bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoid receptor-dependent inhibition of cellular proliferation in dexamethasone-resistant and hypersensitive rat hepatoma cell variants.
Authors: Cook P W , Swanson K T , Edwards C P , Firestone G L
Published: September 2015

Exposure of the Fu5 rat hepatoma cell line to glucocorticoids, such as dexamethasone and hydrocortisone, suppressed the growth rate and final density of cells grown in the presence of serum. This hormonal effect was proportional to receptor occupancy and affinity and, in addition, the glucocorticoid antagonist RU38486 prevented this response. Two classes of dexamethasone-resistant variants that failed to be growth inhibited were recovered from ethyl methylsulfonate-mutagenized populations by continuous culture in the presence of 1 microM dexamethasone. The first class, represented by the EDR3 subclone, was completely glucocorticoid unresponsive and failed to express receptor transcripts. The second class, represented by the EDR1, EDR5, and EDR7 subclones, possessed significant levels of glucocorticoid receptor but were only partially glucocorticoid responsive when stimulated with saturating levels of hormone. Introduction of functional glucocorticoid receptor genes into both classes of dexamethasone-resistant variants by a recombinant retrovirus expression vector restored glucocorticoid responsiveness and suppression of cell growth. A hypersensitive variant (BDS1), recovered by bromodeoxyuridine selection, was fully glucocorticoid responsive, and its inhibition of proliferation was more acutely regulated by dexamethasone. Taken together, our results established that the inhibition of proliferation in Fu5 rat hepatoma cells represents a new glucocorticoid response that requires the expression of a functional glucocorticoid receptor.
High-level expression of c-H-ras1 fails to fully transform rat-1 cells.
Authors: Ricketts M H , Levinson A D
Published: September 2015

Rat-1 cells were transfected with plasmids encoding normal (Gly-12), nonactivated (Pro-12), and activated (Val-12 and Ile-12) p21H-ras in the presence of an amplifiable dihydrofolate reductase marker. The introduced DNA was amplified by selection in methotrexate to establish the relationship between p21H-ras expression and various hallmarks of cellular transformation. The maximum level of p21H-ras (Gly-12) consistent with cell viability was approximately 0.13% of total cell protein (approximately 60,000 molecules per cell); this is 44-fold greater than the level of the endogenous protein. The maximum tolerated level of a second nontransforming form of p21H-ras (pro-12) was about half of this. Amplification in Rat-1 cells of H-ras genes encoding the highly oncogenic Val-12 and Ile-12 forms of p21H-ras could not be achieved by methotrexate selection, providing strong evidence that synthesis of activated p21H-ras above a certain threshold (about 0.02% of total protein) in Rat-1 cells is incompatible with cell viability. Individual cell lines were isolated and their morphology, anchorage-independent growth, tumorigenicity, and response to and production of growth factors were studied. We report that cell lines expressing near-maximum tolerated levels of either the normal or pro-12 form of p21H-ras were not as transformed as cells expressing much more modest levels of the highly oncogenic (Val-12) form, suggesting that the complete elaboration of the transformed phenotype by ras depends, at least in part, on mutations that distinguish the cellular and viral proteins. We found that cells expressing elevated levels of the normal p21(H-ras) could be fully transformed by the activated (Val-12) form and that such cells continued to overexpress p21(H-ras) (Gly-12), arguing against a role for normal ras genes in suppression of the oncogenic potential of their mutationally activated counterparts.
An electrophoretic karyotype of Neurospora crassa.
Authors: Orbach M J , Vollrath D , Davis R W , Yanofsky C
Published: September 2015

A molecular karyotype of Neurospora crassa was obtained by using an alternating-field gel electrophoresis system which employs contour-clamped homogeneous electric fields. The migration of all seven N. crassa chromosomal DNAs was defined, and five of the seven molecules were separated from one another. The estimated sizes of these molecules, based on their migration relative to Schizosaccharomyces pombe chromosomal DNA molecules, are 4 to 12.6 megabases. The seven linkage groups were correlated with specific chromosomal DNA bands by hybridizing transfers of contour-clamped homogeneous electric field gels with radioactive probes specific to each linkage group. The mobilities of minichromosomal DNAs generated from translocation strains were also examined. The methods used for preparation of chromosomal DNA molecules and the conditions for their separation should be applicable to other filamentous fungi.
Structural and functional characterization of mouse U7 small nuclear RNA active in 3' processing of histone pre-mRNA.
Authors: Soldati D , Schümperli D
Published: September 2015

Oligonucleotides derived from the spacer element of the histone RNA 3' processing signal were used to characterize mouse U7 small nuclear RNA (snRNA), i.e., the snRNA component active in 3' processing of histone pre-mRNA. Under RNase H conditions, such oligonucleotides inhibited the processing reaction, indicating the formation of a DNA-RNA hybrid with a functional ribonucleoprotein component. Moreover, these oligonucleotides hybridized to a single nuclear RNA species of approximately 65 nucleotides. The sequence of this RNA was determined by primer extension experiments and was found to bear several structural similarities with sea urchin U7 snRNA. The comparison of mouse and sea urchin U7 snRNA structures yields some further insight into the mechanism of histone RNA 3' processing.
Double minute chromosomes can be produced from precursors derived from a chromosomal deletion.
Authors: Carroll S M , DeRose M L , Gaudray P , Moore C M , Needham-Vandevanter D R , Von Hoff D D , Wahl G M
Published: September 2015

Recent experiments have shown that gene amplification can be mediated by submicroscopic, autonomously replicating, circular extrachromosomal molecules. We refer to those molecules as episomes (S. Carroll, P. Gaudray, M. L. DeRose, J. F. Emery, J. L. Meinkoth, E. Nakkim, M. Subler, D. D. Von Hoff, and G. M. Wahl, Mol. Cell. Biol. 7:1740-1750, 1987). The experiments reported in this paper explore the way episomes are formed and their fate in the cell over time. The data reveal that in our system the episomes are initially 250 kilobases, but gradually enlarge until they become double minute chromosomes. In addition, we show that episomes or double minute chromosomes can integrate into chromosomes. Our results also suggest that episomes can be produced by deletion of the corresponding sequences from the chromosome.
Cloning of the human keratin 18 gene and its expression in nonepithelial mouse cells.
Authors: Kulesh D A , Oshima R G
Published: September 2015

Human keratin 18 (K18) and the homologous mouse protein, Endo B, are intermediate filament subunits of the type I keratin class. Both are expressed in many simple epithelial cell types including trophoblasts, the first differentiated cell type to appear during mouse embryogenesis. The K18 gene was identified and cloned from among the 15 to 20 similar sequences identified within the human genome. The identity of the cloned gene was confirmed by comparing the sequence of the first two exons to the K18 cDNA sequence and transfecting the gene into various murine cell lines and verifying the encoded protein as K18 by immunoprecipitation and partial peptide mapping. The transfected K18 gene was expressed in mouse HR9 parietal endodermal cells and mouse fibroblasts even though the fibroblasts fail to express endogenous Endo B. S1 nuclease protection analysis indicated that mRNA synthesized from the transfected K18 gene is initiated at the same position as authentic K18 mRNA found in both BeWo trophoblastoma cells and HeLa cells. Pulse-chase experiments indicated that the human K18 protein is stable in murine parietal endodermal cells (HR9) which express EndoA, a complementary mouse type II keratin. Surprisingly, however, K18 was degraded when synthesized in cells which lack a type II keratin. This turnover of K18 may be an important mechanism by which epithelial cells maintain equal molar amounts of both type I and II keratins. In addition, the levels of the endogenous type I Endo B in parietal endodermal cells were compensatingly down regulated in the presence of the K18 protein, while the levels of the endogenous type II Endo A were not affected in any of the transfected cell lines.
Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.
Authors: Cizdziel P E , de Mars M , Murphy E C
Published: September 2015

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.
RAP30/74: a general initiation factor that binds to RNA polymerase II.
Authors: Burton Z F , Killeen M , Sopta M , Ortolan L G , Greenblatt J
Published: September 2015

We have previously shown by affinity chromatography that RAP30 and RAP74 are the mammalian proteins that have the highest affinity for RNA polymerase II. Here we show that RAP30 binds to RAP74 and that the RAP30-RAP74 complex (RAP30/74) is required for accurate initiation by RNA polymerase II. RAP30/74 is required for accurate transcription from the following promoters: the adenovirus major late promoter, the long terminal repeat of human immunodeficiency virus, P2 of the human c-myc gene, the mouse beta maj-globin promoter (all of which have TATA boxes), and the mouse dihydrofolate reductase promoter (which lacks a TATA box). RAP30/74 is not required for initiation by RNA polymerase III at the adenovirus virus-associated RNA promoters. Therefore, RAP30/74 is a general initiation factor that binds to RNA polymerase II.
Transformation of Tetrahymena thermophila with hypermethylated rRNA genes.
Authors: Karrer K M , Yao M C
Published: September 2015

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.
Poly(A) shortening and degradation of the 3' A+U-rich sequences of human c-myc mRNA in a cell-free system.
Authors: Brewer G , Ross J
Published: September 2015

The early steps in the degradation of human c-myc mRNA were investigated, using a previously described cell-free mRNA decay system. The first detectable step was poly(A) shortening, which generated a pool of oligoadenylated mRNA molecules. In contrast, the poly(A) of a stable mRNA, gamma globin, was not excised, even after prolonged incubation. The second step, degradation of oligoadenylated c-myc mRNA, generated decay products whose 3' termini were located within the A+U-rich portion of the 3' untranslated region. These products disappeared soon after they were formed, consistent with rapid degradation of the 3' region. In contrast, the 5' region, corresponding approximately to c-myc exon 1, was stable in vitro. The data indicate a sequential degradation pathway in which 3' region cleavages occur only after most or all of the poly(A) is removed. To account for rapid deadenylation, we suggest that the c-myc poly(A)-poly(A)-binding protein complex is readily dissociated, generating a protein-depleted poly(A) tract that is no longer resistant to nucleases.
Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein.
Authors: Vijaya S , Elango N , Zavala F , Moss B
Published: September 2015

Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.
Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.
Authors: Bender M A , Miller A D , Gelinas R E
Published: September 2015

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.
Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor.
Authors: Leong K , Brunet L , Berk A J
Published: September 2015

Extracts of adenovirus-infected HeLa cells have 5- to 10-fold-higher activity for transcription from the major late promoter in vitro than do extracts of mock-infected or E1A mutant-infected cells (K. Leong and A. J. Berk, Proc. Natl. Acad. Sci. USA 83:5844-5848, 1986). In this study, we analyzed extracts from mock-infected cells and from cells infected with an E1A mutant, pm975, which expresses principally the large E1A protein responsible for the stimulation of transcription. These extracts were fractionated by phosphocellulose chromatography, a procedure which separates factors required for transcription from this promoter (J. D. Dignam, B. S. Shastry, and R. G. Roeder, Methods Enzymol. 101:582-589, 1983), allowing the quantitative assay of individual factors (M. Samuels, A. Fire, and P. A. Sharp, J. Biol. Chem. 257:14419-14427, 1982). Fractions eluted with 0.04, 0.35, and 0.6 M KCl, which contained RNA polymerase II, the upstream factor MLTF, and three general polymerase II transcription factors, had similar activities when prepared from virus-infected or from mock-infected cells. The sequence-specific DNA-binding activity of MLTF was also similar in the virus-infected- and mock-infected-cell extracts. In contrast, the 1.0 M KCl fraction prepared from virus-infected cells consistently exhibited activity severalfold higher than that of the equivalent fraction prepared in parallel from mock-infected cells. E1A protein eluted principally (greater than 80%) in the 0.35 M KCl fraction. Results of others (M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985) have shown that the 1.0 M KCl fraction, containing 2 to 5% of the unfractionated protein extract, contains a factor which binds specifically to the major late promoter TATA box. These results, together with a recent genetic analysis of the E1B promoter which demonstrated that the TATA box was required for its efficient transcriptional activation (transactivation) by E1A (L. Wu, D. S. E. Rosser, M. Schmidt, and A. J. Berk, Nature (London) 326:512-515, 1987), are consistent with the model that E1A protein indirectly activates the TATA box transcription factor. Consistent with this model was the finding that mutants of the major late promoter containing only the TATA box and cap site region were transcribed at higher rates with extracts from virus-infected cells than with extracts from mock-infected cells. Other models consistent with the results are also discussed.
Ligand-induced tyrosine kinase activity of the colony-stimulating factor 1 receptor in a murine macrophage cell line.
Authors: Downing J R , Rettenmier C W , Sherr C J
Published: September 2015

Metabolic labeling of simian virus 40-immortalized murine macrophages with 32Pi and immunoblotting with antibodies to phosphotyrosine demonstrated that the c-fms proto-oncogene product (colony-stimulating factor 1 [CSF-1] receptor) was phosphorylated on tyrosine in vivo and rapidly degraded in response to CSF-1. Stimulation of the CSF-1 receptor also induced immediate phosphorylation of several other cellular proteins on tyrosine. By contrast, the mature cell surface glycoprotein encoded by the v-fms oncogene was phosphorylated on tyrosine in the absence of CSF-1, suggesting that it functions as a ligand-independent kinase.
DNA-binding factors of B lymphoid cells are susceptible to limited proteolytic cleavage during nuclear extract preparation.
Authors: Mather E L
Published: September 2015

DNA-binding proteins that interact with the 3' end of the mouse mu immunoglobulin heavy chain gene were identified by the electrophoretic mobility shift assay. Complexes of distinctly different mobilities were formed by extracts prepared from B lymphoid lines representing different stages of maturation. The apparent stage-specific differences are shown to be due to proteolytic events that occurred during extract preparation.
Meiotic expression of human ornithine transcarbamylase in the testes of transgenic mice.
Authors: Kelley K A , Chamberlain J W , Nolan J A , Horwich A L , Kalousek F , Eisenstadt J , Herrup K , Rosenberg L E
Published: September 2015

In an attempt to use mouse metallothionein-I (mMT-I) regulatory sequences to direct expression of human ornithine transcarbamylase in the liver of transgenic animals, fusion genes joining either 1.6 kilobases or 185 base pairs of the mMT-I regulatory region to the human ornithine transcarbamylase protein-coding sequence were used to produce transgenic mice. In mice carrying the fusion gene with 1.6 kilobases of the mMT-I 5'-flanking sequences, transgene expression was observed in a wide range of tissues, but, unexpectedly, expression in liver was never observed. Surprisingly, in mice carrying the fusion gene regulated by only 185 base pairs of the mMT-I 5'-flanking sequences, the transgene was expressed exclusively in male germ cells during the tetraploid, pachytene stage of meiosis.
Retinoic acid increases the sensitivity of the rat embryo fibroblast transformation assay.
Authors: Halazonetis T D , Daugherty C , Leder P
Published: September 2015

The rat embryo fibroblast focus assay is used to evaluate the transforming potential of several oncogenes. The sensitivity of this assay increased fivefold when retinoic acid was added to tissue culture media. Retinoic acid probably acts by selectively inhibiting the proliferation of nontransformed cells.
Structure and expression of germ line immunoglobulin gamma 2b transcripts.
Authors: Lutzker S , Alt F W
Published: September 2015

We have isolated a cDNA copy of a truncated C gamma 2b transcript produced by Abelson murine leukemia virus transformants that spontaneously switch from mu to gamma 2b. The initiation site of this transcript was 2 kilobases 5' to the gamma 2b switch recombination region, demonstrating its germ line origin. Nucleotide sequence analyses suggest that this transcript does not encode a protein. Expression of germ line gamma 2b transcripts in Abelson murine leukemia virus transformants and in normal spleen cells correlated with endogenous gamma 2b class switch activity.
Developmentally regulated expression of a human "finger"-containing gene encoded by the 5' half of the ret transforming gene.
Authors: Takahashi M , Inaguma Y , Hiai H , Hirose F
Published: September 2015

We isolated and sequenced a cDNA clone of the human gene encoded by the 5' half of the ret transforming gene. The nucleotide sequence indicates that it encodes a protein with "finger" structures which represent putative metal- and nucleic acid-binding domains. Transcription of this gene was detected at high levels in a variety of human and rodent tumor cell lines, mouse testis, and embryos. In addition, a unique transcript was observed in testis RNA. When the expression of the unique transcript was examined at different stages of spermatogenesis, a striking increase in mRNA levels accompanied progression from meiotic prophase pachytene spermatocytes to postmeiotic round spermatids. This finger-containing gene may thus function in male germ cell development.
p75, a polypeptide component of karyoskeletal protein-enriched fractions associated with transcriptionally active loci of Drosophila melanogaster polytene chromosomes.
Authors: Benton B M , Berrios S , Fisher P A
Published: September 2015

A 75-kilodalton polypeptide has been identified which copurifies with karyoskeletal protein-enriched fractions prepared from Drosophila melanogaster embryos. Results of indirect immunofluorescence experiments suggest that this protein, here designated p75, is primarily associated with puffed regions of larval salivary gland polytene chromosomes. In nonpolytenized Schneider 2 tissue culture cells, p75 appeared to be localized throughout the nuclear interior during interphase. In mitotic cells, p75 was redistributed diffusely. A possible role for karyoskeletal elements in transcriptional regulation is discussed.
Differential expression of individual members of the histone multigene family due to sequences in the 5' and 3' regions of the genes.
Authors: Levine B J , Liu T J , Marzluff W F , Skoultchi A I
Published: September 2015

Histone proteins are encoded by a multigene family. The H3.2(614) and H2a(614) genes are present as single copies which are expressed at high levels, accounting for 30 to 40% of the H3 and H2a mRNAs, respectively, in different types of mouse cells. The other genes which have been isolated each contribute only a very small amount to the total type-specific mRNA pool. We demonstrate here that the differences in the level of expression of these genes are partly due to differences in their transcription rates. To investigate the sequences responsible for these differences in expression among the members of each family, we carried out DNA-mediated gene transfer experiments with both intact and chimeric histone genes. The 5' region of a highly expressed gene [H3.2(614) or H2a(614)] was attached to the 3' region of a histone gene which was expressed at low levels (H3-221 or H2a-291) and vice versa. The results show that sequences in both the 5' and 3' regions of the H3.2(614) and H2a(614) genes contribute to their high level of mRNA production by two independent mechanisms. The effect of the 3' sequences on mRNA accumulation has been narrowed to a 65-base-pair region including the 3'-terminal palindrome and downstream signal implicated in mRNA processing.
Reconstitution of the Rous sarcoma virus transforming protein pp60v-src into phospholipid vesicles.
Authors: Resh M D
Published: September 2015

An artificial membrane system was developed to study the molecular basis for interaction of pp60v-src, the Rous sarcoma virus transforming protein, with lipid bilayers. pp60v-src was extracted from cell membranes by detergent solubilization and reincorporated into phospholipid vesicles. Reconstituted pp60v-src retained tyrosine kinase activity and was integrally associated with the liposome through a 10-kilodalton (kDa) amino-terminal domain. The same 10-kDa domain was shown to anchor pp60v-src to the plasma membrane of transformed cells. Reconstitution experiments performed with nonmyristylated pp60v-src proteins revealed that these polypeptides did not interact with phospholipid vesicles. In contrast, myristylated, soluble pp60v-src molecules (including a highly purified pp60v-src preparation) could be reconstituted into liposomes, but their interaction with the liposomal bilayer was not mediated by the 10-kDa amino-terminal domain. When membrane proteins were included during reconstitution of purified pp60v-src, binding through the 10-kDa anchor was restored. A model is presented to accommodate the different types of interactions of pp60v-src with liposomes; the model postulates the existence of an additional membrane component that anchors the pp60v-src polypeptide to the phospholipid bilayer.
Three sequence-specific DNA-protein complexes are formed with the same promoter element essential for expression of the rat somatostatin gene.
Authors: Andrisani O M , Pot D A , Zhu Z , Dixon J E
Published: September 2015

We identified three sequence-specific DNA-protein complexes which are formed after in vitro binding of nuclear extracts, derived from neuronal (CA-77, rat brain) or non-neuronal (HeLa) cells, to positions -70 to -29 of the rat somatostatin promoter. The protein(s) responsible for the formation of the three sequence-specific complexes was fractionated from rat brain whole cell extracts by DEAE-Sepharose chromatography. The critical contact residues of the factor(s) in each complex, as determined by methylation interference analyses, are located within positions -59 to -35, which is protected from DNase I digestion; these include the G residues of a TGACGTCA consensus also found in the cAMP-responsive human enkephalin (positions -105 to -76) and E1A-inducible adenovirus type 5 E3 (positions -72 to -42) promoters. Competition assays with these heterologous promoters reveal that the factor(s) of each complex displays approximately 50-fold greater affinity for the somatostatin promoter-binding site. Synthetic oligonucleotides spanning positions -70 to -29 of the somatostatin promoter and containing single-base substitutions of the G residues in the TGACGTCA consensus were utilized in competition assays. The G residues located in the center of the module are the most critical determinants in the formation of the three sequence-specific complexes. Deletions disrupting the TGACGTCA consensus abolish not only formation of the three complexes in vitro but also expression of the somatostatin promoter in vivo, suggesting that formation of one or more of these complexes is essential for transcription of the rat somatostatin gene.
Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.
Authors: Shapiro R A , Herrick D , Manrow R E , Blinder D , Jacobson A
Published: September 2015

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.
Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.
Authors: Lax I , Johnson A , Howk R , Sap J , Bellot F , Winkler M , Ullrich A , Vennstrom B , Schlessinger J , Givol D
Published: September 2015

The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.
Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene.
Authors: Feinbaum R L , Ausubel F M
Published: September 2015

We have cloned an Arabidopsis thaliana chalcone synthase (CHS) gene on the basis of cross-hybridization with a Petroselinum hortense CHS cDNA clone. The protein sequence deduced from the A. thaliana CHS DNA sequence is at least 85% homologous to the CHS sequences from P. hortense, Antirrhinum majus, and Petunia hybrida. Southern blot analysis indicated that CHS is a single-copy gene in A. thaliana. High-intensity light treatment of A. thaliana plants for 24 h caused a 50-fold increase in CHS enzyme activity and an accumulation of visibly detectable levels of anthocyanin pigments in the vegetative structures of these plants. A corresponding increase in the steady-state level of CHS mRNA was detected after high-intensity light treatment for the same period of time. The accumulation of CHS mRNA in response to high-intensity light was due, at least in part, to an increased rate of transcription of the CHS gene as demonstrated by nuclear runoff experiments.
mRNA-decapping enzyme from Saccharomyces cerevisiae: purification and unique specificity for long RNA chains.
Authors: Stevens A
Published: September 2015

An enzyme that hydrolyzes one PPi bond of the cap structure of mRNA, yielding m7GDP and 5'-p RNA was purified from Saccharomyces cerevisiae to a stage suitable for characterization. The specificity of the enzyme was studied, using both yeast mRNA and synthetic RNAs labeled in the cap structure. A synthetic capped RNA (540 nucleotides) was not reduced in size, while as much as 80% was decapped. Yeast mRNA treated with high concentrations of RNase A, nuclease P1, or micrococcal nuclease was inactive as a substrate. The use of synthetic capped RNAs of different sizes (50 to 540 nucleotides) as substrates showed that the larger RNA can be a better substrate by as much as 10-fold. GpppG-RNA was hydrolyzed at a rate similar to that at which 5'-triphosphate end group were not hydrolyzed.
fra-1: a serum-inducible, cellular immediate-early gene that encodes a fos-related antigen.
Authors: Cohen D R , Curran T
Published: September 2015

A set of proteins antigenically related to the c-fos protein (Fos) are induced by serum in fibroblasts. To isolate cDNA clones of genes encoding such proteins, a lambda gt11 expression cDNA library constructed from serum-stimulated rat fibroblasts was screened with antibodies raised against a hydrophilic region (amino acids 127 to 152) of Fos. One of the positive clones identified, termed fra-1 (Fos-related antigen) was characterized. It encoded a protein that shared several regions of extensive amino acid homology with Fos (including the region that showed similarity to both the yeast GCN4 regulatory protein and the protein encoded by the jun oncogene), although its nucleotide sequence was considerably diverged from that of the c-fos gene. Only a subset of the agents and conditions that activated c-fos also induced fra-1. Induction of fra-1 expression following serum stimulation was delayed compared with that of c-fos. However, like c-fos, fra-1 was induced rapidly by serum in the presence of protein synthesis inhibitors. Thus, a family of Fos-related, inducible genes are involved in the cellular immediate-early transcriptional response to extracellular stimuli.
p53 increases experimental metastatic capacity of murine carcinoma cells.
Authors: Pohl J , Goldfinger N , Radler-Pohl A , Rotter V , Schirrmacher V
Published: September 2015

Transfection of a cloned p53 gene into a murine bladder carcinoma cell with a low metastatic capacity led to elevated levels of p53 protein in clonal transfectants. After intravenous inoculation into syngeneic mice, p53-transfected clones showed significantly increased metastatic potential in comparison with control transfectants. The observed change did not seem to be due to a change in growth potential per se since the cell lines showed similar growth properties in vitro.
Infrequent genomic rearrangement and normal expression of the putative RB1 gene in retinoblastoma tumors.
Authors: Goddard A D , Balakier H , Canton M , Dunn J , Squire J , Reyes E , Becker A , Phillips R A , Gallie B L
Published: September 2015

Retinoblastoma (RB) tumors develop when both alleles of a gene (RB1) are mutated and unable to function normally. Recently, Friend et al. [S. H. Friend, R. Bernards, S. Rogelj, R. A. Weinberg, J. M. Rapaport, D. M. Albert, and T. P. Dryja, Nature (London) 32:643-646, 1986] reported the cloning of a gene, 4.7R, with some properties expected for the RB1 gene, namely, a high frequency (30%) of genomic rearrangements in tumors and absence of message in all RB tumors examined. To extend the characterization of this gene, we used 4.7R probes to search for genomic rearrangements of DNA and to study the expression of the 4.7R gene in RB tumors, osteosarcoma (OS) tumors arising in RB patients, and other normal and malignant tissues. In 34 previously unreported RB and OS tumors arising in RB patients, we observed only four (12%) with genomic abnormalities. Transcripts of 4.7R were present in 12 of 17 RB tumors, 2 of 2 OS tumors, and all non-RB tumors and normal tissues tested. We were unable to confirm the high frequency of truncated messages of 4.7R in RB tumors reported by Lee et al. (W. H. Lee, R. Bookstein, F. Hong, L. J. Young, J. Y. Shaw, and E. Y. Lee, Science 235:1394-1399, 1987) and Fung et al. (Y. K. Fung, A. L. Murphree, A. Tang, J. Qian, S. H. Hinrichs, and W. F. Benedict, Science 236:1657-1661, 1987) but did confirm the presence of a truncated transcript in the RB cell line Y79. Of the RB and RB-related OS tumors which appeared normal on Southern blots, 2 of 26 or 12% had abnormal transcripts, giving a combined frequency of 22% abnormalities in the 4.7R gene detectable by Southern and Northern (RNA) blot analyses.
Establishment of composite DNA derived from L factor as a plasmid in mouse embryonal carcinoma (F9) cells.
Authors: Nishimori K , Kohda T , Fujiwara J , Oishi M
Published: September 2015

We have recently reported a mammalian cell plasmid (L factor) whose structure is related to that of polyomavirus (T. Kusano, H. Uehara, H. Saito, K. Segawa, and M. Oishi, Proc. Natl. Acad. Sci. USA 84:1789-1793, 1987). When composite DNA constructed from L factor and a foreign gene was introduced into mouse embryonal carcinoma (F9) cells by transfection, the DNA was reestablished in the cells as a plasmid. The reestablished plasmid DNA in F9 cells could be rescued in Escherichia coli. The plasmid-bearing cells underwent normal in vitro differentiation in response to retinoic acid. The efficiency of plasmid establishment of the L-factor-derived DNA and transcriptional and transient replicational activities were compared with those of similar composite DNA constructed from polyomavirus and an embryonal carcinoma mutant of polyomavirus which is permissive in F9 cells. The results suggest an inverse relationship between the efficiency of the plasmid establishment and the activity of gene expression controlled by the intrinsic enhancer-promoter of the DNA.
mRNA synthesis rates in vivo for androgen-inducible sequences in mouse kidney.
Authors: Watson G , Paigen K
Published: September 2015

A method was developed for measuring in vivo rates of mRNA synthesis in mice by pulse-labeling with the RNA precursor [3H]orotate and then using hybridization to recover specific mRNAs. The efficiency of recovery is determined with synthetic RNAs as internal hybridization standards. The method is particularly applicable to the kidney since this organ shows a strong preferential uptake of the label. Rates of synthesis, expressed as a fraction of total RNA synthesis, were measured for the androgen-inducible mRNAs coding for beta-glucuronidase (GUS), ornithine decarboxylase (ODC), the protein coded by the RP-2 gene, and the so-called kidney androgen-regulated protein (KAP). Control mRNAs coded for beta-actin, phosphoenolpyruvate carboxykinase, and major urinary protein. Testosterone markedly increased the synthesis of the androgen-inducible mRNAs, but not the control mRNAs. Induction was not seen in mutant mice lacking functional androgen receptor protein. For GUS, ODC, and RP-2 mRNAs, the fold induction of synthesis was less than the fold induction of concentration, suggesting that mRNA stabilization also plays a part in the response to androgen. For GUS, ODC, and RP-2 mRNAs, but not KAP mRNA, induction of synthesis was rapidly reversed after testosterone removal. KAP mRNA was also exceptional in that its concentration was disproportionately high compared with its rate of synthesis, implying that it is a particularly stable mRNA.
Transcriptional-translational regulatory circuit in Saccharomyces cerevisiae which involves the GCN4 transcriptional activator and the GCN2 protein kinase.
Authors: Roussou I , Thireos G , Hauge B M
Published: September 2015

GCN4 protein mediates the transcriptional activation of amino acid biosynthetic genes in Saccharomyces cerevisiae by specifically binding to DNA sequences in their 5'-regulatory regions. GCN4 expression is regulated at the level of translation, with translational derepression occurring under conditions of amino acid starvation. The product of the GCN2 gene is essential for translational derepression of GCN4. Sequence analysis of the GCN2 gene reveals that the GCN2 protein has a domain highly homologous to the catalytic domain of all known protein kinases. Furthermore, gcn2 strains are deficient in a protein kinase activity corresponding to a protein with the calculated molecular weight deduced from the GCN2 open reading frame. Therefore it is likely that GCN2 encodes a protein kinase, which may be directly involved in translational regulation of the GCN4 mRNA. Transcription of the GCN2 gene is increased when cells are cultured in amino acid starvation medium. This transcriptional activation is mediated by the GCN4 protein, which binds to the promoter region of the GCN2 gene. Thus, this system is modulated by a transcriptional-translational regulatory circuit, which is activated by amino acid starvation. Activation is not the result of a simple quantitative increase of either one of the identified components of the circuit.
Replication program of active and inactive multigene families in mammalian cells.
Authors: Hatton K S , Dhar V , Brown E H , Iqbal M A , Stuart S , Didamo V T , Schildkraut C L
Published: September 2015

In a comprehensive study, the temporal replication of tissue-specific genes and flanking sequences was compared in nine cell lines exhibiting different tissue-specific functions. Some of the rules we have determined for the replication of these tissue specific genes include the following. (i) Actively transcribed genes usually replicate during the first quarter of the S phase. (ii) Some immunoglobulin genes replicate during the first half of S phase even when no transcriptional activity is detected but appear to replicate even earlier in cell lines where they are transcribed. (iii) Nontranscribed genes can replicate during any interval of S phase. (iv) Multigene families arranged in clusters of 250 kilobases or less define a temporal compartment comprising approximately one-quarter of S phase. While these rules, and others that are discussed, apply to the tissue-specific genes studied here, all tissue-specific genes may not follow this pattern. In addition, housekeeping genes did not follow some of these rules. These results provide the first molecular evidence that the coordinate timing of replication of contiguous sequences within a multigene family is a general property of the mammalian genome. The relationship between replication very early during S phase and the transcriptional activity within a chromosomal domain is discussed.
Structure and transcription of the actin gene of Trypanosoma brucei.
Authors: Ben Amar M F , Pays A , Tebabi P , Dero B , Seebeck T , Steinert M , Pays E
Published: September 2015

In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.
Decreased expression of the stress protein HSP70 is an early event in murine erythroleukemic cell differentiation.
Authors: Hensold J O , Housman D E
Published: September 2015

Two-dimensional protein gels were used to systematically assess changes in gene expression in Friend erythroleukemia cells after exposure to inducers of differentiation. A rapid decrease in expression of the stress protein HSP70 was observed after exposure to inducers. The kinetics of this change suggest that it may be related to the cellular events that regulate the onset of differentiation.