bluuebunny/crossref_metadata_2025 huggingface dataset:
A direct role for C/EBP and the AP-I-binding site in gene expression linked to adipocyte differentiation.
Authors: Herrera R , Ro H S , Robinson G S , Xanthopoulos K G , Spiegelman B M
Published: October 2015

Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein. Using specific deletions and point mutations, we have shown that at least two distinct sequence elements in the aP2 promoter contribute to the expression of the chloramphenicol acetyltransferase gene in chimeric constructions transfected into adipose cells. An AP-I site at -120, shown earlier to bind Jun- and Fos-like proteins, serves as a positive regulator of chloramphenicol acetyltransferase gene expression in adipocytes but is specifically silenced by adjacent upstream sequences in preadipocytes. Sequences upstream of the AP-I site at -140 (termed AE-1) can function as an enhancer in both cell types when linked to a viral promoter but can stimulate expression only in fat cells in the intact aP2 promoter. The AE-1 sequence binds an adipocyte protein identical or very closely related to an enhancer-binding protein (C/EBP) that has been previously implicated in the regulation of several liver-specific genes. A functional role for C/EBP in the regulation of the aP2 gene is indicated by the facts that C/EBP mRNA is induced during adipocyte differentiation and the aP2 promoter is transactivated by cotransfection of a C/EBP expression vector into preadipose cells. These results indicate that sequences that bind C/EBP and the Fos-Jun complex play major roles in the expression of the aP2 gene during adipocyte differentiation and demonstrate that C/EBP can directly regulate cellular gene expression.
RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus.
Authors: Kolodziej P , Young R A
Published: October 2015

To improve our understanding of RNA polymerase II, the gene that encodes its third-largest subunit, RPB3, was isolated from a lambda gt11 DNA library by using antibody probes. The RPB3 DNA sequence predicts a 318-amino-acid protein whose sequence was confirmed, in part, by microsequence analysis of the gel-purified RNA polymerase II subunit. RPB3 was found to be an essential single-copy gene that is tightly linked to HIS6 on chromosome IX. An RPB3 temperature-sensitive mutant that arrested growth after three to four generations at the restrictive temperature was isolated. When the mutant was shifted to the restrictive temperature, RNA polymerase II could no longer assemble, previously assembled functional enzyme was depleted, and mRNA levels were consequently reduced. These results demonstrate that RPB3 is an essential component of the mRNA transcription apparatus. Finally, the RPB3 protein is similar in sequence and length to RPC5, a subunit common to RNA polymerases I and III, suggesting that these subunits may play similar roles in RNA polymerases I, II, and III.
The G protein alpha o subunit alters morphology, growth kinetics, and phospholipid metabolism of somatic cells.
Authors: Bloch D B , Bonventre J V , Neer E J , Seidman J G
Published: October 2015

The physiological role of the alpha o subunit of guanine nucleotide-binding (G) protein was investigated with a murine adrenal cell line (Y1) transfected with a rat alpha o cDNA cloned in a retroviral expression vector. The parental cell line lacked detectable alpha o subunit. Expression of the alpha o cDNA in transfected cell lines was confirmed by Western blot (immunoblot) analysis. The rat alpha o subunit interacted with murine beta and gamma subunits and associated with cell membranes. Y1 cells containing large amounts of alpha o subunit had altered cellular morphology and reduced rate of cell division. In addition, GTP-gamma S-stimulated release of arachidonic acid from these cells was significantly increased compared with that in control cells. The alpha o subunit appears directly or indirectly to regulate cellular proliferation, morphology, and phospholipid metabolism.
Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1.
Authors: Chambers A , Tsang J S , Stanway C , Kingsman A J , Kingsman S M
Published: October 2015

The promoter of the yeast glycolytic gene encoding phosphoglycerate kinase (PGK) contains an upstream activation sequence between bases -538 and -402 upstream of the initiating ATG. The upstream activation sequence contains multiple functional elements, including an essential region called the activator core (AC) sequence and three copies of the pentamer 5'-CTTCC-3'. The AC sequence shows strong homology to the consensus binding sites for the yeast proteins RAP1 (GRF1) and TUF. We have demonstrated that the yeast protein which interacts with the AC sequence is the DNA-binding protein RAP1. Expression of the PGK gene is found to be regulated according to the carbon source in the growth medium. PGK mRNA levels are high in yeast cells grown in glucose medium but low in yeast cells grown in media containing carbon sources such as pyruvate and acetate. This carbon source regulation of transcription was found to be mediated, in part, via regulation of RAP1 binding to the AC sequence. The promoters of many other yeast glycolytic genes also contain consensus RAP1-binding sites and copies of the CTTCC pentamer. This suggests that RAP1 may be involved in transcriptional control of many other glycolytic genes in addition to the PGK gene.
A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.
Authors: Corthésy B , Cardinaux J R , Claret F X , Wahli W
Published: October 2015

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and, as demonstrated by DNA-binding assays, interacts with a liver-specific transcription factor. The second is required in association with the estrogen-responsive element to mediate hormonal induction and is recognized by the Xenopus liver homolog of nuclear factor I.
Histone H1 represses transcription from minichromosomes assembled in vitro.
Authors: Shimamura A , Sapp M , Rodriguez-Campos A , Worcel A
Published: October 2015

We have previously shown that transcription from a Xenopus 5S rRNA gene assembled into chromatin in vitro can be repressed in the absence of histone H1 at high nucleosome densities (one nucleosome per 160 base pairs of DNA) (A. Shimamura, D. Tremethick, and A. Worcel, Mol. Cell. Biol. 8:4257-4269, 1988). We report here that transcriptional repression may also be achieved at lower nucleosome densities (one nucleosome per 215 base pairs of DNA) when histone H1 is present. Removal of histone H1 from the minichromosomes with Biorex under conditions in which no nucleosome disruption was observed led to transcriptional activation. Transcriptional repression could be restored by adding histone H1 back to the H1-depleted minichromosomes. The levels of histone H1 that repressed the H1-depleted minichromosomes failed to repress transcription from free DNA templates present in trans. The assembly of transcription complexes onto the H1-depleted minichromosomes protected the 5S RNA gene from inactivation by histone H1.
The C-terminal domain of the largest subunit of RNA polymerase II and transcription initiation.
Authors: Moyle M , Lee J S , Anderson W F , Ingles C J
Published: October 2015

Monoclonal antibodies specific for the evolutionarily conserved C-terminal heptapeptide repeat domain of the largest subunit of RNA polymerase II inhibited the initiation of transcription from mammalian promoters in vitro. Since these antibodies did not inhibit elongation and randomly initiated transcription, the heptapeptide repeats may function by binding class II transcription initiation factor(s).
DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.
Authors: Budd M E , Wittrup K D , Bailey J E , Campbell J L
Published: October 2015

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.
Functional identification of regulatory elements within the promoter region of platelet-derived growth factor 2.
Authors: Pech M , Rao C D , Robbins K C , Aaronson S A
Published: October 2015

Human platelet-derived growth factor (PDGF) is composed of two polypeptide chains, PDGF-1 and PDGF-2, the human homolog of the v-sis oncogene. Deregulation of PDGF-2 expression can confer a growth advantage to cells possessing the cognate receptor and, thus, may contribute to the malignant phenotype. We investigated the regulation of PDGF-2 mRNA expression during megakaryocytic differentiation of K562 cells. Induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a greater than 200-fold increase in PDGF-2 transcript levels in these cells. Induction was dependent on protein synthesis and was not enhanced by cycloheximide exposure. In our initial investigation of the PDGF-2 promoter, a minimal promoter region, which included sequences extending only 42 base pairs upstream of the TATA signal, was found to be as efficient as 4 kilobase pairs upstream of the TATA signal in driving expression of a reporter gene in uninduced K562 cells. We also functionally identified different regulatory sequence elements of the PDGF-2 promoter in TPA-induced K562 cells. One region acted as a transcriptional silencer, while another region was necessary for maximal activity of the promoter in megakaryoblasts. This region was shown to bind nuclear factors and was the target for trans-activation in normal and tumor cells. In one tumor cell line, which expressed high PDGF-2 mRNA levels, the presence of the positive regulatory region resulted in a 30-fold increase in promoter activity. However, the ability of the minimal PDGF-2 promoter to drive reporter gene expression in uninduced K562 cells and normal fibroblasts, which contained no detectable PDGF-2 transcripts, implies the existence of other negative control mechanisms beyond the regulation of promoter activity.
Control of the Saccharomyces cerevisiae regulatory gene PET494: transcriptional repression by glucose and translational induction by oxygen.
Authors: Marykwas D L , Fox T D
Published: October 2015

The product of the Saccharomyces cerevisiae nuclear gene PET494 is required to promote the translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII). The level of cytochrome c oxidase activity is affected by several different environmental conditions, which also influence coxIII expression. We have studied the regulation of PET494 to test whether the level of its expression might modulate coxIII translation in response to these conditions. A pet494::lacZ fusion was constructed and used to monitor PET494 expression. PET494 was regulated by oxygen availability: expression in a respiration-competent diploid strain grown anaerobically was one-fifth the level of expression in aerobically grown cells. However, since PET494 mRNA levels did not vary in response to oxygen deprivation, regulation by oxygen appears to occur at the translational level. This oxygen regulation was not mediated by heme, and PET494 expression was independent of the heme activator protein HAP2. The regulation of PET494 expression by carbon source was also examined. In cells grown on glucose-containing medium, PET494 was expressed at levels four- to sixfold lower than in cells grown on ethanol and glycerol. However, addition of ethanol to glucose-containing medium induced PET494 expression approximately twofold. PET494 transcript levels varied over a fourfold range in response to different carbon sources. The effects on PET494 expression of mutations in the SNF1, SNF2, SSN6, and HXK2 genes were also determined and found to be minimal.
The C-terminal domain of transcription factor IIIA interacts differently with different 5S RNA genes.
Authors: Xing Y Y , Worcel A
Published: October 2015

DNase I footprints and affinity measurements showed that the C-terminal arm of Xenopus transcription factor IIIA interacts differently with different Xenopus 5S DNAs, forming three distinct types of transcription factor IIIA-5S DNA complexes: a somatic type, a major-oocyte (and pseudogene) type, and a trace-oocyte type. Site-directed mutagenesis on the major-oocyte 5S gene revealed that somatic-type changes at positions 53, 55, and 56 changed the structure of the transcription factor IIIA-5S DNA complex from major-oocyte to somatic, and a single trace-oocyte change at position 56 caused the change from major-oocyte to trace-oocyte complex. We further show that the somatic-type changes are accompanied by a marked enhancement in the rate of 5S RNA transcription, and we discuss the possible biological relevance of these findings.
The adenovirus-inducible factor E2F stimulates transcription after specific DNA binding.
Authors: Yee A S , Raychaudhuri P , Jakoi L , Nevins J R
Published: October 2015

The promoter-specific factor E2F interacts with critical regulatory sequences within the adenovirus E2 promoter. In addition, the level of active factor increases markedly during a virus infection, dependent on E1A function and coincident with the trans activation of E2 transcription. We have purified the E2F factor through a combination of standard biochemical procedures and DNA affinity chromatography. The purified factor was a single polypeptide of 54,000 molecular weight, as determined by UV crosslinking and renaturation of gel-fractionated protein. Addition of affinity-purified factor to an in vitro transcription system resulted in stimulation of transcription from a promoter containing two E2F-binding sites but not promoters lacking binding sites. We thus conclude that E2F is indeed capable of stimulating transcription once it has bound to the promoter.
A ras-dependent pathway abolishes activity of a muscle-specific enhancer upstream from the muscle creatine kinase gene.
Authors: Sternberg E A , Spizz G , Perry M E , Olson E N
Published: October 2015

Differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific genes whose products are required for the specialized functions of the mature muscle fiber. The program for myogenic differentiation is subject to negative control by several peptide growth factors and by the products of mutationally activated ras oncogenes, which persistently activate intracellular cascades normally triggered by specific growth factors. Previously, we reported that induction of the muscle creatine kinase (mck) gene during myogenesis was dependent on a distal upstream enhancer that cooperated with a proximal promoter to direct high levels of expression in developing muscle cells (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909). To investigate the mechanisms whereby ras blocks the induction of muscle-specific genes, we have examined the ability of mck 5' regulatory elements to direct expression of the linked reporter gene for chloramphenicol acetyltransferase (cat) in C2 myoblasts bearing mutant N-ras and H-ras oncogenes. In this paper we report that expression of activated ras alleles abolishes activity of the mck upstream enhancer but does not affect the activity of the mck promoter. The ability of ras to repress the expression of mck-cat fusion genes that have been transfected either transiently or stably into myoblasts suggests that ras may exert its effects on muscle-specific genes through mechanisms independent of chromatin configurations or DNA methylation. These results also suggest that ras blocks establishment of the myogenic phenotype by preventing the accumulation of regulatory factors required for transcriptional induction of muscle-specific genes.
Identification of sequences responsible for transcriptional activation of the allantoate permease gene in Saccharomyces cerevisiae.
Authors: Rai R , Genbauffe F S , Sumrada R A , Cooper T G
Published: October 2015

DAL5 is a constitutively expressed allantoin system gene whose product is required for allantoate transport. Its simple pattern of expression prompted us to use this gene for identifying the element(s) that mediates transcriptional activation of allantoin system genes. Deletion analysis of the DAL5 5'-flanking sequences resulted in identification of two small regions required for DAL5 expression. Analysis of these two regions with synthetic oligonucleotides localized the sequences supporting transcriptional activation to two DNA fragments of 10 to 12 base pairs, each containing one copy of the pentanucleotide 5'-GATAA-3'. The 5'-flanking region of DAL5 contained eight copies of this sequence. Synthetic constructions containing single copies of these fragments were unable to support transcriptional activation, while those containing two or more copies supported high-level activation. The 5'-GATAA-3' sequence was also found beneath the footprint of a DNA-binding protein. These observations are consistent with the suggestion that DNA fragments containing the sequence 5'-GATAA-3' play an important role in DAL5 gene expression, probably representing a portion of the binding site for a transcriptional activation factor.
Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro.
Authors: Tsurimoto T , Stillman B
Published: October 2015

Cell extracts (S100) derived from human 293 cells were separated into five fractions by phosphocellulose chromatography and monitored for their ability to support simian virus 40 (SV40) DNA replication in vitro in the presence of purified SV40 T antigen. Three fractions, designated I, IIA, and IIC, were essential. Fraction IIC contained the known replication factors topoisomerases I and II, but in addition contained a novel replication factor called RF-C. The RF-C activity, assayed in the presence of I, IIA, and excess amounts of purified topoisomerases, was detected in both cytosol and nuclear fractions, but was more abundant in the latter fraction. RF-C was purified from the 293 cell nuclear fraction to near homogeneity by conventional column chromatography. The reconstituted reaction mix containing purified RF-C could replicate SV40 origin-containing plasmid DNA more efficiently than could the S100 extract, and the products were predominantly completely replicated, monomer molecules. Interestingly, in the absence of RF-C, early replicative intermediates accumulated and subsequent elongation was aberrant. Hybridization studies with strand-specific, single-stranded M13-SV40 DNAs showed that in the absence of RF-C, abnormal DNA synthesis occurred preferentially on the lagging strand, and leading-strand replication was inefficient. These products closely resembled those previously observed for SV40 DNA replication in vitro in the absence of proliferating-cell nuclear antigen. These results suggest that an elongation complex containing RF-C and proliferating-cell nuclear antigen is assembled after formation of the first nascent strands at the replication origin. Subsequent synthesis of leading and lagging strands at a eucaryotic DNA replication fork can be distinguished by different requirements for multiple replication components, but we suggest that even though the two polymerases function asymmetrically, they normally progress coordinately.
Chimeric 3-hydroxy-3-methylglutaryl coenzyme A reductase-dihydrofolate reductase genes display bidirectional expression and unidirectional regulation in stably transfected cells.
Authors: Abrams J M , Schimke R T
Published: October 2015

We have constructed hybrid dihydrofolate reductase (DHFR) genes which are controlled by the sterol-responsive hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase promoter. Stable transfection frequencies of these chimeric templates into a DHFR-deficient Chinese hamster cell line indicate that the HMG CoA reductase promoter fragment confers DHFR transformation irrespective of its orientation relative to a downstream murine DHFR cDNA. Sterol-regulated levels of DHFR RNA and protein are detected from hybrid genes which carry a properly oriented promoter fragment. Constructions which invert this HMG CoA reductase promoter, however, generate DHFR RNA levels which do not respond to sterols. In the context of these transfected fusion genes, we present evidence of divergent opposite-strand transcription initiating from the HMG CoA reductase 5' fragment. In contrast, the endogenous HMG CoA reductase promoter region shows no apparent evidence of such bidirectional activity.
The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro.
Authors: Bernstein P , Peltz S W , Ross J
Published: October 2015

Using an in vitro mRNA decay system, we investigated how poly(A) and its associated poly(A)-binding protein (PABP) affect mRNA stability. Cell extracts used in the decay reactions were depleted of functional PABP either by adding excess poly(A) competitor or by passing the extracts over a poly(A)-Sepharose column. Polyadenylated mRNAs for beta-globin, chloramphenicol acetyltransferase, and simian virus 40 virion proteins were degraded 3 to 10 times faster in reactions lacking PABP than in those containing excess PABP. The addition of purified Saccharomyces cerevisiae or human cytoplasmic PABP to PABP-depleted reactions stabilized the polyadenylated mRNAs. In contrast, the decay rates of nonpolyadenylated mRNAs were unaffected by PABP, indicating that both the poly(A) and its binding protein were required for maintaining mRNA stability. A nonspecific single-stranded binding protein from Escherichia coli did not restore stability to polyadenylated mRNA, and the stabilizing effect of PABP was inhibited by anti-PABP antibody. The poly(A) tract was the first mRNA segment to be degraded in PABP-depleted reactions, confirming that the poly(A)-PABP complex was protecting the 3' region from nucleolytic attack. These results indicate that an important function of poly(A), in conjunction with its binding protein, is to protect polyadenylated mRNAs from indiscriminate destruction by cellular nucleases. A model is proposed to explain how the stability of an mRNA could be affected by the stability of its poly(A)-PABP complex.
Inhibition of tyrosine kinase activity of the epidermal growth factor (EGF) receptor by a truncated receptor form that binds to EGF: role for interreceptor interaction in kinase regulation.
Authors: Basu A , Raghunath M , Bishayee S , Das M
Published: October 2015

The tyrosine kinase activity of the epidermal growth factor (EGF) receptor is regulated by a truncated receptor of 100 kilodaltons (kDa) that contains the EGF-binding site but not the kinase domain. The inhibition of kinase is not due to competition for available EGF or for the kinase substrate-binding site. Chemical cross-linking studies suggest that the 100-kDa receptor may form a heterodimer with the intact EGF receptor. Structurally related receptor kinases, such as the platelet-derived growth factor receptor, the insulin receptor, and the Neu receptor, were not inhibited by the 100-kDa receptor. The results indicate that (i) the inhibition was specific for the EGF receptor, (ii) the kinase domain had little or no role in determining target specificity, and (iii) the regulation of kinase may be due to a specific interaction of the 100-kDa receptor with the ligand-binding domain of the EGF receptor kinase.
Physical linkage of mouse lambda genes by pulsed-field gel electrophoresis suggests that the rearrangement process favors proximate target sequences.
Authors: Storb U , Haasch D , Arp B , Sanchez P , Cazenave P A , Miller J
Published: October 2015

The first complete map of a mammalian immunoglobulin gene locus is presented. Mouse lambda genes were mapped by pulsed-field gel electrophoresis. The gene order is V2-Vx-C2-C4-V1-C3-C1. The distance between V2 or Vx and the C2-C4 cluster is 74 or 55 kilobases (kb), respectively, whereas that between V1 and C3-C1 is only 19 kb; V2 and C3-C1 are at least 190 kb apart. Thus, the distances between the lambda subloci are inversely proportional to their frequencies of rearrangement. The related gene lambda 5 is not within the 500 kb of the lambda locus mapped here.
Retrovirus-mediated transfer and expression of the interleukin-3 gene in mouse hematopoietic cells result in a myeloproliferative disorder.
Authors: Wong P M , Chung S W , Dunbar C E , Bodine D M , Ruscetti S , Nienhuis A W
Published: October 2015

A high-titer, recombinant retroviral vector produced in psi 2 packaging cells has been used to introduce the murine interleukin-3 (IL-3) gene into mouse hematopoietic cells. Integration and expression of the IL-3 gene was observed in spleen foci from which could be derived factor-independent, continuously proliferating cell lines. Irradiated or genetically anemic W/Wv recipients of infected hematopoietic cells developed a myeloproliferative syndrome characterized by a marked elevation in leukocyte count, bone marrow hyperplasia, and enlargement of the liver and spleen. The syndrome reflected proliferation of one or more stem cell clones, the progeny of which were capable of repopulating secondary recipients. One animal developed the syndrome primarily by a paracrine mechanism. Endogenous IL-3 production caused amplification of hematopoietic cells but did not appear to alter the maturational or self-renewal potential of these cells.
A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester.
Authors: Muramatsu M , Kaibuchi K , Arai K
Published: October 2015

We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKC alpha with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, delta PKC beta, lacking the coding region for amino acid positions 6 to 159 of PKC beta was also constructed. These mutant kinase genes expressed under the control of the SR alpha promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKC alpha at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. delta PKC beta, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.
Enhancement of major histocompatibility class I protein synthesis by DNA damage in cultured human fibroblasts and keratinocytes.
Authors: Lambert M E , Ronai Z A , Weinstein I B , Garrels J I
Published: October 2015

Exposure of primary human fibroblasts or simian virus 40-transformed human keratinocytes to several different classes of DNA damage, including UV light C (254 nm), resulted in a rapid increase in the expression of human major histocompatibility class I (MHC-I) proteins. MHC-I induction was also detected after exposure to low doses of the protein synthesis inhibitor cycloheximide, suggesting that MHC-I induction by DNA damage may be a component in a derepressible cellular SOS pathway.
Cell cycle regulation of H2b histone octamer DNA-binding activity in Chinese hamster lung fibroblasts.
Authors: Ito M , Sharma A , Lee A S , Maxson R
Published: October 2015

The promoter regions of H2b histone genes contain a 14-base-pair element which includes the octamer ATTTGCAT. Mutational analysis has implicated the octamer element in the cell cycle-dependent expression of H2b histone genes. In this report, we address the question of whether the DNA-binding activity of the octamer transcription factor is itself cell cycle regulated. By using a gel mobility shift assay, we measured the relative amounts of octamer-binding activity during various phases of the cell cycle in serum-synchronized Chinese hamster fibroblasts. We found that the activity increased approximately fivefold between late G1 phase and early S phase and then decreased threefold between late S phase and G2 phase. These cell cycle-dependent changes in octamer DNA-binding activity may in part account for the selective transcription of H2b histone genes in late G1 and S phases.
S-phase-specific transcription regulatory elements are present in a replication-independent testis-specific H2B histone gene.
Authors: Hwang I , Chae C B
Published: October 2015

The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes during meiotic prophase I in the absence of any significant DNA synthesis. Unlike somatic histones, synthesis of testis-specific histones is not affected by inhibitors of DNA synthesis. A genomic rat TH2B gene was cloned by using a DNA fragment derived from TH2B cDNA as a probe. Expression of the cloned TH2B was investigated by gene transfer experiments. From these studies, we found that the 5' upstream region of the cloned TH2B gene contained S-phase-specific transcription elements which regulated expression of a reporter gene in an S-phase-specific manner. The S-phase-regulatory element was found to be located in two regions containing CCAAT elements between -153 and -110 base pairs (bp) and an octamer element (ATTTGCAT) between -109 and -84 bp. The two regions were required for a maximal stimulation of transcription of the cloned TH2B gene in S phase. On the other hand, only the octamer element was reported be important for the S-phase-specific transcription of human H2B gene. Since the synthesis of the TH2B histone is independent of DNA synthesis and specific for pachytene spermatocytes in vivo, the presence of the S-phase-specific transcription regulatory elements in the TH2B gene is surprising.
Expression of the murine alpha B-crystallin gene is not restricted to the lens.
Authors: Dubin R A , Wawrousek E F , Piatigorsky J
Published: October 2015

The murine alpha B-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of alpha B-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse alpha B-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the alpha B-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of alpha B-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.
Deletions within the amino-terminal half of the c-src gene product that alter the functional activity of the protein.
Authors: Nemeth S P , Fox L G , DeMarco M , Brugge J S
Published: October 2015

To examine how amino acid sequences outside of the catalytic domain of pp60c-src influence the functional activity of this protein, we have introduced deletion mutations within the amino-terminal half of pp60c-src. These mutations caused distinct changes in the biochemical properties of the c-src gene products and in the properties of cells infected with retroviruses carrying these mutant c-src genes. Cells expressing the c-srcNX protein, which contains a deletion of amino acids 15 to 89, displayed a refractile, spindle-shaped morphology, formed intermediate-sized, tightly packed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Thus, deletion of amino acids 15 to 89 can activate the kinase activity and transforming potential of the c-src gene product. Deletion of amino acids 112 to 225, however, did not increase the kinase activity or transforming ability of pp60c-src; indeed, deletion of these sequences in c-srcHP suppressed phenotypic alterations induced by pp60c-src. Cells expressing the c-srcNP or c-srcBS gene products (containing deletions of amino acids 15 to 225 and 55 to 169, respectively) displayed a fusiform, refractile morphology and formed diffuse colonies in soft agar; the mutant proteins displayed an increased in vitro protein-tyrosine kinase activity. However, only a few cellular proteins contained elevated levels of phosphotyrosine in vivo. Thus, deletions downstream of amino acid 89 severely restricted the ability of c-src to phosphorylate cellular substrates in vivo without affecting the intrinsic tyrosine kinase activity of the c-src gene product. These results suggest the existence of at least two modulatory regions within the amino-terminal half of pp60c-src that are important for the regulation of tyrosine kinase activity and for the interaction of pp60c-src with cellular substrates.
Nonrandom distribution of long mono- and dinucleotide repeats in Drosophila chromosomes: correlations with dosage compensation, heterochromatin, and recombination.
Authors: Lowenhaupt K , Rich A , Pardue M L
Published: October 2015

Long stretches of (dC-dA)n.(dT-dG)n, abbreviated CA/TG, have a distinctive distribution on Drosophila chromosomes (M.L. Pardue, K. Lowenhaupt, A. Rich, and A. Nordheim, EMBO J. 6:1781-1789, 1987). The distribution of CA/TG suggests a correlation with the overall transcriptional activity of chromosomal regions and with the ability to undergo meiotic recombination. These correlations are conserved among Drosophila species and may indicate one or more chromosomal functions. To test the generality of these findings, we analyzed the distribution of the rest of the six possible mono- and dinucleotide repeats (A/T, C/G, AT/AT, CA/TG, CT/AG, and CG/CG). All but CG/CG were present at significant levels in the genomes of the six Drosophila species studied; however, A/T levels were an order of magnitude lower than those of the other sequences. Data base analyses suggested that the same sequences are present in other eucaryotes. Like CA/TG, both CT/AG and C/G showed increased levels on dosage-compensating chromosomes; however, the individual sites clearly differed for each sequence. In contrast, A/T and AT/AT, although present in Drosophila DNA, could not be detected in situ in polytene chromosomes. We also used in situ hybridization to analyze the neo-Y chromosome of Drosophila miranda, an ancestral autosome that has become attached to the Y chromosome and is now partially heterochromatic. The neo-Y has acquired repeated DNA sequences; we found that the added sequences are as devoid of mono- and dinucleotide repeats as other heterochromatin. The distribution and function of these sequences are likely to result from both their repetitious nature and base contents.
A v-H-ras-dependent hemopoietic tumor model involving progression from a clonal stage of transformation competence to autocrine interleukin 3 production.
Authors: Nair A P , Diamantis I D , Conscience J F , Kindler V , Hofer P , Moroni C
Published: October 2015

Autocrine interleukin 3 (IL-3)-secreting tumors were generated from an IL-3-dependent mouse mast cell line (PB-3c) after introduction of the v-H-ras oncogene. Tumor progression was characterized by four distinct phenotypes. The first corresponded to immortalized mast cells unresponsive to the oncogenic effect of v-H-ras. The second was expressed in a clonable subpopulation of PB-3c cells and was marked by the competence to form v-H-ras-dependent tumors (immortalized transformation competence). The third was a direct effect of v-H-ras expression on all PB-3c cells and was characterized in vitro by a reduced IL-3 requirement. Upon injection of v-H-ras-expressing, transformation-competent cells into mice, the final, fully malignant phenotype developed with a long latency period and was marked in vitro by independence of exogenous IL-3 and by autocrine IL-3 stimulation. Northern (RNA) blot analysis and an RNase A-T1 protection assay showed that IL-3 production was strictly associated with the tumor phenotype. Two of six tumors showed an alteration at the 5' region of the IL-3 gene. We conclude that v-H-ras required complementation by IL-3 gene rearrangement or an alternate event to generate autocrine mastocytomas.
Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells.
Authors: Kaufman R J , Wasley L C , Davies M V , Wise R J , Israel D I , Dorner A J
Published: October 2015

In plasma, antihemophilic factor (factor VIII) exists as a 200-kilodalton heavy-chain polypeptide in a metal ion association with an 80-kilodalton light-chain polypeptide. This complex is bound by hydrophobic and hydrophilic interactions to a large multimeric glycoprotein, von Willebrand factor (vWF). Accumulation of secreted human factor VIII activity expressed in Chinese hamster ovary cells requires the addition of serum in the growth medium, which provides vWF. Here we report that coexpression of vWF with factor VIII in Chinese hamster ovary cells resulted in increased stable accumulation of factor VIII activity in the absence of serum in the growth medium. In the coexpressing cells, the vWF cDNA transcription unit was transcribed to yield mRNA which was efficiently translated. vWF was properly processed and secreted to yield disulfide-bonded high-molecular-weight multimers similar to those observed in vWF secreted from human endothelial cells. Nuclear run-on assays showed that the factor VIII gene was transcribed at a level similar to that of the vWF gene, but the mRNA did not accumulate to high levels in the cytoplasm. In addition, although the translation efficiency of the factor VIII mRNA was similar to that of vWF, the processing and secretion of the factor VIII primary translation product was dramatically reduced compared with vWF. These results demonstrate that in Chinese hamster ovary cells both factor VIII mRNA accumulation and the processing and secretion of the primary factor VIII translation product are inefficient processes.
The BCR-ABL oncogene transforms Rat-1 cells and cooperates with v-myc.
Authors: Lugo T G , Witte O N
Published: October 2015

The tyrosine kinase P210 is the gene product of the rearranged BCR-ABL locus on the Philadelphia chromosome (Ph1), which is found in leukemic cells of patients with chronic myelogenous leukemia. It has a weakly oncogenic effect in immature murine hematopoietic cells and does not transform NIH 3T3 cells. We have found that P210 has a strikingly different effect in Rat-1 cells, another line of established rodent fibroblasts. Stable expression of P210 in Rat-1 cells caused a distinct morphological change and conferred both tumorigenicity and capacity for anchorage-independent growth. The introduction of v-myc into Rat-1 cells expressing P210 led to complete morphological transformation and enhanced tumorigenicity. No such interaction took place in NIH 3T3 cells. Thus, Rat-1 cells can be used to detect cooperation between BCR-ABL and other oncogenes and may prove useful for the identification of secondary oncogenic events in chronic myelogenous leukemia.
DNA strand specificity for UV-induced mutations in mammalian cells.
Authors: Vrieling H , Van Rooijen M L , Groen N A , Zdzienicka M Z , Simons J W , Lohman P H , van Zeeland A A
Published: October 2015

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.
Nucleotide sequence of an unequal sister chromatid exchange site in a mouse myeloma cell line.
Authors: Katzenberg D R , Tilley S A , Birshtein B K
Published: October 2015

The mouse myeloma cell line MPC 11 carries two C gamma 2a immunoglobulin heavy-chain genes on the expressed chromosome, a duplication shown to have occurred through unequal sister chromatid exchange (USCE). In the present report, we present the nucleotide sequence of the USCE joint and show that both breaks occurred within tracts of repeated TC dinucleotides. Additional TC dinucleotide tracts and two oligonucleotide segments (N sequences) were inserted at the USCE site.
Identification and characterization of a chicken alpha-globin enhancer.
Authors: Knezetic J A , Felsenfeld G
Published: October 2015

We identify and describe the properties of an enhancer within the chicken alpha-globin gene cluster. This cluster consists of one gene (pi) expressed only in primitive erythrocytes and two (alpha A and alpha D) expressed in both primitive and definitive cell lineages. The genes are linked together in the order 5'-pi-alpha D-alpha A-3' and occupy a region about 10 kilobase pairs long. The enhancer is located at the 3' end of the cluster, about 750 base pairs 3' to the alpha A translation stop site. When assayed by transfection into either primitive or definitive primary chicken erythrocytes, this element stimulated expression from plasmids containing the alpha D- or alpha A-globulin gene promoters. Except for sites in the alpha-globin promoters, no other stimulatory activity was observed in DNA taken from other regions of the alpha-globin locus. Moderate resolution DNase I hypersensitivity studies as well as DNase I footprinting revealed three regions of protein binding, each containing a similar core DNA sequence within the enhancer element. Gel mobility shift studies demonstrated that all three regions bind the recently identified erythrocyte-specific factor, EryfI, which has binding sites in the regulatory regions of all chicken globin genes. Our data suggest that the enhancer we have identified may act in vivo only on the alpha A gene; expression of the alpha D gene is affected by another EryfI site located in the alpha D promoter. Such a mechanism would be consistent with the observed relative abundances of alpha A- and alpha D-globin in vivo. The simplicity of these regulatory elements may reflect the limited repertoire of expression of these genes during development.
Cell-heritable stages of tumor progression in transgenic mice harboring the bovine papillomavirus type 1 genome.
Authors: Sippola-Thiele M , Hanahan D , Howley P M
Published: October 2015

Tumorigenesis of dermal fibroblasts in a line of transgenic mice carrying the BPV-1 genome was found to involve distinct proliferative stages. Cell cultures derived from normal skin, from benign proliferative fibromatoses, and from malignant fibrosarcomas each evidenced distinguishable, cell-heritable characteristics. The latent viral genome was transcriptionally inactive in normal-appearing skin and was activated in the dermal fibromatoses. Fibrosarcoma cells grew continuously in culture, formed domelike foci, and had a more rounded, anaplastic appearance. Independent cultures derived from the fibromatoses varied in their proliferative characteristics, which correlated well with the levels of viral gene expression. In contrast, progression to malignancy was not accompanied by a further increase in transgene activity, which strongly implicated cellular genetic changes in the later stages of tumorigenesis.