bluuebunny/crossref_metadata_2025 huggingface dataset:
Rat c-raf oncogene activation by a rearrangement that produces a fused protein.
Authors: Ishikawa F , Takaku F , Nagao M , Sugimura T
Published: September 2015

In a previous study, activated rat c-raf was detected by an NIH 3T3 cell transfection assay, and a rearrangement was demonstrated in the 5' half of the sequence of the gene. In the present study, the cDNAs of normal and activated rat c-raf were analyzed. Results showed that the activated c-raf gene is transcribed to produce a fused mRNA, in which the 5' half of the sequence is replaced by an unknown rat sequence. This mRNA codes a fused c-raf protein. The normal and activated c-raf cDNAs were each connected to the long terminal repeat of Rous sarcoma virus and transfected into NIH 3T3 cells. Only the activated form had transforming activity. We conclude that the rearrangement is responsible for the activation of c-raf.
Expression of qa-1F activator protein: identification of upstream binding sites in the qa gene cluster and localization of the DNA-binding domain.
Authors: Baum J A , Geever R , Giles N H
Published: September 2015

The qa-1F regulatory gene of Neurospora crassa encodes an activator protein required for quinic acid induction of transcription in the qa gene cluster. This activator protein was expressed in insect cell culture with a baculovirus expression vector. The activator binds to 13 sites in the gene cluster that are characterized by a conserved 16-base-pair sequence of partial dyad symmetry. One site is located between the divergently transcribed qa-1F and qa-1S regulatory genes, corroborating prior evidence that qa-1F is autoregulated and controls expression of the qa-1S repressor. Multiple upstream sites located at variable positions 5' to the qa structural genes appear to allow for greater transcriptional control by qa-1F. Full-length and truncated activator peptides were synthesized in vitro, and the DNA-binding domain was localized to the first 183 amino acids. A 28-amino acid sequence within this region shows striking homology to N-terminal sequences from other lower-eucaryotic activator proteins. A qa-1F(Ts) mutation is located within this putative DNA-binding domain.
The genome of Trypanosoma cruzi contains a constitutively expressed, tandemly arranged multicopy gene homologous to a major heat shock protein.
Authors: Dragon E A , Sias S R , Kato E A , Gabe J D
Published: September 2015

cDNA libraries have been constructed in the plasmid vector pUC18 with mRNA isolated from both epimastigotes and trypomastigotes of the Peru strain of Trypanosoma cruzi. Pools of randomly selected clones were analyzed by hybridization-selection-translation. Translation products were immunoprecipitated either with normal human sera or with sera from patients with Chagas' disease (chagasic sera), and the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this approach, a cDNA clone (pEC5) was identified which encodes a portion of an 85,000-Mr polypeptide. A genomic clone was subsequently isolated (FG1) by using oligonucleotide probes derived from the DNA sequence of this cDNA clone. A portion of this clone was isolated and sequenced, and the coding region for the protein was identified. Computer analysis of the predicted protein sequence indicates that this protein is closely related to the 83,000-Mr heat shock protein (hsp83) of Drosophila melanogaster, the hsp90 of Saccharomyces cerevisiae, and the hsp90 of chicken. This gene is tandemly organized in the T. cruzi genome as a cluster of 6 to 10 copies.
Human recipient cell for oncogene transfection studies.
Authors: Tainsky M A , Shamanski F L , Blair D , Vande Woude G
Published: September 2015

We used human oncogene DNA to transform the nontumorigenic, revertant, human osteosarcoma cell line HOS TE-85 clone 5 (ATCC CRL 1543) to tumorigenicity in athymic nude mice with latency periods as short as 3 weeks. These cells were also transformed by genetic markers in genomic DNA samples. Because of their low rate of spontaneous tumor formation and the simplicity of culturing them, HOS cells provide a human cell alternative to NIH 3T3 murine fibroblasts for oncogene transfection studies.
Different activities of viral enhancer elements before and after stable integration of transfected DNAs.
Authors: Yoshimura F K , Chaffin K
Published: September 2015

Analysis of the RNA and DNA levels of a selectable gene linked to a murine retroviral enhancer demonstrated a correlation between RNA levels and tissue-specific enhancer activity during transient expression in T cells but not in stably transformed cell lines.
Effect of deletion and insertion on double-strand-break repair in Saccharomyces cerevisiae.
Authors: Struhl K
Published: September 2015

I investigated double-strand-break repair in Saccharomyces cerevisiae cells by measuring the frequencies and types of integration events at the PET56-HIS3-DED1 chromosomal region associated with the introduction of linearized plasmid DNAs containing homologous sequences. In general, the integration frequencies observed in strains containing a wild-type region, a 1-kilobase (kb) deletion, or a 5-kb insertion were similar, provided that the cleavage site in the plasmid DNA was present in the host genome. Cleavage at a plasmid DNA site corresponding to a region deleted in the chromosome caused a 10-fold reduction in the integration frequency even when the site was close to regions of homology. However, although the integration frequency was normal even when cleavage occurred only 25 base pairs (bp) outside the deletion breakpoint, 98% of the events were associated not with the usual heterogenote structure, but instead with a homogenote structure containing two copies of the deletion allele separated by vector sequences. Similarly, when cleavage occurred 80 bp outside the 5-kb substitution breakpoint, 40% of the integration events were associated with homogenote structures. From these observations, I suggest that exonuclease and polymerase activities are not rate-limiting steps in double-strand-break repair, exonuclease activity is coupled to the initiation step, the integration frequency is strongly influenced by the amount of homology near the recombinogenic ends, both ends of a linear DNA molecule might interact with the host chromosome before significant exonuclease or polymerase action, and the average repair tract is about 600 bp.
Translational control of ribosomal protein synthesis during early Dictyostelium discoideum development.
Authors: Steel L F , Jacobson A
Published: September 2015

Throughout the developmental program of Dictyostelium discoideum there are substantial changes in the rates of both ribosome utilization and rRNA transcription and processing. We examined the regulation of ribosomal protein (r-protein) gene expression and found that, at the start of development, expression of these genes was drastically and specifically reduced by a block to translational initiation. An apparently separate event signals a sudden decrease in the relative amount of r-protein mRNA at about 10 h of development, a time when aggregated amoebae are forming tight cell-cell contacts. For the first 9 h of development, the relative amount of r-protein mRNA remained essentially unchanged and comparable to levels detected in growing cells. While the r-protein mRNAs were almost fully loaded on polysomes during vegetative growth, they were specifically excluded from polysomes at the start of development. The translational block was not the result of irreversible structural changes which inactivate the r-protein mRNAs since they remained translatable both in vitro, in wheat germ extracts, and in vivo, where they were recruited onto polysomes in the presence of the elongation inhibitor cycloheximide. In addition, precise measurements of poly(A) tail lengths on individual hybrid-selected mRNA species showed that there is no difference in the poly(A) tail length of r-protein mRNA isolated from growing cells and 1-h developing cells. Therefore, changes in translational efficiency cannot be attributed to cleavage of poly(A) tails.
Amino acid sequences in the alpha 1 domain and not glycosylation are important in HLA-A2/beta 2-microglobulin association and cell surface expression.
Authors: Santos-Aguado J , Biro P A , Fuhrmann U , Strominger J L , Barbosa J A
Published: September 2015

The role of the single carbohydrate moiety present on the HLA-A2 molecule was studied by introducing several amino acid substitutions (by site-directed mutagenesis of the HLA-A2 gene) in the consensus glycosylation sequence Asn-X-Ser. Two different amino acid substitutions of the asparagine residue at position 86 (glutamine and aspartic acid) resulted in the synthesis of ca. 39,000-molecular-weight nonglycosylated heavy chains that were detected in the cytoplasm but not on the surface of mouse L-cell transfectants. However, a low level of surface expression was detected following transfection of human (rhabdomyosarcoma) cells or mouse L cells containing human beta 2-microglobulin. The defect in surface expression was not due to the absence of the glycan moiety, since the substitution of a glycine for a serine at amino acid 88 did not have the same drastic effect in the presence of human beta 2-microglobulin. These and other data suggest that the asparagine residue may play a critical role in the conformation of the HLA heavy chain and its interaction with beta 2-microglobulin. Immunofluorescence microscopy following permeabilization of the transfectants demonstrated that the unglycosylated HLA heavy chains are sequestered in an unidentified cellular compartment that is different from the Golgi structure.
mRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae.
Authors: Altmann M , Handschin C , Trachsel H
Published: September 2015

We have isolated genomic and cDNA clones encoding protein synthesis initiation factor eIF-4E (mRNA cap-binding protein) of the yeast Saccharomyces cerevisiae. Their identity was established by expression of a cDNA in Escherichia coli. This cDNA encodes a protein indistinguishable from purified eIF-4E in terms of molecular weight, binding to and elution from m7GDP-agarose affinity columns, and proteolytic peptide pattern. The eIF-4E gene was isolated by hybridization of cDNA to clones of a yeast genomic library. The gene lacks introns, is present in one copy per haploid genome, and encodes a protein of 213 amino acid residues. Gene disruption experiments showed that the gene is essential for growth.
Nonmitogenic morphoregulatory action of pp60v-src on multicellular epithelial structures.
Authors: Warren S L , Nelson W J
Published: September 2015

Madin-Darby canine kidney (MDCK) cells form polarized, multicellular epithelial structures in vitro. Low-level expression of pp60v-src in MDCK cells elicits plasticity in these multicellular structures. Plasticity was revealed by the displacement of cells from mechanically stressed regions of the epithelial monolayers; however, the two-dimensional relationship between the cells in the remainder of the monolayer was maintained. Electron microscopy of multicellular structures revealed abnormal separation of the lateral membranes of adjacent cells and selective uncoupling of the junctional complex; the zonula adherens was disrupted, but the zonula occludens and desmosomes were retained. Significantly, this result was not accompanied by transformation of the cells, as judged by the absence of anchorage-independent growth potential. These results demonstrate a nonmitogenic biological activity of pp60v-src which is experimentally dissociable from transformation. This morphoregulatory action on higher-order epithelial structures may reflect a function of related cellular tyrosine kinases.
Phosphorylation of the Saccharomyces cerevisiae equivalent of ribosomal protein S6 has no detectable effect on growth.
Authors: Johnson S P , Warner J R
Published: September 2015

The phosphorylation of mammalian ribosomal protein S6 is affected by a variety of agents, including growth factors and tumor promoters, as well as by expressed oncogenes. Its potential role in the regulation of protein synthesis has been the object of much study. We have developed strains of Saccharomyces cerevisiae in which the phosphorylatable serines of the equivalent ribosomal protein (S10) were converted to alanines by site-directed mutagenesis. The S10 of such cells is not phosphorylated. Comparison of these cells with the parental cells, whose genomes differ by only six nucleotides, revealed no differences in the lag phase or logarithmic phase of a growth cycle, in growth on different carbon sources, in sporulation, or in sensitivity to heat shock. We conclude that in S. cerevisiae the phosphorylation of ribosomal protein S10 may play no role in regulating the synthesis of proteins. This conclusion leads one to ask whether certain protein phosphorylations are simply the adventitious, if easily observable, result of the imperfect specificity of one or another protein kinase.
Induction of a rapidly responsive hepatic gene product by thyroid hormone requires ongoing protein synthesis.
Authors: Jacoby D B , Engle J A , Towle H C
Published: September 2015

The regulation of a gene, designated spot 14, which is rapidly induced in rat liver in response to 3,5,3'-triiodo-L-thyronine (T3) was studied as a model for exploring the molecular basis of thyroid hormone action. The time course of induction of the nuclear precursor to spot 14 mRNA after intramuscular injection of T3 displayed a very short lag period of between 10 and 20 min. The rapidity of this effect suggests that the induction in gene expression occurs as a primary response to the hormone-receptor interaction. The protein synthesis inhibitor cycloheximide injected 15 min before T3 completely blocked the accumulation of nuclear precursor RNA 30 min after T3 treatment. Emetine, an inhibitor of protein synthesis which acts by a different mechanism than cycloheximide, also blocked the induction of the spot 14 nuclear precursor RNA. The increased rate of spot 14 gene transcription observed after T3 treatment, as measured by nuclear run-on assay, was similarly completely abolished in the presence of cycloheximide. In addition, ongoing protein synthesis was required for maintaining spot 14 nuclear precursor RNA at induced levels in animals previously treated with T3. On the other hand, cycloheximide had no effect on T3 uptake or binding to the nuclear receptor during the 45-min time frame studied. The paradox of the rapid kinetics of induction and the requirement of ongoing protein synthesis may be explained by a protein with an extremely short half-life which is necessary for T3 induction of the spot 14 gene.
Translation and membrane insertion of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.
Authors: Wilson C , Gilmore R , Morrison T
Published: September 2015

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses is likely in the unusual class of glycoproteins with the amino terminus cytoplasmic and the carboxy terminus lumenal or external to the cell. The properties of the membrane insertion of the HN protein of Newcastle disease virus, a prototype paramyxovirus, were explored in wheat germ extracts containing microsomal membranes. HN protein was inserted into membranes cotranslationally, resulting in a glycosylated protein completely resistant to trypsin and proteinase K digestion. No detectable posttranslation insertion occurred. Insertion required signal recognition particle. Signal recognition particle in the absence of membranes inhibited HN protein synthesis. Comparisons of the trypsin digestion products of the HN protein made in the cell-free system with newly synthesized HN protein from infected cells showed that the cell-free product was in a conformation different from that of the pulse-labeled protein in infected cells. First, trypsin digestion of intact membranes from infected cells reduced the size of the 74,000-dalton HN protein by approximately 1,000 daltons, whereas trypsin digestion of HN protein made in the cell-free system had no effect on the size of the protein. Second, trypsin digestion of Triton X-100-permeabilized membranes isolated from infected cells resulted in a 67,000-dalton trypsin resistant HN protein fragment. A trypsin-resistant core of comparable size was not present in the digestion products of in-vitro-synthesized HN protein. Evidence is presented that the newly synthesized HN protein in infected cels contain intramolecular disulfide bonds not present in the cell-free product.
Transcription of adenovirus 2 major late and peptide IX genes under conditions of in vitro nucleosome assembly.
Authors: Matsui T
Published: September 2015

Plasmid DNA containing adenovirus 2 major late and peptide IX genes was assembled into nucleosomes in vitro, and the assembled nucleosomes were used as a template to study the regulatory mechanism of transcription initiation under these conditions. Neither the major late nor peptide IX genes was transcribed on the already-assembled nucleosomes. However, the major late gene, but not the peptide IX gene, was transcribed efficiently when the DNA was incubated with HeLa cell extracts prior to assembly into nucleosomes. These results indicate that prebinding of some component in the cell extracts to DNA is essential to activate transcription of the major late gene on nucleosomes assembled under the conditions used here. Since gene IX on the nucleosomes was not transcriptionally active regardless of preincubation of DNA with the extracts, some other component or another, different template structure which is not able to be identified in an in vitro system with deproteinized DNA template might be required for activation of peptide IX gene transcription. To know the function of the upstream sequences of the major late gene, effects of the deletion on transcription of nucleosomes were compared with that of deproteinized DNA. The result showed that depression of transcription by deleting the upstream sequences had more effect on nucleosomes than on deproteinized DNA.
Gene recombination in X-ray-sensitive hamster cells.
Authors: Hamilton A A , Thacker J
Published: September 2015

Recombination was measured in Chinese hamster ovary (CHO-K1) cells and in the X-ray-sensitive mutants xrs1 and xrs7, which show a defect in DNA double-strand break repair. To assay recombination, pairs of derivatives of the plasmid pSV2gpt were constructed with nonoverlapping deletions in the gpt gene region and cotransferred into the different cell types. Recombination efficiencies, measured as the transformation frequency with a pair of deletion plasmids relative to that with the complete pSV2gpt plasmid, were about 6% in both CHO-K1 and the xrs mutants for plasmids linearized at a site outside the gpt gene. However, these efficiencies were substantially enhanced by the introduction of a double-strand break into the homologous region of the gpt gene in one of a pair of deletion plasmids before cotransfer. This enhancement was apparently only about half as great for the xrs cells as for CHO-K1, but variation in the data was considerable. A much larger difference between CHO-K1 and the xrs mutants was found when the DNA concentration dependence of transformation was explored. While the transformation frequency of CHO-K1 increased linearly with DNA concentration, no such increase occurred with the xrs mutants irrespective of whether complete plasmids or pairs of deletion plasmids were transferred. The fraction of cells taking up DNA, assayed autoradiographically, was similar in all cell types. Therefore we suggest that while homologous recombination of plasmid molecules may not be substantially reduced in the xrs mutants,processes involved in the stable integration of plasmid DNA into genomic DNA are significantly impaired.
Ingi, a 5.2-kb dispersed sequence element from Trypanosoma brucei that carries half of a smaller mobile element at either end and has homology with mammalian LINEs.
Authors: Kimmel B E , ole-MoiYoi O K , Young J R
Published: September 2015

A dispersed repetitive element named ingi, which is present in the genome of the protozoan parasite Trypanosoma brucei, is described. One complete 5.2-kilobase element and the ends of two others were sequenced. There were no direct or inverted terminal repeats. Rather, the ends consisted of two halves of a previously described 512-base-pair transposable element (G. Hasan, M.J. Turner, and J.S. Cordingley, Cell 37:333-341, 1984). Oligo(dA) tails and possible insertion site duplications suggested that ingi is a retroposon. The sequenced element appears to be a pseudogene copy of an original retroposon with one or more open reading frames occupying most of its length. Significant homologies of the encoded amino acid sequences with reverse transcriptases and mammalian long interpersed nuclear element sequences suggest a remote evolutionary origin for this kind of retroposon.
Glucocorticoids enhance stability of human growth hormone mRNA.
Authors: Paek I , Axel R
Published: September 2015

We have studied the control of expression of the human growth hormone (hGH) gene introduced into the chromosomes of mouse fibroblasts. Cell lines transformed with the hGH gene expressed low levels of intact hGH mRNA and secreted hGH protein into the medium. Although the level of expression of hGH mRNA was low, the gene remained responsive to induction by glucocorticoid hormones. To localize the sequences responsible for induction and to determine the mechanism by which these cis-acting sequences enhance gene expression, we have constructed a series of fusion genes between the hGH gene and the herpes simplex virus (HSV) thymidine kinase (tk) gene. We have demonstrated that a fusion gene in which hGH cDNA is flanked at its 5' terminus by an HSV tk promoter and is flanked at its 3' terminus by 3' HSV tk DNA remains inducible by glucocorticoids. Our studies indicate that the hGH exons contain sequences which are responsible for glucocorticoid hormone induction. Pulse-chase experiments, in vitro nuclear transcription, and approach to steady-state measurements indicate that the mechanisms responsible for induction of the hGH cDNA fusion gene operate posttranscriptionally to enhance the stability of hGH mRNA. Moreover, this increased stability was associated with an increase in the length of the 3' poly(A) tail on hGH mRNA.
Products of in vitro cleavage and polyadenylation of simian virus 40 late pre-mRNAs.
Authors: Sheets M D , Stephenson P , Wickens M P
Published: September 2015

Formation of mRNA 3' termini involves cleavage of an mRNA precursor and polyadenylation of the newly formed end. Cleavage of simian virus 40 late pre-mRNA in a crude nuclear extract generated two RNAs, 5' and 3' half-molecules. These RNAs were unmodified and linear. The 5' half-molecule contained sequences upstream but not downstream of the poly(A) site and ended in a 3'-terminal hydroxyl. The 3' half-molecules comprised a family of RNAs, each of which contains only sequences downstream of the poly(A) site, and ends in a 5'-terminal phosphate. These RNAs differed only in the locations of their 5' terminus. The 3' terminus of the 5' half-molecule was the adenosine 10 nucleotides downstream of AAUAAA, at the +1 position. The 5' terminus of the longest 3' half-molecule was at +2. Thus, these two RNAs contain every nucleoside and phosphate of the precursor. The existence of these half-molecules demonstrates that endonucleolytic cleavage occurs near the poly(A) site. 5' half-molecules generated in the presence of EDTA (which blocks polyadenylation, but not cleavage) ended at the adenosine at position +1 of the precursor. When incubated in the extract under suitable conditions, they became polyadenylated. 5' half-molecules formed in 3'-dATP-containing reactions contained a single 3'-deoxyadenosine (cordycepin) residue added onto the +1 adenosine and were poor polyadenylation substrates. We infer that the +1 adenosine of the precursor becomes the first A of the poly(A) tract and provides a 3' hydroxyl group to which poly(A) is added posttranscriptionally.
Translational control mediated by eucaryotic initiation factor-2 is restricted to specific mRNAs in transfected cells.
Authors: Kaufman R J , Murtha P
Published: September 2015

The translational efficiency of mRNA molecules transcribed from plasmid DNA transfected into COS-1 monkey cells can be increased 10- to 20-fold by the coexpression of the adenovirus virus-associated RNAs I and II. Experiments described here demonstrate a similar increase in translational efficiency by the addition of 2-aminopurine, an inhibitor of double-stranded RNA-activated protein kinase, to the culture medium. Both virus-associated RNA and 2-aminopurine presumably exert their effect by alteration of the functional level of eucaryotic initiation factor-2. The translational stimulation mediated by both means is shown to be restricted to the plasmid-derived mRNAs because there is no qualitative or quantitative alteration in host protein synthesis. The results are consistent with models invoking a localized activation of double-stranded RNA-activated kinase leading to a translational block.
Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.
Authors: Nonet M , Scafe C , Sexton J , Young R
Published: September 2015

We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis.
Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells.
Authors: Cartier M , Chang M W , Stanners C P
Published: September 2015

A new dominant amplifiable selective system for use in bacterium-animal cell shuttle vectors was developed by the insertion of a 2-kilobase genomic fragment containing the cloned Escherichia coli gene for asparagine synthetase (AS) into the pBR322-simian virus 40 recombinant vector pSV2 so as to place the translational initiator codon for the bacterial AS about 1,000 base pairs downstream from the simian virus 40 early promoter. This new construct, pSV2-AS, retains bacterial sequences for transcriptional and translational initiation and so can express AS in bacteria. The construct can also complement AS- mutants of mammalian cells, giving AS+ transfectants capable of growth in medium lacking asparagine, with relatively high efficiency (about 300 colonies per microgram of DNA per 10(6) cells exposed). The vector can be amplified up to 100-fold in such AS+ transfectants by selection in asparagine-free medium containing increasing concentrations of the AS inhibitor beta-aspartyl hydroxamate. AS+ transfectants were found to be much more resistant to a second AS inhibitor, Albizziin, than were normal AS+ animal cell lines. This difference, which may indicate a strong resistance of the bacterial AS enzyme to Albizziin, was exploited to develop an effective selection for bacterial AS transfectants of a number of wild-type AS+ cell lines of rat, Chinese hamster, mouse, and human origin. LR-73 cells, a Chinese hamster AS+ cell line, were transfected with pSV2-AS with an efficiency of about 1,000 colonies per 0.5 microgram of DNA per 10(6) cells. The integrated construct in these cells was amplified by incubation of the transfectants in increasing concentrations of beta-aspartyl hydroxamate. Advantages and disadvantages of this new dominant, selectable, and amplifiable marker over markers commonly used in shuttle vectors are discussed.
Nucleotides in the polyomavirus enhancer that control viral transcription and DNA replication.
Authors: Tang W J , Berger S L , Triezenberg S J , Folk W R
Published: September 2015

The polyomavirus enhancer is required in cis for high-level expression of the viral early region and for replication of the viral genome. We introduced multiple mutations in the enhancer which reduced transcription and DNA replication. Polyomaviruses with these mutant enhancers formed very small plaques in whole mouse embryo cells. Revertants of the viral mutants were isolated and characterized. Reversion occurred by any of the following events: restoration of guanosines at nucleotide (nt) 5134 and nt 5140 within the adenovirus 5 E1A enhancer core AGGAAGTGACT; acquisition of an A----G mutation at nt 5258, which is the same mutation that enables polyomavirus to grow in embryonal carcinoma F9 cells; duplication of mutated sequences between nt 5146 and 5292 (including sequences homologous with immunoglobulin G, simian virus 40, and bovine papillomavirus enhancer elements). Reversion restored both the replicative and transcriptional functions of the viruses. Revertants that acquired the F9 mutation at nt 5258 grew at least 20-fold better than the original mutant in whole mouse embryo cells, but replicated only marginally better than the original mutant in 3T6 cells. Viruses with a reversion of the mutation at nt 5140 replicated equally well in both types of cells. Since individual nucleotides in the polyomavirus enhancer simultaneously altered DNA replication and transcription in specific cell types, it is likely that these processes rely upon a common element, such as an enhancer-binding protein.
Molecular genetics of androgen-dependent and -independent expression of mouse sex-limited protein.
Authors: Stavenhagen J , Loreni F , Hemenway C , Kalff M , Robins D M
Published: September 2015

Genes of the mouse S locus encoding C4 (the fourth component complement) and Slp (sex-limited protein) show extensive homology but are distinct in their function and regulation. In some mouse strains, such as B10.D2, Slp is androgen regulated, whereas in others, such as B10.W7R, expression of Slp is constitutive. We have previously shown that the B10.W7R strain has multiple Slp genes. In this report, we present the structure of the single C4 and four Slp genes of the B10.W7R S locus and compare the upstream flanking regions by partial sequence analysis and function in transfection assays. Of the four Slp genes, three (Slpw7.A, Slpw7.B, and Slpw7.C) have upstream and promoter regions very similar to those of C4. The fourth Slp gene (Slpw7.D) is instead virtually identical to the androgen-regulated allele (Slpd from the B10.D2 mouse) in upstream regions. In particular, far-upstream sequences from both Slpd and Slpw7.D render the bacterial chloramphenicol acetyltransferase gene hormonally responsive upon transfection into mammary carcinoma cell lines. The upstream sequences between 2 to 3 kilobases of the Slp promoter initiate transcription from multiple sites when fused proximal to the chloramphenicol acetyltransferase gene, and these transcripts are threefold more abundant in the presence of androgen. This behavior is similar for Slpd and Slpw7.D, which suggests that Slpw7.D may be androgen regulated but that this is masked in vivo by constitutive expression of the other Slp genes. Nonhomologous recombination is implicated not only in expanding the copy number of C4 and Slp genes in the B10.W7R mouse but also in creating hybrid genes with regulatory features of C4 and structural features of Slp.
Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila.
Authors: Perrot M , Barreau C , Bégueret J
Published: September 2015

The effect of the chromosomal ends of Tetrahymena thermophila on the stability of linear transforming molecules in the filamentous fungus Podospora anserina was tested. A derivative of an integrative vector for this fungus has been constructed, so that after linearization, the ends of the plasmid are the telomeric sequences of T. thermophila. After transformation, this linear molecule was maintained as an extrachromosomal plasmid with no integrated copies in about 50% of the transformants. Under selective conditions, there was approximately one linear molecule per 5 to 10 nuclei, and these extrachromosomal molecules were rapidly lost under nonselective conditions. The circular plasmid carrying an inverted repeat of T. thermophila telomeres could be linearized and processed in vivo.
Structure and expression of the Saccharomyces cerevisiae CRY1 gene: a highly conserved ribosomal protein gene.
Authors: Larkin J C , Thompson J R , Woolford J L
Published: September 2015

The Saccharomyces cerevisiae CRY1 gene encodes ribosomal protein rp59, a component of the 40S ribosomal subunit. Mutations in CRY1 can confer resistance to the alkaloid cryptopleurine, an inhibitor of the elongation step of translation. The nucleotide sequence of the cloned CRY1 gene was determined. The predicted amino acid sequence shows that CRY1 encodes a 14,561-dalton polypeptide that has 88% amino acid sequence homology to the hamster or human S14 ribosomal protein responsible for emetine resistance and 45% homology to Escherichia coli ribosomal protein S11. Analysis of the DNA sequences upstream from CRY1 revealed the presence of three sequences, HOMOL1 (consensus, A/TACATCC/TG/ATA/GCA), RPG (consensus, ACCCA/GTACATT/CT/A), and a thymine-rich sequence, found upstream of more than 20 other cloned yeast genes encoding components of the translational apparatus. We exploited the ability to assay the expression of CRY1 in vivo by using the cryptopleurine resistance phenotype to demonstrate that these three consensus sequences are necessary for the transcription of CRY1. We previously showed that the upstream promoter element of the yeast RP39A gene consists of these identical sequence motifs. Therefore, we suggest that these three sequences define a consensus promoter element for the genes encoding the yeast translational apparatus. CRY1 is one of several hundred yeast genes, including ribosomal protein genes, whose expression is transiently decreased 10-fold upon heat shock. We found that the HOMOL1 and RPG consensus sequences are not necessary for the heat shock response of CRY1.
The adenovirus Ela gene induces differentiation of F9 teratocarcinoma cells.
Authors: Montano X , Lane D P
Published: September 2015

Undifferentiated F9 cells transfected with plasmids encoding adenovirus E1a gene products underwent radical morphological changes. They ceased to express the SSEA-1 stem cell marker antigen and started to express a number of the characteristics of the differentiated state that is induced in F9 cells by treatment with retinoic acid. In particular, they expressed keratin intermediate filaments and acquired the ability to synthesise simian virus 40 tumor antigens after virus infection. The transfected cells expressed the E1a proteins, and this expression was necessary to induce the phenotypic changes, since a coisogenic plasmid encoding only a truncated 70-amino-acid E1a polypeptide and the transfection procedure itself did not detectably after the morphology or marker expression of the F9 stem cells. The phenotypic change was induced by both 13S and 12S cDNA plasmids. We discuss these results in the context of known E1a functions and with reference to the other oncogenes and external factors that can cause F9 cell differentiation.
Specific growth inhibitory sequences in genomic DNA from quiescent human embryo fibroblasts.
Authors: Padmanabhan R , Howard T H , Howard B H
Published: September 2015

We used HeLa cells as recipients in a gene transfer assay to characterize DNA sequences that negatively regulate mammalian cell growth. In this assay, genomic DNA from quiescent human embryo fibroblasts was more inhibitory for HeLa replication than was DNA from either Escherichia coli or HeLa cells. Surprisingly, growth inhibitory activity depended on the growth state of the cells from which genomic DNA was prepared; it was strongest in DNA prepared from serum-deprived, quiescent embryo fibroblasts. This latter observation implies a role for DNA modification(s) in regulating the activity of the inhibitory sequences detected in our assay. The level of the observed growth inhibitory activity was sometimes high, suggesting that the relevant sequences may be abundantly represented in the mammalian genome. We speculate that these findings may provide new insights into the molecular mechanisms involved in cellular quiescence and in vitro senescence.
Chromatin structure of the HLA-DR alpha gene in different functional states of major histocompatibility complex class II gene expression.
Authors: Peterlin B M , Hardy K J , Larsen A S
Published: September 2015

We utilized DNase I hypersensitivity mapping to study chromatin structure within the HLA-DR alpha gene. We found a single DNase I-hypersensitive site coinciding with the HLA-DR alpha gene promoter in all cells studied. Moreover, in cells that constitutively express HLA-DR, two additional DNase I-hypersensitive sites were observed. These lie within the first intron of the HLA-DR alpha gene and encompass DNA sequences that share homologies with regulatory loci of the immunoglobulin and immune response genes, as well as with core enhancer consensus sequences.
Microinjection of ras p21 induces a rapid rise in intracellular pH.
Authors: Hagag N , Lacal J C , Graber M , Aaronson S , Viola M V
Published: September 2015

Quiescent mouse NIH 3T3 cells responded to microinjection of activated ras p21 with a rapid and sustained rise in intracellular pH (approximately 0.17 pH units). The p21-induced pH change was inhibited by amiloride treatment or growth of cells in medium low in sodium, suggesting a role for the Na+/H+ antiporter. Amiloride was found to suppress p21-induced mitosis, also.
In vitro transcription of immunoglobulin genes in a B-cell extract: effects of enhancer and promoter sequences.
Authors: Sen R , Baltimore D
Published: September 2015

Transfection experiments have led to the identification of three DNA sequences that are responsible for the tissue-specific expression of immunoglobulin genes. As a first step toward characterizing these regulatory phenomena at the biochemical level, we report the development of an in vitro transcription system from cells of the B lymphoid lineage. In these extracts, transcription of the MOPC41 kappa promoter is correctly initiated and dependent on the presence of an upstream sequence element located between -44 and -79 base pairs from the cap site. Second, although standard in vitro transcriptions are not affected by the presence or absence of enhancer sequences, we observed that the addition of polyethylene glycol led to a B-cell extract-specific suppression of transcription from a template that carries an immunoglobulin enhancer.
Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange.
Authors: Hattori S , Clanton D J , Satoh T , Nakamura S , Kaziro Y , Kawakita M , Shih T Y
Published: September 2015

The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.
Recombination and ligation of transfected DNA in CHO mutant EM9, which has high levels of sister chromatid exchange.
Authors: Hoy C A , Fuscoe J C , Thompson L H
Published: September 2015

Transformation frequencies were measured in CHO mutant EM9 after transfection with intact or modified plasmid pSV2-gpt. The mutant and wild-type strain behaved similarly under all conditions except when homologous recombination was required to produce an intact plasmid. Therefore, the defect of the mutant which renders it slow in DNA strand break rejoining and high in sister chromatid exchange induction reduces its ability to recombine foreign DNA molecules.
Transfer of human genes conferring resistance to methylating mutagens, but not to UV irradiation and cross-linking agents, into Chinese hamster ovary cells.
Authors: Kaina B , Van Zeeland A A , Backendorf C , Thielmann H W , Van de Putte P
Published: September 2015

Chinese hamster ovary cells were transfected by human DNA ligated to the bacterial gpt (xanthine-guanine-phosphoribosyltransferase) gene which was used either in its native form or after partial inactivation with methylnitrosourea. The gpt+ transfectants were screened for resistance to high doses of N-methyl-N'-nitro-N-nitrosoguanidine. Using this approach, we showed that Chinese hamster ovary cells can acquire N-methyl-N'-nitro-N-nitrosoguanidine resistance upon transfection with DNA from diploid human fibroblasts, that this resistance is transferable by secondary transfection and is specific for methylating mutagens, and that it is not caused by increased removal of O6-methylguanine, 3-methyladenine, and 7-methylguanine from DNA.
The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation.
Authors: Olson E N , Spizz G , Tainsky M A
Published: September 2015

Differentiation of skeletal muscle involves withdrawal of myoblasts from the cell cycle, fusion to form myotubes, and the coordinate expression of a variety of muscle-specific gene products. Fibroblast growth factor and type beta transforming growth factor specifically inhibit myogenesis; however, the transmembrane signaling pathways responsible for suppression of differentiation by these growth factors remain elusive. Because ras proteins have been implicated in the transduction of growth factor signals across the plasma membrane, we used DNA-mediated gene transfer to investigate the potential involvement of this family of regulatory proteins in the control of myogenesis. Transfection of the mouse skeletal muscle cell line C2 with the oncogenic forms of H-ras or N-ras completely suppressed both myoblast fusion and induction of the muscle-specific gene products nicotinic acetylcholine receptor and creatine kinase. Inhibition of differentiation by activated ras genes occurred at the level of muscle-specific mRNA accumulation. In contrast, proto-oncogenic forms of N-ras or H-ras had no apparent effects on the ability of C2 cells to differentiate. Myoblasts transfected with activated ras genes exhibited normal growth properties and ceased proliferating in the absence of mitogens, indicating that ras inhibited differentiation through a mechanism independent of cell proliferation. These results demonstrate that activated ras gene products mimic the inhibitory effects of fibroblast growth factor and type beta transforming growth factor on myogenic differentiation and suggest that each of these regulators of myogenesis may operate through a common intracellular pathway.
Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae.
Authors: Field J , Broek D , Kataoka T , Wigler M
Published: September 2015

In the yeast Saccharomyces cerevisiae, yeast RAS proteins are potent activators of adenylate cyclase. In the present work we measured the activity of adenylate cyclase in membranes from Saccharomyces cerevisiae which overexpress this enzyme. The response of the enzyme to added RAS2 proteins bound with various guanine nucleotides and their analogs suggests that RAS2 proteins are active in their GTP-bound form and are virtually inactive in their GDP-bound form. Also, active RAS2 protein is not inhibited by inactive RAS2, suggesting that the inactive form does not compete with the active form in binding to its effector.
Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP.
Authors: Olempska-Beer Z , Freese E
Published: September 2015

Meiosis and sporulation of Saccharomyces cerevisiae are initiated in a guanine auxotroph by guanine deprivation (E. Bautz Freese, Z. Olempska-Beer, A. Hartig, and E. Freese, Dev. Biol. 102:438-451, 1984). We used this condition to examine a hypothesis (K. Matsumoto, I. Uno, and T. Ishikawa, Cell 32:417-423, 1983) that initiation of meiosis requires a low level of cAMP. We found that, after guanine deprivation, the intracellular concentration of cAMP transiently decreased not more than 20% and not at all if the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to the medium. Under these conditions, at least 76% of the cells sporulated in the absence of IBMX, and almost 100% sporulated in its presence. The sporulating cells continually excreted cAMP and utilized the gluconeogenic carbon source. The cells failed to sporulate efficiently and to form four-spored asci if simultaneously deprived of guanine and carbon. After guanine deprivation in glucose medium, sporulation remained suppressed and intracellular cAMP was unchanged. We conclude that, under conditions of guanine starvation, cAMP deficiency is not required for initiation of meiosis and sporulation, cAMP is produced in excess and excreted to the medium, the cells sporulate better if the cAMP concentration is increased by addition of IBMX, the cells require a gluconeogenic carbon source for complete and efficient sporulation, and suppression of sporulation by glucose is not mediated by cAMP.
Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.
Authors: Himmelfarb H J , Simpson E M , Friesen J D
Published: September 2015

Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain.