bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA.
Authors: Mitsialis S A , Manley J L , Guntaka R V
Published: September 2015

The nucleotide sequences in the long terminal repeat of avian sarcoma virus that are recognized in vitro by HeLa cell RNA polymerase II have been identified. For this purpose, various 5' and 3' deletions were introduced into a cloned long terminal repeat fragment. The effects of these deletions on transcription initiation in HeLa whole-cell extracts were then studied. Three specific transcripts have been identified. The major transcript is initiated at nucleotide +1 (relative to the cap site). Deletion of the upstream sequence between -299 and -55 has no effect on the level of transcription from this start site, whereas deletion of the sequence downstream of -14 drastically reduces the levels of transcription. In contrast, deletion of the sequence downstream from the TATA box has no effect on the initiation or efficiency of synthesis of the two minor RNA species, which are initiated at around nucleotides -260 and -105. The transcription of these RNA products, however, is abolished by an upstream deletion between -299 and -55. These results suggest that HeLa cell RNA polymerase II recognizes in vitro more than one promoter site present in the long terminal repeat of the avian sarcoma virus genome and defines the sequences required for initiation of the major transcript.
Localization of the adenovirus E1Aa protein, a positive-acting transcriptional factor, in infected cells infected cells.
Authors: Feldman L T , Nevins J R
Published: September 2015

The function of the adenovirus E1Aa protein (the product of the 13S E1A mRNA) during a productive viral infection is to activate transcription of the six early viral transcription units. To study the mechanism of action of this protein, a peptide which was 13 amino acids long and had a sequence unique to the protein product of the adenovirus 13S E1A mRNA (pE1Aa) was coupled to keyhole limpet hemocyanin and used to raise an antibody in rabbits. The resulting antiserum was specific to this protein and did not react with the protein product of the 12S E1A mRNA, which shares considerable sequence with the E1Aa protein. This antiserum was used to probe for the E1Aa protein in situ by indirect immunofluorescence and in extracts of infected HeLa cells. We found that the protein was associated with large cellular structures both in the nucleus and in the cytoplasm. The nuclear form of the protein was analyzed further and was found to purify with the nuclear matrix.
Isolation and transcriptional characterization of three genes which function at start, the controlling event of the Saccharomyces cerevisiae cell division cycle: CDC36, CDC37, and CDC39.
Authors: Breter H J , Ferguson J , Peterson T A , Reed S I
Published: September 2015

The genes CDC36, CDC37, and CDC39, thought to function in the cell division control process in Saccharomyces cerevisiae, were isolated from a recombinant plasmid library prepared by partial digestion of S. cerevisiae genomic DNA with Sau3A and insertion into the S. cerevisiae-Escherichia coli shuttle vector YRp7. In each case, S. cerevisiae DNA sequences were identified which could complement mutant alleles of the gene in question and which could direct integration of a plasmid at the chromosomal location known to correspond to that gene. Complementing DNA segments were subcloned to remove extraneous coding regions. The coding regions corresponding to CDC36, CDC37, and CDC39 were then identified and localized by R-loop analysis. The estimated sizes of the three coding regions were 615, 1,400, and 2,700 base pairs, respectively. Transcriptional orientation of the coding regions was established by using M13 vectors to prepare strand-specific probes followed by hybridization to blots of electrophoresed S. cerevisiae mRNA. The intracellular steady-state abundance of the mRNA species corresponding to the genes was estimated by comparing hybridization signals on RNA blots to that of a previously determined standard, the cell cycle start gene CDC28. The quantities calculated for the three mRNA species were low, ranging from 1.5 +/- 1 copies per haploid cell for the CDC36 mRNA to 3.1 +/- 1.5 and 4.6 +/- 2 copies per haploid cell for the CDC37 and CDC39 mRNAs, respectively. The CDC28 mRNA had been previously estimated at 7.0 +/- 2 copies per cell.
Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases.
Authors: Cirullo R E , Dana S , Wasmuth J J
Published: September 2015

We have developed a simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, we have constructed Chinese hamster cell lines that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure. Analysis of these cell lines with Southern blots confirmed the presence of a small number of restriction endonuclease fragments containing human DNA specifically. These cell lines represent a convenient and simple means to clone the human genomic sequences of interest.
Enhanced production of hepatitis B surface antigen in NIH 3T3 mouse fibroblasts by using extrachromosomally replicating bovine papillomavirus vector.
Authors: Wang Y , Stratowa C , Schaefer-Ridder M , Doehmer J , Hofschneider P H
Published: September 2015

We have constructed a recombinant pBR322 plasmid composed of a subgenomic transforming fragment of bovine papillomavirus DNA and the hepatitis B surface antigen gene from cloned hepatitis B virus DNA and used it for transfection of NIH 3T3 mouse fibroblasts. The transformed cells retain the plasmids in extrachromosomal form with a copy number of about 50 to 100 per cell. Expression of the hepatitis B surface antigen gene linked to bovine papillomavirus DNA is independent of its orientation relative to the bovine papillomavirus vector. Cell lines continuously secreting high amounts of hepatitis B surface antigen into the medium could be established. The antigen is released into the culture medium as 22-nm particles, having the same physical properties and constituent polypeptides as those found in the serum of hepatitis B virus-infected patients.
Immunological phenotype of lymphomas induced by avian leukosis viruses.
Authors: Chen L C , Courtneidge S A , Bishop J M
Published: September 2015

The production of immunoglobulin by six cell lines derived from bursal tumors induced by avian leukosis virus follows two general patterns: (i) three cell lines that have been extensively passaged in culture synthesize and secrete light chains only; (ii) three cell lines that are recently isolated produce and secrete monomeric immunoglobulin M in addition to free light chains. All six cell lines synthesize and secrete both glycosylated and unglycosylated forms of light chain. We conclude that the cell lines established from lymphomas induced by avian leukosis virus represent relatively mature, but possibly abnormal, stages in the development of chicken B-lymphocytes. The immunoglobulin M produced by the cell lines failed to form detectable immune complexes with avian leukosis virus. It therefore appears that the immunoglobulin M is not directed against viral antigens and that autogenous antigenic stimulus cannot account for the sustained growth of the neoplastic B-lymphocytes.
Deletion and amplification of the HGPRT locus in Chinese hamster cells.
Authors: Fuscoe J C , Fenwick R G , Ledbetter D H , Caskey C T
Published: September 2015

Somatic cell selective techniques and hybridization analyses with a cloned cDNA probe were used to isolate and identify Chinese hamster cell lines in which the X-linked gene for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) has been altered. Two of 19 HGPRT-deficient mutants selected were found to have major DNA deletions affecting the HGPRT locus. Cytogenetic studies revealed that the X chromosome of each deletion mutant had undergone a translocation event, whereas those from the remaining 17 mutants were normal. Phenotypic revertants of the thermosensitive HGPRT mutant RJK526 were isolated, and amplification of the mutant allele was shown to be the predominant mechanism of reversion. Comparisons of restriction enzyme fragments of DNA from deletion versus amplification strains identified two regions of the Chinese hamster genome that contained homology to the cDNA probe. One was shown to be much larger than the 1,600-nucleotide mRNA for HGPRT and to be comprised of linked fragments that contained the functional HGPRT gene. The second was neither transcribed nor tightly linked to the functional gene. These initial studies of HGPRT alterations at the level of DNA thus identified molecular mechanisms of phenotypic variation.
Enhancement of methotrexate resistance and dihydrofolate reductase gene amplification by treatment of mouse 3T6 cells with hydroxyurea.
Authors: Brown P C , Tlsty T D , Schimke R T
Published: September 2015

We investigated various parameters associated with the initial selection of mouse 3T6 cells for resistance to single concentrations of methotrexate and characterized resistant colonies for the presence of additional (amplified) copies of the dihydrofolate reductase gene. Our results indicate that the frequency of occurrence of dihydrofolate reductase gene amplification varies with the selecting concentration of methotrexate and is highly variable between clonally derived sublines of mouse 3T6 cells. Second, we increased the frequency of occurrence of cells with amplified dihydrofolate reductase genes by transiently inhibiting DNA synthesis with hydroxyurea before the selection of cells in single concentrations of methotrexate. This effect was dependent on the concentration of hydroxyurea, the time of exposure to the drug, and the time interval between the removal of hydroxyurea and the selection of cells in methotrexate.
Proteolysis of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins.
Authors: Nelson W J , Traub P
Published: September 2015

The degradation of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins proceeds in two stages in the form of a limited proteolysis. At first, the reaction is very rapid, with the stepwise and complete removal of a peptide (ca. 9,000 daltons) from the N-terminal of vimentin and desmin. This results in the production of a characteristic "staircase" of degradation products, as seen in two-dimensional polyacrylamide gel electrophoresis. The second stage of proteolysis is characterized by the accumulation of peptides which are resistant to further proteolysis; this is due not to product inhibition but to the fact that these peptides are not substrates for the proteinase and therefore do not protect the latter from inactivation (autodigestion). In vitro phosphorylation of the substrates does not affect proteinase activity, probably because the phosphorylation site is located towards the C-terminal of the molecules. The specific and limited proteolysis of vimentin and desmin results in the deletion of the nucleic acid binding and filament assembly site of these proteins, indicating that the Ca2+-activated proteinase plays a role in regulating the function(s) of these intermediate filament proteins, rather than their simple turnover during the cell cycle.
Neural tissues express high levels of the cellular src gene product pp60c-src.
Authors: Cotton P C , Brugge J S
Published: September 2015

Chicken embryo tissues were examined for the expression of pp60c-src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. Three assays, including a solid-phase radioimmunoassay, a competitive radioimmunoprecipitation assay, and an immune complex protein kinase assay, were employed. Elevated levels of pp60c-src were detected in lysates from several neural tissues, including brain, retina, and spinal ganglia. Other tissues contained 8- to 10-fold-lower levels of pp60c-src, levels comparable to those found in chicken embryo fibroblasts. Expression of pp60c-src in brain tissues was also shown to vary with the developmental stage of the embryo.
Simian virus 40 early- and late-region promoter functions are enhanced by the 72-base-pair repeat inserted at distant locations and inverted orientations.
Authors: Fromm M , Berg P
Published: September 2015

Tandemly repeated 72-base-pair (bp) segments located between nucleotides 107 and 250 of the simian virus 40 genome are essential for early region transcription. The functional requirement for the 72-bp repeat was supplied even when that segment was translocated to several locations distant from, and in different orientation, relative to, the promoter. Regardless of the position of the 72-bp enhancer segment, transcription was initiated at the same locations as with the normal promoter. Translocation of the 72-bp repeat segment to other sites in the genome resulted in the appearance of DNase I hypersensitivity at that site in the intranuclear viral minichromosomes. One of the translocations which did not produce enhancement of early- and late-region expression also failed to create a DNase I-hypersensitive site at the translocated 72-bp segment.
Mutation at autosomal loci of Chinese hamster ovary cells: involvement of a high-frequency event silencing two linked alleles.
Authors: Bradley W E
Published: September 2015

Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.
Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination.
Authors: Babich A , Feldman L T , Nevins J R , Darnell J E , Weinberger C
Published: September 2015

We have studied the adenovirus-induced inhibition of host cell protein synthesis and the effect of infection on the overall metabolism of host cell mRNA during the late phase of adenovirus infection by following the fate of a number of cellular mRNAs complementary to specific cloned DNA segments. At a time in infection when the rate of total cellular protein synthesis is drastically (greater than 90%) reduced, transcription of specific cellular genes is undiminished. However, the transport of newly synthesized cellular mRNA to the cytoplasm is greatly decreased. This decreased appearance of new mRNA in the cytoplasm cannot account for the observed cessation of cell specific protein synthesis, however, since the concentration of several preexisting cellular mRNAs, including the mRNA for actin, remains unchanged throughout the course of infection. The preexisting mRNA is intact, capped, and functional as judged by its ability to direct protein synthesis in vitro in a cap-dependent fashion. The interruption in host translation appears to operate at the level of initiation directly, since we find that fewer ribosomes are associated with a given cellular mRNA after infection than before infection. Furthermore, the in vivo inhibition of cellular protein synthesis does not appear to be the result of competition with viral mRNA, since conditions which prevent the efficient initiation of translation of viral mRNA (infection with a viral mutant) do not result in the recovery of cell translation. Thus, it appears that a late adenovirus gene product directly mediates a shutoff of host protein synthesis.
Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development.
Authors: Bond J F , Farmer S R
Published: September 2015

The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.
Amplification and hormone-regulated expression of a mouse mammary tumor virus-Eco gpt fusion plasmid in mouse 3T6 cells.
Authors: Chapman A B , Costello M A , Lee F , Ringold G M
Published: September 2015

Mouse 3T6 cells were transformed with a chimeric DNA plasmid, pSVMgpt, in which the mouse mammary tumor virus (MMTV) promoter was fused to the Escherichia coli gene encoding xanthine-guanine phosphoribosyl transferase (Eco gpt). The transformants exhibited glucocorticoid-inducible expression of Eco gpt. With limiting xanthine concentrations, conditions were established in which cell growth became hormone dependent. Cells selected for their ability to grow in limiting concentrations of both xanthine and glucocorticoids contained amplified levels of Eco gpt DNA, and expression of Eco gpt remained glucocorticoid inducible in these amplified cells. Thus, amplification of the MMTV promoter region in itself did not abolish hormonal responsiveness of a gene. In addition to increased levels of Eco gpt DNA, some of the selected cells also exhibited increased levels (two- to threefold) of glucocorticoid receptors. Lastly, we found that excessive expression of Eco gpt is toxic to 3T6 cells; by maintaining low hormone levels and, therefore, low levels of expression, we were able to select cells with amplified Eco gpt. Thus, the MMTV promoter may be of general utility in expressing genes whose products may be lethal if they are produced in excessive quantities.
Platelet-derived growth factor-modulated translatable mRNAs.
Authors: Hendrickson S L , Scher C D
Published: September 2015

The treatment of density-arrested BALB/c 3T3 cells with electrophoretically homogeneous or highly purified preparations of the platelet-derived growth factor (PDGF) stimulated the rapid and selective accumulation of several species of abundant mRNA identified by cell-free translation. These translatable mRNAs appeared long before entry into the S phase. Less PDGF was required for selective mRNA accumulation than for PDGF-modulated DNA synthesis. The translatable mRNAs also accumulated after addition of the epidermal growth factor but not after addition of insulin or platelet-poor plasma. Their selective accumulation was blocked by addition of actinomycin D. Three classes of PDGF-modulated mRNAs were defined. An early (primary) RNA appeared within 30 to 60 min of PDGF addition; its accumulation was not blocked by cycloheximide. Another early mRNA also appeared within 60 min, but treatment with both PDGF and cycloheximide was required for optimal accumulation. A third class, secondary RNAs, began to accumulate later at 90 to 120 min; the appearance of this class was inhibited by cycloheximide. One- and two-dimensional gel electrophoresis of translation products demonstrated that a spontaneously transformed BALB/c 3T3 (ST2-3T3) cell line, which does not require PDGF or epidermal growth factor for growth, constitutively accumulated the secondary growth factor-regulated mRNAs. The accumulation of these translatable mRNAs may be required for PDGF-modulated DNA synthesis.
Mouse rDNA: sequences and evolutionary analysis of spacer and mature RNA regions.
Authors: Goldman W E , Goldberg G , Bowman L H , Steinmetz D , Schlessinger D
Published: September 2015

Two regions of mouse rDNA were sequenced. One contained the last 323 nucleotides of the external transcribed spacer and the first 595 nucleotides of 18S rRNA; the other spanned the entire internal transcribed spacer and included the 3' end of 18S rRNA, 5.8S rRNA, and the 5' end of 28S rRNA. The mature rRNA sequences are very highly conserved from yeast to mouse (unit evolutionary period, the time required for a 1% divergence of sequence, was 30 X 10(6) to 100 X 10(6) years). In 18S rRNA, at least some of the evolutionary expansion and increase in G + C content is due to a progressive accretion of discrete G + C-rich insertions. Spacer sequence comparisons between mouse and rat rRNA reveal much more extensive and frequent insertions and substitutions of G + C-rich segments. As a result, spacers conserve overall G + C richness but not sequence (UEP, 0.3 X 10(6) years) or specific base-paired stems. Although no stems analogous to those bracketing 16S and 23S rRNA in Escherichia coli pre-rRNA are evident, certain features of the spacer regions flanking eucaryotic mature rRNAs are conserved and could be involved in rRNA processing or ribosome formation. These conserved regions include some short homologous sequence patterns and closely spaced direct repeats.
Different mRNAs have different nuclear transit times in Dictyostelium discoideum aggregates.
Authors: Mangiarotti G , Zuker C , Chisholm R L , Lodish H F
Published: September 2015

Nuclear processing of mRNA precursors in differentiating multicellular Dictyostelium discoideum aggregates is markedly slower than in growing amoebae. Thus, we have been able to determine the time of nuclear processing of individual mRNA species in postaggregating cells by following the incorporation of 32PO4 into nuclear and cytoplasmic RNA complementary to cloned cDNAs. Precursors of mRNAs synthesized during both growth and differentiation remain in the nucleus for about 25 to 60 min. By contrast, typical mRNAs which are synthesized only by postaggregative cells have nuclear processing times between 50 and 100 min. Depending on the particular mRNA, between 20 and 60% of nuclear transcripts are converted into cytoplasmic mRNA. A third class of mRNAs are transcribed from a set of repetitive DNA segments and are expressed predominantly during differentiation. Nuclear precursors of these mRNAs are extensively degraded within the nucleus or very rapidly after transport to the cytoplasm. Those sequences that are stable in the cytoplasm exit from the nucleus only after a lag of over 2 h. Thus, mRNAs encoded by different genes that are subject to different types of developmental controls display different times of transit to the cytoplasm and different efficiencies of nuclear processing. Differential nuclear processing may contribute to the regulation of the level of individual cytoplasmic mRNAs.
Interferon treatment inhibits pinocytosis.
Authors: Wilcox D K , Whitaker-Dowling P A , Youngner J S , Widnell C C
Published: September 2015

Treating mouse L cells with crude or purified mouse interferon inhibited fluid-phase pinocytosis. Inhibition was maximum at 24 h after treatment with 1,000 U of interferon per ml and was dose dependent and reversible with time. Pinocytosis was inhibited when human and chicken embryo cells were treated with homologous, but not heterologous, interferons.
Mouse and Drosophila genes encoding the major heat shock protein (hsp70) are highly conserved.
Authors: Lowe D G , Fulford W D , Moran L A
Published: September 2015

We used a cloned Drosophila melanogaster hsp70 gene to hybrid-select heat shock-induced mouse mRNA and showed that this mRNA encodes the major mouse heat shock protein. This result suggests that the sequence of the hsp70 gene(s) is highly conserved.
Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.
Authors: Purchio A F , Wells S K , Collett M S
Published: September 2015

pp60v-src, the product of the Rous sarcoma virus src gene, was partially purified by immunoaffinity chromatography from extracts of Rous sarcoma virus-transformed field vole cells. Incubation of this preparation with ATP plus Mg2+ and subsequent repurification by chromatography on hexylamine-agarose resulted in a net increase in the specific activity of the src protein kinase. This increase in phosphotransferase activity was detected by using a variety of substrates including casein, tubulin, and a 34,000-dalton protein presumed to be an in vivo target substrate of pp60v-src. In all cases, the phosphorylation was at tyrosine residues, and the kinase activity was inhibited by preincubation of the enzyme with immunoglobulin G prepared from tumor-bearing rabbit sera. The implications of these results for the regulation and control of pp60v-src-associated kinase activity are discussed.
Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae.
Authors: Finkelstein D B , Strausberg S
Published: September 2015

The HSP90 gene of the yeast Saccharomyces cerevisiae encodes a heat shock-inducible protein with an Mr of 90,000 (hsp90) and unknown function. We fused DNA fragments of a known sequence (namely, either end of a 1.4-kilobase EcoRI fragment which contains the S. cerevisiae TRP1 gene) to an EcoRI site within the coding sequence of the HSP90 gene. When these fusions are introduced into S. cerevisiae they direct the synthesis of unique truncated hsp90 proteins. By determining the size and charge of these proteins we were able to deduce the translational reading frame at the (EcoRI) fusion site. This information allowed us to design and construct a well-defined in-frame fusion between the S. cerevisiae HSP90 gene and the Escherichia coli lacZ gene. When this fused gene is introduced into S. cerevisiae on a multicopy plasmid vector, it directs the heat shock-inducible synthesis of a fused protein, which is an enzymatically active beta-galactosidase. Thus, for the first time, it is possible to quantitate the heat shock response in a eucaryotic organism with a simple enzyme assay.
Strong selection for cells containing new ecotropic recombinant murine leukemia virus provirus after propagation of C57BL/6 radiation-induced thymoma cells in vitro or in vivo.
Authors: Jolicoeur P , Rassart E , Sankar-Mistry P
Published: September 2015

Using the Southern procedure, we have studied the presence of ecotropic-specific murine leukemia viral sequences in genomic DNA isolated from primary X-ray-induced thymomas, from lymphoid cell lines established from them, or from secondary tumors passaged in vivo. We found that primary radiation-induced thymomas and infiltrated spleens do not harbor newly acquired ecotropic provirus. However, additional ecotropic proviruses (which appear recombinant in the gagpol region) could be detected in most of the tumorigenic cell lines established in vitro from them and in tumors arising from subcutaneous transplantation of the primary thymomas. These results suggest that primary radiation-induced thymomas may not be clonal. They also indicate a strong correlation between the presence of ecotropic recombinant proviruses in the genome and the growth ability, both in vitro and in vivo, of specific cells within these thymomas, suggesting a possible mitogenic function for murine leukemia virus.
Saccharomyces cerevisiae killer virus transcripts contain template-coded polyadenylate tracts.
Authors: Hannig E M , Thiele D J , Leibowitz M J
Published: September 2015

The M double-stranded RNA component of type 1 killer strains of the yeast Saccharomyces cerevisiae contains an internal 200-base pair adenine- and uracil-rich region. The plus strands of this viral genomic RNA contain an internal adenine-rich region which allows these strands to bind to polyuridylate-Sepharose as tightly as do polyadenylated RNAs with 3'-terminal polyadenylated tracts of 70 to 100 residues. Internal template coding of an adenine-rich tract in positive polarity in vivo and in vitro transcripts of M double-stranded RNA may serve as an alternate method of transcript polyadenylation. The 3'-terminal residue of the in vitro m transcript is a non-template-encoded purine residue. The 5' terminus of this transcript is involved in a stem-and-loop structure which includes an AUG initiation codon, along with potential 18S and 5.8S rRNA binding sites. Except for the 3'-terminal residue, transcription in in vitro shows complete fidelity.
Isolation and characterization of four adenovirus type 12-transformed human embryo kidney cell lines.
Authors: Whittaker J L , Byrd P J , Grand R J , Gallimore P H
Published: September 2015

Four transformed cell lines were established from cultures of human embryo kidney (HEK) cells microinjected or transfected with cloned adenovirus 12 (Ad12) EcoRI-C DNA (0 through 16.5 map units of the left-hand end of the viral genome). Each cell line showed a different growth pattern. Southern blotting demonstrated that all of the cell lines contained Ad12-specific DNA sequences, but in the microinjected isolates these were at a much lower copy number than in the transfected isolate. Two cell lines (Ad12 HEK 1 and 3) appeared to contain tandemly repeated Ad12 EcoRI-C DNA fragments. Immunoprecipitation and Western blotting confirmed that Ad12 early region 1 (E1) proteins were being expressed by all four of the transformed cell lines, but indicated that E1A polypeptide expression was considerably less than E1B polypeptide expression. All of the Ad12-transformed HEK cell lines were tumorigenic when inoculated intracranially into athymic nude mice.
Cell cycle regulation of mouse H3 histone mRNA metabolism.
Authors: Alterman R B , Ganguly S , Schulze D H , Marzluff W F , Schildkraut C L , Skoultchi A I
Published: September 2015

The mechanisms responsible for the periodic accumulation and decay of histone mRNA in the mammalian cell cycle were investigated in mouse erythroleukemia cells, using a cloned mouse H3 histone gene probe that hybridizes with most or all H3 transcripts. Exponentially growing cells were fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cycle without the use of treatments that arrest growth. Measurements of H3 histone mRNA content throughout the cell cycle show that the mRNA accumulates gradually during S phase, achieving its highest value in mid-S phase when DNA synthesis is maximal. The mRNA content then decreases as cells approach G2. These results demonstrate that the periodic synthesis of histones during S phase is due to changes in the steady-state level of histone mRNA. They are consistent with the conventional view in which histone synthesis is regulated coordinately with DNA synthesis in the cell cycle. The periodic accumulation and decay of H3 histone mRNA appear to be controlled primarily by changes in the rate of appearance of newly synthesized mRNA in the cytoplasm, determined by pulse-labeling whole cells with [3H]uridine. Measurements of H3 mRNA turnover by pulse-chase experiments with cells in S and G2 did not provide evidence for changes in the cytoplasmic stability of the mRNA during the period of its decay in late S and G2. Furthermore, transcription measurements carried out by brief pulse-labeling in vivo and by in vitro transcription in isolated nuclei indicate that the rate of H3 gene transcription changes to a much smaller extent than the steady-state levels of the mRNA or the appearance of newly synthesized mRNA in the cytoplasm. The results suggest that post-transcriptional processes make an important contribution to the periodic accumulation and decay of histone mRNA and that these processes may operate within the nucleus.
An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.
Authors: Richter A , Ozer H L , DesGroseillers L , Jolicoeur P
Published: September 2015

To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39 degrees C) during the early part of the virus cycle (between 0- to 20-h postinfection) greatly inhibited virus production. This effect was not observed in revertant or wild-type cells. Molecular studies by the Southern transfer procedure of the unintegrated viral DNA synthesized in these cells at a permissive (33 degrees C) or nonpermissive temperature revealed that the levels of linear double-stranded viral DNA (8.8 kilobase pairs) were nearly identical in mutant or revertant cells incubated at 33 or 39 degrees C. However, the levels of two species of supercoiled viral DNA (with one or two long terminal repeats) were significantly lower in mutant cells incubated at 39 degrees C than in mutant cells incubated at 33 degrees C or in revertant cells incubated at 39 degrees C. Pulse-chase experiments showed that linear viral DNA made at 39 degrees C could not be converted into supercoiled viral DNA in mutant cells after a shift down to 33 degrees C. In contrast, such conversion was observed in revertant cells. Restriction endonuclease analysis did not detect differences in the structure of linear viral DNA made at 39 degrees C in mutant cells as compared to linear viral DNA isolated from the same cells at 33 degrees C. However, linear viral DNA made at 39 degrees C in mutant cells was poorly infectious in transfection assays. Taken together, these results strongly suggest that this X-linked gene, affecting mouse cell DNA synthesis, is operating in the early phase of murine leukemia virus replication. It seems to affect the level of production of unintegrated linear viral DNA only slightly while greatly reducing the infectivity of these molecules. In contrast, the accumulation of supercoiled viral DNA and subsequent progeny virus production are greatly reduced. Our pulse-chase experiments suggest that the apparent, but not yet identified, defect in linear viral DNA molecules might be responsible for their subsequent impaired circularization.
Expression of the intermediate-filament-associated protein synemin in chicken lens cells.
Authors: Granger B L , Lazarides E
Published: September 2015

Synemin, a 230-kilodalton polypeptide component of avian muscle and erythrocyte intermediate filaments, is also found in association with the vimentin filaments of lens tissue. In chicken lens cells, synemin is bound to the core vimentin polymer with the same 180-nm periodicity that it exhibits in erythrocytes. Its solubility properties are characteristic of those of intermediate filaments in general and similar to those of synemin in muscle cells and erythrocytes. Synemin appears at an early stage of lens development and undergoes a dramatic accumulation as the epithelial cells elongate and differentiate into fiber cells. In contrast to synemin in cultured skeletal muscle, lens synemin is not confined to postmitotic, terminally differentiating cells but is present in proliferative cells as well. It is lost from the fibers near the center of the lens, as are many other cellular structures including intermediate filaments. These findings provide new information about the occurrence and expression of avian synemin and new insight regarding its presumptive role as a modulator of intermediate-filament function.
Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.
Authors: Yocum R R , Hanley S , West R , Ptashne M
Published: September 2015

We present the DNA sequence of a 914-base pair fragment from Saccharomyces cerevisiae that contains the GAL1-GAL10 divergent promoter, 140 base pairs of GAL10 coding sequence, and 87 base pairs of GAL1 coding sequence. From this fragment, we constructed four pairs of GAL1-lacZ and GAL10-lacZ fusions on various types of yeast plasmid vectors. On each type of vector, the fused genes were induced by galactose and repressed by glucose. The response of a GAL1-lacZ fusion to gal4 and gal80 regulatory mutations was similar to the response of intact chromosomal GAL1 and GAL10 genes. A set of deletions that removed various portions of the GAL10 regulatory sequences from a GAL10-CYC1-lacZ fusion was constructed in vitro. These deletions defined a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for full-strength galactose induction and an adjacent guanine-cytosine rich 55 base pairs that contained sequences sufficient for weak induction.
Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit.
Authors: Mok M , Maderious A , Chen-Kiang S
Published: September 2015

We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication.
Inhibition of protein synthesis stabilizes histone mRNA.
Authors: Stimac E , Groppi V E , Coffino P
Published: September 2015

The inhibition of protein synthesis in exponentially growing S49 cells leads to a specific fivefold increase in histone mRNA in 30 min. The rate of transcription of histone mRNA, measured in intact or digitonin-permeabilized cells, is increased slightly, if at all, by cycloheximide inhibition of protein synthesis. Both approach-to-equilibrium labeling and pulse-chase experiments show that cycloheximide prolongs histone mRNA half-life from approximately 30 min to greater than 2 h. Histone mRNA made before the addition of cycloheximide becomes stable after the inhibition of protein synthesis, whereas removal of the inhibitor is followed by rapid degradation of histone mRNA. This suggests that the increased stability of histone mRNA during inhibition of protein synthesis results not from alteration of the structure of the mRNA, but from the loss of an activity in the cell which regulates histone mRNA turnover.
DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells.
Authors: Flatau E , Gonzales F A , Michalowsky L A , Jones P A
Published: September 2015

A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 microM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 10T1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines.
Loss of chloroplast DNA methylation during dedifferentiation of Chlamydomonas reinhardi gametes.
Authors: Sano H , Grabowy C , Sager R
Published: September 2015

In Chlamydomonas reinhardi the chloroplast DNA (ch;DNA) of mating type plus cells undergoes cyclical methylation and demethylation during the life cycle. Methylation occurs during gametogenesis, and fully differentiated gametes can be dedifferentiated back to vegetative cells which contain nonmethylated chlDNA by the addition of a nitrogen source for growth. We examined the dedifferentiation process and found that the mating ability of gametes was lost rapidly after the start of dedifferentiation at a time when the chlDNA was still methylated. The enzymatic activity of the 200-kilodalton DNA methyltransferase was lost at a rate consistent with the rate of dilution during cell division. Methylation of chlDNA decreased at a slower rate than was expected from cell division alone but was consistent with the continuing activity of the preexisting methyltransferase so long as it was present. These results support the hypothesis that demethylation of chlDNA occurs by dilution out of enzymatic methylating activity rather than by enzymatic demethylation.
Isolation of cDNA clones for genes exhibiting reduced expression after differentiation of murine teratocarcinoma stem cells.
Authors: Levine R A , LaRosa G J , Gudas L J
Published: September 2015

In the absence of retinoic acid, PSA-G teratocarcinoma stem cells spontaneously differentiate at a moderate frequency into fibroblast-like cells. In the presence of retinoic acid and dibutyryl cyclic AMP, PSA-G stem cells differentiate into parietal endoderm cells. We prepared a cDNA library from undifferentiated PSA-G teratocarcinoma stem cells; this cDNA library was then screened for gene sequences which exhibit a reduction in expression during the differentiation of these stem cells. From ca. 1,000 clones screened, eight independent sequences were isolated. The level of expression of these cloned genes decreases by 3.0-fold to more than 10-fold after differentiation of PSA-G cells into fibroblast-like cells. After treatment of either PSA-G or F9 teratocarcinoma cells with retinoic acid and dibutyryl cyclic AMP for 72 h, the expression of seven genes is inhibited by two- to fourfold. This decrease of clone-specific transcripts can be detected within 12 h after the addition of retinoic acid. Hybridization-selection and in vitro translation experiments identified the proteins encoded by three of the cloned genes: pST 6-23 codes for a 89,000-dalton protein, pST 7-105 codes for a 41,000-dalton protein, and pST 9-31 codes for a 34,000-dalton protein. The 89,000-dalton protein encoded by pST 6-23 is a heat shock protein. In vitro transcription experiments demonstrate that the retinoic acid-mediated decrease in pST 6-135- and pST 1-68-specific RNA occurs at the transcriptional level and that dibutyryl cyclic AMP acts posttranscriptionally to further depress the levels of these RNAs.
Calcitonin/calcitonin gene-related peptide transcription unit: tissue-specific expression involves selective use of alternative polyadenylation sites.
Authors: Amara S G , Evans R M , Rosenfeld M G
Published: September 2015

Different 3' coding exons in the rat calcitonin gene are used to generate distinct mRNAs encoding either the hormone calcitonin in thyroidal C-cells or a new neuropeptide referred to as calcitonin gene-related peptide in neuronal tissue, indicating the RNA processing regulation is one strategy used in tissue-specific regulation of gene expression in the brain. Although the two mRNAs use the same transcriptional initiation site and have identical 5' terminal sequences, their 3' termini are distinct. The polyadenylation sites for calcitonin and calcitonin gene-related peptide mRNAs are located at the end of the exons 4 and 6, respectively. Termination of transcription after the calcitonin exon does not dictate the production of calcitonin mRNA, because transcription proceeds through both calcitonin and calcitonin gene-related peptide exons irrespective of which mRNA is ultimately produced. In isolated nuclei, both polyadenylation sites appear to be utilized; however, the proximal (calcitonin) site is preferentially used in nuclei from tissues producing calcitonin mRNA. These data suggest that the mechanism dictating production of each mRNA involves the selective use of alternative polyadenylation sites.