bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulatory myosin light-chain genes of Caenorhabditis elegans.
Authors: Cummins C , Anderson P
Published: September 2015

We have cloned and analyzed the Caenorhabditis elegans regulatory myosin light-chain genes. C. elegans contains two such genes, which we have designated mlc-1 and mlc-2. The two genes are separated by 2.6 kilobases and are divergently transcribed. We determined the complete nucleotide sequences of both mlc-1 and mlc-2. A single, conservative amino acid substitution distinguishes the sequences of the two proteins. The C. elegans proteins are strongly homologous to regulatory myosin light chains of Drosophila melanogaster and vertebrates and weakly homologous to a superfamily of eucaryotic calcium-binding proteins. Both mlc-1 and mlc-2 encode abundant mRNAs. We mapped the 5' termini of these transcripts by using primer extension sequencing of mRNA templates. mlc-1 mRNAs initiate within conserved hexanucleotides at two different positions, located at -28 and -38 relative to the start of translation. The 5' terminus of mlc-2 mRNA is not encoded in the 4.8-kilobase genomic region upstream of mlc-2. Rather, mlc-2 mRNA contains at its 5' end a short, untranslated leader sequence that is identical to the trans-spliced leader sequence of three C. elegans actin genes.
Transcription termination at the chicken beta H-globin gene.
Authors: Pribyl T M , Martinson H G
Published: September 2015

We characterized the transcription termination region of the chicken beta H-globin gene. First we located the region by nuclear runon transcription in vitro. Then we sequenced and subcloned it into a chloramphenicol acetyltransferase (CAT) expression vector for assay in vivo. The region of beta H termination contains two interesting elements located about 1 kilobase downstream of the beta H gene poly(A) site. Either element alone can block CAT expression if inserted between the promoter and the poly(A) site of the cat gene in pRSVcat. The first element in the termination region is an unusually large inverted repeat in the DNA (delta G = -71 kcal). The second element, 200 base pairs further downstream, is an RNA polymerase II promoter which directs transcription back upstream on the complementary strand. This transcription converges on and collides with that from the beta H gene at or near the inverted repeat where transcription from both directions stops. We propose that the inverted repeat is a strong pause site which positions the converging polymerases for mutual site-specific termination.
Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation.
Authors: Banks G R , Taylor S Y
Published: September 2015

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.
Expression and regulation of the pituitary- and placenta-specific human glycoprotein hormone alpha-subunit gene is restricted to the pituitary in transgenic mice.
Authors: Fox N , Solter D
Published: September 2015

Expression of the glycoprotein hormone alpha subunit occurs in both the pituitary and placenta in humans. However, this study found that expression of this subunit is restricted to the pituitary in mice. An interspecies analysis of human alpha-subunit gene regulation was undertaken, using the transgenic-mouse approach. In mice transgenic for a genomic clone containing the complete human alpha-subunit gene and several kilobases of 5'- and 3'-flanking sequences, cell-type-specific expression and hormonal regulation of the human alpha-subunit transgene occurred in the mouse pituitary, whereas no expression of the transgene was detectable in the mouse placenta. These findings provide strong evidence that a common trans-acting factor(s) regulates glycoprotein hormone alpha-subunit gene expression in the human and mouse pituitaries; however, this factor(s) or a unique factor(s), though functional in the human placenta, is either nonfunctional or absent in the mouse placenta.
The epidermal growth factor receptor from prostate cells is dephosphorylated by a prostate-specific phosphotyrosyl phosphatase.
Authors: Lin M F , Clinton G M
Published: September 2015

Human prostatic acid phosphatase (PAcP) has been found to have phosphotyrosyl-protein phosphatase activity (H. C. Li, J. Chernoff, L. B. Chen, and A. Kirschonbaun, Eur. J. Biochem. 138:45-51, 1984; M.-F. Lin and G. M. Clinton, Biochem. J. 235:351-357, 1986) and has been suggested to negatively regulate phosphotyrosine levels, at least in part, by inhibition of tyrosine protein kinase activity (M.-F. Lin and G. M. Clinton, Adv. Protein Phosphatases 4:199-228, 1987; M.-F. Lin, C. L. Lee, and G. M. Clinton, Mol. Cell. Biol. 6:4753-4757, 1986). We investigated the molecular interaction of PAcP with a specific tyrosine kinase, the epidermal growth factor (EGF) receptor, from prostate carcinoma cells. Of several proteins phosphorylated in membrane vesicles from prostate carcinoma cells, PAcP selectively dephosphorylated the EGF receptor. The prostate EGF receptor was more efficiently dephosphorylated by PAcP than by another phosphotyrosyl phosphatase, potato acid phosphatase. Further characterization of the interaction of PAcP with the EGF receptor revealed that the optimal rate of dephosphorylation occurred at neutral rather than at acid pH. Thus, the enzyme that we formerly referred to as PAcP we now call prostatic phosphotyrosyl-protein phosphatase. Hydrolysis of phosphate from tyrosine residues in the immunoprecipitated EGF receptor catalyzed by purified prostatic phosphotyrosyl-protein phosphatase caused a 40 to 50% decrease in the receptor tyrosine kinase activity with angiotensin as the substrate. In contrast, autophosphorylation of the receptor was associated with an increase in tyrosine kinase activity.
The human transforming growth factor alpha promoter directs transcription initiation from a single site in the absence of a TATA sequence.
Authors: Jakobovits E B , Schlokat U , Vannice J L , Derynck R , Levinson A D
Published: September 2015

Transforming growth factor alpha (TGF-alpha) is a transformation-responsive mitogenic polypeptide that is expressed in the brain, epithelial cells, and activated macrophages. We isolated and characterized the TGF-alpha promoter and localized the 5' end of the TGF-alpha transcript to a unique position. Surprisingly, no apparent TATA box was present in the promoter sequence, suggesting that transcription from mammalian genes can initiate at unique and specific positions from promoters lacking this sequence motif.
T-cell activation signals and human T-cell leukemia virus type I-encoded p40x protein activate the mouse granulocyte-macrophage colony-stimulating factor gene through a common DNA element.
Authors: Miyatake S , Seiki M , Yoshida M , Arai K
Published: September 2015

Activation of T cells by an antigen, a mitogen, or a combination of a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]) and a calcium ionophore (A23187) leads to induction of a set of lymphokine genes. Treatment of human T-cell leukemia line Jurkat by a mitogen or p40x, a transactivator protein encoded by human T-cell leukemia virus type I, activates many transfected lymphokine genes in a transient transfection assay. To study the mechanism of lymphokine gene induction, we examined the effects of mitogen stimulation and p40x on the gene for the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) in Jurkat cells. Deletion and mutation analyses showed that the 5'-flanking region of the gene for the GM-CSF is composed of two types of regulatory elements. One sequence, located at positions -95 to -73, determines response to stimulation by either TPA-A23187 or p40x. This region contains conserved lymphokine element 2, which appears in the gene for interleukin 3 (IL-3) and is followed by a GC-rich stretch. This GC-rich stretch alone specifies inducible response to p40x but not to TPA-A23187. Another sequence, located at positions -113 to -96 upstream of a TATA-like sequence, mediates inducible response to p40x but not to TPA-A23187. This sequence includes conserved lymphokine element 1, which appears in several lymphokine-cytokine genes, such as those for IL-3, G-CSF, and IL-2. We previously showed that the simian virus 40 early region promoter was also induced by a mitogen or p40x in Jurkat cells. Deletion analysis showed that the minimum region require for stimulation by both signals are identical. These results, which indicate that p40(x) stimulates transcription of the gene for the GM-CSF or the simian virus 40 early region promoter through the same DNA element or an overlapping DNA element required for induction by a mitogen, lend further support to the notion that p40(x) can exert its function by activating a component(s) of the T-cell signal transduction pathway which is activated by an antigen or a mitogen.
Expression of the rat myosin light-chain 2 gene in transgenic mice: stage specificity, developmental regulation, and interrelation with the endogenous gene.
Authors: Shani M , Dekel I , Yoffe O
Published: September 2015

The expression of the rat skeletal myosin light-chain 2 gene in two transgenic strains was tissue specific and stage specific. However, the temporal regulation during development of the transgene was different from that of the endogenous gene. Surprisingly, in one strain, the expression of the transgene was associated with a significant down-regulation of the endogenous gene. The possible mechanisms to account for the suppression of the endogenous gene and the potential implications of this suppression are discussed.
Characterization of N-myc amplification units in human neuroblastoma cells.
Authors: Zehnbauer B A , Small D , Brodeur G M , Seeger R , Vogelstein B
Published: September 2015

A set of DNA clones comprising 48 independent HindIII fragments (215 kilobases of sequence) was derived from the N-myc amplification unit of the neuroblastoma cell line NGP. These clones were used to investigate N-myc amplification units in NGP cells and 12 primary neuroblastoma tumors. Three parameters were evaluated: (i) the number of rearrangements from germ line configuration that had occurred during the amplification process; (ii) the homogeneity of amplification units within individual tumors; and (iii) the conservation of amplified sequences among different tumors. The results indicated that remarkably few rearrangements had occurred during amplification, that the amplification units within any one tumor were quite homogeneous, and that although each tumor contained a unique pattern of amplified DNA fragments, there was considerable similarity between the amplification units of different tumors. In particular, the amplification units were strikingly similar over a contiguous domain of at least 140 kilobases surrounding the N-myc structural gene.
An integral membrane protein antigen associated with the membrane attachment sites of actin microfilaments is identified as an integrin beta-chain.
Authors: Maher P A , Singer S J
Published: September 2015

A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.
Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: alternative exon usage predicts two different precursor proteins.
Authors: Rorsman F , Bywater M , Knott T J , Scott J , Betsholtz C
Published: September 2015

The human platelet-derived growth factor (PDGF) A-chain locus was characterized by restriction endonuclease analysis, and the nucleotide sequence of its exons was determined. Seven exons were identified, spanning approximately 22 kilobase pairs of genomic DNA. Alternative exon usage, identified by cDNA cloning, occurs in a human glioblastoma cell line and may give rise to two types of A-chain precursors with different C termini. The exon-intron arrangement was similar to that of the PDGF B-chain/sis locus and seemed to divide the precursor proteins into functional domains. Southern blot analysis of genomic DNA showed that a single PDGF A-chain gene was present in the human genome.
Expansions and contractions of the genetic map relative to the physical map of yeast chromosome III.
Authors: Symington L S , Petes T D
Published: September 2015

To examine the relationship between genetic and physical chromosome maps, we constructed a diploid strain of the yeast Saccharomyces cerevisiae heterozygous for 12 restriction site mutations within a 23-kilobase (5-centimorgan) interval of chromosome III. Crossovers were not uniformly distributed along the chromosome, one interval containing significantly more and one interval significantly fewer crossovers than expected. One-third of these crossovers occurred within 6 kilobases of the centromere. Approximately half of the exchanges were associated with gene conversion events. The minimum length of gene conversion tracts varied from 4 base pairs to more than 12 kilobases, and these tracts were nonuniformly distributed along the chromosome. We conclude that the chromosomal sequence or structure has a dramatic effect on meiotic recombination.
Transcriptional properties of BmX, a moderately repetitive silkworm gene that is an RNA polymerase III template.
Authors: Wilson E T , Condliffe D P , Sprague K U
Published: September 2015

We analyzed the transcriptional properties of a repetitive sequence element, BmX, that belongs to a large gene family (approximately 2 x 10(4) copies) in the genome of the Bombyx mori silkworm. We discovered BmX elements because of their ability to direct transcription by polymerase III in vitro and used them to test the generality of the properties of previously identified silkworm polymerase III control elements. We found that the signals that act in cis to control BmX transcription strongly resemble those that direct transcription of other silkworm polymerase III templates. As with silkworm tRNA and 5S RNA genes, transcription of BmX requires sequence signals located both upstream and downstream from the site of transcription initiation. The critical upstream sequences are structurally as well as functionally similar in the three kinds of templates. The downstream control region of BmX resembles the corresponding part of a silkworm alanine tRNA gene in that it provides a large (greater than 100 base pairs) region that influences transcription factor binding. Moreover, the factor-binding regions of both tRNA(Ala) and BmX genes are remarkable in that under certain conditions, key elements within them (the B boxes, for example) appear dispensable. This behavior can be understood if, in both of these templates, the downstream control region acts as a large target for interaction with a multifactor complex.
Elevated expression of pp60c-src alters a selective morphogenetic property of epithelial cells in vitro without a mitogenic effect.
Authors: Warren S L , Handel L M , Nelson W J
Published: September 2015

Madin-Darby canine kidney (MDCK) cells are highly differentiated and have retained the morphogenetic properties necessary to form polarized, multicellular epithelial structures (cysts) in vitro that resemble epithelial tissues in vivo. We introduced the c-src gene into MDCK cells to elevate the level of the plasma membrane-associated cellular tyrosine kinase, pp60c-src, to levels two- to ninefold higher than that expressed in parent MDCK cells. Our results revealed a highly discriminatory biological action of pp60c-src on the morphogenetic properties of MDCK cells. Elevated expression of pp60c-src conferred on MDCK cells the ability to undergo dramatic changes of cell shape that includes the formation of long cell processes (100 to 200 microns), never observed in control MDCK cells. The morphogenesis of multicellular epithelial cysts was altered by elevated levels of pp60c-src and led to predictable distortions of their three-dimensional architecture. However, these cells established morphologically normal cell polarity, formed adhesive epithelial cell-cell contacts indistinguishable from those of control MDCK cells, and exhibited neither focus-forming ability or anchorage-independent growth potential. Finally, we showed that MDCK cells expressing elevated levels of pp60c-src exhibit increased phosphorylation of a more limited number of phosphotyrosine-containing proteins than MDCK cells expressing pp60v-src. We suggest that a natural function of pp60c-src is to regulate the morphogenetic properties which determine the shape of differentiated cells and multicellular structures.
Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site.
Authors: Forsburg S L , Guarente L
Published: September 2015

We analyzed upstream activation sequence 2 (UAS2), one of two independent UAS elements in the CYC1 gene of Saccharomyces cerevisiae. Deletions and linker scanning mutations across the 87 base pairs previously defined as UAS2 showed two separate functional elements required for full activity. Region 1, from -230 to -200, contains the principal activation site and responds to the trans-acting regulatory loci HAP2 and HAP3. A portion of region 1 is homologous to two other HAP2-HAP3-responsive UASs and includes the G----A transition mutation UP1, which increases UAS2 activity. This consensus sequence TNATTGGT bears striking similarity to several CAAT box sequences of higher cells. Region 2, from -192 to -178, substantially enhances the activity of region 1, yet has little activity by itself. These regions bind distinct proteins found in crudely fractionated yeast extracts.
The HAP3 regulatory locus of Saccharomyces cerevisiae encodes divergent overlapping transcripts.
Authors: Hahn S , Pinkham J , Wei R , Miller R , Guarente L
Published: September 2015

Activation of the CYC1 upstream activation site, UAS2, and transcription of several other genes encoding respiratory functions requires the product of the regulatory gene HAP2. We report here the isolation and characterization of a second UAS2 regulatory gene, HAP3. Like mutations in HAP2, a mutation in HAP3 abolishes the activity of UAS2 and prevents growth on nonfermentable carbon sources. The HAP3 gene was cloned and, surprisingly, was found to encode two divergently transcribed, overlapping transcripts: a 570-base RNA and a 3-kilobase (kb) RNA. Chromosomal disruption experiments defined the critical region for HAP3 function to a 1.3-kb segment in which the two transcripts overlap. Analysis of the HAP3 DNA sequence showed that the 570-base transcript could encode a protein of 144 amino acids. Synthesis of the 144-amino-acid protein under regulatory control in vivo demonstrated that this protein is essential for activity of UAS2 as well as for growth on nonfermentable carbon sources. The largest open reading frame in the critical region of the 3-kb transcript is only 86 amino acids. Using site-directed mutagenesis, we demonstrated that the 86-amino-acid open reading frame was not involved in UAS2 activity. The possible role of this 3-kb antisense RNA in HAP3 expression or function is discussed.
ATR1, a Saccharomyces cerevisiae gene encoding a transmembrane protein required for aminotriazole resistance.
Authors: Kanazawa S , Driscoll M , Struhl K
Published: September 2015

In Saccharomyces cerevisiae, 3-amino-1,2,4-triazole (aminotriazole) competitively inhibits the activity of imidazoleglycerolphosphate dehydratase, the product of the HIS3 gene. Wild-type strains are able to grow in the presence of 10 mM aminotriazole because they induce the level of imidazoleglycerolphosphate dehydratase. However, strains containing gcn4 mutations are unable to grow in medium containing aminotriazole because they lack the GCN4 transcriptional activator protein necessary for the coordinate induction of HIS3 and other amino acid biosynthetic genes. Here, we isolated a new gene, designated ATR1, which when present in multiple copies per cell allowed gcn4 mutant strains to grow in the presence of aminotriazole. In wild-type strains, multiple copies of ATR1 permitted growth at extremely high concentrations of aminotriazole (80 mM), whereas a chromosomal deletion of ATR1 caused growth inhibition at very low concentrations (5 mM). When radioactive aminotriazole was added exogenously, cells with multiple copies of ATR1 accumulated less aminotriazole than wild-type cells, whereas cells with the atr1 deletion mutation retained more aminotriazole. Unlike the mammalian mdr or yeast PDR genes that confer resistance to many drugs, ATR1 appears to confer resistance only to aminotriazole. Genetic analysis, mRNA mapping, and DNA sequencing revealed that (i) the primary translation product of ATR1 contains 547 amino acids, (ii) ATR1 transcription is induced by aminotriazole, and (iii) the ATR1 promoter region contains a binding site for the GCN4 activator protein. The deduced amino acid sequence suggests that ATR1 protein is very hydrophobic with many membrane-spanning regions, has several potential glycosylation sites, and may contain an ATP-binding site. We suggest that ATR1 encodes a membrane-associated component of the machinery responsible for pumping aminotriazole (and possibly other toxic compounds) out of the cell.
Premature translation termination mediates triosephosphate isomerase mRNA degradation.
Authors: Daar I O , Maquat L E
Published: September 2015

We characterized an anemia-inducing mutation in the human gene for triosephosphate isomerase (TPI) that resulted in the production of prematurely terminated protein and mRNA with a reduced cytoplasmic half-life. The mutation converted a CGA arginine codon to a TGA nonsense codon and generated a protein of 188 amino acids, instead of the usual 248 amino acids. To determine how mRNA primary structure and translation influence mRNA stability, in vitro-mutagenized TPI alleles were introduced into cultured L cells and analyzed for their effect on TPI RNA metabolism. Results indicated that mRNA stability is decreased by all nonsense and frameshift mutations. To determine the relative contribution of the changes in mRNA structure and translation to the altered half-life, the effects of individual mutations were compared with the effects of second-site reversions that restored translation termination to normal. All mutations that resulted in premature translation termination reduced the mRNA half-life solely or mainly by altering the length of the mRNA that was translated. The only mutation that altered translation termination and that reduced the mRNA half-life mainly by affecting the mRNA structure was an insertion that shifted termination to a position downstream of the normal stop codon.
Nematode repetitive DNA with ARS and segregation function in Saccharomyces cerevisiae.
Authors: Felsenstein K M , Emmons S W
Published: September 2015

Several members of a repetitive DNA family in the nematode Caenorhabditis elegans have been shown to express ARS and centromeric function in Saccharomyces cerevisiae. The repetitive family, denoted CeRep3, consists of dispersed repeated elements about 1 kilobase in length, present 50 to 100 times in the nematode genome. Three elements were sequenced and found to contain DNA sequences homologous to yeast ARS and CEN consensus sequences. Nematode DNA segments containing these repeats were tested for ARS and CEN (or SEG) function after ligation to shuttle vectors and introduction into yeast cells. Such nematode segments conferred ARS function to the plasmid, as judged by an increased frequency of transformation compared with control plasmids without ARS function. Some, but not all, also conferred to the plasmid increased mitotic stability, increased frequency of 2+:2- segregation in meiosis, and decreased plasmid copy number. These effects are similar to those of yeast centromeric DNA. In view of these results, we suggest that the CeRep3 repetitive family may have replication and centromeric functions in C. elegans.
Constitutive expression of a c-myb cDNA blocks Friend murine erythroleukemia cell differentiation.
Authors: Clarke M F , Kukowska-Latallo J F , Westin E , Smith M , Prochownik E V
Published: September 2015

A full-length human c-myb cDNA clone has been isolated from a CCRF-CEM leukemia cell cDNA library. The plasmid vector contains simian virus 40-derived promotor, splice, and polyadenylation sequences as well as a transcription unit for a dihydrofolate reductase cDNA. We have introduced this construct into Friend erythroleukemia (F-MEL) cells and have isolated a number of clones which contain intact and transcriptionally active human c-myb sequences. F-MEL clones expressing the highest levels of the human c-myb mRNA differentiate poorly in response to dimethyl sulfoxide. Two clones which initially expressed low levels of human c-myb transcripts and which differentiated normally were subsequently inhibited in their ability to differentiate when grown in successively higher concentrations of methotrexate, due to amplification and enhanced expression of plasmid sequences. The inhibitory effect on F-MEL differentiation appeared to be independent of the early decline in c-myc transcripts which were normally regulated in all cases examined. Our results indicate that constitutive expression of a nontruncated human c-myb cDNA can exert profound effects on erythroid differentiation and argue for a causal role of c-myb in the F-MEL differentiation process.
Chicken homolog of the mos proto-oncogene.
Authors: Schmidt M , Oskarsson M K , Dunn J K , Blair D G , Hughes S , Propst F , Vande Woude G F
Published: September 2015

We compared the sequence and properties of the chicken mos homolog with the previously characterized mouse and human c-mos genes. Sequence analysis revealed one major open reading frame of 1,047 base pairs encoding a protein of 349 amino acids. Both the nucleotide sequence and the deduced amino acid sequence showed 62% overall homology to mouse and human c-mos, but regions of higher conservation (approximately 70%) occurred in the putative ATP-binding and kinase domains. We detected mos transcripts by Northern (RNA) analyses in RNA prepared from chicken and quail ovaries and testes. Evidence for low levels of mos RNA expression in adult chicken heart, kidney, and spleen and in the entire embryo was obtained by S1 nuclease protection experiments. In contrast to the low transforming efficiency of human c-mos when linked to a mouse retroviral long terminal repeat element, chicken c-mos transformed NIH 3T3 cells as efficiently as mouse c-mos did. We also show that chicken primary embryo fibroblasts were morphologically altered when infected with an avian retroviral vector containing the chicken c-mos coding region.
Suppression of chromosomal mutations affecting M1 virus replication in Saccharomyces cerevisiae by a variant of a viral RNA segment (L-A) that encodes coat protein.
Authors: Uemura H , Wickner R B
Published: September 2015

For the maintenance of "killer" M1 double-stranded RNA in Saccharomyces cerevisiae, more than 30 chromosomal genes are required. The requirement for some of these genes can be completely suppressed by a cytoplasmic element, [B] (for bypass). We have isolated a mutant unable to maintain [B] (mab) and found that it is allelic to MAK10, one of the three chromosomal MAK genes required for the maintenance of L-A. The heat curing of [B] always coincided with the loss of L-A. To confirm that [B] is located on L-A, we purified viral particles containing either L-A or M1 from strains with or without [B] activity and transfected these purified particles into a strain which did not have either L-A or M1. The transfectants harboring L-A and M1 from a [B] strain showed the [B] phenotype, but the transfectants with L-A and M1 from a [B-o] strain did not show the [B] phenotype. Furthermore, the transfectants having L-A from a [B] strain and M1 from a [B-o] strain also showed the [B] phenotype. Therefore, we concluded that [B] is a property of a variant of L-A. In the transfection experiment, we also proved that the superkiller phenotype of the [B] strain is a property of L-A and that L-A with [B] activity can maintain a higher copy number of M1 regardless of the source of M1 viruslike particles. These data suggest that MAK genes whose mutations are suppressed by [B] are concerned with the protection of M1 (+) single-stranded RNA or the formation of M1 viruslike particles and that an L-A with more efficient production of M1 viruslike particles can completely dispense with the requirement for those MAK genes.
Localization of a repressive sequence contributing to B-cell specificity in the immunoglobulin heavy-chain enhancer.
Authors: Weinberger J , Jat P S , Sharp P A
Published: September 2015

The immunoglobulin heavy-chain enhancer is a cis-acting element which activates transcription of nearby genes only in cells of the lymphoid lineage. To identify the minimal sequences necessary to impart cell type transcriptional specificity, we tested the activity of several deletions and internal mutations in the mu enhancer. Experiments involving measurement of both chloramphenicol acetyltransferase activity and RNA levels indicated the presence of a dominant repressor element within the mu enhancer. This repressive activity was detected in fibroblasts but not in myeloma cells. Removal or disruption of this repressor element revealed the presence of elements within the mu enhancer that activate transcription in fibroblasts. Thus, enhancer tissue specificity is in part due to the composite of both constitutive activation and cell-type-specific repressive activity. The possible biological roles of this phenomenon are discussed.
Localization of a highly divergent mammalian testicular alpha tubulin that is not detectable in brain.
Authors: Hecht N B , Distel R J , Yelick P C , Tanhauser S M , Driscoll C E , Goldberg E , Tung K S
Published: September 2015

Sequence analysis of a mouse testicular alpha-tubulin partial cDNA, pRD alpha TT1, reveals an isotype that differs from both the somatic and the predominant testicular alpha tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-Gly-Glu-Glu, found in the carboxyl termini of many alpha tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis alpha-tubulin cDNA, we have have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular alpha tubulin that is not detectable in brain alpha tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple alpha-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.
A retrovirus expressing the 12S adenoviral E1A gene product can immortalize epithelial cells from a broad range of rat tissues.
Authors: Cone R D , Grodzicker T , Jaramillo M
Published: September 2015

An epithelial cell-transforming virus could be of great use, both in the culture of epithelial cell lines and in the study of carcinogenesis. Since the adenoviral E1A gene has been shown to partially transform some epithelial cells from primary rat cell cultures, we constructed retrovirus vectors containing either the 12S or 13S E1A cDNA sequences to facilitate the transfer of these genes into a variety of primary cell types. The 12S E1A virus induced proliferation and immortalization of epithelial cells in rat kidney, liver, heart, pancreas, and thyroid primary cultures. In the two cases tested, heart and liver cultures, E1A-immortalized cells were nontumorigenic, but could be completely transformed by subsequent introduction of the ras oncogene. To our surprise, the 13S virus had a greatly reduced immortalization potential. We discuss these data in light of the model of Spindler et al. (K. R. Spindler, C. Y. Eng, and A.-J. Berk, J. Virol. 53:742-750, 1985), in which the 12S E1A protein is required for the complete induction of the cellular DNA replication machinery in the quiescent human epithelial cells in which adenoviruses normally replicate.
Functional characterization of a pyrimidine-rich element in the 5'-noncoding region of the yeast iso-1-cytochrome c gene.
Authors: McNeil J B
Published: September 2015

A predominantly pyrimidine-rich sequence (purine in the template strand, 32 of 37 bases) is located between a functional TATA element and the corresponding transcription start site region of the Saccharomyces cerevisiae iso-1-cytochrome c (CYC1) gene. By using linker deletions and gene fusion techniques, the functional characteristics of this pyrimidine sequence were examined. Results indicate that the function of this element is to limit the accumulation of full-length mRNAs with 5' ends which map upstream of the pyrimidine-rich sequence. Data suggest that the 5'-noncoding region of the CYC1 gene possesses signals for mRNA 3'-end processing.
Functional analysis of the sea urchin U7 small nuclear RNA.
Authors: Gilmartin G M , Schaufele F , Schaffner G , Birnstiel M L
Published: September 2015

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.
The stability of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells varies with growth rate.
Authors: Rao T R , Slobin L I
Published: September 2015

The decay rates of eucaryotic elongation factor Tu (eEF-Tu) mRNA and eucaryotic initiation factor 4A (eIF-4A) mRNA in Friend erythroleukemia (FEL) cells were determined under several different growth conditions. In FEL cells which were no longer actively dividing (stationary phase), eEF-Tu mRNA was found to be rather stable, with a t1/2 of about 24 h. In rapidly growing FEL cells eEF-Tu mRNA was considerably less stable, with a t1/2 of about 9 h. In both cases a single rate of mRNA decay was observed. However, when stationary-phase cells resumed growth after treatment with fresh medium, we observed that eEF-Tu mRNA decay followed a biphasic process. The faster of the two decay rates involved approximately 50% of the eEF-Tu mRNA and had a t1/2 of about 1 h. The decay rates for eIF-4A (t1/2 = 2 h) and total poly(A)+ RNA (t1/2 = 3 h) were unaffected by changes in growth conditions. The t1/2 for polysomal eEF-Tu mRNA was found to be about 8 h when stationary FEL cells were treated with fresh medium. Previous work in this laboratory has shown (T. R. Rao and L. I. Slobin, Mol. Cell. Biol. 7:687-697, 1987) that when FEL cells are allowed to grow to stationary phase, approximately 60% of the mRNA for eEF-Tu is found in a nontranslating postpolysomal messenger ribonucleoprotein (mRNP) particle. eEF-Tu mRNP was rapidly cleared from stationary cells after treatment with fresh medium. The data presented in this report indicate that the stability of eEF-Tu mRNP is rapidly altered and the particle is targeted for degradation when stationary FEL cells resume growth.
Two functional estrogen response elements are located upstream of the major chicken vitellogenin gene.
Authors: Burch J B , Evans M I , Friedman T M , O'Malley P J
Published: September 2015

We used a transient-expression assay to identify two estrogen response elements (EREs) associated with the major chicken vitellogenin gene (VTGII). Each element was characterized by its ability to confer estrogen responsiveness when cloned in either orientation next to a chimeric reporter gene consisting of the herpes simplex virus thymidine kinase promoter and the chloramphenicol acetyl transferase-coding region. Deletion analyses indicated that sequences necessary for the distal ERE resided within the region from -626 to -613 (nucleotide positions relative to the VTGII start site) whereas those necessary for the proximal ERE were within the region from -358 to -335. These distal and proximal elements contain, respectively, a perfect copy and an imperfect copy of the 13-base-pair sequence that is an essential feature of the EREs associated with two frog vitellogenin genes. These chicken VTGII EREs mapped near regions that were restructured at the chromatin level when the endogenous VTGII gene was expressed in the liver in response to estradiol. These data suggest a model for the tissue-specific expression of this estrogen-responsive gene.
Expression of the yeast UB14 gene increases in response to DNA-damaging agents and in meiosis.
Authors: Treger J M , Heichman K A , McEntee K
Published: September 2015

The polyubiquitin gene, UB14, of Saccharomyces cerevisiae is regulated by a variety of environmental stresses and physiological conditions. After exposure of rapidly growing yeast cells to DNA-damaging agents (4-nitroquinoline-1-oxide and N-methyl-N'-nitro-N-nitrosoguanidine), intracellular levels of UB14 transcript increased rapidly. Induction of UB14 transcripts occurred within 30 to 60 min of exposure to 4-nitroquinoline-1-oxide in RAD+, rad52, and rad6 repair-deficient yeast strains. In high-density RAD+ cultures, the effect of alkylating agents on UB14 transcript levels is attenuated, in part because of significant increases in the basal level of this message in untreated cells. We also observed that the levels of UB14 transcripts increased significantly when diploid cells were exposed to sporulation conditions. Maximal levels of UB14 transcripts were reached after 6 to 8 h in sporulation medium. Accumulation of UB14 transcripts occurred in a/alpha diploids that undergo meiosis but not in asporogenous alpha/alpha diploids exposed to the same nutritional conditions.
Molecular cloning and biological characterization of a human gene, ERCC2, that corrects the nucleotide excision repair defect in CHO UV5 cells.
Authors: Weber C A , Salazar E P , Stewart S A , Thompson L H
Published: September 2015

The UV-sensitive Chinese hamster ovary (CHO) cell line UV5, which is defective in the incision step of nucleotide excision repair, was used to identify and clone a complementing human gene, ERCC2, and to study the repair process. Genomic DNA from a human-hamster hybrid cell line was sheared and cotransferred with pSV2gpt plasmid DNA into UV5 cells to obtain five primary transformants. Transfer of sheared DNA from one primary transformant resulted in a secondary transformant expressing both gpt and ERCC2. The human repair gene was identified with a probe for Alu-family repetitive sequences. For most primary, secondary, and cosmid transformants, survival after UV exposure showed a return to wild-type levels of resistance. The levels of UV-induced mutation at the aprt locus for secondary and cosmid transformants varied from 50 to 130% of the wild-type level. Measurements of the initial rate of UV-induced strand incision by alkaline elution indicated that, whereas the UV5 rate was 3% of the wild-type level, rates of cosmid-transformed lines were similar to that of the wild type, and the secondary transformant rate was about 165% of the wild-type rate. Analysis of overlapping cosmids determined that ERCC2 is between 15.5 and 20 kilobases and identified a closely linked gpt gene. Cosmids were obtained with functional copies of both ERCC2 and gpt. ERCC2 corrects only the first of the five CHO complementation groups of incision-defective mutants.
Organization of multiple regulatory elements in the control region of the adenovirus type 2-specific VARNA1 gene: fine mapping with linker-scanning mutants.
Authors: Railey J F , Wu G J
Published: September 2015

The adenovirus type 2-specific virus-associated RNA 1 (VARNA1) gene is transcribed by eucaryotic RNA polymerase III. Previous studies using deletion mutants for transcription have shown that the VARNA1 gene has a large control region which is composed of several regulatory elements. Twenty-five exact linker-scanning mutations in the control region, from -33 to +77, of this gene were used for definition of the number and boundaries of these elements. The effects of these mutations on transcription and competition for transcription factors in human KB cell extracts revealed five positive regulatory elements. The essential element, which coincided with the B block, was absolutely required for both transcription and formation of stable complexes. A second element, which included the A block, was also required for both transcription and formation of stable complexes. Although this element is not as essential as the B-block element, together with the B-block element it may be necessary for formation of the most basal form of transcription machinery. Therefore, these two elements are the promoter elements in this gene. In addition, one possible element in the interblock region and two elements in the 5' flanking region were also required for efficient transcription, but they were moderately required for formation of stable complexes. Transcription of these mutants and the wild-type gene using an extract of 293 cells was stimulated at least threefold over that with the KB cell extract, as expected. Similar regulatory elements of this gene were revealed, however, when the 293 cell extract was used for transcription of these mutants, suggesting that the E1A-mediated specific transcription factors act on the transcription machinery in a sequence-nonspecific manner.
Concerted evolution of human amylase genes.
Authors: Gumucio D L , Wiebauer K , Caldwell R M , Samuelson L C , Meisler M H
Published: September 2015

Cosmid clones containing 250 kilobases of genomic DNA from the human amylase gene cluster have been isolated. These clones contain seven distinct amylase genes which appear to comprise the complete multigene family. By sequence comparison with the cDNAs, we have identified two pancreatic amylase genes and three salivary amylase genes. Two truncated pseudogenes were also recovered. Intergenic distances of 17 to 22 kilobases separate the amylase gene copies. Within the past 10 million years, duplications, gene conversions, and unequal crossover events have resulted in a very high level of sequence similarity among human amylase gene copies. To identify sequence elements involved in tissue-specific expression and hormonal regulation, the promoter regions of the human amylase genes were sequenced and compared with those of the corresponding mouse genes. The promoters of the human and mouse pancreatic amylase genes are highly homologous between nucleotide -160 and the cap site. Two sequence elements thought to influence pancreas-specific expression of the rodent genes are present in the human genes. In contrast, similarity in the 5' flanking sequences of the salivary amylase genes is limited to several short sequence elements whose positions and orientations differ in the two species. Some of these sequence elements are also associated with other parotid-specific genes and may be involved in their tissue-specific expression. A glucocorticoid response element and a general enhancer element are closely associated in several of the amylase promoters.