bluuebunny/crossref_metadata_2025 huggingface dataset:
Immortalization by c-myc, H-ras, and Ela oncogenes induces differential cellular gene expression and growth factor responses.
Authors: Kelekar A , Cole M D
Published: September 2015

Early-passage rat kidney cells were immortalized or rescued from senescence with three different oncogenes: viral promoter-driven c-myc, H-ras (Val-12), and adenovirus type 5 E1a. The normal c-myc and H-ras (Gly-12) were unable to immortalize cells under similar conditions. Quantitation of RNA in the ras-immortalized lines demonstrated that the H-ras oncogene was expressed at a level equivalent to that of the normal H-ras gene in established human or rat cell lines. Cell lines immortalized by different oncogenes were found to have distinct growth responses to individual growth factors in a short-term assay. E1a-immortalized cells were largely independent of serum growth factors, whereas c-myc-immortalized cells responded to serum better than to epidermal growth factor and insulin. H-ras-immortalized cells responded significantly to insulin alone and gave a maximal response to epidermal growth factor and insulin. Several cellular genes associated with platelet-derived growth factor stimulation, including c-myc, were expressed at high levels in the H-ras-immortalized cells, and c-myc expression was deregulated, suggesting that the H-ras oncogene has provided a "competence" function. H-ras-immortalized cells could not be morphologically transformed by secondary transfection with a long terminal repeat-c-myc oncogene, but secondary transfection of the same cells with H-ras (Val-12) produced morphologically transformed colonies that had 20- to 40-fold higher levels of H-ras oncogene expression. Thus, transformation in this system is dependent on high levels of H-ras oncogene expression rather than on the presence of activated H-ras and c-myc oncogenes in the same cell.
Fertilization triggers unmasking of maternal mRNAs in sea urchin eggs.
Authors: Grainger J L , Winkler M M
Published: September 2015

Fertilization of sea urchin eggs results in a large increase in the rate of protein synthesis which is mediated by the translation of stored maternal mRNA. The masked message hypothesis suggests that messenger ribonucleoprotein particles (mRNPs) from unfertilized eggs are translationally inactive and that fertilization results in alterations of the mRNPs such that they become translationally active. Previous workers have isolated egg mRNPs by sucrose gradient centrifugation and have assayed their translational activity in heterologous cell-free systems. The conflicting results they obtained are probably due to the sensitivity of mRNPs to artifactual activation and inactivation. Previously, we demonstrated that unfractionated mRNPs in a sea urchin cell-free translation system were translationally inactive. Now, using large-pore gel filtration chromatography, we partially purified egg mRNPs while retaining their translationally repressed state. Polysomal mRNPs from fertilized eggs isolated under the same conditions were translationally active. The changes in the pattern of proteins synthesized by fractionated unfertilized and fertilized mRNPs in vitro were similar to those changes observed in vivo. Treatment of egg mRNPs with buffers containing high salt and EDTA, followed by rechromatography, resulted in the activation of the mRNPs and the release of an inhibitor of translation from the mRNPs. Analysis of the inhibitory fraction on one-dimensional sodium dodecyl sulfate gels indicated that this fraction contains a complex set of proteins, several of which were released from high-salt-EDTA-activated mRNPs and not from inactive low-salt control mRNPs. One of the released proteins may be responsible for the repression of egg mRNPs in vitro and be involved in the unmasking of mRNPs at fertilization.
The int-1 proto-oncogene products are glycoproteins that appear to enter the secretory pathway.
Authors: Papkoff J , Brown A M , Varmus H E
Published: September 2015

The int-1 proto-oncogene encodes a primary product of 370 amino acids, is normally expressed in mid-gestational embryos and adult testis, and is activated by proviral insertions during mammary carcinogenesis. Polyclonal and monoclonal antibodies directed against int-1-specific synthetic peptides immunoprecipitate up to five forms of int-1 protein, ranging in size from 36,000 to 44,000 Mr, from cell lines that express cloned int-1 DNA introduced by transfection or infection with retroviral vectors. Pulse-chase labeling experiments and glycosidase digestions suggested that the smallest of the int-1 proteins is the primary translation product lacking its signal peptide and that it is modified to produce the larger species of sequential glycosylation. Subcellular fractionations demonstrated that all immunoprecipitable forms of int-1 are mainly associated with membranes. int-1 proteins in crude microsomal preparations are resistant to proteolysis and extractable at elevated pH, suggesting that they are sequestered within cytoplasmic vesicles in a manner consistent with the behavior of secretory products. However, we were unable to identify secreted int-1 products in extracellular fluids.
Molecular cloning of gene sequences regulated during squamous differentiation of tracheal epithelial cells and controlled by retinoic acid.
Authors: Smits H L , Floyd E E , Jetten A M
Published: September 2015

A cDNA library was constructed from polyadenylated RNA present in squamous differentiated rabbit tracheal epithelial cells. Screening of the cDNA library was aimed at identifying RNAs that were abundant in squamous cells and expressed at low levels in undifferentiated cells. Two different recombinants were obtained containing inserts, 0.86 and 0.77 kilobases (kb) in size, that hybridized to mRNAs 1.0 and 1.25 kb in length. These RNAs were present at approximately 50-fold higher levels in squamous cells than in proliferative or confluent retinoic acid-treated cells. The increase in the levels of the 1.0- and 1.25-kb RNAs correlated closely with the onset of squamous differentiation and was not related to induction of terminal cell division. Treatment of rabbit tracheal epithelial cells with transforming growth factor beta, which induces squamous differentiation in these cells, also resulted in elevated levels of the 1.0- and 1.25-kb RNAs. The increased levels of these RNAs in squamous cells appeared to a large extent to be regulated at a posttranscriptional level. Retinoic acid not only inhibited the increase in the levels of the 1.0- and 1.25-kb RNAs but also reversed the expression of these RNAs in squamous cells. These results suggest that retinoic acid affects, directly or indirectly, molecular events that induce alterations in the posttranscriptional processing of the transcripts corresponding to the 1.0- and 1.25-kb RNAs.
Tissue-specific activity of the pro-opiomelanocortin gene promoter.
Authors: Jeannotte L , Trifiro M A , Plante R K , Chamberland M , Drouin J
Published: September 2015

The pro-opiomelanocortin (POMC) gene is specifically expressed in corticotroph cells of the anterior pituitary. To define the POMC promoter sequences responsible for tissue-specific expression, we assessed POMC promoter activity by gene transfer into POMC-expressing pituitary tumor cells (AtT-20) and fibroblast L cells. The rat POMC promoter was only efficiently utilized and correctly transcribed in AtT-20 cells. 5'-End deletion analysis revealed two promoter regions required for activity in AtT-20 cells. When tested by fusion to a heterologous promoter, DNA fragments corresponding to both regions exhibited tissue-specific activity, suggesting the presence of at least two tissue-specific DNA sequence elements within the promoter. In summary, POMC promoter sequences from -480 to -34 base pairs appear sufficient to mimic the specificity of anterior pituitary expression.
Negative and positive regulation by a short segment in the 5'-flanking region of the human cytomegalovirus major immediate-early gene.
Authors: Nelson J A , Reynolds-Kohler C , Smith B A
Published: September 2015

To analyze the significance of inducible DNase I-hypersensitive sites occurring in the 5'-flanking sequence of the major immediate-early gene of human cytomegalovirus (HCMV), various deleted portions of the HCMV immediate-early promoter regulatory region were attached to the chloramphenicol acetyltransferase (CAT) gene and assayed for activity in transiently transfected undifferentiated and differentiated human teratocarcinoma cells, Tera-2. Assays of progressive deletions in the promoter regulatory region indicated that removal of a 395-base-pair portion of this element (nucleotides -750 to -1145) containing two inducible DNase I sites which correlate with gene expression resulted in a 7.5-fold increase in CAT activity in undifferentiated cells. However, in permissive differentiated Tera-2, human foreskin fibroblast, and HeLa cells, removal of this regulatory region resulted in decreased activity. In addition, attachment of this HCMV upstream element to a homologous or heterologous promoter increased activity three- to fivefold in permissive cells. Therefore, a cis regulatory element exists 5' to the enhancer of the major immediate-early gene of HCMV. This element negative modulates expression in nonpermissive cells but positively influences expression in permissive cells.
Multiple functional motifs in the chicken U1 RNA gene enhancer.
Authors: Roebuck K A , Walker R J , Stumph W E
Published: September 2015

The DNA sequence requirements of chicken U1 RNA gene expression have been examined in an oocyte transcription system. An enhancer region, which was required for efficient U1 RNA gene expression, is contained within a region of conserved DNA sequences spanning nucleotide positions -230 to -183, upstream of the transcriptional initiation site. These DNA sequences can be divided into at least two distinct subregions or domains that acted synergistically to provide a greater than 20-fold stimulation of U1 RNA synthesis. The first domain contains the octamer sequence ATGCAAAT and was recognized by a DNA-binding factor present in HeLa cell extracts. The second domain (the SPH domain) consists of conserved sequences immediately downstream of the octamer and is an essential component of the enhancer. In the oocyte, the DNA sequences of the SPH domain were able to enhance gene expression at least 10-fold in the absence of the octamer domain. In contrast, the octamer domain, although required for full U1 RNA gene activity, was unable to stimulate expression in the absence of the adjacent downstream DNA sequences. These findings imply that sequences 3' of the octamer play a major role in the function of the chicken U1 RNA gene enhancer. This concept was supported by transcriptional competition studies in which a cloned chicken U4B RNA gene was used to compete for limiting transcription factors in oocytes. Multiple sequence motifs that can function in a variety of cis-linked configurations may be a general feature of vertebrate small nuclear RNA gene enhancers.
Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.
Authors: Markland W , Smith A E , Roberts B L
Published: September 2015

An in vitro nuclear translocation system is described in which isolated rat liver nuclei were incubated in a defined buffered medium containing radiolabeled or fluorescently labeled exogenous proteins. The nuclei were rapidly recovered, extracted, and analyzed for the presence of associated radiolabeled or fluorescently labeled proteins. The isolated nuclei exhibited the same specificity for protein uptake as seen previously in vivo, accumulating simian virus 40 wild-type large-T antigen and p53 while excluding a cytoplasmic variant of large-T antigen (d10) and bovine serum albumin. The rapid nuclear accumulation of wild-type large-T antigen was shown to be selective and dependent upon the recognition of a wild-type nuclear location signal, ATP and temperature dependent, and unidirectional. Taken together, the data suggest that in our in vitro system the nuclear translocation of wild-type large-T antigen exhibits some of the characteristics of an active transport process.
Alternative processing of RNA transcribed from NMYC.
Authors: Stanton L W , Bishop J M
Published: September 2015

NMYC is a gene whose amplification and overexpression have been implicated in the generation of certain human malignancies. Little is known of how the expression of NMYC is normally controlled. We have therefore characterized transcription from the gene and the structure and stability of the resulting mRNAs. Transcription from NMYC is exceptionally complex: it initiates at numerous sites that may be grouped under the control of two promoters, and the multiplicity of initiation sites combines with alternative splicing to engender two forms of mRNA. The mRNAs have different 5' leader sequences (alternative first exons of the gene) but identical bodies (the second and third exons of the gene). Both forms of mRNA are unstable, with half-lives of ca. 15 min. Both encode the previously identified 65,000 and 67,000-dalton products of NMYC. However, the alternative first exons contain distinctive open reading frames that may diversify the coding potential of NMYC. The complexities in transcription of NMYC expand the means by which expression of the gene might be controlled.
Synthesis of U1 RNA in isolated mouse cell nuclei: initiation and 3'-end formation.
Authors: Lobo S M , Marzluff W F
Published: September 2015

The transcription of U1 RNA genes was studied in isolated nuclei from mouse myeloma cells. Using a cloned U1b gene as a probe, we showed that isolated nuclei synthesize both U1b and U1a RNA. The U1 RNAs were initiated in vitro, as measured by incorporation of adenosine 5'-O-(2-thiotriphosphate) into U1 RNA. There was transcription of the 3'-flanking region but no transcription of regions directly 5' to the U1 genes. In addition to U1 RNAs of the correct length which were released from the nuclei, there were larger RNAs, presumably resulting from transcription into the 3'-flanking region, which were retained in the nuclei. Chase experiments showed that these longer transcripts were not precursors to mature U1 RNA, a finding consistent with the idea that 3'-end formation is coincident with transcription. During the chase, there was maturation of the 3' ends of U1a and U1b RNAs from slightly longer precursors. In addition to accurate transcription of U1 RNA, there was also synthesis of U2 and U3 RNA. All three of these RNAs were transcribed by RNA polymerase II, as measured by their sensitivity to alpha-amanitin.
Autogenous regulation of histone mRNA decay by histone proteins in a cell-free system.
Authors: Peltz S W , Ross J
Published: September 2015

We tested the hypothesis that histone mRNA turnover is accelerated in the presence of free histone proteins. In an in vitro mRNA decay system, histone mRNA was degraded four- to sixfold faster in reaction mixtures containing core histones and a cytoplasmic S130 fraction than in reaction mixtures lacking these components. The decay rate did not change significantly when histones or S130 was added separately, suggesting either that the histones were modified and thereby activated by S130 or that additional factors besides histones were required. RecA, SSB (single-stranded binding), and histone proteins all formed complexes with histone mRNA, but only histones induced accelerated histone mRNA turnover. Therefore, the effect was not the result of random RNA-protein interactions. Moreover, histone proteins did not induce increased degradation of gamma globin mRNA, c-myc mRNA, or total poly(A)- or poly(A)+ polysomal mRNAs. This autoregulatory mechanism is consistent with the observed accumulation of cytoplasmic histone proteins in cells after DNA synthesis stops, and it can account, in part, for the rapid disappearance of histone mRNA at the end of S phase.
Identification of upstream activator sequences that regulate induction of the beta-galactosidase gene in Kluyveromyces lactis.
Authors: Leonardo J M , Bhairi S M , Dickson R C
Published: September 2015

Transcription of the Kluyveromyces lactis beta-galactosidase gene, LAC4, is inducible by galactose and lactose. We examined the effects of deletion mutations within the LAC4 promoter on the expression of beta-galactosidase activity. The results of these experiments indicate that at least two upstream activator sequences (UAS) mediate maximum induction by galactose. These UAS sequence elements are homologous to UAS that regulate induction of the melibiose-galactose regulon of Saccharomyces cerevisiae. We also show that a synthetic copy of one of the K. lactis UAS restores the inducibility of a deleted, noninducible LAC4 promoter. Since the uninduced or basal level of LAC4 expression was increased in several promoter deletion strains and in deletion strains carrying one or two synthetic UAS, we examined the contribution of the LAC9 positive regulatory protein to this effect. The LAC9 protein is thought to bind to UAS and activate transcription of LAC4 (L.V. Wray, M.M. Witte, R.C. Dickson, and M.I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987). Our results demonstrate that LAC9 protein plays a role in setting the uninduced level of gene expression, but other factors also participate. For example, in a lac9 background a LAC4 promoter deletion mutant with two copies of a synthetic 17-base-pair UAS yields a sevenfold higher level of uninduced LAC4 expression than the same strain with one UAS. These and other data indicate that the basal level of gene expression is strongly influenced by the base sequence of the promoter.
Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.
Authors: Breunig K D , Kuger P
Published: September 2015

As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription.
Allelic variation and differential expression at the 27-kilodalton zein locus in maize.
Authors: Das O P , Messing J W
Published: September 2015

Allelic variation between inbred lines at the 27-kilodalton zein gene locus in maize has been used to study gene expression in developing endosperm. The inbred lines W22 and W23 contain two genes for this protein within two tandem repeats; the individual genes are virtually identical, with 99.9% homology in the 5'-flanking regions. Using gene-specific oligonucleotide probes, we have shown that transcripts of the downstream gene are found at a 2.5-fold-higher level than those of the upstream gene. Another inbred line, BSSS53, has one copy of the gene which is a recombinant of the duplicated genes at the 3'-flanking region. This line has been used in reciprocal crosses to demonstrate dosage effects for the overexpression of the downstream gene and to show that the overexpression of mRNA is reflected in a corresponding increase in the protein level. The accumulation of the protein through development does not, however, always correspond to the difference in mRNA levels.
Binding of multiple nuclear factors to the 5' upstream regulatory element of the murine major histocompatibility class I gene.
Authors: Shirayoshi Y , Miyazaki J , Burke P A , Hamada K , Appella E , Ozato K
Published: September 2015

Transcription of mouse major histocompatibility complex class I genes is controlled by the conserved class I regulatory element (CRE) in the 5' flanking region. The CRE, approximately 40 base pairs long, acts as a negative control element in undifferentiated F9 embryonal carcinoma cells which do not express the major histocompatibility complex genes. The same element, however, acts as a positive control element in cells expressing the genes at high levels. To investigate the molecular basis of the regulatory role of the CRE, we studied the binding of nuclear proteins to the CRE of the H-2Ld gene by gel mobility shift and methylation interference experiments. Nuclear extracts from L fibroblasts and LH8 T lymphocytes revealed three distinct factors that bind discrete sequences within the CRE. The three sequences correspond to the inverted and direct repeats within the CRE. In contrast, F9 extracts exhibited factor binding to only two of the three sequences and lack a major factor detected in the above two cell types. Protein-binding sites within each of the three sequences were identified by methylation interference experiments. These data were in full agreement with results obtained by a competition assay performed with a series of mutant oligonucleotides containing a few nucleotide substitutions in each of the three regions. The results illustrate complex DNA-protein interactions in which several independent proteins bind to overlapping sequences in the CRE in a cell type-specific fashion.
Identical genomic footprints of the adenovirus EIIa promoter are detected before and after EIa induction.
Authors: Devaux B , Albrecht G , Kedinger C
Published: September 2015

Genomic DNase I footprinting was used to compare specific protein binding to the adenovirus type 5 early, EIa-inducible, EIIa promoter. Identical protection patterns of the promoter region were observed whether EIIa transcription was undetectable or fully induced. These results suggest that EIa-mediated transcriptional induction does not increase binding of limiting transcription factors to the promoter but rather that transactivation results from the proper interactions between factors already bound to their cognate sequences.
A mutant epidermal growth factor receptor with defective protein tyrosine kinase is unable to stimulate proto-oncogene expression and DNA synthesis.
Authors: Honegger A M , Szapary D , Schmidt A , Lyall R , Van Obberghen E , Dull T J , Ullrich A , Schlessinger J
Published: September 2015

Cultured NIH-3T3 cells devoid of endogenous epidermal growth factor (EGF) receptors were transfected with cDNA expression constructs encoding either normal human EGF receptor or a receptor mutated in vitro at Lys-721, a residue that is thought to function as part of the ATP-binding site of the kinase domain. Unlike the wild-type EGF-receptor expressed in these cells, which exhibited EGF-dependent protein tyrosine kinase activity, the mutant receptor lacked protein tyrosine kinase activity and was unable to undergo autophosphorylation and to phosphorylate exogenous substrates. Despite this deficiency, the mutant receptor was normally expressed on the cell surface, and it exhibited both high- and low-affinity binding sites. The addition of EGF to cells expressing wild-type receptors caused the stimulation of various responses, including enhanced expression of proto-oncogenes c-fos and c-myc, morphological changes, and stimulation of DNA synthesis. However, in cells expressing mutant receptors, EGF was unable to stimulate these responses, suggesting that the tyrosine kinase activity is essential for EGF receptor signal transduction.
Heat-induced expression of albumin during early stages of rat embryo development.
Authors: Srinivas U K , Revathi C J , Das M R
Published: September 2015

An examination of heat-induced expression of proteins in tissues from adult and embryonic liver in rats shows that albumin, which is constitutively expressed in adult liver and is not synthesized in embryos before 16 days of gestation, appears in liver cells at earlier stages of development upon heat shock. On the basis of available evidence for the expression of heat shock proteins at distinct stages of development and on the basis of our findings, it may be argued that there could be common molecular events taking place during development and as a result of heat shock. We suggest also that one of the consequences of heat shock could be an internal change of pH within the cell which, in turn, might trigger alterations in gene expression.
Transient response of amplified metallothionein genes in CHO cells to induction by alpha interferon.
Authors: Morris S , Huang P C
Published: September 2015

Alpha interferon treatment of CHO cells elicits the rapid synthesis of many gene products, including metallothionein (MT), a protein which avidly binds heavy metals such as zinc, cadmium, and copper. Since MTs appear to have a pleiotropic role in the cell, ranging from metal detoxification to free-radical scavenging, interferon treatment may trigger a generalized defense mechanism. Activation by interferon, however, was transient, with MT mRNA being maximally detectable by a cytodot procedure within the first hour. Subsequent addition of interferon was ineffective until 7 h after the initial treatment. The action of zinc, a potent inducer of MT, however, remained independent of alpha interferon induction. The transient nature of induction by interferon was examined for altered rate of MT mRNA turnover.
Physical analysis of the COR region: a cluster of six genes in Saccharomyces cerevisiae.
Authors: Barry K , Stiles J I , Pietras D F , Melnick L , Sherman F
Published: September 2015

Six genes, CYC1, UTR1, UTR3, OSM1, tRNAGly, and RAD7, have been localized within an 8-kilobase region on chromosome X of the yeast Saccharomyces cerevisiae. The physical structures and the transcripts of these genes were identified by analyzing a normal strain and six deletion mutants by genomic blotting, transcriptional analysis, and gene disruption procedures. The well-studied CYC1 gene encodes iso-1-cytochrome c; the tRNAGly gene encodes a tRNA; deletion of OSM1 and RAD7 causes sensitivity to hypertonic medium and UV irradiation, respectively. There were no observable phenotypes in strains having deletions of the UTR1, UTR3, and tRNAGly gene. The high density of transcripts, with little or almost no intragenic regions, indicates that the chromosomal organization of S. cerevisiae resembles the chromosomal organization of procaryotes rather than higher eucaryotes.
Expression of hamster P-glycoprotein and multidrug resistance in DNA-mediated transformants of mouse LTA cells.
Authors: Deuchars K L , Du R P , Naik M , Evernden-Porelle D , Kartner N , van der Bliek A M , Ling V
Published: September 2015

The overexpression of a plasma membrane glycoprotein, P-glycoprotein, is strongly correlated with the expression of multidrug resistance. This phenotype (frequently observed in cell lines selected for resistance to a single drug) is characterized by cross resistance to many drugs, some of which are used in cancer chemotherapy. In the present study we showed that DNA-mediated transformants of mouse LTA cells with DNA from multidrug-resistant hamster cells acquired the multidrug resistance phenotype, that the transformants contained hamster P-glycoprotein DNA sequences, that these sequences were amplified whereas the recipient mouse P-glycoprotein sequences remained at wild-type levels, and that the overexpressed P-glycoprotein in these cells was of hamster origin. Furthermore, we showed that the hamster P-glycoprotein sequences were transfected independently of a group of genes that were originally coamplified and linked within a 1-megabase-pair region in the donor hamster genome. These data indicate that the high expression of P-glycoprotein is the only alteration required to mediate multidrug resistance.
Characterization of the rearranged tpr-met oncogene breakpoint.
Authors: Dean M , Park M , Vande Woude G F
Published: September 2015

We determined the nucleotide sequence of the rearranged trp-met genomic locus and the corresponding portions of the unrearranged tpr and met genomic fragments. The breakpoints occur at one end of a stretch of 21 A residues that follow an Alu repetitive sequence in the tpr locus and within a group of 3 A residues in the met proto-oncogene locus. We conclude that the fusion between the tpr locus on chromosome 1 and the met locus on chromosome 7 resulted from a recombination event.
The excised leader of human cytochrome c oxidase subunit I mRNA which contains the origin of mitochondrial DNA light-strand synthesis accumulates in mitochondria and is polyadenylated.
Authors: Gaines G , Rossi C , Attardi G
Published: September 2015

We identified a polyadenylated RNA species which contains the origin of human mitochondrial DNA light-strand synthesis and the surrounding complementary sequences of the four light-strand-encoded tRNAs. This RNA (RNA 9L) is probably derived from the leader portion of RNA 6 which is excised during the formation of the mature cytochrome c oxidase subunit mRNA (RNA 9). The high degree of secondary structure of this RNA is presumably responsible for its anomalous electrophoretic behavior in denaturing polyacrylamide gels.
Primary structure polymorphism at amino acid residue 72 of human p53.
Authors: Matlashewski G J , Tuck S , Pim D , Lamb P , Schneider J , Crawford L V
Published: September 2015

We analyzed p53 cDNA and genomic clones from a variety of normal and transformed cells. Sequence analysis of these clones revealed that amino acid residue 72 can be an arginine, proline, or cysteine. This single codon difference results in electrophoretically distinct forms of human p53 seen in normal and transformed cells.
Cell cycle regulation of a mouse histone H4 gene requires the H4 promoter.
Authors: Seiler-Tuyns A , Paterson B M
Published: September 2015

The mouse histone H4 gene, when stably transformed into L cells on the PSV2gpt shuttle vector, is cell cycle regulated in parallel with the endogenous H4 genes. This was determined in exponentially growing pools of transformants fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cell cycle without the use of treatments that arrest growth. Linker additions in the 5' noncoding region of the H4 RNA or in the coding region of the gene did not affect the cell cycle-regulated expression of the modified H4 gene even though the overall level of expression was altered. However, replacing the H4 promoter with the human alpha-2 globin promoter, so that the histone transcript produced by the chimeric gene remains essentially unchanged, resulted in the constitutive expression of H4 mRNA during all phases of the cell cycle with no net increase in H4 mRNA levels during the G1-to-S transition. From these results we conclude that all the information necessary for the cell cycle-regulated expression of the H4 gene is contained in the 5.2-kilobase subclone used in these studies with 228 nucleotides of 5'-flanking DNA and that the increase in H4 mRNA during the G1-to-S transition in the cell cycle is mediated by the H4 promoter and not by the increased stability of the H4 RNA.
Nonsense mutations of the ornithine decarboxylase structural gene of Neurospora crassa.
Authors: Davis R H , Hynes L V , Eversole-Cire P
Published: September 2015

Ornithine decarboxylase (ODC) (EC 4.1.1.17) is an early enzyme of polyamine synthesis, and its activity rises quickly at the onset of growth and differentiation in most eucaryotes. Some have speculated that the enzyme protein may have a role in the synthesis of rRNA in addition to its role in catalyzing the decarboxylation of ornithine (G. D. Kuehn and V. J. Atmar, Fed. Proc. 41:3078-3083, 1982; D. H. Russell, Proc. Natl. Acad. Sci. USA 80:1318-1321, 1983). To test this possibility, we sought mutational evidence for the indispensability of the ODC protein for normal growth of Neurospora crassa. We found three new, ODC-deficient mutants that lacked ODC protein. Among these and by reversion analysis of an earlier set of mutants, we found that two ODC-deficient mutants carried nonsense mutations in the ODC structural gene, spe-1. Allele LV10 imparted a complete deficiency for enzyme activity (less than 0.006% of normal) and had no detectable ODC antigen. Allele PE4 imparted a weak activity to cells (0.1% of derepressed spe+ cultures) and encoded a lower-molecular-weight ODC subunit (Mr = 43,000) in comparison to that of the wild-type strain (Mr = 53,000). Strains carrying either mutation, like other spe-1 mutants, grew at a normal rate in exponential culture if the medium was supplemented with spermidine, the main end product of the polyamine pathway in N. crassa. Unless an antigenically silent, N-terminal fragment with an indispensable role persists in the LV10-bearing mutant, we conclude that the ODC protein has no role in the vegetative growth of this organism other than the synthesis of polyamines. The data extend earlier evidence that spe-1 is the structural gene for ODC in N. crassa. The activity found in mutants bearing allele PE4 suggests that the amino acids nearest the carboxy terminus do not contribute to the active site of the enzyme.
Concerted deletions and inversions are caused by mitotic recombination between delta sequences in Saccharomyces cerevisiae.
Authors: Rothstein R , Helms C , Rosenberg N
Published: September 2015

Deletions of a tyrosine tRNA suppressor gene, SUP4-o, are mediated by recombination between short repeated delta sequences in Saccharomyces cerevisiae. The arrangement of the five solo delta sequences that surround the SUP4 locus was established by DNA sequence analysis. Seven deletion classes were identified by genomic blotting. DNA sequence analysis also showed that the delta sequences within a 6.5-kilobase region of the SUP4 locus were the endpoints of these events. In three of these classes, an adjacent interval surrounded by delta sequences was inverted in concert with the deletion. The frequency of all deletion classes decreased in strains that contained mutations in the recombination and repair gene RAD52. We present two gene conversion mechanisms by which these rearrangements could have been generated. These models may also explain deletions between repeated sequences in other systems.
Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing.
Authors: Hurt D J , Wang S S , Lin Y H , Hopper A K
Published: September 2015

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.
Transcription of the ADH2 gene in Saccharomyces cerevisiae is limited by positive factors that bind competitively to its intact promoter region on multicopy plasmids.
Authors: Irani M , Taylor W E , Young E T
Published: September 2015

Transcription of the ADH2 gene in the yeast Saccharomyces cerevisiae was inhibited by excess copies of its own promoter region. This competition effect was promoter specific and required the upstream activation sequence of ADH2 as well as sequences 3' to the TATA box. Introducing excess copies of ADR1, an ADH2-specific regulatory gene, did not alleviate the competition that was observed in these circumstances during both constitutive and derepressed ADH2 expression. Excess copies of the upstream region did not release ADH2 from glucose repression, consistent with the view that ADH2 is regulated by positive trans-acting factors.
Initiation factor protein modifications and inhibition of protein synthesis.
Authors: Duncan R F , Hershey J W
Published: September 2015

The protein covalent modification state of eucaryotic initiation factors eIF-2 and eIF-4B in HeLa cells was examined after they were exposed to a variety of conditions or treatments that regulate protein synthesis. A few factors (e.g., variant pH and sodium fluoride) altered the phosphorylation state of the initiation factor proteins, but the majority (hypertonic medium, ethanol, dimethyl sulfoxide sodium selenite, sodium azide, and colchicine) had no effect on either protein. While initiation factor phosphorylation may regulate protein synthesis in response to many physiological situations, other pathways can regulate protein synthesis under nonphysiological circumstances.
Saccharomyces cerevisiae mutants unresponsive to alpha-factor pheromone: alpha-factor binding and extragenic suppression.
Authors: Jenness D D , Goldman B S , Hartwell L H
Published: September 2015

Mutations in six genes that eliminate responsiveness of Saccharomyces cerevisiae a cells to alpha-factor were examined by assaying the binding of radioactively labeled alpha-factor to determine whether their lack of responsiveness was due to the absence of alpha-factor receptors. The ste2 mutants, known to be defective in the structural gene for the receptor, were found to lack receptors when grown at the restrictive temperature; these mutations probably affect the assembly of active receptors. Mutations in STE12 known to block STE2 mRNA accumulation also resulted in an absence of receptors. Mutations in STE4, 5, 7, and 11 partially reduced the number of binding sites, but this reduction was not sufficient to explain the loss of responsiveness; the products of these genes appear to affect postreceptor steps of the response pathway. As a second method of distinguishing the roles of the various STE genes, we examined the sterile mutants for suppression. Mating of the ste2-3 mutant was apparently limited by its sensitivity to alpha-factor, as its sterility was suppressed by mutation sst2-1, which leads to enhanced alpha-factor sensitivity. Sterility resulting from each of four ste4 mutations was suppressed partially by mutation sst2-1 or by mutation bar1-1 when one of three other mutations (ros1-1, ros2-1, or ros3-1) was also present. Sterility of the ste5-3 mutant was suppressed by mutation ros1-1 but not by sst2-1. The ste7, 11, and 12 mutations were not suppressed by ros1 or sst2. Our working model is that STE genes control the response to alpha-factor at two distinct steps. Defects at one step (requiring the STE2 gene are suppressed (directly or indirectly) by mutation sst2-1, whereas defects at the other step (requiring the STE5 gene) are suppressed by the ros1-1 mutation. The ste4 mutants are defective for both steps. Mutation ros1-1 was found to be allelic to cdc39-1. Map positions for genes STE2, STE12, ROS3, and FUR1 were determined.
Repression of quiescence-specific polypeptides in chicken heart mesenchymal cells transformed by Rous sarcoma virus.
Authors: Bédard P A , Balk S D , Gunther H S , Morisi A , Erikson R L
Published: September 2015

Chicken heart mesenchymal cells do not proliferate in medium of physiological composition containing plasma (S. Balk, Proc. Natl. Acad. Sci. USA 77:6606-6610, 1980). To understand the molecular events involved in cell quiescence and in the initiation of cell division under physiological conditions, we examined the differences in the patterns of protein synthesis of quiescent, hormone-stimulated, and Rous sarcoma virus-transformed chicken heart mesenchymal cells. We describe the expression of a 20,000-kilodalton (kDa) polypeptide actively synthesized by quiescent cells but not by their transformed counterparts. Normal chicken heart mesenchymal cells stimulated with epidermal growth factor and insulin also repressed the synthesis of the 20,000-kDa polypeptide while actively growing but synthesized increasing amounts of the protein at high cell density (confluence). The synthesis of the 20,000-kDa protein is not restricted to chicken heart mesenchymal cells, since confluent, density-arrested chicken embryo fibroblasts also expressed high levels of the protein. Transformed chicken heart mesenchymal cells and embryo fibroblasts did not synthesize the protein even at high cell density. The 20,000-kDa polypeptide accumulated in the culture medium.
Inactivation of a transfected gene in human fibroblasts can occur by deletion, amplification, phenotypic switching, or methylation.
Authors: Gebara M M , Drevon C , Harcourt S A , Steingrimsdottir H , James M R , Burke J F , Arlett C F , Lehmann A R
Published: September 2015

Plasmids containing the bacterial gpt gene under control of the simian virus 40 promoter were transfected into a simian virus 40-transformed human fibroblast line. Two transfectants, E2 and C10, which contain stably integrated single copies of the gpt gene, were isolated. These two lines produce Gpt- variants spontaneously with a frequency of about 10(-4). We carried out a detailed molecular analysis of the spectrum of alterations which gave rise to the Gpt- phenotype in these variants. DNA from 14 of 19 Gpt- derivatives of one of the cell lines (E2) contains deletions or rearrangements of gpt-containing sequences. In four of the remaining five lines, the Gpt- phenotype was correlated with reduced levels of expression rather than with changes in the gross structure of the gpt gene, and it was possible to reactivate the gpt gene. In one Gpt- line, gpt mRNA was present at normal levels, but no active enzyme was produced. Spontaneous Gpt- derivatives of the other cell line (C10) produced a completely different spectrum of alterations. Very few deletions were found, but several derivatives contained additional extrachromosomal gpt sequences, and, remarkably, in two other Gpt- lines, gpt-containing sequences were amplified more than 100-fold. The phenotypes of the majority of the Gpt- derivatives of C10 could be attributed to alterations in gene expression caused by methylation.
Sequestration analysis for RNA polymerase I transcription factors with various deletion and point mutations reveals different functional regions of the mouse rRNA gene promoter.
Authors: Nagamine M , Kishimoto T , Aono J , Kato H , Kominami R , Muramatsu M
Published: September 2015

We compared the ability of various deletion and substitution mutants of the mouse rRNA gene promoter to bind essential factors required for accurate transcription initiation by RNA polymerase I. Different amounts of a competitor template were first incubated with a mouse cell extract containing the whole complement of factors and RNA polymerase I, and then a tester template was added for the second incubation. Transcription was started by adding nucleoside triphosphates (one labeled), and the accurate transcripts were determined on a gel. The results indicated that the ability of 5' deletion mutants to sequester essential factors decreased almost concurrently with the impairment of in vitro transcription activity, whereas when the promoter sequence was removed from the 3' side, the transcription activity decreased earlier and more drastically than the sequestration ability. Similar, though not identical, results were obtained by preincubation with fraction D separated on a phosphocellulose column, indicating that the major factor which was sequestered was TFID, the species-dependent transcription initiation factor that binds first to the promoter in the initiation reaction (H. Kato, M. Nagamine, R. Kominami, and M. Muramatsu, Mol. Cell. Biol. 6:3418-3427, 1986). Compilation of the data suggests that a region inside the 5' half of the core promoter (-40 to -1) is essential for the binding of TFID. The 3' half of the promoter (-1 to downstream) is not essential for the binding of TFID but is highly important for an efficient transcription initiation. A strong down-mutant with a one-base substitution at -16 (G to A) had a reduced ability to bind to TFID, whereas a null mutant with a single base substitution at -7 (G to A) showed a binding ability similar to that of the wild-type promoter when tested with whole-cell extract. This null mutant, however, could not sequester the TFID well when incubated with fraction D alone, suggesting that the binding of TFID with this mutant is unstable in the absence of another factor(s) present in cell extract. The factor is not TFIA, which binds after TFID, because the addition of fraction A containing TFIA did not cause TFID to bind to the mutant. The availability of different mutants having lesions at different steps of transcription initiation will provide a powerful tool for the dissection of the initiation reaction of the RNA gene.
Heat-inducible human factor that binds to a human hsp70 promoter.
Authors: Kingston R E , Schuetz T J , Larin Z
Published: September 2015

A factor found in nuclear extracts of human cells bound to the heat shock element of a human heat shock protein 70 gene. The level of this factor was significantly increased after heat shock. This induction was rapid and was not blocked by cycloheximide, suggesting that an initial event in the response of a human cell to heat is the activation of a preexisting regulatory factor.
Human retroviral sequences on the Y chromosome.
Authors: Silver J , Rabson A , Bryan T , Willey R , Martin M A
Published: September 2015

Novel endogenous human retroviral sequences were cloned by low-stringency hybridization, using the pol gene of endogenous human retrovirus 51-1. One clone, lambda NP-2, contained gag, pol, env, and long terminal repeat sequences related to the corresponding portions of clone 51-1 and the closely related full-length endogenous human retrovirus 4-1. The sequence of the env gene of NP-2 was 73% homologous to that of 4-1. Genomic Southern blots of male and female DNAs showed that NP-2 is located on the Y chromosome and that the Y chromosome also contains one other sequence closely related to the env and 3' flanking regions of NP-2. Conservation of flanking DNA suggests that the second Y chromosome copy of the NP-2 env sequence arose by gene duplication rather than provirus insertion.