bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342.
Authors: McCracken A A , Kruse K B , Brown J L
Published: October 2015

Human alpha-1-proteinase inhibitor (A1PI) deficiency, associated with the Z-variant A1PI (A1PI/Z) gene, results from defective secretion of the inhibitor from the liver. The A1PI/Z gene exhibits two point mutations which specify amino acid substitutions, Val-213 to Ala and Glu-342 to Lys. The functional importance of these substitutions in A1PI deficiency was investigated by studying the secretion of A1PI synthesized in COS cells transfected with A1PI genes altered by site-directed mutagenesis. This model system correctly duplicates the secretion defect seen in individuals homozygous for the A1PI/Z allele and shows that the substitution of Lys for Glu-342 alone causes defective secretion of A1PI. The substitution of Lys for Glu-342 eliminates the possibility for a salt bridge between residues 342 and 290, which may decrease the conformational stability of the molecule and thus account for the secretion defect. However, when we removed the potential to form a salt bridge from the wild-type inhibitor by changing Lys-290 to Glu (A1PI/SB-290Glu), secretion was not reduced to the 19% of normal level seen for A1PI/Z-342Lys; in fact, 75% of normal secretion was observed. When the potential for salt bridge formation was returned to A1PI/Z-342Lys by changing Lys-290 to Glu, only 46% of normal secretion was seen. These data indicate that the amino acid substitution at position 342, rather than the potential to form the 290-342 salt bridge, is the critical alteration leading to the defect in A1PI secretion.
A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells.
Authors: Bender M A , Gelinas R E , Miller A D
Published: October 2015

Murine bone marrow was infected with a high-titer retrovirus vector containing the human beta-globin and neomycin phosphotransferase genes. Anemic W/Wv mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human beta-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human beta-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human beta-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide. DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active beta-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.
Dynamic interaction between a Drosophila transcription factor and RNA polymerase II.
Authors: Price D H , Sluder A E , Greenleaf A L
Published: October 2015

We have purified factor 5, a Drosophila RNA polymerase II transcription factor. Factor 5 was found to be required for accurate initiation of transcription from specific promoters and also had a dramatic effect on the elongation properties of RNA polymerase II. Kinetic studies suggested that factor 5 stimulates the elongation rate of RNA polymerase II on a dC-tailed, double-stranded template by reducing the time spent at the numerous pause sites encountered by the polymerase. The factor was found to be composed of two polypeptides (34 and 86 kilodaltons). Both subunits bound tightly to pure RNA polymerase II but were not bound to polymerase in elongation complexes. Our results suggest that factor 5 interacts briefly with the paused polymerase molecules and catalyzes a conformational change in them such that they adopt an elongation-competent conformation.
Sea urchin early and late H4 histone genes bind a specific transcription factor in a stable preinitiation complex.
Authors: Tung L , Morris G F , Yager L N , Weinberg E S
Published: October 2015

Early embryonic H4 (EH4) and H2B (EH2B) and late embryonic H4 (LH4) histone genes were transcribed in vitro in a nuclear extract from hatching blastula embryos of the sea urchin Strongylocentrotus purpuratus. The extract was prepared by slight modifications of the methods of Morris et al. (G. F. Morris, D. H. Price, and W. F. Marzluff, Proc. Natl. Acad. Sci. USA 83:3674-3678, 1986) that have been used to obtain a cell-free transcription system from embryos of the sea urchin Lytechinus variegatus. Achievement of maximum levels of transcription of the EH4 and LH4 genes required a 5- to 10-min preincubation of template with extract in the absence of ribonucleoside triphosphates. This preincubation allowed the formation of a stable complex which was preferentially transcribed compared with a second EH4 or LH4 template that was added 10 min later. Although the EH4 gene inhibited both EH4 and LH4 gene transcription in this assay and although the LH4 gene inhibited both EH4 and LH4 genes, neither of these genes inhibited transcription of the EH2B gene. Preincubation with the EH2B gene had no effect on the transcription of subsequently added EH4 or LH4 genes. Using this template commitment assay, we showed that the site of binding of at least one essential factor required for transcription of both EH4 and LH4 genes was located between positions -102 and -436 relative to the 5' terminus of the EH4 mRNA. Moreover, deletion of this region resulted in a reduction in EH4 gene transcription in vitro. The sea urchin gene-specific trans-acting factors, in the analysis of the cis-acting sequences with which they interact, and in biochemical studies on the formation of stable transcription complexes.
Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation.
Authors: Runge K W , Zakian V A
Published: October 2015

The termini of Saccharomyces cerevisiae chromosomes consist of tracts of C1-3A (one to three cytosine and one adenine residue) sequences of approximately 450 base pairs in length. To gain insights into trans-acting factors at telomeres, high-copy-number linear and circular plasmids containing tracts of C1-3A sequences were introduced into S. cerevisiae. We devised a novel system to distinguish by color colonies that maintained the vector at 1 to 5, 20 to 50, and 100 to 400 copies per cell and used it to change the amount of telomeric DNA sequences per cell. An increase in the number of C1-3A sequences caused an increase in the length of telomeric C1-3A repeats that was proportional to plasmid copy number. Our data suggest that telomere growth is inhibited by a limiting factor(s) that specifically recognizes C1-3A sequences and that this factor can be effectively competed for by long tracts of C1-3A sequences at telomeres or on circular plasmids. Telomeres without this factor are exposed to processes that serve to lengthen chromosome ends.
Characterization and purification of H1TF2, a novel CCAAT-binding protein that interacts with a histone H1 subtype-specific consensus element.
Authors: Gallinari P , La Bella F , Heintz N
Published: October 2015

Definition of mechanisms regulating human histone H1 gene transcription during the cell cycle requires the isolation and biochemical characterization of protein factors which interact with specific promoter elements. Two distinct binding activities have been identified in nuclear extracts from HeLa cells and mapped within a 180-base-pair (bp) region of a cell cycle-regulated H1 gene promoter. H1TF1 bound to an H1-specific A + C-rich sequence (AC box), 100 bp upstream of the cap site; H1TF2 interacted with the H1 subtype-specific consensus element and was dependent on the presence of an intact CCAAT box for binding. H1TF2 was purified through a combination of ion-exchange and oligonucleotide affinity chromatographies. Analysis of purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV crosslinking showed that H1TF2 was a single polypeptide of 47 kilodaltons. This factor was distinct from previously characterized CCAAT-binding proteins in both molecular size and binding properties. Fractions containing H1TF2 activity activated transcription in vitro only if programmed with an H1 DNA template carrying an intact H1TF2-binding site.
Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases.
Authors: Ellis S R , Hopper A K , Martin N C
Published: October 2015

Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.
Human retinoblastoma gene: long-range mapping and analysis of its deletion in a breast cancer cell line.
Authors: Bookstein R , Lee E Y , Peccei A , Lee W H
Published: October 2015

Mutational inactivation of the retinoblastoma (RB) gene is considered a fundamental event in the formation of several types of human cancer. A substantial proportion of RB gene mutations are partial or complete deletions that extend an unknown distance beyond one or both ends of the gene. To provide a framework for measuring the extent of these deletions, we have constructed a long-range restriction map of SfiI sites spanning 850 kilobases around the RB gene. This map was applied in a molecular analysis of RB gene deletion in breast cancer cell line MB468. A previous study of this cell line demonstrated deletion of the entire RB gene except for exons 1 and 2 (E. Y.-H. P. Lee, H. To, J.-Y. Shew, R. Bookstein, P. Scully, and W.-H. Lee, Science 241:218-221, 1988). Genomic clones containing the deletion junction were isolated from a library made from MB468 DNA. A probe obtained from the far side of the deletion junction was used to localize and clone the unknown 3' endpoint, demonstrating that the chromosomal mutation in this case was a simple deletion spanning 200 kilobases. Sequence analysis of the deletion junction indicated a conservative deletion with no loss or gain of nucleotides. The deletion endpoints had no sequence homology to each other or to any repetitive sequence family, such as Alu, so the recombination event was illegitimate. Structural analysis of this and other RB gene deletions is important for understanding molecular mechanisms of recessive oncogenesis.
The Xenopus laevis U2 gene distal sequence element (enhancer) is composed of four subdomains that can act independently and are partly functionally redundant.
Authors: Tebb G , Mattaj I W
Published: October 2015

The sequences involved in enhancement of transcription of the Xenopus U2 small nuclear RNA gene by the distal sequence element (DSE) of its promoter were analyzed in detail by microinjection of mutant genes into Xenopus oocytes. The DSE was shown to be roughly 60 base pairs long. Within this region, four motifs were found to contribute to DSE function: an ATGCAAAT octamer sequence, an SpI binding site, and two additional motifs which, since they are related in sequence, may bind the same transcription factor. These motifs were named D2 (for DSE; U2). Both the octamer sequence and the SpI site bound nuclear factors in vitro, but no factor binding to the D2 motifs was detected. All four elements were independently capable of enhancing transcription of the U2 gene to some extent. Furthermore, when assayed under both competitive and noncompetitive conditions, the individual units of the DSE displayed functional redundancy.
Functional heterogeneity of proto-oncogene tyrosine kinases: the C terminus of the human epidermal growth factor receptor facilitates cell proliferation.
Authors: Velu T J , Vass W C , Lowy D R , Beguinot L
Published: October 2015

Previous reports have indicated that the C termini of the membrane-associated tyrosine kinases encoded by c-src and c-fms proto-oncogenes have a negative effect on their biological activity and that this effect is mediated by their C-terminal tyrosine residue. To determine whether this was true for the human epidermal growth factor (EGF) receptor, which is also a membrane-associated tyrosine kinase proto-oncogene, we have constructed two premature termination mutants, dc19 and dc63, that delete the C-terminal 19 and 63 amino acids, respectively, from the human full-length receptor (hEGFR). The smaller deletion removes the C-terminal tyrosine residue, while the larger deletion removes the two most C-terminal tyrosines; similar deletions are found in v-erbB. As previously shown for the gene encoding the full-length EGF receptor, the two C-terminal mutants induced EGF-dependent focal transformation and anchorage-independent growth of NIH 3T3 cells. However, both dc19 and dc63 were quantitatively less efficient than the gene encoding the full-length receptor, with dc63 being less active than dc19. Although the C-terminal mutants displayed lower biological activity than the gene encoding the full-length receptor, the mutant receptors were found to be similar in several respects to the full-length receptor. These parameters included receptor localization, stability in the absence of EGF, receptor half-life in the presence of EGF, EGF binding, extent of EGF-dependent autophosphorylation in vitro, and EGF-dependent phosphorylation of an exogenous substrate in vitro. Therefore, the C-terminal 63 amino acids of the human receptor have no detectable influence on EGF-dependent early events. We conclude that in contrast
Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice.
Authors: Kim E , Waters S H , Hake L E , Hecht N B
Published: October 2015

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.
Regulation of the mRNA for monocyte-derived neutrophil-activating peptide in differentiating HL60 promyelocytes.
Authors: Kowalski J , Denhardt D T
Published: October 2015

A cDNA library was constructed from HL60 human promyelocyte poly(A)+ RNA harvested 3 h after induction of macrophage differentiation with 12-O-tetradecanoyl phorbol-13-acetate in the presence of cycloheximide. We isolated from this library a 1.6-kilobase full-length clone designated b4 whose corresponding mRNA was greatly increased in abundance in cytoplasmic RNA under these conditions. Dideoxy sequencing revealed that this mRNA encoded MONAP (monocyte-derived neutrophil-activating peptide), a 10-kilodalton monokine with neutrophil-specific chemotactic and enzyme-releasing activities. The 3' untranslated region of this mRNA was found to be 1.2 kilobases long and possessed nine copies of the AUUUA sequence known to be associated with regulation of mRNA stability. Actinomycin D chase experiments yielded evidence that cytoplasmic stabilization was one of the means of regulation of MONAP expression. Analysis of cytoplasmic poly(A)- RNA revealed the presence of several discrete truncated species that shared a common 5' end and appeared to be intermediates of degradation. S1 mapping showed that the 3' ends of these molecules were distributed throughout the 3' untranslated region, preferentially in A + U-rich regions, broadly correlating with the distribution of AUUUA sites. Nuclear run-on experiments indicated that transcriptional induction accounted for less than 15% of the accumulation of MONAP mRNA. This mRNA was induced in HL60 cells by treatment with several differentiation-inducing agents: 12-O-tetradecanoyl phorbol-13-myristate alone, sodium butyrate, vitamin D3, and dimethyl sulfoxide. It was also induced in quiescent diploid lung fibroblasts stimulated to divide by serum, and it was constitutively overexpressed by some human tumor lines.
Hygromycin resistance as a selectable marker in Dictyostelium discoideum.
Authors: Egelhoff T T , Brown S S , Manstein D J , Spudich J A
Published: October 2015

We have constructed an expression cartridge which has the bacterial hygromycin resistance gene (hph) fused to the Dictyostelium discoideum actin 15 promoter, with a segment of 3'-flanking DNA from the actin 15 locus placed downstream of the hph gene to serve as a transcription terminator. The plasmid pDE109, which contained this cartridge and a Dictyostelium origin of replication, transformed D. discoideum with high efficiency under hygromycin selection. The availability of this selectable marker circumvents the previous limitation of having G418 resistance as the only selectable marker for this organism; secondary transformation can now be used to introduce DNA into previously transformed cell lines.
Expression of avian Ca2+-ATPase in cultured mouse myogenic cells.
Authors: Karin N J , Kaprielian Z , Fambrough D M
Published: October 2015

cDNA encoding Ca2+-ATPase was cloned from a chicken skeletal muscle library. The cDNA (termed FCa) comprised 3,239 base pairs, including an open reading frame encoding 994 amino acids which showed the highest degree of homology with the adult rabbit fast-twitch Ca2+-ATPase isoform (C. J. Brandl, S. de Leon, D. R. Martin, and D. H. MacLennan, J. Biol. Chem. 262:3768-3774, 1987). Radiolabeled FCa hybridized to a 3.2-kilobase transcript in chicken skeletal muscle RNA but not to cardiac muscle RNA, which confirmed its identity as encoding the fast Ca2+-ATPase isoenzyme. FCa was transfected into the mouse myogenic line C2C12, from which a protein of 100 kilodaltons was immunopurified by using a monoclonal antibody specific for the avian fast Ca2+-ATPase. Immunofluorescence microscopy of a line (designated C2FCa2) stably expressing the avian Ca2+-ATPase localized the protein to the nuclear envelope and a population of cytoplasmic vesicles. A similar pattern was observed when C2FCa2 cells were stained with DiOC6(3), a cyanine dye that labels endoplasmic reticulum and mitochondria (M. Terasaki, J. Song, J. R. Wong, M. J. Weiss, and L. B. Chen, Cell 38:101-108, 1984). We conclude that the avian Ca2+-ATPase fast isoform is expressed and correctly targeted to the endoplasmic reticulum in mouse C2C12 cells.
Three breakpoints of variant t(2;8) translocations in Burkitt's lymphoma cells fall within a region 140 kilobases distal from c-myc.
Authors: Henglein B , Synovzik H , Groitl P , Bornkamm G W , Hartl P , Lipp M
Published: October 2015

The variant translocations t(2;8) in Burkitt's lymphoma cells join band q24 of chromosome 8, distal from c-myc, to the Igkappa locus, with considerable variation in the location of the breakpoints on chromosome 8. We report the cloning and molecular characterization of a chromosome 8 region, distal from the c-myc locus, which encompasses the breakpoints of the Burkitt's lymphoma cell lines BL64, BL21, and LY91 within 11 kilobase pairs, termed provisionally bvr-1 (Burkitt's variants' rearranging region 1). Using probes from the c-myc, the bvr-1, and the human pvt-1 loci obtained by chromosome walking coupled with pulsed-field gel electrophoresis, we have constructed a physical map of the region 3' of c-myc. We map bvr-1 and pvt-1 about 140 and 260 kilobase pairs, respectively, distal from c-myc.
Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells.
Authors: Axelrod J H , Reich R , Miskin R
Published: October 2015

The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities.
A transcriptional cascade governs entry into meiosis in Saccharomyces cerevisiae.
Authors: Smith H E , Mitchell A P
Published: October 2015

Two signals activate meiosis in yeast: starvation and expression of the a1 and alpha 2 products of the mating-type locus. Prior studies suggest that these signals stimulate expression of an activator of meiosis, the IME1 (inducer of meiosis) product. We have cloned a gene, IME2, with properties similar to those of IME1: both genes are required for meiosis, and both RNAs are induced in meiotic cells. Elevated dosage of IME1 or IME2 stimulates the meiotic recombination pathway without starvation; thus, the IME products may be part of the switch that activates meiosis. IME1 was found to be required for IME2 expression, and a multicopy IME2 plasmid permitted meiosis in an ime1 deletion mutant. Accordingly, we propose that the IME1 product stimulates meiosis mainly through activation of IME2 expression.
Glucose-regulated protein (GRP94 and GRP78) genes share common regulatory domains and are coordinately regulated by common trans-acting factors.
Authors: Chang S C , Erwin A E , Lee A S
Published: October 2015

We isolated the promoter of the human gene encoding the 94,000-dalton glucose-regulated protein (GRP94). The 5'-flanking region important for its expression was identified by deletion analysis. Comparison of the promoters of the genes for GRP78 and GRP94 derived from human, rat, and chicken cells revealed a common domain of 28 base pairs within the putative regulatory regions of both genes. This domain has been shown to interact with protein factors in the promoter of the gene for GRP78. Since the genes for GRP94 and GRP78 are transcriptionally regulated with similar kinetics under a variety of stress conditions, we are interested in examining the possible mechanisms for their coordinated expression. Through in vitro and in vivo competition assays, we found that the protein factors which interact with the promoter of the gene for GRP94 also have affinity for the conserved domain of the promoter of the gene for GRP78. These findings suggest that the genes for GRP94 and GRP78 are coordinately regulated through common trans-acting factors which recognize a common regulatory domain of glucose-regulated protein gene promoters.
Differentiation in vitro of a leukemia virus-induced B-cell lymphoma into macrophages.
Authors: Hanecak R , Zovich D C , Pattengale P K , Fan H
Published: October 2015

Cells of the hemopoietic system arise by proliferation and differentiation of progenitor cells. This process begins with multipotential stem cells which can self-renew and also undergo progressive differentiation to progenitor cells committed to particular lineages, ultimately yielding mature blood cells (D. Metcalf and M. A. S. Moore, Haematopoietic Cells, 1971). Early commitment of lymphoid progenitors is generally believed to separate the lymphoid lineage from the myeloid and erythroid lineages, whose progenitors are separated late in differentiation (Metcalf and Moore, 1971). We recently developed a derivative of Moloney murine leukemia virus (M-MuLV) in which the enhancer sequences from simian virus 40 were substituted into the M-MuLV long terminal repeat. This recombinant virus (delta Mo + SV M-MuLV) induces pre-B and B lymphoid leukemia with long latency after inoculation of 2-day-old NIH Swiss mice (R. Hanecak, P. K. Pattengale, and H. Fan, J. Virol. 62:2427-2436, 1988). In this report, we describe the derivation of a permanent, virus-producing cell line with the phenotypic characteristics of mature macrophages from a B-cell-derived lymphoblastic lymphoma induced by delta Mo + SV M-MuLV. Comparison studies of immunoglobulin heavy-chain gene rearrangements and also delta Mo + SV M-MuLV proviral integration sites confirmed that the macrophage cell line was derived from the original B-lymphoblastic lymphoma. Moreover, inoculation of the macrophage cell line into animals resulted in histiocytic sarcomas of the macrophage type, thus reflecting stable conversion of B-lymphoid tumor cells to the macrophage phenotype. These results suggest a closer relationship between lymphoid and myeloid cells than previously believed.
Involvement of second messengers in regulation of the low-density lipoprotein receptor gene.
Authors: Auwerx J H , Chait A , Wolfbauer G , Deeb S S
Published: October 2015

Transcription of the low-density lipoprotein receptor (LDL-R) gene in the human monocytic leukemic cell line THP-1 and in the human hepatocarcinoma cell line Hep-G2 is regulated by second messengers of the diacylglycerol-protein kinase C (DAG-PKC), inositol 1,4,5-triphosphate-Ca2+, and cyclic AMP pathways. Exogenous phospholipase C (which releases DAG and inositol 1,4,5-triphosphate), PKC activators (phorbol esters and DAG), Ca2+ ionophores, and a cyclic AMP analog all transiently induced accumulation of LDL-R mRNA. The effects of these three signal-transducing pathways were to a large extent additive. Furthermore, PKC stimulation effected an increase in LDL binding, which suggested that the increase in LDL-R mRNA resulted in an increase in functional cell surface receptor activity. These results suggest that uptake of cholesterol by these cells is under control of both intracellular cholesterol levels and external signals.
Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1.
Authors: Thukral S K , Tavianini M A , Blumberg H , Young E T
Published: October 2015

ADR1 is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae. ADR1 has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UAS1) in the ADH2 promoter. A functional dissection of ADR1 was performed by using a series of amino- and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli beta-galactosidase. These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression. Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo. Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding. Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding. The smallest fusion protein able to activate ADH2 transcription, containing ADR1 amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids 1 to 642 of ADR1. In addition, multiple regions of the ADR1 polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified. An ADR1-beta-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADR1 was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADR1 influence the expression of the gene fusion.
The upstream muscle-specific enhancer of the rat muscle creatine kinase gene is composed of multiple elements.
Authors: Horlick R A , Benfield P A
Published: October 2015

A series of constructs that links the rat muscle creatine kinase promoter to the bacterial chloramphenicol acetyltransferase gene was generated. These constructs were introduced into differentiating mouse C2C12 myogenic cells to localize sequences that are important for up-regulation of the creatine kinase gene during myogenic differentiation. A muscle-specific enhancer element responsible for induction of chloramphenicol acetyltransferase expression during myogenesis was localized to a 159-base-pair region from 1,031 to 1,190 base pairs upstream of the transcription start site. Analysis of transient expression experiments using promoters mutated by deletion indicated the presence of multiple functional domains within this muscle-specific regulatory element. A DNA fragment spanning this region was used in DNase I protection experiments. Nuclear extracts derived from C2 myotubes protected three regions (designated E1, E2, and E3) on this fragment from digestion, which indicated there may be three or more trans-acting factors that interact with the creatine kinase muscle enhancer. Gel retardation assays revealed that factors able to bind specifically to E1, E2, and E3 are present in a wide variety of tissues and cell types. Transient expression assays demonstrated that elements in regions E1 and E3, but not necessarily E2, are required for full enhancer activity.
In vitro activation and nuclear translocation of NF-kappa B catalyzed by cyclic AMP-dependent protein kinase and protein kinase C.
Authors: Shirakawa F , Mizel S B
Published: October 2015

We have examined whether a precursor form of NF-kappa B, a DNA-binding protein that plays a role in the transcriptional control of several genes, including kappa immunoglobulin light chain and interleukin-2 receptor alpha subunit, could be activated in vitro by protein kinases. DNA-binding activity of NF-kappa B was induced in the cytosolic fraction of unstimulated 70Z/3 murine pre-B cells by incubation with the catalytic subunit of cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). In contrast, PKA and PKC did not activate NF-kappa B in nuclear extracts from unstimulated cells. Identical results were obtained with the human natural killer-like cell line YT, which can be induced to express the interleukin-2 receptor alpha subunit in response to interleukin-1, cyclic AMP, or phorbol 12-myristate 13-acetate. Furthermore, when nuclei from unstimulated cells were incubated with PKA- or PKC-treated cytosolic fraction for 30 min at 30 degrees C, NF-kappa B was translocated into the nuclei. This translocation did not occur at 4 degrees C and was inhibited by wheat germ agglutinin but not by concanavalin A. Our findings support the conclusion that NF-kappa B exists in the cytoplasm of unstimulated cells in an inactive form that can be converted by exposure to PKA or PKC to an active DNA-binding form that can translocate to the nucleus.
Some of the signals for 3'-end formation in transcription of the Saccharomyces cerevisiae Ty-D15 element are immediately downstream of the initiation site.
Authors: Yu K , Elder R T
Published: October 2015

Fragments from the Ty-D15 element of Saccharomyces cerevisiae were assayed for the ability to direct 3'-end formation for RNA initiated by the GAL1 promoter. The delta, the direct repeat at each end of the element, was capable of forming 3' ends at two sites, an inefficient upstream site and an efficient downstream site near the end of the delta. Different sequences were required for 3'-end formation at these sites. For the efficient site, all transcripts had 3' ends in the delta and no downstream transcription was detected, which suggested that these sequences terminate transcription. Surprisingly, the delta region downstream of the initiation site for Ty RNA comprised part of this major site and terminated more than 50% of the transcripts that read into it. Sequences necessary for the efficient site were localized to two small regions. Both regions were upstream of the 3' end and contained similarities to a tripartite consensus sequence that has been proposed as a terminator element. Sequences near the position of the 3' end could also affect termination; a short G + C-rich sequence inserted just downstream changed an efficient terminator to an inefficient one. Initiation in the delta had no effect on the efficiency or positions or termination in that delta. A new initiation site was seen when the same delta terminated transcription, but transcriptional interference did not occur, since the amount of initiation was not decreased.
Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos.
Authors: Fujii M , Shalloway D , Verma I M
Published: October 2015

A promoter of the nuclear proto-oncogene fos was activated by cotransfection with the viral src gene. Ability to transactivate the c-fos promoter was dependent on tyrosine kinase activity, because (i) src mutants which have reduced tyrosine kinase activity due to mutation of Tyr-416 to Phe showed lower promoter activation, (ii) pp60c-src mutants which have increased tyrosine kinase activity due to mutation of Tyr-527 to Phe also augmented c-fos promoter induction, and (iii) mutation in the ATP-binding site of pp60v-src strongly suppressed c-fos promoter activation. Tyrosine kinase activity alone, however, was not sufficient for promoter activation, because of pp60v-src mutant which lacked its myristylation site and consequently membrane association showed no increased c-fos promoter activation. Both the tyrosine kinase- and membrane-association-defective mutants were also unable to induce transformation. Therefore, phosphorylation of membrane-associated substrates appears to be required for both gene expression and cellular transformation by the src protein. Two regions of the c-fos promoter located between positions -362 and -324 and positions -323 and -294 were responsive to src stimulation. We believe that protein tyrosine phosphorylation represents an important step of signal transduction from the membrane to the nucleus.
Transcriptional and posttranscriptional regulation contributes to the sex-regulated expression of two sequence-related genes at the janus locus of Drosophila melanogaster.
Authors: Yanicostas C , Vincent A , Lepesant J A
Published: October 2015

We investigated the structure and developmental pattern of expression of two genes clustered at the janus locus, located at 99D.3R. Data obtained from genomic and cDNA sequencing and from a combination of S1 mapping and primer extension experiments indicated a very unusual organization of this locus, which appeared to be composed of two partially overlapping genes, designated janA and janB. These two genes were found to be transcribed in the same direction. janA encoded one minor and two major transcripts. The 5' end of the janB mRNA mapped within the 3' untranslated region of the janA transcribed sequence. The overlapping region was 118 bases long. Similarities observed between these two genes with respect to both peptidic sequence and intron position strongly suggested that this locus originated from the duplication of an ancestral transcription unit. However, each of the resulting genes has acquired its own specificity of expression linked to sex determination. The janB transcript was detected only in males, and its expression at the adult stage was restricted to germ line cells. The janA gene displayed a much more complex expression; one of the major mRNAs was found in both sexes and at all stages, whereas the two other janA transcripts were expressed only in males.
Purification of a yeast centromere-binding protein that is able to distinguish single base-pair mutations in its recognition site.
Authors: Cai M J , Davis R W
Published: October 2015

A centromere-specific DNA-binding protein has been purified to homogeneity by a combination of conventional and sequence-affinity chromatography from the yeast Saccharomyces cerevisiae. This protein (designated CBP-I) has an apparent molecular weight of 16,000. It binds specifically to the CDEI (centromere DNA element I) region of yeast centromere DNA, as shown by the electrophoretic mobility retardation assay and DNase I protection analysis, but does not bind specifically to other regions of yeast centromere DNA such as CDEII and CDEIII. The relative binding affinity of purified CBP-I to five different point mutations of CDEI correlates directly with the previously determined ability of each point mutation to convey centromere function in a mitotic chromosome segregation assay (J. H. Hegemann, J. H. Shero, G. Cottarel, P. Philippsen, and P. Hieter, Mol. Cell. Biol. 8:2523-2535, 1988). This supports the authenticity of CBP-I as a functional component of the yeast kinetochore.
The first intron of the 4F2 heavy-chain gene contains a transcriptional enhancer element that binds multiple nuclear proteins.
Authors: Karpinski B A , Yang L H , Cacheris P , Morle G D , Leiden J M
Published: October 2015

We utilized the human 4F2 heavy-chain (4F2HC) gene as a model system to study the regulation of inducible gene expression during normal human T-cell activation. Previous studies have demonstrated that 4F2HC gene expression is induced during normal T-cell activation and that the activity of the gene is regulated, at least in part, by the interaction of a constitutively active 5'-flanking housekeeping promoter and a phorbol ester-responsive transcriptional attenuator element located in the exon 1-intron 1 region of the gene. We now report that 4F2HC intron 1 contains a transcriptional enhancer element which is active on a number of heterologous promoters in a variety of murine and human cells. This enhancer element has been mapped to a 187-base-pair RsaI-AluI fragment from 4F2HC intron 1. DNase I footprinting and gel mobility shift analyses demonstrated that this fragment contains two nuclear protein-binding sites (NF-4FA and NF-4FB) which flank a consensus binding site for the inducible AP-1 transcription factor. Deletion analysis showed that the NF-4FA, NF-4FB, and AP-1 sequences are each necessary for full enhancer activity. Murine 4F2HC intron 1 displayed enhancer activity similar to that of its human counterpart. Comparison of the sequences of human and murine 4F2HC intron 1s demonstrated that the NF-4FA, NF-4FB, and AP-1 sequence motifs have been highly conserved during mammalian evolution.
Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein.
Authors: Hickey E , Brandon S E , Smale G , Lloyd D , Weber L A
Published: October 2015

Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89 alpha and hsp89 beta) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89 alpha, is induced by the adenovirus E1A gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987). We have isolated a human hsp89 alpha gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a beta-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89 alpha protein sequence differed from the human hsp89 beta sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89 alpha gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89 alpha mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.
Multiple proto-oncogene activations in avian leukosis virus-induced lymphomas: evidence for stage-specific events.
Authors: Clurman B E , Hayward W S
Published: October 2015

We have examined avian leukosis virus-induced B-cell lymphomas for multiple, stage-specific oncogene activations. Three targets for viral integration were identified: c-myb, c-myc, and a newly identified locus termed c-bic. The c-myb and c-myc genes were associated with different lymphoma phenotypes. The c-bic locus was a target for integration in one class of lymphomas, usually in conjunction with c-myc activation. The data indicate that c-myc and c-bic may act synergistically during lymphomagenesis and that c-bic is involved in late stages of tumor progression.
Transformation of NIH 3T3 fibroblasts by an activated form of p59hck.
Authors: Ziegler S F , Levin S D , Perlmutter R M
Published: October 2015

Phosphorylation of a tyrosine residue near the carboxy terminus of src-family protein tyrosine kinases is believed to regulate the biological activity of these gene products. Conversion of this tyrosine in p59hck (Tyr-501) to a phenylalanine residue by using oligonucleotide-directed mutagenesis yielded a product (p59hckF501) with very potent transforming activity. Quantitative analysis by a soft-agar cloning assay revealed that p59hckF501 was more than 100-fold more effective than a closely related transforming element, p56lckF505, in colony formation. Cells bearing p59hckF501 had increased levels of protein phosphotyrosine. The ability of p59hckF501 to transform NIH 3T3 cells was abolished by a second mutation believed to destroy the ATP-binding domain.
RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth.
Authors: Woychik N A , Young R A
Published: October 2015

RPB4 encodes the fourth-largest RNA polymerase II subunit in Saccharomyces cerevisiae. The RPB4 gene was cloned and sequenced, and its identity was confirmed by amino acid sequence analysis of tryptic peptides from the purified subunit. The RPB4 DNA sequence predicted a protein of 221 amino acids with a molecular mass of 25,414 daltons. The central 100 amino acids of the RPB4 protein were found to be similar to a segment of the major sigma subunit in Escherichia coli RNA polymerase. Deletion of RPB4 produced cells that were heat and cold sensitive but could grow, albeit slowly, at intermediate temperatures. RNA polymerase II lacking the RPB4 subunit exhibited markedly reduced activity in crude extracts in vitro. The RPB4 subunit, although not essential for mRNA synthesis or enzyme assembly, was essential for normal levels of RNA polymerase II activity and indispensable for cell viability over a wide temperature range.
Nonsense mutations in the dihydrofolate reductase gene affect RNA processing.
Authors: Urlaub G , Mitchell P J , Ciudad C J , Chasin L A
Published: October 2015

Steady-state dihydrofolate reductase (dhfr) mRNA levels were decreased as a result of nonsense mutations in the dhfr gene. Thirteen DHFR-deficient mutants were isolated after treatment of Chinese hamster ovary cells with UV irradiation. The positions of most point mutations were localized by RNA heteroduplex mapping, the mutated regions were isolated by cloning or by enzymatic amplification, and base changes were determined by DNA sequencing. Two of the mutants suffered large deletions that spanned the entire dhfr gene. The remaining 11 mutations consisted of nine single-base substitutions, one double-base substitution, and one single-base insertion. All of the single-base substitutions took place at the 3' position of a pyrimidine dinucleotide, supporting the idea that UV mutagenesis proceeds through the formation of pyrimidine dimers in mammalian cells. Of the 11 point mutations, 10 resulted in nonsense codons, either directly or by a frameshift, suggesting that the selection method favored a null phenotype. An examination of steady-state RNA levels in cells carrying these mutations and a comparison with similar data from other dhfr mutants (A. M. Carothers, R. W. Steigerwalt, G. Urlaub, L. A. Chasin, and D. Grunberger, J. Mol. Biol., in press) showed that translation termination mutations in any of the internal exons of the gene gave rise to a low-RNA phenotype, whereas missense mutations in these exons or terminations in exon 6 (the final exon) did not affect dhfr mRNA levels. Nuclear run-on experiments showed that transcription of the mutant genes was normal. The stability of mature dhfr mRNA also was not affected, since (i) decay rates were the same in wild-type and mutant cells after inhibition of RNA synthesis with actinomycin D and (ii) intronless minigene versions of cloned wild-type and nonsense mutant genes were expressed equally after stable transfection. We conclude that RNA processing has been affected by these nonsense mutations and present a model in which both splicing and nuclear transport of an RNA molecule are coupled to its translation. Curiously, the low-RNA mutant phenotype was not exhibited after transfer of the mutant genes, suggesting that the transcripts of transfected genes may be processed differently than are those of their endogenous counterparts.