bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells.
Authors: DeClue J E , Martin G S
Published: September 2015

The cytoskeletal protein talin was found to undergo enhanced phosphorylation at tyrosine residues in chicken embryo fibroblasts following transformation by Rous sarcoma virus. An increase in the tyrosine phosphorylation of talin was also observed within 6 h in cells infected by the temperature-sensitive mutant tsNY68 after a shift from the nonpermissive to the permissive temperature. The overall extent of phosphorylation was 0.07 mol of phosphate per mol of talin and was not appreciably altered by transformation. In uninfected cells talin was shown to be phosphorylated at multiple sites by tryptic peptide mapping. Following transformation most of these sites remained phosphorylated, to the same or to a lesser extent, while novel, phosphotyrosine-containing phosphopeptides appeared. Talin was phosphorylated at tyrosine in cells infected by Rous sarcoma virus mutants which induce altered or partial transformation morphologies; thus the increased phosphorylation of talin at tyrosine occurred irrespective of the morphology induced. Transformation by Y73 also induced elevated levels of phosphotyrosine in talin, whereas transformation by the avian erythroblastosis and Fujinami sarcoma viruses did not.
Human globin gene promoter sequences are sufficient for specific expression of a hybrid gene transfected into tissue culture cells.
Authors: Rutherford T , Nienhuis A W
Published: September 2015

The contribution of the human globin gene promoters to tissue-specific transcription was studied by using globin promoters to transcribe the neo (G418 resistance) gene. After transfection into different cell types, neo gene expression was assayed by scoring colony formation in the presence of G418. In K562 human erythroleukemia cells, which express fetal and embryonic globin genes but not the adult beta-globin gene, the neo gene was expressed strongly from a fetal gamma- or embryonic zeta-globin gene promoter but only weakly from the beta promoter. In murine erythroleukemia cells which express the endogenous mouse beta genes, the neo gene was strongly expressed from both beta and gamma promoters. In two nonerythroid cell lines, human HeLa cells and mouse 3T3 fibroblasts, the globin gene promoters did not allow neo gene expression. Globin-neo genes were integrated in the erythroleukemia cell genomes mostly as a single copy per cell and were transcribed from the appropriate globin gene cap site. We conclude that globin gene promoter sequences extending from -373 to +48 base pairs (bp) (relative to the cap site) for the beta gene, -385 to +34 bp for the gamma gene, and -555 to +38 bp for the zeta gene are sufficient for tissue-specific and perhaps developmentally specific transcription.
The viral Ki-ras gene must be expressed in the G2 phase if ts Kirsten sarcoma virus-infected NRK cells are to proliferate in serum-free medium.
Authors: Durkin J P , Whitfield J F
Published: September 2015

NRK cells infected with a temperature-sensitive Kirsten sarcoma virus (ts371 KSV) are transformed at 36 degrees C, but are untransformed at 41 degrees C which inactivates the abnormally thermolabile oncogenic p21Ki product of the viral Ki-ras gene. At 41 degrees C, tsKSV-infected NRK cells were arrested in G0/G1 when incubated in serum-free medium, but could then be stimulated to transit G1, replicate DNA, and divide by adding serum at 41 degrees C or dropping the temperature to a p21-activating 36 degrees C without adding serum. When quiescent cells at 41 degrees C were stimulated to transit G1 in serum-free medium by activating p21 at 36 degrees C and then shifted back to the p21-inactivating 41 degrees C in the mid-S phase, they continued replicating DNA but could not transit G2. Reactivating p21 in the G2-arrested cells by once again lowering the temperature to 36 degrees C stimulated a rapid entry into mitosis. By contrast, while serum-stimulated quiescent G0 cells at 41 degrees C replicate DNA and divide, serum did not induce G2-arrested cells to enter mitosis, indicating that serum growth factors may trigger events in the G1 phase that ultimately determine G2 transit. These observations made with the viral ras product suggest that cellular ras proto-oncogene products have a role in G2 transit of normal cells.
Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor.
Authors: Mann S K , Firtel R A
Published: September 2015

We examined two sets of genes expressed early in the developmental cycle of Dictyostelium discoideum that appear to be regulated by cyclic AMP (cAMP). The transcripts of both sets of genes were not detectable in vegetative cells. During normal development on filter pads, RNA complementary to these genes could be detected at about 2 h, peaked around 6 to 8 h, and decreased gradually thereafter. Expression of these genes upon starvation in shaking culture was stimulated by pulsing the cells with nanomolar levels of cAMP, a condition that mimics the in vivo pulsing during normal aggregation. Expression was inhibited by caffeine or by continuous levels of cAMP, a condition found later in development when in vivo expression of these genes decreased. The inhibition of caffeine could be overcome by pulsing cells with cAMP. These results suggest that the expression is mediated via the cell surface cAMP receptor, but does not require a rise in intracellular cAMP. mRNA from a gene of the second class was induced upon starvation, peaked by 2.5 h of development, and then declined. In contrast to the other genes, its expression was maintained by continuous levels of cAMP and repressed by cAMP pulses. These and other results on a number of classes of developmentally regulated genes indicates that changing levels of cAMP, acting via the cell surface cAMP receptor, are involved in controlling these groups of genes. We also examined the structure and partial sequence of the cAMP pulse-induced genes. The two tandemly duplicated M3 genes were almost continuously homologous over the sequenced portion of the protein-coding region except for a region near the N-terminal end. The two M3 genes had regions of homology in the 5' flanking sequence and showed slight homology to the same regions in gene D2, another cAMP pulse-induced gene. D2 showed extremely significant homology over its entire sequenced length to an acetylcholinesterase. The results presented here and by others suggest that expression of many early genes in D. discoideum is regulated via the cell surface cAMP receptor. We expect that many of these genes may play essential roles in early Dictyostelium development and could code for elements of the cAMP signal transduction pathway involved in aggregation.
Microinjection of transforming ras protein induces c-fos expression.
Authors: Stacey D W , Watson T , Kung H F , Curran T
Published: September 2015

Microinjection of p21ras induced c-fos protein accumulation in three types of 3T3 cells. The induction was rapid and efficient and persisted for many hours. In addition, anti-ras antibody dramatically reduced c-fos accumulation after serum stimulation of injected cells. However, cells which expressed p21ras continuously did not maintain a high level of c-fos expression.
Transcriptional analyses of interferon-inducible mRNAs.
Authors: Kusari J , Sen G C
Published: September 2015

Using nuclear runoff transcription assays we demonstrated that alpha interferon-mediated induction of transcription of four mRNAs in HeLa monolayer cells needed ongoing protein synthesis and that such a need could be obviated by pretreating the cells with gamma interferon which, by itself, did not induce transcription of these mRNAs. In another human cell line, RD-114, synthesis of alpha interferon-inducible mRNA 561 did not need ongoing protein synthesis. In this line, however, in which interferon inhibits replication of some viruses but not of others, transcription of two of the six interferon-inducible mRNAs that we examined was not appreciably enhanced by interferon.
A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay.
Authors: Akusjärvi G , Svensson C , Nygård O
Published: September 2015

The mechanism by which adenovirus virus-associated RNAI stimulates translational efficiency in a transient-expression assay in 293 cells was investigated. We showed that DNA transfection leads to activation of a protein kinase that phosphorylates the alpha subunit of eucaryotic initiation factor 2 and, as a consequence, inhibition of polypeptide chain initiation. Cotransfection of a plasmid encoding adenovirus type 2 virus-associated RNAI recovered the translational capacity by preventing activation of the kinase.
Functional domains of a T-DNA promoter active in crown gall tumors.
Authors: Bruce W B , Gurley W B
Published: September 2015

Promoter domains required for transcriptional expression of the 780 gene of T-right (pTi15955) were identified by deletion mutagenesis. Accurate quantitation of transcriptional activity of a series of 5' and internal deletion mutants was achieved by using a double gene vector containing a reference 780 gene as an internal standard. Results of the 5' deletions delineated an activator element located between -440 and -229 base pairs (bp) from the start of transcription. Removal of this region resulted in a 100-fold decrease in promoter activity. Two relatively small internal deletion/substitution mutations at positions -74 to -76 and -98 to -112 reduced promoter activity to 38 and 42%, respectively. In most cases large-scale internal deletions (38 to 151 bp) occurring in various locations from positions -12 to -348 bp caused a significant loss in major promoter activity. However, three internal deletions starting at position -37 and extending upstream as far as -153 bp either had little effect on transcriptional activity or resulted in increased activity. Removal of the TATA motif drastically reduced promoter activity to less than 0.1% of the wild type. A minor start of transcription was detected 60 bases upstream from the major transcriptional start site. This minor promoter shares the same activator element as the major promoter for full activity. Deletion and insertion mutations downstream of the minor promoter TATA demonstrated the role of the TATA box in positioning the start of transcription.
A factor present in fetal calf serum enhances oncogene-induced transformation of rodent fibroblasts.
Authors: Hsiao W L , Lopez C A , Wu T , Weinstein I B
Published: September 2015

Our previous studies indicated that addition of the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) or teleocidin to Dulbecco modified Eagle medium supplemented with calf serum enhanced T24-induced focus formation in both the murine C3H 10T1/2 and rat 6 embryo fibroblast cell lines. In the present studies we have found that fetal calf serum (FCS) is more potent than 12-O-tetradecanoylphorbol-13-acetate in enhancing T24-induced focus formation, in terms of the number and size of the foci, in both C3H 10T1/2 and rat 6 cells. Time course studies indicate that FCS can exert this enhancing effect when it is added several days after the transfection with T24 DNA. In rat 6 cells, an 11-fold increase in T24-induced focus formation occurred when the transfected cultures were maintained for only 1 day in 5% FCS, starting 4 days after the transfection. Several known growth factors, including epidermal growth factor, transforming growth factors alpha and beta, insulin, and platelet-derived growth factor, did not enhance T24-induced transformation in these cell systems. Fractionation studies indicate that the factor present in FCS has a molecular weight of about 1,300, is not lipid soluble, and is acid, base, and heat stable. These findings suggest that a factor(s) normally present in serum may enhance the emergence of tumor cells in vivo, by acting in concert with an activated oncogene, during the multistage carcinogenic process.
Isolation of the human gene that complements a temperature-sensitive cell cycle mutation in BHK cells.
Authors: Ittmann M , Greco A , Basilico C
Published: September 2015

We have cloned the human genomic DNA and the corresponding cDNA for the gene which complements the mutation of tsBN51, a temperature-sensitive (Ts) cell cycle mutant of BHK cells which is blocked in G1 at the nonpermissive temperature. After transfecting human DNA into TsBN51 cells and selecting for growth at 39.5 degrees C, Ts+ transformants were identified by their content of human AluI repetitive DNA sequences. Following two additional rounds of transfection, a genomic library was constructed from a tertiary Ts+ transformant and a recombinant phage containing the complementing gene isolated by screening for human AluI sequences. A genomic probe from this clone recognized a 2-kilobase mRNA in human and tertiary transformant cell lines, and this probe was used to isolate a biologically active cDNA from the Okayama-Berg cDNA expression library. Sequencing of this cDNA revealed a single open reading frame encoding a polypeptide of 395 amino acids. The deduced BN51 gene product has a high proportion of acidic and basic amino acids which are clustered in four hydrophilic domains spaced at 60- to 80-amino-acid intervals. These domains have strong sequence homology to each other. Thus, the tsBN51 protein consists of periodic repetitive clusters of acidic and basic amino acids.
The two gene pairs encoding H2A and H2B play different roles in the Saccharomyces cerevisiae life cycle.
Authors: Norris D , Osley M A
Published: September 2015

We have isolated Saccharomyces cerevisiae mutants bearing deletions of one or the other of the two divergently transcribed gene pairs encoding H2A and H2B. The deletions produced diverse effects on the yeast life cycle. Deletion of TRT1, one of the H2A-H2B gene pair sets, affected mitotic growth, sporulation, spore germination, the heat shock response, and exit from the stationary phase; deletion of TRT2, the other H2A-H2B gene pair set, had negligible effects on these same processes. Using a genetic complementation assay, we found that the differential effects of the deletions could be attributed to two features of the gene sets: first, the expression of the TRT1 gene pair, but not the TRT2 gene pair, could compensate for the absence of its partner; second, the protein subtypes encoded by the two gene pairs appear to have different functions in the heat shock response.
c-fos sequence necessary for basal expression and induction by epidermal growth factor, 12-O-tetradecanoyl phorbol-13-acetate and the calcium ionophore.
Authors: Fisch T M , Prywes R , Roeder R G
Published: September 2015

We have investigated the sequence requirements for induction of the human c-fos gene by epidermal growth factor (EGF), 12-O-tetradecanoyl-13-acetate (TPA), and the calcium ionophore A23187 by transfecting c-fos promoter mutants into HeLa and A431 cells. Induction by both EGF and TPA in HeLa cells required the presence of the c-fos enhancer located at -317 to -298 relative to the mRNA cap site. A23187, however, did not induce expression of the transfected gene, even though it strongly induced expression of the endogenous gene, suggesting that it has different requirements for induction than do EGF and TPA. We have also investigated the role of promoter sequences downstream of the enhancer in general expression and induction of c-fos. A sequence between -97 and -76, which includes an 8-base-pair perfect direct repeat, was needed for efficient general expression but not for induction of the gene. A factor in nuclear extracts that bound specifically to this sequence was detected by a gel mobility shift assay. A 7-base-pair sequence, located between -63 and -57 relative to the mRNA cap site and previously shown to be important for general expression of mouse c-fos, was also important for general expression of the human gene. In addition, this element was important for inducibility by EGF and TPA, since induction was significantly reduced when internal deletion mutants that retained the enhancer but lacked the -63 to -57 sequence element were analyzed in transfecting assays.
Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative.
Authors: Seidman M M
Published: September 2015

Intermolecular recombination in mammalian cells was studied by coinfecting African green monkey cells in culture with two shuttle vector plasmids, each carrying an incomplete but overlapping portion of the gene for neomycin resistance. The region of homology between the two plasmids was about 0.6 kilobases. Recombination between the homology regions could reconstruct the neomycin resistance gene, which was monitored by analysis of progeny plasmids in bacteria. The individual plasmids carried additional markers which, in combination with restriction analysis, allowed the determination of the frequency of formation of the heterodimeric plasmid which would be formed in a conservative recombination reaction between the homologous sequences. Reconstruction of the neomycin resistance gene was readily observed, but only 1 to 2% of the neomycin resistance plasmids had the structure of the conservative heterodimer. Treatment of the plasmids which enhanced the frequency of the neomycin resistance gene reconstruction reaction did not significantly increase the relative frequency of conservative product plasmids. The results support nonconservative models for recombination of these sequences.
Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation.
Authors: Sisodia S S , Sollner-Webb B , Cleveland D W
Published: September 2015

To analyze the specificity of RNA processing reactions, we constructed hybrid genes containing RNA polymerase III promoters fused to sequences that are normally transcribed by polymerase II and assessed their transcripts following transfection into human 293 cells. Transcripts derived from these chimeric constructs were analyzed by using a combined RNase H and S1 nuclease assay to test whether RNAs containing consensus 5' and 3' splicing signals could be efficiently spliced in intact cells, even though they were transcribed by RNA polymerase III. We found that polymerase III-derived RNAs are not substrates for splicing. Similarly, we were not able to detect poly(A)+ RNAs derived from genes that contained a polymerase III promoter linked to sequences that were necessary and sufficient to direct 3'-end cleavage and polyadenylation when transcribed by RNA polymerase II. Our findings are consistent with the view that in vivo splicing and polyadenylation pathways are obligatorily coupled to transcription by RNA polymerase II.
Guanine analog-induced differentiation of human promyelocytic leukemia cells and changes in queuine modification of tRNA.
Authors: Kretz K A , Katze J R , Trewyn R W
Published: September 2015

Treatment of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient human promyelocytic leukemia (HL-60) cells with 6-thioguanine results in growth inhibition and cell differentiation. 6-Thioguanine is a substrate for the tRNA modification enzyme tRNA-guanine ribosyltransferase, which normally catalyzes the exchange of queuine for guanine in position 1 of the anticodon of tRNAs for asparagine, aspartic acid, histidine, and tyrosine. During the early stages of HGPRT-deficient HL-60 cell differentiation induced by 6-thioguanine, there was a transient decrease in the queuine content of tRNA, and changes in the isoacceptor profiles of tRNA(His) indicate that 6-thioguanine was incorporated into the tRNA in place of queuine. Reversing this structural change in the tRNA anticodon by addition of excess exogenous queuine reversed the 6-thioguanine-induced growth inhibition and differentiation. Similar results were obtained when 8-azaguanine (another inhibitor of queuine modification of tRNA that can be incorporated into the anticodon) replaced 6-thioguanine as the inducing agent. The data suggest a primary role for the change in queuine modification of tRNA in mediating the differentiation of HGPRT-deficient HL-60 cells induced by guanine analogs.
Genomic characterization of a gamma-interferon-inducible gene (IP-10) and identification of an interferon-inducible hypersensitive site.
Authors: Luster A D , Ravetch J V
Published: September 2015

The genomic organization of a gamma-interferon-inducible gene, IP-10, reveals three introns that interrupt the transcribed sequence into four functional domains. Comparison of the intron-exon structure of this gene to the gene for an homologous chemotactic platelet protein, platelet factor 4, establishes that both genes are interrupted in precisely the same positions within homologous codons; this demonstrates that they belong to a gene family that evolved from a common ancestor. IP-10 and PF4 are two members of a newly described gene family that is likely to include the homologous chemotactic and mitogenic platelet basic proteins (connective tissue-activating protein III and beta-thromboglobulin), the transformation-related protein 9E3, and 310c, a mitogen-stimulated leukocyte protein. A DNase I-hypersensitive site has been found in responsive cells in a region upstream of the RNA initiation site. This hypersensitive site is induced by gamma interferon and thus provides a structural basis for the transcriptional activation seen for this gene by gamma interferon.
Phorbol ester enhances human immunodeficiency virus-promoted gene expression and acts on a repeated 10-base-pair functional enhancer element.
Authors: Kaufman J D , Valandra G , Roderiquez G , Bushar G , Giri C , Norcross M A
Published: September 2015

T-cell activation pathways are involved in the regulation of human immunodeficiency virus (HIV) expression. Phorbol 12-myristate 13-acetate (PMA) is a potent inducer of T-cell immune functions and has recently been demonstrated to increase viral replication in cell lines infected with HIV. To define sequences required for viral induction by PMA. T-cell lines were transiently transfected with viral long terminal repeat (LTR) sequences directing chloramphenicol acetyltransferase (CAT) gene expression. PMA added to transfected cell cultures 24 h before harvest reproducibly increased both CAT mRNA and enzyme expression 2- to 2-fold. Sequences necessary for basal and PMA-induced levels of CAT expression were determined by deletion and enhancer reconstitution constructs with fragments and oligonucleotides from the original LTR-CAT expression plasmid. PMA-inducible and basal activity required tandem repeats of a core enhancer element (GGGACTTTCC) located in the LTR between -105 and -82 relative to the RNA start site. The enhancerlike sequence could be inserted at a site distant to the CAT gene open reading frame and functioned in a position- and orientation-independent manner. The data thus define a transcriptionally active regulatory-enhancer element critical to the control of HIV gene expression.
Transient expression of the beta interferon promoter in human cells.
Authors: Xanthoudakis S , Alper D , Hiscott J
Published: September 2015

A human transient expression assay was used to examine the inducible transcriptional activation of beta interferon (IFN-beta) and IFN-alpha 1 promoters in a homologous cellular environment. Use of 293 cells, an adenovirus DNA-transformed human embryonic kidney cell line, permitted Sendai virus-inducible expression of IFN-beta-CAT hybrid gene. Introduction of the simian virus 40 (SV40) enhancer 5' or 3' to the IFN-CAT gene increased basal (uninduced) levels of chloramphenicol acetyltransferase (CAT) activity; in one construct the SV40 enhancer--IFN-beta regulatory region combination increased the induced CAT activity 50- to 100-fold, suggesting that this may be a generally useful inducible enhancer-promoter combination. No expression from the IFN-alpha-CAT hybrid gene was detected in 293 cells, indicating that human epithelioid cells lack a factor required for expression of the IFN-alpha promoter. However, when the IFN-alpha regulatory region was combined with the SV40 enhancer, a low level of inducible CAT activity was detected in the human transient system.
Murine Hox-1.7 homeo-box gene: cloning, chromosomal location, and expression.
Authors: Rubin M R , King W , Toth L E , Sawczuk I S , Levine M S , D'Eustachio P , Nguyen-Huu M C
Published: September 2015

A new murine homeo-box, called Hox-1.7, has been identified in a rare cDNA from F9 teratocarcinoma stem cells. The Hox-1.7 homeo-box is 68 and 72% homologous to the Drosophila antennapedia (Antp) and iab-7 homeo-boxes, respectively. A major 2.5-kilobase transcript and several minor transcripts were detected by Northern blot (RNA blot) analysis in adult tissues as well as in midgestational embryos. The posterior spinal cord was found to be a major site of Hox-1.7 expression in 12.5-day-old embryos. Somatic cell hybrids were used to map the Hox-1.7 gene to mouse chromosome 6. Restriction fragment length polymorphisms associated with either the Hox-1.7 gene or the previously known Hox-1 complex were identified. Their distribution patterns in recombinant inbred mouse strains were used to determine the linkage between the two loci as well as to other loci on chromosome 6. This maps Hox-1 and Hox-1.7 close to two mouse loci that affect morphogenesis, postaxial hemimelia (px) and hypodactyly (Hd).
Posttranslational translocation of influenza virus hemagglutinin across microsomal membranes.
Authors: Chao C C , Bird P , Gething M J , Sambrook J
Published: September 2015

The biosynthesis of influenza virus hemagglutinin (HA) and its translocation across microsomal membranes were studied in a mammalian cell-free system. All forms of HA could be cotranslationally translocated with high efficiency. However, only truncated forms of HA were translocated after protein synthesis has been terminated. The efficiency of this posttranslational translocation was dependent on the extent of the truncation. Posttranslational translocation was ribosome dependent and occurred only in the presence of a functional N-terminal signal sequence. The molecular mechanism of protein targeting and translocation across the membrane of the endoplasmic reticulum is discussed.
Characterization of a centromere-linked recombination hot spot in Saccharomyces cerevisiae.
Authors: Neitz M , Carbon J
Published: September 2015

A 1.5-kilobase-pair SalI-HindIII (SH) restriction fragment from the region of Saccharomyces cerevisiae chromosome XIV immediately adjacent to the centromere appears to contain sequences that act as a hot spot for mitotic recombination. The presence of SH DNA on an autonomously replicating plasmid stimulates homologous genetic exchange between yeast genomic sequences and those present on the plasmid. In all recombinants characterized, exchange occurs in plasmid yeast sequences adjacent to rather than within the SH DNA. Hybridization analyses reveal that SH-containing plasmids are present in linear as well as circular form in S. cerevisiae and that linear forms are generated by cleavage at specific sites. Presumably, it is the linear form of the plasmid that is responsible for the stimulation of genetic exchange. Based on these observations, it is proposed that this DNA fragment contains a centromere-linked recombination hot spot and that SH-stimulated recombination occurs via a mechanism similar to double-strand-gap repair (J. W. Szostak, T. Orr-Weaver, J. Rothstein, and F. Stahl, Cell 33:25-35 1983).
Differential methylation of the ornithine carbamoyl transferase gene on active and inactive mouse X chromosomes.
Authors: Mullins L J , Veres G , Caskey C T , Chapman V
Published: September 2015

Ornithine carbamoyl transferase (Oct) is an X-linked gene which exhibits tissue-specific expression. To determine whether methylation of specific CpG sequences plays a role in dosage compensation or tissue-specific expression of the gene, 13 potentially methylatable sites were identified over a 30-kilobase (kb) region spanning from approximately 15 kb upstream to beyond exon II. Fragments of the Mus hortulanus Oct gene were used as probes to establish the degree of methylation at each site. By considering the methylation status in liver (expressing tissue) versus kidney (nonexpressing tissue) from male and female mice, the active and inactive genes could be investigated on active and inactive X-chromosome backgrounds. One MspI site, 12 kb 5' of the Oct-coding region, was cleaved by HpaII in liver DNA from males but not in kidney DNA from males and thus exhibited complete correlation with tissue-specific expression of the gene. Six other sites showed partial methylation, reflecting incomplete correlation with tissue-specific expression.
TFIIIA binds to different domains of 5S RNA and the Xenopus borealis 5S RNA gene.
Authors: Sands M S , Bogenhagen D F
Published: September 2015

We have established the conditions for the reassociation of 5S RNA and TFIIIA to form 7S particles. We tested the ability of altered 5S RNAs to bind TFIIIA, taking advantage of the slower mobility of 7S particles compared with free 5S RNA in native polyacrylamide gels. Linker substitution mutants were constructed encompassing the entire gene, including the intragenic control region. In vitro transcripts of the linker substitution mutants were tested for their ability to bind TFIIIA to form 7S ribonucleoprotein particles. Altered 5S RNAs with base changes in or around helices IV and V, which would interfere with the normal base pairing of that region, showed decreased ability to bind TFIIIA. The transcripts of some mutant genes that were efficiently transcribed (greater than 50% of wild-type efficiency) failed to bind TFIIIA in this gel assay. In contrast, the RNA synthesized from a poorly transcribed mutant, LS 86/97, in which residues 87 to 96 of the RNA were replaced in the single-stranded loop at the base of helix V, bound TFIIIA well. The data indicate that TFIIIA binds to different domains in the 5S RNA gene and 5S RNA.
Yeast pre-mRNA splicing requires a minimum distance between the 5' splice site and the internal branch acceptor site.
Authors: Thompson-Jäger S , Domdey H
Published: September 2015

We have generated several deletions within the intron of a yeast actin gene construct which have lead to different splicing efficiencies as measured by Northern blot (RNA blot) and primer extension analyses. Our data especially demonstrate that a minimum distance from the 5' splice site to the internal branch acceptor site is required for accurate and efficient splicing. In a construct in which splicing was completely abolished, splicing could be restored by expanding the distance from the 5' splice site to the internal branch acceptor site with heterologous sequences. Alternative splicing, i.e., exon skipping and the use of a cryptic 5' splice site, was observed when the mRNA precursor was derived from a tandem repeat of a truncated intron with flanking exon sequences.
Reconstitution of the U1 small nuclear ribonucleoprotein particle.
Authors: Patton J R , Patterson R J , Pederson T
Published: September 2015

Although the U1 small nuclear ribonucleoprotein particle (snRNP) was the first mRNA-splicing cofactor to be identified, the manner in which it functions in splicing is not precisely understood. Among the information required to understand how U1 snRNP participates in splicing, it will be necessary to know its structure. Here we describe the in vitro reconstitution of a particle that possesses the properties of native U1 snRNP. 32P-labeled U1 RNA was transcribed from an SP6 promoter-human U1 gene clone and incubated in a HeLa S100 fraction. A U1 particle formed which displayed the same sedimentation coefficient (approximately 10S) and buoyant density (1.40 g/cm3) as native U1 snRNP. The latter value reflects the ability to withstand isopycnic banding in Cs2SO4 without prior fixation, a property shared by native U1 snRNP. The reconstituted U1 particle reacted with both the Sm and RNP monoclonal antibodies, showing that these two classes of snRNP proteins were present. Moreover, the reconstituted U1 snRNP particle was found to display the characteristic Mg2+ switch of nuclease sensitivity previously described for native U1 snRNP: an open, nuclease-sensitive conformation at a low Mg2+ concentration (3 mM) and a more compact, nuclease-resistant organization at a higher concentration (15 mM). The majority of the U1 RNA in the reconstituted particle did not contain hypermethylated caps, pseudouridine, or ribose 2-O-methylation, showing that these enigmatic posttranscriptional modifications are not essential for reconstitution of the U1 snRNP particle. The extreme 3' end (18 nucleotides) of U1 RNA was required for reconstitution, but loop II (nucleotides 64 to 77) was not. Interestingly, the 5' end (15 nucleotides) of U1 RNA that recognizes pre-mRNA 5' splice sites was not required for U1 snRNP reconstruction.
Multiple 5'-flanking regions of the human alpha-skeletal actin gene synergistically modulate muscle-specific expression.
Authors: Muscat G E , Kedes L
Published: September 2015

Transfection into myogenic and nonmyogenic cell lines was used to investigate the transcriptional regulation of the human alpha-skeletal actin gene. We demonstrated that 1,300 base pairs of the 5'-flanking region directed high-level transient expression of the bacterial chloramphenicol acetyltransferase gene in differentiated mouse C2C12 and rat L8 myotubes but not in mouse nonmuscle L.TK- and HuT-12 cells. Unidirectional 5' deletion analysis and heterologous promoter stimulation experiments demonstrated that at least three transcription-regulating subdomains lie in this 1,300-base-pair region. A proximal cis-acting transcriptional element located between positions -153 and -87 relative to the start of transcription at +1 was both sufficient and necessary for muscle-specific expression and developmental regulation during myogenesis in the two myogenic cell systems. The region 3' of position -87 interacted with factors present in both myogenic and fibroblastic cells and appeared to define, or to be a major component of, the basal promoter. In C2C12 myotubes, but not in L8 myotubes, a distal sequence domain between positions -1300 and -626 and the proximal sequence domain between positions -153 and -87 each induced transcription about 10-fold and synergistically increased CAT expression 100-fold over levels achieved by the sequences 3' of position -87. Furthermore, these cis-acting elements independently and synergistically modulated an enhancerless, heterologous simian virus 40 promoter in a tissue-specific manner. DNA fragments which included the proximal domain displayed classical enhancerlike properties. The central region between positions -626 and -153, although required in neither cell line, had a positive, two- to threefold, additive role in augmenting expression in L8 cells but not in C2C12 cells. This suggests that certain elements between positions -1300 and -153 appear to be differentially utilized for maximal expression in different myogenic cells and that the particular combination of domains used is dependent on the availability, in kind or amount, of trans-acting, transcription-modulating factors present in each cell type. Thus, multiple myogenic factors that vary qualitatively and quantitatively may be responsible for the different and complex modulatory programs of actin gene expression observed during in vivo muscle differentiation.
An altered form of pp60c-src is expressed primarily in the central nervous system.
Authors: Le Beau J M , Wiestler O D , Walter G
Published: September 2015

The expression of two forms of pp60c-src, pp60 and pp60+, was measured in the central nervous system (CNS) and the peripheral nervous system. Both forms were expressed in the CNS, whereas only pp60 was primarily detected in the peripheral nervous system. Our findings suggest that pp60+ may play a role in events important to the CNS.
Towards determination of the structure of the Saccharomyces cerevisiae a-factor: an acylated pentadecapeptide blocks a-factor activity.
Authors: Becker J M , Marcus S , Kundu B , Shenbagamurthi P , Naider F
Published: September 2015

Putative a-factor peptides YIIKGVFWADP, YIIKGVFWANP, YIIKGLFWADP, YIIKGLFWANP, YIIKGVFWDPA, and YIIKGVFWDPACVIA and several peptide derivatives were synthesized and were found to be inactive in growth arrest assays, yet they blocked the activity of biological a-factor. Antagonism was greatest with YIIKGVFWDPAC(palmitoyl)VIA. Thus, the structure of a-factor may be a lipopeptide resembling this palmitoylated pentadecapeptide.
A complex translocation at the murine kappa light-chain locus.
Authors: Shapiro M A , Weigert M
Published: September 2015

We have previously reported that a segment of DNA from a murine plasmacytoma comprises DNA from three chromosomes, the immunoglobulin kappa light-chain locus on chromosome 6, the S mu locus on chromosome 12, and a region on chromosome 15. We now report that the reciprocal product contains DNA from only the kappa locus and chromosome 15 and not from S mu. We conclude that a complex series of events, including both a transposition of DNA and a translocation between chromosomes, generated these imperfect reciprocal products.
A human tRNA(iMet) gene produces multiple transcripts.
Authors: Vnencak-Jones C L , Wahab S Z , Zehner Z E , Holmes W M
Published: September 2015

A third nonallelic locus of the human methionyl-tRNA multigene family (tRNA(iMet-3) was isolated. This gene, unlike two other tRNA(iMet) loci, lacks a remarkable run of T and C residues which functions as a termination of transcription signal. Instead, three tandem termination signals, each containing no more than four thymidylate residues, function as relatively inefficient termination signals. As a result, polymerase readthrough generates at least three transcripts in vitro. The efficiency of apparent termination varies significantly at these sites. All resulting transcripts appear to be processed in vitro.
Purification and characterization of chicken ovalbumin gene upstream promoter transcription factor from homologous oviduct cells.
Authors: Bagchi M K , Tsai S Y , Tsai M J , O'Malley B W
Published: September 2015

Previous studies established that the chicken ovalbumin gene upstream promoter (COUP) sequence, which lies between -70 and -90 base pairs upstream from the cap site, is essential for the efficient transcription of the ovalbumin gene. A transcription factor which binds to this sequence has been purified from the homologous chicken oviduct cells. The purification scheme starting from oviduct nuclear extract involved a combination of conventional column and sequence-specific DNA affinity chromatography steps. Using gel retardation and DNase I footprinting techniques to assay COUP-binding activity, we achieved extensive purification of this factor. Binding competition studies with the purified factor indicated that it bound specifically to the COUP sequence and that the binding could be competed for only by the promoter DNA fragments or synthetic oligonucleotides containing the COUP sequence. The purified protein preparation showed multiple polypeptide bands on polyacrylamide gel electrophoresis. Renaturation of separated polypeptides after extraction from the gel matrix was carried out. The majority of renatured polypeptides exhibited specific binding to the COUP sequence.
Complex protein binding within the mouse immunoglobulin heavy-chain enhancer.
Authors: Peterson C L , Calame K L
Published: September 2015

We have begun to purify and characterize several proteins which bind to the mouse immunoglobulin heavy-chain enhancer to understand the molecular interactions important for enhancer activity. Three proteins which bind to different sites on the immunoglobulin heavy-chain enhancer have been chromatographically separated and partially purified. One protein binds a site which has not been reported previously and does not bind to other reported protein-binding sites on the immunoglobulin heavy-chain enhancer. Binding-site boundaries for the three partially purified proteins have been precisely mapped by methylation interference, DNase I footprinting, and orthophenanthroline/copper chemical nuclease footprinting. We have also characterized these three proteins with respect to dissociation rate constants.
Regulation of pyrimidine deoxyribonucleotide metabolism by substrate cycles in dCMP deaminase-deficient V79 hamster cells.
Authors: Bianchi V , Pontis E , Reichard P
Published: September 2015

A mutant V79 hamster fibroblast cell line lacking the enzyme dCMP deaminase was used to study the regulation of deoxynucleoside triphosphate pools by substrate cycles between pyrimidine deoxyribosides and their 5'-phosphates. Such cycles were suggested earlier to set the rates of cellular import and export of deoxyribosides, thereby influencing pool sizes (V. Bianchi, E. Pontis, and P. Reichard, Proc. Natl. Acad. Sci. USA 83:986-990, 1986). While normal V79 cells derived more than 80% of their dTTP from CDP reduction via deamination of dCMP, the mutant cells had to rely completely on UDP reduction for de novo synthesis of dTTP, which became limiting for DNA synthesis. Because of the allosteric properties of ribonucleotide reductase, CDP reduction was not diminished, leading to a large expansion of the dCTP pool. The increase of this pool was kept in check by a shift in the balance of the deoxycytidine/dCMP cycle towards the deoxynucleoside, leading to massive excretion of deoxycytidine. In contrast, the balance of the deoxyuridine/dUMP cycle was shifted towards the nucleotide, facilitating import of extracellular deoxynucleosides.
Replication and segregation of plasmids containing cis-acting regulatory sites of silent mating-type genes in Saccharomyces cerevisiae are controlled by the SIR genes.
Authors: Kimmerly W J , Rine J
Published: September 2015

In Saccharomyces cerevisiae, two cis-acting regulatory sites called E and I flank the silent mating-type gene, HMRa, and mediate SIR-dependent transcriptional repression of the a1-a2 promoters. It has been shown previously that the E and I sites have plasmid replicator (ARS) activity. We show in this report that the ARS activity of the E and I sites is governed by the SIR genotype of the cell. In wild-type cells, a plasmid carrying the E site from HMRa (HMR E) in the vector YIp5 exhibited very high mitotic stability at a copy number of approximately 25 per cell. However, in sir2, sir3, or sir4 mutants, plasmids with HMR E had the low mitotic stability characteristic of plasmids containing ARS1, a SIR-independent replicator. Elevated mitotic stability of plasmids that carry HMR E is due to a segregation mechanism provided by SIR and HMR E. In sir2 and sir4 mutants, the plasmid copy number was significantly lowered, suggesting that these gene products also participate in the replication of plasmids carrying HMR E. The phenotype of point mutations introduced at an 11-base-pair ARS consensus sequence present at HMR E indicated that this sequence is functional but not absolutely required for autonomous replication of the plasmid and that it is not required for SIR-dependent mitotic stabilization. A plasmid carrying both a centromere and HMR E exhibited reduced mitotic stability in wild-type cells. This destabilization appeared to be due to antagonism between the segregation functions provided by the centromere and by HMR E.
Stimulation of tubulin gene transcription by deciliation of sea urchin embryos.
Authors: Gong Z Y , Brandhorst B P
Published: September 2015

Deciliation by hypertonic shock of embryos of the sea urchin Lytechinus pictus resulted in an increase in synthesis of alpha- and beta-tubulins, the consequence of an increased concentration of RNA encoding the tubulins. RNA run-on assays in isolated nuclei indicated that this response is due to a transient increase in the rate of synthesis of tubulin RNA beginning within 5 min of deciliation. This enhancement of tubulin gene transcription also occurred in deciliated embryos treated with the microtubule-depolymerizing agent colcemid; thus the reaction to deciliation is not a response to a reduction in concentration of unpolymerized tubulin utilized for ciliogenesis. In deciliated embryos treated with colcemid, the elevated level of tubulin RNA declined rapidly, due to its destabilization by the elevated concentration of unpolymerized tubulin. The increased transcription of tubulin genes is a response to the loss of cilia, not to the hypertonic shock, and occurs even when cilium regeneration is prevented. Inhibition of protein synthesis with puromycin or emetine did not prevent the transcriptional enhancement but stabilized tubulin mRNA, resulting in increased accumulation of tubulin mRNA after deciliation.
Identification of an estrogen-responsive element from the 5'-flanking region of the rat prolactin gene.
Authors: Maurer R A , Notides A C
Published: September 2015

The DNA sequences which interact with the estrogen receptor and which mediate the estrogenic regulation of prolactin gene transcription have been investigated by the use of receptor-DNA-binding experiments and gene transfer studies. Nitrocellulose filter binding assays using highly purified estrogen receptor and cloned fragments of the 5'-flanking region of the rat prolactin gene demonstrate that the receptor selectively binds to DNA sequences located between nucleotides -1713 and -1532 with respect to the transcription initiation site. The binding of the estrogen receptor to this region of the prolactin gene was strongly dependent on receptor concentration, suggesting that receptor dimers may be important in DNA binding. These data demonstrate that the selective binding of purified estrogen receptor to specific sequences of the rat prolactin gene is an intrinsic property of the receptor and is not due to the interaction of receptor with other proteins. The role of specific prolactin gene sequences in mediating the estrogenic regulation of prolactin gene transcription was confirmed by the use of prolactin-chloramphenicol acetyltransferase fusion genes. These studies demonstrated that sequences upstream of position -1532 are required for estrogen responsiveness. Furthermore, the region of the prolactin gene at -1713 to -1495 was able to confer estrogen responsiveness on the thymidine kinase promoter. Exonuclease III protection experiments further localized the receptor-binding sequences to positions -1587 to -1563. Comparison of the nucleotide sequence of the region of the prolactin gene which binds the estrogen receptor with the sequence of other estrogen-responsive genes suggested the presence of the conserved sequence [sequence in text], which shows similarity to sequences thought to mediate glucocorticoid receptor effects on transcription.