bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of UV light on small nuclear RNA synthesis: increased inhibition during postirradiation cell incubation.
Authors: Morra D S , Eliceiri B P , Eliceiri G L
Published: September 2015

It has been shown previously that the synthesis of small nuclear RNAs (snRNAs) U1, U2, U3, U4, and U5, in contrast to that of all other RNA species tested, decreases markedly within 2 h of cell incubation after exposure to UV light (254 nm), while pyrimidine dimers are being removed from DNA. We examined the possibility that the postirradiation cell incubation-dependent, UV light-induced inhibition of snRNA synthesis might reflect hypersensitivity of the snRNA transcriptional domains to single-stranded DNA nicks or relaxation of DNA torsional stress or both that occur during DNA repair. This late suppression of snRNA biosynthesis was as pronounced in UV light-irradiated (DNA incision-deficient) xeroderma pigmentosum fibroblasts (complementation group A) as in irradiated normal human fibroblasts. The synthesis of snRNAs was not preferentially sensitive to gamma radiation (which produces single-stranded DNA breaks) or novobiocin or nalidixic acid (which induce DNA relaxation). Neither of these two drugs prevented the UV light-induced inhibition of snRNA synthesis observed during postirradiation cell incubation. These results suggest that the late suppression of snRNA synthesis does not result from hypersensitivity of snRNA transcriptional domains to single-stranded DNA cleavages or relaxation of DNA torsional strain. The UV light-induced late inhibition of snRNA synthesis: shows an inactivation curve whose slope differs from that observed immediately after irradiation; is seen in untransformed cells as well as established cells lines; and has been conserved between birds and mammals.
Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src.
Authors: Kamps M P , Sefton B M
Published: September 2015

All 15 protein kinases whose amino acid sequence is known contain a lysine residue at a position homologous to that of lysine-295 in p60src, the transforming protein of Rous sarcoma virus. The ATP analog p-fluorosulfonyl 5'-benzoyl adenosine inactivates both p60src and the catalytic subunit of the cyclic AMP-dependent protein kinase by modification of this lysine. We used oligonucleotide-directed mutagenesis to examine the possible functions of this residue. Lysine-295 in p60src was replaced with a glutamic acid, an arginine, or a histidine residue, and mutant p60src proteins were characterized in chicken cells infected by mutant viruses. None of these three mutant p60src proteins had tyrosine protein kinase activity in vitro, and none induced morphological transformation of infected cells. Since neither a histidine nor an arginine residue can replace the function of lysine-295, we suggest that it carries out the specialized function of proton transfer in the phosphotransferase reaction. All three mutant viruses underwent reversion to wild type during passage in tissue culture. Because the rate with which this occurred differed significantly among the mutants, reversion appears to have resulted from errors in transcription, rather than from recombination with the cellular src gene.
Structural characterization of a heterogeneous family of rat brain mRNAs.
Authors: Tsou A P , Lai C , Danielson P , Noonan D J , Sutcliffe J G
Published: September 2015

A large heterogeneous family of RNAs derived from a single rat gene contains members that differ from each other at one or more of three positions. Their 5' ends are nested and transcription can begin at 22 or more sites covering 265 nucleotides. Many of the 5' ends are detectable only in brain RNAs, and even 5' ends common with other tissues appear with different absolute and relative abundances in brain RNA. The central portions of the RNAs are of two forms, differing only by the presence or absence of 17 nucleotides; these forms are probably produced by alternative splicing. Polyadenylation occurs at either of two sites. This complicated family of 88 RNAs encodes two novel putative proteins that differ at their C termini.
Quantitative analysis of gene suppression in integrated retrovirus vectors.
Authors: Emerman M , Temin H M
Published: September 2015

Previously, we described a retrovirus vector that contained two genes: a 5' gene under transcriptional control of the viral long terminal repeat and a 3' gene under transcriptional control of the herpes simplex virus thymidine kinase promoter. By using a biological assay, we found that expression of the 5' gene is suppressed when there was selection for the 3' gene and expression of the 3' gene is suppressed when there is selection for the 5' gene (M. Emerman and H. M. Temin, Cell 39:459-467, 1984). In the present study, we replaced the thymidine kinase promoter with stronger promoters, and we measured expression of the genes in the retrovirus vectors by enzyme activity and RNA analysis. We found that all of the vectors displayed gene suppression when analyzed biochemically, although not when analyzed biologically. The suppressed genes produced about 10 to 50% as much product as when they were selected. The amount of suppression depended on whether the suppressed gene was 5' or 3' to the selected gene and from which promoter the suppressed gene was transcribed. The amount of suppression correlated with a decrease in the amount of steady-state RNA transcribed from the suppressed gene's promoter.
Methylation of the mouse hprt gene differs on the active and inactive X chromosomes.
Authors: Lock L F , Melton D W , Caskey C T , Martin G R
Published: September 2015

It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt gene.
Eel electric organ: hyperexpressing calmodulin system.
Authors: Munjaal R P , Connor C G , Turner R , Dedman J R
Published: September 2015

The electroplax of the electric eel Electrophorus electricus is the most abundant source of the calcium-binding protein calmodulin. The electroplax has 250 times the amount of calmodulin and its mRNA than eel skeletal muscle. Our data suggest that there is no major difference in gene copies, the degree of methylation, or genome rearrangement of the calmodulin gene in DNAs from eel electroplax and muscle. Differences in the calmodulin-binding proteins in electroplax and muscle suggest a differential role for the functional expression of calmodulin in cellular regulation.
Structural diversity of the major surface antigen of Plasmodium falciparum merozoites.
Authors: Schwarz R T , Riveros-Moreno V , Lockyer M J , Nicholls S C , Davey L S , Hillman Y , Sandhu J S , Freeman R R , Holder A A
Published: September 2015

The structures of the major merozoite surface antigen of Plasmodium falciparum and the gene encoding it were indistinguishable for the Wellcome strain and the Thai clone T9/94 but different for clones T9/96, T9/98, and T9/101. The central portion of the gene is subject to the greatest variation in structure. The protein from all five lines was found to be posttranslationally modified by covalent addition of both carbohydrate and fatty acid.
Monoclonal antibody Y13-259 recognizes an epitope of the p21 ras molecule not directly involved in the GTP-binding activity of the protein.
Authors: Lacal J C , Aaronson S A
Published: September 2015

The p21 products of ras proto-oncogenes are GTP-binding proteins with associated GTPase activity. Recent studies have indicated that ras p21 may be required for the initiation of normal cell DNA synthesis, since microinjection of a monoclonal antibody, Y13-259, blocks serum stimulation of DNA synthesis in quiescent cell cultures (L. S. Mulcahy, M.R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We localized the structural domain within the p21 molecule recognized by the Y13-259 monoclonal antibody. By analysis of a series of bacterially expressed p21 deletion mutants, the monoclonal antibody was found to interact with a region between positions 70 and 89 in the p21 amino acid sequence. By comparison of the coding sequences of different p21 proteins recognized by this monoclonal antibody, a highly conserved amino acid region between positions 70 and 81 was found to be the most likely site for the epitope detected by the Y13-259 antibody. This monoclonal antibody was further shown not to interfere directly with in vitro biochemical functions of the molecule, including GTP binding, GTPase, and autokinase activities.
The Drosophila melanogaster gypsy transposable element encodes putative gene products homologous to retroviral proteins.
Authors: Marlor R L , Parkhurst S M , Corces V G
Published: September 2015

We determined the complete nucleotide sequence of the gypsy element present at the forked locus of Drosophila melanogaster in the f1 allele. The gypsy element shares more homology with vertebrate retroviruses than with the copia element of D. melanogaster or the Ty element of Saccharomyces cerevisiae, both in overall organization and at the DNA sequence level. This transposable element is 7,469 base pairs long and encodes three putative protein products. The long terminal repeats are 482 nucleotides long and contain transcription initiation and termination signals; sequences homologous to the polypurine tract and tRNA primer binding site of retroviruses are located adjacent to the long terminal repeats. The central region of the element contains three different open reading frames. The second one encodes a putative protein which shows extensive amino acid homology to retroviral proteins, including gag-specific protease, reverse transcriptase, and DNA endonuclease.
Tandemly duplicated upstream control sequences mediate copper-induced transcription of the Saccharomyces cerevisiae copper-metallothionein gene.
Authors: Thiele D J , Hamer D H
Published: September 2015

Transcription of the Saccharomyces cerevisiae copper-metallothionein gene, CUP1, inducible by copper. By analyzing deletion and fusion mutants in the CUP1 5'-flanking region, we identified two closely related, tandemly arranged copper regulatory elements. A synthetic version of one of these elements conferred efficient copper induction on a heterologous promoter when present in two tandem copies.
The t-unique coding domain is important to the transformation maintenance function of the simian virus 40 small t antigen.
Authors: Bikel I , Mamon H , Brown E L , Boltax J , Agha M , Livingston D M
Published: September 2015

The small t antigen (t) of simian virus 40, a 174-amino-acid-containing protein, when present together with the other early viral protein, large T antigen (T), plays an important role in the maintenance of simian virus 40-induced neoplastic phenotype in certain cells. Indeed, each protein functions in a complementary manner in this process. The t coding unit is composed of two segments, a 5' region of 246 nucleotides which is identical to that of the corresponding 5' region of the T coding unit and a 3' segment of 276 nucleotides which is unique. Two mutant, t-encoding genomes, one bearing a missense and the other a nonsense mutation at the same point in the t-unique coding region were constructed in vitro and found to be defective in their ability to dissolve the actin cytoskeleton of rat fibroblasts and to complement T in the growth of mouse fibroblasts in soft agar. Therefore, the unique segment of the t gene encodes a portion of the t molecule which is essential to its transformation maintenance function.
Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis.
Authors: Naumovski L , Friedberg E C
Published: September 2015

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.
Morphological transformation of established rodent cell lines by high-level expression of the adenovirus type 2 E1a gene.
Authors: Senear A W , Lewis J B
Published: September 2015

When a strong promoter derived from the mouse metallothionein gene was substituted for the homologous adenovirus type 2 E1a promoter, leading to enhanced levels of E1a RNAs and proteins in cells transfected with the chimeric gene, the E1a gene alone was able to induce in established cell lines alterations in cellular morphology and growth properties similar to those produced by the combined action of E1a and E1b genes. The qualitative effects of E1a gene expression upon cellular properties thus depend on the level of expression of the E1a gene. Furthermore, E1a may be the primary transforming gene of adenoviruses, since it produced many of the characteristics of transformed cells that had previously been attributed to E1b.
Deletions in the C-terminal coding region of the v-sis gene: dimerization is required for transformation.
Authors: Hannink M , Sauer M K , Donoghue D J
Published: September 2015

The v-sis gene encodes chain B of platelet-derived growth factor. However, this gene codes for additional amino acids at both the N terminus and the C terminus of its gene product which are not present in the amino acid sequence of platelet-derived growth factor. We constructed a series of deletion mutants with deletions in the v-sis gene in order to define the C-terminal limit of the v-sis gene product which is required for transformation. Deletion mutants of the v-sis gene which encoded truncated gene products up to 57 residues shorter than the v-siswt gene product were still able to transform cells. The minimal transforming region of the v-sis gene product contained six residues fewer than were present in chain B of platelet-derived growth factor. Only 10 residues, including the sequence Cys-Lys-Cys, separated the smallest transforming gene product from the largest nontransforming gene product. These cysteine residues were also important for dimerization of the v-sis gene product, since all of the nontransforming v-sis deletions were unable to form dimers when they were analyzed under nonreducing conditions. Our results suggest that there is a strong connection between transformation and dimerization.
Orientation of the v-erb-B gene product in the plasma membrane.
Authors: Schatzman R C , Evan G I , Privalsky M L , Bishop J M
Published: September 2015

The retroviral oncogene v-erb-B encodes a truncated version of the receptor for epidermal growth factor. To define the disposition of the v-erb-B protein within cells and across the plasma membrane, we raised antibodies against defined epitopes in the protein and used these in immunofluorescence to analyze cells transformed by v-erb-B. A small fraction of the v-erb-B protein was found on the plasma membrane in a clustered configuration. The bulk of the protein was located in the endoplasmic reticulum and Golgi apparatus. Epitopes near the amino terminus of the v-erb-B protein were displayed on the surface of the cell, whereas epitopes in the protein kinase domain were located exclusively within cells. We conclude that the v-erb-B protein spans the plasma membrane in a manner similar or identical to that of the epidermal growth factor receptor, even though the viral transforming protein does not possess the signal peptide that is thought to direct insertion of the receptor into the membrane.
An active chromatin structure acquired by translocated c-myc genes.
Authors: Kakkis E , Prehn J , Calame K
Published: September 2015

We used general sensitivity to DNase I digestion to analyze the chromatin structure of c-myc genes in seven murine plasmacytomas. In every case, the 3' portion of c-myc juxtaposed with C alpha displayed a much more DNase I-sensitive chromatin structure than untranslocated c-myc or, in one case analyzed, the reciprocally translocated 5' portion. Our data suggest the presence of regulatory sequences near the C alpha gene segment.
Simultaneous expression of salivary and pancreatic amylase genes in cultured mouse hepatoma cells.
Authors: Darlington G J , Tsai C C , Samuelson L C , Gumucio D L , Meisler M H
Published: September 2015

The tissue-specific expression of two types of mouse amylase genes does not overlap in vivo; the Amy-1 locus is transcribed in the parotid gland and the liver, while expression of Amy-2 is limited to the pancreas. We identified a mouse hepatoma cell line, Hepa 1-6, in which both amylase genes can be simultaneously expressed. Amy-1 is constitutively active in these cells and is inducible by dexamethasone at the level of mRNA. We demonstrated that the liver-specific promoter of Amy-1 is utilized by the dexamethasone-treated hepatoma cells, and that glucocorticoid consensus sequences are present upstream of this promoter. Amy-2 is not detectable constitutively, but can be activated if the cells are cultured in serum-free medium containing dexamethasone. Expression of Amy-2 in a nonpancreatic cell type has not previously been observed. We speculate that induction of Amy-1 and activation of Amy-2 may involve different regulatory mechanisms. Hepa 1-6 cells provide an experimental system for molecular analysis of these events.
Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.
Authors: Dron M , Modjtahedi N , Brison O , Tovey M G
Published: September 2015

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.
DNA methyltransferase induced by PBCV-1 virus infection of a Chlorella-like green alga.
Authors: Xia Y N , Van Etten J L
Published: September 2015

A DNA methyltransferase was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1. The enzyme recognized the sequence GATC and methylated deoxyadenosine solely in GATC sequences. Host DNA, which contains GATC sequences, but not PBCV-1 DNA, which contains GmATC sequences, was a good substrate for the enzyme in vitro. The DNA methyltransferase activity was first detected about 1 h after viral infection; PBCV-1 DNA synthesis and host DNA degradation also began at about this time. The appearance of the DNA methyltransferase activity required de novo protein synthesis, and the enzyme was probably virus encoded. Methylation of DNAs with the PBCV-1-induced methyltransferase conferred resistance of the DNAs to a PBCV-1-induced restriction endonuclease enzyme described previously (Y. Xia, D. E. Burbank, L. Uher, D. Rabussay, and J. L. Van Etten, Mol. Cell. Biol. 6:1430-1439). We propose that the PBCV-1-induced methyltransferase protects viral DNA from the PBCV-1-induced restriction endonuclease and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.
Identification of primary transcriptional start sites of mouse mitochondrial DNA: accurate in vitro initiation of both heavy- and light-strand transcripts.
Authors: Chang D D , Clayton D A
Published: September 2015

The major transcriptional control sequences of vertebrate mitochondrial DNA lie within the displacement loop region. Transcription events initiating in the displacement loop sequence of the mouse genome were identified by 5' end mapping of primary transcripts by S1 nuclease protection and primer extension techniques. Light-strand transcription initiates at a single site, 165 nucleotides upstream of the major heavy-strand origin of replication. Transcription of the heavy strand occurs at two distinct sites, 5 and 13 nucleotides upstream of the gene for phenylalanyl-tRNA, the first heavy-strand-encoded gene. This spatial relationship of the two transcriptional start sites with each other and with the origin of heavy-strand replication and the gene for tRNAPhe is quite similar to that for human mitochondrial DNA. The predominant form of primary heavy-strand transcript in mouse is a short, ca. 75-nucleotide, RNA containing the sequences of tRNAPhe and a few additional nucleotides at the 5' end of tRNAPhe, suggesting that the processing of tRNA involves independent cleavages at the 5' and 3' ends of tRNA sequences.
The memory gene dunce+ encodes a remarkable set of RNAs with internal heterogeneity.
Authors: Davis R L , Davidson N
Published: September 2015

We have previously isolated the region of the Drosophila melanogaster X chromosome which contains the dunce+ gene and mapped the dunce2 mutation by recombination to a 10- to 12-kilobase (kb) interval (R. L. Davis and N. Davidson, Mol. Cell. Biol. 4:358-367, 1984). Here, we examine the expression of the dunce+ chromosomal region and identify the dunce+ gene within that region. A region of ca. 25 kb which contains the 10- to 12-kb interval to which dunce2 was mapped codes for polyadenylated RNAs of 9.6, 7.4, 7.2, 7.0, 5.4, and 4.5 kb in adult flies. These transcripts are encoded by the same DNA strand and share sequences of some exons, indicating that the transcripts arise from the same gene. Some genome probes internal to the ca. 25-kb coding region show transcript-specific hybridization, demonstrating alternate usage of exonic sequence information in the formation of the mature transcripts. The basis for this internal heterogeneity in RNAs is most likely alternative splicing. Two dunce mutants examined show aberrant RNA expression from this coding region, confirming that this region is the dunce gene. The developmental expression of these transcripts has been examined. The 5.4-kb RNA is present at all developmental stages. The 9.6-, 7.4-, 7.2-, and 7.0-kb RNAs are not expressed at detectable levels in embryos, but are detected in late embryogenesis and in later developmental stages. The 4.5-kb species is found in early embryos and adults, but not in intermediate stages. We discuss the remarkable transcript heterogeneity and expression pattern with respect to the important function this gene performs in neurobiological and other physiological processes.
Proto-oncogene c-ros codes for a molecule with structural features common to those of growth factor receptors and displays tissue specific and developmentally regulated expression.
Authors: Neckameyer W S , Shibuya M , Hsu M T , Wang L H
Published: September 2015

A recombinant DNA clone containing cellular sequences homologous to the transforming sequence, v-ros, of avian sarcoma virus UR2 was isolated from a chicken genomic DNA library. Heteroduplex mapping and nucleotide sequencing reveal that the v-ros sequences are distributed in nine exons ranging from 65 to 204 nucleotides on cellular ros (c-ros) DNA over a range of 11 kilobases. Comparison of the deduced amino acid sequences of c-ros and v-ros shows two differences: v-ros contains a three-amino-acid insertion within the hydrophobic domain presumed to be involved in membrane association, and (ii) the carboxyl 12 amino acids of v-ros are completely different from those of the deduced c-ros sequence. The deduced amino acid sequence of c-ros bears striking structural features similar to those of insulin and epidermal growth factor receptors, including the presumed hydrophobic membrane binding domain, amino acids flanking the domain, and the distance between the domain and the catalytic region of the kinase activity. The expression of c-ros appears to be under a very stringent control. When tissues at various stages of chicken development were analyzed, only kidney was found to contain a significant level of c-ros RNA. The level of c-ros RNA in kidney tissue is most abundant in 7- to 14-day-old chickens. Finally, nucleotide sequences of c-ros DNA and UR2-associated helper viral genome at regions corresponding to the gag ros recombination site suggest that the junction has been formed by RNA splicing.
A novel general approach to eucaryotic mutagenesis functionally identifies conserved regions within the adenovirus 13S E1A polypeptide.
Authors: Kimelman D
Published: September 2015

A new approach to the isolation of mutations in mammalian genes was developed which permits both the selection of infrequently occurring mutants that alter the cellular morphology of recipient cells and the rapid reisolation of the mutant gene. The adenovirus type 5 13S early region 1a (E1a) gene was mutagenized in vitro with sodium bisulfite and then efficiently transferred into cells with a retrovirus shuttle vector. Three classes of mutants of the 13S E1a gene product were isolated, each of which induced a distinct morphological alteration. The mutant E1a gene was reisolated from each cell line, and the precise nucleotide changes were determined. The E1a-induced morphological alterations were further examined by the construction of single and double point mutations within different regions of the polypeptides by utilizing the amino acid substitutions obtained from the original mutants. The results suggest that each of the three regions of highly conserved amino acids within the E1a 13S polypeptide has a distinct role in the alteration of cellular morphology and the activation of gene expression.
Molecular cloning of suppressor of sable, a Drosophila melanogaster transposon-mediated suppressor.
Authors: Chang D Y , Wisely B , Huang S M , Voelker R A
Published: September 2015

A hybrid dysgenesis-induced allele [su(s)w20] associated with a P-element insertion was used to clone sequences from the su(s) region of Drosophila melanogaster by means of the transposon-tagging technique. Cloned sequences were used to probe restriction enzyme-digested DNAs from 22 other su(s) mutations. None of three X-ray-induced or six ethyl methanesulfonate-induced su(s) mutations possessed detectable variation. Seven spontaneous, four hybrid dysgenesis-induced, and two DNA transformation-induced mutations were associated with insertions within 2.0 kilobases (kb) of the su(s)w20 P-element insertion site. When the region of DNA that included the mutational insertions was used to probe poly(A)+ RNAs, a 5-kb message was detected in wild-type RNA that was present in greatly reduced amounts in two su(s) mutations. By using strand-specific probes, the direction of transcription of the 5-kb message was determined. The mutational insertions lie in DNA sequences near the 5' end of the 5-kb message. Three of the seven spontaneous su(s) mutations are associated with gypsy insertions, but they are not suppressible by su(Hw).
Effects of progressive depletion of TCM1 or CYH2 mRNA on Saccharomyces cerevisiae ribosomal protein accumulation.
Authors: Nam H G , Fried H M
Published: September 2015

When present in excess, the mRNAs for Saccharomyces cerevisiae ribosomal proteins L3 and L29 are translated less efficiently, so that synthesis of these proteins remains commensurate with that of other ribosomal proteins (N.J. Pearson, H.M. Fried, and J.R. Warner, Cell 29:347-355, 1982; J.R. Warner, G. Mitra, W.F. Schwindinger, M. Studeny, and H.M. Fried, Mol. Cell. Biol. 5:1512-1521, 1985). We used a yeast strain with a conditionally transcribed derivative of the L3 gene to deplete cells progressively of L3 mRNA. In this case translation of L3 mRNA did not become more efficient so that L3 was not maintained at a normal level. Even when there was an initial excess of L3 mRNA, interruption of its further transcription produced an immediate drop in L3 synthesis, suggesting that the translational efficiency of preexisting mRNA cannot be altered. Lack of L3 synthesis afforded an opportunity to examine coordinate accumulation of other ribosomal proteins. Without L3, apparent synthesis of several 60S subunit proteins diminished, and 60S subunits did not assemble. A similar phenomenon occurred when, in a second strain, synthesis of ribosomal protein L29 was prevented. Loss of 60S subunit assembly was accompanied by a destabilization of some 60S ribosomal protein mRNAs. These data suggest that synthesis of some S. cerevisiae ribosomal proteins may be regulated posttranscriptionally as a function of the extent to which they are assembled.
Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA.
Authors: Tseng R W , Acheson N H
Published: September 2015

We devised a strategy to measure the efficiency of transcription termination in vivo by RNA polymerase on polyomavirus DNA. Pulse-labeled nuclear RNA was hybridized with a single-stranded polyomavirus DNA fragment which spans the transcription initiation region. Hybrids were treated with RNase, bound to nitrocellulose filters, eluted with S1 nuclease, and analyzed by gel electrophoresis. The ratio of full-length to less-than-full-length DNA-RNA hybrids was used to calculate transcription termination frequency. We found that 50% of the polymerases terminated per traverse of the L DNA strand during the late phase of infection. The method for mapping in vivo pulse-labeled RNAs which we developed is potentially useful for studying unstable cellular or viral RNAs.
Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis.
Authors: Ornelles D A , Fey E G , Penman S
Published: September 2015

Cytochalasin D was shown to be a reversible inhibitor of protein synthesis in HeLa cells. The inhibition was detectable at drug levels typically used to perturb cell structure and increased in a dose-dependent manner. The drug also released mRNA from the cytoskeletal framework in direct proportion to the inhibition of protein synthesis. The released mRNA was unaltered in its translatability as measured in vitro but was no longer translated in the cytochalasin-treated HeLa cells. The residual protein synthesis occurred on polyribosomes that were reduced in amount but displayed a normal sedimentation distribution. The results support the hypothesis that mRNA binding to the cytoskeletal framework is necessary although not sufficient for translation. Analysis of the cytoskeletal framework, which binds the polyribosomes, revealed no alterations in composition or amount of protein as a result of treatment with cytochalasin D. Electron microscopy with embedment-free sections shows the framework in great detail. The micrographs revealed the profound reorganization effected by the drug but did not indicate substantial disaggregation of the cytoskeletal elements.
Sequences near the 3' secretion-specific polyadenylation site influence levels of secretion-specific and membrane-specific IgG2b mRNA in myeloma cells.
Authors: Kobrin B J , Milcarek C , Morrison S L
Published: September 2015

The expressed immunoglobulin gamma 2b (IgG2b) heavy-chain gene of 4T001 was cloned into the shuttle vector pSV2-gpt and transfected into myeloma J558L and lymphoma A20.2J. Northern blots indicated that the transfected gamma 2b gene was processed in a manner similar to the endogenous heavy chain in both lymphoma and myeloma cells. To identify sequences important for immunoglobulin mRNA processing, we constructed deletions around the secretion-specific polyadenylation site and introduced the deleted genes into J558L cells. The BAL deletion lacked 670 base pairs of intervening sequence between secreted and membrane regions; the Kpn deletion lacked 830 base pairs in this region. J558L cells transfected with either the entire gamma 2b gene or the delta BAL vector produced predominantly secretion-specific gamma 2b mRNA and protein. J558L cells transfected with the delta Kpn vector produced approximately equimolar amounts of secretion-specific and membrane-specific gamma 2b mRNA. Both 55,000-dalton secreted and 62,000-dalton putative surface IgG2b proteins were detected in the delta Kpn transfectants. We conclude that sequences absent in the Kpn deletion but present in the BAL deletion exert an important role in the production of secretion-specific mRNA. The Kpn deletion removes the normal site of cleavage and poly(A) addition, and it is possible that it is the absence of this site which changes the processing pattern. Alternatively, it is possible that sequences absent in the Kpn deletion but present in the BAL deletion function in regulating the production of predominantly secretion-specific mRNA in myeloma cells. The possible role of a highly conserved sequence found in this region is discussed.
Nucleotide sequence of the two rat cellular rasH genes.
Authors: Ruta M , Wolford R , Dhar R , Defeo-Jones D , Ellis R W , Scolnick E M
Published: September 2015

We present the nucleotide sequence of the coding region of the rat c-rasH-1 gene and a partial sequence analysis of the rat c-rasH-2 gene. By comparing these sequences with the Harvey murine sarcoma virus ras gene, we predict that the p21 protein encoded by the Harvey virus differs from the cellular c-rasH-1-encoded p21 at only two amino acids; those at positions 12 and 59. Alterations at each of these positions may play a role in activating the viral p21 protein. The c-rasH-2 gene is likely to be a nonfunctional pseudogene because it lacks introns, cannot be activated to transform NIH 3T3 cells, and differs in sequence from both c-rasH-1 and v-rasH at several base pair positions.
Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae.
Authors: McIntosh E M , Haynes R H
Published: September 2015

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).
Somatic excision of transposable element Tc1 from the Bristol genome of Caenorhabditis elegans.
Authors: Harris L J , Rose A M
Published: September 2015

We investigated the ability of the transposable element Tc1 to excise from the genome of the nematode Caenorhabditis elegans var. Bristol N2. Our results show that in the standard lab strain (Bristol), Tc1 excision occurred at a high frequency, comparable to that seen in the closely related Bergerac strain BO. We examined excision in the following way. We used a unique sequence flanking probe (pCeh29) to investigate the excision of Tc1s situated in the same location in both strains. Evidence of high-frequency excision from the genomes of both strains was observed. The Tc1s used in the first approach, although present in the same location in both genomes, were not known to be identical. Thus, a second approach was taken, which involved the genetic manipulation of a BO variant, Tc1(Hin). The ability of this BO Tc1(Hin) to excise was retained after its introduction into the N2 genome. Thus, we conclude that excision of Tc1 from the Bristol genome occurs at a high frequency and is comparable to that of Tc1 excision from the Bergerac genome. We showed that many Tc1 elements in N2 were apparently functionally intact and were capable of somatic excision. Even so, N2 Tc1s were prevented from exhibiting the high level of heritable transposition displayed by BO elements. We suggest that Bristol Tc1 elements have the ability to transpose but that transposition is heavily repressed in the gonadal tissue.
Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L cell genome.
Authors: Kato S , Anderson R A , Camerini-Otero R D
Published: September 2015

We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk- cells were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail by nucleotide sequencing and hybridization techniques. These plasmids contained a total of seven cellular-exogenous DNA junctions. In all cases, there was no sequence homology between the exogenous and cellular DNA sequences adjacent to the joining sites, and no specific exogenous or cellular sequences occurred at the junctions. Rearrangement or deletion of Ltk- DNA was always associated with the integration of exogenous DNA. All of the assignable cellular sequences at the junctions were repetitive sequences. Two of these sequences were from the MIF-1 repetitive sequence family, and a third consisted of a 40-base pair simple copolymer of alternating deoxyadenosine-deoxythymidine. Our results suggest that repetitive sequences are relatively favorable sites for the integration of exogenous DNA.
Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides.
Authors: Chang C N , Matteucci M , Perry L J , Wulf J J , Chen C Y , Hitzeman R A
Published: September 2015

Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion.
New positive and negative regulators for general control of amino acid biosynthesis in Saccharomyces cerevisiae.
Authors: Greenberg M L , Myers P L , Skvirsky R C , Greer H
Published: September 2015

The biosynthesis of most amino acids in Saccharomyces cerevisiae is coregulated. Starvation for a single amino acid results in the derepression of amino acid biosynthetic enzymes in many unrelated pathways. This phenomenon, known as general control, is mediated by both positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the identification and characterization of several new regulatory genes for this system, GCN6, GCN7, GCN8, GCN9, and GCD5. A mutation in the negative regulator GCD5 was isolated on the basis of its suppression of a gcn2 mutation. The effect of gcd5 is a posttranscriptional increase in histidine biosynthetic enzyme activity. Suppressors of gcd5 which are deficient in derepression were in turn isolated. Eight such mutations, defining four new positive regulatory genes (GCN6 through GCN9), were obtained. These mutations are recessive, confer sensitivity to multiple amino acid analogs, and result in decreased mRNA levels for genes under general control. The GCN6 and GCN7 gene products were shown to be positive regulators for transcription of the GCN4 gene, the most direct-acting positive regulator thus far identified. The interaction of GCN6 and GCN7 with GCN4 is fundamentally different from that of previously isolated GCN genes. It should also be noted that these gcn selections gave a completely different nonoverlapping set of mutations from earlier selections which relied on analog sensitivity. Thus, we may have identified a new class of GCN genes which are functionally distinct from GCN1 through GCN5.
Proviral integration site Mis-1 in rat thymomas corresponds to the pvt-1 translocation breakpoint in murine plasmacytomas.
Authors: Villeneuve L , Rassart E , Jolicoeur P , Graham M , Adams J M
Published: September 2015

Two loci independently implicated in T-and B-lymphocyte neoplasia are shown to be equivalent. The Mis-1 locus is a common proviral integration site in retrovirally induced rat T lymphomas, while the pvt-1 locus on murine chromosome 15 frequently translocates to the kappa locus in plasmacytomas bearing 6;15 translocations. By comparing cloned sequences, we show that pvt-1 is the murine homolog of Mis-1.
A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene.
Authors: Papageorge A G , Willumsen B M , Johnsen M , Kung H F , Stacey D W , Vass W C , Lowy D R
Published: September 2015

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.