bluuebunny/crossref_metadata_2025 huggingface dataset:
2,3,7,8-Tetrachlorodibenzo-p-dioxin-inducible aryl hydrocarbon receptor-mediated change in CYP1A1 chromatin structure occurs independently of transcription.
Authors: Durrin L K , Whitlock J P
Published: October 2015

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces, in a receptor-dependent fashion, an increase in the accessibility of CYP1A1 chromatin to restriction endonucleases. The 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced change in chromatin structure occurs rapidly and does not require ongoing RNA or protein synthesis. The increased accessibility of chromatin DNA may facilitate its subsequent interaction with other transcription factors.
Metal-specific posttranscriptional control of human metallothionein genes.
Authors: Sadhu C , Gedamu L
Published: October 2015

During the initial 4 h of treatment, copper and zinc similarly activated the rates of transcription and mRNA accumulation from the two human metallothionein (MT) genes, viz., MTI-G and MTII-A, in the hepatoblastoma cell line HepG2. The levels of copper-induced MT mRNAs remained at a plateau for up to 15 h. In contrast, the levels of zinc-induced MT mRNAs gradually declined after about 4 h, despite substantial transcription. The decrease in the zinc-induced MT mRNA half-life is probably due to a posttranscriptional event(s).
Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity.
Authors: Stanley P
Published: October 2015

The production of glycoproteins with carbohydrates of defined structure and minimal heterogeneity is important for functional studies of mammalian carbohydrates. To facilitate such studies, several Chinese hamster ovary mutants that carry between two and four glycosylation mutations were developed. All of the lines grew readily in culture despite the drastic simplification of their surface carbohydrates. Therefore, both endogenous glycoproteins and those introduced by transfection can be obtained with specifically tailored carbohydrates. The lectin resistance properties of the mutants showed that each line expresses a novel array of cell surface carbohydrates useful for identifying specific roles for carbohydrates in cellular interactions. In addition, they showed that the epistatic relationships among different glycosylation mutations are not entirely predictable, providing insight into the complexity of the carbohydrate structures at the Chinese hamster ovary cell surface.
Context affects nuclear protein localization in Saccharomyces cerevisiae.
Authors: Nelson M , Silver P
Published: October 2015

Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus. We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase. Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase. A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae. From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.
Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product.
Authors: Romanczuk H , Wormington W M
Published: October 2015

Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.
Selection and characterization of ricin toxin A-chain mutations in Saccharomyces cerevisiae.
Authors: Frankel A , Schlossman D , Welsh P , Hertler A , Withers D , Johnston S
Published: October 2015

A DNA sequence encoding the A chain of ricin toxin (RTA) from the castor bean plant, Ricinus communis, was placed under GAL1 promoter control and transformed into Saccharomyces cerevisiae. Induction of expression of RTA was lethal. This lethality was the basis for a selection of mutations in RTA which inactivated the toxin. A number of mutant alleles which encoded cross-reactive material were sequenced. Eight of the first nine mutant RTAs studied showed single-amino-acid changes involving residues located in the proposed active-site cleft.
High-resolution mapping of replication fork movement through the amplified dihydrofolate reductase domain in CHO cells by in-gel renaturation analysis.
Authors: Leu T H , Hamlin J L
Published: October 2015

Utilizing an in vivo labeling method on synchronized cultures, we have previously defined a 28-kilobase (kb) replication initiation locus in the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) (N. H. Heintz and J. L. Hamlin, Proc. Natl. Acad. Sci. USA 79:4083-4087, 1982; N. H. Heintz and J. L. Hamlin, Biochemistry 22:3552-3557, 1983; N. H. Heintz, J. D. Milbrandt, K. S. Greisen, and J. L. Hamlin, Nature [London] 302:439-441, 1983). To locate the origin of replication in this 243-kb amplicon with more precision, we used an in-gel renaturation procedure (I. Roninson, Nucleic Acids Res. 11:5413-5431, 1983) to examine the labeling pattern of restriction fragments from the amplicon in the early S phase. This method eliminates background labeling from single-copy sequences and allows quantitation of the relative radioactivity in individual fragments. We used this procedure to follow the movement of replication forks through the amplicons, to roughly localize the initiation locus, and to estimate the rate of fork travel. We also used a slight modification of this method (termed hybridization enhancement) to illuminate the labeling pattern of smaller restriction fragments derived solely from the initiation locus itself, thereby increasing resolution. Our preliminary results suggest that there are actually two distinct initiation sites in the amplicon that are separated by approximately 22 kb.
Viral DNA synthesis in nonpermissive rat F-111 cells and its role in neoplastic transformation by polyomavirus.
Authors: Hacker D L , Fluck M M
Published: October 2015

We have investigated the occurrence and role of polyomavirus DNA synthesis in neoplastic transformation by this virus. We show that after infection of Fischer rat F-111 cells at 37 degrees C, there is two- to threefold increase in the level of viral DNA as compared with the input signal, with a peak observed between 5 and 7 days postinfection. Viral DNA synthesis is about 10 times higher at 33 degrees C and increases up to 15 days postinfection. Most of the viral DNA produced is supercoiled (form I DNA). On the basis of in situ hybridization, it appears that viral replication is restricted to a small fraction of the population. At the lower temperature, more cells are permissive for viral DNA synthesis and the level of synthesis per permissive cell is higher. The DNA synthesis observed is large T-antigen dependent, and the increase in viral DNA synthesis at 33 degrees C is paralleled by an increase in the expression of this viral protein. When large T antigen is inactivated, the half-life of de novo-synthesized viral DNA is less than 12 h, suggesting that large T antigen may be responsible for the stability of the viral genomes as well as their synthesis. Surprisingly, at early times postinfection (0 to 48 h), when the essential function of large T antigen in transformation is expressed (as demonstrated in shift-up experiments with tsa mutants), the level of large T antigen is below the detection level and is at least 10-fold lower than the levels observed in permissive infections at the start of viral DNA synthesis. The difference in viral DNA at 37 and 33 degrees C allowed us to study its effect on transformation. Although an increase in transformation frequency is observed in wild-type A2 infections carried at 33 degrees C (frequencies two to three times higher than at 37 degrees C), this increase appears to be unrelated to the increase in viral DNA synthesis. Furthermore, the overall level of viral DNA and large T antigen in F-111 cells may not affect the integration of the viral genome, since the patterns of integration in cells transformed by wild-type A2 at 33 and 37 degrees C appear similar. The results are compatible with a role for large T antigen in integration-transformation which is not simply to amplify the viral genome to enhance the probability of its integration.
NF-kappa B binds within a region required for B-cell-specific expression of major histocompatibility complex class II gene E alpha d.
Authors: Blanar M A , Burkly L C , Flavell R A
Published: October 2015

A region upstream of the murine major histocompatibility complex gene, E alpha d, has been shown previously to be required for B-cell expression. Binding of the B-cell-specific factor, NF-kappa B, to a site within this region is indistinguishable from that observed with the kappa enhancer binding site. NF-kappa B may be responsible for E alpha d B-cell expression.
Detection of a Myc-associated protein by chemical cross-linking.
Authors: Gillespie D A , Eisenman R N
Published: October 2015

A single nuclear protein (Myc-associated protein) can be specifically cross-linked to avian Myc proteins by treatment of nuclei or cells with the reversible cross-linker dimethyl 3,3'-dithiobis-propionimidate. Myc-associated protein has a molecular weight of approximately 500,000, is not detectably phosphorylated and, in contrast to Myc, has a long apparent half-life of greater than 3 h.
Sequence and structural requirements of a mitochondrial protein import signal defined by saturation cassette mutagenesis.
Authors: Bedwell D M , Strobel S A , Yun K , Jongeward G D , Emr S D
Published: October 2015

The Saccharomyces cerevisiae F1-ATPase beta subunit precursor contains redundant mitochondrial protein import information at its NH2 terminus (D. M. Bedwell, D. J. Klionsky, and S. D. Emr, Mol. Cell. Biol. 7:4038-4047, 1987). To define the critical sequence and structural features contained within this topogenic signal, one of the redundant regions (representing a minimal targeting sequence) was subjected to saturation cassette mutagenesis. Each of 97 different mutant oligonucleotide isolates containing single (32 isolates), double (45 isolates), or triple (20 isolates) point mutations was inserted in front of a beta-subunit gene lacking the coding sequence for its normal import signal (codons 1 through 34 were deleted). The phenotypic and biochemical consequences of these mutations were then evaluated in a yeast strain deleted for its normal beta-subunit gene (delta atp2). Consistent with the lack of an obvious consensus sequence for mitochondrial protein import signals, many mutations occurring throughout the minimal targeting sequence did not significantly affect its import competence. However, some mutations did result in severe import defects. In these mutants, beta-subunit precursor accumulated in the cytoplasm, and the yeast cells exhibited a respiration defective phenotype. Although point mutations have previously been identified that block mitochondrial protein import in vitro, a subset of the mutations reported here represents the first single missense mutations that have been demonstrated to significantly block mitochondrial protein import in vivo. The previous lack of such mutations in the beta-subunit precursor apparently relates to the presence of redundant import information in this import signal. Together, our mutants define a set of constraints that appear to be critical for normal activity of this (and possibly other) import signals. These include the following: (i) mutant signals that exhibit a hydrophobic moment greater than 5.5 for the predicted amphiphilic alpha-helical conformation of this sequence direct near normal levels of beta-subunit import (ii) at least two basic residues are necessary for efficient signal function, (iii) acidic amino acids actively interfere with import competence, and (iv) helix-destabilizing residues also interfere with signal function. These experimental observations provide support for mitochondrial protein import models in which both the structure and charge of the import signal play a critical role in directing mitochondrial protein targeting and import.
Mitogen-induced genes are subject to multiple pathways of regulation in the initial stages of T-cell activation.
Authors: Irving S G , June C H , Zipfel P F , Siebenlist U , Kelly K
Published: October 2015

The delivery of a mitogenic signal to T cells via any one of several cell surface molecules elicits a variety of intracellular responses, some or all of which regulate subsequent gene expression events. The expression of nine novel mitogen-induced genes in response to various T-cell-activating agents was examined to evaluate the diversity of pathways which regulate such genes. The relative contribution of distinct secondary signals, individually or together, to mitogen-stimulated gene induction and the capability of individual genes to respond to the sometimes divergent signals generated from different cell surface structures is addressed. The activation of T cells with mitogenic monoclonal antibodies directed against the CD2 or CD3 cell surface molecules, or with phytohemagglutinin, induced all nine genes. Thus, stimulation by fully mitogenic agents regardless of cell surface-binding specificity correlated with the expression of all of the genes studied. However, heterogeneous patterns of gene expression, encompassing five regulatory classes, were revealed by the use of phorbol 12-myristate 13-acetate, calcium ionophore, and anti-CD28 monoclonal antibody, agents which mediated only a subset of intracellular events and thus an incomplete mitogenic signal. Interleukin-2 and two novel lymphokines represented one regulatory class that appeared to require unique transcriptional activation signals relative to the other mitogen-induced genes. As demonstrated in the accompanying paper (P. F. Zipfel, S. G. Irving, K. Kelly, and U. Siebenlist, Mol. Cell. Biol. 9:1041-1048, 1989), the immediate transcriptional response of T cells to mitogenic stimulation is quite complex, involving numerous genes beyond those which have been previously described. Furthermore, the discrimination of several regulatory phenotypes among these nine genes suggests that a multiplicity of signaling pathways extends from the cell surface to the level of transcription.
Complexity of the primary genetic response to mitogenic activation of human T cells.
Authors: Zipfel P F , Irving S G , Kelly K , Siebenlist U
Published: October 2015

We describe the isolation and characterization of more than 60 novel cDNA clones that constitute part of the immediate genetic response to resting human peripheral blood T cells after mitogen activation. This primary response was highly complex, both in the absolute number of inducible genes and in the diversity of regulation. Although most of the genes expressed in activated T cells were shared with the activation response of normal human fibroblasts, a significant number were more restricted in tissue specificity and thus likely encode or effect the differentiated functions of activated T cells. The activatable genes could be further differentiated on the basis of kinetics of induction, response to cycloheximide, and sensitivity to the immunosuppressive drug cyclosporin A. It is of note that cyclosporin A inhibited the expression of more than 10 inducible genes, which suggests that this drug has a broad genetic mechanism of action.
Regulation of 2',5'-oligoadenylate synthetase gene expression by interferons and platelet-derived growth factor.
Authors: Garcia-Blanco M A , Lengyel P , Morrison E , Brownlee C , Stiles C D , Rutherford M , Hannigan G , Williams B R
Published: October 2015

In murine BALB/c 3T3 cell cultures, either beta interferon or platelet-derived growth factor (PDGF) enhanced expression of the 2',5'-oligoadenylate synthetase mRNA and protein. The time course of induction in response to beta interferon was similar to that in response to PDGF. Of several growth factors known to be present in clotted blood serum (i.e., epidermal growth factor, transforming growth factor beta, and PDGF), only PDGF enhanced expression of 2',5'-oligoadenylate synthetase. The linkage of an interferon response element-containing segment from the 5'-flanking region of a human or murine 2',-5'-oligoadenylate synthetase gene made a heterologous gene responsive to interferon. The expression of such a gene construct in transfected cells was also induced by PDGF. Induction by PDGF was inhibited by mono- or polyclonal antibodies to murine interferon, which suggested that induction by PDGF requires interferon. Both PDGF and interferon induced nuclear factors that bound to this interferon response element-containing segment in vitro.
Accumulation of viruslike particles in a yeast mutant lacking a mitochondrial pore protein.
Authors: Dihanich M , van Tuinen E , Lambris J D , Marshallsay B
Published: October 2015

The lack of mitochondrial porin is not lethal in Saccharomyces cerevisiae, but it impairs some respiratory functions and, therefore, growth on nonfermentable carbon sources such as glycerol. However, after a lag phase porinless mutant cells adapt to growth on glycerol, accumulating large amounts of an 86-kilodalton (kDa) protein (M. Dihanich, K. Suda, and G. Schatz, EMBO J. 6:723-728, 1987) and of a 5-kilobase RNA. Immunogold labeling localized the 86 kDa-protein exclusively to the cytosol fraction, although most of it cosedimented with the microsome fraction in earlier cell fractionations. This discrepancy was resolved when the 86-kDa protein was identified as the major coat protein in viruslike particles (VLPs) which is encoded by a double-stranded RNA (L-A RNA). Elimination of VLPs in the original porinless strain by introduction of the mak10 or the mak3 mutation increased the respiratory defect and prolonged its lag phase on nonfermentable carbon sources. The fact that the simultaneous loss of VLPs and respiratory functions are the introduction of mak10 or mak3 occurred even in some porin-containing wild-type strains suggests that there is a link between VLP and mitochondrial functions.
Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein.
Authors: Ryden T A , Beemon K
Published: October 2015

DNA-protein interactions involving enhancer and promoter sequences within the U3 regions of several avian retroviral long terminal repeats (LTRs) were studied by DNase I footprinting. The rat CCAAT/enhancer-binding protein, C/EBP, bound to all four viral LTRs examined. The Rous sarcoma virus binding site corresponded closely to the 5' limit of the LTR enhancer; nucleotides -225 to -188 were protected as a pair of adjacent binding domains. The Fujinami sarcoma virus LTR bound C/EBP at a single site at nucleotides -213 to -195. C/EBP also bound to the promoter region of the enhancerless Rous-associated virus-0 LTR at nucleotides -77 to -57. The avian myeloblastosis virus LTR bound C/EBP at three sites: nucleotides -262 to -246, -154 to -134, and -55 to -39. We have previously observed binding of C/EBP to an enhancer in the gag gene of avian retroviruses. A heat-treated nuclear extract from chicken liver bound to all of the same retroviral sequences as did C/EBP. Alignment of the avian retroviral binding sequences with the published binding sites for C/EBP in two CCAAT boxes and in the simian virus 40, polyoma, and murine sarcoma virus enhancers suggested TTGNNGCTAATG as a consensus sequence for binding of C/EBP. When two bases of this consensus sequence were altered by site-specific mutagenesis of the Rous sarcoma virus LTR, binding of the heat-stable chicken protein was eliminated.
p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor.
Authors: Hudziak R M , Lewis G D , Winget M , Fendly B M , Shepard H M , Ullrich A
Published: October 2015

The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been show to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.
Sequences within the spacer region of yeast rRNA cistrons that stimulate 35S rRNA synthesis in vivo mediate RNA polymerase I-dependent promoter and terminator activities.
Authors: Mestel R , Yip M , Holland J P , Wang E , Kang J , Holland M J
Published: October 2015

Sequences within the spacer region of yeast rRNA cistrons stimulate synthesis of the major 35S rRNA precursor in vivo 10- to 30-fold (E. A. Elion and J. R. Warner, Cell 39:663-673, 1984). Spacer sequences that mediate this stimulatory activity are located approximately 2.2 kilobases upstream from sequences that encode the 5' terminus of the 35S rRNA precursor. By utilizing a centromere-containing plasmid carrying a 35S rRNA minigene, a 160-base-pair region of spacer rDNA was identified by deletion mapping that is required for efficient stimulation of 35S rRNA synthesis in vivo. A 22-base-pair sequence, previously shown to support RNA polymerase I-dependent selective initiation of transcription in vitro, was located 15 base pairs upstream from the 3' boundary of the stimulatory region. A 77-base pair region of spacer DNA that mediates transcriptional terminator activity in vivo was identified immediately downstream from the 5' boundary of the stimulatory region. Deletion mutations extending downstream from the 5' boundary of the 160-base-pair stimulatory region simultaneously interfere with terminator activity and stimulation of 35S rRNA synthesis from the minigene. The terminator region supported termination of transcripts initiated by RNA polymerase I in vivo. The organization of sequences that support terminator and promoter activities within the 160-base-pair stimulatory region is similar to the organization of rDNA gene promoters in higher organisms. Possible mechanisms for spacer-sequence-dependent stimulation of yeast 35S rRNA synthesis in vivo are discussed.
Enhanced jun gene expression is an early genomic response to transforming growth factor beta stimulation.
Authors: Pertovaara L , Sistonen L , Bos T J , Vogt P K , Keski-Oja J , Alitalo K
Published: October 2015

Transforming growth factor beta (TGF beta) is a multifunctional polypeptide that regulates proliferation, differentiation, and other functions of many cell types. The pathway of TGF beta signal transduction in cells is unknown. We report here that an early effect of TGF beta is an enhancement of the expression of two genes encoding serum- and phorbol ester tumor promoter-regulated transcription factors: the junB gene and the c-jun proto-oncogene, respectively. This stimulation was observed in human lung adenocarcinoma A549 cells which were growth inhibited by TGF beta, AKR-2B mouse embryo fibroblasts which were growth stimulated by TGF beta, and K562 human erythroleukemia cells, which were not appreciably affected in their growth by TGF beta. The increase in jun mRNA occurred with picomolar TGF beta concentrations within 1 h of TGF beta stimulation, reached a peak between 1 and 5 h in different cells, and declined gradually to base-line levels. This mRNA response was followed by a large increase in the biosynthesis of the c-jun protein (AP-1), as shown by metabolic labeling and immunoprecipitation analysis. However, differential and cell type-specific regulation appeared to determine the timing and magnitude of the response of each jun gene in a given cell. In AKR-2B and NIH 3T3 cells, only junB was induced by TGF beta, evidently in a protein synthesis-independent fashion. The junB response to TGF beta was maintained in c-Ha-ras and neu oncogene-transformed cells. Thus, one of the earliest genomic responses to TGF beta may involve nuclear signal transduction and amplification by the junB and c-jun transcription factors in concert with c-fos, which is also induced. The differential activation of the jun genes may explain some of the pleiotropic effects of TGF beta.
Cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mRNAs.
Authors: Nover L , Scharf K D , Neumann D
Published: October 2015

In heat-shocked tomato cell cultures, cytoplasmic heat shock granules (HSGs) are tightly associated with a specific subset of mRNAs coding mainly for the untranslated control proteins. This messenger ribonucleoprotein complex was banded in a CsCl gradient after fixation with formaldehyde (approximately 1.30 g/cm3). It contains all the heat shock proteins and most of the RNA applied to the gradient. During heat shock, a reversible aggregation of HSGs from 15S precursor particles can be shown. These pre-HSGs are not identical to the 19S plant prosomes. Ultrastructural analysis supports the ribonucleoprotein nature of HSGs and their composition of approximately 10-nm precursor particles. A model summarizes our results. It gives a reasonable explanation for the striking conservation of untranslated mRNAs during heat shock and may apply also to animal cells.
Cell-specific activity of the modulator region in the human cytomegalovirus major immediate-early gene.
Authors: Lubon H , Ghazal P , Hennighausen L , Reynolds-Kohler C , Lockshin C , Nelson J
Published: October 2015

In this paper we demonstrate that modulator sequences upstream of the enhancer of the major immediate-early promoter of human cytomegalovirus exert a differential effect on the level of transcription in a variety of cells and that this region has the capacity to interact with specific nuclear protein. Depending on the cell type, these modulator sequences increased or decreased transcriptional activation from the IE1 gene promoter-enhancer. The cell lines identified in this report should be useful to study the molecular mechanism of cell-specific transcriptional repression and activation exerted by the major immediate-early promoter upstream region.
Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.
Authors: Lambowitz A M , Sabourin J R , Bertrand H , Nickels R , McIntosh L
Published: October 2015

Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species.
Fine mapping of a mouse metallothionein gene metal response element.
Authors: Culotta V C , Hamer D H
Published: October 2015

Metal-regulated transcription of metallothionein (MT) genes in higher eucaryotes involves multiple copies of a highly conserved 17-base-pair metal-regulatory element (MRE). We have assayed by transient transfection the ability of mouse MT-I element d (MREd) to confer metal responsivity to constructs containing the mouse MT-I TATA box and the bacterial chloramphenicol acetyltransferase indicator gene. A single copy of MREd works bidirectionally to afford a three- to fourfold induction, and dual copies act cooperatively to yield a 10- to 20-fold response. Element d responds to the same spectrum of heavy metals as doses the complete MT gene promoter. The sequences involved in induction by metals were delineated by analyzing point mutations in MREd. While nucleotides of the highly conserved core sequence TGCPuCXC are critical, substitutions in the less conserved regions affect the induction response only marginally. These sequences include residues of a potential Sp1-binding site, suggesting that if Sp1 binds to MREd, it has little if any role in induction by metals.
Transcriptional regulation of acetyl coenzyme A carboxylase gene expression by tumor necrosis factor in 30A-5 preadipocytes.
Authors: Pape M E , Kim K H
Published: October 2015

Acetyl coenzyme A (acetyl-CoA) carboxylase activity, amount, and mRNA levels increase during the differentiation of 30A-5 preadipocytes to adipocytes. Tumor necrosis factor (TNF) completely prevents this differentiation, with concomitant inhibition of acetyl-CoA carboxylase mRNA accumulation. To investigate the mechanisms by which TNF prevents acetyl-CoA carboxylase mRNA accumulation, we determined the effect of TNF on the transcription rate of the carboxylase gene and the half-life of carboxylase mRNA. Nuclear runoff transcription assays revealed no differences in the number of RNA polymerase molecules actively engaged in transcription of the acetyl-CoA carboxylase gene in preadipocytes, adipocytes, TNF-treated preadipocytes, or at any time during the course of differentiation. However, changes in adipsin, glycerophosphate dehydrogenase, and actin mRNAs, whose levels are also differentiation dependent, can be accounted for in part by changes in the number of polymerase complexes on their respective genes. To determine whether TNF caused a decrease in the stability of carboxylase RNA transcripts, we measured the rate of decay of prelabeled acetyl-CoA carboxylase mRNA. Control and TNF-treated cells showed no difference between the apparent half-lives of acetyl-CoA carboxylase mRNAs (9 h). However, the rate of acetyl-CoA carboxylase mRNA synthesis in vivo was decreased three- to fourfold in the presence of TNF. These data demonstrate that TNF prevents accumulation of acetyl-CoA carboxylase mRNA during preadipocyte differentiation by decreasing the rate of acetyl-CoA carboxylase gene transcription. However, transcriptional control is not due to a change in the number of RNA polymerase complexes actively engaged in carboxylase transcript elongation which could be measured by a number runoff assay. Instead, transcriptional control may be related to the rate at which RNA polymerase traverses the acetyl-CoA carboxylase gene.
Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and alpha 1-antitrypsin genes.
Authors: Costa R H , Grayson D R , Darnell J E
Published: October 2015

Transthyretin (TTR) and alpha 1-antitrypsin (alpha 1-AT) are expressed at high levels in the liver and also in at least one other cell type. We report here a detailed analysis of the proximal regulatory region of the TTR gene, which has uncovered two new DNA-binding factors that are present mainly (or only) in hepatocytes. One of these new factors, hepatocyte nuclear factor 3 (HNF-3), binds to two sites that are crucial in TTR expression as well as to two additional sites in the alpha 1-AT proximal enhancer region. The second new factor, HNF-4, binds to two sites in TTR that are required for gene activity. We had previously identified binding sites for another hepatocyte-enriched DNA-binding protein (C/EBP or a relative thereof), and additional promoter-proximal sites for that protein in both TTR and alpha 1-AT are also reported here. From these results it seems clear that cell-specific expression is not simply the result of a single cell-specific factor for each gene but the result of a combination of such factors. The variation and distribution of such factors among different cell types could be an important basis for the coordinate expression of the TTR and alpha 1-AT genes in the liver or the discordant transcriptional activation of these genes in a few other cell types. The identification of such cell-enriched factors is a necessary prelude to understanding the basis for cell specificity.
Intragenic revertants of yeast invertase variants with secretion-defective leader sequences.
Authors: Preuss D , Botstein D
Published: October 2015

Several secretion-defective variants of invertase from Saccharomyces cerevisiae were generated by replacement of the wild-type signal sequence codons with DNA fragments with random sequences. Strains encoding these proteins failed to grow on medium containing sucrose as the sole source of carbon. The invertase that was made in these strains was found to fractionate with soluble, cytoplasmic proteins, and indirect immunofluorescence confirmed that the mutant invertase was located throughout the cytoplasm. To define the defects in the secretion-defective leader sequences, we selected revertants by requiring growth on sucrose. Surprisingly, most of the reversion events consisted of point changes and duplications in the upstream noncoding portion of the gene. Each of these changes introduced several hydrophobic residues into the nonfunctional leader sequences, suggesting that the defective random leader peptides might simply lack adequate hydrophobicity to be effective signal peptides.
Demethylation enhances removal of pyrimidine dimers from the overall genome and from specific DNA sequences in Chinese hamster ovary cells.
Authors: Ho L , Bohr V A , Hanawalt P C
Published: October 2015

We have examined the effects of changes in cytosine methylation on DNA repair in UV-irradiated Chinese hamster ovary (CHO) cells. A hypomethylated derivative of the CHO K1B11 line, B11aza, was established by passaging B11 cells over several months in increasing concentrations of 5-azacytidine; greater than 60% demethylation was consistently demonstrated in these conditioned cells. Following a UV dose of 10 J/m2, the amount of repair replication performed within 24 h was approximately twofold higher in B11aza cells than in control B11 cells. Removal of T4 endonuclease V-sensitive sites (ESS) from specific restriction fragments within and around the dihydrofolate reductase (DHFR) gene was then examined in B11aza cells and compared with that in B11 cells. Although demethylation had little or no effect on repair in the 5' half of the DHFR gene, within a nontranscribed sequence immediately downstream from the gene, or within an extragenic region further downstream from the DHFR gene, significant increases in repair were observed at the 3' end of the DHFR gene and within an extragenic region upstream of the DHFR gene. However, the increases in DNA repair were not accompanied by any changes in overall cellular resistance to UV when colony-forming ability was assayed. We suggest that the level of DNA methylation may play an indirect role in the regulation of DNA repair, perhaps through an effect on chromatin structure or transcriptional activity.
Phosphatidylinositol kinase activity associates with viral p60src protein.
Authors: Fukui Y , Hanafusa H
Published: October 2015

Immunoprecipitates of p60v-src proteins from chicken embryo fibroblasts infected with Rous sarcoma virus were assayed for phosphatidylinositol (PI) kinase activity in the absence of detergents. The product of the PI kinase reaction, phosphatidylinositol monophosphate (PIP), migrated slightly slower than did the authentic phosphatidylinositol-4-monophosphate marker in thin-layer chromatography and was indistinguishable from phosphatidylinositol-3-monophosphate produced by PI kinase type I. Furthermore, the deacylated product comigrated with glycerophosphoinositol-3-phosphate in high-performance liquid chromatography. Both sucrose gradient fractionation and the heat stability of PI kinase activity from cells infected with temperature-sensitive mutants suggest that the PI kinase activity is not intrinsic to p60v-src but is a property of another molecule complexed with p60v-src. All transforming variants of p60src were associated with PI kinase activity, whereas this enzyme activity was hardly detectable in immunoprecipitates from cells infected with nontransforming viruses encoding p60c-src or an enzymatically inactive variant. However, PI kinase activity was found in p60src immunoprecipitates from cells infected with nonmyristylated, nontransforming mutants as well as temperature-sensitive mutants at the nonpermissive temperature, which indicated that simple association of PI kinase activity with p60src is not sufficient for cell transformation.
Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.
Authors: Jat P S , Sharp P A
Published: October 2015

The thermolabile large T antigen, encoded by the simian virus 40 early-region mutant tsA58, was used to establish clonal cell lines derived from rat embryo fibroblasts. These cell lines grew continuously at the permissive temperature but upon shift-up to the nonpermissive temperature showed rapidly arrested growth. The growth arrest occurred in either the G1 or G2 phase of the cell cycle. After growth arrest, the cells remained metabolically active as assayed by general protein synthesis and the ability to exclude trypan blue. The inability of these cell lines to divide at the nonpermissive temperature was not readily complemented by the exogenous introduction of other nuclear oncogenes. This finding suggests that either these genes establish cells via different pathways or that immortalization by one oncogene results in a finely balanced cellular state which cannot be adequately complemented by another establishment gene.
Purification and characterization of multiple nuclear factors that bind to the TAX-inducible enhancer within the human T-cell leukemia virus type 1 long terminal repeat.
Authors: Tan T H , Horikoshi M , Roeder R G
Published: October 2015

Within the human T-cell leukemia virus type I promoter, there are three copies of a 21-base-pair repeat (hereafter called the tax-responsive element [TRE]) that both contributes to basal promoter activity and mediates induction by the viral activator TAX. We have identified and separated three nuclear proteins that interact with the TRE. The TRE-binding protein designated TREB-3 bound more avidly to a multimerized TRE than to a single-copy TRE, while the other two TRE-binding proteins, TREB-1 and TREB-2, bound equally well to either TRE. TREB-1 has been purified to near homogeneity, and binding activity was localized to a protein of 35 to 43 kilodaltons. The affinity-purified TREB-1 activated transcription from the human T-cell leukemia virus type I promoter in vitro. The purified TREB-1 fraction contained activating transcription factor binding activity and showed a cooperative interaction with the TATA-binding factor (TFIID) on the adenovirus E4 promoter.
Heat shock and developmental regulation of the Drosophila melanogaster hsp83 gene.
Authors: Xiao H , Lis J T
Published: October 2015

In contrast to the hsp70 gene, whose expression is normally at a very low level and increases by more than 2 orders of magnitude during heat shock, the hsp83 gene in Drosophila melanogaster is expressed at high levels during normal development and increases only severalfold in response to heat shock. Developmental expression of the hsp83 gene consists of a high level of tissue-general, basal expression and a very high level of expression in ovaries. We identified regions upstream of the hsp83 gene that were required for its developmental and heat shock-induced expression by assaying beta-galactosidase activity and mRNA levels in transgenic animals containing a series of 5' deletion and insertion mutations of an hsp83-lacZ fusion gene. Deletion of sequences upstream of the overlapping array of a previously defined heat shock consensus (HSC) sequence eliminated both forms of developmental expression of the hsp83 gene. As a result, the hsp83 gene with this deletion mutation was regulated like that of the hsp70 gene. Moreover, an in vivo polymer competition assay revealed that the overlapping HSC sequences of the hsp83 gene and the nonoverlapping HSC sequences of the hsp70 gene had similar affinities for the factor required for heat induction of the two heat shock genes. We discuss the functional similarity of hsp70 and hsp83 heat shock regulation in terms of a revised view of the heat shock-regulatory sequence.
H-ras mutants lacking the epitope for the neutralizing monoclonal antibody Y13-259 show decreased biological activity and are deficient in GTPase-activating protein interaction.
Authors: Srivastava S K , Di Donato A , Lacal J C
Published: October 2015

We have generated deletion mutants of the H-ras p21 protein which lack residues 58 to 63 or 64 to 68 and contain either the normal glycine or an activating mutation, arginine, at position 12. None of the deleted proteins were recognized by monoclonal antibody Y13-259, and those mutants with activating mutations showed at least a 100-fold reduction in their transforming activities compared with the activities of their nondeleted counterparts. Alterations observed in the in vitro GTPase or GTP interchange properties of the deletion mutants were not consistent with the decrease in their transforming activities. Moreover, each mutant showed normal membrane localization, which is essential for its biological activity. Recently, a newly identified protein, designated GTPase-activating protein (GAP), was found to markedly increase GTPase activity of the normal ras p21 but not of p21 mutants bearing activating lesions (H. Adari, D. R. Lowy, B. M. Willumsen, C. J. Der, and F. McCormick, Science 240:518-521, 1988). We showed that GAP had no effect on the in vitro GTPase activity of the deletion mutants of the normal p21 protein. Since similar deletions in mutants with activating lesions at position 12 or 59 or both showed decreased transforming activity, our results suggest that the recognition site for Y13-259 within the ras p21 molecule influences directly or indirectly the interaction of ras p21 with GAP and that this interaction is critical for biological activity of ras proteins.
Conserved pattern of antisense overlapping transcription in the homologous human ERCC-1 and yeast RAD10 DNA repair gene regions.
Authors: van Duin M , van Den Tol J , Hoeijmakers J H , Bootsma D , Rupp I P , Reynolds P , Prakash L , Prakash S
Published: October 2015

We report that the genes for the homologous Saccharomyces cerevisiae RAD10 and human ERCC-1 DNA excision repair proteins harbor overlapping antisense transcription units in their 3' regions. Since naturally occurring antisense transcription is rare in S. cerevisiae and humans (this is the first example in human cells), our findings indicate that antisense transcription in the ERCC-1-RAD10 gene regions represents an evolutionarily conserved feature.
Interaction of nuclear proteins with a positive cis-acting element of rat embryonic myosin heavy-chain promoter: identification of a new transcriptional factor.
Authors: Yu Y T , Nadal-Ginard B
Published: October 2015

A DNA fragment of the rat embryonic myosin heavy-chain promoter (MHCemb) has been found to specifically bind a nuclear factor (NFe) present in extracts prepared from mouse C2 myoblasts, myotubes, and HeLa cells. The nucleotide sequence of the binding site (BSe) has been identified as 5'-GTGTCAGTCA-3' and was located between -93 and -84. Transient expression studies on MHCemb promoter deletion constructs in C2 myoblasts and C2 myotubes suggested that NFe is a transcriptional factor. Deletion of the NFe-binding site resulted in four- to sixfold and twofold reduction of promoter activity in C2 myotubes and C2 myoblasts, respectively. Furthermore, point mutations at the BSe not only abolished the NFe-binding activity of the MHCemb promoter but also resulted in reduction of the promoter activity to levels similar to those of the deletion constructs in C2 myotubes, myoblasts, and Hela cells (four- to sixfold). Although BSe and the binding site of the recently identified transcriptional factors AP-1 and ATF share significant homology, the results from competition binding assays indicated that NFe is different from both AP-1 and ATF.
Molecular cloning of a gene selectively induced by gamma interferon from human macrophage cell line U937.
Authors: Fan X D , Stark G R , Bloom B R
Published: October 2015

A cDNA clone encoding a gamma interferon (IFN-gamma)-inducible mRNA in human cells of the macrophage lineage was isolated and characterized. The corresponding gene, gamma.1, was selectively induced by IFN-gamma, responding a hundredfold better to IFN-gamma than to IFN-alpha. The induction was rapid and transient, with maximal mRNA accumulation at about 3 h and decline to the basal level after 48 h. Transcriptional activation could be detected as early as 5 min after IFN-gamma stimulation and accounted entirely for the mRNA accumulation. The induction of gamma.1 by IFN-gamma was cell-type restricted, being seen only in macrophages and endothelial cells. In addition, phorbol ester-induced differentiation of promyelocytic HL-60 cells and promonocytic THP-1 cells rendered the gamma.1 gene inducible by IFN-gamma. The 1.0-kilobase gamma.1 cDNA sequence encoded a small predicted polypeptide of 38 amino acids and had a conserved sequence associated with rapidly turning over mRNAs. In vitro translation of the gamma.1 transcript yielded a 4,000-dalton polypeptide.