bluuebunny/crossref_metadata_2025 huggingface dataset:
Domains of beta-tubulin essential for conserved functions in vivo.
Authors: Fridovich-Keil J L , Bond J F , Solomon F
Published: September 2015

The relationship between the primary sequence of tubulins and their properties in cells was studied by gene transfection experiments. Previously, we studied a chimeric beta-tubulin formed from chicken beta-tubulin-2 sequences in the amino-terminal portion and the highly divergent Saccharomyces cerevisiae TUB2 sequences in the carboxy-terminal 25% of the molecule. In the cytoplasm of cultured animal cells, this protein incorporates into all microtubule structures and assembles with the same efficiency as endogenous tubulin. We show that the protein products of chimeric genes with an increasing proportion of yeast sequence, extending 5' of the carboxy-terminal 25%, are abnormal in two ways. First, they assemble with a significantly lower efficiency than the original chimeric protein or the endogenous tubulins. Second, they are less stable in the cytoplasm. The results suggest that the position of the yeast sequences is crucial in determining the properties of the molecule. Results of analyses of 1 deletion mutation and 10 linker insertions in the original chimeric tubulin suggest that those changes made outside the carboxyl terminus completely disrupt assembly activity, while those made in the carboxyl terminus do not.
Insertions of up to 17 amino acids into a region of alpha-tubulin do not disrupt function in vivo.
Authors: Schatz P J , Georges G E , Solomon F , Botstein D
Published: September 2015

Microtubules in yeasts are essential components of the mitotic and meiotic spindle and are necessary for nuclear movement during cell division and mating. The yeast Saccharomyces cerevisiae has two alpha-tubulin genes, TUB1 and TUB3, either of which alone is sufficient for these processes when present in a high enough copy number. Comparisons of sequences from several species reveals the presence of a variable region near the amino terminus of alpha-tubulin proteins. We perturbed the structure of this region in TUB3 by inserting into it 3, 9, or 17 amino acids and tested the ability of these altered proteins to function as the only alpha-tubulin protein in yeast cells. We found that each of these altered proteins was sufficient on its own for mitotic growth, mating, and methods of yeast. We conclude that this region can tolerate considerable variation without losing any of the highly conserved functions of alpha-tubulin. Our results suggest that variability in this region occurs because it can be tolerated, not because it specifies an important function for the protein.
Regulation by the yeast mating-type locus of STE12, a gene required for cell-type-specific expression.
Authors: Fields S , Herskowitz I
Published: September 2015

The STE12 gene of the yeast Saccharomyces cerevisiae is necessary for RNA synthesis from two sets of cell-type-specific genes. We isolated a recombinant plasmid that carries the STE12 gene by complementation of the mating defect of ste12 cells. The DNA of the cloned gene was used to disrupt the chromosomal STE12 gene and to identify the STE12 transcript. We show that the STE12 transcript level is repressed 5- to 10-fold in a/alpha cells. The STE12 product thus acts to promote diploidy by activating expression of the two sets of genes necessary for mating, and then its synthesis is repressed by products unique to the diploid cell type.
Developmentally regulated expression of a truncated myosin light-chain 1F/3F gene.
Authors: Garfinkel L I , Davidson N
Published: September 2015

Fast skeletal muscle myosin light-chain I (MLC1f) and myosin light-chain 3 (MLC3f) mRNAs are both derived from a single rat MLC1/3f gene. MLC1f mRNA begins at the first exon of the gene, while MLC3f mRNA begins with exon 2, 10 kilobases downstream. Both mRNAs require alternate splicing of internal exons for accurate expression. We showed that a truncated rat MLC1f/3f gene lacking exon 1 and the first 6.3 kilobases of the intron separating exons 1 and 2 produced rat MLC3f mRNA in a developmentally regulated manner after introduction into myogenic mouse cells, thus demonstrating in vivo the presence of a functional promoter associated with exon 2. Correctly spliced mRNA was produced after transfer of this truncated gene into both myogenic and nonmyogenic cells, indicating that the pattern of splicing of this complex transcript was due to a structural features of the RNA and was independent of cell type.
Induction of cell cycle progression by adenovirus E1A gene 13S- and 12S-mRNA products in quiescent rat cells.
Authors: Nakajima T , Masuda-Murata M , Hara E , Oda K
Published: September 2015

Rat 3Y1 cell lines that express either adenovirus type 12 E1A 13S mRNA or 12S mRNA in response to dexamethasone treatment were established by introduction of recombinant vector DNA containing the E1A 13S- or 12S-mRNA cDNA placed downstream of the hormone-inducible promoter of mouse mammary tumor virus. These cell lines were growth arrested, and the induction of cell cycle progression was analyzed by flow cytometry after switch on of the cDNA by the addition of dexamethasone. The results indicate that the 13S- or 12S-mRNA product alone has the ability to cause progression of the cell cycle at a similar rate. The simultaneous addition of epidermal growth factor accelerated the rate of cell cycle progression in the transition from the G0/G1 phase to the S phase.
H5 gene specific trans-activation by nuclear extracts from avian erythroid cells.
Authors: Wigley P L , Wells J R
Published: September 2015

Nuclear extracts from chicken erythroid cells selectively stimulate transcription of the chicken histone H5 gene (and not of other chicken histone genes) after coinjection into frog oocytes. This effect is shown to involve an enhancerlike activity, and a region of the H5 gene sufficient to mediate trans-activation is defined.
Expression of HLA-DR antigen in human class II mutant B-cell lines by double infection with retrovirus vectors.
Authors: Yang Z , Korman A J , Cooper J , Pious D , Accolla R S , Mulligan R C , Strominger J L
Published: September 2015

A new retrovirus vector containing the gene for hygromycin B resistance (hyg) as a selectable marker under the control of an internal simian virus 40 promoter was constructed. It was used, together with an analogous previously described vector, DO1, which contains the gene for G418 resistance, to introduce and express the genes for the two chains of a human class II major histocompatibility complex antigen in NIH 3T3 cells. In addition, these vectors were used to express DR antigens in two human mutant B-lymphoblastoid cell lines, one of which was deleted for both alleles of the DR alpha gene and the other of which expressed no class II antigens because of a genetic defect in a putative trans-acting regulatory factor.
Expression of transfected vimentin genes in differentiating murine erythroleukemia cells reveals divergent cis-acting regulation of avian and mammalian vimentin sequences.
Authors: Ngai J , Bond V C , Wold B J , Lazarides E
Published: September 2015

We studied the expression of transfected chicken and hamster vimentin genes in murine erythroleukemia (MEL) cells. MEL cells normally repress the levels of endogenous mouse vimentin mRNA during inducermediated differentiation, resulting in a subsequent loss of vimentin filaments. Expression of vimentin in differentiating MEL cells reflects the disappearance of vimentin filaments during mammalian erythropoiesis in vivo. In contrast, chicken erythroid cells express high levels of vimentin mRNA and vimentin filaments during terminal differentiation. We demonstrate here that chicken vimentin mRNA levels increase significantly in differentiating transfected MEL cells, whereas similarly transfected hamster vimentin genes are negatively regulated. In conjunction with in vitro nuclear run-on transcription experiments, these results suggest that the difference in vimentin expression in avian and mammalian erythropoiesis is due to a divergence of cis-linked vimentin sequences that are responsible for transcriptional and posttranscriptional regulation of vimentin gene expression. Transfected chicken vimentin genes produce functional vimentin protein and stable vimentin filaments during MEL cell differentiation, further demonstrating that the accumulation of vimentin filaments is determined by the abundance of newly synthesized vimentin.
Tissue-specific enhancer of the human glycoprotein hormone alpha-subunit gene: dependence on cyclic AMP-inducible elements.
Authors: Delegeane A M , Ferland L H , Mellon P L
Published: September 2015

We identified and characterized elements which confer tissue specificity and cyclic AMP (cAMP) responsiveness to the human glycoprotein alpha-subunit gene. An enhancer containing an 18-base-pair repeat conferred cAMP responsiveness in a non-tissue-specific fashion. DNase I protection assays revealed DNA-binding factors that bound to this element in both placental and nonplacental cells. It also enhanced the alpha-subunit promoter in a tissue-specific manner but had a negligible effect on a heterologous promoter. A unique element found upstream of this enhancer had no independent activity but, in combination with the cAMP-responsive enhancer, distinctly increased the tissue-specific activity of both the alpha-subunit promoter and a heterologous promoter. A factor that bound to this upstream element was found in placental but not nonplacental cells. We conclude that this novel element acts, perhaps through a specific trans-acting factor, in concert with a cAMP-responsive enhancer to confer tissue specificity to the alpha-subunit gene.
Amino acid sequences that determine the nuclear localization of yeast histone 2B.
Authors: Moreland R B , Langevin G L , Singer R H , Garcea R L , Hereford L M
Published: September 2015

Histone-beta-galactosidase protein fusions were used to identify the domain of yeast histone 2B, which targets this protein to the nucleus. Amino acids 28 to 33 in H2B were required for nuclear localization of such fusion proteins and thus constitute a nuclear localization sequence. The amino acid sequence in this region (Gly-29 Lys Lys Arg Ser Lys Ala) is similar to the nuclear location signal in simian virus 40 large T antigen (Pro-126 Lys Lys Lys Arg Lys Val) (D. Kalderon, B.L. Roberts, W.D. Richardson, and A.E. Smith, Cell 39:499-509, 1984). A point mutation changing lysine 31 to methionine abolished nuclear localization of an H2B-beta-galactosidase fusion protein containing amino acids 1 to 33 of H2B. However, an H2B-beta-galactosidase fusion protein containing both this point mutation and the H2A interaction domain of H2B was nuclear localized. These results suggest that H2A and H2B may be cotransported to the nucleus as a heterodimer.
Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly.
Authors: Rich B E , Steitz J A
Published: September 2015

cDNA clones encoding three antigenically related human ribosomal phosphoproteins (P-proteins) P0, P1, and P2 were isolated and sequenced. P1 and P2 are analogous to Escherichia coli ribosomal protein L7/L12, and P0 is likely to be an analog of L10. The three proteins have a nearly identical carboxy-terminal 17-amino-acid sequence (KEESEESD(D/E)DMGFGLFD-COOH) that is the basis of their immunological cross-reactivity. The identities of the P1 and P2 cDNAs were confirmed by the strong similarities of their encoded amino acid sequences to published primary structures of the homologous rat, brine shrimp, and Saccharomyces cerevisiae proteins. The P0 cDNA was initially identified by translation of hybrid-selected mRNA and immunoprecipitation of the products. To demonstrate that the coding sequences are full length, the P0, P1, and P2 cDNAs were transcribed in vitro by bacteriophage T7 RNA polymerase and the resulting mRNAs were translated in vitro. The synthetic P0, P1, and P2 proteins were serologically and electrophoretically identical to P-proteins extracted from HeLa cells. These synthetic P-proteins were incorporated into 60S but not 40S ribosomes and also assembled into a complex similar to that described for E. coli L7/L12 and L10.
Pleiotropic plasma membrane ATPase mutations of Saccharomyces cerevisiae.
Authors: McCusker J H , Perlin D S , Haber J E
Published: September 2015

We isolated a large number of mutations in the structural gene for the plasma membrane ATPase (PMA1) of Saccharomyces cerevisiae. These mutations were selected by their resistance to the aminoglycoside antibiotic hygromycin B. Biochemical analysis of purified membrane preparations showed that the plasma membrane ATPase activity of the mutants was reduced as much as 75%. Intragenic complementation of pma1 mutants suggested that the yeast plasma membrane ATPase was a multimeric enzyme. The pma1 mutants were apparently defective in maintaining internal pH; more than half of the mutants were unable to grow either at a low pH or in the presence of a weak acid. Most pma1 mutants were also osmotic pressure sensitive. At a very low temperature (5 degrees C) many pma1 mutants were unable to grow and were arrested as unbudded cells. The three most severely affected mutants were also unable to grow in the presence of NH4+. The most extreme mutant exhibited a severe defect in progression through the cell cycle; on synthetic medium, the cells progressively accumulated nucleus-containing small buds that generally failed to complete bud enlargement and cytokinesis. Most of the pleiotropic phenotypes of pma1 mutants could be suppressed by the addition of 50 mM KCl but not NaCl to the medium.
Differential patterns of transcript accumulation during human myogenesis.
Authors: Gunning P , Hardeman E , Wade R , Ponte P , Bains W , Blau H M , Kedes L
Published: September 2015

We evaluated the extent to which muscle-specific genes display identical patterns of mRNA accumulation during human myogenesis. Cloned satellite cells isolated from adult human skeletal muscle were expanded in culture, and RNA was isolated from low- and high-confluence cells and from fusing cultures over a 15-day time course. The accumulation of over 20 different transcripts was compared in these samples with that in fetal and adult human skeletal muscle. The expression of carbonic anhydrase 3, myoglobin, HSP83, and mRNAs encoding eight unknown proteins were examined in human myogenic cultures. In general, the expression of most of the mRNAs was induced after fusion to form myotubes. However, several exceptions, including carbonic anhydrase and myoglobin, showed no detectable expression in early myotubes. Comparison of all transcripts demonstrated little, if any, identity of mRNA accumulation patterns. Similar variability was also seen for mRNAs which were also expressed in nonmuscle cells. Accumulation of mRNAs encoding alpha-skeletal, alpha-cardiac, beta- and gamma-actin, total myosin heavy chain, and alpha- and beta-tubulin also displayed discordant regulation, which has important implications for sarcomere assembly. Cardiac actin was the only muscle-specific transcript that was detected in low-confluency cells and was the major alpha-actin mRNA at all times in fusing cultures. Skeletal actin was transiently induced in fusing cultures and then reduced by an order of magnitude. Total myosin heavy-chain mRNA accumulation lagged behind that of alpha-actin. Whereas beta- and gamma-actin displayed a sharp decrease after initiation of fusion and thereafter did not change, alpha- and beta-tubulin were transiently induced to a high level during the time course in culture. We conclude that each gene may have its own unique determinants of transcript accumulation and that the phenotype of a muscle may not be determined so much by which genes are active or silent but rather by the extent to which their transcript levels are modulated. Finally, we observed that patterns of transcript accumulation established within the myotube cultures were consistent with the hypothesis that myoblasts isolated from adult tissue recapitulate a myogenic developmental program. However, we also detected a transient appearance of adult skeletal muscle-specific transcripts in high-confluence myoblast cultures. This indicates that the initial differentiation of these myoblasts may reflect a more complex process than simple recapitulation of development.
Trans-acting regulatory mutations that alter transcription of Saccharomyces cerevisiae histone genes.
Authors: Osley M A , Lycan D
Published: September 2015

Using a Saccharomyces cerevisiae strain containing an integrated copy of an H2A-lacZ fusion gene, we screened for mutants which overexpressed beta-galactosidase as a way to identify genes which regulate transcription of the histone genes. Five recessive mutants with this phenotype were shown to contain altered regulatory genes because they had lost repression of HTA1 transcription which occurs upon inhibition of chromosome replication (D. E. Lycan, M. A. Osley, and L. Hereford, Mol. Cell. Biol. 7:614-621, 1987). Periodic transcription was affected in the mutants as well, since the HTA1 gene was transcribed during the G1 and G2 phases of the cell cycle, periods in the cell cycle when this gene is normally not expressed. A similar loss of cell cycle-dependent transcription was noted for two of the three remaining histone loci, while the HO and CDC9 genes continued to be expressed periodically. Using isolated promoter elements inserted into a heterologous cycl-lacZ fusion gene, we demonstrated that the mutations fell in genes which acted through a negative site in the TRT1 H2A-H2B promoter.
Induction of glucagon sensitivity in a transformed kidney cell line by prostaglandin E2 and its inhibition by epidermal growth factor.
Authors: Lin M C , Darfler F J , Beckner S K
Published: September 2015

A model system using a transformed dog kidney cell line (Madin-Darby canine kidney), has been established for studying the process of differentiation. Glucagon responsiveness can be restored to these transformed cells by various differentiation inducers, including prostaglandin E2. Glucagon response was measured in terms of the ability of glucagon to stimulate cAMP production. Induction of glucagon sensitivity seems to be mediated by cAMP. The ability of various prostaglandin analogs to elevate the cAMP level correlates closely with their ability to induce glucagon sensitivity. In fact, 8-Br-cAMP is also a potent inducer. To define the nature of this cAMP-mediated process, we identified several inhibitors of this induction process. These differentiation inhibitors include serum, phorbol ester, and epidermal growth factor. These inhibitors do not have a direct effect on cAMP production by cells in the presence or absence of hormones. Furthermore, induction by 8-Br-cAMP is also inhibited by these agents. Therefore, the site of inhibition is located beyond the point of cAMP production. Possible interaction between cAMP- and epidermal growth factor-dependent phosphorylations is discussed.
Posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability.
Authors: Theodorakis N G , Morimoto R I
Published: September 2015

We have examined the posttranscriptional regulation of hsp70 gene expression in two human cell lines, HeLa and 293 cells, which constitutively express high levels of HSP70. HSP70 mRNA translates with high efficiency in both control and heat-shocked cells. Therefore, heat shock is not required for the efficient translation of HSP70 mRNA. Rather, the main effect of heat shock on translation is to suppress the translatability of non-heat shock mRNAs. Heat shock, however, has a marked effect on the stability of HSP70 mRNA; in non-heat-shocked cells the half-life of HSP70 mRNA is approximately 50 min, and its stability increases at least 10-fold upon heat shock. Moreover, HSP70 mRNA is more stable in cells treated with protein synthesis inhibitors, suggesting that a heat shock-sensitive labile protein regulates its turnover. An additional effect on posttranscriptional regulation of hsp70 expression can be found in adenovirus-infected cells, in which HSP70 mRNA levels decline precipititously late during infection although hsp70 transcription continues unabated.
Identification of healed terminal DNA fragments in linear minichromosomes of Schizosaccharomyces pombe.
Authors: Matsumoto T , Fukui K , Niwa O , Sugawara N , Szostak J W , Yanagida M
Published: September 2015

The minichromosome Ch16 of the fission yeast Schizosaccharomyces pombe is derived from the centromeric region of chromosome III. We show that Ch16 and a shorter derivative, Ch12, made by gamma-ray cleavage, are linear molecules of 530 and 280 kilobases, respectively. Each minichromosome has two novel telomeres, as shown by genomic Southern hybridization with an S. pombe telomere probe. Comparison by hybridization of the minichromosomes and their chromosomal counterparts showed no signs of gross rearrangement. Cosmid clones covering the ends of the long arms of Ch16 and Ch12 were isolated, and subcloned fragments that contained the breakage sites were identified. They are apparently unique in the genome. By hybridization and Bal 31 digestion, the ends appear to consist of the broken-end sequences directly associated with short stretches (about 300 base pairs) of new DNA that hybridizes to a cloned S. pombe telomere. They do not contain the telomere-adjacent repeated sequences that are present in the normal chromosomes. The sizes of the short telomeric stretches are roughly the same as those of the normal chromosomes. Our results show that broken chromosomal ends in S. pombe can be healed by the de novo addition of the short telomeric repeats. The formation of Ch16 must have required two breakage-healing events, whereas a single cleavage-healing event in the long arm of Ch16 yielded Ch12.
Proline utilization in Saccharomyces cerevisiae: sequence, regulation, and mitochondrial localization of the PUT1 gene product.
Authors: Wang S S , Brandriss M C
Published: September 2015

The PUT1 gene of Saccharomyces cerevisiae, believed to encode proline oxidase, has been completely sequenced and contains an open reading frame capable of encoding a polypeptide of 476 amino acids in length. The amino terminus of the protein deduced from the DNA sequence has a characteristic mitochondrial import signal; two PUT1-lacZ gene fusions were constructed that produced mitochondrially localized beta-galactosidase in vivo. The transcription initiation and termination sites of the PUT1 mRNA were determined. By using a PUT1-lacZ gene fusion that makes a cytoplasmic beta-galactosidase, the regulation of the PUT1 gene was studied. PUT1 is inducible by proline, responds only slightly to carbon catabolite repression, and is not regulated by the cytochrome activator proteins HAP1 and HAP2. The PUT1 gene is under oxygen regulation; expression in anaerobically grown cells is 10-fold lower than in aerobically grown cells. Oxygen regulation is abolished when cells are respiratory deficient. PUT1 expression in a [rho-] strain grown either aerobically or anaerobically is as high as that seen in a [rho+] strain grown aerobically. Studies on PUT1 promoter deletions define a region between positions -458 and -293 from the translation initiation site that is important for full expression of the PUT1 gene and required for oxygen regulation.
Interferon-responsive regulatory elements in the promoter of the human 2',5'-oligo(A) synthetase gene.
Authors: Benech P , Vigneron M , Peretz D , Revel M , Chebath J
Published: September 2015

The interferon (IFN)-activated human 2',5'-oligo(A) synthetase E gene contains 11 RNA starts and lacks TATA and CAAT signals. DNA sequences around the promoter make the expression of the chloramphenicol acetyltransferase gene (CAT) inducible over 20-fold by IFN. A 72-base-pair segment (E-IRS) immediately upstream of the RNA starts was defined as being required for IFN-activated expression of the E-gene promoter-CAT constructs and acts in a position-independent manner. It also confers IFN-activated enhancement to the herpes simplex virus thymidine kinase promoter. On this promoter, the 5' part of the E-IRS functions as a constitutive enhancer, while the last 16 base pairs of the E-IRS is sufficient to give IFN-induced expression. On the E-gene promoter, the constitutive enhancer and the IFN-activated sequence are both needed but can be separated. In addition, promoter competition experiments indicate a third regulatory region which helps to repress expression of the E gene in uninduced cells.
Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.
Authors: Ng R , Carbon J
Published: September 2015

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.
The stem-loop structure at the 3' end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability.
Authors: Pandey N B , Marzluff W F
Published: September 2015

Chimeric genes were made by fusing mouse histone genes with a human alpha-globin gene. The genes were introduced into mouse L cells and the stability of the chimeric mRNAs was measured when DNA synthesis was inhibited. An mRNA containing all the globin coding sequences and the last 30 nucleotides of the histone mRNA was degraded at the same rate as histone mRNA.
Negative and positive regulation of human leukocyte antigen class I gene transcription in K562 leukemia cells.
Authors: Chen E , Karr R W , Ginder G D
Published: September 2015

The mechanism of transcriptional activation of human leukocyte antigen class I genes by gamma interferon and 5-azacytidine was studied in K562 human leukemia cells. Nuclear run-on transcription assays with various protein and RNA synthesis inhibitors yield evidence for both stimulation of a positive regulatory factor and inhibition of an mRNA that codes for a labile repressor. A novel mechanism is proposed to explain how 5-azacytidine can activate repressed genes without affecting DNA methylation.
RNA polymerase II transcription factors H4TF-1 and H4TF-2 require metal to bind specific DNA sequences.
Authors: Dailey L , Roberts S B , Heintz N
Published: September 2015

Specific DNA-binding and in vitro transcription activities of H4TF-1 and H4TF-2 are inactivated by chelating agents. Binding activity is restored by addition of Zn2+, and H4TF-2 is also reactivated by Fe2+. In contrast, preformed factor-DNA complexes are resistant to chelators. Therefore, metal ions are a required component of the H4TF-1 and H4TF-2 DNA-binding domains.
Molecular analysis of G+C-rich upstream sequences regulating transcription of the human carbonic anhydrase II gene.
Authors: Shapiro L H , Venta P J , Tashian R E
Published: September 2015

The upstream promoter sequences of the human carbonic anhydrase II (CA II) gene have been studied by 5' deletion analysis. Promoter activity was assayed by transfection and chloramphenicol acetyltransferase assay in both human HeLa cells and murine L cells. This investigation showed that the CA II promoter is comparable in activity to that of the simian virus 40 early-region promoter and enhancer and that the CA II upstream sequences exert a different pattern of control in the two cell lines.
Sequence and nuclear localization of the Saccharomyces cerevisiae HAP2 protein, a transcriptional activator.
Authors: Pinkham J L , Olesen J T , Guarente L P
Published: September 2015

Activation of the CYC1 upstream activation site (UAS2) and other Saccharomyces cerevisiae genes encoding respiratory functions requires the products of the regulatory loci HAP2 and HAP3. We present here the DNA sequence of the yeast HAP2 gene and an initial investigation into the function of its product. The DNA sequence indicated that HAP2 encoded a 265-amino-acid protein whose carboxyl third was highly basic. Also found in the sequence was a polyglutamine tract spanning residues 120 to 133. Several experiments described herein suggest that HAP2 encodes a direct activator of transcription. First, a bifunctional HAP2-beta-galactosidase fusion gene was localized to the yeast nucleus. Second, a lexA-HAP2 fusion gene was capable of activating transcription when bound to a lexA operator site. The additional requirement for the HAP3 product in activation is discussed.
Augmentation of c-fos mRNA expression by activators of protein kinase C in fresh, terminally differentiated resting macrophages.
Authors: Radzioch D , Bottazzi B , Varesio L
Published: September 2015

Expression of c-fos mRNA was investigated in fresh, normal peritoneal macrophages (M phi), which are terminally differentiated, nonproliferating cells. The levels of c-fos mRNA were dramatically increased by stimulation with phorbol myristate acetate (PMA), calcium ionophore, or 1-oleoyl-2-acetoyl glycerol (OAG). Induction of c-fos mRNA by all the above agents followed similar kinetics, with a peak of mRNA 30 min after stimulation. These results demonstrate that c-fos mRNA can be augmented in fresh, terminally differentiated cells. Since the stimuli increasing c-fos mRNA are direct or indirect activators of protein kinase C, our data suggest that in M phi c-fos mRNA is controlled by protein kinase C activation. PMA, calcium ionophore, and OAG were biologically active in M phi. PMA and calcium ionophore induced respiratory burst and tumoricidal activity, respectively, whereas OAG and PMA were chemotactic for M phi. Interferons beta and gamma, potent M phi activators eliciting tumoricidal activity, did not alter the levels of c-fos mRNA. These results indicate that c-fos mRNA augmentation is a stimulus-specific rather than a function-specific response connected to activation of protein kinase C.
Metal-responsive elements act as positive modulators of human metallothionein-IIA enhancer activity.
Authors: Karin M , Haslinger A , Heguy A , Dietlin T , Cooke T
Published: September 2015

The human metallothionein IIA (hMT-IIA) gene contains two enhancer elements whose activity is induced by heavy-metal ions such as Cd2+. To determine the nature of the relationship between the metal-responsive elements and the element(s) responsible for the basal activity of the enhancers, the basal-level enhancer element(s), the hMT-IIA enhancers were subjected to mutational analysis. We show that deletion of the metal-responsive elements had no effect on the basal activity of the enhancer but prevented further induction by Cd2+. On the other hand, replacement of the basal-level enhancer element with linker DNA led to inactivation of the enhancer both before and after treatment with Cd2+. Therefore, the metal-responsive elements seems to act as a positive modulator of enhancer function in the presence of heavy-metal ions. In addition to the two enhancers, the hMT-IIA promoter contained one other element, the GC box, required for its basal expression. Unlike deletion of the basal-level enhancer element, replacement of the GC box with linker DNA had no effect on the ability of the promoter to be induced by Cd2+.
Induction of urokinase-type plasminogen activator by UV light in human fetal fibroblasts is mediated through a UV-induced secreted protein.
Authors: Rotem N , Axelrod J H , Miskin R
Published: September 2015

Plasminogen activator was previously shown to be induced by UV light in human cells with low capacity to repair UV-induced DNA lesions. We now show that in human fetal fibroblasts UV light enhanced the two mRNA species coding for the urokinase-type plasminogen activator (uPA) and the tissue-type plasminogen activator, but immunological analysis revealed exclusively uPA activity. Several independent and complementary experiments indicated that induction of uPA was mediated, apparently entirely, through a UV-induced, secreted protein (UVIS) in the growth medium of irradiated cells. First, elevation of uPA mRNA after irradiation was severely blocked by cycloheximide. Second, replacement of conditioned medium in irradiated cells while the rate of plasminogen activator induction was maximal rapidly and completely stopped any further increase in uPA activity. Third, addition of the same removed conditioned medium to nonirradiated cells mimicked UV light in enhancing the level of uPA activity as well as that of uPA mRNA. Fourth, UVIS activity was completely lost by treating the conditioned medium with trypsin but not with nucleases. Kinetic measurements indicated that the accumulation of UVIS rather than the induction of uPA by UVIS conferred the rate-limiting step in the overall process of uPA induction. Both UV light and UVIS acted synergistically with inhibitors of DNA repair for uPA induction. Based on these results, a model is proposed implicating relaxation of DNA torsional stress of an as yet undefined DNA sequence(s) in the induction of UVIS, which is then responsible for activation of the uPA gene.
Stimulation of T cells through the CD3/T-cell receptor complex: role of cytoplasmic calcium, protein kinase C translocation, and phosphorylation of pp60c-src in the activation pathway.
Authors: Ledbetter J A , Gentry L E , June C H , Rabinovitch P S , Purchio A F
Published: September 2015

Stimulation of T cells or the Jurkat T-cell line with soluble antibodies to the CD3/T-cell receptor complex causes mobilization of cytoplasmic Ca2+, which is blocked by pertussis toxin but not by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and translocation of protein kinase C activity from the cytoplasm to the membrane. Such stimulation also causes phosphorylation of pp60c-src at an amino-terminal serine residue. These activities are consistent with induction of phosphatidylinositol metabolism after antibody binding. Anti-CD3 stimulation with antibody in solution, however, does not cause Jurkat cells to release interleukin 2 and blocks rather than induces proliferation of T cells. Induction of interleukin 2 production by Jurkat cells and proliferation by normal T cells requires anti-CD3 stimulation with antibody on a solid support, such as Sepharose beads or a plastic dish. Thus, we examined phosphorylation of pp60c-src after stimulation of Jurkat cells with anti-CD3 in solution or on solid phase. Both of these caused serine phosphorylation of pp60c-src that was indistinguishable even after 4 h of stimulation. These results indicate that the mode of anti-CD3 stimulation (in solution or on solid phase) controls a cellular function that modifies the consequences of signal transduction through phosphatidylinositol turnover.
Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells.
Authors: Rao T R , Slobin L I
Published: September 2015

When Friend erythroleukemia cells were allowed to grow to stationary phase (2 X 10(6) to 3 X 10(6) cells per ml), approximately 60% of the mRNA for eucaryotic elongation factor Tu (eEF-Tu) sedimented at less than or equal to 80S, and most of the remaining factor mRNA was associated with small polysomes. Under the same growth conditions, greater than 90% of the mRNA for eucaryotic initiation factor 4A remained associated with polysomes. The association of eEF-Tu mRNA with polysomes changed dramatically when stationary-phase cells were treated with fresh medium. After 1 h in fresh medium, approximately 90% of eEF-Tu mRNA in Friend cells was found in heavy polysomes. Associated with the shift of eEF-Tu mRNA into heavy polysomes, we found at least a 2.6-fold increase in the synthesis of eEF-Tu in vivo as well as a remarkable 40% decrease in the total amount of eEF-Tu mRNA per cell. Our data raise the possibility that eEF-Tu mRNA that has accumulated in ribonucleoprotein particles in stationary-phase cells is degraded rather than reutilized for eEF-Tu synthesis.
Recognition of mutant and cryptic 5' splice sites by the U1 small nuclear ribonucleoprotein in vitro.
Authors: Chabot B , Steitz J A
Published: September 2015

We examined the ability of U1 small nuclear ribonucleoproteins (U1 snRNPs) to recognize mutant and cryptic 5' splice sites on beta-globin pre-mRNA substrates using an RNase T1 protection assay. When U1 snRNPs were prebound to anti-(U1)RNP antibodies, we detected binding to mutant but not to cryptic 5' splice sites on several substrates. By contrast, in a splicing extract at 0 degree C, neither the mutated nor cryptic 5' splice sites of a human beta-globin transcript were selected as protected fragments with the same antibodies. However, after incubation of the transcript in the extract to yield splicing intermediates, fragments that included a cryptic 5' splice site were detected. The results of our analyses suggest that U1 snRNPs are involved in recognizing cryptic 5' splice sites but that interactions with other splicing components are required to stabilize the association.
Firefly luciferase gene: structure and expression in mammalian cells.
Authors: de Wet J R , Wood K V , DeLuca M , Helinski D R , Subramani S
Published: September 2015

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.
Metaphase protein phosphorylation in Xenopus laevis eggs.
Authors: Lohka M J , Kyes J L , Maller J L
Published: September 2015

Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior.
Dissociation of c-fos induction from macrophage differentiation in human myeloid leukemic cell lines.
Authors: Calabretta B
Published: September 2015

Treatment of five human myeloid leukemic cell lines (KG1, ML3, HL-60, U-937, and HEL) with TPA was followed by macrophage differentiation and was accompanied by an early and transient increase in the mRNA level of c-fos proto-oncogene. The induction of c-fos was also observed in human cell lines K562 and K-Gla that did not respond to TPA with terminal macrophage differentiation. The treatment of HL-60 and U-937 cell lines with 1-oleoyl-2-acetylglycerol, a synthetic analog of diacylglycerol that, like TPA, stimulates protein kinase C activity, was followed by early and transient induction of c-fos mRNA in the absence of terminal macrophage differentiation. Finally, treatment of HL-60 with TPA in the presence of retinal, an inhibitor of protein kinase C, drastically reduced the induction of c-fos mRNA but had no effect on the terminal macrophage differentiation that is induced in this cell line by TPA. These results indicate that the induction of c-fos and terminal macrophage differentiation in response to TPA treatment can be dissociated in the in vitro models provided by human myeloid leukemic cell lines. Moreover, these findings suggest that the induction of c-fos is not only insufficient but may also be unnecessary for the differentiation along the monocyte-macrophage pathway.
Cloning of a human S-phase cell cycle gene: use of transient expression for screening.
Authors: Fainsod A , Diamond G , Marcus M , Ruddle F H
Published: September 2015

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.
Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.
Authors: Kelly R , Shaw D R , Ennis H L
Published: September 2015

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.
Expression of H-ras correlates with metastatic potential: evidence for direct regulation of the metastatic phenotype in 10T1/2 and NIH 3T3 cells.
Authors: Egan S E , McClarty G A , Jarolim L , Wright J A , Spiro I , Hager G , Greenberg A H
Published: September 2015

Using three independent approaches, we studied the effects of H-ras on metastasis formation. Analysis of five in vitro-ras-transfected 10T1/2 clones with either flat or refractile morphologies revealed a relationship between metastatic potential, H-ras expression, and anchorage-independent growth. Four metastatic variants derived from a poorly metastatic, low-H-ras-expressing line all expressed high levels of H-ras RNA and grew efficiently in soft agar. Activation of H-ras expression in the metastatic tumors had occurred through amplification and rearrangement of H-ras sequences. In addition, preinduction of p21 synthesis in NIH 3T3 line 433, which contains v-H-ras under transcriptional control of the glucocorticoid-sensitive mouse mammary tumor virus long terminal repeat, significantly increased metastatic efficiency. Glucocorticoid treatment of normal or pEJ-transformed NIH 3T3 cells did not affect metastatic potential. These data reveal a direct relationship between ras expression and metastasis formation and suggest that metastatic and transformed phenotypes may be coregulated in ras-transformed 10T1/2 and NIH 3T3 cells.