bluuebunny/crossref_metadata_2025 huggingface dataset:
Secreted int-1 protein is associated with the cell surface.
Authors: Papkoff J , Schryver B
Published: October 2015

The murine int-1 proto-oncogene has been implicated in neural development and, when transcriptionally activated by mouse mammary tumor virus, contributes to the genesis of mammary tumors. To understand the function of the int-1 gene product in these processes, we have characterized the biochemical properties of int-1 protein expressed in a neuroendocrine cell line transfected with int-1 cDNA. Here we provide evidence that int-1 protein is secreted and associates with the cell surface. int-1 protein was very efficiently processed and secreted through the constitutive secretory pathway, although no int-1 protein could be immunoprecipitated from the culture medium. Treatment with suramin effectively released mature int-1 proteins into the culture fluid, which suggests that secreted int-1 protein associates with the cell surface or extracellular matrix. We have also shown directly, by radioiodination of intact cells and by surface antibody adsorption, that secreted int-1 proteins can be detected on the cell surface. These data support a model in which int-1 protein is secreted and functions locally in cell-to-cell signaling.
Two distinct enzymes contribute to biphasic S6 phosphorylation in serum-stimulated chicken embryo fibroblasts.
Authors: Sweet L J , Alcorta D A , Erikson R L
Published: October 2015

Serum stimulation of quiescent chicken embryo fibroblasts resulted in a time-dependent, biphasic activation of S6 kinase activity. Chromatographic fractionation of serum-stimulated cell lysates resolved two distinct S6 kinase activities. Anti-Xenopus S6 kinase II antiserum immunoprecipitated a 90,000-Mr S6 kinase but did not cross-react with a smaller, 65,000-Mr S6 kinase. Phosphopeptide analysis confirmed that the 90,000- and 65,000-Mr proteins were structurally unrelated and established that the 65,000-Mr protein isolated by the current protocol was the same serum-stimulated chicken embryo fibroblast S6 kinase as that previously characterized (J. Blenis, C. J. Kuo, and R. L. Erikson, J. Biol. Chem. 262:14373-14376, 1987). These results demonstrate the contribution of two distinct S6 kinases to total serum-stimulated ribosomal protein S6 phosphorylation.
Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription.
Authors: Ponticelli A S , Struhl K
Published: October 2015

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.
GAL4 protein: purification, association with GAL80 protein, and conserved domain structure.
Authors: Chasman D I , Kornberg R D
Published: October 2015

Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.
The yeast KRE5 gene encodes a probable endoplasmic reticulum protein required for (1----6)-beta-D-glucan synthesis and normal cell growth.
Authors: Meaden P , Hill K , Wagner J , Slipetz D , Sommer S S , Bussey H
Published: October 2015

Yeast kre mutants define a pathway of cell wall (1----6)-beta-D-glucan synthesis, and mutants in genes KRE5 and KRE6 appear to interact early in such a pathway. We have cloned KRE5, and the sequence predicts the product to be a large, hydrophilic, secretory glycoprotein which contains the COOH-terminal endoplasmic reticulum retention signal, HDEL. Deletion of the KRE5 gene resulted in cells with aberrant morphology and extremely compromised growth. Suppressors to the KRE5 deletions arose at a frequency of 1 in 10(7) to 1 in 10(8) and permitted an analysis of deletions which were found to contain no alkali-insoluble (1----6)-beta-D-glucan. These results indicate a role for (1----6)-beta-D-glucan in normal cell growth and suggest a model for sequential assembly of (1----6)-beta-D-glucan in the yeast secretory pathway.
Evidence that replication initiates at only some of the potential origins in each oligomeric form of bovine papillomavirus type 1 DNA.
Authors: Schvartzman J B , Adolph S , Martín-Parras L , Schildkraut C L
Published: October 2015

In a subclone of ID13 mouse fibroblasts latently infected with bovine papillomavirus type 1 (BPV-1) DNA, the viral genome occurred as a mixture of extrachromosomal circular monomers and oligomers. Multiple copies were also associated with the host cell genome, predominantly at a single site in a head-to-tail tandem array. We examined the replicative intermediates of extrachromosomal forms of BPV-1 DNA by using two-dimensional gel electrophoresis. The results obtained indicate that initiation of DNA replication occurred near the center of the EcoRI-BamHI 5.6-kilobase fragment. In some molecules, however, this fragment was replicated from one end to the other by means of a single fork initiated elsewhere. Termination also occurred within this fragment. The EcoRI-BamHI 2.3-kilobase fragment replicated as a DNA molecule containing a termination site for DNA replication and also by means of a single fork traversing the fragment from one end to the other. Thus, replication forks proceeded through these fragments in different manners, apparently depending on whether they were part of a monomer, a dimer, a trimer, or higher oligomers. These observations lead to the conclusion that initiation of DNA replication in BPV-1 DNA takes place at or close to plasmid maintenance sequence 1. From this point, replication proceeds bidirectionally and termination occurs approximately 180 degrees opposite the origin. The results obtained are consistent with one or more replication origins being quiescent in BPV-1 DNA oligomers.
Molecular and developmental characterization of the heat shock cognate 4 gene of Drosophila melanogaster.
Authors: Perkins L A , Doctor J S , Zhang K , Stinson L , Perrimon N , Craig E A
Published: October 2015

The Drosophila heat shock cognate gene 4 (hsc4), a member of the hsp70 gene family, encodes an abundant protein, hsc70, that is more similar to the constitutively expressed human protein than the Drosophila heat-inducible hsp70. Developmental expression revealed that hsc4 transcripts are enriched in cells active in endocytosis and those undergoing rapid growth and changes in shape.
A and T homopolymeric stretches mediate a DNA inversion in Plasmodium falciparum which results in loss of gene expression.
Authors: Pologe L G , de Bruin D , Ravetch J V
Published: October 2015

Ring-infected erythrocyte surface antigen-negative isolates of Plasmodium falciparum demonstrate a complex DNA rearrangement with inversion of 5' coding sequences, deletion of upstream and flanking sequences, and healing of the truncated chromosome by telomere addition. An inversion intermediate that results in the telomeric gene structure for RESA has been identified in the pathway. This inversion creates a mitotically stable substrate for the sequence-specific addition of telomere repeats at the deletion breakpoint.
Transformation of NIH 3T3 cells by overexpression of the normal coding sequence of the rat neu gene.
Authors: Di Marco E , Pierce J H , Knicley C L , Di Fiore P P
Published: October 2015

While the normal human erbB-2 gene is potently transforming when overexpressed in NIH 3T3 cells, its rat homolog, the neu gene, seems to acquire transforming properties only upon alteration of its coding sequence. In this study, we compared the effects of different levels of expression of normal erbB-2 and neu in NIH 3T3 cells. Our results revealed that the normal rat neu gene acts as a potent oncogene when sufficiently overexpressed in NIH 3T3 cells.
A pharmacologically distinct cyclic AMP receptor is responsible for the regulation of gp80 expression in Dictyostelium discoideum.
Authors: Ma P C , Siu C H
Published: October 2015

The EDTA-resistant cell-cell adhesion expressed at the aggregation stage of Dictyostelium discoideum is mediated by a cell surface glycoprotein of Mr 80,000 (gp80). The expression of gp80 is developmentally regulated by cyclic AMP (cAMP). In vitro nuclear run-on experiments show that transcription of the gp80 gene is initiated soon after the onset of development. The basal level of gp80 transcription is significantly augmented by exogenous cAMP pulses. Interestingly, in analog studies, 2'-deoxy-cAMP, 8-bromo-cAMP, and N6-monobutyryl-cAMP are all capable of inducing a rapid accumulation of gp80 mRNA, suggesting the presence of a unique cAMP receptor that responds equally well to these analogs. To determine whether intracellular cAMP plays a role in the regulation of gp80 expression, caffeine was used to block cAMP-induced receptor-mediated adenylate cyclase activation. Expression of gp80 mRNA was blocked in caffeine-treated cells but could be substantially restored by treatment with exogenous cAMP pulses, suggesting that adenylate cyclase activation is not required. gp80 expression was also examined in the signal transduction mutants synag 7 and frigid A. In both mutants, gp80 was expressed at the basal level. Pulses of cAMP as well as 2'-deoxy-cAMP and N6-monobutyryl-cAMP were capable of restoring the normal level of gp80 expression in synag 7 cells. These results, taken together, indicate bimodal regulation of gp80 expression during development and the involvement of a novel cAMP receptor in the transmembrane signalling pathway that regulates gp80 gene expression.
Chromatin structures of the rat tyrosine aminotransferase gene relate to the function of its cis-acting elements.
Authors: Nitsch D , Stewart A F , Boshart M , Mestril R , Weih F , Schütz G
Published: October 2015

The relationship between DNase I-hypersensitive sites (HSs) and transcriptional enhancers of the rat tyrosine aminotransferase (TAT) gene was examined by comparing HSs in and around the TAT gene with the activity of the corresponding DNA sequences in transient transfection assays. In this manner, we identified two HSs as liver-specific enhancers. Of three hepatoma cell lines examined, only one sustained TAT mRNA levels comparable to those of liver. In this cell line, both enhancers were strongly active, and strong hypersensitivity in chromatin over the enhancers was evident. The other two hepatoma cell lines had reduced levels of TAT mRNA and no or altered hypersensitivity over either the enhancers or the promoter. One of these lines carried a negative regulator of the TAT gene, the tissue specific extinguisher Tse-1. This cell line exhibited all HSs characteristic of the strongly active gene except at the promoter; however, one enhancer was inactive even though hypersensitive in chromatin. In a TAT-nonexpressing cell line, inactivity of both enhancers correlated with absence of the respective HSs. We conclude that although hypersensitivity in chromatin necessarily accompanies cell-type-specific enhancer activity, the occurrence of cell-type-specific HSs does not imply that the underlying sequences harbor enhancers active in transient transfection assays.
Cyclic AMP-dependent protein kinase regulates transcription of the phosphoenolpyruvate carboxykinase gene but not binding of nuclear factors to the cyclic AMP regulatory element.
Authors: Quinn P G , Granner D K
Published: October 2015

We have examined the binding of factors in rat liver nuclear extracts to the phosphoenolpyruvate carboxykinase (PEPCK) gene cyclic AMP (cAMP) response element (CRE) and other CREs and have isolated a rat liver CRE-binding protein (CREBP) cDNA. In addition, we have examined the influence of altering the phosphorylation state of nuclear factors on both CRE binding and in vitro transcription. Specific binding to the PEPCK CRE was measured in a mobility shift assay. CRE sequences of the PEPCK, somatostatin, and glycoprotein hormone alpha subunit genes competed equally for binding of rat liver nuclear factors to the PEPCK CRE, whereas mutant PEPCK CRE sequences did not compete for binding. Oligonucleotides complementary to rat pheochromocytoma CREBP (Gonzalez et al., Nature [London] 337:749-752, 1989) were used to prime rat liver and brain cDNA in the polymerase chain reaction. The predominant CREBP molecule obtained was identical to the rat pheochromocytoma CREBP except for a 14-amino-acid deletion in the N-terminal half that was also present in a human placental cDNA (Hoeffler et al., Science 242:1430-1433, 1988). The regulation of transcription by cAMP was examined by coincubation of rat liver nuclear extract with the purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A). Although binding to the CRE was unaffected, in vitro transcription directed by the PEPCK promoter was stimulated by catalytic subunit, and this effect was blocked by protein kinase inhibitor peptide. In contrast, when nuclear extract was coincubated with phosphatase, there was substantial inhibition of in vitro transcription directed by the PEPCK promoter, but there was no effect on binding to the CRE. The major effects of catalytic subunit were exerted through the CRE, but residual stimulation was evident in promoter fragments containing only the TATA element. These data suggest that factors are bound to the CRE at constitutively high levels and that their capacity for transcriptional activation is regulated by phosphorylation.
Nucleocytoplasmic transport and processing of small nuclear RNA precursors.
Authors: Neuman de Vegvar H E , Dahlberg J E
Published: October 2015

We have analyzed the structures and locations of small nuclear RNA (snRNA) precursors at various stages in their synthesis and maturation. In the nuclei of pulse-labeled Xenopus laevis oocytes, we detected snRNAs that were longer than their mature forms at their 3' ends by up to 10 nucleotides. Analysis of the 5' caps of these RNAs and pulse-chase experiments showed that these nuclear snRNAs were precursors of the cytoplasmic pre-snRNAs that have been observed in the past. Synthesis of pre-snRNAs was not abolished by wheat germ agglutinin, which inhibits export of the pre-snRNAs from the nucleus, indicating that synthesis of these RNAs is not obligatorily coupled to their export. Newly synthesized U1 RNAs could be exported from the nucleus regardless of the length of the 3' extension, but pre-U1 RNAs that were elongated at their 3' ends by more than about 10 nucleotides were poor substrates for trimming in the cytoplasm. The structure at the 3' end was critical for subsequent transport of the RNA back to the nucleus. This requirement ensures that truncated and incompletely processed U1 RNAs are excluded from the nucleus.
Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.
Authors: Muscarella D E , Ellison E L , Ruoff B M , Vogt V M
Published: October 2015

A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains.
The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator.
Authors: Lemaire P , Vesque C , Schmitt J , Stunnenberg H , Frank R , Charnay P
Published: October 2015

The mouse gene Krox-24 is transiently activated during cell cycle reentry. It encodes a protein with three zinc fingers similar to those of the transcription factor Sp1. Here we present a biochemical characterization of the gene products. Krox-24 mRNA is translated into two proteins of 82 and 88 kilodaltons, designated p82Krox-24 and p88Krox-24, respectively. p82Krox-24 is initiated at the first AUG codon of the open reading frame, whereas synthesis of p88Krox-24 starts at a non-AUG codon located upstream. Both proteins were synthesized in HeLa cells infected with recombinant vaccinia viruses expressing Krox-24 cDNAs. Under these conditions, they were found phosphorylated on serine residues and glycosylated. The availability of the proteins made possible the determination of the DNA recognition sequence. In vitro, Krox-24 bound specifically to the sequence 5'-GCG(C/G)GGGCG-3'. This sequence is similar but not identical to the Sp1 target sequence. Insertion of an oligomer for the binding site in cis, close to the herpes simplex virus thymidine kinase promoter, rendered this promoter responsive to Krox-24. Krox-24 is therefore a sequence-specific transcriptional activator. Krox-24-binding sites were found upstream of several serum-inducible genes, raising the possibility that Krox-24 is involved in the regulation of these genes.
TEC1, a gene involved in the activation of Ty1 and Ty1-mediated gene expression in Saccharomyces cerevisiae: cloning and molecular analysis.
Authors: Laloux I , Dubois E , Dewerchin M , Jacobs E
Published: October 2015

Ty and Ty-mediated gene expression observed in haploid cells of Saccharomyces cerevisiae depends on several determinants, some of which are required for the expression of haploid-specific genes. We report here the cloning and molecular analysis of TEC1. TEC1 encodes a 486-amino-acid protein that is a trans-acting factor required for full Ty1 expression and Ty1-mediated gene activation. However, mutation or deletion of the TEC1 gene had little effect on total Ty2 transcript levels. Our analysis provides clear evidence that TEC1 is not involved in mating or sporulation processes. Unlike most of the proteins involved in Ty and adjacent gene expression, the product of TEC1 has no known cellular function. Although there was no mating-type effect on TEC1 expression, our results indicate that the TEC1 and the a/alpha diploid controls on Ty1 expression are probably not cumulative.
Transformation of murine bone marrow cells with combined v-raf-v-myc oncogenes yields clonally related mature B cells and macrophages.
Authors: Principato M , Cleveland J L , Rapp U R , Holmes K L , Pierce J H , Morse H C , Klinken S P
Published: October 2015

Murine bone marrow cells infected with replication-defective retroviruses containing v-raf alone or v-myc alone yielded transformed pre-B cell lines, while a retroviral construct containing both v-raf and v-myc oncogenes produced clonally related populations of mature B cells and mature macrophages. The genealogy of these transformants demonstrates that mature myeloid cells were derived from cells with apparent B-lineage commitment and functional immunoglobulin rearrangements. This system should facilitate studies of developmental relationships in hematopoietic differentiation and analysis of lineage determination.
Structural analysis of the mouse mdr1a (P-glycoprotein) promoter reveals the basis for differential transcript heterogeneity in multidrug-resistant J774.2 cells.
Authors: Hsu S I , Cohen D , Kirschner L S , Lothstein L , Hartstein M , Horwitz S B
Published: October 2015

In multidrug-resistant mouse J774.2 cells, the differential overproduction of functionally distinct phosphoglycoprotein isoforms reflects the amplification or transcriptional activation or both of two mdr gene family members, mdr1a and mdr1b. The mdr1a gene is a complex transcriptional unit whose expression is associated with multiple transcript sizes. Independently selected multidrug-resistant J774.2 cell lines differentially overexpress either 4.6- and 5.0-kilobase (kb) or 4.7- and 5.1-kb mdr1a transcripts. However, abundant overproduction of the mdr1a gene product was observed only in cell lines which overexpressed the 4.6- and 5.0-kb mRNAs. In order to determine the basis for mdr1a transcript heterogeneity and the relationship between transcript size and steady-state mdr1a protein levels, genomic and cDNA sequence analyses of the 5' and 3' ends of the mdr1a gene were carried out. Promoter sequence analysis and primer extension mapping indicated that mdr1a transcripts were differentially initiated from two putative promoters to generate either 5.1- and 4.7-kb or 5.0- and 4.6-kb transcripts in four multidrug-resistant J774.2 cell lines. Sequence analysis of 3' cDNA variants and a 3' genomic fragment revealed that the 5.1- and 5.0-kb mRNAs had identical 3'-untranslated regions which differed from those of the 4.7- and 4.6-kb mRNAs as a result of the utilization of a more downstream alternative poly(A) addition signal. Transcript initiation from the putative upstream promoter correlated with a 70 to 85% decrease in steady-state mdr1a protein levels relative to transcript levels. In addition, the identification of putative AP-1 and AP-2 promoter elements suggests a possible role for protein kinase A and protein kinase C in the regulation of mdr1a. The implications of these findings for mdr gene expression and regulation are discussed.
A downstream-element-binding factor facilitates assembly of a functional preinitiation complex at the simian virus 40 major late promoter.
Authors: Ayer D E , Dynan W S
Published: October 2015

Recent work has shown that many promoters recognized by eucaryotic RNA polymerase II contain essential sequences located downstream of the transcriptional initiation site. We show here that the activity of a promoter element centered 28 base pairs downstream of the simian virus 40 major late initiation site appears to be mediated by a DNA-binding protein, which was isolated by affinity chromatography from HeLa cell nuclear extracts. In the absence of the other components of the transcriptional machinery, the protein bound specifically but weakly to its recognition sequence, with a Kd of approximately 10(-8) M. Analysis of kinetic data showed that mutation of the downstream element decreased the number of functional preinitiation complexes assembled at the promoter without significantly altering the time required for half the complexes to assemble. This suggests that in the absence of the downstream activating protein, preinitiation complexes are at least partially assembled but are not transcriptionally competent.
Structures of human genes coding for cytokine LD78 and their expression.
Authors: Nakao M , Nomiyama H , Shimada K
Published: October 2015

LD78 is a member of a newly identified superfamily of small inducible proteins involved in inflammatory responses, wound healing, and tumorigenesis. Southern blot analysis of the EcoRI-digested human genomic DNAs, using previously isolated LD78 cDNA as a probe, showed that in each individual there are 4.2- and 4.8-kilobase-pair (kb) fragments and that some have an additional 6.5-kb fragment. The 4.2-kb fragment contained genomic DNA sequences corresponding to the LD78 cDNA and was named the LD78 alpha gene. The 4.8-kb fragment contained similar sequences, showing 94% homology to the LD78 alpha gene, and was named the LD78 beta gene. The LD78 alpha gene was present in a single or a few copies per haploid genome, whereas the copy number of the LD78 beta gene and of the 6.5-kb fragment hybridizable to LD78 cDNA varied among the samples tested. Treatment of human myeloid cell lines HL-60 and U937 with phorbol 12-myristate 13-acetate (PMA) increased within 2 h cellular levels of the RNA hybridizable to LD78 cDNA. The human glioma cell line U105MG and primary culture of human fibroblasts also expressed the hybridizable RNA in response to PMA. Addition of cycloheximide had no apparent effect on this response in U937 cells and inhibited the response in fibroblasts, whereas it stimulated the response in HL-60 and U105MG cells. mRNA phenotyping experiments revealed that the LD78 alpha and LD78 beta genes were both transcribed in PMA-stimulated U937 cells.
Structure and transcription of the mouse erythropoietin receptor gene.
Authors: Youssoufian H , Zon L I , Orkin S H , D'Andrea A D , Lodish H F
Published: October 2015

The complete gene encoding the mouse erythropoietin receptor was isolated by using a cDNA probe derived from a mouse erythroleukemia (MEL) cell library. The gene spans approximately 5 kilobases and is present in a single copy per haploid genome. It contains eight exons, and the nucleotide sequence of the coding region from the genomic DNA is identical to the sequence of one of the MEL cDNA clones except for a single amino acid substitution (Leu----Val) at codon 163. There is a cluster of three major transcriptional start sites approximately 150 nucleotides upstream of the initiator ATG codon which is conserved in erythropoietin-dependent and -independent erythroleukemic cells, in MEL cells at different stages of differentiation, and in normal bone marrow cells. The promoter region contains a potential binding site for Sp1, erythroid-specific transcription factor GF-1, and several CACCC boxes, but not typical TATA or CAAT sequences. A fusion gene containing 452 nucleotides of 5' noncoding sequence linked to a promoterless human growth hormone gene directed the transcription of the latter in MEL cells but not in mouse fibroblasts, T cells, B cells, or macrophagelike cells, suggesting that this promoter functions in an erythroid-specific manner.
Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe.
Authors: Sunnerhagen P , Seaton B L , Nasim A , Subramani S
Published: October 2015

We have cloned the rad1 gene of Schizosaccharomyces pombe by complementation of the rad1-1 mutant, which is deficient in DNA repair and recombination. The coding region of the gene is 582 base pairs long and contains no introns. The predicted product is a strongly acidic, 22-kilodalton protein containing 194 amino acid residues. This gene does not exhibit significant homology to any other known repair gene. The major transcription start site is at 27 base pairs upstream of the putative start codon. Insertion mutagenesis revealed that besides the coding region, at least 151 base pairs of 5'-flanking sequence are required for full complementing activity. A strain carrying a null allele of rad1 was constructed and found to have a phenotype closely similar to that of the rad1-1 mutant. Expression in Escherichia coli of the coding region yielded a protein product of a size close to that predicted from the DNA sequence. This product reacted with antibodies raised against a synthetic peptide with a sequence from that predicted for the protein product. We have localized the rad1 gene to NotI fragment E of the S. pombe genome.
Identification of a G1-S-phase-regulated region in the human thymidine kinase gene promoter.
Authors: Roehl H H , Conrad S E
Published: October 2015

We have identified a regulatory region in the human thymidine kinase gene promoter. A set of promoter deletion mutants was constructed, linked to the bacterial neomycin resistance gene, and stably transfected into Rat3 cells. It was shown that the region between 135 and 67 base pairs upstream of the cap site is required for conveying G1-S-phase regulation to the linked neo gene. In addition, primer extension assays demonstrated that the same transcriptional start sites were used in G1- and S-phase cells and in the various deletion mutants tested.
Synergy between the NF-E1 erythroid-specific transcription factor and the CACCC factor in the erythroid-specific promoter of the human porphobilinogen deaminase gene.
Authors: Frampton J , Walker M , Plumb M , Harrison P R
Published: October 2015

A 114-base-pair promoter fragment of the human porphobilinogen deaminase gene functioned in an erythroid-specific manner in transient transfection experiments. Site-directed mutagenesis of the binding site for the erythroid-specific transcription factor (NF-E1) or an adjacent CACCC motif abolished the promoter activity. Increasing the spacing between these sites progressively reduced promoter activity, but there was no evidence that a critical alignment of the two factors on the DNA helix was required.
Identification of an octamer-binding site in the human kappa light-chain enhancer.
Authors: Nelms K , Van Ness B
Published: October 2015

Octamer motifs contribute to the function and tissue specificity of immunoglobulin heavy- and light-chain gene promoters and the heavy-chain enhancer. A variant octamer-binding site within a conserved region of the human kappa light-chain gene enhancer which contributes to the function of this enhancer has been identified.
Periodic biosynthesis of the human M-phase promoting factor catalytic subunit p34 during the cell cycle.
Authors: McGowan C H , Russell P , Reed S I
Published: October 2015

The product of the CDC2Hs gene is the protein kinase subunit of the M-phase promoting factor, which is required for entry into mitosis. The activity of this kinase is regulated in a cell cycle-dependent manner by reversible phosphorylation and through association with other proteins. We report here that in HeLa cells, the abundance of the CDC2Hs mRNA and the rate of synthesis of the encoded protein, p34, vary in a cell cycle-dependent manner.
Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein.
Authors: Mitts M R , Grant D B , Heideman W
Published: October 2015

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remains unknown.
Transcriptional potentiation of the vitellogenin B1 promoter by a combination of both nucleosome assembly and transcription factors: an in vitro dissection.
Authors: Corthésy B , Léonnard P , Wahli W
Published: October 2015

The Xenopus laevis vitellogenin B1 promoter was assembled into nucleosomes in an oocyte extract. Subsequent RNA polymerase II-dependent transcription from these DNA templates fully reconstituted in chromatin in a HeLa nuclear extract was increased 50-fold compared with naked DNA. Remarkably, under specific conditions, production of a high level of transcripts occurred at very low DNA (1 ng/microliter) and HeLa nuclear protein (1.6 micrograms/microliters) concentrations. When partially reconstituted templates were used, transcription efficiency was intermediate between that of fully reconstituted and naked DNA. These results implicate chromatin in the process of the transcriptional activation observed. Depletion from the oocyte assembly extract of an NF-I-like factor which binds in the promoter region upstream of the TATA box (-114 to -101) or deletion from the promoter of the region interacting with this factor reduced the transcriptional efficiency of the assembled templates by a factor of 5, but transcription of these templates was still 10 times higher than that of naked DNA. Together, these results indicate that the NF-I-like factor participates in the very efficient transcriptional potentiation of the vitellogenin B1 promoter which occurs during nucleosome assembly.
Differential trans activation associated with the muscle regulatory factors MyoD1, myogenin, and MRF4.
Authors: Yutzey K E , Rhodes S J , Konieczny S F
Published: October 2015

Expression of the mammalian muscle regulatory factors MyoD1, myogenin, and MRF4 will convert C3H10T1/2 fibroblasts to stable muscle cell lineages. Recent studies have shown that MyoD1 and myogenin also trans-activate expression of a number of cotransfected contractile protein genes, suggesting that these muscle regulatory factors are involved in controlling terminal differentiation events. The extent and specificity of trans activation by the muscle regulatory factors, however, have not been compared directly. In this study, we found that MyoD1, myogenin, and MRF4 exhibited different trans-activation capacities. In contrast to MyoD1 and myogenin, MRF4 was inefficient in trans-activating most of the genes tested, although conversion of C3H10T1/2 fibroblasts to a myogenic lineage was observed at similar frequencies with all three factors. Addition of basic fibroblast growth factor to cells expressing exogenous muscle regulatory factors inhibited the transcriptional activation of cotransfected genes, demonstrating that MyoD1, myogenin, or MRF4 proteins alone are not sufficient to produce a terminally differentiated phenotype. In all cases, trans activation was dependent on signal transduction pathways that are regulated by fibroblast growth factor. Our observations, coupled with previous studies showing differences in the temporal expression and protein structure of MyoD1, myogenin, and MRF4, suggest that the individual members of the muscle regulatory factor family have distinct biological roles in controlling skeletal muscle development.
The cytokine response element of the rat alpha 1-acid glycoprotein gene is a complex of several interacting regulatory sequences.
Authors: Won K A , Baumann H
Published: October 2015

Expression of the rat alpha 1-acid glycoprotein gene is stimulated by interleukin-1 (IL-1) and interleukin-6 (IL-6) and is synergistically enhanced by the combination of the two. The distal regulatory element (DRE), a 142-base-pair (bp) sequence located 5 kilobase pairs upstream of the transcriptional start site, appears to be crucial for this cytokine response. The cytokine-specific regulatory sequences within the DRE have been identified by inserting individual DRE subregions, selected combinations of these, or a few linker mutated fragments into a plasmid containing an enhancerless simian virus 40 promoter linked to the chloramphenicol acetyltransferase gene. The regulatory activity was determined in transiently transfected human and rat hepatoma cells. The IL-1 response region was confined to the 5'-most 62 bp of the DRE, and its function seemed to depend on at least two separate components. The same region was also responsive to phorbol ester treatment. The IL-6 regulatory function was dependent on a 54-bp sequence located within the 3' half of the DRE. When the IL-1 response region was recombined with the IL-6 regulatory region of the DRE or with IL-6 response elements of other plasma protein genes, a strong cooperative action by IL-1 and IL-6 was achieved. The functional DRE sequences were recognized by nuclear proteins extracted from rat liver and hepatoma cells. However, no cytokine-inducible binding activity was detectable, which suggests that transcriptional regulation through the DRE might be controlled by posttranslational modification of constitutively bound trans-acting factors.
Regulation of the Discoidin I gamma gene in Dictyostelium discoideum: identification of individual promoter elements mediating induction of transcription and repression by cyclic AMP.
Authors: Vauti F , Morandini P , Blusch J , Sachse A , Nellen W
Published: October 2015

We dissected the promoter of the developmentally induced and cyclic AMP-repressed discoidin I gamma gene and identified a sequence element essential for developmental induction. Transfer of the element to an inactive heterologous promoter demonstrated that this sequence is sufficient to confer expression in axenically growing cells and to induce gene activity in development after growth on bacteria. A 16-base-pair sequence within this element was shown to be sufficient for induction in the discoidin promoter context and was used to reactivate different truncated promoter constructs. This led to the localization of an element necessary for down regulation of gene expression by extracellular cyclic AMP.
N-myc mRNA forms an RNA-RNA duplex with endogenous antisense transcripts.
Authors: Krystal G W , Armstrong B C , Battey J F
Published: October 2015

Nuclear runoff transcription studies revealed nearly equivalent sense and antisense transcription across exon 1 of the N-myc locus. Antisense primary transcription initiates at multiple sites in intron 1 and gives rise to stable polyadenylated and nonpolyadenylated transcripts. This pattern of antisense transcription, which is directed by RNA polymerase II, is independent of gene amplification and cell type. The nonpolyadenylated antisense transcripts have 5' ends which are complementary to the 5' ends of the N-myc sense mRNA. We determined, by using an RNase protection technique designed to detect in vivo duplexes, that most of the cytoplasmic nonpolyadenylated antisense RNA exists in an RNA-RNA duplex with approximately 5% of the sense N-myc mRNA. Duplex formation appeared to occur with only a subset of the multiple forms of the N-myc mRNA, with the precise transcriptional initiation site of the RNA playing a role in determining this selectivity. Cloning of each strand of the RNA-RNA duplex revealed that most duplexes included both exon 1 and intron 1 sequences, suggesting that duplex formation could modulate RNA processing by preserving a population of N-myc mRNA which retains intron 1.
Human trk oncogenes activated by point mutation, in-frame deletion, and duplication of the tyrosine kinase domain.
Authors: Coulier F , Kumar R , Ernst M , Klein R , Martin-Zanca D , Barbacid M
Published: October 2015

Malignant activation of the human trk proto-oncogene, a member of the tyrosine protein kinase receptor family, has been implicated in the development of certain human cancers, including colon and thyroid papillary carcinomas. trk oncogenes have also been identified in cultured cells transfected with various DNAs. In this study, we report the characterization of three in vitro-generated trk oncogenes, trk2, trk4, and trk5 (R. Oskam, F. Coulier, M. Ernst, D. Martin-Zanca, and M. Barbacid, Proc. Natl. Acad. Sci. USA 85:2964-2968, 1988), in an effort to understand the spectrum of mutational events that can activate the human trk gene. Nucleotide sequence analysis of cDNA clones of trk2 and trk4 revealed that these oncogenes were generated by a head-to-tail arrangement of two trk tyrosine protein kinase domains connected by a purine-rich region. These oncogenes code for cytoplasmic molecules of 67,000 (p67trk2) and 69,000 (p69trk4) daltons. In contrast, the product of the trk5 oncogene, gp95trk5, is a cell surface glycoprotein of 95,000 daltons. This oncogene was generated by a 153-base-pair in-frame deletion within sequences coding for the extracellular domain of the trk receptor. This activating deletion encompasses a triplet coding for one of the nine cysteine residues that the trk receptor shares with the product of the highly related trkB tyrosine protein kinase gene. Introduction of a single point mutation (TGT----AGT) in this codon resulted in a novel trk oncogene whose product, gp140S345, differs from the nontransforming trk proto-oncogene receptor in a single amino acid residue, Ser-345 instead of Cys-345. These results illustrate that multiple molecular mechanisms, including point mutation, internal deletion, and kinase domain duplication, can result in the malignant activation of the human trk proto-oncogene.
Complementation by BCL2 and C-HA-RAS oncogenes in malignant transformation of rat embryo fibroblasts.
Authors: Reed J C , Haldar S , Croce C M , Cuddy M P
Published: October 2015

The BCL2 (B cell lymphoma/leukemia-2) and C-HA-RAS oncogenes encode membrane-associated proteins of 26 and 21 kilodaltons, respectively. Although RAS proteins have long been known for their ability to bind and hydrolyze GTP, recent investigations suggest that BCL2 encodes a novel GTP-binding protein (S. Haldar, C. Beatty, Y. Tsujimoto, and C. M. Croce, Nature [London] 342:195-198, 1989). Cotransfection of BCL2 and HA-RAS oncogenes resulted in morphological transformation of early-passage rodent fibroblasts, rendering these cells tumorigenic in animals and enabling them to grow in semisolid medium. In contrast, cotransfection of BCL2 with oncogenes that encode nuclear proteins (E1A and C-MYC) did not produce malignant transformation, whereas HA-RAS did complement with these genes. These findings suggest that proteins encoded by oncogenes such as BCL2 and HA-RAS, although having similar subcellular locations and perhaps similar biochemical properties, can regulate distinct complementary pathways involved in cellular transformation.
A DNA signal from the Thy-1 gene defines de novo methylation patterns in embryonic stem cells.
Authors: Szyf M , Tanigawa G , McCarthy P L
Published: October 2015

Although DNA can be extensively methylated de novo when introduced into pluripotent cells, the CpG island in the Thy-1 gene does not become methylated either in the mouse embryo or in embryonic stem cells. A 214-base-pair region near the promoter of the Thy-1 gene protects itself as well as heterologous DNA sequences from de novo methylation. We propose that this nucleotide sequence is representative of a class of important signals that limits de novo methylation in the embryo and establishes the pattern of hypomethylated CpG dinucleotides found in somatic tissues.
The cytomegalovirus enhancer: a pan-active control element in transgenic mice.
Authors: Schmidt E V , Christoph G , Zeller R , Leder P
Published: October 2015

In an effort to identify widely active positive regulatory elements, we have examined the action of the cytomegalovirus enhancer-promoter in transgenic mice. These elements activated expression in 24 of 28 tissues tested. The greatest expression was observed in the heart, kidney, brain, and testis. Maximum expression further localized to specific cells within the heart and kidney.
Serum amyloid A protein-related mRNA expression in herpes simplex virus type 2-transformed hamster cells.
Authors: Gervais C , Suh M
Published: October 2015

A nearly full-length cDNA very similar to murine serum amyloid A protein was cloned from herpes simplex virus type 2-transformed hamster cells. The expression of mRNA was constitutive in these cells and could be superinduced by conditioned medium. Higher rates of nuclear runoff transcription in transformed and induced cells indicated some regulation at the transcriptional level.
Cooperative activation of transcription by bovine papillomavirus type 1 E2 can occur over a large distance.
Authors: Thierry F , Dostatni N , Arnos F , Yaniv M
Published: October 2015

The viral transcriptional factors encoded by the E2 open reading frame bind to the specific DNA sequence elements ACCGNNNNCGGT, allowing activation or repression of transcription. We have analyzed bovine papillomavirus type 1 E2 transactivation using recombinant genes containing E2-binding sites inserted at either 3' or 5' positions relative to the heterologous transcriptional initiation site of the herpes simplex virus thymidine kinase gene. In these hybrid plasmids, strong transactivation required the presence of a minimum of two E2-binding sites in close proximity to the promoter or five binding sites at a distance. The presence of a single E2-binding motif 5', close to the initiation site, increased the efficiency of E2 transactivation from a distance in a more-than-additive manner. Since each E2-binding site bound a dimer of the E2 protein, these experiments suggest that transactivation by E2 requires the interaction between several E2 dimers with other essential transcription factors. This interaction may be facilitated by DNA looping, which would bring E2 molecules close to the promoter.