bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells.
Authors: Peterson T C , Killary A M , Fournier R E
Published: September 2015

The structural gene encoding liver-specific tyrosine aminotransferase (TAT; EC 2.6.1.5) was assigned to mouse chromosome 8 by screening a series of hybrid cell lines for retention of murine Tat-1 gene sequences by genomic Southern blotting. This assignment demonstrated that the Tat-1 structural gene was not syntenic with Tse-1, a chromosome 11-linked locus that negatively regulates TAT expression in trans (A. M. Killary and R. E. K. Fournier, Cell 38:523-534, 1984). We also showed that the fibroblast Tat-1 gene was systematically activated in hepatoma X fibroblast hybrids retaining fibroblast chromosomes 8 in the absence of chromosome 11 but was extinguished in cells retaining both fibroblast chromosomes. Thus, the TAT structural genes of both parental cell types were coordinately regulated in the intertypic hybrids, and the TAT phenotype of the cells was determined by the presence or absence of fibroblast Tse-1.
Modulation of transcriptional activity and stable complex formation by 5'-flanking regions of mouse tRNAHis genes.
Authors: Morry M J , Harding J D
Published: September 2015

We determined the nucleotide sequences of three mouse tRNAHis genes and a tRNAGly gene present in two different lambda clones. One lambda clone contained two tRNAHis genes 600 base pairs (bp) apart in opposite orientations. The other clone contained a tRNAHis and a tRNAGly gene 569 bp apart in the same orientation. The coding regions of the three tRNAHis genes were identical to sequenced mammalian tRNAHis if posttranscriptional modifications are not considered. Notably, the three tRNAHis genes and a fourth gene previously sequenced by us contained within the flanking regions, various amounts of short, conserved 5' leader sequences and 3' trailer sequences directly abutting the coding regions. Otherwise the flanking regions were not homologous. Deletion mutants of one of the tRNAHis genes were constructed which contained 228, 99, 9, and 3 bp of the wild-type 5'-flanking region, respectively. Deletion of 5'-flanking sequences from positions -9 to -4 reduced transcriptional activity substantially (ca. fivefold) in a HeLa cell S-100 lysate. This effect was independent of the vector sequences in the deletion clone, implying that the region from -4 to -9 of the intact gene contains a positive modulatory element for transcription in vitro. The deletion mutant containing 3 bp of wild-type 5'-flanking sequence also had a greatly reduced ability to inhibit the transcription of a second tRNA gene in a competition assay. Thus, the normal 5'-flanking region influences the ability of the gene to form stable complexes with transcription factors. These data further indicate that a mammalian transcription extract is sensitive to 5'-flanking-region effects if a suitable tRNA gene is assayed.
Cell-type-specific synthesis of murine immunoglobulin mu RNA from an adenovirus vector.
Authors: Ruether J E , Maderious A , Lavery D , Logan J , Fu S M , Chen-Kiang S
Published: September 2015

The mouse immunoglobulin heavy-chain mu constant region gene was cloned into the early region 1B of an adenovirus type 5 vector to allow reproducible kinetics of expression of the mu gene in the presence of continuous host protein synthesis after infection by the recombinant. The immunoglobulin-adenovirus recombinant is helper independent in infecting human fibroblastic and B- and T-cell lines and expresses mu in a cell-type-specific manner. By Northern blot analysis, correctly polyadenylated and spliced E1B-mu S and E1B-mu m mRNAs are found to be equally abundant at steady state in fibroblasts. In contrast, and appropriately, only E1B-mu S mRNAs accumulate in a lambda light-chain-secreting myeloma cell line. Analysis of nascent transcripts pulse labeled in isolated nuclei demonstrates equimolar polymerase loading throughout the mu region in all cell types infected by mu-Ad. Thus, correct polyadenylation and splicing of E1B-mu S and E1B-mu m in fibroblasts does not require transcription termination in the region separating the mu S and mu m polyadenylation sites. Furthermore, differential expression of mu transcripts in the background of myeloma cells is regulated at the level of RNA processing and does not require the presence of the immunoglobulin heavy-chain enhancer or promoter element.
Sequence-specific activation of transcription by adenovirus EIa products is observed in HeLa cells but not in 293 cells.
Authors: Leff T , Chambon P
Published: September 2015

The adenovirus EIa gene products activate transcription from the viral EIII and EIIaE promoters. We studied the mechanism of this stimulation by constructing a series of chimeric promoter recombinants containing the upstream regions of the EIII and EIIaE promoters linked to the TATA box-start-site regions of the viral major late and EIIa late promoters. By introducing these recombinants into HeLa cells together with recombinants producing the EIa gene products, we demonstrated that the induction of EIII and EIIaE transcription by EIa 13S and 12S mRNA products is dependent on sequences located in the upstream region (approximately -40 to -250) of these promoters. In addition, we showed that the major late and EIIa late upstream promoter regions do not contain such EIa-responsive sequence elements. In contrast, after transfection of these chimeric promoter recombinants into 293 cells (which constitutively express the EIa proteins), we found that their relative levels of transcription are similar and markedly different from those observed when they are cotransfected into HeLa cells with EIa protein-producing recombinants. We conclude that the efficiency of transcription from a given promoter in 293 cells is not necessarily related to the presence of a specific EIa-responsive element.
Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally.
Authors: Chang D D , Hixson J E , Clayton D A
Published: September 2015

Human mitochondrial DNA is transcribed from two distinct, strand-specific promoters located in the displacement loop region of the genome. The transcriptional control sequences identified by deletion mapping and site-directed mutagenesis studies span short regions surrounding the initiation sites and bear no obvious sequence homology to any nuclear or procaryotic promoters. In vitro transcription analyses also revealed several minor initiation sites that are characterized by a pyrimidine-rich region followed by a purine-rich region, a feature that is shared by the two major promoters. In this paper, we report a new class of minor promoters in human mitochondrial DNA. These minor promoters were localized to the same duplex DNA sequences that direct major transcriptional events, but they had transcriptional polarity opposite to that of the major promoters. Furthermore, nucleotide changes that affected the major form of transcription similarly affected transcription in the opposite direction. For one of these minor promoters, a corresponding in vivo RNA species initiating from the same site was identified. These observations indicate that the major transcriptional promoters in human mitochondria can function bidirectionally both in vivo and in vitro.
Binding of alpha-factor pheromone to Saccharomyces cerevisiae a cells: dissociation constant and number of binding sites.
Authors: Jenness D D , Burkholder A C , Hartwell L H
Published: September 2015

The number of alpha-factor binding sites on yeast MATa cells (8,000) and the equilibrium dissociation constant (6 X 10(-9) M) were determined from direct binding experiments. These values correct our previously reported estimates (D. D. Jennes, A. C. Burkholder, and L. H. Hartwell, Cell 35:521-529, 1983) that were based on indirect isotope dilution studies, and they lead to a revised rate constant for the association process (kon = 3 X 10(5) mol-1 s-1).
Sensitive and specific DNA probe for detection of Plasmodium falciparum.
Authors: Enea V
Published: September 2015

The isolation and some characteristics of a very sensitive DNA probe for the detection of Plasmodium falciparum are described. The probe is species specific and represents a large, albeit variable, fraction of the genome in all the strains tested. In addition to its immediate practical uses for the detection and quantitation of parasites, the probe defines an interesting family of repeated sequences.
Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis.
Authors: Hirsch J P , Henry S A
Published: September 2015

The INO1 gene of Saccharomyces cerevisiae encodes the regulated enzyme inositol-1-phosphate synthase, which catalyzes the first committed step in the synthesis of inositol-containing phospholipids. The expression of this gene was analyzed under conditions known to regulate phospholipid synthesis. RNA blot hybridization with a genomic clone for INO1 detected two RNA species of 1.8 and 0.6 kb. The abundance of the 1.8-kb RNA was greatly decreased when the cells were grown in the presence of the phospholipid precursor inositol, as was the enzyme activity of the synthase. Complementation analysis showed that this transcript encoded the INO1 gene product. The level of INO1 RNA was repressed 12-fold when the cells were grown in medium containing inositol, and it was repressed 33-fold when the cells were grown in the presence of inositol and choline together. The INO1 transcript was present at a very low level in cells containing mutations (ino2 and ino4) in regulatory genes unlinked to INO1 that result in inositol auxotrophy. The transcript was constitutively overproduced in cells containing a mutation (opi1) that causes constitutive expression of inositol-1-phosphate synthase and results in excretion of inositol. The expression of INO1 RNA was also examined in cells containing a mutation (cho2) affecting the synthesis of phosphatidylcholine. In contrast to what was observed in wild-type cells, growth of cho2 cells in medium containing inositol did not result in a significant decrease in INO1 RNA abundance. Inositol and choline together were required for repression of the INO1 transcript in these cells, providing evidence for a regulatory link between the synthesis of inositol- and choline-containing lipids. The level of the 0.6-kb RNA was affected, although to a lesser degree, by many of the same factors that influence INO1 expression.
Expression of a secreted transplantation antigen gene during murine embryogenesis.
Authors: Stein P , Barra Y , Jay G , Strickland S
Published: September 2015

We examined the midgestation mouse embryo for transcripts related to the secreted transplantation antigen Q10 and show here that this gene is transcribed in the endoderm of the visceral yolk sac. Its level of expression is highest at day 14 and then declines as development proceeds. Concurrently with the decrease in yolk sac expression, the amount of transcripts accumulating in the fetal liver increases during late embryogenesis.
Pyrimidine dimers block simian virus 40 replication forks.
Authors: Berger C A , Edenberg H J
Published: September 2015

UV light produces lesions, predominantly pyrimidine dimers, which inhibit DNA replication in mammalian cells. The mechanism of inhibition is controversial: is synthesis of a daughter strand halted at a lesion while the replication fork moves on and reinitiates downstream, or is fork progression itself blocked for some time at the site of a lesion? We directly addressed this question by using electron microscopy to examine the distances of replication forks from the origin in unirradiated and UV-irradiated simian virus 40 chromosomes. If UV lesions block replication fork progression, the forks should be asymmetrically located in a large fraction of the irradiated molecules; if replication forks move rapidly past lesions, the forks should be symmetrically located. A large fraction of the simian virus 40 replication forks in irradiated molecules were asymmetrically located, demonstrating that UV lesions present at the frequency of pyrimidine dimers block replication forks. As a mechanism for this fork blockage, we propose that polymerization of the leading strand makes a significant contribution to the energetics of fork movement, so any lesion in the template for the leading strand which blocks polymerization should also block fork movement.
Growth factor requirements of oncogene-transformed NIH 3T3 and BALB/c 3T3 cells cultured in defined media.
Authors: Zhan X , Goldfarb M
Published: September 2015

We report conditions for the efficient growth of NIH 3T3 and BALB/c 3T3 cells cultured in a defined medium supplemented with either platelet-derived growth factor (PDGF) or pituitary-derived fibroblast growth factor (FGF). The oncogenes v-mos, v-src, v-sis, and c-H-ras Val 12 can induce morphological transformation of these cells and can release them from the mitogen requirement for growth, while the oncogene v-fos cannot abrogate the PDGF-FGF requirement. The radically different behavior of normal and transformed NIH 3T3 cells in PDGF-FGF-free defined medium can form the basis of a sensitive new fibroblast transformation assay.
Coinfection with viruses carrying the v-Ha-ras and v-myc oncogenes leads to growth factor independence by an indirect mechanism.
Authors: Vogt M , Lesley J , Bogenberger J , Volkman S , Haas M
Published: September 2015

The concomitant expression of certain oncogenes can transform normal diploid rodent cells into transplantable tumorigenic cells. The mechanism by which these oncogenes collaborate is unclear. Recent findings (M. Oshimura, T. M. Gilmer, and J. C. Barrett, Nature [London] 316:636-639, 1985) raise the possibility that karyotypic changes, including monosomy for chromosome 15, are required to induce tumorigenicity in Syrian hamster embryo cells transfected in vitro with v-Ha-ras and v-myc DNAs. We studied the effect of the oncogenes v-Ha-ras and v-myc, introduced by viral infection, on murine hematopoietic cells. The induction of growth factor independence by the two oncogenes was used as an in vitro correlate of tumorigenicity. After a period of reduced growth rate reminiscent of the growth rate of cells in crisis, the doubly infected cells became growth factor independent. These cells showed a great variability in their karyotypes.
A transcriptional enhancer and an interferon-responsive sequence in major histocompatibility complex class I genes.
Authors: Vogel J , Kress M , Khoury G , Jay G
Published: September 2015

The major histocompatibility complex class I antigens play an indispensable role in cell-cell interactions. Perturbation of their expression has been shown to have deleterious physiological consequences, including the escape of transformed cells from immune detection. In an attempt to understand how class I genes are regulated, we dissected the Ld gene to identify potential control regions. By using a test vector containing the simian virus 40 early promoter placed upstream of the bacterial chloramphenicol acetyltransferase (cat) gene, we demonstrated the presence of a transcriptional enhancer within the 5'-flanking region. The sequence is functional in both orientations and has been mapped within 350 base pairs upstream of the Ld transcriptional start site. Although human adenovirus 12 can suppress endogenous class I genes, it cannot down-regulate the activity of the transiently transfected cat gene which has been placed under the control of the Ld enhancer and promoter. Our results suggested that if the human adenovirus 12-induced function regulates the expression of class I genes by a trans mechanism, then its target site must not be within 1.9 kilobases of the 5'-flanking region. Treatment of cells with interferon increases the accumulation of class I transcripts. Expression of the cat gene under the control of the Ld enhancer and promoter also can be up-regulated by interferon. Our study shows that the target sequence required for this enhancement resides, at least in part, within the same 350-base pair segment which contains the transcriptional enhancer.
Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation.
Authors: Helfman D M , Cheley S , Kuismanen E , Finn L A , Yamawaki-Kataoka Y
Published: September 2015

The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites.
Product of Saccharomyces cerevisiae nuclear gene PET494 activates translation of a specific mitochondrial mRNA.
Authors: Costanzo M C , Fox T D
Published: September 2015

The product of Saccharomyces cerevisiae nuclear gene PET494 is known to be required for a posttranscriptional step in the accumulation of one mitochondrial gene product, subunit III of cytochrome c oxidase (coxIII). Here we show that the PET494 protein probably acts in mitochondria by demonstrating that both a PET494-beta-galactosidase fusion protein and unmodified PET494 are specifically associated with mitochondria. To define the PET494 site of action, we isolated mutations that suppress a pet494 deletion. These mutations were rearrangements of the mitochondrial gene oxi2 that encodes coxIII. The suppressor oxi2 genes had acquired the 5'-flanking sequences of other mitochondrial genes and gave rise to oxi2 transcripts carrying the 5'-untranslated leaders of their mRNAs. These results demonstrate that in wild-type cells PET494 specifically promotes coxIII translation, probably by interacting with the 5'-untranslated leader of the oxi2 mRNA.
Crystallin gene expression and lentoid body formation in quail embryo neuroretina cultures transformed by the oncogenic retrovirus Mill Hill 2 or Rous sarcoma virus.
Authors: Simonneau L , Crisanti P , Lorinet A M , Alliot F , Courtois Y , Calothy G , Pessac B
Published: September 2015

The lens-specific proteins alpha and delta crystallins and lentoid bodies, structures that follow a differentiation pathway similar to that of the lens, regularly appear after 4 to 5 weeks in quail embryo neuroretina monolayer cultures. We have investigated the effects of the avian oncogenic retroviruses Mill Hill 2 and Rous sarcoma virus on this process. Quail embryo neuroretina cells transformed by Mill Hill 2 virus were established into permanent cultures that synthesized alpha and delta crystallins and contained stem cells for the production of lentoid bodies. In contrast, transformation with the Rous sarcoma virus mutant tsNY-68 blocked the appearance of mRNA crystallins, but cytoplasmic alpha and delta crystallin mRNA and alpha crystallin appeared 44 h after a shift to the nonpermissive temperature. However, delta crystallins and lentoid bodies were only present after 7 days. The crystallins of transformed quail neuroretina cultures were immunologically indistinguishable from those of quail lenses and of normal quail embryo neuroretina cultures.
Variants of vaccinia virus hemagglutinin altered in intracellular transport.
Authors: Shida H
Published: September 2015

Two classes of revertants were isolated from a vaccinia virus mutant whose hemagglutinins (HAs) accumulate on nuclear envelopes and rough endoplasmic reticulums. The HAs of one of the revertants had the same phenotype as the wild type, i.e., rapid and efficient movement to the cell surface. The HAs of the second class had biphasic transport: rapid export to the cell surface as in the wild type and slow movement to the medial cisternae of the Golgi apparatus. Biochemical and nucleotide sequence analyses showed that the HAs of all the mutants examined that have defects in transport from the rough endoplasmic reticulum to the Golgi apparatus have altered cytoplasmic domains and that the HAs of the second class of revertants lack the whole cytoplasmic domain, while the HAs of the first class of revertants have a wild-type cytoplasmic domain.
Characterization of two nonallelic pairs of late histone H2A and H2B genes of the sea urchin: differential regulation in the embryo and tissue-specific expression in the adult.
Authors: Kemler I , Busslinger M
Published: September 2015

Two nonallelic pairs of late H2A and H2B genes of the sea urchin Psammechinus miliaris were isolated on two different cosmid clones. The genes of cosmid PmL1 are separated by 11 kilobases of DNA and code for the late H2A-2 and H2B-2 variants. The genes of clone PmL2 are divergently transcribed with 1,060 base pairs of intergenic spacer DNA and code for novel variants of the H2A-2 and H2B-2 type. A comparison of the promoter sequences revealed little homology upstream of the TATA box with the exception of a 24-base-pair-long conserved sequence which is present at the same position in both late H2B promoters and part of which is identical with the "H2B-specific" 5' element. The mRNAs of the H2A and H2B genes of cosmid PmL1 reach their maximal levels early in the mesenchyme blastula embryo, whereas the transcripts of both genes of clone PmL2 accumulate maximally only later in the pluteus larva. In the adult sea urchin all four mRNAs are present in the tube foot but not in the intestine and lantern muscle. This pattern of differential expression in the embryo and tissue-specific expression in the adult suggests cell lineage-specific regulation of the late H2A-2 and H2B-2 genes. Another class of late histone genes represented by the H2A-3 and H2B-1 genes was shown to be expressed in all three adult tissues tested, whereas transcripts of the late H2A-1 genes could not be detected, suggesting that these genes are active exclusively during sea urchin development.
Mating type-like conversion promoted by the 2 micrograms circle site-specific recombinase: implications for the double-strand-gap repair model.
Authors: Jayaram M
Published: September 2015

Double-strand breaks in DNA are known to promote recombination in Saccharomyces cerevisiae. Yeast mating type switching, which is a highly efficient gene conversion event, is apparently initiated by a site-specific double-strand break. The 2 micrograms circle site-specific recombinase, FLP, has been shown to make double-strand breaks in its substrate DNA. By using a hybrid 2 micrograms circle::Tn5 plasmid, a portion of which resembles, in its DNA organization, the active (MAT) and the silent (HML) yeast mating type loci, it is shown that FLP mediates a conversion event analogous to mating type switching. Whereas the FLP site-specific recombination is not dependent on the RAD52 gene product, the FLP-induced conversion is abolished in a rad52 background. The FLP-promoted conversion in vivo can be faithfully reproduced by making a double-stranded gap in vitro in the vicinity of the FLP site and allowing the gap to be repaired in vivo.
Rat-brain Na,K-ATPase beta-chain gene: primary structure, tissue-specific expression, and amplification in ouabain-resistant HeLa C+ cells.
Authors: Mercer R W , Schneider J W , Savitz A , Emanuel J , Benz E J , Levenson R
Published: September 2015

We deduced the complete amino acid sequence of the rat brain Na,K-ATPase beta-subunit from cDNA. The rat brain beta-subunit exhibits a high degree of primary sequence and secondary structural homology with the human and Torpedo beta-subunit polypeptides. Analysis of rat tissue RNA reveals that the beta-subunit gene encodes four separate mRNA species which are expressed in a tissue-specific fashion. In ouabain-resistant HeLa C+ cells, beta-subunit DNA sequences are amplified (approximately 20-fold) and beta-subunit mRNAs are overproduced relative to levels in parental HeLa cells. These results suggest that the beta-subunit plays an important role in Na,K-ATPase structure-function and in the mechanism underlying cellular resistance to the cardiac glycosides.
Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells.
Authors: Bullock P , Miller J , Botchan M
Published: September 2015

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.
Induction and repression of the urea amidolyase gene in Saccharomyces cerevisiae.
Authors: Genbauffe F S , Cooper T G
Published: September 2015

The DUR1,2 gene from Saccharomyces cerevisiae has been isolated on recombinant plasmids along with all DNA between the DUR1,2 and MET8 loci. DUR1,2 was found to encode a 5.7-kilobase transcript, which is consistent with our earlier suggestion that the DUR1 and DUR2 loci are two domains of a single multifunctional gene. Steady-state levels of the DUR1,2 transcript responded to induction and nitrogen catabolite repression in the same way as urea amidolyase activity. dal81 mutants (grown with inducer) contained barely detectable amounts of DUR1,2 RNA, whereas dal80 mutants (grown without inducer) contained the same amount as a wild-type induced culture. These observations support our earlier hypothesis that DUR1,2 is transcriptionally regulated, with control being mediated by the DAL80 and DAL81 gene products. We cloned the DUR1,2-Oh mutation and found it to be a Ty insertion near sequences required for complementation of dur1,2 mutations. The ROAM phenotype of the DUR1,2-Oh mutation is sharply different from that of cis-dominant, DUR80 mutations, which enhance DUR1,2 expression but do not affect the normal control pattern of the gene. There is evidence that DUR80 mutations may also be Ty insertions, which generate phenotypes that are different from those in DUR1,2-Oh mutations.
Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression.
Authors: Ma H , Botstein D
Published: September 2015

Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabolite repression of three different genes in yeasts: SUC2, CYC1, and GAL10. The results indicate that hxk1 or hxk2 single null mutants can ferment fructose but that hxk1 hxk2 double mutants cannot. The hxk2 single mutant, as well as the double mutant, failed to show catabolite repression in all three systems, while the hxk1 null mutation had little or no effect on catabolite repression.
Different tissue-specific expression of the amylase gene Amy-1 in mice and rats.
Authors: Sierra F , Pittet A C , Schibler U
Published: September 2015

We cloned the rat alpha-amylase gene Amy-1 and compared its structure and expression with its mouse counterpart. The results showed that the general organization of the transcriptionally active rat Amy-1 gene was similar to that of its mouse counterpart; i.e., the rat gene also contained two independent transcriptional promoters. The distance between the two promoters in the rat gene was, however, more than double (6 kilobases) that measured in the mouse gene (2.8 kilobases). In addition, the rat genome also contained an independent, orphonlike version of the weaker Amy-1 promoter, which was transcriptionally silent. In spite of the similar overall organization of the Amy-1 genes in mouse and rat cells, an interesting difference was observed in the expression of the weak promoter in these two closely related rodents. In rats this promoter was significantly active only in liver cells, while in mice it was utilized with similar efficiencies in parotid, liver, and pancrease cells. Moreover, the transcripts produced in rat liver had a very heterogeneous population of 5' ends, located between 180 and 220 nucleotides upstream of the two homologous start sites observed for this promoter in mouse liver, even though the sequences around this region were strongly conserved between the two species.
T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.
Authors: Smale S T , Tjian R
Published: September 2015

We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication.
Accumulation of c-src mRNA is developmentally regulated in embryonic neural retina.
Authors: Vardimon L , Fox L E , Moscona A A
Published: September 2015

Accumulation of c-src mRNA gradually increased during early development of the neural retina in chicken embryos and reached a peak by days 11 to 13 of embryonic life. Thereafter, its amount declined to a low level which persisted also in adult retina. The early increase in c-src mRNA correlated inversely with the decrease in the amount of H3.2 replication histone mRNA and with the decline in the rate of cell growth. The accumulation profile of c-src mRNA corresponded to that of pp60c-src protein, suggesting that the latter is regulated at the level of transcription.
Transcriptional and posttranscriptional control of c-myc gene expression in WEHI 231 cells.
Authors: Levine R A , McCormack J E , Buckler A , Sonenshein G E
Published: September 2015

Incubation of WEHI 231 cells, derived from a murine B-cell lymphoma, with antisera directed against its surface immunoglobulin results in the inhibition of growth within 24 h. Previously, we demonstrated that this treatment selectively affects cytoplasmic levels of c-myc mRNA (J. E. McCormack, V. H. Pepe, R. B. Kent, M. Dean, A. Marshak-Rothstein, and G. E. Sonenshein, Proc. Natl. Acad. Sci. USA 81:5546-5550, 1984). An initial increase in the cytoplasmic mRNA level is followed by a precipitous drop. We now show that the early increase results from a dramatic increase in the rate of c-myc gene transcription, as well as from partial stabilization of the mRNA in the cytoplasm. The later decrease results from a shutdown in transcription of the c-myc gene and a return to the normal lability of the cytoplasmic c-myc mRNA. Treatment with phorbol ester, like treatment with anti-immunoglobulin sera, inhibited WEHI 231 cell growth and caused similar changes in cytoplasmic c-myc mRNA levels, which can also be related to alterations in c-myc gene transcription. These results indicate that the control of c-myc gene expression in B cells is effected through regulation at multiple levels.
Chromatin structure of a P-element-transduced hsp-28 gene in Drosophila melanogaster.
Authors: Eissenberg J C , Elgin S C
Published: September 2015

The Drosophila hsp-28 gene was heat inducible when transduced to novel chromosomal sites even when no direct selection for transduced gene expression was imposed. The pattern of DNase I-hypersensitive sites 5' to the wild type and transduced copy of hsp-28 was similar. In addition, DNase I-hypersensitive sites occurred within the P-element sequences flanking transduced loci.
Dissociated expression of c-myc and a fos-related competence gene during cardiac myogenesis.
Authors: Schneider M D , Payne P A , Ueno H , Perryman M B , Roberts R
Published: September 2015

Cardiac myocytes irreversibly lose their proliferative capacity soon after birth, and cardiac DNA synthesis becomes uncoupled from mitotic division. Therefore, we examined cardiac muscle for developmental down regulation of inducible proto-oncogenes associated with cell proliferation. c-myc mRNA decreased continuously from day 13 of embryonic development and was dissociated from expression of the fos-related gene r-fos, which decreased precipitously between days 3 and 7 after birth.
Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein.
Authors: Kato J Y , Takeya T , Grandori C , Iba H , Levy J B , Hanafusa H
Published: September 2015

We have previously shown that Rous sarcoma virus variants that carry the cellular homolog (c-src) of the viral src gene (v-src) do not transform chicken embryo fibroblasts. We also have shown that replacement of sequences upstream or downstream from the BglI site of the cellular src gene with the corresponding regions of v-src restored transforming activity to the hybrid genes. Since there are only six amino acid changes between p60c-src and p60v-src within the sequences upstream from BglI, we constructed chimeric molecules involving v-src and c-src to determine the effect of each amino acid substitution on the biological activities of the gene product. We found that the change from Thr to Ile at position 338 or the replacement of a fragment of c-src containing Gly-63, Arg-95, and Thr-96 with a corresponding fragment of v-src containing Asp-63, Trp-95, and Ile-96 converted p60c-src into a transforming protein by the criteria of focus formation, anchorage-independent growth, and tumor formation in newborn chickens. These mutations also resulted in elevation of the protein kinase activity of p60c-src.
Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA.
Authors: Peterson C L , Orth K , Calame K L
Published: September 2015

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.
Expression of a human cytomegalovirus late gene is posttranscriptionally regulated by a 3'-end-processing event occurring exclusively late after infection.
Authors: Goins W F , Stinski M F
Published: September 2015

A phenomenon of posttranscriptional regulation has been previously identified in cytomegalovirus-infected human fibroblast cells (Wathen and Stinski, J. Virol. 41:462, 1982). A region typifying this phenomenon has been located within the large unique component of the viral genome (map units 0.408 to 0.423). Even though this transcriptional unit was highly transcribed at early times after infection, mRNAs from this region were only detectable on the polyribosomes after viral DNA replication. Thus, this region is believed to code for a late gene. Single-strand-specific nuclease mapping experiments of viral transcripts established that the transcriptional initiation sites and the 5' ends of a downstream exon were identical at early and late times. However, the late transcripts differed from the early transcripts by the processing of the 3' end of the viral RNAs. This involved either the removal of a distinct region of the transcript by the selection of an upstream cleavage and polyadenylation site or the differential splicing of the RNA molecule. The upstream cleavage and polyadenylation site was identified by nuclease mapping analyses and DNA sequencing. The 3'-end processing of these transcripts is necessary for the detection of these viral RNAs within the cytoplasm of the infected cell. We propose that human cytomegalovirus either codes for a factor(s) that is involved in the 3'-end-processing event at late times after infection or stimulates the synthesis of a host cell factor(s) involved in this complex regulatory event. This level of regulation may have an influence on the types of cells that permit productive cytomegalovirus replication.
Multiple protein-binding sites in the 5'-flanking region regulate c-fos expression.
Authors: Gilman M Z , Wilson R N , Weinberg R A
Published: September 2015

We tested sequences flanking the mouse c-fos gene for the ability to form specific DNA-protein complexes with factors present in crude nuclear extracts prepared from mammalian cells. Three such complexes were detected. One complex formed in a region necessary for the induction of c-fos expression by serum growth factors. Two additional complexes formed at sequences that contribute to basal c-fos promoter activity in vivo. These complexes represent three novel sequence-specific DNA-binding activities which appear to participate in the regulation of c-fos transcription.
Modulation of novel-length DOPA decarboxylase transcripts by 20-OH-ecdysone in a Drosophila melanogaster Kc cell subline.
Authors: Swiderski R E , O'Connor J D
Published: September 2015

The induction of DOPA decarboxylase (DDC) activity by 20-OH-ecdysone (20-OHE) in a subline of Drosophila melanogaster Kc cells was investigated. Cells cultured in the continuous presence of the steroid hormone exhibited a 96-h temporal lag prior to a peak of DDC enzyme activity while arrested in the G2 phase of the cell cycle. The concentration of Ddc RNA increased sixfold between 72 and 96 h after initial exposure to hormone. Similarly, this increase was correlated temporally with a 26-fold increase in DDC enzyme activity. The Kc Ddc primary transcript, processing intermediate, and mature mRNA all were approximately 500 nucleotides longer than the corresponding transcripts observed for newly eclosed adult D. melanogaster. In vitro translation of poly(A)+ RNA from Kc cells resulted in an immunoprecipitable polypeptide which exhibited similar mobility on sodium dodecyl sulfate gels to that of DDC synthesized in vitro by larval epidermal poly(A)+ RNA.
Human proto-oncogene N-myc encodes nuclear proteins that bind DNA.
Authors: Ramsay G , Stanton L , Schwab M , Bishop J M
Published: September 2015

N-myc is a gene whose amplification has been implicated in the genesis of several malignant human tumors. We have identified two proteins with molecular weights of 65,000 and 67,000 encoded by N-myc. The abundance of these proteins in tumor cells was consonant with the extent of amplification of N-myc. The two proteins apparently arose from the same mRNA, were phosphorylated, were exceptionally unstable, were located in the nucleus of cells, and bound to both single- and double-stranded DNA. These properties suggest that the products of N-myc and of the related proto-oncogene c-myc may have similar biochemical functions and that N-myc may be a regulatory gene. Our findings sustain the view that inordinate expression of N-myc may contribute to the genesis of several different human tumors.
Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src.
Authors: Cooper J A , King C S
Published: September 2015

Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.
Myoglobin expression: early induction and subsequent modulation of myoglobin and myoglobin mRNA during myogenesis.
Authors: Weller P A , Price M , Isenberg H , Edwards Y H , Jeffreys A J
Published: September 2015

We showed that myoglobin gene transcription and the appearance of myoglobin occur very early in myogenesis, in both humans and mice. In contrast to the contractile protein genes, there is a subsequent increase of 50- to 100-fold in myoglobin mRNA and protein levels during later muscle development. Myoglobin and myoglobin mRNA are present at elevated levels in fetal heart and are also detectable at low levels in adult smooth muscle. The absolute level of myoglobin mRNA in highly myoglobinized seal muscle is very high [2.8% of the total population of poly(A)+ RNAs]. Levels of myoglobin in seal skeletal muscle and in various human muscle types appear to be determined by the size of the myoglobin mRNA pool. In contrast, low levels of myoglobin in mouse skeletal muscle are not apparently correlated with low levels of myoglobin mRNA. As expected from the early appearance of myoglobin mRNA in embryonic skeletal muscle, both rat and mouse embryonic myoblasts accumulate myoglobin mRNA on fusion and differentiation in vitro.
Two distant and precisely positioned domains promote transcription of Xenopus laevis rRNA genes: analysis with linker-scanning mutants.
Authors: Windle J J , Sollner-Webb B
Published: September 2015

To examine the internal organization of the promoter of the Xenopus laevis rRNA gene, we constructed a series of linker-scanning mutants that traverse the rDNA initiation region. The mutant genes, which have 3 to 11 clustered base substitutions set within an otherwise unaltered rDNA promoter sequence, were injected into Xenopus oocyte nuclei, and their transcriptional capacity was assessed by S1 nuclease analysis of the resultant RNA. The data demonstrate that there are two essential promoter domains, the distal boundaries of which coincide with the promoter boundaries established previously by analysis of 5' and 3' deletion mutants. The upstream promoter domain is relatively small and extends from residues ca. -140 to -128. The downstream domain is considerably larger, encompassing residues ca. -36 to +10, and exactly corresponds in both size and position to the mammalian minimal promoter region. The Xenopus rDNA sequence between these two essential domains has a much smaller effect on the level of transcriptional initiation. In light of the fact that a large portion of this intervening region consists of a segment (residues -114 to -72) that is duplicated many times in the upstream spacer to form an rDNA enhancer sequence, it is noteworthy that a "-115/-77 linker scanner," in which virtually this entire segment is replaced by a polylinker sequence, has full promoter activity in the injected Xenopus borealis oocytes. Analysis of a parallel series of spacing change linker-scanning mutants revealed the unexpected result that the relative positions of the upstream and downstream promoter domains are very critical: all spacing alterations of more than 2 base pairs within this 100-base-pair region virtually abolish promoter activity. We conclude that the factors that bind to these two distant promoter domains must interact in a very precise stereospecific manner.