bluuebunny/crossref_metadata_2025 huggingface dataset:
An excision event that may depend on patchy homology for site specificity.
Authors: Bourgaux-Ramoisy D , Gendron D , Chartrand P , Bourgaux P
Published: September 2015

In mouse cells transformed by a mutant polyomavirus genome, recombination between integrated viral DNA and flanking cellular DNA resulted in the excision of two readily amplifiable chimeras, designated RmI and RmII. The crossing-over that generated RmII was unique in that it involved a simple cellular sequence in which the triplet 5'-CTG-3' was repeated many times. We show that the sequence across the junction resulting from excision was identical in several molecules of RmII, as if the cross-over generating this junction always involved exactly the same two sites on the viral and cellular DNA. We also show that the cellular site mapped where the replacement of a G by an A in one of many successive 5'-CTG-3' triplets generated a homology of five nucleotides (5'-CTACT-3') with the viral site. Oligonucleotides on both sides of these sites are probably involved in matching the two DNAs prior to recombination.
Anemia induces accumulation of erythropoietin mRNA in the kidney and liver.
Authors: Bondurant M C , Koury M J
Published: September 2015

Regulation of the production of erythropoietin occurs in the kidney and liver largely through control of accumulation of erythropoietin mRNA. Erythropoietin mRNA was first detected in kidneys at 1.5 h postanemia and reached a plateau value at least 200-fold above the control value by 4 to 8 h. A 20-base sequence immediately upstream from the reported erythropoietin mRNA initiation site is complementary to a hypervariable sequence in 18S rRNA.
Modulation of p36 phosphorylation in human cells: studies using anti-p36 monoclonal antibodies.
Authors: Isacke C M , Trowbridge I S , Hunter T
Published: September 2015

We have characterized two monoclonal antibodies which recognize human p36. These have been used to examine the sites and extent of serine and tyrosine phosphorylation of p36 in human cells treated with epidermal growth factor and platelet-derived growth factor and in human cells transformed with viruses whose oncogenes encode protein-tyrosine kinases.
Identification of a second trans-acting gene controlling maltose fermentation in Saccharomyces carlsbergensis.
Authors: Dubin R A , Perkins E L , Needleman R B , Michels C A
Published: September 2015

Maltose fermentation in Saccharomyces spp. requires the presence of a dominant MAL locus. The MAL6 locus has been cloned and shown to encode the structural genes for maltose permease (MAL61), maltase (MAL62), and a positively acting regulatory gene (MAL63). Induction of the MAL61 and MAL62 gene products requires the presence of maltose and the MAL63 gene. Mutations within the MAL63 gene produce nonfermenting strains unable to induce the two structural gene products. Reversion of these mal63 nonfermenters to maltose fermenters nearly always leads to the constitutive expression of maltase and maltose permease, and constitutivity is always linked to MAL6. We demonstrated that for one such revertant, strain C2, constitutivity did not require the MAL63 gene, since deletion disruption of this gene did not affect the constitutive expression of the structural genes. In addition, constitutivity was trans acting. Deletion disruption of the MAL6-linked structural genes for maltase and maltose permease in this strain did not affect the constitutive expression of a second, unlinked maltase structural gene. We isolated new maltose-fermenting revertants of a nonfermenting strain which carried a deletion disruption of the MAL63 gene. All 16 revertants isolated expressed maltase constitutively. In one revertant studied in detail, strain R10, constitutive expression was demonstrated to be linked to MAL6, semidominant, trans acting, and residing outside the MAL63-MAL61-MAL62 genes. From these studies we propose the existence of a second trans-acting regulatory gene at the MAL6 locus. We call this new gene MAL64. We mapped the MAL64 gene 2.3 centimorgans to the left of MAL63. The role of the MAL64 gene product in maltose fermentation is discussed.
Mitogens and protein synthesis inhibitors induce ornithine decarboxylase gene transcription through separate mechanisms in the BC3H1 muscle cell line.
Authors: Olson E N , Spizz G
Published: September 2015

Ornithine decarboxylase (ODCase), the rate-limiting enzyme in polyamine biosynthesis, exhibits dramatic fluctuations in activity in response to a variety of hormones and growth factors and has been shown to be down-regulated during myogenesis. In the present study, the molecular mechanisms involved in expression of ODCase mRNA were examined in cells of the BC3H1 muscle line. Proliferating, undifferentiated cells in medium with 20% fetal calf serum displayed high levels of ODCase mRNA and enzyme activity. The transfer of proliferating cells to medium containing 0.5% serum resulted in their withdrawal from the cell cycle and a 20- to 50-fold reduction in the steady-state level of ODCase mRNA within 24 h. Down-regulation of ODCase mRNA was paralleled by a decrease in ODCase enzyme activity and ODCase gene transcription. ODCase mRNA was rapidly reinduced by exposure of quiescent, differentiated cells to medium with 20% serum or by inhibition of protein synthesis with cycloheximide. The accumulation of ODCase mRNA after mitogenic stimulation or protein synthesis inhibition was accompanied by an increase in ODCase gene transcription. The mechanisms whereby mitogens and protein synthesis inhibitors induced ODCase transcription appeared to be different since cycloheximide potentiated the effects of mitogens, resulting in superinduction of ODCase transcription to a level significantly greater than in the presence of mitogens alone. These results indicate that ODCase down-regulation during myogenesis is controlled primarily at the level of ODCase gene transcription. These data also demonstrate that ODCase expression is regulated by antagonistic signals, positive signals for transcription elicited by mitogens and negative signals from endogenous protein repressors that influence ODCase transcription.
Cellular proteins homologous to the viral yes gene product.
Authors: Sudol M , Hanafusa H
Published: September 2015

We raised antibodies in rabbits against the amino-terminal portion of the viral yes protein produced in bacteria with the use of an expression vector based on the lac operon. The anti-yes serum thus obtained precipitated P90gag-yes from Yamaguchi 73 virus-transformed chicken embryo fibroblasts, and this immunoprecipitation was blocked by the purified antigen. The anti-yes serum did not recognize viral src, fps, or fgr proteins. Affinity-purified anti-yes immunoglobulin G (IgG) precipitated two proteins of 59 and 62 kilodaltons from lysates of normal chicken embryo fibroblasts. Two-dimensional tryptic peptide mapping showed that these proteins are closely related to P90gag-yes and that they are different from pp60c-src. Similar to P90gag-yes, the 59- and 62-kilodalton proteins were phosphorylated exclusively on tyrosine in an in vitro kinase reaction, whereas in vivo they were phosphorylated on serine and, to a lesser extent, on tyrosine as well. Expression of the 59- and 62-kilodalton proteins, determined by the immune complex kinase assay, was relatively high in brain, retina, kidney, and liver. The presence in normal chicken embryo fibroblasts and in chicken kidney of two transcripts, 3.7 and 3.9 kilobases in length, that hybridize with a yes-specific DNA probe, as well as the two proteins recognized by anti-yes IgG, suggests either differential splicing of cellular yes gene transcripts or the existence of another yes-related gene.
Transcriptional regulation of the muscle creatine kinase gene and regulated expression in transfected mouse myoblasts.
Authors: Jaynes J B , Chamberlain J S , Buskin J N , Johnson J E , Hauschka S D
Published: September 2015

The muscle-specific form of creatine kinase (MCK) is induced in differentiating myoblast cultures, and a dramatic increase in mRNA levels precedes and parallels the increase in MCK protein. To study this induction, the complete MCK gene was cloned and characterized. The transcription unit was shown to span 11 kilobases and to contain seven introns. The splice junctions were identified and shown to conform to the appropriate consensus sequences. Close homology with branchpoint consensuses was found upstream of the 3' splice sites in six of seven cases. Transcriptional regulation of the gene in differentiating myoblast cultures was demonstrated by nuclear run-on experiments; increases in transcription accounted for a major part of the increased mRNA levels. Regulated expression of a transfected MCK gene containing the entire transcription unit with 3.3 kilobases of 5'-flanking sequence was also demonstrated during differentiation of the MM14 mouse myoblast cell line. The MCK 5'-flanking region was sufficient to confer transcriptional regulation to a heterologous structural gene, since chloramphenicol acetyl transferase activity was induced during differentiation of cultures transfected with an MCK-chloramphenicol acetyl transferase fusion construct. Examination of the DNA sequence immediately upstream of the transcription start site revealed a 17-nucleotide element which occurred three times. Comparisons with other muscle-specific genes which are also transcriptionally regulated during myogenesis revealed upstream homologies in the alpha-actin and myosin heavy chain genes, but not in the myosin light-chain genes, with the regions containing these repeats. We suggest that coordinate control of a subset of muscle genes may occur via recognition of these common sequences.
Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells.
Authors: McConlogue L , Dana S L , Coffino P
Published: September 2015

We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. All showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four- to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA.
Construction of a helper-free recombinant adenovirus that expresses polyomavirus large T antigen.
Authors: Massie B , Gluzman Y , Hassell J A
Published: September 2015

Adenovirus-polyomavirus recombinant viruses were constructed in vitro by inserting a hybrid transcription unit composed of the adenovirus type 2 major late promoter and the early coding region of polyomavirus into the adenovirus type 5 vector Ad5 delta E1/dl309. The vector lacks the E1a and E1b transcription units and contains a unique restriction endonuclease cleavage site in their place. The polyomavirus genomic insert contained a small deletion which precluded the synthesis of functional small and middle T antigen but allowed for the synthesis of large T antigen. One recombinant virus, Ad5PyR39, which contained the hybrid transcription unit in the opposite transcriptional orientation from the overall direction of late-gene transcription, was studied in detail. Ad5PyR39 replicated efficiently without a helper virus in human 293 cells and expressed hybrid mRNAs of the expected size and composition that were translated to yield large T antigen. The large T antigen synthesized in 293 cells was the same size as that produced in mouse 3T6 cells lytically infected with polyomavirus, and this protein bound efficiently and specifically to the large-T-antigen-binding sites in polyomavirus DNA. Moreover, the large T antigen encoded by the recombinant virus proved capable of catalyzing the replication in mouse 3T6 cells of a plasmid containing the polyomavirus origin for DNA replication. Comparison of the amount of large T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells infected with Ad5PyR39, under optimal conditions for each system, revealed at least a fivefold greater yield of the protein on a per cell basis in the latter system compared with the former. Ad5PyR39 should prove to be useful to isolate large quantities of functional polyomavirus large T antigen for structural and biochemical studies.
Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product.
Authors: Tratschin J D , Tal J , Carter B J
Published: September 2015

We previously described use of the human parvovirus, adeno-associated virus (AAV), as a vector for transient expression in mammalian cells of the gene for chloramphenicol acetyltransferase (CAT). In the AAV vector, pTS1, the CAT gene is expressed under the control of the major AAV promoter p40. This promoter is embedded within the carboxyl-terminal region of an open reading frame (orf-1) which codes for a protein (rep) required for AAV DNA replication. We show here that the rep product has additional trans-acting properties to regulate gene expression. First, deletion or frame-shift mutations in orf-1, which occurred far upstream of p40, increased expression of CAT in human 293 (adenovirus-transformed) cells. This increased CAT expression was abolished when such mutant AAV vectors were transfected into 293 cells together with a second AAV vector which could supply the wild-type AAV rep product in trans. Thus, an AAV rep gene product was a negative regulator, in trans, of expression of CAT in uninfected 293 cells. In adenovirus-infected 293 cells, the function of the AAV rep product was more complex, but in some cases, it appeared to be a trans activator of the expression from p40. In HeLa cells, only trans activation by rep was seen in the absence or presence of adenovirus. Neither activation nor repression by the rep product required replication per se of the AAV vector DNA. Thus, trans-acting negative or positive regulation of gene expression by the AAV rep gene is modulated by factors in the host cell and by the helper adenovirus.
Nucleosome phasing on a DNA fragment from the replication origin of simian virus 40 and rephasing upon cruciform formation of the DNA.
Authors: Nobile C , Nickol J , Martin R G
Published: September 2015

Nucleosomes were reconstituted in vitro from a fragment of DNA spanning the simian virus 40 minimal replication origin. The fragment contains a 27-base-pair palindrome (perfect inverted repeat). DNA molecules with stable cruciform structures were generated by heteroduplexing this DNA fragment with mutants altered within the palindromic sequence (C. Nobile and R. G. Martin, Int. Virol., in press). Analyses of the structural features of the reconstituted nucleosomes by the DNase I footprint technique revealed two alternative DNA-histone arrangements, each one accurately phased with respect to the uniquely labeled DNA ends. As linear double-stranded DNA, a unique core particle was formed in which the histones strongly protected the regions to both sides of the palindrome. The cruciform structure seemed to be unable to associate with core histones and, therefore, an alternative phasing of the histone octamer along the DNA resulted. Thus, nucleosome positioning along a specific DNA sequence appears to be influenced in vitro by the secondary structure (linear or cruciform) of the 27-base-pair palindrome. The formation of cruciform structures in vivo, if they occur, might therefore represent a molecular mechanism by which nucleosomes are phased.
Cloning and characterization of a variant surface glycoprotein expression site from Trypanosoma equiperdum.
Authors: Raibaud A , Buck G , Baltz T , Eisen H
Published: September 2015

Variant surface glycoprotein (VSG) genes of African trypanosomes are expressed when they are inserted into one of several telomere-linked expression sites. We cloned and characterized an 11-kilobase (kb) DNA fragment located upstream of an expressed VSG gene. A DNA sequence of 1.8 kb that is located immediately upstream of the inserted VSG gene contains sequences homologous to the 76-base-pair repeats described as being upstream of VSG genes in Trypanosoma brucei (D. A. Campbell, M. P. Van Bree, and J. C. Boothroyd, Nucleic Acids Res. 12:2759-2774). There are no such sequences elsewhere in the 11-kb cloned region. Southern blot analysis using probes from the cloned region revealed multiple unlinked copies of the same or very similar regions. At least three of these are located near telomeres, and two have been shown to be used for the expression of known Trypanosoma equiperdum VSG genes. Like VSG genes, the upstream sequences themselves can be duplicated and deleted. The choice of expression site to be used by a duplicated VSG gene is nonrandom; the site used for expression of the parental VSG gene is strongly favored for use in the daughter variant. Furthermore, even when the parental expression site is not used, the VSG gene occupying it is replaced. Thus, an active expression site is a preferential target for gene conversion in the next variation event.
Expression of a novel high-affinity purine nucleobase transport function in mutant mammalian T lymphoblasts.
Authors: Aronow B , Toll D , Patrick J , Hollingsworth P , McCartan K , Ullman B
Published: September 2015

The single nucleoside transport function of mouse S49 lymphoblasts also transports purine bases (B. Aronow and B. Ullman, J. Biol. Chem. 261:2014-2019, 1986). This transport of purine bases by S49 cells is sensitive to inhibition by dipyridamole (DPA) and 4-nitrobenzylthioinosine, two potent inhibitors of nucleoside transport. Therefore, wild-type S49 cells cannot salvage low hypoxanthine concentrations in the presence of 10 microM DPA and 11 microM azaserine; the latter is a potent inhibitor of purine biosynthesis. Among a mutagenized wild-type population, a cell line, JPA2, was isolated which could proliferate in 50 microM hypoxanthine-11 microM azaserine-10 microM DPA. The basis for the survival of JPA2 cells under these selective conditions was expression of a unique, high-affinity purine nucleobase transport function not present in wild-type cells. JPA2 cells could transport 5 microM concentrations of hypoxanthine, guanine, and adenine 15- to 30-fold more efficiently than parental cells did. Kinetic analyses revealed that the affinity of the JPA2 transporter for all three purine bases was much greater than that of the wild-type nucleobase transport system. Moreover, nucleobase transport in JPA2 cells, unlike that in parental cells, was insensitive to inhibition by DPA, 4-nitrobenzylthioinosine, sulfhydryl reagents, and nucleosides. No alterations in nucleoside transport capability, phosphoribosylpyrophosphate levels, or purine phosphoribosyltransferase enzymes were detected in JPA2 cells. Thus, JPA2 cells express a novel nucleobase transport capability which can be distinguished from the nucleoside transport function by multiple biochemical parameters.
Beta-adrenergic stimulation of c-fos gene expression in the mouse submandibular gland.
Authors: Barka T , Gubits R M , van der Noen H M
Published: September 2015

Isoproterenol (IPR), a beta-adrenergic agonist, induces division of acinar cells in the parotid and submandibular glands of adult rodents and produces hyperplastic and hypertrophic enlargements of these organs. We analyzed the effects of IPR on thymidine incorporation, c-fos mRNA levels, and the immunocytochemical localization of c-fos protein in the submandibular glands of adult and of 5- and 14-day-old mice. In the glands of untreated mice c-fos transcripts were not detectable. In all experimental groups, administration of IPR led to a rapid, transient increase in the c-fos mRNA level. Propranolol blocked the IPR effect, while treatment with IPR and cycloheximide led to superinduction. We observed no correlation between the effect of IPR on cell replication or organ growth and stimulation of c-fos expression, and conclude that the latter is the result of beta-adrenergic receptor-IPR interaction. The c-fos protein was localized immunocytochemically in both the cytoplasm and the nuclei of acinar cells and in the nuclei of duct cells.
Human c-ros-1 gene homologous to the v-ros sequence of UR2 sarcoma virus encodes for a transmembrane receptorlike molecule.
Authors: Matsushime H , Wang L H , Shibuya M
Published: September 2015

We isolated a human gene (designated c-ros-1) homologous to the v-ros sequence of UR2 sarcoma virus. Ten exons, 1,414 base pairs spanning 26 kilobases, contained a tyrosine kinase domain, a transmembrane domain, and a part of an extracellular domain carrying an N glycosylation site which was not acquired by UR2 sarcoma virus. The predicted structure of c-ros-1 is unique among the src family and clearly distinct from the human insulin receptor.
Nonhomologous recombination in the parvovirus chromosome: role for a CTATTTCT motif.
Authors: Hogan A , Faust E A
Published: September 2015

The mechanism of nonhomologous recombination in murine cells infected with the parvovirus minute virus of mice (MVM) has been investigated by analysis of DNA sequences at recombination junctions in naturally occurring deletion variants of the virus. We report here that nonhomologous recombination in the MVM chromosome is characterized by short homologies, by insertion at recombination junctions of foreign DNA sequences that are enriched for preferred eucaryotic topoisomerase I cleavage sites, and by an association with a common DNA sequence motif of the type 5'-CTATTTCT-3'. Additional analyses of broken MVM chromosomes provided evidence for specific enzymatic cleavage within 5'-CTTATC-3' and 5'-CTATTC-3' sequences. The results indicate that the 5'-CTATTTCT-3' motif is an important genetic element for nonhomologous recombination in the parvovirus chromosome.
Isolation and sequencing of a cDNA clone homologous to the v-sis oncogene from human endothelial cells.
Authors: Tong B D , Levine S E , Jaye M , Ricca G , Drohan W , Maciag T , Deuel T F
Published: September 2015

A clone containing the 3' end of the mRNA for the human c-sis gene (homologous to the B chain of platelet-derived growth factor) was isolated from a cDNA library derived from human umbilical vein endothelial cells and then sequenced. The analysis of possible translation products in all three reading frames indicated that the A chain of platelet-derived growth factor was not coded for within the 3' end of the c-sis mRNA. The 3' end of the mRNA for c-sis is contained in or adjacent to exon 6.
Induction of specific transcription by RNA polymerase III in transformed cells.
Authors: Carey M F , Singh K , Botchan M , Cozzarelli N R
Published: September 2015

RNA polymerase III (pol III) transcripts of the highly repeated mouse B2 gene family are increased in many oncogenically transformed murine cell lines. In cells transformed by simian virus 40, the small, cytoplasmic B2 RNAs are present at 20-fold-higher levels than in normal cells (M. R. D. Scott, K. Westphal, and P. W. J. Rigby, Cell 34:557-567, 1983; K. Singh, M. Carey, S. Saragosti, and M. Botchan, Nature [London] 314:553-556). We found that transcripts of the highly repeated B1 gene family are also increased 20-fold upon simian virus 40 transformation and showed that these RNAs result from pol III transcription. In contrast, transcripts from less highly repeated pol III templates such as the 5S, 7SL, 7SK, 4.5SI, tRNAMet, and tRNAPro genes are unaffected. The expression of the B2 RNAs in isolated nuclei shows that the augmentation is due mainly to an increased rate of transcription by pol III. There is thus specific transformation-inducible pol III transcription. We developed an in vitro transcription assay which utilizes genomic DNA as a template to study the transcription of all members of a repetitive gene family in their native context. This assay reproduces the low cytoplasmic levels of B1 compared with B2 RNAs suggesting that this ratio is dictated by intrinsic signals in the DNA.
Evolutionarily selected replication origins: functional aspects and structural organization.
Authors: Lee-Chen G J , Woodworth-Gutai M
Published: September 2015

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.
Characterization of an activated human ros gene.
Authors: Birchmeier C , Birnbaum D , Waitches G , Fasano O , Wigler M
Published: September 2015

A human oncogene, mcf3, previously detected by a combination of DNA-mediated gene transfer and a tumorigenicity assay, derives from a human homology of the avian v-ros oncogene. Both v-ros and mcf3 can encode a protein with homology to tyrosine-specific protein kinases, and both mcf3 and v-ros encode a potential transmembrane domain N terminal to the kinase domain. mcf3 probably arose during gene transfer from a normal human ros gene by the loss of a putative extracellular domain. There do not appear to be any other gross rearrangements in the structure of mcf3.
Transfection of the EJ rasHa gene into keratinocytes derived from carcinogen-induced mouse papillomas causes malignant progression.
Authors: Harper J R , Roop D R , Yuspa S H
Published: September 2015

The development of malignant tumors in carcinogen-treated mouse skin appears to involve several genetic changes. Genetic changes which initiate the process are believed to induce alterations in the normal pattern of epidermal differentiation, resulting in the formation of benign tumors, i.e., epidermal papillomas. Subsequent changes appear to be required for the malignant conversion of papillomas to epidermal, squamous-cell carcinomas. Activation of the rasHa gene occurs frequently in chemically induced benign skin papillomas as well as squamous cell carcinomas and thus may represent one mechanism to achieve the initiation step. In the present study, we analyzed several cell lines derived from chemically induced mouse skin papillomas for the presence of transforming oncogenes by transfection of their DNA into NIH 3T3 cells. These papilloma cell lines exhibit an altered differentiation program, i.e., the ability to proliferate under culture conditions favoring terminal differentiation. When DNA from six separate cell lines was tested in the NIH 3T3 transfection assay, active transforming activity was not detected. However, when the EJ rasHa gene was introduced into three of the papilloma cell lines by DNA transfection, transfectants showed an enhanced capacity to proliferate under differentiating culture conditions and formed rapidly growing, anaplastic carcinomas in nude mice. Our findings suggest that in some papilloma cells, a genetic change distinct from rasHa activation may produce an altered differentiation program associated with the initiation step, and this genetic alteration may act in a cooperating fashion with an activated ras gene to result in malignant progression.
Negative regulatory gene for general control of amino acid biosynthesis in Saccharomyces cerevisiae.
Authors: Myers P L , Skvirsky R C , Greenberg M L , Greer H
Published: September 2015

In Saccharomyces cerevisiae, many amino acid biosynthetic pathways are coregulated by a complex general control system: starvation for a single amino acid results in the derepression of amino acid biosynthetic genes in multiple pathways. Derepression of these genes is mediated by positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the isolation and characterization of a previously unreported negative regulatory gene, GCD3. A gcd3 mutation is recessive to wild type, confers resistance to multiple amino acid analogs, and results in overproduction and partially constitutive elevation of mRNA levels for amino acid biosynthetic genes. Furthermore, a gcd3 mutation can overcome the derepression-deficient phenotype of mutations in the positive regulatory GCN1, GCN2, and GCN3 genes. However, the gcd3 mutation cannot overcome the derepression-deficient phenotype of a gcn4 mutation, suggesting that GCD3 acts as a negative regulator of the important GCN4 gene. Northern blot analysis confirmed this conclusion, in that the steady-state levels of GCN4 mRNA are greatly increased in a gcd3 mutant. Thus, the negative regulatory gene GCD3 plays a central role in derepression of amino acid biosynthetic genes.
Human growth hormone as a reporter gene in regulation studies employing transient gene expression.
Authors: Selden R F , Howie K B , Rowe M E , Goodman H M , Moore D D
Published: September 2015

The human growth hormone (hGH) transient assay system described here is based on the expression of hGH directed by cells transfected with hGH fusion genes. Levels of secreted hGH in the medium, measured by a simple radioimmunoassay, are proportional to both levels of cytoplasmic hGH mRNA and the amount of transfected DNA. The system is extremely sensitive, easy to perform, and is qualitatively different from other transient expression systems in that the medium is assayed and the cells themselves are not destroyed. The hGH transient assay system is appropriate for analyses of regulation of gene expression and was utilized here to investigate the effect of the simian virus 40 enhancer on the herpes simplex virus thymidine kinase promoter and the effect of zinc on the mouse metallothionein-I promoter. The expression of hGH can also be used as an internal control to monitor transfection efficiency along with any other transient expression system. All cell types tested thus far (including AtT-20, CV-1, GC, GH4, JEG, L, and primary pituitary cells) were able to secrete hGH into the medium.
A consensus sequence polymer inhibits in vivo expression of heat shock genes.
Authors: Xiao H , Lis J T
Published: September 2015

Promoter function for hsp70 gene expression in Drosophila melanogaster was studied with an in vivo competition assay. A polymer of 40 tandem copies of the pair of regulatory elements of the hsp70 gene was constructed and cloned into a plasmid vector. Various marked genes were cotransfected with the polymer plasmid into Schneider line 2 cells, and their expression was determined by enzyme activity measurements. The polymer dramatically inhibited expression of cotransfected hsp70, hsp26, and hsp83 genes, but not cotransfected copia and histone genes. Our results indicate that in vivo, a trans-acting, positive regulatory factor, which can be titrated by heat shock consensus sequences, is required for activation of heat shock genes and is specific for these genes; the coordinate induction of different heat shock genes appears to be mediated by similar, but not identical, interactions of the trans-acting induction factor and the cis-acting heat shock consensus sequences; and the uninduced or basal level expression of the transfected hsp70 gene is also due to interaction of the consensus sequence with a positively acting factor.
Immunologically distinct p53 molecules generated by alternative splicing.
Authors: Arai N , Nomura D , Yokota K , Wolf D , Brill E , Shohat O , Rotter V
Published: September 2015

Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full-length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421-PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96-bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo.
Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila.
Authors: Bannon G A , Perkins-Dameron R , Allen-Nash A
Published: September 2015

The presence of specific proteins (known as immobilization antigens) on the surface of the ciliated protozoan Tetrahymena thermophila is under environmental regulation. There are five different classes (serotypes) of surface proteins which appear on the cell surface when T. thermophila is cultured under different conditions of temperature or incubation medium; three of these are temperature dependent. The appearance of these proteins on the cell surface is mutually exclusive. We used polyclonal antibodies raised against 30 degrees C (designated SerH3)- and 40 degrees C (designated SerT)-specific surface antigens to study their structure and expression. We showed that these surface proteins contain at least one disulfide bridge. On sodium dodecyl sulfate-denaturing polyacrylamide gels, the nonreduced 30 degrees C- and 40 degrees C-specific surface proteins migrated with molecular sizes of 69 and 36 kilodaltons, respectively. The reduced forms of the proteins migrated with molecular sizes of 58 and 30 kilodaltons, respectively. The synthesis of the surface proteins responded rapidly and with a time course similar to that of the incubation temperature. The synthesis of each surface protein was greatly reduced within 1 h and undetectable by 2 h after a shift to the temperature at which the protein is not expressed. Surface protein synthesis resumed by the end of 1 h after a shift to the temperature at which the protein is expressed. The temperature-dependent induction of these surface proteins appears to be dependent on the synthesis of new mRNA, as indicated by a sensitivity to actinomycin D. Surface protein syntheses were mutually exclusive except at a transition temperature. At 35 degrees C both surface proteins were synthesized by a cell population. These data support the potential of this system as a model for the study of the effects of environmental factors on the genetic regulation of cell surface proteins.
Expression of multiple proliferin genes in mouse cells.
Authors: Wilder E L , Linzer D I
Published: September 2015

Several copies of the prolactin-related proliferin gene were detected in the mouse genome, encoding at least two distinct forms of proliferin. These forms correspond to proliferin cDNA clones derived from BALB/c 3T3 tissue culture and BALB/c placental RNA. Tissue culture and placental cells were each found to express both forms of proliferin.
High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion.
Authors: Gyuris J , Duda E G
Published: September 2015

After a new transformation procedure, 10% of Saccharomyces cerevisiae cells were found to contain transforming DNA sequences. We used direct transfer of plasmid molecules by fusing bacterial minicell protoplasts to yeast protoplasts. Since the procedure significantly reduces the toxic effect of procaryotic protoplasm on the eucaryotic organism, it might be generally applicable in other systems in which transformation is inefficient or impossible.
Extrachromosomal recombination in mammalian cells as studied with single- and double-stranded DNA substrates.
Authors: Lin F L , Sperle K M , Sternberg N L
Published: September 2015

We have previously proposed a model to account for the high levels of homologous recombination that can occur during the introduction of DNA into mammalian cells (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984). An essential feature of that model is that linear molecules with ends appropriately located between homologous DNA segments are efficient substrates for an exonuclease that acts in a 5'----3' direction. That process generates complementary single strands that pair in homologous regions to produce an intermediate that is processed efficiently to a recombinant molecule. An alternative model, in which strand degradation occurs in the 3'----5' direction, is also possible. In this report, we describe experiments that tested several of the essential features of the model. We first confirmed and extended our previous results with double-stranded DNA substrates containing truncated herpesvirus thymidine kinase (tk) genes (tk delta 5' and tk delta 3'). The results illustrate the importance of the location of double-strand breaks in the successful reconstruction of the tk gene by recombination. We next transformed cells with pairs of single-stranded DNAs containing truncated tk genes which should anneal in cells to generate the recombination intermediates predicted by the two alternative models. One of the intermediates would be the favored substrate in our original 5'----3' degradative model and the other would be the favored substrate in the alternative 3'----5' degradative model. Our results indicate that the intermediate favored by the 3'----5' model is 10 to 20 times more efficient in generating recombinant tk genes than is the other intermediate.
Residual nitrobenzylthioinosine-resistant nucleoside transport in a transport mutant (AE1) of S49 murine T-lymphoma cells.
Authors: Plagemann P G , Woffendin C
Published: September 2015

The uptake of various nucleosides by S49 mouse T-lymphoma cells and that by a single-step nucleoside transport-defective mutant thereof (AE1) were compared. Residual nucleoside entry into AE1 cells occurred via two routes, nonmediated permeation and saturable, non-concentrative transport with broad substrate specificity and a Michaelis-Menten constant approximating that for thymidine transport in wild-type cells. However, in contrast to nucleoside transport in wild-type cells, residual nucleoside transport in AE1 cells was resistant to inhibition by nitrobenzylthioinosine. In its properties the latter resembled nitrobenzylthioinosine-resistant nucleoside transport observed in other types of mammalian cells. It amounted to less than 1% of the total nucleoside transport activity of wild-type S49 cells. The results indicate that nitrobenzylthioinosine-resistant and -sensitive nucleoside transports are genetically distinguishable. In wild-type cells, the salvage of thymidine, when present at concentrations higher than 1 to 10 microM, was limited by phosphorylation, because of the saturation of thymidine kinase. In AE1 cells, entry into the cells mainly limited thymidine salvage, but at high thymidine concentrations the combined entry via residual transport and nonmediated permeation was sufficiently rapid to support intracellular thymidine phosphorylation at rates comparable to those observed in wild-type cells.
DNA mismatch repair detected in human cell extracts.
Authors: Glazer P M , Sarkar S N , Chisholm G E , Summers W C
Published: September 2015

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.
Nucleotide sequence analysis of a cloned DNA fragment from human cells reveals homology to retrotransposons.
Authors: Flügel R M , Maurer B , Bannert H , Rethwilm A , Schnitzler P , Darai G
Published: September 2015

During molecular cloning of proviral DNA of human spumaretrovirus, various recombinant clones were established and analyzed. Blot hybridization revealed that one of the recombinant plasmids had the characteristic features of a member of the long interspersed repetitive sequences family. The DNA element was analyzed by restriction mapping and nucleotide sequencing. It showed a high degree of amino acid sequence homology of 54.3% when compared with the 5'-terminal part of the pol gene product of the murine retrotransposon LIMd. The 3' region of the cloned DNA element encodes proteins with an even higher degree of homology of 67.4% in comparison to the corresponding parts of a member of the primate KpnI sequence family.
Ty1 sequence with enhancer and mating-type-dependent regulatory activities.
Authors: Errede B , Company M , Hutchison C A
Published: September 2015

Some insertion mutations in Saccharomyces cerevisiae activate the expression of adjacent structural genes. The CYC7-H2 mutation is a Ty1 insertion 5' to the iso-2-cytochrome c coding region of CYC7. The Ty1 insertion causes a 20-fold increase in CYC7 expression in a and alpha haploid cell types of S. cerevisiae. This activation is repressed in the a/alpha diploid cell type. Previous computer analysis of the CYC7-H2 Ty1 activator region identified two related sequences with homology both to mammalian enhancers and to a yeast a/alpha control site. A 112-base-pair (bp) DNA fragment encompassing one of these blocks of homology functioned as one component of the Ty1 activator. A 28-bp synthetic oligonucleotide with the wild-type homology block sequence was also functional. A single base pair mutation within the enhancer core of the synthetic 28-bp regulatory element reduced its activation ability to near background amounts. In addition, the 112-bp Ty1 fragment by itself functioned as a target for repression of adjacent gene expression in a/alpha diploid cells.
Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes.
Authors: Chabot B , Steitz J A
Published: September 2015

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.
Gene expression of the chondroitin sulfate proteoglycan core protein PG19.
Authors: Bourdon M A , Shiga M , Ruoslahti E
Published: September 2015

We have examined genomic sequences and mRNA species hybridizing to a cDNA clone of a yolk sac carcinoma chondroitin sulfate proteoglycan designated PG19. Genomic blot hybridizations with cDNAs covering the majority of the PG19 mRNA sequence revealed 15 to 17 gene fragments. Similar analysis with probes representing either the propeptide or the combined core protein COOH-terminal domain and 3' untranslated sequences revealed single genomic fragments indicating that a single gene codes for the PG19 proteoglycan. Genomic blot analysis with cDNA sequences coding for the serine-glycine repeat of the core protein identified the same gene fragments observed with the entire PG19 cDNA, indicating that this coding region is homologous with sequences present in multiple genes. The same probes were also used to examine mRNA expression. In addition to the PG19 mRNA, several PG19-related mRNAs could be seen. These PG19-related mRNAs had homology with the serine-glycine coding sequence of the PG19 cDNA. These mRNAs may be coding for proteoglycans. The mRNA coding for PG19 appeared to be uniquely expressed in parietal yolk sac and mast cell lineages. The PG19 mRNA existed in different forms in parietal yolk sac and mast cell lines due to cell-type-specific differences in the length of the 5' untranslated sequences. These results indicate that expression of the PG19 proteoglycan gene is regulated both in terms of cell-type-specific transcription and selection of a transcriptional start site.