bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells.
Authors: Brugge J S , Jarosik G , Andersen J , Queral-Lustig A , Fedor-Chaiken M , Broach J R
Published: September 2015

The Rous sarcoma virus (RSV) pp60v-src protein was expressed in Saccharomyces cerevisiae cells either from a plasmid vector carrying the v-src gene or in yeast cells containing a single-copy v-src gene chromosomally integrated. In both yeast strains, v-src gene transcription is regulated by the galactose-inducible GAL10 promoter. Growth in galactose-containing medium resulted in constitutive expression of pp60v-src in the integrated strain and transient expression of higher levels of pp60v-src in the plasmid-bearing strain. The concentration of pp60v-src in the plasmid-bearing strain at its peak of expression was approximately threefold lower than that found in RSV-transformed mammalian cells. pp60v-src synthesized in yeast cells was phosphorylated in vivo on sites within the amino and carboxyl halves of the molecule. In immune complex kinase assays, the yeast pp60v-src was autophosphorylated on tyrosine and was able to phosphorylate exogenous substrates such as casein and enolase. The specific activity of pp60v-src synthesized in yeast cells was approximately 5- to 10-fold higher than that made in mammalian cells. Induction of pp60v-src caused the death of the plasmid-bearing yeast strain and transient inhibition of growth of the single-copy strain. Concomitantly, this induction resulted in high levels of tyrosine phosphorylation of yeast cell proteins. This indicates that pp60v-src functions as a tyrosine-specific phosphotransferase in yeast cells and suggests that hyperphosphorylation of yeast proteins is inimical to cell growth.
Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions.
Authors: Brouillette S , Chartrand P
Published: September 2015

We present an intermolecular recombination assay for mammalian cells that does not involve the reconstitution of a selectable marker. It is based on the generation of a shuttle vector by recombination between a bacterial and a mammalian vector. The recombinants can thus be amplified in mammalian cells, isolated by plasmid rescue in an Escherichia coli RecA- host, and identified by in situ hybridization, by using mammalian vector sequences as probes. Since both parental molecules can share defined lengths of homology, this assay permits a direct comparison between homologous and nonhomologous intermolecular recombination. Our results indicate that the dominant intermolecular recombination mechanism is a nonhomologous one. The relative frequency of homologous to nonhomologous recombination was influenced by the length of shared homology between parental molecules and the replicative state of the parental molecules, but not by the introduction of double-strand breaks per se. Finally, almost all of the recombinants with a homologous junction did not have the reciprocal homologous junction but instead had a nonhomologous one. We propose a model to account for the generation of these recombinants.
Identification of a human gene (HCK) that encodes a protein-tyrosine kinase and is expressed in hemopoietic cells.
Authors: Quintrell N , Lebo R , Varmus H , Bishop J M , Pettenati M J , Le Beau M M , Diaz M O , Rowley J D
Published: September 2015

We have isolated cDNAs representing a previously unrecognized human gene that apparently encodes a protein-tyrosine kinase. We have designated the gene as HCK (hemopoietic cell kinase) because its expression is prominent in the lymphoid and myeloid lineages of hemopoiesis. Expression in granulocytic and monocytic leukemia cells increases after the cells have been induced to differentiate. The 57-kilodalton protein encoded by HCK resembles the product of the proto-oncogene c-src and is therefore likely to be a peripheral membrane protein. HCK is located on human chromosome 20 at bands q11-12, a region that is affected by interstitial deletions in some acute myeloid leukemias and myeloproliferative disorders. Our findings add to the diversity of protein-tyrosine kinases that may serve specialized functions in hemopoietic cells, and they raise the possibility that damage to HCK may contribute to the pathogenesis of some human leukemias.
Regulatory function of the Saccharomyces cerevisiae RAS C-terminus.
Authors: Marshall M S , Gibbs J B , Scolnick E M , Sigal I S
Published: September 2015

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.
Expression of rat transforming growth factor alpha mRNA during development occurs predominantly in the maternal decidua.
Authors: Han V K , Hunter E S , Pratt R M , Zendegui J G , Lee D C
Published: September 2015

Previous studies have shown that transforming growth factor alpha is expressed during rodent development. To establish the site(s) of transforming growth factor alpha mRNA expression during rat embryogensis, we performed in situ hybridization and Northern blot analyses on samples of embryonic and maternal tissues at various gestational ages. Our results indicate that the high levels of transforming growth factor alpha mRNA that are observed during early development are the result of expression in the maternal decidua and not in the embryo. Decidual expression appears to be induced after implantation, peaks at day 8, and then slowly declines through day 15 at which time the decidua is being resorbed. Expression of transforming growth factor alpha mRNA is highest in that region of the decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and other maternal tissues. The developmentally regulated expression of transforming growth factor alpha mRNA in the decidua, together with the presence of epidermal growth factor receptors in this tissue, suggests that transforming growth factor alpha stimulates proliferation locally through an autocrine mechanism. Since epidermal growth factor receptors are present in the embryo and placenta, transforming growth factor alpha produced in the decidua may also act on these tissues through paracrine or endocrine mechanisms.
The ras-like yeast YPT1 gene is itself essential for growth, sporulation, and starvation response.
Authors: Segev N , Botstein D
Published: September 2015

The Saccharomyces cerevisiae gene YPT1 encodes a protein that exhibits significant homology to the mammalian ras proteins. Using gene disruption techniques, we have shown that the intact YPT1 gene is required for spore viability. Lethality caused by loss of YPT1 function, unlike that caused by loss of the yeast ras homologs RAS1 and RAS2 function, is not suppressed by the bcy1 mutation, suggesting that YPT1 does not act through the adenylate cyclase regulatory system. A cold-sensitive allele, ypt1-1, was constructed. At the nonpermissive temperature, mutants died, exhibiting aberrant nuclear morphology, as well as abnormal distribution of actin and tubulin. The mutant cells died without exhibiting classical cell-cycle-specific arrest; nevertheless, examination of cellular DNA content suggests that the YPT1 function is required, particularly after S phase. Cells carrying the ypt1-1 mutation died upon nitrogen starvation even at a temperature permissive for growth; diploid cells homozygous for ypt1-1 did not sporulate. The YPT1 gene is thus involved in nutritional regulation of the cell cycle as well as in normal progression through the mitotic cell cycle.
Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes.
Authors: Rettenmier C W , Roussel M F , Ashmun R A , Ralph P , Price K , Sherr C J
Published: September 2015

NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions.
Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.
Authors: Heard J M , Herbomel P , Ott M O , Mottura-Rollier A , Weiss M , Yaniv M
Published: September 2015

The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription.
Pentapeptide nuclear localization signal in adenovirus E1a.
Authors: Lyons R H , Ferguson B Q , Rosenberg M
Published: September 2015

The adenovirus E1a gene products are nuclear proteins important in transcriptional control of viral functions during infection. By producing normal E1a proteins and derivatives of E1a in bacteria and microinjecting these proteins into cultured cells, we were able to examine their ability to localize to the nucleus. We showed that a short peptide sequence at the carboxyl terminus of E1a is necessary for the rapid (30-min) nuclear localization of that protein. Additionally, we showed that just the last five amino acids of E1a are sufficient to direct nuclear accumulation of a heterologous protein, Escherichia coli galactokinase, with the same kinetics as native E1a. The mechanism by which this pentamer mediates rapid nuclear localization was examined by testing the ability of a galactokinase derivative which has no signal pentamer to exit the nucleus, as well as to enter it. Because neither free entry nor exit was detected, the effect of the signal is unlikely to be through increased nuclear retention of freely diffusible proteins but rather by enhancement of entry into the nucleus.
Expression of transfected mutant beta-actin genes: alterations of cell morphology and evidence for autoregulation in actin pools.
Authors: Leavitt J , Ng S Y , Aebi U , Varma M , Latter G , Burbeck S , Kedes L , Gunning P
Published: September 2015

Two different mutant human beta-actin genes have been introduced into normal diploid human (KD) fibroblasts and their immortalized derivative cell line, HuT-12, to assess the impact of an abnormal cytoskeletal protein on cellular phenotypes such as morphology, growth characteristics, and properties relating to the neoplastic phenotype. A mutant beta-actin containing a single mutation (Gly-244----Asp-244) was stable and was incorporated into cytoskeletal stress fibers. Transfected KD cells which expressed the stable mutant beta-actin in excess of normal beta-actin were morphologically altered. In contrast, a second mutant beta-actin gene containing two additional mutations (Gly-36----Glu-36 and Glu-83----Asp-83, as well as Gly-244----Asp-244) did not alter cell morphology when expressed at high levels in transfected cells, but the protein was labile and did not accumulate in stress fibers. In both KD and HuT-12 cells, endogenous beta- and gamma-actin decreased in response to high-level expression of the stable mutant beta-actin, in a manner consistent with autoregulatory feedback of actin concentrations. Since the percent decreases in the endogenous beta- and gamma-actins were equal, the ratio of net beta-actin (mutant plus normal) to gamma-actin was significantly increased in the transfected cells. Antisera capable of distinguishing the mutant from the normal epitope revealed that the mutant beta-actin accumulated in stress fibers but did not participate in the formation of the actin filament-rich perinuclear network. These observations suggest that different intracellular locations differentially incorporate actin into cytoskeletal microfilaments. The dramatic impact on cell morphology and on beta-actin/gamma-actin ratios in the transfected diploid KD cells may be related to the acquisition of some of the characteristics of cells that underwent the neoplastic transformation event that originally led to the appearance of the beta-actin mutations.
Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae.
Authors: Percival-Smith A , Segall J
Published: September 2015

We found that the introduction into a yeast cell of a high-copy-number plasmid containing the 5' end of the SPS2 gene, a sporulation-specific gene of Saccharomyces cerevisiae, led to a reduction in the efficiency of spore formation. The plasmid pAP290, which contains the sequence from -138 to +152 of the SPS2 gene, caused a fivefold reduction in spore formation; the presence of the plasmid had no effect on transcription of the chromosomal SPS2 gene. A plasmid containing only the sequence upstream of the TATA box of the SPS2 gene (-350 to -68) was unable to inhibit the completion of sporulation, whereas the downstream sequence, from -70 to +404, although unable by itself to inhibit sporulation, could do so when provided with an upstream fragment containing the CYC1 upstream activation sequence. Deletion of 22 base pairs from pAP290, which introduced a frameshift after codon 17 of the SPS2 gene and reduced the open reading frame to 26 amino acids, generated a plasmid (pAP290 delta Pst) which could no longer inhibit sporulation. The SPS2 inserts of pAP290 and pAP290 delta Pst were found to direct equivalent levels of sporulation-specific transcription. We conclude from these results that the presence of both the SPS2 promoter (or a substitute promoter) and the initial coding sequence of the SPS2 gene is required in the high-copy-number plasmid to generate the asporogenous phenotype. We speculate that the accumulation of a protein containing the amino-terminal portion of the SPS2 gene product, synthesized from the transcripts of the truncated plasmid-borne copies of the SPS2 gene, prevents ascus formation.
Evolutionary changes of sequences and factors that direct transcription termination of human and mouse ribsomal genes.
Authors: Bartsch I , Schoneberg C , Grummt I
Published: September 2015

We have analyzed the sequences required for termination of human rDNA transcription. The human ribosomal transcription unit is shown to extend about 350 nucleotides into the 3'-terminal spacer and ends immediately upstream of a region with a distinct sequence heterogeneity. This heterogeneous region contains a cluster of conserved 10-base pair sequence elements which exert a striking homology to the proximal part of the 18-base pair murine rDNA transcription termination signal sequence, termed SalI box. Exonuclease III protection assays and in vitro transcription experiments with both homologous and heterologous human-mouse minigene constructs, and extracts from HeLa or Ehrlich ascites cells, reveal a functional analogy of the human sequence to the mouse SalI box. It mediates binding of a nuclear protein which functions as a transcription termination factor. The murine signal sequence is recognized by the human factor but not vice versa. The different sequence specificities and electrophoretic properties of the functionally equivalent protein factors suggest that a molecular coevolution has taken place between the termination signal sequences and the genes coding for the termination factors.
In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA.
Authors: Gampel A , Tzagoloff A
Published: September 2015

A region of the Saccharomyces cerevisiae mitochondrial cytochrome b gene encompassing the entire terminal intron plus flanking exonic sequences has been cloned in an SP6 vector. A runoff transcript prepared from this construct as well as the native cytochrome b pre-mRNA containing the terminal intervening sequence were found to act as substrates for the autocatalytic excision of the intervening sequence in vitro. This reaction proceeds under conditions previously shown by Cech and co-workers to promote protein-independent excision of the Tetrahymena rRNA intervening sequence. The 5' and 3' termini of the excised intervening sequence, determined by S1 nuclease mapping and sequence analysis, are consistent with the known sequence of the cytochrome b mRNA. The same region of the cytochrome b gene from a yeast mutant, defective in splicing due to a mutation in a critical sequence inside the terminal intron, has also been cloned in an SP6 vector. The mutant transcript fails to self-splice in the in vitro assay. These observations provide strong presumptive evidence that in vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA occurs by an autocatalytic mechanism analogous to that shown for other group I introns. In vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA, however, exhibits complete dependence on a protein factor previously shown to be encoded by the nuclear gene CBP2.
The small nuclear ribonucleoprotein SS-B/La binds RNA with a conserved protease-resistant domain of 28 kilodaltons.
Authors: Chan E K , Tan E M
Published: September 2015

SS-B/La is a nuclear protein of 48 kilodaltons with two structural domains of Mr 28,000 and Mr 23,000 generated by proteolytic cleavage. UV irradiation was used to cross-link preexisting intracellular La-RNA complexes. Subsequent protease digestion and diagonal gel electrophoresis showed that the RNA-binding site resided in the nonphosphorylated, methionine-rich 28-kilodalton domain.
Human HER2 (neu) promoter: evidence for multiple mechanisms for transcriptional initiation.
Authors: Tal M , King C R , Kraus M H , Ullrich A , Schlessinger J , Givol D
Published: September 2015

We localized the 5' region of the human gene HER2 in a cloned fragment of genomic DNA. This clone contained exons 1 to 4 of HER2, spanning the coding sequence for the first 191 amino acids. The promoter region of HER2 was identified upstream to exon 1 by nuclease S1 mapping and by a functional assay in which the promoter region drives the expression of a chloramphenicol acetyltransferase gene. The HER2 promoter is different from the promoter of the epidermal growth factor receptor gene (HER1), and the GC boxes which are typical of the promoter of the epidermal growth factor receptor gene are absent from the HER2 promoter. One major and two minor RNA start sites located at nucleotides 178, 244, and 257 upstream to the initiator ATG were identified. The first one is 21 and 70 base pairs downstream from typical TATAA and CAAT boxes, respectively. This indicates that transcription of HER2/neu can be regulated by a mechanism involving a TATA box, as well as by other unidentified regulatory elements.
Transient expression of foreign genes in lymphoid cells is enhanced by phorbol ester.
Authors: Holbrook N J , Luethy J D , Kin J
Published: September 2015

In lymphoid cells and an erythroleukemia cell line, phorbol myristate acetate significantly enhanced (up to 90-fold) expression of chloramphenicol acetyltransferase driven by four unrelated viral transcriptional units. The results suggest that phorbol myristate acetate acts via a general mechanism to enhance gene expression in these cells and provides a convenient means for increasing transient expression of foreign DNA.
Binding of a cellular protein to the gibbon ape leukemia virus enhancer.
Authors: Quinn J P , Holbrook N , Levens D
Published: September 2015

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.
Aplysia californica neurons express microinjected neuropeptide genes.
Authors: DesGroseillers L , Cowan D , Miles M , Sweet A , Scheller R H
Published: September 2015

Neuropeptide genes are expressed in specific subsets of large polyploid neurons in Aplysia californica. We have defined the transcription initiation sites of three of these neuropeptide genes (the R14, L11, and ELH genes) and determined the nucleotide sequence of the promoter regions. The genes contain the usual eucaryotic promoter signals as well as other structures of potential regulatory importance, including inverted and direct repeats. The L11 and ELH genes, which are otherwise unrelated, have homology in the promoter regions, while the R14 promoter was distinct. When cloned plasmids were microinjected into Aplysia neurons in organ culture, transitions between supercoiled, relaxed circular, and linear DNAs occurred along with ligation into high-molecular-weight species. About 20% of the microinjected neurons expressed the genes. The promoter region of the R14 gene functioned in expression of the microinjected DNA in all cells studied. When both additional 5' and 3' sequences were included, the gene was specifically expressed only in R14, suggesting that the specificity of expression is generated by a multicomponent repression system. Finally, the R14 peptide could be expressed in L11, demonstrating that it is possible to alter the transmitter phenotype of these neurons by introduction of cloned genes.
Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes.
Authors: Lugo T G , Handelin B , Killary A M , Housman D E , Fournier R E
Published: September 2015

We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcell fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.
Molecular cloning of gene sequences regulated by tumor promoters and mitogens through protein kinase C.
Authors: Johnson M D , Housey G M , Kirschmeier P T , Weinstein I B
Published: September 2015

cDNA clones representing genes whose expression is modulated by treatment with mitogens and tumor promoters were isolated and characterized. TPA-S1 corresponds to an mRNA species whose abundance was increased markedly within 1 h of exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), and TPA-R1 represents an mRNA that was decreased in TPA-treated cells. The induction of TPA-S1 was blocked by actinomycin D but was not affected by cycloheximide, and it was specific for phorbol esters with tumor-promoting activity. The role of protein kinase C in the induction of TPA-S1 is supported by the following lines of evidence. (i) Agents that activated protein kinase C (TPA, platelet-derived growth factor, and diacylglycerol) also increased TPA-S1 mRNA levels. (ii) A potent PKC inhibitor blocked the induction of TPA-S1. (iii) Down-regulation of PKC activity, by treatment of cells with TPA for 24 h, resulted in a loss of responsiveness to TPA-S1 induction by subsequent TPA treatment. DNA sequence analysis of TPA-S1 predicts a cysteine-rich, secreted protein with a molecular weight of 22.6 X 10(3) that exhibits homology with sequences representing a protein with human erythroid-potentiating activity and protease inhibitory activity.
Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines.
Authors: Hinds P W , Finlay C A , Frey A B , Levine A J
Published: September 2015

A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.
U1 small nuclear ribonucleoproteins are required early during spliceosome assembly.
Authors: Zillmann M , Rose S D , Berget S M
Published: September 2015

U1 small nuclear ribonucleoproteins (snRNPs) are required for in vitro splicing of pre-mRNA. Sequences within U1 RNA hybridize to, and thus recognize, 5' splice junctions. We have investigated the mechanism of association of U1 snRNPs with the spliceosome. U1-specific antibodies detected U1 association with precursor RNA early during assembly. Removal of the 5' terminal sequences of U1 RNA by oligo-directed cleavage or removal of U1 snRNPs by immunoprecipitation prior to the addition of precursor RNA depressed the association of all snRNPs with precursor RNA as detected by immunoprecipitation of splicing complexes by either Sm or U1-specific antibodies. Assembly of the spliceosome as monitored by gel electrophoresis was also depressed after cleavage of U1 RNA. The dependency of Sm precipitability of precursor RNA upon the presence of U1 snRNPs suggests that U1 snRNPs participate in the early recognition of substrate RNAs by U2 to U6 snRNPs. Although removal of the 5'-terminal sequences of U1 depressed U1 snRNP association with precursor RNA, it did not eliminate it, suggesting semistable association of U1 snRNPs with the assembling spliceosome in the absence of U1 RNA hybridization. This association was not dependent upon 5' splice junction sequences but was dependent upon 3' intronic sequences, indicating that U1 snRNPs interact with factors recognizing 3' intronic sequences. Mutual dependence of 5' and 3' recognition factors suggests significant snRNP-snRNP communication during early assembly.
Selective induction of human heat shock gene transcription by the adenovirus E1A gene products, including the 12S E1A product.
Authors: Simon M C , Kitchener K , Kao H T , Hickey E , Weber L , Voellmy R , Heintz N , Nevins J R
Published: September 2015

We have previously shown that the human 70-kilodalton heat shock protein gene (hsp70) is induced by the adenovirus E1A gene product and during the S-G2 phase of the cell cycle. In this study, we investigated the effect of E1A on the expression of other human hsp genes. A gene encoding one form of the hsp89 protein (hsp89 alpha) was activated during an adenovirus infection with kinetics similar to those of activation of hsp70. The induction required a functional E1A gene. However, the hsp89 transcript was not cell cycle regulated. Genes encoding another form of hsp89 and the hsp27 protein were not induced by E1A or during the cell cycle. Further examination of hsp70 expression revealed a greater complexity than previously seen. S1 nuclease analysis using an hsp70 cDNA as well as a distinct hsp70 genomic clone demonstrated three related hsp70 transcripts; two were induced by E1A, and one was not. Both of the E1A-inducible genes were regulated during the cell cycle. All three were induced by heat shock. These results suggest common aspects of control among certain members of this family of cellular genes distinct from heat shock control. Finally, using viruses that express the individual E1A proteins, we found that the hsp70 gene is induced by the 12S and the 13S E1A products. The efficiency of induction by the 12S product was somewhat less than that by the 13S product but only by a factor of less than 2. This is in contrast to the induction of early viral genes, for which the 13S product is considerably more efficient than the 12S product.
Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.
Authors: Hoekema A , Kastelein R A , Vasser M , de Boer H A
Published: September 2015

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.
Identification of the principal promoter sequence of the c-H-ras transforming oncogene: deletion analysis of the 5'-flanking region by focus formation assay.
Authors: Honkawa H , Masahashi W , Hashimoto S , Hashimoto-Gotoh T
Published: September 2015

A number of deletion mutants were isolated, including 5', 3', and internal deletions in the 5'-flanking region of the human cellular oncogene related to the Harvey sarcoma virus (c-H-ras), and their transforming activities were examined in NIH 3T3 cells. DNA sequences which could not be detected without losing transforming activity were localized to a relatively short stretch upstream of the region which showed homology to the 5'-flanking region of v-H-ras oncogene. S1 nuclease analysis indicated that there were two clusters of mRNA start sites at positions that were about 1,371 and 1,298 base pairs upstream of the first coding ATG. The minimum region required for promoter function was estimated to be a 51-base-pair-long (or less) DNA segment. The promoter was GC rich (78%) and did not contain the consensus sequences that are usually observed in PolII-directed promoters but contained a GC box within which one of the mRNA start sites was included. In addition, two sets of positive and negative elements seemed to be located between the promoter and the protein-coding region, which appeared to influence positively and negatively, respectively, the efficiency of transformation with the c-H-ras oncogene.
The rat elastase I regulatory element is an enhancer that directs correct cell specificity and developmental onset of expression in transgenic mice.
Authors: Hammer R E , Swift G H , Ornitz D M , Quaife C J , Palmiter R D , Brinster R L , MacDonald R J
Published: September 2015

A total of 134 base pairs of the 5' flanking sequence of the elastase I gene is sufficient and necessary to direct expression of the passive human growth hormone gene (hGH) to the exocrine pancreas. We demonstrate that this elastase I regulatory region contains a transcriptional enhancer which directs acinar cell-specific expression in transgenic animals. The elastase I enhancer specifies correct expression of the linked hGH gene in an orientation- and position-independent manner and can activate a heterologous promoter. The enhancer also directs the appropriate temporal activation of the hGH gene in the developing pancreas. Transcription is initiated correctly for the elastase I or hGH promoter, and the transcripts are correctly processed regardless of the enhancer position within or outside the fusion gene. The elastase I enhancer generates coincident DNase I-hypersensitive sites in pancreatic chromatin when moved 3 kilobases upstream or within the first intron of the hGH gene and when associated with the hGH promoter.
Changes in repeat number, sequence, and reading frame in S-antigen genes of Plasmodium falciparum.
Authors: Saint R B , Coppel R L , Cowman A F , Brown G V , Shi P T , Barzaga N , Kemp D J , Anders R F
Published: September 2015

The S antigens from different isolates of Plasmodium falciparum exhibit extensive size, charge, and serological diversity. We show here that the S-antigen genes behave as multiple alleles of a single locus. The size heterogeneity results from different numbers, lengths, and/or sequences of tandem repeat units encoded within the S-antigen genes. Two genes studied here encode antigenically different S antigens but nevertheless have closely related tandem repeat sequences. We show that antigenic differences can arise because repeats are translated in different reading frames.
Differential promoter utilization by the c-myc gene in mitogen- and interleukin-2-stimulated human lymphocytes.
Authors: Broome H E , Reed J C , Godillot E P , Hoover R G
Published: September 2015

Transcription of the c-myc gene is initiated from two principal promoters, P1 and P2. We demonstrate here that a shift in promoter utilization occurred with time in human peripheral blood mononuclear cells (PBMC) that had been stimulated to proliferate. The P1/P2 ratio reached a maximum of approximately 1.3 at 4 h after phytohemagglutinin stimulation and a minimum of 0.31 at 48 h. Actinomycin decay experiments demonstrated that both P1 and P2 transcripts had similar half-lives at early and late times after mitogen stimulation, indicating that the shift in promoter utilization was probably not posttranscriptionally regulated. Addition of interleukin-2 to previously activated PBMC increased c-myc mRNA, but unlike increases after mitogen stimulation, the P1/P2 ratio stayed less than 0.5. Our findings demonstrated that there was a difference between mitogen- and interleukin-2-stimulated increases in c-myc RNA in PBMC.
Different early-signaling pathways coupled to transcriptional and posttranscriptional regulation of gene expression during mitogenic activation of T lymphocytes.
Authors: White M W , Oberhauser A K , Kuepfer C A , Morris D R
Published: September 2015

Two categories of mitogen-induced mRNAs were defined in T lymphocytes. The type 1 messages (represented by c-myc) were regulated transcriptionally, and their expression seemed to be calmodulin dependent. The type 2 messages (ornithine decarboxylase, actin, and alpha-tubulin) were regulated posttranscriptionally through activation of protein kinase C.
In situ protein-DNA interactions at a dioxin-responsive enhancer associated with the cytochrome P1-450 gene.
Authors: Durrin L K , Whitlock J P
Published: September 2015

We used an in situ exonuclease III protection technique (C. Wu, Nature [London] 309:229, 1984) to analyze protein-DNA interactions at a dioxin-responsive enhancer. Our results imply that the 2,3,7,8-tetrachlorodibenzo-p-dioxin-receptor complex interacts with the dioxin-responsive enhancer to activate transcription of the cytochrome P1-450 gene.
Transient correction of genetic defects in cultured animal cells by introduction of functional proteins.
Authors: Ortiz D , Baldwin M M , Lucas J J
Published: September 2015

Material was introduced into cultures of cells by using the method of scrape loading, in which cells are simply rubbed from the surface of a plastic tissue culture dish by a rubber-tipped rod in the presence of a macromolecule of interest. The volume of solution introduced into cells was comparable to that generally injected in the direct microinjection method with glass capillaries, that is, about 50 to 100 fl per cell. Genetic defects (lack of hypoxanthine-guanine phosphoribosyltransferase and thymidine kinase) in several cell lines were transiently corrected by scraping the cells in the presence of crude cell extracts prepared from wild-type cells.
Adenovirus E1A requires synthesis of a cellular protein to establish a stable transcription complex in injected Xenopus laevis oocytes.
Authors: Richter J D , Hurst H C , Jones N C
Published: September 2015

The Escherichia coli-expressed adenovirus E1A 13S mRNA product injected into Xenopus oocytes was active, as assessed by its ability to stimulate the transcription of an injected gene which is normally responsive to E1A in mammalian cells. In the presence of the protein synthesis inhibitors pactamycin or cycloheximide, E1A was correctly posttranslationally modified (phosphorylated) and transported to the nucleus; but it failed to stimulate the transcription of an injected gene containing the human heat shock protein 70 promoter. The basal (unstimulated) level of transcription of the gene was unaffected by these inhibitors. If oocytes were cultured in the presence of cycloheximide after E1A stimulated transcription, however, the high level of transcription was maintained for several hours without new protein synthesis. Results of competition studies with the same promoter (the heat shock protein 70 promoter) linked to two marked genes demonstrated that once the induction of transcription by E1A took place, the stimulated levels of transcription were maintained, even when they were challenged with excess competitor DNA. Results of these studies suggest that E1A requires the synthesis of a cellular protein to form a stable transcription complex.
Complex regulation of the muscle-specific contractile protein (troponin I) gene.
Authors: Konieczny S F , Emerson C P
Published: September 2015

A cloned quail troponin I contractile protein gene, stably transfected into a mouse myogenic cell line, exhibits appropriate developmental activation and quantitative expression during myoblast differentiation. Deletion mutagenesis analyses reveal that the troponin I gene has two distinct cis regulatory elements required for its developmental expression, as measured by mRNA accumulation and nuclear runoff transcription assays. One element in the 5' flanking region is required for maximum quantitative expression, and a second larger regulatory element (1.5 kilobases) within the first intron is responsible for differentiation-specific transcription. The upstream region is highly sensitive to negative repression by interaction with pBR322 sequences. The larger intragenic region retains some activity when moved to the 5' and 3' flanking regions and when inverted but is maximally active in its native intragenic site. The concerted activities of these two regulatory regions produce a 100- to 200-fold transcriptional activation during myoblast differentiation. The conserved 5' exon-intron organization of troponin I and other contractile protein genes suggests a possible mechanism by which intragenic control elements coordinate contractile protein gene regulation during skeletal myogenesis.
The testis-specific phosphoglycerate kinase gene pgk-2 is a recruited retroposon.
Authors: Boer P H , Adra C N , Lau Y F , McBurney M W
Published: September 2015

In both humans and mice, two genes encode phosphoglycerate kinase, a key enzyme in the glycolytic pathway. The pgk-1 gene is expressed in all somatic cells, is located on the X chromosome, and contains 10 introns. The pgk-2 gene is expressed only in sperm cells, is located on an autosome, and has no introns. The nucleotide sequence of the pgk-2 gene suggests that it arose from pgk-1 more than 100 million years ago by RNA-mediated gene duplication. The pgk-2 gene may, then, be a transcribed retroposon. Thus, gene duplication by retroposition may have been used as a mechanism for evolutionary diversification.
Molecular cloning and sequence of a cholesterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway.
Authors: Clarke C F , Tanaka R D , Svenson K , Wamsley M , Fogelman A M , Edwards P A
Published: September 2015

Differential hybridization and molecular cloning have been used to isolate CR39, a cDNA which hybridizes to a 1.2-kilobase (kb) mRNA in rat liver. The level of CR39 mRNA was increased seven- to ninefold over normal levels by dietary cholestyramine and mevinolin and decreased about fourfold compared with normal levels by cholesterol feeding or administration of mevalonate. Similar changes in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase were observed under the various conditions. In vitro translation of either CR39 hybrid selected RNA or 1.2-kb CR39 RNA generated by an SP6 in vitro transcription system produced a polypeptide of 39,000 daltons. As deduced from the nucleotide sequence of a full-length CR39 cDNA, the rat CR39 polypeptide contained 344 amino acids and had a molecular weight of 39,615. The predicted amino acid composition and submit molecular weight of the rat CR39 were very similar to those of prenyltransferases isolated from chicken, pig, and human. The sequence of amino acid residues 173 through 203 in the rat CR39 polypeptide showed that 17 out of 30 matched an active-site peptide of avian liver prenyltransferase. Thus, alterations in the rate of cholesterogenesis resulted in the coordinate regulation of three mRNAs encoding HMG-CoA reductase, HMG-CoA synthase, and CR39, the latter being tentatively identified as prenyltransferase.
Isolation of telomere DNA from Neurospora crassa.
Authors: Schechtman M G
Published: September 2015

The most distal known gene on Neurospora crassa linkage group VR, his-6, was cloned. A genomic walk resulted in isolation of the telomere at VR. It was obtained from a library in which the endmost nucleotides of the chromosome had not been removed by nuclease treatment before being cloned, and mapping indicates that the entire chromosome end has probably been cloned. Sequences homologous to the terminal 2.5 kilobases of DNA from VR from these Oak Ridge N. crassa strains are found at other sites in the genome. To characterize these sites, I crossed an Oak Ridge-derived his-6 strain with a wild-type strain of different genetic background (Mauriceville) and characterized the hybridization patterns seen in the progeny. It appears that the sequences homologous to the VR terminus are found at genetically different sites in the two parental strains, and no hybridization to the VR telomere from Mauriceville was detected. The other genomic copies identified in the Oak Ridge parent were not telomeres. I suggest that any repeating sequence blocks found immediately adjacent to the VR terminus in Oak Ridge strains must be small and that the repeating element identified in that background may be an N. crassa transposable element integrated near the the chromosome end at VR.