bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenovirus VA RNAI: a positive regulator of mRNA translation.
Authors: Svensson C , Akusjärvi G
Published: September 2015

We have developed a sensitive transient expression assay in 293 cells to study the effect of VA RNAs on the translation of adenovirus mRNAs. Monolayers of 293 cells were transfected with mixtures of recombinant plasmids encoding adenovirus-specific transcription units and plasmids encoding VA RNAs. Transfected cells were labeled with [35S]methionine for ca. 15 h, and labeled cell extracts were prepared. Changes in the protein expression caused by VA RNA cotransfection were measured by immunoprecipitation, using monospecific antisera prepared against adenovirus-specific polypeptides. Using this experimental design, we demonstrate that VA RNAI stimulates the translation of both early and late adenovirus mRNAs. Synthesis of the E3 19,000-dalton glycoprotein and the E2A 72,000-dalton DNA binding protein was stimulated between 10 and 20 times by VA RNAI cotransfection. Synthesis of the late hexon polypeptide was also stimulated, although translation of hexon was from an aberrant mRNA lacking the second and third segments of the common tripartite leader attached to late adenovirus mRNAs. VA RNAII, although very homologous to VA RNAI, does not function as a translational stimulator.
Superkiller mutations in Saccharomyces cerevisiae suppress exclusion of M2 double-stranded RNA by L-A-HN and confer cold sensitivity in the presence of M and L-A-HN.
Authors: Ridley S P , Sommer S S , Wickner R B
Published: September 2015

In an mktl host, L-A-HN double-stranded RNA excludes M2 double-stranded RNA at 30 degrees C but not at 20 degrees C. Recessive mutations suppressing the exclusion of M2 by L-A-HN in an mktl host include six ski (superkiller) genes, three of which (ski6, ski7 and ski8) are new genes. The dominant mutations in one gene (MKS50) and recessive mutations in at least two genes (mks1 and mks2) suppress M2 exclusion by L-A-HN but do not show other characteristics of ski mutations and thus define a new class of killer-related chromosomal genes. Mutations in ski2, ski3, ski4, ski6, ski7, and ski8 result in increased M copy number at 30 degrees C and prevent the cells from growing at 8 degrees C. Elimination of M double-stranded RNA from a cold-sensitive ski- strain results in the loss of cold sensitivity. ski- [KIL-sd1] strains lack L-A-HN, carry L-A-E, and have a lower M1 copy number than do ski- [KIL-k1] strains and are only slightly cold sensitive. The LTS5 (=MAK6) product is required both for low temperature growth and for M1 maintenance or replication. We propose that the elevated levels of M in ski- strains divert the host LTS5 product away from the host and to the M replication process. We also suggest that the essential role of L-A in M replication is protection of M double-stranded RNA from the negative influence of SKI+ products.
Recessive constitutive mutant Chinese hamster ovary cells (CHO-K1) with an altered A system for amino acid transport and the mechanism of gene regulation of the A system.
Authors: Moffett J , Englesberg E
Published: September 2015

Chinese hamster ovary cells (CHO-K1) starved for 24 h for amino acids show a severalfold increase in velocity of proline transport through the A system (Vmax is five times that of unstarved cells). This increase is inhibited by cycloheximide, actinomycin D, N-methyl-alpha-amino isobutyric acid (MeAIB, a non-metabolizable specific A system amino acid analog), and by other amino acids that are generally transported by the A system. However, transport by the A system is not a prerequisite for this repression, and all compounds that have affinity for the A system do not necessarily act as "co-repressors." The addition of proline, MeAIB, or other amino acids, as described above, to derepressed cells results in a rapid decrease in A system activity. As shown with proline and MeAIB, this decrease in activity is in part due to a rapid trans-inhibition and a slow, irreversible inactivation of the A system. Neither process is inhibited by cycloheximide or actinomycin D. Alanine antagonizes the growth of CHO-K1 pro cells by preventing proline transport, and alanine-resistant mutants (alar) have been isolated (Moffett et al., Somatic Cell Genet. 9:189-213, 1983). alar2 and alar4 are partial and full constitutive mutants for the A system and have two and six times the Vmax for proline uptake by the A system, respectively. The A system in alar4 is also immune to the co-repressor-induced inactivation. Both alar2 and alar4 phenotypes are recessive. Alar3 shows an increase in Vmax and Km for proline transport through the A system, and this phenotype is codominant. All three mutants have a pleiotropic effect, producing increases in activity of the ASC and P systems of amino acid transport. This increase is not due to an increase in the Na+ gradient. The ASC and P phenotypes behave similarly to the A system in hybrids. A model has been proposed incorporating these results.
Transcription of the mouse ribosomal spacer region.
Authors: Wood K M , Bowman L H , Thompson E A
Published: September 2015

This paper describes experiments designed to test the hypothesis that DNA sequences upstream from the mouse rRNA promoter are transcribed in vivo or in vitro. Plasmid pB28 contains a SalI restriction fragment that extends from -169 to -1,894 base pairs, with respect to the origin of transcription of pre-rRNA. Labeled RNA synthesized in intact cells does not hybridize to this region. Neither S1 nuclease mapping nor RNA dot blot hybridization revealed the presence of sequences complementary to this region. Transcriptional studies carried out in vitro indicated that this region is not transcribed under conditions that are optimal for utilization of the authentic rRNA promoter. Moreover, this region does not appear to form stable transcription complexes with RNA polymerase I transcription components. These data indicate that the mouse rDNA repeating unit differs from those of Xenopus spp. and Drosophila melanogaster in that reduplicated RNA polymerase I promoters are not found in the mouse rDNA spacer region.
Independent regulation by sodium butyrate of gonadotropin alpha gene expression and cell cycle progression in HeLa cells.
Authors: Darnell R B
Published: September 2015

Sodium butyrate alters the growth and gene expression of a variety of differentiating and neoplastic cell types. For example, addition of 5 mM butyrate to HeLa cells is reported to both induce gonadotropin alpha subunit biosynthesis and block cell cycling in G1. We have studied these two actions of butyrate on HeLa cells and found that they are regulated in distinct ways. The induction of alpha subunit synthesis was due to an increase in the rate of transcription of the alpha gene. Using synchronized populations of HeLa cells, we determined that butyrate stimulates alpha transcription throughout the cell cycle. In contrast, treated cells arrest in G1 only if exposed to butyrate for a discrete period during the previous S phase. We conclude that butyrate inhibits DNA synthesis through a cell cycle-specific action that is independent from its direct action to stimulate transcription of the gonadotropin alpha gene.
Amino acid alterations within a highly conserved region of the Rous sarcoma virus src gene product pp60src inactivate tyrosine protein kinase activity.
Authors: Bryant D L , Parsons J T
Published: September 2015

Bisulfite mutagenesis techniques have been used to introduce single-point mutations within a region of the Rous sarcoma virus src gene defined by a BglI restriction endonuclease cleavage site. The mutants of Rous sarcoma virus that are produced by these techniques encode src proteins which contain single amino acid changes within a highly conserved amino acid sequence encompassing residues 430 to 433. DNA from the mutants CHpm26 ( Ala430 to Val), CHpm9 ( Pro431 to Ser), CHpm6 ( Glu432 to Lys), and CHpm65 ( Ala433 to Thr) each failed to transform chicken cells upon transfection, whereas DNA from CHpm59 (a third base alteration in the codon for Glu432 ) readily transformed chicken cells. Analysis of immune complexes containing the altered src proteins indicates that these proteins have decreased tyrosine protein kinase activity in vitro. In vivo labeling of cells infected with the mutant virus revealed diminished levels of the tyrosine-phosphorylated 34,000-molecular-weight protein. These data indicate that mutations within the sequence Ala430 - Pro431 - Glu432 - Ala433 lead to alterations in pp60src-specific tyrosine protein kinase activity and a concomitant loss of transforming potential of the mutant virus.
Induction of alpha-fetoprotein synthesis in differentiating F9 teratocarcinoma cells is accompanied by a genome-wide loss of DNA methylation.
Authors: Young P R , Tilghman S M
Published: September 2015

F9 teratocarcinoma cells can be grown as monolayers or aggregates, and upon treatment with retinoic acid they will differentiate into parietal or visceral endoderm, respectively. Visceral endoderm specifically synthesizes alpha-fetoprotein and albumin mRNAs, which are not found in parietal endoderm. In contrast, both endoderms produce enhanced levels of the major histocompatibility antigen (H2) mRNA compared with F9 cells. F9 cells contain highly methylated DNA as judged by restriction enzyme digestion. However, upon differentiation into visceral endoderm, there is a genome-wide loss of methylation in induced, silent, and constitutively expressed genes. Experiments in which methylation loss is induced via the methyltransferase inhibitor 5-azacytidine result in no induction of alpha-fetoprotein mRNA and no morphological differentiation, suggesting that methylation loss alone is not sufficient to induce the visceral endoderm phenotype. Likewise, 5-azacytidine treatment of differentiated cells does not result in enhanced expression of alpha-fetoprotein mRNA. However, the patterns of loss of DNA methylation at all sites examined after differentiation or 5-azacytidine treatment were remarkably similar, suggesting that the two occur by a similar mechanism, the inhibition of DNA methyltransferase activity. These results argue that the specificity for methylation loss at a given site is an inherent property of aggregated F9 cell chromatin. This system provides a model for studying a tissue-specific change in DNA methylation upon differentiation.
Polyamine starvation prolongs the S and G2 phases of polyamine-dependent (arginase-deficient) CHO cells.
Authors: Anehus S , Pohjanpelto P , Baldetorp B , Långström E , Heby O
Published: September 2015

This study analyzes the effects of polyamine starvation on cell cycle traverse of an arginase-deficient CHO cell variant (CHO-A7). These cells grow well in serum-free medium, provided that it contains ornithine or polyamines or both. In the absence of ornithine or polyamines or both, the CHO-A7 cells develop severe polyamine deficiency and, as a consequence, grow more slowly. When grown to a stationary phase in the presence of ornithine or putrescine or both, the CHO-A7 cells became arrested in G0/early G1. However, when starved for ornithine and polyamines, they accumulated in the S and G2 phases. Ornithine and polyamine starvation of CHO-A7 cells causes an increase in ornithine decarboxylase activity. When this increase was prevented by treatment with DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, growth was further suppressed, and a greater fraction of cells were found in the S and G2 phases of the cell cycle.
Lack of retrovirus gene expression in somatic cell hybrids of friend cells and teratocarcinoma cells with a teratocarcinoma phenotype.
Authors: Asche W , Colletta G , Warnecke G , Nobis P , Pennie S , King R M , Ostertag W
Published: September 2015

Two types of hybrids between cells with erythroid phenotype (Friend cells) and teratocarcinoma cells can be distinguished: cell hybrids with an erythroid phenotype, which release or can be induced to release large amounts of Friend spleen focus-forming virus (F-SFFV) on exposure to bromodeoxyuridine and cell hybrids with a teratocarcinoma phenotype, which do not release Friend virus and are not inducible for F-SFFV release. In this paper, we attempted to relate these differences to the expression of F-SFFV and Friend murine leukemia virus (F-MuLV) functions. Teratocarcinoma phenotype hybrids retained F-SFFV-and F-MuLV-related provirus sequences. They did not express F-SFFV- or F-MuLV-related RNA or proteins. The hybrids differentiated to endoderm-like cells on exposure to retinoic acid or hexamethylene-bis -acetamide. These cells, in contrast to the teratocarcinoma phenotype (uninduced) cells expressing SSEA-1-like antigens, did not express SSEA-1-like antigens; they formed typical, prekeratin-staining cytoskeletal structures and could be induced to release mouse interferon. The differentiating cells, but not the uninduced teratocarcinoma hybrids, were infected productively with F-MuLV or the F-MuLV--F-SFFV complex. They, however, did not express endogenous F-SFFV. Endogenous F-SFFV functions could not be rescued by infection with F-MuLV. Induction of teratocarcinoma hybrids with retinoic acid did not activate endogenous F-MuLV or F-SFFV transcription or protein synthesis. These data demonstrated two control mechanisms of Friend virus repression: one which acted trans during formation of the cell hybrids and was maintained only in teratocarcinoma phenotype cells and the other which acted cis and was still operative during induction of endodermal differentiation.
Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites.
Authors: Montell C , Fisher E F , Caruthers M H , Berk A J
Published: September 2015

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.
Cellular DNA region involved in induction of thymic lymphomas (Mlvi-2) maps to mouse chromosome 15.
Authors: Tsichlis P N , Strauss P G , Kozak C A
Published: September 2015

Two cellular DNA regions representing common domains for proviral DNA integration ( Mlvi -1 and Mlvi -2) have been identified in Moloney murine leukemia virus-induced rat thymic lymphomas. Cellular sequences which were free of repeated DNA derived from a clone that defines the Mlvi -2 integration domain (lambda Cl228 ) were found to be highly conserved in a variety of vertebrate species that we examined, including mice, hamsters, cats, and humans. In this study, we identified the chromosomal map location of the Mlvi -2 homologous sequences in mice by using hamster-mouse somatic cell hybrids. The results show that Mlvi -2 is present on mouse chromosome 15 but is unrelated to the c-myc and c-sis proto-oncogenes, which map on the same chromosome. Since aberrations on chromosome 15 have been observed reproducibly in mouse thymomas, our data suggest that Mlvi -2 may define a novel sequence involved in the induction or progression of murine thymic lymphomas.
Genetic analysis of nucleoside transport in Leishmania donovani.
Authors: Iovannisci D M , Kaur K , Young L , Ullman B
Published: September 2015

Genetic dissection of nucleoside transport in Leishmania donovani indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 microM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 microM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.
Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process.
Authors: Lin F L , Sperle K , Sternberg N
Published: September 2015

We have constructed phage lambda and plasmid DNA substrates (lambda tk2 and ptk2) that contain two defective herpesvirus thymidine kinase (tk) genes that can be used to detect homologous recombination during the transfer of DNA into mouse L cells deficient in thymidine kinase activity. The recombination event reconstructs a wild-type tk gene and is scored because it converts Tk- cells to Tk+. Using this system, we have shown that (i) both intramolecular and intermolecular homologous recombination can be detected after gene transfer; (ii) the degree of recombination decreases with decreasing tk gene homology; and (iii) the efficiency of recombination can be stimulated 10- to 100-fold by cutting the tk2 DNA with restriction enzymes at appropriate sites relative to the recombining sequences. Based on the substrate requirements for these recombination events, we propose a model to explain how recombination might occur in mammalian cells. The essential features of the model are that the cut restriction site ends are substrates for cellular exonucleases that degrade DNA strands. This process exposes complementary strands of the two defective tk genes, which then pair. Removal of unpaired DNA at the junction between the paired and unpaired regions permits a gap repair process to reconstruct an intact gene.
Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.
Authors: Dreyfuss G , Choi Y D , Adam S A
Published: September 2015

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.
Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.
Authors: Manos M M , Gluzman Y
Published: September 2015

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.
Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates.
Authors: Collett M S , Belzer S K , Purchio A F
Published: September 2015

When analyzed from transformed cell lysates, pp60v-src, the product of the Rous sarcoma virus src gene, typically appears as a single polypeptide of 60,000 molecular weight, phosphorylated at two major sites, an amino-terminal region serine residue and carboxy-terminal region tyrosine residue. We describe here the identification of variant forms of pp60v-src present in transformed cell lysates that exhibited an altered electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. This change in migration appeared to be the result of some alteration in the amino-terminal portion of the molecule and paralleled the appearance of extensive amino-terminal region tyrosine phosphorylation on the pp60v-src molecule. These structural modifications were further correlated with a dramatic increase in the protein kinase-specific activity of pp60v-src. The detection of these variant forms of pp60v-src depended on the prior treatment of the transformed cell cultures with vanadium ions or the inclusion in the cell disruption buffer of Mg2+ or ATP-Mg2+. The implications is that modified, highly active forms of the pp60v-src protein exist in transformed cells, but are transient and rapidly converted to stable forms, possibly by specific dephosphorylation. We suggest that amino-terminal region tyrosine phosphorylation of pp60v-src, presumably the result of autophosphorylation, serves to greatly enhance src protein enzymatic activity, but that much of the regulation of this transforming protein's function may involve a phosphotyrosyl protein phosphatase.
Regulation of basal and induced levels of the MEL1 transcript in Saccharomyces cerevisiae.
Authors: Post-Beittenmiller M A , Hamilton R W , Hopper J E
Published: September 2015

The MEL1 gene in Saccharomyces cerevisiae is required for the production of alpha-galactosidase and for the catabolism of melibiose. Production of alpha-galactosidase is induced by galactose or melibiose and repressed by glucose. Inducibility is controlled by the positive and negative regulatory proteins GAL4 and GAL80, respectively. We have cloned the MEL1 gene to study its transcriptional expression and regulation. Evidence is presented that the MEL1 gene encodes alpha-galactosidase and that mel0 is a naturally occurring allele which lacks the alpha-galactosidase-coding sequences. RNAs prepared from wild-type cells and from cells carrying either the noninducible gal4-2 or GAL80S-100 allele grown on three different carbon sources were examined by Northern hybridization analyses. In wild-type cells under noninducing conditions, such as growth on glycerol-lactic acid, the MEL1 transcript was detected at a basal level which was 1 to 2% of the fully induced level. The basal level of expression was diminished in cells carrying the gal4-2 mutant allele but not in cells carrying the GAL80S-100 allele. The basal and induced RNA levels are repressed by glucose. Size determinations of the MEL1 transcripts detected in glycerol-lactic acid- and galactose-grown cells provided no evidence for two distinct transcripts.
Carbohydrate-binding protein 35: identification of the galactose-specific lectin in various tissues of mice.
Authors: Crittenden S L , Roff C F , Wang J L
Published: September 2015

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured mouse 3T3 fibroblasts. In this study, antibodies directed against CBP35 were used to screen for cross-reactive proteins in various cultured cells and in various organs and tissues of mice. Cross-reactive proteins of the same molecular weight (Mr, 35,000) were found in human, mouse, and chicken fibroblasts and in a macrophage-like cell line, P388D1. Similarly, cross-reactive proteins were also found in the embryonic liver, lung, spleen, thymus, skin, and muscle tissue and in the lung, artery, thymus, and spleen of the adult mouse. Fractionation of extracts of mouse lung on affinity columns of asialofetuin-Sepharose yielded a protein whose molecular weight, carbohydrate-binding specificity, and immunological properties suggest that it is CBP35 derived from the lung, hereafter designated CBP35 (lung). The binding of 125I-labeled CBP35 (lung) to rabbit erythrocytes was quantitated in the presence and absence of various carbohydrates. It was found that only carbohydrates containing galactose were inhibitors of the binding; the disaccharide lactose was 100-fold more potent as an inhibitor than was the monosaccharide galactose. When extracts of the adult mouse liver were fractionated by asialofetuin-Sepharose chromatography, only a protein corresponding to CBP16 was isolated; no CBP35 was found. These results corroborate the immunoblotting data, which indicated that CBP35 was not detectable in the adult mouse liver.
Integration and expression of several molecular forms of Rous sarcoma virus DNA used for transfection of mouse cells.
Authors: Luciw P A , Oppermann H , Bishop J M , Varmus H E
Published: September 2015

To assess the factors required for integration and expression of retroviral DNA, we have examined viral DNA, RNA, and protein in NIH/3T3 mouse cells transformed by transfection with various forms of cloned Rous sarcoma virus (RSV) DNA. Linear RSV DNA molecules, derived from circular DNA containing two long terminal repeats (LTRs) and permuted by cleavage at the SacI restriction endonuclease site in the leader sequence, were integrated near the ends of the linear molecule, with the LTRs on the 3' side of the src gene. Integration of a subgenomic RSV DNA fragment containing the viral src gene without intact LTRs also occurred near the ends of the linear molecule. Head-to-tail tandem arrays of RSV DNA species were observed in some transformed cell lines that received fully digested DNA and in all cell lines that received DNA ligated to produce oligomers before transfection. Closed circular RSV DNA, with one or two LTRs, integrated without apparent specificity within several regions of the viral genome. After transfection with SacI-permuted RSV DNA still linked to arms of the lambda bacteriophage vector DNA, bacteriophage sequences were joined to host DNA. Transformed cell lines produced by transfection with the various forms of RSV DNA produced similar levels of viral src protein, although the efficiency of successful transformation varied by at least two orders of magnitude. Analyses of viral polyadenylated RNA, together with the patterns of viral DNA in transformed cells, indicated that viral DNA can be integrated and expressed without regard to LTR sequences, with adjacent host DNA presumably supplying signals required for the promotion and processing of functional src mRNA.
Loss of a consensus splice signal in a mutant immunoglobulin gene eliminates the CH1 domain exon from the mRNA.
Authors: Brandt C R , Morrison S L , Birshtein B K , Milcarek C
Published: September 2015

A series of mouse myeloma mutants, derived from a cell line of the murine MPC-11 tumor (gamma 2b, kappa), resemble human heavy-chain disease in their loss of an internal domain (exon). In these mutants, most of the gamma 2b CH1 exon was present in the nuclear RNA but was removed during splicing to form the mature cytoplasmic RNA. Amino acid sequence studies of one mutant (10.1) are consistent with the loss of the complete CH1 domain. A second mutant cell line (I17) derived from 10.1 and containing the same CH1 alteration was shown by S1 nuclease protection experiments to have an additional mRNA deletion spanning the CH2-CH3 domain boundary. This second deletion was shown to result from a genomic alteration that provided a marker for the isolation of the expressed H-chain allele. To determine the basis of the CH1 splicing defect, the 117 genome-expressed gamma 2b constant region DNA was cloned. Sequence studies showed a deletion of 99 nucleotides around the 3' end of the CH1 domain, which removed the splice site and flanking DNA, apparently causing the aberrant splicing of the RNA transcript. The sequence deleted in the mutant is flanked by short repeats of the octameric sequence CCAGCCAG in the wild-type gene. In the mutant, one copy of the repeat, in addition to the sequences between the repeats, has been lost.
An antibody to a synthetic peptide recognizes polyomavirus middle-T antigen and reveals multiple in vitro tyrosine phosphorylation sites.
Authors: Harvey R , Oostra B A , Belsham G J , Gillett P , Smith A E
Published: September 2015

Antibodies were raised against three synthetic peptides corresponding to sequences surrounding tyrosine 315, a putative in vitro phosphorylation site in polyomavirus middle-T antigen. Only one of the peptides (called C and corresponding to residues 311 to 330) elicited antibodies that recognized middle-T efficiently. Middle-T present in immunoprecipitates formed with purified anti-C serum still accepted phosphate on tyrosine in an in vitro kinase reaction. This implies that tyrosines other than 315 and 322 that lie within the antibody binding region are phosphorylated under these conditions. This conclusion was supported by the altered partial V8 proteolysis fingerprint of the labeled middle-T. Two-dimensional tryptic fingerprint analysis of 32P-labeled middle-T showed that several tryptic peptides identified as including tyrosine 315 and 322 were missing from middle-T labeled in anti-C immunoprecipitates compared with middle-T labeled in immunoprecipitates made by using anti-tumor cell serum. However, one major labeled peptide remained. This peptide was also present in fingerprints of 32P-labeled middle-T coded by M45, dl23, pAS131, and dl1013, but a peptide with altered mobility was present in dl8 middle-T. This identified the peptide as including tyrosine 250. We deduce from these data that (i) the presence of the antibody against peptide C inhibits phosphorylation of tyrosines 315 and 322; (ii) middle-T labeled in the kinase reaction after immunoprecipitation with anti-C serum is phosphorylated on tyrosine 250; and (iii) when anti-tumor cell serum is used in the in vitro kinase reaction, middle-T is phosphorylated at multiple sites, including residues 250, 315, and 322.
Isolation and characterization of the Beadex locus of Drosophila melanogaster: a putative cis-acting negative regulatory element for the heldup-a gene.
Authors: Mattox W W , Davidson N
Published: September 2015

We isolated recombinant lambda phage clones spanning 49 kilobases of DNA which contain the Beadex and heldup-a loci of Drosophila melanogaster. These cloned DNAs were used to analyze the structure of eight dominant mutant alleles of the Beadex locus which show increased gene activity. A region, only 700 base pairs in length, is altered in each of these mutants. Six of the mutations have DNA insertions within this segment. Most of these insertions resemble retrovirus-like transposable elements. In one case (Beadex2) the inserted sequences are homologous to the gypsy transposon family. The other two Beadex alleles were induced by hybrid dysgenesis and suffered deletions which included at least part of the 700-base-pair segment. These deletions appear to have resulted from imprecise excision or deletion of a nearby P element found in the wild-type parental strain. Analysis of one heldup-a allele (heldup-aD30r) indicates that a similar P element-mediated event is responsible for this lesion. In this mutant, deletion of sequences no more than 1,600 base pairs from the Beadex locus accompanies the loss of heldup-a function. The deleted sequences in heldup-aD30r include the entire 700-base-pair segment within which at least part of the Beadex locus resides, yet these flies have no Beadex phenotype. This indicates that a functional heldup-a gene is necessary for expression of the Beadex phenotype. Together, these results suggest that the Beadex functional domain is contained within a short segment of DNA near the heldup-a gene and support the hypothesis that the Beadex locus functions as a cis-acting negative regulatory element for the heldup-a gene.
Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.
Authors: Covey L , Choi Y , Prives C
Published: September 2015

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.
Identification of regulatory regions within the Ty1 transposable element that regulate iso-2-cytochrome c production in the CYC7-H2 yeast mutant.
Authors: Errede B , Cardillo T S , Teague M A , Sherman F
Published: September 2015

The CYC7-H2 mutation in the yeast Saccharomyces cerevisiae was caused by insertion of a Ty1 transposable element in front of the iso-2-cytochrome c structural gene, CYC7. The Ty1 insertion places iso-2-cytochrome c production under control of regulatory signals that are normally required for mating functions in yeast cells. We have investigated the regions of the Ty1 insertion that are responsible for the aberrant production of iso-2-cytochrome c in the CYC7-H2 mutant. Five alterations of the CYC7-H2 gene were obtained by specific restriction endonuclease cleavage of the cloned DNA and ligation of appropriate fragments. The CYC7+, CYC7-H2, and modified CYC7-H2 genes were each inserted into the yeast vector YIp5 and used to transform a cytochrome c-deficient yeast strain. Expression and regulation of each allele integrated at the CYC7 locus have been compared in vivo by determination of the amount of iso-2-cytochrome c produced. These results show that distal regions of the Ty1 element are not essential for the CYC7-H2 overproducing phenotype. In contrast, alterations in the vicinity of the proximal Ty1 junction abolish the CYC7-H2 expression and give rise to different phenotypes.
Expression of type I and III collagen genes during differentiation of embryonic chicken myoblasts in culture.
Authors: Gerstenfeld L C , Crawford D R , Boedtker H , Doty P
Published: September 2015

Expression of type I and III procollagen genes was studied in embryonic chicken myoblast cell cultures, obtained from thigh muscles of 11-day-old embryos. Differentiation initiated by the addition of ovotransferrin (30 micrograms/ml) was followed visually by phase-contrast microscopy. Myoblast fusion and myotube formation were detected by day 3 and appeared to be complete by day 7. The synthesis of procollagens was monitored by labeling cell cultures for 1 h with [3H]proline and determining the radioactivity in procollagen chains by scanning densitometry of the fluorograms of the sodium dodecyl sulfate-polyacrylamide gels. A 10- to 20-fold increase in the rate of pro alpha-1(I), pro alpha-2(I), and pro alpha-1(III) collagen synthesis was observed, with the greatest increase occurring between days 3 and 9. Collagen mRNA levels in the myoblast cultures were examined by Northern blot and dot blot hybridization assays. The 10- to 20-fold increased rate of protein synthesis was accompanied by a 15-fold increase in the steady-state levels of pro alpha-1(I) and pro alpha-2(I) mRNAs and a 10-fold increase in the steady-state levels of pro alpha-1(III). As a correlate to the studies of collagen expression during myoblast differentiation, the expression of actin mRNAs was examined. Although alpha actin could be detected by day 4, a complete switch from lambda and beta to alpha actin was not observed in the time periods examined. Similar results were obtained in the analysis of RNA extracted from embryonic legs at days 12 and 17 of gestation. Myoblast differentiation is manifested by the accumulation of both muscle-specific mRNAs, such as actin, and type I and III procollagen mRNAs.
DNA replication and chromatin structure of simian virus 40 insertion mutants.
Authors: Innis J W , Scott W A
Published: September 2015

Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replication efficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin of replication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfection into COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure was retained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the ori site. New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves within the ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficient function of the viral origin of replication is correlated with its proximity to an altered chromatin structure.
Most highly repeated dispersed DNA families in the mouse genome.
Authors: Bennett K L , Hill R E , Pietras D F , Woodworth-Gutai M , Kane-Haas C , Houston J M , Heath J K , Hastie N D
Published: September 2015

The construction of a small library of mouse repetitive DNA has been previously reported (Pietras et al., Nucleic Acids Res. 11:6965-6983, 1983). Here we report that the 35 plasmids in this library corresponding to highly repeated (greater than 30,000 copies per genome) dispersed DNA sequences can be grouped into no more than 5 distinct families. These families together comprise 8 to 10% of the mouse genome. They include the previously described small elements B1, B2, and R and the large MIF-1 element. Twelve of the 35 clones contain evolutionarily conserved (EC) sequences. One EC clone in our library mostly consists of alternating dCdT residues; another consists of tandem repeats of the sequence CCTCT. The majority of B1s and B2s in the genome appear to be homogeneous, whereas R sequences, ECs, and MIF-1s are heterogeneous. Two earlier reports showed highly repeated mammalian DNA sequences in the herpesvirus genome (Peden et al., Cell 31:71-80, 1982; Puga et al., Cell 31:81-87, 1982). We show that sequences homologous to our EC clones are present in the herpesvirus genome, although these polypyrimidine stretches are not detected in poxvirus, adenovirus, and simian virus 40 genomes. We detect transcripts containing homology to all of these sequences in a nuclear transcription assay. Also, we show that small, polyadenylated RNA molecules homologous to B2 sequences are expressed in undifferentiated embryonal carcinoma cells but not in their differentiated derivatives. The significance of these findings is discussed.
A transcriptionally active monkey genomic segment homologous to the regulatory region of simian virus 40 is associated with DNase I-hypersensitive sites.
Authors: Lord S T , Singer M F
Published: September 2015

Segments of monkey genomic DNA that are homologous to the control region around the origin of replication of simian virus 40 were previously cloned and characterized (Queen et al., Mol. Cell Biol. 1:1061-1068, 1981). We describe here two DNase I-hypersensitive sites that map in the region of monkey chromatin around one such ori-like segment. One of these sites lies within the simian virus 40 homologous segment which is also a site from which transcription initiates bidirectionally (J. Saffer and M. Singer, submitted for publication).
Short-term, high-efficiency expression of transfected DNA.
Authors: Sussman D J , Milman G
Published: September 2015

We have achieved high-efficiency uptake and expression of foreign DNA in mouse Ltk- cells by modifying the DEAE-dextran-mediated transfection method of McCutchan and Pagano (J. Natl. Cancer Inst. 42:351-357, 1968) to include an initial incubation at elevated pH followed by a shock treatment with dimethyl sulfoxide. Up to 80% of mouse Ltk- cells transfected with the herpes simplex virus thymidine kinase gene expressed thymidine kinase as measured by autoradiography.
Transfer of herpes simplex virus thymidine kinase synthesized in bacteria by a high-expression plasmid to tissue culture cells by protoplast fusion.
Authors: Waldman A S , Milman G
Published: September 2015

The introduction of a protein into living tissue culture cells may permit the in vivo study of functions of the protein. We have previously described a high-efficiency-expression plasmid, pHETK2, containing the herpes simplex virus type 1 thymidine kinase (TK) gene which, upon temperature induction, causes TK to be synthesized as greater than 4% of the bacterial protein. In this report we show that enzymatically active TK was transferred to mouse Ltk- cells by polyethylene glycol-mediated fusion with protoplasts prepared from bacteria containing induced levels of TK. The presence of TK in the Ltk- cells was detected by the incorporation of [3H]thymidine into cell nuclei as measured by autoradiography.
Expression of simian virus 40 large T antigen in embryonal carcinoma cell hybrids.
Authors: Tunnacliffe A , Crawford L V , Goodfellow P
Published: September 2015

Previous work has shown that murine embryonal carcinoma cells are refractory to infection with various viruses, including simian virus 40. Thus, large T and small t antigens, the products of the simian virus 40 early region, are not produced when the virus infects embryonal carcinoma cells, in contrast to other cell types. We show, by qualitative and quantitative analyses, that embryonal carcinoma cell hybrids, containing a simian virus 40 early region integrated into human DNA, are capable of producing viral large T antigen.