bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of plasmid R388-encoded type II dihydrofolate reductase as a dominant selective marker in Saccharomyces cerevisiae.
Authors: Miyajima A , Miyajima I , Arai K , Arai N
Published: September 2015

The R388 plasmid-encoded drug-resistant type II dihydrofolate reductase gene (R . dhfr) was expressed in Saccharomyces cerevisiae by fusing the R . dhfr coding sequence to the yeast TRP5 promoter. Yeast cells harboring these recombinant plasmids grew in media with 10 micrograms of methotrexate per ml and 5 mg of sulfanilamide per ml, a condition which inhibits the growth of wild-type cells. Addition of a 390-base-pair fragment from the 3'-noncoding region of TRP5 downstream from R . dhfr increased expression. Presumably, the added segment promoted termination or polyadenylation or both of the R . dhfr transcript. The activity of the plasmid-encoded dihydrofolate reductase and the copy number of the R . dhfr plasmid in cells grown in drug-selective media were higher by one order of magnitude than those grown in nutrition-selective media. Plasmid copy number, as well as the plasmid-encoded enzyme level, decreased when cells were selected for prototrophy. In drug-selective media, the plasmid-encoded enzyme level and the content of R . dhfr transcripts were nearly constant in cells harboring R . dhfr plasmids containing different yeast promoters. In contrast, the plasmid copy number and beta-lactamase activity encoded in cis by plasmids were much higher when R . dhfr was associated with the weak TRP5 promoter than when it was fused to the strong ADC1 promoter. These results indicate that plasmid copy number, i.e., gene dosage of R . dhfr, correlates inversely with the strength of the promoter associated with R . dhfr, and cells with a higher plasmid copy number were enriched in drug-selective media. The transformation efficiency of R . dhfr fused to the ADC1 promoter was almost the same on drug-selective plates as on nutrition-selective plates, indicating that R . dhfr is suitable as a dominant selective transformation marker in S. cerevisiae.
Size-variant pp60src proteins of recovered avian sarcoma viruses interact with adhesion plaques as peripheral membrane proteins: effects on cell transformation.
Authors: Krueger J G , Garber E A , Chin S S , Hanafusa H , Goldberg A R
Published: September 2015

We have shown previously that the membrane association of the src proteins of recovered avian sarcoma viruses (rASVs) 1702 (56 kilodaltons) and 157 (62.5 kilodaltons), whose size variations occur within 8 kilodaltons of the amino terminus, is salt sensitive and that, in isotonic salt, these src proteins fractionate as soluble cytoplasmic proteins. In contrast, wild-type Rous sarcoma virus pp60src behaves as an integral plasma membrane protein in cellular fractionation studies and shows prominent membrane interaction by immunofluorescence microscopy. In this study we have examined the distribution of these size-variant src proteins between free and complexed forms, their subcellular localization by immunofluorescence microscopy, and their ability to effect several transformation-related cell properties. Glycerol gradient sedimentation of extracts from cells infected either with rASV 1702 or rASV 157 showed that soluble src proteins of these viruses were distributed between free and complexed forms as has been demonstrated for wild-type Rous sarcoma virus pp60src. Pulse-chase studies with rASV pp60src showed that, like wild-type Rous sarcoma virus pp60src, it was transiently found in a complexed form. Indirect immunofluorescence showed that size-variant pp60src proteins are localized in adhesion plaques and regions of cell-to-cell contact in rASV 1702- or 157-infected cells. This result is in contrast with the generalized localization of pp60src in plasma membranes of control rASV-infected cells which produce pp60src. Chicken embryo fibroblasts infected by rASVs 1702 and 157 display a partial-transformation phenotype with respect to (i) transformation-related morphology, (ii) cell surface membrane changes, and (iii) retained extracellular fibronectin. It is possible that the induction of a partial-transformation phenotype may be the result of the unique interaction of the src proteins encoded by these viruses with restricted areas of the plasma membrane.
Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.
Authors: Lobaton C D , Moreno A , Oxender D L
Published: September 2015

We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20.
Co-amplification of double minute chromosomes, multiple drug resistance, and cell surface P-glycoprotein in DNA-mediated transformants of mouse cells.
Authors: Robertson S M , Ling V , Stanners C P
Published: September 2015

A genetic system comprised of mammalian cell mutants which demonstrate concomitant resistance to a number of unrelated drugs has been described previously. The resistance is due to reduced cell membrane permeability and is correlated with the presence of large amounts of a plasma membrane glycoprotein termed P-glycoprotein. This system could represent a model for multiple drug resistance which develops in cancer patients treated with chemotherapeutic drugs. We demonstrate here that the multiple drug resistance phenotype can be transferred to mouse cells with DNA from a drug-resistant mutant and then amplified quantitatively by culture in media containing increasing concentrations of drug. The amount of P-glycoprotein was correlated directly with the degree of drug resistance in the transformants and amplified transformants. In addition, the drug resistance and expression of P-glycoprotein of the transformants were unstable and associated quantitatively with the number of double minute chromosomes. We suggest that the gene for multiple drug resistance and P-glycoprotein is contained in these extrachromosomal particles and is amplified by increases in double minute chromosome number. The potential use of this system for manipulation of mammalian genes in general is discussed.
Temporal analysis of general control of amino acid biosynthesis in Saccharomyces cerevisiae: role of positive regulatory genes in initiation and maintenance of mRNA derepression.
Authors: Penn M D , Thireos G , Greer H
Published: September 2015

In Saccharomyces cerevisiae, starvation for a single amino acid results in the derepression of enzyme activities in multiple amino acid biosynthetic pathways. Derepression is a consequence of increased transcription of the genes encoding these enzymes. Analysis of the kinetics of mRNA elevation established that derepression occurs within 5 min of a shift of the culture from rich medium to starvation medium. Any starvation condition was sufficient to trigger an initial high mRNA elevation; however, it was the severity of starvation which determined the steady-state mRNA levels that were subsequently established. The products of the positive regulatory genes AAS101, AAS103, and AAS2 were shown to be required in the initiation phase of this response, whereas the AAS102 gene product was required to maintain the new elevated steady-state mRNA levels. The AAS101 and AAS102 genes were cloned. Consistent with their respective roles in initiation and maintenance of derepression. AAS101 mRNA was found to be expressed at high levels in both rich and starvation media, whereas AAS102 mRNA was derepressed only under starvation conditions. The derepression of AAS102 mRNA is dependent on the AAS101 gene product.
Isolation and characterization of a sperm-specific gene family in the nematode Caenorhabditis elegans.
Authors: Klass M R , Kinsley S , Lopez L C
Published: September 2015

The major sperm protein (MSP) of the nematode Caenorhabditis elegans is a low-molecular-weight (15,000) basic protein implicated in the pseudopodial movement of mature spermatozoa. Its synthesis occurs in a specific region of the gonad and is regulated at the level of transcription (M. Klass and D. Hirsh, Dev. Biol. 84:299-312, 1981; S. Ward and M. Klass, Dev. Biol. 92:203-208, 1982; Klass et al., Dev. Biol. 93:152-164, 1982). A developmentally regulated gene family has been identified that codes for this MSP. Whole genomic blots, as well as analysis of genomic clone banks, indicate that there are between 15 and 25 copies of the MSP gene in the nematode genome. Southern blot analysis also indicates that there is no rearrangement or amplification within the MSP gene family during development. No evidence was found of methylation at various restriction sites surrounding the MSP gene family, and similarly, no correlation between methylation and expression was observed. Three distinct members of this MSP gene family have been cloned, and their nucleotide sequences have been determined. Differential screening of a cDNA clone bank made from polyadenylated mRNA from adult males yielded 45 male-specific clones, 32 of which were clones of MSP genes. One of these cDNA clones was found to contain the entire nucleotide sequence for the MSP, including part of the 5' leader and all of the 3' trailing sequence. Genomic clones bearing copies of the MSP genes have been isolated. At least one of the members of this gene family is a pseudogene. Another member of the MSP gene family that has been cloned from genomic DNA contains the entire uninterrupted structural sequence for the MSP in addition to a 5' flanking sequence containing a promoter-like region with the classic TATA box, a sequence resembling the CAAT box, and a putative ribosome-binding sequence. The 3' trailing sequences of the genomic and the cDNA clones contain an AATAAA polyadenylation site.
DNase I sensitivity of integrated simian virus 40 DNA.
Authors: Blanck G , Chen S , Pollack R
Published: September 2015

We undertook an analysis of integrated simian virus 40 (SV40) DNA to learn whether the DNase I-sensitive region is retained in the integrated array of mouse transformants. Our results indicate that full-length integrated SV40 chromatin retains a DNase I-hypersensitive region at the same point as in nonintegrated SV40 chromatin. Thus, the lack of a DNase I-hypersensitive region is not likely to be the reason for nonpermissivity of SV40 in mouse cells. In addition, results reported here indicate that a deletion of about 200 base pairs of DNA in the region of the DNase I-hypersensitive site severely reduces the sensitivity of integrated SV40 chromatin. This result is similar to a previously reported result obtained with deletion mutants of SV40 analyzed in the lytic cycle. It is the first report of a DNA lesion affecting DNase I hypersensitivity of a mammalian chromosome.
Differential expression of the Xenopus laevis tadpole and adult beta-globin genes when injected into fertilized Xenopus laevis eggs.
Authors: Bendig M M , Williams J G
Published: September 2015

Xenopus laevis tadpole and adult beta-globin genes were injected into fertilized X. laevis eggs. Both injected genes replicated and were retained in the developing embryos with equal efficiency. Transcripts of the injected adult gene were detectable at gastrulation and reached a maximum level shortly thereafter. In contrast, transcripts of the injected tadpole gene were not detected until much later stages of development. The level of expression of both the injected genes was low compared with the level of expression of the chromosomal genes during erythropoiesis.
Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.
Authors: Steele R E , Bakken A H , Reeder R H
Published: September 2015

We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of site-specific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.
Saccharomyces cerevisiae CDC8 gene and its product.
Authors: Birkenmeyer L G , Hill J C , Dumas L B
Published: September 2015

The product of the Saccharomyces cerevisiae CDC8 gene is essential for normal cellular DNA replication; the determination of the structure of the gene and the identification of its product would facilitate the examination of its role in this process. We have cloned a 1,000-base-pair fragment of the S. cerevisiae genome carrying the functional gene. The nucleotide sequence includes one long open reading frame; it is flanked by sequences typical of other S. cerevisiae genes. This sequence predicts a polypeptide chain product of 216 amino acids with a molecular weight of 24,600. A polyadenylated RNA transcript of this sequence was identified by hybridization; in vitro translation of RNA samples enriched for this transcript produced a specific polypeptide chain of apparent molecular weight between 24,000 and 25,000. Thus the reading frame identified represents the authentic CDC8 gene, and the amino acid sequence of its product has been deduced. Our observations differ from two previous reports of the identification of the putative CDC8 protein based upon in vitro complementation assays.
Barriers to nuclease Bal31 digestion across specific sites in simian virus 40 chromatin.
Authors: Scott W A , Walter C F , Cryer B L
Published: September 2015

A portion of the nucleoprotein containing viral DNA extracted from cells infected by simian virus (SV40) is preferentially cleaved by endonucleases in a region of the genome encompassing the origin of replication and early and late promoters. To explore this nuclease-sensitive structure, we cleaved SV40 chromatin molecules with restriction enzymes and digested the exposed termini with nuclease Bal31. Digestion proceeded only a short distance in the late direction from the MspI site, but some molecules were degraded 400 to 500 base pairs in the early direction. By comparison, BglI-cleaved chromatin was digested for only a short distance in the early direction, but some molecules were degraded 400 to 450 base pairs in the late direction. These barriers to Bal31 digestion (bracketing the BglI and the MspI sites) define the borders of the same open region in SV40 chromatin that is preferentially digested by DNase I and other endonucleases. In a portion of the SV40 chromatin, Bal31 could not digest through the nuclease-sensitive region and reached barriers after digesting only 50 to 100 base pairs from one end or the other. Chromatin molecules that contain barriers in the BglI to MspI region are physically distinct from molecules that are open in this region as evidenced by partial separation of the two populations on sucrose density gradients.
In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin.
Authors: Moehring T J , Danley D E , Moehring J M
Published: September 2015

Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups from S-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem. 255:10710-10716, 1980).
Developmental changes in translatable RNA species associated with meiosis and spore formation in Saccharomyces cerevisiae.
Authors: Weir-Thompson E M , Dawes I W
Published: September 2015

During Saccharomyces cerevisiae sporulation distinct changes in translatable mRNA species have been detected by two-dimensional gel electrophoresis of the polypeptides produced in a messenger-dependent, cell-free rabbit reticulocyte lysate primed with RNA prepared from a/alpha, a/a, and alpha/alpha isogenic diploids at different stages of sporulation. The availability of functional mRNA increased by about 25% during the first 4 h after transfer of either sporulating (a/alpha), or nonsporulating (a/a and alpha/alpha) diploids to sporulation medium. Thereafter functional mRNA decreased such that in the a/alpha strain after 24 h there was only about 50% of the amount in vegetative cells; a less marked decrease was observed in the a/a and alpha/alpha strains. Of 750 mRNA species detected, 43 underwent alterations only during sporulation in the a/alpha strain, whereas 36 changes were common to all three strains and one mRNA specific to alpha/alpha vegetative cells was detected. Only four of the sporulation-specific changes were due to the de novo appearance of translatable species, and two of these became predominant species of the total population. The majority of the specific changes were due to either permanent or transient increases in the concentration of individual mRNA species; 11 decreases were found. Changes were found at most stages of sporulation, although many occurred in either of two stages, one early (before 2 h) and the other later (between 6 and 8 h) when commitment to meiotic segregation was beginning. The results provide evidence for both quantitative and, to a lesser extent, qualitative transcriptional control of gene expression during sporulation.
Requirement for distal upstream sequences for maximal transcription in vitro of early region IV of adenovirus.
Authors: Handa H , Sharp P A
Published: September 2015

A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested for transcription activity in vitro. At least three elements were found to be important for maximal transcription in HeLa whole-cell extracts. Deletion of the TATA box drastically reduced the transcription activity from the EIV promoter. Sequences between nucleotides -58 and -44 are also important for efficient transcription since deletion of this region reduced activity by 50%. More importantly, sequences residing upstream from -140 critically influence the level of EIV transcription. Deletion of sequences between nucleotides -325 (the right terminus of adenovirus genome) and -140 reduced the level of transcription more than 10-fold. It is possible that a specific cellular factor stimulates EIV transcription by recognition of these upstream sequences. The dependence of transcription from the EIV promoter on a distal upstream element may explain some aspects of the regulation of this promoter.
Spontaneous and 5-azacytidine-induced reexpression of ornithine carbamoyl transferase in hepatoma cells.
Authors: Delers A , Szpirer J , Szpirer C , Saggioro D
Published: September 2015

Rat hepatoma cells that do not synthesize the hepatic enzyme ornithine carbamoyl transferase spontaneously give rise to producing cells at a low frequency. Reexpression of this differentiation trait is strongly increased by 5-azacytidine treatment, suggesting that hypermethylation plays a critical role in the impaired expression of the ornithine carbamoyl transferase gene in hepatoma cells.
Three sea urchin actin genes show different patterns of expression: muscle specific, embryo specific, and constitutive.
Authors: Garcia R , Paz-Aliaga B , Ernst S G , Crain W R
Published: September 2015

The expression of three different actin genes in the sea urchin, Strongylocentrotus purpuratus, was monitored in embryos and adult tissues by using untranslated mRNA sequences as specific hybridization probes. Three distinct patterns of expression were found: muscle specific, embryo specific, and constitutive (i.e., present in all tissues examined). The actin genes encoding the muscle-specific and constitutively expressed genes were each found to be present once in the haploid genome. The embryo-specific probe could derive from either a single gene or a small subset of actin genes. These data demonstrate that at least three members of the sea urchin actin gene family are expressed in distinct ways and thus are probably associated with different regulatory programs of gene expression necessary for development of this metazoan.
Expression of the chicken c-src gene in COS cells.
Authors: Gilmer T M
Published: September 2015

The cellular homolog of the Rous sarcoma virus transforming gene (v-src) was cloned into a plasmid containing the simian virus 40 origin of replication and transcriptional signals. This recombinant plasmid, designated pSVOHCS11 , directs the synthesis of relatively high levels of c-src mRNA and c-src protein ( pp60c -src), when the plasmid is studied 48 to 72 h after calcium phosphate-mediated DNA transfection of COS (monkey) cells. The level of c-src mRNA synthesis is 50-fold higher than the amount of c-src RNA produced in uninfected chicken embryo fibroblasts. Furthermore, the level of pp60c -src expressed in pSVOHCS11 -transfected COS cells is approximately the same as that of pp60v -src in Rous sarcoma virus-transformed cells. Using this recombinant plasmid, we demonstrated that c-src mRNA contains sequences which map 3' to the previously identified c-src-v-src regions of homology. In view of the small amount of c-src mRNA and protein that can be isolated from uninfected cells, this transient expression system offers a convenient source of material for further analyses of the c-src gene product.
Biosynthesis of calmodulin in normal and virus-transformed chicken embryo fibroblasts.
Authors: Zendegui J G , Zielinski R E , Watterson D M , Van Eldik L J
Published: September 2015

We report here that the higher levels of calmodulin in transformed chicken embryo fibroblasts are due to an increase in the rate of synthesis of calmodulin that results from an increased amount of calmodulin-specific mRNA in transformed cells. Transformation of several types of eucaryotic cells by oncogenic viruses results in a two- to threefold increase in the intracellular levels of calmodulin. We used the normal chicken embryo fibroblast and its Rous sarcoma virus-transformed counterpart to examine the biosynthesis of calmodulin. We show that the higher levels of calmodulin found in transformed fibroblasts appear to be the consequence of a selective increase in the rate of synthesis of calmodulin above that of total soluble or total cellular protein. A significant difference in the rate of degradation of calmodulin or total protein between transformed and normal cells was not detected. We also examined the mechanism of the increased synthesis rate of calmodulin and show that the levels of calmodulin mRNA are increased in transformed fibroblasts as measured by both translational activity and hybridization to a calmodulin cDNA probe. It is suggested by these data that the higher levels of calmodulin in transformed cells may result from a specific increase in the rate of either calmodulin gene transcription or mRNA processing.
cis-Dominant mutations which dramatically enhance DUR1,2 gene expression without affecting its normal regulation.
Authors: Chisholm G , Cooper T
Published: September 2015

We have isolated three cis-dominant mutations which dramatically enhance DUR1 ,2 gene expression in Saccharomyces cerevisiae. The mutant phenotype, which is expressed both in haploid and MATa/MAT alpha diploid strains, does not appear to be an alteration of the normal control system for this gene because its expression remained fully inducible and sensitive to nitrogen catabolite repression. Instead, we found much higher levels of DUR1 ,2-specific RNA under both uninduced and induced conditions, i.e., the overproduction trait was superimposed on normal regulation of the gene. The mutations seemed to affect gene expression in a unidirectional manner or to be specific for DUR1 ,2 gene expression, because other genes in proximity to the mutations were not affected. We feel that these mutations may alter the chromatin structure in the vicinity of the DUR1 ,2 upstream control sequences or, alternatively, may be Ty insertions which no longer possess the ROAM characteristics reported by others and ourselves.
Tyrosyl and cyclic AMP-dependent protein kinase activities in BHK cells that express viral pp60src.
Authors: Clinton G M , Roskoski R
Published: September 2015

Protein kinase activities were measured in Rous sarcoma virus-infected baby hamster kidney (BHK) cells that express v-src (BHK [v-src]) and compared with those of revertant and control BHK cells. We observed about a fivefold-higher tyrosine phosphorylating activity in BHK (v-src) cell extracts, which was due to src but not other cellular tyrosyl kinase activities since preincubation with anti-src serum reduced the activity to control cell levels. The cyclic AMP-dependent protein kinase activity was also altered when v-src was expressed. Resolution of the two cyclic AMP-dependent isozymes from the detergent-soluble fraction of cells revealed that the type I activity was selectively decreased about fivefold in BHK (v-src) cells.
Expression of the src and abl cellular oncogenes during development of Drosophila melanogaster.
Authors: Lev Z , Leibovitz N , Segev O , Shilo B Z
Published: September 2015

Transcription of the Drosophila homologs of the abl and src cellular oncogenes, termed Dash and Dsrc , respectively, was studied. Despite the fact that Dash and Dsrc share sequence homology, they have distinct patterns of transcription during development. The Dash transcript, 6.2 kilobases long, is found in maternal RNA stored in unfertilized eggs and in embryos up to 4 h after egg laying. In contrast, Dsrc transcripts are 3.2, 4.8, and 5.2 kilobases long. They are detected in eggs and embryos and to a lesser extent in larvae and adult flies. The relative amount of each of the three Dsrc transcripts changes during the different stages of development.
Overproduction of discoidin I by a temperature-sensitive motility mutant of Dictyostelium discoideum.
Authors: Biswas S , Kayman S C , Clarke M
Published: September 2015

Dictyostelium discoideum MC2 is a temperature-sensitive motility mutant of AX3. Mutant cells are incapable of growth, phagocytosis, and migration under restrictive conditions (Kayman et al., J. Cell Biol. 92:705-711, 1982). We show here that at the restrictive temperature MC2 cells grown axenically or on bacteria synthesized excessive quantities of the lectin discoidin I. By two-dimensional gel electrophoresis and peptide mapping, the proteins overproduced by MC2 cells were indistinguishable from discoidin I synthesized at lower levels in AX3 cells. At least two of the three species of discoidin I were overproduced. This protein family constituted 9% of the total protein in cells that were incubated overnight at 27 degrees C in axenic medium. Although MC2 cells are defective in nutrient uptake under restrictive conditions, the overproduction of discoidin I did not appear to be part of a pleiotropic response to starvation. We propose that transcription of the coordinately regulated discoidin I genes is altered in mutant cells. This alteration may be related to the motility defects manifested by MC2.
UV radiation facilitates methotrexate resistance and amplification of the dihydrofolate reductase gene in cultured 3T6 mouse cells.
Authors: Tlsty T D , Brown P C , Schimke R T
Published: September 2015

Pretreatment of 3T6 murine cells with the carcinogen UV radiation or N-acetoxy-N-acetylaminofluorene increased the number of methotrexate-resistant colonies. This carcinogen-induced enhancement was seen only at low toxicities. The enhancement was transient and was observed at its maximum when cells were subjected to methotrexate selection 12 to 24 h after treatment. The addition of a tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate, during or after carcinogen treatment further enhanced this effect. A large proportion of the resistant colonies had an increase in the dihydrofolate reductase gene copy number and the relative proportions of colonies with amplified genes were similar, regardless of whether selected cells were untreated, treated with carcinogen, or treated with carcinogen plus promoter. We discuss some of the variables which both enhance the generation and improve the detection of methotrexate-resistant colonies, as well as certain implications of our results for the generation and mechanism of gene amplification.
Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences.
Authors: Firzlaff J M , Diggelmann H
Published: September 2015

In mouse Ltk- cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the transcribed MMTV DNA as well as on the flanking mouse DNA sequences. We found that the specific increase in the polymerase loading after hormone treatment is proportional to the increase in the amount of stable MMTV mRNA. When the DNA sequences which are responsible for hormone-receptor binding and for the increased MMTV mRNA levels were deleted, no increase in RNA polymerase II loading on MMTV DNA was observed. Nuclear RNA chains which were transcribed in response to hormone treatment were detected not only from the transfected MMTV DNA but also from the mouse DNA sequences adjacent to the 3' end of the provirus.
Alterations of transcription and translation in HeLa cells exposed to amino acid analogs.
Authors: Thomas G P , Mathews M B
Published: September 2015

Amino acid analogs, like other effectors of the stress response, induce in mammalian cells the same gene products that are induced upon heat shock; incorporation of the analog into protein is required for induction. We show here that induction by analogs involves controls operating at the levels of both transcription and translation. The electrophoretic patterns of newly made mRNAs simplify with time such that the putative stress protein mRNAs are the only species transported from the nucleus. Concomitantly, the patterns of protein synthesis simplify such that the stress proteins become nearly exclusive polypeptide products. Although the normal mRNAs are either not used or used with greatly reduced efficiency, they are not degraded and retain translatability when transferred to cell-free systems. Soon after the stress response has been induced, there follows a defect in the initiation of polypeptide chains, as evidenced by examination of polysome profiles. Upon prolonged exposure, polysomes are recovered, and although they give rise to stress proteins almost exclusively, the normal mRNAs are still present in these structures. Thus, in addition to the initiation defect, a lesion in elongation may also be involved. The extreme sensitivity of protein synthesis to the inhibition of RNA synthesis, together with the parallel simplifications in the patterns of newly made mRNAs and polypeptides, may imply that only newly made mRNAs are efficiently translated in analog-treated cells.
Structure and expression of the oncogene c-myc in fresh tumor material from patients with hematopoietic malignancies.
Authors: Rothberg P G , Erisman M D , Diehl R E , Rovigatti U G , Astrin S M
Published: September 2015

c-myc is the cellular gene homologous to the transforming sequence of MC29, an acute avian retrovirus. The human c-myc gene was cloned and used to study the structure and expression of c-myc in a variety of human hematopoietic malignancies. In a careful study of 106 patients, c-myc RNA was found to be expressed at elevated levels in tumor cells of 17 leukemia patients and five lymphoma patients. The c-myc gene was found to be rearranged in two lymphomas, an African Burkitt's lymphoma and a non-Hodgkins lymphoma in leukemic phase. The Burkitt's rearrangement involved the insertion of new DNA sequences upstream from the c-myc 5' coding region, presumably replacing the normal c-myc transcriptional promoter. None of the other 104 patients, including 20 with elevated myc expression, exhibited any evidence of a genetic rearrangement involving the c-myc gene. Our results show that there is a subset of hematopoietic malignancies characterized by elevated expression of c-myc. This elevated expression in most cases is not due to obvious genetic changes (rearrangement, amplification) at the c-myc locus nor to chromosomal translocations in the vicinity of this gene.
Conversion through homologous recombination of the gene encoding Simian virus 40 115,000-molecular-weight super T antigen to a gene encoding a normal-size large T antigen variant.
Authors: May P , Resche-Rigon M , Borde J , Breugnot C , May E
Published: September 2015

We have previously cloned the gene encoding a 115,000-Mr super T antigen (115K super T antigen), an elongated form of the Simian virus 40 large T antigen, originating from the rat cell line V 11 F1 clone 1, subclone 7 (May et al., J. Virol. 45:901-913, 1983). DNA sequence analysis has shown that the 115K super T antigen gene contains notably an in-phase duplication of a sequence located in the region of tsA mutations. We have also shown that the 115K super T antigen gene is able to induce the formation of transformed foci in transfected rat cells. After rat cell cultures were transfected with the cloned gene encoding 115K super T antigen, we obtained a large number of transformants as reported in this paper. In these transformants, we detected a very high frequency of new T antigen variants, as shown by immunoprecipitation of the cell extracts with anti-simian virus 40 tumor serum followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Based on these results and all of the data presently available, it appears likely that the input plasmid or cosmid DNAs containing the cloned gene were first subjected to recombination events that yield new variant T antigen genes before these recombinant genes become integrated. The new variant T antigens observed in the transformants were predominantly those comigrating with normal-size large T antigen. In fact, these latter variants appeared to be indistinguishable from wild-type large T antigen as judged by restriction mapping by Southern blotting of the total genomic DNA of the transformants. Models of intermolecular or intramolecular homologous recombination occurring between or within the input plasmid or input cosmid DNA molecules are proposed to account for the formation of such revertants.
UV Irradiation induces an activity which stimulates Simian virus 40 rescue upon cell fusion.
Authors: Nomura S , Oishi M
Published: September 2015

UV irradiation of African green monkey cells greatly stimulated efficiency of simian virus 40 induction from simian virus 40-transformed Syrian hamster cells after cell fusion. The maximum inducing activity was observed at 15 to 20 h after irradiation but remained only transiently. The addition of cycloheximide after UV irradiation eliminated the stimulation of the activity.
Location of 64K collagen producer chondrocytes in developing chicken embryo tibiae.
Authors: Capasso O , Tajana G , Cancedda R
Published: September 2015

The synthesis of a new low-molecular-weight collagen by cultured chicken embryo chondrocytes has been recently demonstrated (Capasso et al., Exp. Cell Res. 142:197-206, 1982; Gibson et al., J. Cell Biol. 93:767-774, 1982; Schmid and Conrad, J. Biol. Chem. 257:12444-12450, 1982). In this paper we report results on the location of chondrocytes synthesizing this new collagen (64K collagen) in the developing chicken embryo. The 64K collagen is synthesized in very large amounts by cells concentrated at the diaphysis of 9-day-old and at the epiphysis of 17-day-old embryo tibiae. These regions are characterized by a remodeling of the cartilage matrix leading to the replacement of the cartilage with bone tissue; therefore, this collagen appears to be a marker of a specific developmental stage of chondrocytes. The origin of cells competent for the synthesis of the 64K collagen is also discussed.
Enhanced transformation of human cells by UV-irradiated pSV2 plasmids.
Authors: Spivak G , Ganesan A K , Hanawalt P C
Published: September 2015

Irradiating the plasmid pSV2-gpt with UV (254 nm) doses up to 200 J m-2 caused a dose-dependent increase in the yield of Gpt+ transformants when the plasmid was introduced into human cells by calcium phosphate coprecipitation. UV doses greater than 1 kJ m-2 were required to reduce the efficiency of transformation below that obtained with unirradiated DNA.
Cellular src gene product detected in the freshwater sponge Spongilla lacustris.
Authors: Barnekow A , Schartl M
Published: September 2015

Serum from Rous sarcoma virus tumor-bearing rabbits immunoprecipitated from extracts of the freshwater sponge Spongilla lacustris a tyrosine-specific protein kinase with characteristics similar to the chicken pp60c-src kinase activity. An immune competition assay confirmed the relationship between the protein from sponges and viral pp60v-src.
Flagellar regeneration of the trypanosome Crithidia fasciculata involves post-translational modification of cytoplasmic alpha tubulin.
Authors: Russell D G , Gull K
Published: September 2015

Deflagellation of Crithidia fasciculata stimulated formation of new flagella and maximized production of alpha 3 tubulin. Continuous labeling during reflagellation revealed that alpha 1, 2, and 3 tubulins were formed, whereas the polyadenylated RNA translation products lacked alpha 3 isoform. Pulse-chase labeling experiments demonstrated that alpha 3 was a post-translational modification of cytoplasmic alpha tubulin.
Phosphorylation of histones in cells treated with hypertonic and acidic media.
Authors: Pantazis P , West M H , Bonner W M
Published: September 2015

Factors in the extracellular environment, specifically hypertonic or acidic growth media, are shown to alter the modification of histones in several cell lines. For histone 2A, changes in modification were visible in the mass pattern and were found to be primarily changes in phosphorylation. The increased modification of the core histones was quickly reversed when cells were returned to normal medium.
Induction of a step in carcinogenesis that is normally associated with mutagenesis by nonmutagenic concentrations of 5-azacytidine.
Authors: Bouck N , Kokkinakis D , Ostrowsky J
Published: September 2015

The permanent cell line BHK-21/cl 13 can be transformed by mutagenic carcinogens as the result of the induction of a recessive somatic mutation. Yet when these cells were treated with 5-azacytidine under conditions in which no mutants resistant to either ouabain or 6-thioguanine could be detected, they were transformed efficiently. These transformants were induced, not selected. 6-Azacytidine was ineffective at transforming BHK cells; 2'-deoxy-5-azacytidine was exceptionally effective. When tested by cell fusion, transformants induced by 5-azacytidine fell into the same complementation group as those induced by highly mutagenic carcinogens, but they were phenotypically distinct in that they were unstable during prolonged passage and rarely displayed the temperature-limited phenotypes so common among BHK transformants induced by strongly mutagenic carcinogens. These results raise the possibility that a cell can be induced by either genetic or epigenetic means to traverse the same single step in carcinogenesis.
Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.
Authors: Klessig D F , Brough D E , Cleghon V
Published: September 2015

The DNA-binding protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central role in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by DBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DBP is toxic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50- to 200-fold, and within 8 h its synthesis from the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells.
Effect of adenovirus infection on expression of human histone genes.
Authors: Flint S J , Plumb M A , Yang U C , Stein G S , Stein J L
Published: September 2015

The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA synthesis, observed when DNA replication is inhibited by a variety of drugs, is not maintained after adenovirus infection.
Differential regulation of the 70K heat shock gene and related genes in Saccharomyces cerevisiae.
Authors: Ellwood M S , Craig E A
Published: September 2015

Saccharomyces cerevisiae contains a family of genes related to Hsp70, the major heat shock gene of Drosophila melanogaster. The transcription of three of these genes, which show no conservation of sequences 5' to the protein-coding region, was analyzed. The 5' flanking regions from the three genes were fused to the Escherichia coli beta-galactosidase structural gene and introduced into yeasts on multicopy plasmids, putting the beta-galactosidase production under yeast promoter control. Analysis of beta-galactosidase mRNA and protein production in these transformed strains revealed that transcription from the three promoters is differentially regulated. The number of transcripts from one promoter is vastly increased for a brief period after heat shock, whereas mRNA from another declines. Transcripts from a third gene are slightly enhanced upon heat shock; however, multiple 5' ends of the mRNA are found, and a minor species increases in amount after heat shock. Transcription of these promoters in their native state on the chromosome appears to be modulated in the same manner.
Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants.
Authors: Stoker A W , Enrietto P J , Wyke J A
Published: September 2015

Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp60src. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp60src domains that are required for transformation but do not grossly affect tyrosine kinase activity.