bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the affected A gamma globin gene associated with Greek nondeletion hereditary persistence of fetal hemoglobin.
Authors: Stoeckert C J , Metherall J E , Yamakawa M , Eisenstadt J M , Weissman S M , Forget B G
Published: September 2015

The overexpressed A gamma globin gene in the Greek type of nondeletion hereditary persistence of fetal hemoglobin has a unique single-base substitution located at position -117 relative to the site of transcription initiation. This gene and its normal counterpart were transferred into cultured cell lines by using a retroviral vector. The only difference in expression between the transferred normal and mutant gamma genes was observed in the human erythroleukemia cell line KMOE after exposure of the cells to cytosine arabinoside, a condition that resulted in an adult pattern of endogenous globin gene expression by the cells and was associated with increased expression of the mutant gene.
rRNA genes of Naegleria gruberi are carried exclusively on a 14-kilobase-pair plasmid.
Authors: Clark C G , Cross G A
Published: September 2015

An extrachromosomal DNA was discovered in Naegleria gruberi. The 3,000 to 5,000 copies per cell of this 14-kilobase-pair circular plasmid carry all the 18S, 28S, and 5.8S rRNA genes. The presence of the ribosomal DNA of an organism exclusively on a circular extrachromosomal element is without precedent, and Naegleria is only the third eucaryotic genus in which a nuclear plasmid DNA has been found.
Two developmental stages of Neurospora crassa utilize similar mechanisms for responding to heat shock but contrasting mechanisms for recovery.
Authors: Plesofsky-Vig N , Brambl R
Published: September 2015

At the heat shock temperature of 45 degrees C, there is a transient induction of the synthesis of heat shock proteins and repression of normal protein synthesis in cells of Neurospora crassa. Both conidiospores and mycelial cells resume normal protein synthesis after 60 min at high temperature. At the RNA level, however, these two developmental stages responded with different kinetics to elevated temperature. Heat shock RNAs (for hsp30 and hsp83) accumulated and declined more rapidly in spores than in mycelia, and during recovery spores accumulated mRNA that encoded a normal protein (the proteolipid subunit of the mitochondrial ATPase), whereas mycelia showed no increase in this normal RNA (for at least 120 min). Therefore, the resumption of normal protein synthesis in spores may depend upon accumulation of new mRNAs. In contrast, mycelial cells appeared to change their translational preference during continued incubation at elevated temperature, from a discrimination against normal mRNAs to a resumption of their translation into normal cellular proteins, exemplified by the ATPase proteolipid subunit whose synthesis was measured in the heat-shocked cells.
Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis.
Authors: Clanton D J , Lu Y Y , Blair D G , Shih T Y
Published: September 2015

Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.
Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells.
Authors: Leonard D G , Ziff E B , Greene L A
Published: September 2015

Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons.
Cell-type-dependent gene activation by yeast transposon Ty1 involves multiple regulatory determinants.
Authors: Company M , Errede B
Published: September 2015

Ty transposable element insertion mutations of Saccharomyces cerevisiae can cause cell-type-dependent activation of adjacent gene expression. Several cis-acting regulatory regions within Ty1 that are responsible for these effects were identified. A 211-base-pair (bp) region functions as an activator. This region includes the so-called U5 domain of delta and 145 bp of adjacent epsilon sequences. Unlike activation by the intact Ty1, activation by the 211-bp Ty1 subfragment is cell-type independent. The presence of a 112-bp fragment from a more distal region of Ty1 confers cell-type specificity to the activator. The 112-bp fragment includes sequences with homology to mammalian enhancers and to a yeast a/alpha control site. In addition, Ty1 regions that exert negative effects on gene expression were identified. These results demonstrate that the Ty1 transcriptional control region consists of multiple components with distinct regulatory functions.
Molecular cloning of fused, a gene required for normal segmentation in the Drosophila melanogaster embryo.
Authors: Mariol M C , Preat T , Limbourg-Bouchon B
Published: September 2015

Using the chromosomal walk technique, we isolated recombinant lambda bacteriophage and cosmid clones spanning 250 kilobases (kb) in the 17C-D region of the X chromosome of Drosophila melanogaster. This region was known to contain the segment polarity gene fused. Several lethal fused mutations were used to define more precisely the localization of this locus. Southern analysis of genomic DNA revealed that all of them were relatively large deficiencies, the smallest one being 40 kb long. None of the 12 viable fused mutations examined possessed detectable alterations. We isolated a cosmid containing an insertion covering the entire smallest fused deletion (40 kb). We injected this DNA into fused mutant embryos and obtained a partial phenotypic rescue of the embryonic pattern, indicating that this region contained all the sequences necessary for the embryonic expression of the fu+ gene. Within this DNA, a subclone of 14 kb codes for poly(A)+ RNAs of 3.5, 2.5, 1.6, and 1.3 kb detected in embryos from various developmental stages as well as in adults. All these transcripts showed the same developmental expression. This transcribed region was injected into fused mutant embryos, and once again we obtained a partial rescue of the embryonic phenotype, confirming that this region contained at least the fused gene.
Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA.
Authors: Zhang F , Cole C N
Published: September 2015

Cleavage and polyadenylation of substrate RNAs containing the herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene polyadenylation signal region were examined in HeLa cell nuclear extract. 3'-End RNA processing was accurate and efficient and required ATP and Mg2+. Cleavage, but not polyadenylation, occurred in the presence of EDTA or when ATP was replaced with 3' dATP (cordycepin) or AMP(CH2)PP, a nonhydrolyzable analog of ATP. Processing in vitro and in vivo showed the same signal element requirements: a series of substrates containing linker scanning, internal deletion, and small insertion mutations was processed with the same relative efficiencies and at the same sites in vitro and in vivo. A complex involved in 3'-end RNA processing was identified by gel mobility shift analysis. This complex formed rapidly, reached a maximum level after 20 to 30 min, and was much reduced after 2 h. Very little complex was formed at 0 degree C or with substrates lacking a polyadenylation signal. Entry of 32P-labeled tk substrate into the complex could be prevented by addition of excess 35S-labeled tk or adenovirus L3 precursor RNAs. Competition was not observed with tk RNAs lacking a complete polyadenylation signal.
The yeast acid phosphatase can enter the secretory pathway without its N-terminal signal sequence.
Authors: Silve S , Monod M , Hinnen A , Haguenauer-Tsapis R
Published: September 2015

The repressible Saccharomyces cerevisiae acid phosphatase (APase) coded by the PHO5 gene is a cell wall glycoprotein that follows the yeast secretory pathway. We used in vitro mutagenesis to construct a deletion (delta SP) including the entire signal sequence and four amino acids of the mature sequence of APase. An APase-deficient yeast strain was transformed with a high-copy-number plasmid carrying the PHO5/delta SP gene. When expressed in vivo, the PHO5/delta SP gene product accumulated predominantly as an inactive, unglycosylated form located inside the cell. A large part of this unglycosylated precursor underwent proteolytic degradation, but up to 30% of it was translocated, core glycosylated, and matured by the addition of mannose residues, before reaching the cell wall. It appears, therefore, that the signal sequence is important for efficient translocation and core glycosylation of yeast APase but that it is not absolutely necessary for entry of the protein into the yeast secretory pathway. mRNA obtained by in vitro transcription of PHO5 and PHO5/delta SP genes were translated in vitro in the presence of either reticulocyte lysate and dog pancreatic microsomes or yeast lysate and yeast microsomes. The PHO5 gene product was translocated and core glycosylated in the heterologous system and less efficiently in the homologous system. We were not able to detect any translocation or glycosylation of PHO5/delta SP gene product in the heterologous system, but a very small amount of core suppression of glycosylated material could be evidenced in the homologous system.
Amplification of the IMP dehydrogenase gene in Chinese hamster cells resistant to mycophenolic acid.
Authors: Collart F R , Huberman E
Published: September 2015

The regulation of IMP dehydrogenase (IMPDH) was analyzed in Chinese hamster V79 cell variants that exhibit different degrees of resistance to the cytotoxic effect of mycophenolic acid, a specific inhibitor of IMPDH. Western blot (immunoblot) analysis with an IMPDH antiserum revealed a 14- to 27-fold increase in the amount of enzyme in the mycophenolic acid-resistant cells. The antiserum was also used to screen for a phage containing the IMPDH cDNA sequence from a lambda gt11 expression library. Northern blot (RNA blot) analyses of total cellular and poly(A)+ RNA showed that an IMPDH cDNA probe hybridized to a 2.2-kilobase transcript, the amount of which was associated with increased resistance. Southern blotting with the probe indicated an amplification of the IMPDH gene in the mycophenolic acid-resistant cells. Our findings suggest that the acquired mycophenolic acid resistance of the V79 cell variants is associated with increases in the amount and activity of IMPDH and the number of IMPDH gene copies.
Developmental regulation and identification of an isotype encoded by altB, an alpha-tubulin locus in Physarum polycephalum.
Authors: Green L L , Schroeder M M , Diggins M A , Dove W F
Published: September 2015

A subcloned portion of the 5' nontranslated sequence from a Physarum alpha-tubulin cDNA is specific for a single alpha-tubulin locus, altB, of Physarum polycephalum. We find that this locus is expressed only in the plasmodium and encodes at least an alpha 1-tubulin isotype, which we have designated alpha 1B. Hybridization patterns of other subclones of this cDNA reveal two sequences for alpha-tubulin at the altB locus.
Unique DNA repair properties of a xeroderma pigmentosum revertant.
Authors: Cleaver J E , Cortés F , Lutze L H , Morgan W F , Player A N , Mitchell D L
Published: September 2015

A group A xeroderma pigmentosum revertant with normal sensitivity was created by chemical mutagenesis. It repaired (6-4) photoproducts normally but not pyrimidine dimers and had near normal levels of repair replication, sister chromatid exchange, and mutagenesis from UV light. The rate of UV-induced mutation in a shuttle vector, however, was as high as the rate in the parental xeroderma pigmentosum cell line.
One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes.
Authors: Fusco A , Berlingieri M T , Di Fiore P P , Portella G , Grieco M , Vecchio G
Published: September 2015

A system of epithelial cells is described in which it is possible to study the number and the nature of genes capable of conferring the malignant phenotype. Two fully differentiated, hormone-responsive cell lines from rat thyroid glands are presented which are susceptible to one-step or two-step transformation upon infection with several murine acute retroviruses. After infection, both cell lines became independent from their thyrotropic hormone requirement for growth. However, complete transformation was achieved with one of the cell lines (FRTL-5 Cl 2), whereas the other cell line (PC Cl 3) failed to grow in agar and to give rise to tumors in vivo. The latter cell line was susceptible to complete transformation upon cooperation of the v-ras-Ha and the human c-myc oncogenes.
Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species.
Authors: Leto T L , Fortugno-Erikson D , Barton D , Yang-Feng T L , Francke U , Harris A S , Morrow J S , Marchesi V T , Benz E J
Published: September 2015

The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.
DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins.
Authors: Safer B , Cohen R B , Garfinkel S , Thompson J A
Published: September 2015

A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. 32P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of 32P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.
Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms.
Authors: White M K , Weber M J
Published: September 2015

Transformation of both rat and chicken fibroblasts by the src oncogene leads to a four- to fivefold increase in the rate of glucose transport and in the level of the glucose transporter protein. We have previously shown that, with chicken embryo fibroblasts, transformation leads to a reduction in the rate of degradation of the transporter, with little or no increase in the rate of its biosynthesis. We now show that, with the rat-1 cell line, the opposite result was obtained. src-induced transformation led to an increase in transporter biosynthesis, with little effect on turnover. A src-induced increase in transporter mRNA entirely accounted for the increase in biosynthesis of the protein. By contrast, in chicken embryo fibroblasts, the level of transporter mRNA was low and was not induced to rise by src transformation. Thus, src induced an increase in the level of the glucose transport protein by fundamentally different mechanisms in chicken embryo fibroblasts and rat-1 cells. To test whether this difference was due to rat-1 cells being an immortalized cell line, we measured transporter mRNA levels in primary fibroblast cultures from rat embryos and in parallel cultures transformed by src. Transporter mRNA was inducible by src in these cells. Thus, the difference in mRNA inducibility between chicken and rat cells is not due to immortalization.
A developmentally regulated membrane protein gene in Dictyostelium discoideum is also induced by heat shock and cold shock.
Authors: Maniak M , Nellen W
Published: September 2015

We have analyzed the expression of the Dictyostelium gene P8A7 which had been isolated as a cDNA clone from an early developmentally regulated gene. The single genomic copy generated two mRNAs which were subject to different control mechanisms: while one mRNA (P8A7S) was regulated like the cell-type-nonspecific late genes, the other one (P8A7L) was induced during development, when cells were allowed to attach to a substrate, and when cells were subjected to stress, such as heat shock and cadmium. Interestingly the same induction was also observed with cold shock. RNA processing was inhibited by heat and cold shock, leading to nuclear accumulation of a precursor. The translated region of the cDNA was common to both mRNAs and encoded an unusually hydrophobic peptide with the characteristics of a membrane protein.
Cloning and characterization of a cDNA encoding transformation-sensitive tropomyosin isoform 3 from tumorigenic human fibroblasts.
Authors: Lin C S , Leavitt J
Published: September 2015

We isolated a cDNA clone from the tumorigenic human fibroblast cell line HuT-14 that contains the entire protein coding region of tropomyosin isoform 3 (Tm3) and 781 base pairs of 5'- and 3'-untranslated sequences. Tm3, despite its apparent smaller molecular weight than Tm1 in two-dimensional gels, has the same peptide length as Tm1 (284 amino acids) and shares 83% homology with Tm1. Tm3 cDNA hybridized to an abundant mRNA of 1.3 kilobases in fetal muscle and cardiac muscle, suggesting that Tm3 is related to an alpha fast-tropomyosin. The first 188 amino acids of Tm3 are identical to those of rat or rabbit skeletal muscle alpha-tropomyosin, and the last 71 amino acids differ from those of rat smooth muscle alpha-tropomyosin by only 1 residue. Tm3 therefore appears to be encoded by the same gene that encodes the fast skeletal muscle alpha-tropomyosin and the smooth muscle alpha-tropomyosin via an alternative RNA-splicing mechanism. In contrast to Tm4 and Tm5, Tm3 has a small gene family, with, at best, only one pseudogene.
Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing.
Authors: Kaye F , Battey J , Nau M , Brooks B , Seifter E , De Greve J , Birrer M , Sausville E , Minna J
Published: September 2015

We analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs there is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.
Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.
Authors: Wang Y Y , Maher V M , Liskay R M , McCormick J J
Published: September 2015

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.
Expression and biosynthetic variation of the epidermal growth factor receptor in human hepatocellular carcinoma-derived cell lines.
Authors: Carlin C R , Simon D , Mattison J , Knowles B B
Published: September 2015

Expression of the epidermal growth factor (EGF) was analyzed in six human hepatocellular carcinoma-derived and one human hepatoblastoma-derived cell line, each of which retained the differentiated phenotype and functions of the parenchymal hepatocyte. The level of receptor expression of each hepatoma cell line was similar to that of the normal human fibroblast, approximately 10(5) molecules per cell. However, NPLC/PRF/5, a subline of the PLC/PRF/5 cell line obtained following reestablishment of a xenograft tumor in vitro, was found to express 4 x 10(6) high-affinity EGF receptor molecules per cell. Proliferation of the NPLC/PRF/5 cell line was inhibited in the presence of nanomolar quantities of ligand. Receptor overexpression was found to result from EGF receptor gene amplification without apparent rearrangement of the EGF receptor coding sequences. Although cell-specific variability in posttranslational processing of EGF receptor N-linked oligosaccharides in the hepatoma cell lines was found, no difference between the receptors in PLC/PRF/5 and NPLC/PRF/5 was observed and no aberrant receptor-related species were detected. EGF receptor gene amplification in the NPLC/PRF/5 cell line is probably a reflection of genome instability and selection of variants with augmented growth potential in limiting concentrations of EGF in vivo. When viewed in this light, EGF receptor overexpression could represent a manifestation of tumor progression in the EGF-responsive hepatocyte.
Transcriptional and posttranscriptional control of c-fos gene expression in human monocytes.
Authors: Sariban E , Luebbers R , Kufe D
Published: September 2015

We examined the mechanisms that are responsible for the regulation of c-fos gene expression in human monocytes. Levels of c-fos mRNA were low or undetectable in resting monocytes. Results of run-on transcription assays, however, demonstrated that both the first two and last two exons of the c-fos gene were transcribed at similar rates, and that only the sense strand of this gene was transcribed. These findings suggest that the level of c-fos transcripts in resting human monocytes is controlled at a posttranscriptional level. Activation of resting monocytes with phorbol ester was associated with a rapid and transient increase in c-fos mRNA levels. This increase in c-fos transcripts was related to an enhanced rate of c-fos transcription. Moreover, exposure of resting monocytes to inhibitors of protein synthesis induced (i) a rapid and marked (300-fold) increase in c-fos mRNA levels, despite only a 9-fold increase in c-fos transcription, and (ii) a prolongation of the half-life of c-fos mRNA. Thus, while posttranscriptional control was responsible for the down-regulation of c-fos transcripts in both resting and activated human monocytes, transcriptional mechanisms were responsible for the transient increase in c-fos expression induced by phorbol ester. Furthermore, the marked increases in c-fos mRNA associated with inhibition of protein synthesis were regulated by both transcriptional and posttranscriptional mechanisms. These findings may be related to recent observations which indicate that both positive and negative factors transcriptionally regulate c-fos gene expression and that sequences found in the 3'-untranslated region of the c-fos mRNA are responsible for the stability of this transcript.
A specific domain of the adenovirus EIV promoter is necessary to maintain susceptibility of the integrated promoter to EIA transactivation.
Authors: Nishigaki T , Hanaka S , Kingston R E , Handa H
Published: September 2015

We constructed a series of mutations that delete sequences in the promoter region of the early-region IV (EIV) promoter of adenovirus type 5. We fused these promoter mutations to the coding sequences of either the chloramphenicol acetyltransferase or the dihydrofolate reductase (DHFR) gene and tested the ability of a cotransfected EIa gene to stimulate EIV expression. All of the mutations tested were stimulated in these assays, implying that no specific sequence is required for stimulation. Two mutant promoters, deleted for either the TATA box or the region residing between -39 and -177 upstream from the cap site of EIV mRNA, did show a reduced level of stimulation by the EIa products. To assess the effects of the EIA gene products on expression from an EIV promoter integrated into the chromosome, we isolated CHO cell lines containing EIV-DHFR chimeric genes. After introduction of the EIa gene with a second selectable marker, expression from all mutant EIV-DHFR genes was increased. Surprisingly, one mutant promoter, deleted for sequences between -39 and -177, lost the ability to respond to the EIa region on passage of cells, although deletions in any part of the region still retained this ability. These results demonstrate that multiple elements residing between -39 and -177 in the EIV promoter are necessary to maintain susceptibility of the integrated promoter to regulation.
Hepatocyte-stimulating factor, beta 2 interferon, and interleukin-1 enhance expression of the rat alpha 1-acid glycoprotein gene via a distal upstream regulatory region.
Authors: Prowse K R , Baumann H
Published: September 2015

The rat alpha 1-acid glycoprotein (AGP) gene is transcriptionally regulated by dexamethasone, interleukin 1 (IL-1), hepatocyte-stimulating factor, and beta 2 interferon. The steroid and peptide hormones stimulate expression of the AGP gene synergistically as well as independently. The regulatory sequence responsible for dexamethasone-stimulated expression has been localized previously to a region that is 120 to 64 base pairs (bp) upstream of the transcription start site (H. Baumann and L. E. Maquat, Mol. Cell. Biol. 6:2551-2561, 1986). To identify the regulatory sequence that is responsive to the peptide hormones, different lengths of the AGP gene 5'-flanking DNA were linked to the chloramphenicol acetyltransferase gene and then assayed for hormone-inducible chloramphenicol acetyltransferase gene expression in transiently transfected HepG2 cells. We demonstrate that an enhancer region that is responsive to IL-1, hepatocyte-stimulating factor, and beta 2 interferon lies within a 142-bp sequence located 5,300 to 5,150 bp upstream of the transcription start site. This distal regulatory region can confer hormone inducibility to a heterologous promoter; exert its affect in either orientation; and function, to a lesser degree, in nonhepatic but IL-1-responsive cells.
Sequence and expression of a type II keratin, K5, in human epidermal cells.
Authors: Lersch R , Fuchs E
Published: September 2015

We report here the cDNA and amino acid sequences of a human 58-kilodalton type II keratin, K5, which is coexpressed with a 50-kilodalton type I keratin partner, K14, in stratified squamous epithelia. Using a probe specific for the 3'-noncoding portion of this K5 cDNA, we demonstrated the existence of a single human gene encoding this sequence. Using Northern (RNA) blot analysis and in situ hybridization with cRNA probes for both K5 and K14, we examined the expression of these mRNAs in the epidermis and in cultured epidermal cells. Our results indicate that the mRNAs for K5 and K14 are coordinately expressed and abundant in the basal layer of the epidermis. As cells undergo a commitment to terminally differentiate, the expression of both mRNAs seems to be downregulated.
SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae.
Authors: Wilson R B , Tatchell K
Published: September 2015

sra5 mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km cyclic AMP (cAMP) phosphodiesterase activity. We have cloned SRA5 by complementation. It maps to the right arm of chromosome XV, tightly linked to PRT1, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase. Disruptions of SRA5 allowed ras1 ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP. sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP.
An adenylate cyclase from Saccharomyces cerevisiae that is stimulated by RAS proteins with effector mutations.
Authors: Marshall M S , Gibbs J B , Scolnick E M , Sigal I S
Published: September 2015

Conservative amino acid substitutions were introduced into the proposed effector regions of both mammalian Ha-ras (residues 32 to 40) and Saccharomyces cerevisiae RAS2 (residues 39 to 47) proteins. The RAS2[Ser 42] protein had reduced biological function in the yeast S. cerevisiae. A S. cerevisiae strain with a second-site suppressor mutation, SSR2-1, was isolated which could grow on nonfermentable carbon sources when the endogenous RAS2 protein was replaced by the RAS2[Ser 42] protein. The SSR2-1 mutation was mapped to the structural gene for adenylate cyclase (CYR1), and the gene containing SSR2-1 was cloned and sequenced. SSR2-1 corresponded to a point mutation that would create an amino acid substitution of a tyrosine residue for an aspartate residue at position 1547. The SSR2-1 gene encodes an adenylate cyclase that is dependent on ras proteins for activity, but is stimulated by Ha-ras and RAS2 mutant proteins that are unable to stimulate wild-type adenylate cyclase.
Effect of heat shock on ribosome synthesis in Drosophila melanogaster.
Authors: Bell J , Neilson L , Pellegrini M
Published: September 2015

In Drosophila tissue culture cells, the synthesis of ribosomal proteins was inhibited by a 1-h 37 degrees C heat shock. Ribosomal protein synthesis was repressed to a greater extent than that of most other proteins synthesized by these cells at 25 degrees C. After a 1-h heat shock, when the cells were returned to 25 degrees C, the ribosomal proteins were much slower than most other 25 degrees C proteins to return to pre-heat shock levels of synthesis. Relative to one another, all the ribosomal proteins were inhibited and later recovered to normal levels of synthesis at the same rate and to the same extent. Unlike the ribosomal proteins, the precursor to the large rRNAs was continually synthesized during heat shock, although at a slightly reduced level, but was not processed. It was rapidly degraded, with a half-life of approximately 16 min. Pre-heat shock levels of synthesis, stability, and correct processing were restored only when ribosomal protein synthesis returned to at least 50% of that seen in non-heat-shocked cells.
Reduction of endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells.
Authors: Dorner A J , Krane M G , Kaufman R J
Published: September 2015

GRP78 is localized in the endoplasmic reticulum and associates with improperly folded or underglycosylated proteins. The role of GRP78 in secretion was studied in Chinese hamster ovary cells expressing a tissue plasminogen activator (tPA) variant which lacks potential N-linked glycosylation site sequences because of mutagenesis. The expression of variant tPA resulted in elevated levels of GRP78 and its stable association with tPA. The introduction of antisense GRP78 genes resulted in a two- to threefold reduction in GRP78 levels compared with those of the original cells. Cells with reduced levels of GRP78 secreted two- to threefold-higher levels of tPA activity. tPA expressed in these cells displayed reduced association with GRP78, and a greater proportion was processed to the mature form and secreted. These results demonstrate that reduction of GRP78 level can improve the secretion of an associated protein.
Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures.
Authors: Young J C , Witte O N
Published: September 2015

The BCR/ABL gene, formed by the Philadelphia chromosome translocation (Ph1) of human chronic myelogenous leukemia, encodes an altered ABL gene product, P210. P210 is strongly implicated in the malignant process of chronic myelogenous leukemia, but it precise role is unknown. Infection of long-term bone marrow cultures enriched for B-lymphoid cell types with a Moloney murine leukemia virus retroviral vector containing the BCR/ABL cDNA resulted in clonal outgrowths of immature B-lymphoid cells which expressed abundant P210 kinase activity. Surprisingly, infection of long-term myeloid lineage-enriched cultures also resulted in clonal outgrowths of immature B-lymphoid cells. The P210-expressing lymphoid cell lines resulting from either type of culture were resistant to the lethal effects of corticosteroids. These findings indicate that high levels of P210 expressed from a Moloney murine leukemia virus long terminal repeat preferentially stimulate the growth of immature B-lineage cells, and this effect is apparent even in myeloid lineage-enriched cultures, in which few if any lymphoid cells can be detected prior to infection.
mRNA decay rates in late-developing Dictyostelium discoideum cells are heterogeneous, and cyclic AMP does not act directly to stabilize cell-type-specific mRNAs.
Authors: Manrow R E , Jacobson A
Published: September 2015

We reevaluated the use of 32PO4 pulse-chases for analyzing mRNA decay rates in late-developing Dictyostelium cells. We found that completely effective PO4 chases could not be obtained in developing cells and that, as a consequence, the decay rates exhibited by some mRNAs were influenced by the rates at which they were transcribed. In developing cells disaggregated in the presence of cyclic AMP, the poly(A)+ mRNA population turned over with an apparent half-life of 4 h, individual mRNA decay rates were heterogeneous, and some prestalk and prespore mRNAs appeared to decay with biphasic kinetics. In cells disaggregated in the absence of cyclic AMP, all prestalk and prespore mRNAs decayed with biphasic kinetics. During the first 1 to 1.5 h after disaggregation in the absence of cyclic AMP, the cell-type-specific mRNAs were selectively degraded, decaying with half-lives of 20 to 30 min; thereafter, the residual prestalk and prespore mRNA molecules decayed at rates that were similar to those measured in the presence of cyclic AMP. This short-term labilization of cell-type-specific mRNAs was observed even for those species not requiring cyclic AMP for their accumulation in developing cells. The observation that cell-type specific mRNAs can decay at similar rates in disaggregated cells with or without cyclic AMP indicates that this compound does not act directly to stabilize prestalk and prespore mRNAs during development and that its primary role in the maintenance of cyclic-AMP-dependent mRNAs is likely to be transcriptional.
A common factor regulates skeletal and cardiac alpha-actin gene transcription in muscle.
Authors: Muscat G E , Gustafson T A , Kedes L
Published: September 2015

The skeletal and cardiac alpha-actin genes are coexpressed in muscle development but exhibit distinctive tissue-specific patterns of expression. We used an in vivo competition assay and an in vitro electrophoretic mobility shift assay to demonstrate that both genes interact with a common trans-acting factor(s). However, there was at least one gene-specific cis-acting sequence in the skeletal alpha-actin gene that interacted with a trans-acting factor which was not rate limiting in the expression of the cardiac alpha-actin gene. The common factor(s) interacted with several cis-acting regions that corresponded to sequences that are required for the transcriptional modulation of these sarcomeric alpha-actin genes in muscle cells. These regulatory regions contained the sequence motif CC(A + T-rich)6GG, which is known as a CArG box. Results of in vivo competition assays demonstrated that the factor(s) bound by the skeletal alpha-actin gene is also essential for the maximal activity of the cardiac alpha-actin, simian virus 40 (SV40), alpha 2(I)-collagen, and the beta-actin promoters in muscle cells. In contrast, fibroblastic cells contained functionally distinct transcription factor(s) that were used by the SV40 enhancer but that did not interact with the sarcomeric alpha-actin cis-acting sequences. The existence of functionally different factors in these cell types may explain the myogenic specificity of these sarcomeric alpha-actin genes. Results of in vitro studies suggested that both the sarcomeric alpha-actin genes interact with the CArG box-binding factor CBF and that the skeletal alpha-actin promoter contains multiple CBF-binding sites. In contrast, CBF did not interact in vitro with a classical CAAT box, the SV40 enhancer, or a linker scanner mutation of an alpha-actin CArG box. Furthermore, methylation interference and DNase I footprinting assays demonstrated the precise sites of interaction of CBF with three CArG motifs at positions -98, -179, and -225 in the human skeletal alpha-actin gene.
Expression of the troponin complex genes: transcriptional coactivation during myoblast differentiation and independent control in heart and skeletal muscles.
Authors: Bucher E A , Maisonpierre P C , Konieczny S F , Emerson C P
Published: September 2015

We compared the developmental regulation of the three troponin genes that encode the proteins of the Ca2+ regulatory complex in striated muscles of the Japanese quail. Nuclear run-on transcription and RNA protection analyses showed that the fast skeletal troponin I, the fast skeletal troponin T, and the slow skeletal-cardiac troponin C genes were transcriptionally coactivated and that transcripts rapidly accumulated within 6 to 12 h after the initiation of myoblast differentiation. The fast-isoform mRNAs of troponin I and troponin T were coexpressed at similar levels in different skeletal muscles, whereas the slow-cardiac troponin C mRNA varied independently and was the only one of these genes expressed in embryonic and adult heart. We conclude that these troponin genes are transcriptionally coactivated during skeletal myoblast differentiation, indicating that their transcription is under precise temporal control. However, this troponin C gene is regulated independently is specialized striated muscles.
Gene induction by interferons and double-stranded RNA: selective inhibition by 2-aminopurine.
Authors: Tiwari R K , Kusari J , Kumar R , Sen G C
Published: September 2015

Transcription of several interferon-inducible human genes is also induced by double-stranded RNA. The nature and the mechanism of action of signals generated by interferons and by double-stranded RNA which mediate the induction of these genes are under investigation. Here we report that 2-aminopurine, a known inhibitor of protein kinases, could selectively block this induction process. Induction of mRNAs 561 and 6-16 in HeLaM cells by double-stranded RNA was completely inhibited by 10 mM 2-aminopurine, whereas cellular protein and RNA syntheses as well as the induction of metallothionein mRNA by CdCl2 were unaffected by this inhibitor. In addition, 2-aminopurine blocked the induction of the same two mRNAs and of mRNAs 2-5(A) synthetase, 2A, and 1-8 by alpha interferon and of mRNAs 2A and 1-8 by gamma interferon in HeLaM cells. The observed inhibition was at the level of transcription, and for establishing efficient inhibition, the 2-aminopurine treatment had to begin at early stages of interferon treatment. In GM2767 cells, 2-aminopurine inhibited induction of mRNAs 561 and 6-16 by double-stranded RNA but not by alpha interferon. These results suggest that double-stranded RNA-induced signal 2 is distinct from the interferon-alpha-induced signal 2 (R. K. Tiwari, J. Kusari, and G. C. Sen, EMBO J. 6:3373-3378, 1987) and that 2-aminopurine can block the former but not the latter. Moreover, it appeared that 2-aminopurine could block the production of signal 1 by interferons. This was confirmed by experiments in which we separately tested the effects of 2-aminopurine on signal 1 and signal 2 production by interferons in HeLaM cells. Although no direct experimental evidence is available as yet, our results are consistent with the hypothesis that the functioning of a protein kinase activity may be necessary for transcriptional induction of genes by double-stranded RNA and for gene induction by interferons in those cells in which signal 1 production is needed.
In vitro synthesis of pp60v-src: myristylation in a cell-free system.
Authors: Deichaite I , Casson L P , Ling H P , Resh M D
Published: September 2015

Covalent attachment of myristic acid to pp60v-src, the transforming protein of Rous sarcoma virus, was studied in a cell-free system. Using a synthetic peptide containing the first 11 amino acids of the mature pp60v-src polypeptide sequence as a substrate, we probed lysates from a variety of cells and tissues for N-myristyl transferase (NMT) activity. Nearly every eucaryotic cell type tested contained NMT, including avian, mammalian, insect, and plant cells. Since NMT activity was detected in rabbit reticulocyte lysates, we took advantage of the translational capability of these lysates to determine the precise point during translation at which myristate is attached to pp60v-src. src mRNA, transcribed from cloned v-src DNA, was translated in reticulocyte lysates which had been depleted of endogenous myristate. Addition of [3H]myristate to lysates 10 min after the start of synchronized translation resulted in a dramatic decrease in the incorporation of radiolabeled myristate into pp60v-src polypeptide chains. These results imply that although myristate can be attached posttranslationally to synthetic peptide substrates, myristylation in vivo is apparently a very early cotranslational event which occurs before the first 100 amino acids of the nascent polypeptide chain are polymerized.
Formation of an inverted duplication can be an initial step in gene amplification.
Authors: Ruiz J C , Wahl G M
Published: September 2015

We have developed a gene transfer approach to facilitate the identification and isolation of chromosomal regions which are prone to high-frequency gene amplification. Such regions are identified by assaying for transformants which show high-frequency resistance to PALA and/or methotrexate by amplification of a vector containing the genes which encode the enzyme targets of these antiproliferative agents. We identified 2 of 47 transformants which displayed high-frequency amplification of the transfected genes, and in this report we describe the analysis of one of them (L46). Molecular analysis of the integration site in transformant L46 revealed that the donated genes were at the center of an inverted duplication which spanned more than 70 kilobase pairs and consisted largely of host DNA. The data suggest that integration of the transfected sequences generates a submicroscopic molecule containing the inverted duplication and at least 750 kilobases of additional sequences. The donated sequences and the host sequences were readily amplified and lost in exponentially growing cultures in the absence of drug selection, which suggests that the extrachromosomal elements are acentric. In contrast to the instability of this region following gene insertion, the preinsertion site was maintained at single copy level under growth conditions which produced copy number heterogeneity in L46. The implications of our results for mechanisms of genetic instability and mammalian gene amplification are discussed.