bluuebunny/crossref_metadata_2025 huggingface dataset:
Human genes for U2 small nuclear RNA are tandemly repeated.
Authors: Van Arsdell S W , Weiner A M
Published: September 2015

We found that the genes for human U2 small nuclear RNA (snRNA) are organized as a nearly perfect tandem array of 10 to 20 copies per haploid genome. Although the coding region for the mature form of U2 RNA was only 188 base pairs (bp) long, the basic repeating unit of the tandem array was 6 kilobase pairs in length. Comparison of DNA sequences immediately upstream from human U1 and U2 genes revealed two regions of strong homology: region I (15 bp long) lay upstream of region II (20 bp long) and was separated from it by about the same distance in U1 genes (25 bp) as in U2 genes (21 bp); however, region I and region II were located 174 bp further upstream from the 5' end of the snRNA coding sequence in U1 genes than in U2 genes. Homologs of region II were also found upstream of the snRNA coding region in a mouse U2 gene and two rat U1 genes. Murphy et al. (Cell 29:265-274, 1982) have found that sequences within region II may function as the equivalent of a TATA box for initiation by RNA polymerase II in vitro at a position 183 bp upstream from the 5' end of the human U1 snRNA coding region. In light of the data reported here, this result suggests that region II does indeed play a role in transcription but that its position relative to the actual initiation site can vary.
Mutations affecting a surface glycoprotein, gp80, of Dictyostelium discoideum.
Authors: Murray B A , Wheeler S , Jongens T , Loomis W F
Published: September 2015

We isolated two independent mutations in Dictyostelium discoideum that result in the absence of the antigenic determinant recognized by monoclonal antibody E28D8. This antibody reacts with a post-translational modification on the surface glycoprotein gp80 and several other proteins. Both of the mutations occur in the same locus, modB, which was mapped to linkage group VI. The modB mutations result in sufficient alteration of gp80 that it is absent or unrecognizable by two-dimensional gel electrophoresis. Strains carrying modB mutations exhibit "contact sites A"-mediated cell-cell adhesion although more weakly than do wild-type strains and develop to fruiting bodies carrying viable spores. Although gp80 has been implicated in the mechanism of cell-cell adhesion in D. discoideum, it is clear from the behavior of these mutant strains that the determinant on gp80 recognized by E28D8 is not necessary for either morphogenesis or reduced EDTA-resistant adhesion.
Changes in structure and methylation pattern in a cluster of thymidine kinase genes.
Authors: Christy B A , Scangos G A
Published: September 2015

Cell line 101 is a thymidine kinase (TK)-positive derivative of Ltk- which contains ca. 20 copies of the herpes simplex virus TK gene organized in a tandem array. DNA methylation at three sites within the gene and flanking sequences was inversely correlated with expression: the sites were unmethylated in line 101, methylated in each of 4 TK-negative derivatives of 101, and unmethylated in each of 21 TK-positive derivatives derived from them. The same three sites were affected in most of the 20 copies of the TK gene, whereas other sites between them were not affected. Although the entire gene cluster was never lost, indicating that integration into the genome was stable, internal rearrangements occurred at a high frequency. The rearrangements had no obvious correlation with the state of methylation or with the expression of the genes.
Effects of 5-azacytidine on methylation and expression of specific DNA sequences in C3H 10T1/2 cells.
Authors: Hsiao W L , Gattoni-Celli S , Kirschmeier P , Weinstein I B
Published: September 2015

The present study indicates that the transient exposure of C3H 10T1/2 mouse embryo fibroblasts to 5-azacytidine leads to extensive loss of methylation of the protooncogene c-mos and the beta-globin locus at the cell population level and in at least 40 isolated subclones. These changes persisted, even when the cells were serially passaged for many generations without further exposure to the drug. Even though the amount of demethylation, assessed through differential digestion by the restriction enzymes HpaII and MspI, was quite extensive, neither locus was transcribed at a detectable level. This nonselective analysis suggests, therefore, that loss of DNA methylation is not sufficient per se to induce the expression of certain loci. Presumably, the expression of these loci requires additional factors, some of which may be related to cell lineage and differentiation.
Fatty acid-acylated proteins in secretory mutants of Saccharomyces cerevisiae.
Authors: Wen D , Schlesinger M J
Published: September 2015

Yeast secretory (sec) mutants that are blocked in the transport of secretory proteins and accumulate membrane organelles were used to study the biosynthesis of fatty acid-acylated proteins. Four proteins were labeled with [3H]palmitate in sec mutants accumulating endoplasmic reticulum membranes. Three of these (molecular weights approximately equal to 20,000, 50,000, and 120,000) were N-linked glycoproteins, based on their ability to be labeled with [3H]mannose and their sensitivity to endoglycosidase H. The fourth protein (molecular weight approximately equal to 30,000) also was labeled with [3H]mannose but was insensitive to endoglycosidase H; it appeared to contain O-linked sugars. In sec mutants accumulating Golgi membranes or post-Golgi vesicles, a 35-kilodalton protein was labeled with [3H]palmitate. Analysis of Staphylococcus aureus protease V8 digests and pulse-chase experiments indicated that the 30-kilodalton protein was a precursor of 35 kilodaltons. None of these proteins was labeled with [3H]palmitate in a sec mutant that blocked the penetration of nascent polypeptides into endoplasmic reticulum; thus, acylation occurred in endoplasmic reticulum. All four proteins could be recovered from fractions enriched for yeast membranes. Fatty acids were not released from proteins by boiling in sodium dodecyl sulfate or extraction with organic solvents but were recovered as methyl esters after proteins were treated with KOH-methanol, a reaction characteristic of an acyl ester linkage.
Expression from an internal AUG codon of herpes simplex thymidine kinase gene inserted in a retrovirus vector.
Authors: Bandyopadhyay P K , Temin H M
Published: September 2015

We identified structural features that affect the expression of an exogenous gene inserted into a retrovirus vector constructed by using spleen necrosis virus, an avian retrovirus. The thymidine kinase gene from herpes simplex virus type 1 containing deletions in the promoter and terminal sequences of the mRNA was inserted into spleen necrosis virus. We found that synthesis of thymidine kinase by the recovered virus was apparently initiated from internal AUG residues. At least in some cases, however, the level of expression depended on the number of AUGs and the nucleotide sequence around the AUGs that preceded the initiator codon of the thymidine kinase gene.
Polyoma middle T antigen requires cooperation from another gene to express the malignant phenotype in vivo.
Authors: Asselin C , Gélinas C , Branton P E , Bastin M
Published: September 2015

The oncogenic potential of polyomavirus in newborn hamsters can be expressed by a recombinant encoding only the middle T protein. However, polyoma middle T requires the cooperation from small T to induce tumors in newborn rats. Similar complementary functions such as cocarcinogens or tumor promotors can be exerted by the simian virus 40 T antigens as well as by one or several products of the early region 1A of adenovirus 2.
Transformation of protoplasted yeast cells is directly associated with cell fusion.
Authors: Harashima S , Takagi A , Oshima Y
Published: September 2015

The frequency of cell fusion during transformation of yeast protoplasts with various yeast plasmids with a chromosome replicon (YRp or YCp) or 2 mu DNA (YEp) was estimated by two methods. In one method, a mixture of protoplasts of two haploid strains with identical mating type and complementary auxotrophic nuclear markers with or without cytoplasmic markers was transformed. When the number of various phenotypic classes of transformants for the nuclear markers was analyzed by equations derived from binominal distribution theory, the frequency of nuclear fusion among the transformants was 42 to 100% in transformations with the YRp or YCp plasmids and 28 to 39% with the YEp plasmids. In another method, a haploid bearing the sir mutation, which allows a diploid (or polyploid) homozygous for the MAT (mating type) locus to sporulate by the expression of the silent mating-type loci HML and HMR, was transformed with the plasmids. Sporulation ability was found in 43 to 95% of the transformants with the YRp or YCp plasmids, and 26 to 31% of the YEp transformants. When cytoplasmic mixing was included with the nuclear fusion, 96 to 100% of the transformants were found to be cell fusants. Based upon these observations, we concluded that transformation of yeast protoplasts is directly associated with cell fusion.
Tubulin heterogeneity in the trypanosome Crithidia fasciculata.
Authors: Russell D G , Miller D , Gull K
Published: September 2015

The interphase cell of Crithidia fasciculata has three discrete tubulin populations: the subpellicular microtubules, the axonemal microtubules, and the nonpolymerized cytoplasmic pool protein. These three tubulin populations were independently and selectively purified, yielding, in each case, microtubule protein capable of self-assembly. All three preparations polymerized to form ribbons and sheets rather than the more usual microtubular structures. Analyses of the tubulin by two-dimensional polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping indicated that the beta-tubulin complex remained constant regardless of source but that some heterogeneity was present in the alpha subunit. Cytoplasmic pool alpha tubulins (alpha 1/alpha 2) were the only alpha isotypes in the cytoplasm and also formed most of the alpha tubulin species in the pellicular fraction. Flagellar alpha tubulin (alpha 3) was the sole alpha isotype in the flagella; it appeared in small amounts in the pellicular fraction but was completely absent from the cytoplasm. In vitro translation products from polyadenylated RNA from C. fasciculata were also examined by two-dimensional polyacrylamide gel electrophoresis and possessed a protein corresponding to alpha 1/alpha 2 tubulin but lacked any alpha 3 tubulin. The alpha 3 polypeptide arose from a post-translational modification of a precursor polypeptide not identifiable by two-dimensional polyacrylamide gel electrophoresis as alpha 3. Peptide mapping data indicated that cytoplasmic alpha tubulin is the most likely precursor. These results demonstrate alpha-tubulin heterogeneity in this organism and also how close the relationship between flagellar and cytoskeletal tubulins can be among lower eucaryotes.
The number of ribosomes on simian virus 40 late 16S mRNA is determined in part by the nucleotide sequence of its leader.
Authors: Barkan A , Mertz J E
Published: September 2015

The size distributions of polyribosomes containing each of three simian virus 40 late 16S mRNA species that differ in nucleotide sequence only within their leaders were determined. The two 16S RNA species with shorter leaders were incorporated into polysomes that were both larger (on average) and narrower in size distribution than was the predominant wild-type 16S RNA. Therefore, the nucleotide sequence of the leader can influence the number of ribosomes present on the body of an mRNA molecule. We propose a model in which the excision from leaders of sizeable translatable regions permits more frequent utilization of internally located translation initiation signals, thereby enabling genes encoded within the bodies of polygenic mRNAs to be translated at higher rates. In addition, the data provide the first direct evidence that VP1 can, indeed, be synthesized in vivo from the species of 16S mRNA that also encodes the 61-amino acid leader protein.
N-terminal amino acid sequences of the polyoma middle-size T antigen are important for protein kinase activity and cell transformation.
Authors: Templeton D , Eckhart W
Published: September 2015

We constructed deletion mutations which removed N-terminal coding sequences of various lengths from a cloned polyoma middle-size T antigen (MT antigen) gene. We introduced the MT antigen genes into a simian virus 40 expression vector so that they were expressed at high levels under the control of the simian virus 40 late promoter in COS-1 cells. The deletion mutant genes synthesized truncated MT antigens whose size was consistent with the deletion of either 70 or 106 amino acids from N termini, owing to initiation of translation at internal methionine codons in the MT antigen-coding region. The truncated MT antigens were found in cell membrane fractions but failed to show MT antigen-associated protein kinase activity. The cloned deletion mutant DNAs failed to transform rat F2408 or mouse NIH 3T3 cells. Therefore, N-terminal amino acid sequences of the polyoma MT antigen, as well as C-terminal sequences, are important for protein kinase activity and cell transformation.
Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.
Authors: Lin F L , Sternberg N
Published: September 2015

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.
Glucose represses transcription of Saccharomyces cerevisiae nuclear genes that encode mitochondrial components.
Authors: Szekely E , Montgomery D L
Published: September 2015

By Northern blot hybridization analysis, we demonstrated that the steady-state levels of mRNAs specifying the alpha subunit of ATPase, the beta subunit of ATPase, and the ATP/ADP translocator are all reduced in cells grown in glucose-rich medium. The extent to which glucose represses the levels of alpha, beta, and translocator mRNAs varies from strain to strain, from 2.5- to 7-fold. Furthermore, by hybridization experiments with an excess of DNA, we showed that glucose represses the rates of synthesis of these mRNAs. The kinetics of repression and depression of transcription were also studied. Finally, a mutant was characterized which appears to be defective in depression of transcription of the genes encoding the alpha and beta ATPase subunits as well as the ATP/ADP translocator.
Drosophila melanogaster has only one myosin alkali light-chain gene which encodes a protein with considerable amino acid sequence homology to chicken myosin alkali light chains.
Authors: Falkenthal S , Parker V P , Mattox W W , Davidson N
Published: September 2015

A chimeric lambda DNA molecule containing the myosin alkali light-chain gene of Drosophila melanogaster was isolated. The encoded amino acid sequence was determined from the nucleic acid sequence of a cDNA homologous to the genomic clone. The identity of the encoded protein was established by two criteria: (i) sequence homology with the chicken alkali light-chain proteins and (ii) comparison of the two-dimensional gel electrophoretic pattern of the peptides synthesized by in vitro translation of hybrid-selected RNA to that of myosin alkali light-chain peptides extracted from Drosophila myofibrils. There is only one myosin alkali light-chain in D. melanogaster; its chromosomal location is region 98B . This gene is abundantly expressed during the development of larval as well as adult muscles. The Drosophila protein appears to contain one putative divalent cation-binding domain (an EF hand) as compared with the three EF hands present in chicken alkali light chains.
Chromosomal mapping of murine c-fes and c-src genes.
Authors: Blatt C , Harper M E , Franchini G , Nesbitt M N , Simon M I
Published: September 2015

The murine homologs of two viral oncogenes associated with tyrosine-specific kinase activity have been assigned to different loci in the mouse genome. The segregation of restriction site polymorphisms, as detected by probes that are specific for endogenous c-fes and c-src sequences, was followed in the DNA of recombinant inbred strains. The c-fes gene was mapped to the proximal portion of chromosome 7, very close to the Gpi-1 locus, whereas c-src was linked to the Psp locus on the distal half of chromosome 2.
5-Methylcytosine is not detectable in Saccharomyces cerevisiae DNA.
Authors: Proffitt J H , Davie J R , Swinton D , Hattman S
Published: September 2015

We examined the DNA of Saccharomyces cerevisiae by both HpaII-MspI restriction enzyme digestion and high-performance liquid chromatography analysis for the possible presence of 5-methylcytosine. Both of these methods failed to detect cytosine methylation within this yeast DNA; i.e., there is less than 1 5-methylcytosine per 3,100 to 6,000 cytosine residues.
Regional localization of two human cellular Kirsten ras genes on chromosomes 6 and 12.
Authors: Sakaguchi A Y , Zabel B U , Grzeschik K H , Law M L , Ellis R W , Scolnick E M , Naylor S L
Published: September 2015

Human cellular Kirsten ras1 and ras2 genes were localized to chromosomes 6p23 ----q12 and 12p12 .05----pter, respectively, using human-rodent cell hybrids. Thus, the short arms of human chromosomes 11 (encoding lactate dehydrogenase-A and the proto-oncogene c-Ha- ras1 ) and 12 (encoding lactate dehydrogenase B and c-Ki- ras2 ) share at least two pairs of genes that probably evolved from common ancestral genes.
Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts.
Authors: Nelson R L , Branton P E
Published: September 2015

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.
La antigen recognizes and binds to the 3'-oligouridylate tail of a small RNA.
Authors: Mathews M B , Francoeur A M
Published: September 2015

The La antigen is a cellular protein which interacts with many RNA species that are products of RNA polymerase III, including the adenovirus virus-associated (VA) RNAs. We demonstrate that the efficiency of antigen binding in vitro is determined by the number of U residues at the RNA 3' terminus. Forms of VA RNAI with more than two terminal U residues are fully bound, forms with two U residues are partially bound, and forms with fewer than two U residues are not bound at all. The antigen can be covalently linked to VA RNA by UV irradiation, and the site of cross-linking is shown to contain the 3' terminus of the RNA. We conclude that the antigen recognizes the U-rich 3' tail of VA RNA, and presumably that of other polymerase III products, and that it binds at or close to this site.
DNA-mediated cotransfer of excision repair capacity and drug resistance into chinese hamster ovary mutant cell line UV-135.
Authors: MacInnes M A , Bingham J M , Thompson L H , Strniste G F
Published: September 2015

We have investigated DNA-mediated transfer of aminopterin resistance conferred by plasmid and UV resistance conferred by genomic DNA to the Chinese hamster ovary (CHO) cell line UV-135, a UV-sensitive mutant defective in nucleotide excision repair. Plasmid pSV2gpt-CaPO4 coprecipitates induced aminopterin resistance with equal efficiency in the 6-thioguanine-resistant, aminopterin-sensitive, repair-proficient parental line AA8-4(tg-1) and in UV-135(tg-2). Genetic and molecular evidence for genomic DNA-mediated transformation of UV-135(tg-2) cells with a putative excision repair gene were obtained by demonstrating that: (i) UV resistance transformation is dependent upon and specific for genomic DNA from excision repair-competent CHO cells: (ii) UV and drug coresistant colonies are bona fide transferants as verified by hybridization and Southern blotting analysis of pSV2gpt sequences in their genomic DNAs: (iii) confirmed transferants exhibit partial to near normal UV resistances for colony formation: and (iv) UVr transferants have near normal levels of excision repair capacity. The overall frequency of drug and UV resistance cotransformation was 8 X 10(8) per cell plated. This frequency was ca. 200- to 500-fold greater than that expected from coincident but independent UVr reversion and plasmid gene transfer events. DNA transfer techniques with this CHO system will be useful for further analysis of the essential structural DNA sequences, gene cloning, and expression of functional excision repair genes.
New procedure for DNA transfection with polycation and dimethyl sulfoxide.
Authors: Kawai S , Nishizawa M
Published: September 2015

A new procedure for DNA transfection has been developed in a system of chicken embryo fibroblast cells and cloned Rous sarcoma virus DNA by using a polycation reagent as a mediator to adsorb DNA to the cell surface and dimethyl sulfoxide as an agent to facilitate the uptake of adsorbed DNA by the cells. In this new, simple, and convenient polycation-dimethyl sulfoxide transfection, which requires no carrier DNA even with small amounts of DNA, the number of transformed cell foci induced by Rous sarcoma virus DNA was proportional to the dose of the transfecting DNA, and chicken embryo fibroblast cells were successfully transformed by v-src-containing subgenomic DNA as well.
Expression of a proto-oncogene (proto-myb) in hemopoietic tissues of mice.
Authors: Sheiness D , Gardinier M
Published: September 2015

This study addressed the possibility that proto-myb (also called c-myb), the cellular homolog of a retroviral transforming gene, plays a role in hemopoiesis, particularly during maturation of T cells. By gel blot hybridization, we confirmed previous reports that proto-myb transcripts are found at much higher levels in thymic lymphocytes and cells of the erythroid lineage than in other tissue sources. Using dot blot hybridizations, we demonstrated further that similar levels of proto-myb expression are found in thymic lymphocytes taken from young mice with active thymuses and from old mice whose thymuses have undergone involution and that the extent of proto-myb expression decreases at least 10-fold as T cells progress from immature cortical thymocytes to the mature, resting T cells taken from lymph nodes. These results suggest that the protein product of proto-myb functions during T-cell differentiation.
Regulation, linkage, and sequence of mouse metallothionein I and II genes.
Authors: Searle P F , Davison B L , Stuart G W , Wilkie T M , Norstedt G , Palmiter R D
Published: September 2015

The mouse metallothionein II (MT-II) gene is located approximately 6 kilobases upstream of the MT-I gene. A comparison of the sequences of mouse MT-I and MT-II genes (as well as those of other mammals) reveals that the coding regions are highly conserved even at "silent" positions but that the noncoding regions and introns are extremely divergent between primates and rodents. There are four blocks of conserved sequences in the promoters of mouse MT-I, mouse MT-II, and human MT-IIA genes; one includes the TATAAA sequence, and another has been implicated in regulation by heavy metals. Mouse MT-I and MT-II mRNAs are induced to approximately the same extent in vivo in response to cadmium, dexamethasone, or lipopolysaccharide. Mouse MT-I and MT-II genes are regulated by metals but not by glucocorticoids after transfection into HeLa cells.
Structure of the SAD mutation and the location of control sites at silent mating type genes in Saccharomyces cerevisiae.
Authors: Hicks J , Strathern J , Klar A , Ismail S , Broach J
Published: September 2015

The SAD mutation, an extra mating type cassette, has been shown to arise from an unequal mitotic crossover between the MAT and HMR loci, resulting in the formation of a hybrid cassette and a duplication of the MAT-HMR interval. The SAD cassette contains the "a" information and left-hand flanking regions from the parental HMRa cassette and the right-hand flanking sequences of the parental MAT cassette. This arrangement of flanking sequences causes a leaky but reproducible mating phenotype correlated with a low-level expression of the cassette as measured by RNA blotting. This weak expression is attributed to the loss of one flanking control site normally present at the silent HM storage loci.
Gap filling and not replication fork progression is the rate-limiting step in the replication of UV-damaged simian virus 40 DNA.
Authors: White J H , Dixon K
Published: September 2015

We have analyzed the structural characteristics of simian virus 40 replicative intermediate DNA produced after UV irradiation and the kinetics of conversion of this intermediate DNA into form I DNA. Replicative intermediate DNA isolated at 30 or 60 min after UV irradiation consists primarily of two species of molecules that sediment in neutral sucrose gradients as either Cairns theta structures or relaxed monomeric circles. Replication forks on the Cairns intermediate DNA are symmetrically located with respect to the origin of replication, ruling out the possibility of asymmetric pauses or blocks to replication fork progression at damage sites. The relaxed circles contain at least one randomly located discontinuity in the daughter strand. Pulse-chase experiments demonstrated that a UV fluence-dependent fraction of the Cairns intermediate DNA progresses through the relaxed circular intermediate before being converted to completed form I molecules. Disappearance of Cairns intermediate DNA occurs at the same rate in irradiated and unirradiated cells, whereas completion of the relaxed circular intermediate DNA occurs at a slow rate, relatively independent of UV fluence. These data support a model for replication of UV-damaged DNA in which replication rapidly continues past damage sites via a gap formation event.
Transcription control region within the protein-coding portion of adenovirus E1A genes.
Authors: Osborne T F , Arvidson D N , Tyau E S , Dunsworth-Browne M , Berk A J
Published: September 2015

A single-base deletion within the protein-coding region of the adenovirus type 5 early region 1A (E1A) genes, 399 bases downstream from the transcription start site, depresses transcription to 2% of the wild-type rate. Complementation studies demonstrated that this was due to two effects of the mutation: first, inactivation of an E1A protein, causing a reduction by a factor of 5; second, a defect which acts in cis to depress E1A mRNA and nuclear RNA concentrations by a factor of 10. A larger deletion within the protein-coding region of E1A which overlaps the single-base deletion produces the same phenotype. In contrast, a linker insertion which results in a similar truncated E1A protein does not produce the cis-acting defect in E1A transcription. These results demonstrate that a critical cis-acting transcription control region occurs within the protein coding sequence in adenovirus type 5 E1A. The single-base deletion occurs in a sequence which shows extensive homology with a sequence from the enhancer regions of simian virus 40 and polyomavirus. This region is not required for E1A transcription during the late phase of infection.
Differential expression of two neural cell-specific beta-tubulin mRNAs during rat brain development.
Authors: Bond J F , Robinson G S , Farmer S R
Published: September 2015

We recently demonstrated that beta-tubulin mRNA expression is regulated during rat brain development. This is manifested by a dramatic decrease in both 1.8- and 2.9-kilobase (kb) mRNAs when extensive neurite elongation is occurring. Coincident with these decreases is the increased production of a 2.5-kb mRNA. (J.F. Bond and S.R. Farmer, Mol. Cell. Biol. 3:1333-1342, 1983). In the present study, we have isolated and characterized three different cDNAs corresponding to beta-tubulin mRNAs (R beta T.1, R beta T.2, and R beta T.3). Hybridization of 3' untranslated region subclones of R beta T.1 and R beta T.2 cDNAs to RNA from a variety of rat tissues and cells revealed that these two cDNAs are neural cell specific. R beta T.1 corresponds to an abundant 1.8-kb mRNA expressed only at early stages of rat brain development. R beta T.2 corresponds to the 2.5-kb mRNA expressed at later stages. These data strongly suggest that there is differential expression of the beta-tubulin multigene family during rat brain development.
Positive regulatory interactions of the HIS4 gene of Saccharomyces cerevisiae.
Authors: Lucchini G , Hinnebusch A G , Chen C , Fink G R
Published: September 2015

The role of cis- and trans-acting elements in the expression of HIS4 has been examined by using HIS4-lacZ fusions in which lacZ expression is dependent upon the HIS4 5' noncoding region. The cis-acting sequences involved in regulation were defined by studying the effects of the wild-type and various deletions and their revertants on regulation via the general control of amino acid biosynthesis. The role of trans-acting genes was analyzed by studying the regulation of the HIS4-lacZ fusions in strains carrying mutations in the GCN (AAS) or GCD (TRA) genes and in strains carrying the GCN genes on high-copy-number plasmids. These studies have led to the following conclusions. (i) HIS4 is positively regulated by the general control. (ii) At least one copy of the 5'TGACTC3' repeat at -136 is required in cis for this regulation. (iii) Both the GCN4 gene and at least one copy of the repeated sequence are required for expression at the repressed level. (iv) The open reading frames in the 5' noncoding region are not required in either cis or trans for the regulation of HIS4.
Structure and arrangement of the beta-tubulin genes of Leishmania tropica.
Authors: Huang P L , Roberts B E , Pratt D M , David J R , Miller J S
Published: September 2015

We studied the organization and arrangement of the genes encoding beta-tubulin in the protozoan parasite Leishmania tropica and examined the structure and orientation of the beta-tubulin mRNA relative to the gene. There were found to be eight to nine beta-tubulin genes arranged in an array of direct tandem repeat units with a length of 3.8 kilobase pairs, and they were extremely homologous, if not identical, in sequence. These repeat units did not contain the alpha-tubulin genes. The transcribed sequences within the beta-tubulin genes were localized, and the orientation of the major alpha-tubulin mRNA was mapped on the gene by S1 nuclease analysis.
Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences.
Authors: Wolf D , Rotter V
Published: September 2015

Analysis of Abelson murine leukemia virus-transformed L12 cells which lack the p53 cellular encoded tumor antigen revealed alterations in the p53-specific genomic DNA sequences. The active p53 gene, usually contained in a 16-kilobase EcoRI DNA fragment of p53 producer cells, went through major alterations leading to the appearance of a substantially larger 28.0-kilobase p53-specific EcoRI fragment. Detailed restriction enzyme analysis, with genomic probes spanning throughout the whole active p53 gene, indicated that the L12 p53 altered gene contains all the exons and principal introns of the normal p53 16.0-kilobase gene. However, its structure was interrupted by the integration of a novel DNA segment into the noncoding intervening sequences of the first p53 intron. Analysis of the inserted sequences revealed close homology to Moloney murine leukemia virus. This Moloney leukemia murine virus-like particle resides in a 5' to 3' transcriptional orientation, similar to the p53 gene, permitting the transcription of aberrant fused mRNA molecules detected in these cells.
Conformation takes precedence over sequence in adeno-associated virus DNA replication.
Authors: Lefebvre R B , Riva S , Berns K I
Published: September 2015

Deletion of an 11-base symmetrical sequence in the inverted terminal repetition of the adeno-associated virus 2 genome inhibits DNA replication. Substitution of either an 8- or a 12-base symmetrical sequence unrelated to the original 11-base sequence restores DNA replication.
Expression of the Rous sarcoma virus src gene in avian macrophages fails to elicit transformed cell phenotype.
Authors: Lipsich L , Brugge J S , Boettiger D
Published: September 2015

Infection of avian macrophages with Rous sarcoma virus does not induce any changes in the morphology, growth behavior, or expression of macrophage-specific proteins. The absence of cellular transformation does not result from a block in the synthesis of viral proteins, since infectious viruses are released from a majority of cells in the culture. In this report, we examine the synthesis, processing, and functional activity of pp60src in Rous sarcoma virus-infected macrophages to determine whether the absence of transformation is due to an alteration in the functional expression of pp60src. Although the absolute level of pp60src was reduced compared with fibroblasts, the protein exhibited the same phosphorylation pattern and subcellular distribution and was able to phosphorylate immunoglobulin in the immune complex-protein kinase assay. These results imply that the failure of Rous sarcoma virus to transform macrophage may be due to a restriction in the cellular response to a functional src protein, perhaps due to the absence of cellular products which are essential for mediating pp60src-induced transformation.
Plasmid-directed synthesis of genuine adenovirus 2 early-region 1A and 1B proteins in Escherichia coli.
Authors: Ko J L , Harter M L
Published: September 2015

The transforming region of human adenovirus 2 is located in the left 11.2% of the viral genome and is comprised of two distinct genetic units termed E1A and E1B. cDNAs containing the entire nucleotide sequence of the mature E1A 13S and E1B 22S mRNAs that are complementary to these genetic units have been introduced into bacterial plasmids a short distance downstream from the Escherichia coli lac promoter. Upon transformation into appropriate E. coli hosts, one of these plasmids, pKHAO, directed the synthesis of a 45-kilodalton (kd) protein, and the other, pKHBO, synthesized a protein of 54.9 kd. Both of these plasmid-encoded proteins constituted 0.1 to 0.3% of the total cellular protein and were virtually identical to the authentic adenovirus 2 E1A 42- to 50-kd and E1B 53- to 58-kd tumor antigens (T antigen) as determined by gel electrophoresis, immunoprecipitation, and tryptic fingerprint analysis. With the use of our pKHBO expression plasmid we were also able to demonstrate that the second AUG sequence appearing in the E1B 22S mRNA corresponded to the start of the gene encoding the large adenovirus 2 T antigen. This confirms theoretical deductions based on DNA sequencing analysis that translation of the large T antigen initiates translation at an internal ATG rather than at the 5'-proximal AUG.
Cardiac actin is the major actin gene product in skeletal muscle cell differentiation in vitro.
Authors: Bains W , Ponte P , Blau H , Kedes L
Published: September 2015

We examined the expression of alpha-skeletal, alpha-cardiac, and beta- and gamma-cytoskeletal actin genes in a mouse skeletal muscle cell line (C2C12) during differentiation in vitro. Using isotype-specific cDNA probes, we showed that the alpha-skeletal actin mRNA pool reached only 15% of the level reached in adult skeletal muscle and required several days to attain this peak, which was then stably maintained. However, these cells accumulated a pool of alpha-cardiac actin six times higher than the alpha-skeletal actin mRNA peak within 24 h of the initiation of differentiation. After cells had been cultured for an additional 3 days, this pool declined to 10% of its peak level. In contrast, over 95% of the actin mRNA in adult skeletal muscle coded for alpha-actin. This suggests that C2C12 cells express a pattern of sarcomeric actin genes typical of either muscle development or regeneration and distinct from that seen in mature, adult tissue. Concurrently in the course of differentiation the beta- and gamma-cytoskeletal actin mRNA pools decreased to less than 10% of their levels in proliferating cells. The decreases in beta- and gamma-cytoskeletal actin mRNAs are apparently not coordinately regulated.
Sequences on the 3' side of hexanucleotide AAUAAA affect efficiency of cleavage at the polyadenylation site.
Authors: Sadofsky M , Alwine J C
Published: September 2015

The hexanucleotide AAUAAA has been demonstrated to be part of the signal for cleavage and polyadenylation at appropriate sites on eucaryotic mRNA precursors. Since this sequence is not unique to polyadenylation sites, it cannot be the entire signal for the cleavage event. We have extended the definition of the polyadenylation cleavage signal by examining the cleavage event at the site of polyadenylation for the simian virus 40 late mRNAs. Using viable mutants, we have determined that deletion of sequences between 3 and 60 nucleotides on the 3' side of the AAUAAA decreases the efficiency of utilization of the normal polyadenylation site. These data strongly indicate a second major element of the polyadenylation signal. The phenotype of these deletion mutants is an enrichment of viral late transcripts longer than the normally polyadenylated RNA in infected cells. These extended transcripts appear to have an increased half-life due to the less efficient cleavage at the normal polyadenylation site. The enriched levels of extended transcripts in cells infected with the deletion mutants allowed us to examine regions of the late transcript which normally are difficult to study. The extended transcripts have several discrete 3' ends which we have analyzed in relation to polyadenylation and other RNA processing events. Two of these ends map to nucleotides 2794 and 2848, which lie within a region of extensive secondary structure which marks the putative processing signal for the formation of the simian virus 40-associated small RNA. A third specific 3' end reveals a cryptic polyadenylation site at approximately nucleotides 2980 to 2985, more than 300 nucleotides beyond the normal polyadenylation site. This site appears to be utilized only in mutants with debilitated normal sites. The significance of sequences on the 3' side of an AAUAAA for efficient polyadenylation at a specific site is discussed.
Amplification and expression of human alpha-globin genes in Chinese hamster ovary cells.
Authors: Lau Y F , Lin C C , Kan Y W
Published: September 2015

We studied the effects of gene amplification on human globin gene expression in Chinese hamster ovary cells. The normal human alpha-globin gene (N alpha 2) and a hybrid gene (M alpha G) containing the 5' promoter-regulator region of the mouse metallothionein gene and the human structural alpha 2-globin gene were linked to a modular SV2-cDNA dihydrofolate reductase (DHFR) gene. The recombinant DNA molecules were introduced into Chinese hamster ovary cells by calcium phosphate precipitation. After initial selection to retain the DHFR and linked sequences, the cells were cultured in increasing concentrations of methotrexate up to 0.2 mM. Southern blot analysis of total cellular DNA showed an approximately 500- to 1,000-fold increase in the number of copies of DHFR and human alpha-globin genes. The transcription of the alpha-globin and DHFR genes increased as their copy number within the cells increased. The transcription of the amplified hybrid M alpha G gene was also inducible with cadmium treatments. Both mature mRNA and "read-through" transcripts were observed. DHFR constituted approximately 10% of pulse-labeled cellular proteins in these cells, but no human alpha-globin was detected. In vitro translation of polyadenylated RNA from these cells showed that alpha-globin mRNA transcribed from the amplified alpha-globin genes was functional and directed alpha-globin chain synthesis. In situ hybridization of 3H-labeled alpha-globin and DHFR DNA probes in chromosome preparations from the two cell lines indicated that both genes were coamplified in the same chromosomal locations in each cell type. These results indicate that gene amplification enhances human globin gene expression in cultured Chinese hamster ovary cells.
Regulated transcription of c-Ki-ras and c-myc during compensatory growth of rat liver.
Authors: Goyette M , Petropoulos C J , Shank P R , Fausto N
Published: September 2015

We examined the transcription of six cellular oncogenes during the process of compensatory growth in rat liver after partial hepatectomy. We have previously reported that transcripts of c-rasH are elevated during regenerative growth of the liver. We now report that transcripts of c-rasK and c-myc genes are significantly elevated after partial hepatectomy, whereas transcripts of c-abl and c-src are essentially unchanged and transcripts of c-mos are undetectable in either normal or regenerating rat liver. In liver regeneration after partial hepatectomy or chemical injury, changes in c-myc transcripts occur before DNA synthesis. The elevation of c-myc and c-ras transcripts is sequential in that highest levels of c-myc transcripts were detected 12 to 18 h after partial hepatectomy, whereas the levels of c-rasH and c-rasK were maximal by 36 to 48 h. Transcripts of all three activated oncogenes returned to their basal levels by 96 h.
Evidence that the penton base of adenovirus is involved in potentiation of toxicity of Pseudomonas exotoxin conjugated to epidermal growth factor.
Authors: Seth P , Fitzgerald D , Ginsberg H , Willingham M , Pastan I
Published: September 2015

When KB cells are incubated for 1 h with human adenovirus type 2 or type 5 (1 microgram/ml) and a conjugate of epidermal growth factor and Pseudomonas exotoxin (EGF-PE), protein synthesis is inhibited by 80 to 90%. Under these conditions, neither adenovirus nor EGF-PE alone has any effect on host protein synthesis. Thus, adenovirus enhances the toxicity of EGF-PE. A number of antibodies to intact virus and capsid components were tested for their ability to block the enhancing activity and virus uptake. At appropriate dilutions, antibodies prepared against intact virus and penton base blocked the enhancing activity without affecting virus uptake. Antibodies against hexon and fiber blocked virus uptake and enhancing activity in parallel. These studies suggest that the penton base is important in lysis of the vesicles which contain adenovirus and EGF-PE, and this base allows virus and toxin to enter the cytoplasm.
BK virus-plasmid expression vector that persists episomally in human cells and shuttles into Escherichia coli.
Authors: Milanesi G , Barbanti-Brodano G , Negrini M , Lee D , Corallini A , Caputo A , Grossi M P , Ricciardi R P
Published: September 2015

We describe a novel expression vector, pBK TK-1, that persists episomally in human cells that can be shuttled into bacteria. This vector includes sequences from BK virus (BKV), the thymidine kinase (TK) gene of herpes simplex virus type 1, and plasmid pML-1. TK+-transformed HeLa and 143 B cells contained predominantly full-length episomes. There were typically 20 to 40 (HeLa) and 75 to 120 143 B vector copies per cell, although some 143 B transformants contained hundreds. Low-molecular-weight DNA from TK+-transformed cells introduced into Escherichia coli were recovered as plasmids that were indistinguishable from the input vector. Removal of selective pressure had no apparent effect upon the episomal status of pBK TK-1 molecules in TK+-transformed cells. BKV T antigen may play a role in episomal replication of pBK TK-1 since this viral protein was expressed in TK+ transformants and since a plasmid that contained only the BKV origin of replication was highly amplified in BKV-transformed human cells that synthesize BKV T antigen.
Analysis of the reduced growth factor dependency of simian virus 40-transformed 3T3 cells.
Authors: Powers S , Fisher P B , Pollack R
Published: September 2015

We have measured in a defined serum-free medium the platelet-derived growth factor (PDGF) and insulin requirements of normal Swiss 3T3 cells, simian virus 40-transformed 3T3 cells, and partial revertants of simian virus 40-transformed 3T3 cells. Swiss 3T3 cells displayed strong requirements for both PDGF and insulin. Both of these requirements were significantly diminished in simian virus 40-transformed 3T3 cells. Analysis of the PDGF and insulin requirements of the revertants indicated that the loss of either of these two growth factor requirements was not necessarily linked to the other; rather, the growth factor requirements were specifically associated with other parameters of transformation. The reacquisition of a PDGF requirement cosegregated with reversion to density-dependent growth inhibition, whereas reacquisition of a normal insulin requirement cosegregated with reversion to a normal growth dependence on calf serum. Anchorage dependence was dissociable from both growth factor requirements. The relationship between the PDGF requirement and density-dependent growth inhibition was further analyzed in normal 3T3 cells by measuring the PDGF requirement at different cell densities. At high cell densities, the requirement for PDGF became significantly greater. We suggest that at least in part the ability of transformed cells to grow to high saturation densities results from their loss of a requirement for PDGF.