments with these bidomain mutants demonstrated that the UPE was required for the rescue of the CPE mutants. We also examined the hypothesis that this interaction might require a stereospecific alignment of the promoter elements. Our results indicate that the promoter consists of several domains with differing responses to mutations that alter the distance between, or within, the promoter elements. For example, the insertion or deletion of half-multiples of the helical repeat distance between -167 and -147 had no significant effect on transcription. On the other hand, some sites were sensitive to deletions of any size but not to insertions of up to 20 bp. The analyses of two sites yielded results suggesting that they lay between domains of the promoter that must be on the same side of the DNA helix for promoter activity. The first of these sites mapped between -106 and -95.(ABSTRACT TRUNCATED AT 250 WORDS)
Embryonal long terminal repeat-binding protein is a murine homolog of FTZ-F1, a member of the steroid receptor superfamily.
Authors: Tsukiyama T , Ueda H , Hirose S , Niwa O
Published: October 2015

The embryonal long terminal repeat-binding protein, ELP, is present in undifferentiated mouse embryonal carcinoma cells. It binds to and suppresses transcription of the Moloney leukemia virus long terminal repeat in undifferentiated murine embryonal carcinoma cells. We report here that ELP is a mouse homolog of Drosophila FTZ-F1, which positively regulates transcription of the fushi tarazu gene in blastoderm-stage embryos of the fly. As members of the steroid receptor superfamily, ELP and FTZ-F1 have both DNA binding and putative ligand binding domains which are well conserved between the two. ELP and FTZ-F1 function in cells in the extremely early stage of development. A high degree of conservation between the two transcription factors during the evolution of these species indicates the importance of their functions in early-stage embryogenesis. In addition, the sequence elements they recognize do not contain repeat units, in contrast to other steroid receptors, which usually bind to either palindromic or direct repeat sequences.
Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated.
Authors: Fishman-Lobell J , Rudin N , Haber J E
Published: October 2015

HO endonuclease-induced double-strand breaks in Saccharomyces cerevisiae can undergo recombination by two distinct and competing pathways. In a plasmid containing a direct repeat, in which one repeat is interrupted by an HO endonuclease cut site, gap repair yields gene conversions while single-strand annealing produces deletions. Consistent with predictions of the single-strand annealing mechanism, deletion formation is not accompanied by the formation of a reciprocal recombination product. Deletions are delayed 60 min when the distance separating the repeats is increased by 4.4 kb. Moreover, the rate of deletion formation corresponds to the time at which complementary regions become single stranded. Gap repair processes are independent of distance but are reduced in rad52 mutants and in G1-arrested cells.
MEI4, a meiosis-specific yeast gene required for chromosome synapsis.
Authors: Menees T M , Ross-MacDonald P B , Roeder G S
Published: October 2015

The MEI4 gene product is required for meiotic induction of recombination and viable spore production in the yeast Saccharomyces cerevisiae. DNA sequence analysis shows that the MEI4 gene encodes a 450-amino-acid protein bearing no homology to any previously identified protein. The MEI4 coding region is interrupted by a small intron located near the 5' end of the gene. Efficient splicing of the MEI4 transcript is not dependent on the MER1 protein, which is required for splicing the transcript of another meiotic gene, MER2. Expression of a mei4::lacZ fusion gene is meiosis-specific and depends on both heterozygosity at the mating-type locus and nutrient limitation. Northern (RNA) blot hybridization analysis suggests that MEI4 gene expression is regulated at the level of transcription. A functional MEI4 gene is not required for meiotic induction of transcription of the MER1, MER2, MEK1, RED1, SPO11, or RAD50 gene. Cytological analysis of mei4 mutant strains during meiotic prophase demonstrates that the chromosomes form long axial elements that fail to undergo synapsis. The meiosis II division is delayed in mei4 strains.
The core promoter region of the tumor necrosis factor alpha gene confers phorbol ester responsiveness to upstream transcriptional activators.
Authors: Leitman D C , Mackow E R , Williams T , Baxter J D , West B L
Published: October 2015

Activators of protein kinase C, such as 12-O-tetradecanoylphorbol 13-acetate (TPA), are known to regulate the expression of many genes, including the tumor necrosis factor alpha (TNF) gene, by affecting the level or activity of upstream transcription factors. To investigate the mechanism whereby TPA activates the TNF promoter, a series of 5'-deletion mutants of the human TNF promoter linked to chloramphenicol acetyltransferase was transfected into U937 human promonocytic cells. TPA produced a 7- to 11-fold activation of all TNF promoters tested, even those promoters truncated to contain only the core promoter with no upstream enhancer elements. The proximal TNF promoter containing only 28 nucleotides upstream and 10 nucleotides downstream of the RNA start site confers TPA activation to a variety of unrelated upstream enhancer elements and transcription factors, including Sp1, CTF/NF1, cyclic AMP-response element, GAL-E1a, and GAL-VP16. The level of activation by TPA depends on the TATA box structure, since the TPA response is greater in promoters containing the sequence TATAAA than in those containing TATTAA or TATTTA. These findings suggest that the core promoter region is a target for gene regulation by second-messenger pathways.
Differential regulation of the rat phosphoenolpyruvate carboxykinase gene expression in several tissues of transgenic mice.
Authors: Eisenberger C L , Nechushtan H , Cohen H , Shani M , Reshef L
Published: October 2015

The selective expression of a unique copy gene in several mammalian tissues has been approached by studying the regulatory sequences needed to control expression of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene in transgenic mice. A transgene containing the entire PEPCK gene, including 2.2 kb of the 5'-flanking region and 0.5 kb of the 3'-flanking region, exhibits tissue-specific expression in the liver, kidney, and adipose tissue, as well as the hormonal and developmental regulation inherent to endogenous gene expression. Deletions of the 5'-flanking region of the gene have shown the need for sequences downstream of position -540 of the PEPCK gene for expression in the liver and sequences downstream of position -362 for expression in the kidney. Additional sequences upstream of position -540 (up to -2200) are required for expression in adipose tissue. In addition, the region containing the glucocorticoid-responsive elements of the gene used by the kidney was identified. This same sequence was found to be needed specifically for developmental regulation of gene expression in the kidney and, together with upstream sequences, in the intestine. The apparently distinct sequence requirements in the various tissues indicate that the tissues use different mechanisms for expression of the same gene.
High-level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3' untranslated region.
Authors: Dale T C , Krnacik M J , Schmidhauser C , Yang C L , Bissell M J , Rosen J M
Published: October 2015

The high-level expression of the rat whey acidic protein (WAP) gene in transgenic mice depends on the interaction of 5'-flanking promoter sequences and intragenic sequences. Constructs containing 949 bp of promoter sequences and only 70 bp of 3'-flanking DNA were expressed at uniformly high levels, comparable to or higher than that of the endogenous gene. Although this WAP transgene was developmentally regulated, it was expressed earlier during pregnancy than was the endogenous WAP gene. Replacement of 3' sequences, including the WAP poly(A) addition site, with simian virus 40 late poly(A) sequences resulted in an approximately 20-fold reduction in the expression of WAP mRNA in the mammary gland during lactation. Nevertheless, position-independent expression of the transgene was still observed. Further deletion of 91 bp of conserved WAP 3' untranslated region (UTR) led to integration site-dependent expression. Position independence was restored following reinsertion of the WAP 3' UTR into the deleted construct at the same location, but only when the insertion was in the sense orientation. The marked differences observed between the expression levels of the 3'-end deletion constructs in transgenic mice were not seen in transfected CID 9 mammary epithelial cells. In these cells, expression of the endogenous WAP gene was dependent on the interaction of these cells with a complex extracellular matrix. In contrast, the transfected WAP constructs were not dependent on extracellular matrix for expression. Thus, both the abnormal expression of WAP in cells cultured on plastic and the precocious developmental expression of WAP in transgenic mice may reflect the absence of a negative control element(s) within these recombinant constructs.
Regulation of tetradecanoyl phorbol acetate-induced responses in NIH 3T3 cells by GAP, the GTPase-activating protein associated with p21c-ras.
Authors: Nori M , L'Allemain G , Weber M J
Published: October 2015

Proteins of the ras family of oncogenes have been implicated in signal transduction pathways initiated by protein kinase C (PKC) and by tyrosine kinase oncogenes and receptors, but the role that ras plays in these diverse signalling systems is poorly defined. The activity of ras proteins has been shown to be controlled in part by a cellular protein, GAP (GTPase-activating protein), that negatively regulates p21c-ras by enhancing its intrinsic GTPase activity. Thus, overexpression of GAP provides a tool for determining the step(s) in signal transduction dependent on p21c-ras activity. In this paper, we report that overexpression of GAP blocks the phorbol ester (tetradecanoyl phorbol acetate [TPA])-induced activation of p42 mitogen-activated protein kinase (p42mapk), c-fos expression, and DNA synthesis. GAP overexpression did not block responses to serum or fluoroaluminate. Moreover, not all biochemical events elicited by TPA were affected by GAP overexpression, as increased glucose uptake and phosphorylation of MARCKS, a major PKC substrate, occurred normally. Reduction of GAP expression to near normal levels restored the ability of the cells to activate p42mapk in response to TPA. These findings suggest that ras and GAP together play a key role in a PKC-dependent signal transduction pathway which leads to p42mapk activation and cell proliferation.
Differential expression of oocyte-type class III genes with fraction TFIIIC from immature or mature oocytes.
Authors: Reynolds W F , Johnson D L
Published: October 2015

The Xenopus OAX genes can be expressed in oocytes but are virtually inactive in somatic tissues. The tRNA(Met1) (tMET) genes also appear to be developmentally regulated. We have examined the reason for the differential expression of these class III genes. Analysis of the transcriptional activities of extracts derived from immature and mature oocytes revealed that the developmental regulation of these genes can be reproduced in vitro. We have partially purified the required transcription factors B and C from these extracts to ascertain the components responsible for this differential activity. The immature oocyte C fraction activates the tMET and OAX genes when reconstituted with either the immature or mature oocyte-derived B fraction. In contrast, the mature oocyte C fraction fails to activate these genes regardless of which B fraction is used. Both C fractions activated the somatic 5S gene. Purification of the oocyte C fractions by phosphocellulose or B box DNA affinity chromatography failed to separate additional activities responsible for the differential expression of OAX or tMET. By using template exclusion assays, the inability of the mature oocyte C fraction to activate transcription was correlated with an inability to form stable transcription complexes with the tMET or OAX gene.
Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.
Authors: von Pawel-Rammingen U , Aström S , Byström A S
Published: October 2015

The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.
Drugs affecting microtubule dynamics increase alpha-tubulin mRNA accumulation via transcription in Tetrahymena thermophila.
Authors: Stargell L A , Heruth D P , Gaertig J , Gorovsky M A
Published: October 2015

In cultured mammalian cells, an increase in the amount of tubulin monomer due to treatment with a microtubule-depolymerizing agent results in a rapid decline in tubulin synthesis. This autoregulatory response is mediated through a posttranscriptional mechanism which decreases the stability of tubulin message with no change in transcriptional activity of tubulin genes. Conversely, treatment with a microtubule-polymerizing drug, such as taxol, results in a slight increase in the synthesis of tubulin. Surprisingly, we find that two microtubule-depolymerizing agents, colchicine and oryzalin, actually cause an increase in alpha-tubulin synthesis and alpha-tubulin message in starved Tetrahymena thermophila. This increase is paralleled by an increase in transcription of alpha-tubulin sequences measured by run-on transcription, while the half-life of tubulin message measured by decay in the presence of actinomycin D does not change appreciably. Treatment of starved cells with taxol also produces an increase in alpha-tubulin synthesis via an increase in message abundance due to an increase in transcription of the alpha-tubulin gene. These results indicate that tubulin synthesis in T. thermophila is regulated very differently than in cultured mammalian cells.
A ubiquitous factor (HF-1a) and a distinct muscle factor (HF-1b/MEF-2) form an E-box-independent pathway for cardiac muscle gene expression.
Authors: Navankasattusas S , Zhu H , Garcia A V , Evans S M , Chien K R
Published: October 2015

Recent studies have identified a conserved 28-bp element (HF-1) within the rat cardiac MLC-2 gene which confers cardiac muscle-specific and inducible expression during myocardial cell hypertrophy. Utilizing a combination of independent experimental approaches, this study characterizes two cardiac nuclear factors which bind to HF-1, a ubiquitous factor (HF-1a), and an A + T-rich binding factor (HF-1b) which is preferentially expressed in differentiated cardiac and skeletal muscle cells. The HF-1a binding site is located in a core region of the 28-bp conserved element, immediately upstream from the A + T-rich HF-1b site, which is homologous to the MEF-2 site found in a number of muscle genes. By a number of separate criteria (gel mobility shift, competition, and mutagenesis studies), HF-1b and MEF-2 appear to be indistinguishable and thus are either identical or closely related muscle factors. Transient assays of luciferase reporter genes containing point mutations throughout the 28-bp HF-1 regulatory element document the importance of both the HF-1a and HF-1b sites in transient assays in ventricular muscle cells. In the native 250-bp MLC-2 promoter fragment, mutations in the single E box had little effect on cardiac muscle specificity, while point mutations in either the HF-1a or HF-1b binding site significantly reduced promoter activity, underscoring the importance of both the HF-1a and HF-1b sites in the transcriptional activation of this cardiac muscle gene. Thus, this study provides evidence that a novel, ubiquitous factor (HF-1a) and a muscle factor (HF-1b/MEF-2) can form a novel, E-box-independent pathway for muscle-specific expression in ventricular cardiac muscle cells.
ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230.
Authors: Denis C L , Fontaine S C , Chase D , Kemp B E , Bemis L T
Published: October 2015

Four ADR1c mutations that occur close to Ser-230 of the Saccharomyces cerevisiae transcriptional activator ADR1 and which greatly enhance the ability of ADR1 to activate ADH2 expression under glucose-repressed conditions have been shown to reduce or eliminate cyclic AMP-dependent protein kinase (cAPK) phosphorylation of Ser-230 in vitro. In addition, unregulated cAPK expression in vivo blocks ADH2 depression in an ADR1-dependent fashion in which ADR1c mutations display decreased sensitivity to unregulated cAPK activity. Taken together, these data have suggested that ADR1c mutations enhance ADR1 activity by blocking cAPK phosphorylation and inactivation of Ser-230. We have isolated and characterized an additional 17 ADR1c mutations, defining 10 different amino acid changes, that were located in the region defined by amino acids 227 through 239 of ADR1. Three observations, however, indicate that the ADR1c phenotype is not simply equivalent to a lack of cAPK phosphorylation. First, only some of these newly isolated ADR1c mutations affected the ability of yeast cAPK to phosphorylate corresponding synthetic peptides modeled on the 222 to 234 region of ADR1 in vitro. Second, we observed that strains lacking cAPK activity did not display enhanced ADH2 expression under glucose growth conditions. Third, when Ser-230 was mutated to a nonphosphorylatable residue, lack of cAPK activity led to a substantial increase in ADH2 expression under glucose-repressed conditions. Thus, while cAPK controls ADH2 expression and ADR1 is required for this control, cAPK acts by a mechanism that is independent of effects on ADR1 Ser-230. It was also observed that deletion of the ADR1c region resulted in an ADR1c phenotype. The ADR1c region is, therefore, involved in maintaining ADR1 in an inactive form. ADR1c mutations may block the binding of a repressor to ADR1 or alter the structure of ADR1 so that transcriptional activation regions become unmasked.
In vitro transfection of fresh thymocytes and T cells shows subset-specific expression of viral promoters.
Authors: Novak T J , Yoshimura F K , Rothenberg E V
Published: October 2015

We describe conditions under which exogenous DNA templates can be introduced for transient expression into primary murine T lymphocytes. T cells at various stages of development, including concanavalin A-activated splenic T cells, immature pre-T cells, and even small cortical thymocytes, could be successfully transfected. A variety of model DNA constructs were compared in which different viral promoter regions were used to drive expression of the chloramphenicol acetyltransferase (CAT) reporter gene. All showed enhanced expression in cells that had been acutely stimulated with the Ca2+ ionophore A23187 and phorbol ester as chemical proxies for T-cell receptor-mediated signals. In addition, splenocytes but not thymocytes required prior treatment with a mitogen and interleukin-2 in order to express these constructs, implying that even postmitotic thymocytes may be held in a quasiactivated state. A most striking result was the finding that the viral regulatory sequences in the Rous sarcoma virus long terminal repeat and the simian virus 40 early region were subject to sharply differential regulation, with a rank order that changed depending on the developmental stage of the T cells. The most immature thymic blasts and several lymphoma cell lines expressed the pRSV-Cat and pSV2-Cat constructs similarly, but cortical thymocytes exhibited a strong preference for pSV2-Cat. Splenic concanavalin A-stimulated blasts, on the other hand, slightly preferred pRSV-Cat, a tendency which became exaggerated in factor-dependent T-cell lines. The ratio of pRSV-Cat to pSV2-Cat expression varied according to cell type by as much as 500-fold. These results argue against a trivial linkage of promoter preference to cell cycle status but instead provide evidence that activation of T cells at distinct stages of differentiation results in the expression of different ensembles of nuclear regulatory proteins. In contrast to the simian virus 40 and Rous sarcoma virus promoter regions, the long terminal repeats of the retroviruses mink cell focus-forming virus and Akv were expressed well in all primary T-lineage cells. Thus, they represent excellent model promoters for engineering developmental stage-independent expression of exogenous genes in murine T cells.
Induction of HSP70 gene expression by the antiproliferative prostaglandin PGA2: a growth-dependent response mediated by activation of heat shock transcription factor.
Authors: Holbrook N J , Carlson S G , Choi A M , Fargnoli J
Published: October 2015

Prostaglandins (PG) of the A series are potent inhibitors of cell proliferation. Recently, it was shown that PGA2-induced growth arrest was associated with the increased synthesis of stress proteins encoded by the HSP70 gene family. In this study, we have examined the molecular basis for this increases HSP70 expression. Northern (RNA) blot analysis and nuclear run-on assays demonstrated that induction of high levels of HSP70 mRNA results from an increase in the rate of transcription. High-level induction is specific to the HSP70 family of heat shock proteins and is rapid, reversible, dose dependent, and specific for PGs capable of growth-arresting HeLa cells. In addition, the response was found to be highly dependent on the growth state of the cells, as induction occurs in growing but not in confluent nongrowing cell populations. Induction is dependent on the activation of heat shock factor. Cycloheximide pretreatment, which inhibits the antiproliferative effects of PGA2, prevents activation of the heat shock factor and induction of HSP70 mRNA by PGA2. These results support a role for HSP70 in mediating the antiproliferative effects of PGA2.
A collection of mRNA species that are inducible in the RAW 264.7 mouse macrophage cell line by gamma interferon and other agents.
Authors: Farber J M
Published: October 2015

To identify genes induced during macrophage activation, a cDNA library was prepared from cultures of the RAW 264.7 mouse macrophage cell line that had been treated with conditioned medium from mitogen-stimulated spleen cells, and the cDNA library was screened by differential plaque hybridization. Eleven cDNA clones, designated CRG-1 through CRG-11, corresponding to mRNA species inducible in RAW 264.7 cells by the spleen cell conditioned medium, were isolated. Inductions were not blocked by cycloheximide. All of the mRNAs were inducible by gamma interferon, and some were also inducible by alpha and beta interferons, by lipopolysaccharide, by phorbol 12-myristate 13-acetate, and by the calcium ionophore A23187. Sequencing of the cDNAs revealed that CRG-1, CRG-3, and CRG-5 are cDNAs of recently identified transcription factors IRF-1, zif/268, and LRF-1 respectively. As previously reported, CRG-2 and CRG-10 (MIG) encode new members of the platelet factor 4 family of cytokines. CRG-6 corresponds to a new member of a family of interferon-inducible genes clustered on mouse chromosome 1, CRG-9 corresponds to a prostaglandin synthase homolog, CRG-8 corresponds to beta 2-microglobulin, and CRG-4 corresponds to metallothionein II. CRG-11 contains sequences of a truncated L1Md repetitive element as well as nonrepetitive sequences. The nonrepetitive sequence of CRG-11 as well as the sequences of CRG-7 are not closely related to published sequences. The CRG genes and proteins are of interest because of their involvement in macrophage activation, because of their roles as mediators of the effects of gamma interferon and other pleiotropic agents, and because of their usefulness as tools for studying the signal pathways through which gamma interferon and other inducers exert their effects on gene and protein expression.
Formation of heteroduplex DNA during mammalian intrachromosomal gene conversion.
Authors: Bollag R J , Elwood D R , Tobin E D , Godwin A R , Liskay R M
Published: October 2015

We have studied intrachromosomal gene conversion in mouse Ltk- cells with a substrate designed to provide genetic evidence for heteroduplex DNA. Our recombination substrate consists of two defective chicken thymidine kinase genes arranged so as to favor the selection of gene conversion products. The gene intended to serve as the recipient in gene conversion differs from the donor sequence by virtue of a palindromic insertion that creates silent restriction site polymorphisms between the two genes. While selection for gene conversion at a XhoI linker insertion within the recipient gene results in coconversion of the nearby palindromic site in more than half of the convertants, 4% of convertant colonies show both parental and nonparental genotypes at the polymorphic site. We consider these mixed colonies to be the result of genotypic sectoring and interpret this sectoring to be a consequence of unrepaired heteroduplex DNA at the polymorphic palindromic site. DNA replication through the heteroduplex recombination intermediate generates genetically distinct daughter cells that comprise a single colony. We believe that the data provide the first compelling genetic evidence for the presence of heteroduplex DNA during chromosomal gene conversion in mammalian cells.
Characterization of the cDNA and genomic sequence of a G protein gamma subunit (gamma 5).
Authors: Fisher K J , Aronson N N
Published: October 2015

A cDNA from human placenta and liver tissues that contained both sequence for the lysosomal glycosidase di-N-acetylchitobiase and sequence homologous to the gamma subunit of GTP-binding proteins was previously isolated. Here we have shown that the gamma-subunit-homologous portion of this unusual cDNA is derived from a member of the gamma-subunit multigene family. The partial human gamma-subunit sequence was used to isolate the corresponding full-length cDNA clones from bovine and rat livers. The two cDNAs encode identical 68-amino-acid proteins (7.3 kDa) homologous to previously cloned G protein gamma subunits. The bovine gene sequence encoding this new gamma-subunit isoform (gamma 5) was determined and found to have an intron-exon structure consistent with the original human chitobiase-gamma 5-subunit hybrid mRNA being a product of alternative splicing. Genomic cloning also resulted in the isolation of a human gamma 5 pseudogene.
Involvement of cDNA in homologous recombination between Ty elements in Saccharomyces cerevisiae.
Authors: Melamed C , Nevo Y , Kupiec M
Published: October 2015

Strains carrying a marked Ty element (TyUra) in the LYS2 locus were transformed with plasmids bearing a differently marked Ty1 element (Ty1Neo) under the control of the GAL promoter. When these strains were grown in glucose, a low level of gene conversion events involving TyUra was detected. Upon growth on galactose an increase in the rate of gene conversion was seen. This homologous recombination is not the consequence of increased levels of transposition. When an intron-containing fragment was inserted into Ty1Neo, some of the convertants had the intron removed, implying an RNA intermediate. Mutations that affect reverse transcriptase or reverse transcription of Ty1Neo greatly reduce the induction of recombination in galactose. Thus, Ty cDNA is involved in homologous gene conversion with chromosomal copies of Ty elements. Our results have implications about the way families of repeated sequences retain homogeneity throughout evolution.
Evidence that USF can interact with only a single general transcription complex at one time.
Authors: Adami G , Babiss L E
Published: October 2015

By in vitro analysis, the major late promoter (MLP) of adenovirus has been shown to be a simple promoter requiring two elements for efficient transcription: a minimal promoter element (MPE), where the general transcription factor-polymerase II complex binds, and a single functional upstream promoter element (UPE) which interacts with USF. Two hundred bases upstream of the MLP cap site and divergently oriented is the IVa2 promoter. This promoter has its own MPE but shares the MLP UPE, suggesting the possibility that these promoters are coordinately regulated. To determine mechanistically how this might function, we replaced the weak IVa2 minimal promoter with a strong MPE (from the viral E1A gene) and observed mutual inhibition of both promoters and unstable transcription factor binding. Only by duplication of the UPE could this inhibition be relieved. When tested independently, both promoters were shown to require the USF site for maximal activity. These results are compatible with a model in which USF can stably interact with only one transcription complex at a time. When two divergently oriented general transcription complexes compete efficiently for binding of USF, transcription is reduced to the same levels as if the USF site were absent.
Fine-structure map of the human ribosomal protein gene RPS14.
Authors: Diaz J J , Roufa D J
Published: October 2015

We have used polymerase chain reaction-mediated chemical mutagenesis (J.-J. Diaz, D. D. Rhoads, and D. J. Roufa, BioTechniques 11:204-211, 1991) to analyze the genetic fine structure of a human ribosomal protein gene, RPS14. Eighty-three DNA clones containing 158 random single-base substitution mutations were isolated. Mutant RPS14 alleles were tested for biological activity by transfection into cultured Chinese hamster cells. The resulting data permitted us to construct a map of the S14-coding sequence that is comparable to available fine-structure genetic maps of many prokaryotic and lower eukaryotic gene loci. As predicted from the multiplicity of protein-protein and protein-RNA interactions required for ribosomal protein transport and assembly into functional ribosomal subunits, the distribution of null mutations indicated that S14 is composed of multiple, functionally distinct polypeptide domains. Two of the protein's internal domains, designated domains B and D, were essential for S14 biological activity. In contrast, mutations which altered or deleted S14's amino-terminal 20 amino acid residues (domain A) had no observable effect on the protein's assembly and function in mammalian ribosomes. Interestingly, S14 structural domains deduced by in vitro mutagenesis correlate well with the RPS14 gene's exon boundaries.
MET4, a leucine zipper protein, and centromere-binding factor 1 are both required for transcriptional activation of sulfur metabolism in Saccharomyces cerevisiae.
Authors: Thomas D , Jacquemin I , Surdin-Kerjan Y
Published: October 2015

Inactivation of the centromere-binding factor 1 (CBF1) gene results in yeast strains that require methionine for growth. This auxotrophy is due to the inability of such strains to concentrate and assimilate sulfate from the medium. Northern (RNA) blot experiments reveal that the CBF1 protein is required for full induction of MET25 and MET16 gene transcription. However, we show that induction of the sulfate assimilation pathway is not achieved solely by CBF1. This induction also requires the integrity of a positive trans-acting factor, encoded by the MET4 gene. The MET4 gene was cloned, and its sequence reveals that it encodes a protein related to the family of the bZIP transcriptional activators. Evidence that MET4 is a transcriptional activator was provided by demonstrating that DNA-bound LexA-MET4 fusion proteins stimulate expression of a nearby promoter. The use of LexA-MET4 fusion proteins also reveals that the leucine zipper of MET4 is required for the recognition of the MET25 promoter. Moreover, an 18-bp fragment of the MET25 5' upstream region was found to confer S-adenosylmethionine-dependent regulation of a fusion gene. This regulation was shown to depend on both MET4 and CBF1. The obtained results suggest that the binding of CBF1 to its cognate sequences increases the ability of MET4 to stimulate transcription of the MET genes.
Complex intrachromosomal rearrangement in the process of amplification of the L-myc gene in small-cell lung cancer.
Authors: Sekido Y , Takahashi T , Mäkelä T P , Obata Y , Ueda R , Hida T , Hibi K , Shimokata K , Alitalo K , Takahashi T
Published: October 2015

The L-myc gene was first isolated from a human small-cell lung cancer (SCLC) cell line on the basis of its amplification and sequence similarity to c-myc and N-myc. A new mechanism of L-myc activation which results from the production of rlf-L-myc fusion protein was recently reported. On the basis of our earlier observation of a rearrangement involving amplified L-myc in an SCLC cell line, ACC-LC-49, we decided to investigate this rearrangement in detail along with the structure of L-myc amplification units in five additional SCLC cell lines. We report here the identification of a novel genomic region, termed jal, which is distinct from rlf and is juxtaposed to and amplified with L-myc during the process of DNA amplification of the region encompassing L-myc. Long-range analysis using pulsed-field gel electrophoresis revealed that the amplified L-myc locus is involved in highly complex intrachromosomal rearrangements with jal and/or rlf. Our results also suggest that the simultaneous presence of rearrangements both in rlf intron 1 and in regions immediately upstream of L-myc may be necessary for the expression of rlf-L-myc chimeric transcripts.
The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes.
Authors: German M S , Moss L G , Wang J , Rutter W J
Published: October 2015

The pancreatic beta cell makes several unique gene products, including insulin, islet amyloid polypeptide (IAPP), and beta-cell-specific glucokinase (beta GK). The functions of isolated portions of the insulin, IAPP, and beta GK promoters were studied by using transient expression and DNA binding assays. A short portion (-247 to -197 bp) of the rat insulin I gene, the FF minienhancer, contains three interacting transcriptional regulatory elements. The FF minienhancer binds at least two nuclear complexes with limited tissue distribution. Sequences similar to that of the FF minienhancer are present in the 5' flanking DNA of the human IAPP and rat beta GK genes and also the rat insulin II and mouse insulin I and II genes. Similar minienhancer constructs from the insulin and IAPP genes function as cell-specific transcriptional regulatory elements and compete for binding of the same nuclear factors, while the beta GK construct competes for protein binding but functions poorly as a minienhancer. These observations suggest that the patterns of expression of the beta-cell-specific genes result in part from sharing the same transcriptional regulators.
Measurements of excision repair tracts formed during meiotic recombination in Saccharomyces cerevisiae.
Authors: Detloff P , Petes T D
Published: October 2015

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplexes are formed at a high frequency between HIS4 genes located on homologous chromosomes. Using mutant alleles of the HIS4 gene that result in poorly repaired mismatches in heteroduplex DNA, we find that heteroduplexes often span a distance of 1.8 kb. In addition, we show that about one-third of the repair tracts initiated at well-repaired mismatches extend 900 bp.