bluuebunny/crossref_metadata_2025 huggingface dataset:
Independent regulation of transcription of the two strands of the c-myc gene.
Authors: Kindy M S , McCormack J E , Buckler A J , Levine R A , Sonenshein G E
Published: September 2015

Previously we demonstrated the existence of transcripts from the noncoding strand of a rearranged, truncated c-myc gene in murine plasmacytomas in which this oncogene is translocated to an immunoglobulin constant-region gene element (M. Dean, R. B. Kent, and G. E. Sonenshein, Nature [London] 305:443-446, 1983). Here we report on the transcription of the two strands of a normal, unrearranged c-myc gene. We examined the effects of gene rearrangements, growth state transitions, and differentiation on the relative levels of usage of the two strands. Transcription from intron 1 to exon 3 of the murine c-myc gene was studied in in vitro nuclear runoff assays. The level of transcription of the noncoding strand across this region of a germ line c-myc gene in a murine B-cell lymphoma line was comparable to the level observed in plasmacytomas with translocated c-myc genes. Rapid changes in transcription of the coding strand of the c-myc gene could be seen during growth arrest of WEHI 231 cells and during activation of splenic T lymphocytes. Transcription of the noncoding strand was constitutive during these growth state transitions and during activation of primary cultures of quiescent calf aortic smooth muscle cells as well. In contrast, differentiation of murine erythroleukemia cells was accompanied by an early drop in transcription of the two strands of this gene. The ramifications of these findings with respect to measurements of c-myc gene transcription and to the regulation of this gene are discussed.
Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA.
Authors: Parker K A , Steitz J A
Published: September 2015

The human U3 ribonucleoprotein (RNP) has been analyzed to determine its protein constituents, sites of protein-RNA interaction, and RNA secondary structure. By using anti-U3 RNP antibodies and extracts prepared from HeLa cells labeled in vivo, the RNP was found to contain four nonphosphorylated proteins of 36, 30, 13, and 12.5 kilodaltons and two phosphorylated proteins of 74 and 59 kilodaltons. U3 nucleotides 72-90, 106-121, 154-166, and 190-217 must contain sites that interact with proteins since these regions are immunoprecipitated after treatment of the RNP with RNase A or T1. The secondary structure was probed with specific nucleases and by chemical modification with single-strand-specific reagents that block subsequent reverse transcription. Regions that are single stranded (and therefore potentially able to interact with a substrate RNA) include an evolutionarily conserved sequence at nucleotides 104-112 and nonconserved sequences at nucleotides 65-74, 80-84, and 88-93. Nucleotides 159-168 do not appear to be highly accessible, thus making it unlikely that this U3 sequence base pairs with sequences near the 5.8S rRNA-internal transcribed spacer II junction, as previously proposed. Alternative functions of the U3 RNP are discussed, including the possibility that U3 may participate in a processing event near the 3' end of 28S rRNA.
Ankyrin-bound fatty acid turns over rapidly at the erythrocyte plasma membrane.
Authors: Staufenbiel M
Published: September 2015

The membrane skeletal protein ankyrin was shown to be continuously acylated and deacylated with long-chain fatty acids in mature erythrocytes. At least a fraction of the lipid bound to ankyrin turned over rapidly (half-life, approximately 50 min) compared with the polypeptide backbone, which was stable throughout the erythrocyte life. This indicates a regulatory significance of the fatty acid modification for the function of ankyrin.
Chromosome-mediated transfer of the murine Na,K-ATPase alpha subunit confers ouabain resistance.
Authors: Fallows D , Kent R B , Nelson D L , Emanuel J R , Levenson R , Housman D E
Published: September 2015

We transferred murine NIH 3T3 metaphase chromosomes into monkey CV-1 cells to investigate the different ouabain sensitivities of rodent and primate cells. In 16 ouabain-resistant transferents, the mouse Na,K-ATPase alpha 1 subunit gene was detected, suggesting that structural differences between the rodent and primate alpha 1 subunits determine the different ouabain sensitivities.
Plasminogen activator inhibitor type 1 biosynthesis and mRNA level are increased by dexamethasone in human fibrosarcoma cells.
Authors: Andreasen P A , Pyke C , Riccio A , Kristensen P , Nielsen L S , Lund L R , Blasi F , Danø K
Published: September 2015

Dexamethasone increases type 1 plasminogen activator inhibitor (PAI-1) activity released from the human fibrosarcoma cell line HT-1080. We demonstrated that dexamethasone caused about 10-fold increases in the intracellular and extracellular levels of PAI-1 protein, as measured by an enzyme-linked immunosorbent assay, in the rate of PAI-1 biosynthesis, and in the PAI-1 mRNA level. The effects on PAI-1 biosynthesis and mRNA level were detectable within 4 h and were maximal 16 to 24 h after the addition of dexamethasone. Cycloheximide did not inhibit the dexamethasone-induced increases in the capacity of the cells to synthesize PAI-1 and in the PAI-1 mRNA level.
Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans.
Authors: Boylan M T , Mirabito P M , Willett C E , Zimmerman C R , Timberlake W E
Published: September 2015

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA, and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting the expression of nonregulated genes. The three genes code for poly(A)+ RNAs that begin to accumulate at different times during conidiation. The brlA- and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.
Characterization of an ATP-dependent DNA strand transferase from human cells.
Authors: Ganea D , Moore P , Chekuri L , Kucherlapati R
Published: September 2015

We have characterized an enzymatic activity from human cell nuclei which is capable of catalyzing strand exchange between homologous DNA sequences. The strand exchange activity was Mg2+ dependent and required ATP hydrolysis. In addition, it was capable of promoting reannealing of homologous DNA sequences and could form nucleoprotein networks in a fashion reminiscent of purified bacterial RecA protein. Using an in vitro recombination assay, we also showed that the strand exchange activity was biologically important. The factor(s) responsible for the activity has been partially purified.
Accurate and efficient 3' processing of U2 small nuclear RNA precursor in a fractionated cytoplasmic extract.
Authors: Kleinschmidt A M , Pederson T
Published: September 2015

The small nuclear RNAs U1, U2, U4, and U5 are cofactors in mRNA splicing and, like the pre-mRNAs with which they interact, are transcribed by RNA polymerase II. Also like mRNAs, mature U1 and U2 RNAs are generated by 3' processing of their primary transcripts. In this study we have investigated the in vitro processing of an SP6-transcribed human U2 RNA precursor, the 3' end of which matches that of authentic human U2 RNA precursor molecules. Although the SP6-U2 RNA precursor was efficiently processed in an ammonium sulfate-fractionated HeLa cytoplasmic S100 extract, the product RNA was unstable. Further purification of the processing activity on glycerol gradients resolved a 7S activity that nonspecifically cleaved all RNAs tested and a 15S activity that efficiently processed the 3' end of pre-U2 RNA. The 15S activity did not process the 3' end of a tRNA precursor molecule. As demonstrated by RNase protection, the processed 3' end of the SP6-U2 RNA maps to the same nucleotides as does mature HeLa U2 RNA.
Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.
Authors: Erikson E , Stefanovic D , Blenis J , Erikson R L , Maller J L
Published: September 2015

Ribosomal protein S6 becomes highly phosphorylated during progesterone- or insulin-induced maturation of Xenopus laevis oocytes. We have previously purified an Mr 92,000 protein as one of the major S6 kinases from Xenopus unfertilized eggs. In this paper we confirm by renaturation of activity from a sodium dodecyl sulfate-polyacrylamide gel that this protein is an S6 kinase. This enzyme, termed S6 kinase II (S6 K II), was used for the preparation of polyclonal antiserum. Immunocomplexes formed with the antiserum and purified S6 K II were able to express kinase activity with the same substrate specificity as that of the purified enzyme, including autophosphorylation of S6 K II itself. The antiserum did not react with S6 kinase I, another major S6 kinase present in Xenopus eggs, which is chromatographically distinct from S6 K II. The administration of progesterone to oocytes resulted in a 20- to 25-fold increase in S6 kinase activity in extracts of these cells. Immunocomplex kinase assays done on extracts revealed that anti-S6 K II serum reacted with S6 kinase from progesterone-treated oocytes. This antiserum also reacted with the activated S6 kinase from insulin-stimulated oocytes. In addition, anti-S6 K II serum reacted with activated S6 kinase from chicken embryo fibroblasts stimulated with serum or transformed by Rous sarcoma virus. These results indicate that S6 K II or an antigenically related S6 kinase(s) is subject to regulation by mitogenic stimuli in various cell types.
Comparison of tRNA gene transcription complexes formed in vitro and in nuclei.
Authors: Huibregtse J M , Evans C F , Engelke D R
Published: September 2015

The nucleoprotein structure of single-copy tRNA genes in yeast nuclei was examined by DNase I footprinting and compared with that of complexes formed in vitro between the same genes and transcription factor C. Transcription factor C bound to both the 5' and 3' intragenic promoters of the tRNA(SUP53Leu) gene in vitro, protecting approximately 30 base pairs at the 3' promoter (B block) and 40 base pairs at the 5' promoter (A block) and causing enhanced DNase I cleavages between the protected regions. Binding to the two sites was independent of the relative orientation of the two sites on the helix and was eliminated by a single point mutation in the 3' promoter. The chromosomal tRNA(SUP53Leu) and tRNA(UCGSer) genes showed a pattern of protection and enhanced cleavages similar to that observed in vitro, indicating that the stable complexes formed in vitro accurately reflect at least some aspects of the nucleoprotein structure of the genes in chromatin.
Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen.
Authors: Beauchemin N , Benchimol S , Cournoyer D , Fuks A , Stanners C P
Published: September 2015

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.
The first intron in the human c-abl gene is at least 200 kilobases long and is a target for translocations in chronic myelogenous leukemia.
Authors: Bernards A , Rubin C M , Westbrook C A , Paskind M , Baltimore D
Published: September 2015

The c-abl protooncogene is unusual in two respects; it has multiple, widely space N-terminal coding exons transcribed by different promoters, and it is the target of the translocations that form the Philadelphia chromosome found in cells of chronic myelogenous leukemia patients. To understand the organization of the gene in normal and chronic myelogenous leukemia patient DNA we have mapped c-abl by pulsed field gradient gel electrophoresis. We find that one of the alternative 5' exons of the gene lies at least 200 kilobases upstream of the remaining c-abl exons, posing formidable transcription and splicing problems. The 5'-most c-abl exon includes an unusually long 1,276-base-pair segment that contains 15 ATG codons and multiple short open reading frames, upstream of the abl initiator codon. Its peculiar structure suggests that c-abl may be decapitated in most chronic myelogenous leukemia patients, and we demonstrate that this is the case in the chronic myelogenous leukemia cell line K562.
Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector.
Authors: Landau N R , Schatz D G , Rosa M , Baltimore D
Published: September 2015

The role of terminal deoxynucleotidyl transferase (TdT) in the insertion of N regions into the junctional sites of immunoglobulin genes was investigated. Pre-B-cell lines capable of continuous rearrangement of immunoglobulin light-chain genes and differing only in the presence or apparent absence of TdT were derived by infecting cells with a TdT retroviral expression vector or a control vector. The cell lines were then superinfected with a retrovirus-based artificial immunoglobulin gene rearrangement substrate. The substrate was allowed to rearrange in the cell lines and the rearranged proviruses were rescued from the cell lines. Nucleotide sequence analysis of the V-J junctions of the proviral rearranged genes showed a fivefold greater frequency of N-region insertion in proviruses rescued from the TdT+ cell lines than in those rescued from the TdT- cell lines, so that at least 50% of the rearrangements that occurred in the presence of TdT had N regions. It is thus evident that TdT can stimulate N-region insertion, and the enzyme is presumably directly responsible for adding nucleotides at V-J and other immunoglobulin and T-cell receptor gene junctions.
Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation.
Authors: Prezant T , Pfeifer K , Guarente L
Published: September 2015

Regulation of the CYC7 gene of Saccharomyces cerevisiae, encoding iso-2-cytochrome c, was studied. Expression was induced about 20-fold by heme and derepressed 4- to 8-fold by a shift from glucose medium to one containing a nonfermentable carbon source. Deletion analysis showed that induction by heme depends upon sequences between -250 and -228 (from the coding sequence) and upon the HAP1 activator gene, previously shown to be required for CYC1 expression (L. Guarente et al., Cell 36:503-511, 1984). Thus, HAP1 coordinates expression of CYC7 and CYC1, the two genes encoding isologs of cytochrome c in S. cerevisiae. HAP1-18, a mutant allele of HAP1, which increased CYC7 expression more than 10-fold, also acted through sequences between -250 and -228. In vitro binding studies showed that the HAP1 product binds to these sequences (see also K. Pfeifer, T. Prezant, and L. Guarente, Cell 49:19-28, 1987) and an additional factor binds to distal sequences that lie between -201 and -165. This latter site augmented CYC7 expression in vivo. Derepression of CYC7 expression in a medium containing nonfermentable carbon sources depended upon sequences between -354 and -295. The interplay of these multiple sites and the factors that bind to them are discussed.
A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.
Authors: Sachs A B , Davis R W , Kornberg R D
Published: September 2015

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.
Primate brain-specific cytoplasmic transcript of the Alu repeat family.
Authors: Watson J B , Sutcliffe J G
Published: September 2015

A 200-nucleotide RNA homologous to the left monomer of Alu elements was expressed in monkey and human brain and in cell lines but not in nonneural monkey tissues. Similar brain-specific transcription of identifier sequences was observed in rats. Thus, expression of selected repetitive DNA families is a conserved process in mammalian brain.
Effect of protein synthesis inhibition on gene expression during early development of Dictyostelium discoideum.
Authors: Singleton C K , Manning S S , Feng Y
Published: September 2015

Several genes which are deactivated on the initiation of development of Dictyostelium discoideum were identified by differential screening of various cDNA libraries. These genes have in common a decrease in the steady-state levels of their corresponding mRNAs on the onset of development and as development proceeds. When development was carried out in the absence of protein synthesis by inhibition with cycloheximide, the decrease in mRNA levels for most genes (V genes) was normal or slightly accelerated. For about 5% of the genes (H genes), however, cycloheximide caused an apparent induction of expression, as revealed by a slight or dramatic increase in mRNA levels, instead of the normal decrease. This effect was due to inhibition of protein synthesis and not to cycloheximide per se. The induction was found to be due to an enhancement of the transcription rate; normal rates of transcription for the H genes were dependent on continued protein synthesis during vegetative growth and development. Thus, two general regulatory classes exist for deactivation of gene expression on initiation of development, one of which is dependent on and one of which is independent of protein synthesis. Analysis of expression of these genes in mutant strains which are aggregation deficient allowed the classes to be subdivided further. Taken together, these characterizations allow several distinct regulatory mechanisms to be identified that are involved in the deactivation of gene expression on the onset of development in D. discoideum.
The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons.
Authors: Xiong Y , Eickbush T H
Published: September 2015

Two types of insertion elements, R1 and R2 (previously called type I and type II), are known to interrupt the 28S ribosomal genes of several insect species. In the silkmoth, Bombyx mori, each element occupies approximately 10% of the estimated 240 ribosomal DNA units, while at most only a few copies are located outside the ribosomal DNA units. We present here the complete nucleotide sequence of an R1 insertion from B. mori (R1Bm). This 5.1-kilobase element contains two overlapping open reading frames (ORFs) which together occupy 88% of its length. ORF1 is 461 amino acids in length and exhibits characteristics of retroviral gag genes. ORF2 is 1,051 amino acids in length and contains homology to reverse transcriptase-like enzymes. The analysis of 3' and 5' ends of independent isolates from the ribosomal locus supports the suggestion that R1 is still functioning as a transposable element. The precise location of the element within the genome implies that its transposition must occur with remarkable insertion sequence specificity. Comparison of the deduced amino acid sequences from six retrotransposons, R1 and R2 of B. mori, I factor and F element of Drosophila melanogaster, L1 of Mus domesticus, and Ingi of Trypanosoma brucei, reveals a relatively high level of sequence homology in the reverse transcriptase region. Like R1, these elements lack long terminal repeats. We have therefore named this class of related elements the non-long-terminal-repeat (non-LTR) retrotransposons.
A novel in vivo transcription assay demonstrates the presence of globin-inducing trans-acting factors in uninduced murine erythroleukemia cells.
Authors: Wrighton N , Grosveld F
Published: September 2015

We report the development of a novel in vivo transcription assay for trans-acting factors regulating the human gamma- and beta-globin genes. A cDNA coding for the human tissue-type plasminogen activator (t-PA) was inserted into the globin genes. Simian virus 40 small T-antigen splice and polyadenylation signals were included to produce a mature transcript coding for t-PA, whose activity can be detected in single cells by a fibrin-agarose plaque assay. Stable murine L-cell transfectants of the gamma.t-PA and beta.t-PA hybrid genes were fused to various cell lines to show that t-PA expression is increased specifically by erythroid MEL, HEL, and K562 cell fusion. The analogous H-2Kb.t-PA construct was not inducible under the same conditions. Interestingly, uninduced MEL cells increased beta.t-PA expression to the same extent as induced MEL cells. Chemiosmotic permeabilization of the beta-globin tester cell line and exposure to nuclear extracts were used to assay for trans-acting factors capable of stimulating beta.t-PA expression. Such factors were shown to be present in the nuclei of uninduced MEL cells.
Identification and characterization of two functional domains within the murine heavy-chain enhancer.
Authors: Kiledjian M , Su L K , Kadesch T
Published: September 2015

We have investigated the effect of polymerizing defined segments of the immunoglobulin heavy-chain enhancer on the activity of a single, linked transcription unit. Transient assays in lymphoid cells have led to the following observations. First, polymerizing the entire enhancer led to an increase in overall transcription. Second, polymerizing defined DNA segments revealed two distinct functional domains within the enhancer. Although each domain alone possessed only partial enhancer activity, greater than wild-type levels of activity could be obtained upon polymerization. One of these domains contains three regions thought to be involved in protein binding in vivo and in vitro (E motifs E1, E2, and E3). The other domain contains the fourth E motif (E4) and the conserved octanucleotide, ATTTGCAT. We have tested the functional importance of these motifs by determining the effect of mutating these elements singly or in combination in the context of the isolated domains. Although E2, E3, E4, and the octanucleotide are clearly important for enhancer function, mutation of the E1 motif did not appear to have an effect on enhancer activity in our assay. Transient assays in mouse L cells indicate that nonlymphoid cells are able to use a distinct subset of these motifs.
Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.
Authors: Debatisse M , Saito I , Buttin G , Stark G R
Published: September 2015

In a previous study of three independent families of mutants selected for overproduction of adenylate deaminase (AMPD), we were not able to isolate a cDNA probe for the gene and so could not demonstrate its amplification directly. In addition to overproduction of AMPD, four proteins of unknown function, designated W, X, Y1, and Y2, accumulated, and by using the corresponding cDNA probes, we demonstrated amplification of all four genes. In independent mutant clones, sometimes all and sometimes only a subset of these genes were amplified. Assuming that all five genes are linked, the pattern of their coamplification suggested a genetic map in which AMPD lies between W and Y1. We show here that a two-step chromosome walk joins the W and Y1 genes, that the AMPD gene is the only expressed sequence between them, and that its amplification is indeed responsible for overproduction of the AMPD protein. In the course of this work, we cloned and studied two novel joints which mark rearrangements on either side of the AMPD gene. Each joint was generated independently in a single first-step mutant at single or low copy number. Remarkably, each joint was amplified preferentially in every second- and third-step mutant derived from the first-step line in which it was originally present, suggesting that the two independent rearrangements each generated amplification-prone structures.
Tyrosine phosphorylations in vivo associated with v-fms transformation.
Authors: Morrison D K , Browning P J , White M F , Roberts T M
Published: September 2015

The role of tyrosine-specific phosphorylation in v-fms-mediated transformation was examined by immunoblotting techniques together with a high-affinity antibody that is specific for phosphotyrosine. This antiphosphotyrosine antibody detected phosphorylated tyrosine residues on the gp140v-fms molecule, but not gP180v-fms or gp120v-fms, in v-fms-transformed cells. This antibody also identified a number of cellular proteins that were either newly phosphorylated on tyrosine residues or showed enhanced phosphorylation on tyrosine residues as a result of v-fms transformation. However, the substrates of the v-fms-induced tyrosine kinase activity were not the characterized pp60v-src substrates. The phosphorylation of some of these cellular proteins and of the gp140fms molecule was found to correlate with the ability of v-fms/c-fms hybrids to transform cells. In addition, immunoblotting with the phosphotyrosine antibody allowed a comparison to be made of the substrates phosphorylated on tyrosine residues in various transformed cell lines. This study indicates that the pattern of tyrosine phosphorylation in v-fms-transformed cells is strikingly similar to that in v-sis-transformed cells.
Regulation of rat liver maturation in vitro by glucocorticoids.
Authors: Chou J Y , Wan Y J , Sakiyama T
Published: September 2015

The biochemistry of liver maturation was studied by using the RLA209-15 fetal rat hepatocyte line that is temperature sensitive for maintenance of the differentiated fetal liver phenotype. At 33 degrees C these cells were dedifferentiated; but at 40 degrees C they were phenotypically differentiated and, like normal fetal hepatocytes, synthesized moderate levels of albumin and transferrin, high levels of authentic (69,000 and 73,000 molecular weight) rat fetal alpha-fetoprotein (AFP), and low levels of a 65,000-molecular-weight variant AFP. Our results indicated that administration of glucocorticoid hormones to RLA209-15 cells at 40 degrees C induced a series of events associated with normal hepatocyte maturation; synthesis of fetal AFP was inhibited, whereas the synthesis of variant AFP, albumin, transferrin, tyrosine aminotransferase, and alpha 1-acid glycoprotein was induced. The variant AFP was produced by RLA209-15 cells at both temperatures and was encoded by an mRNA of 1.7 kilobases (kb). The fetal AFP was encoded by an mRNA of 2.2 kb. Normal adult rat liver contained three AFP mRNAs of 2.2 (minor), 1.7, and 1.5 kb. The 1.7-kb adult liver AFP mRNA comigrated with the RNA found in RLA209-15 cells, and both directed the synthesis of a 50,000-molecular-weight precursor polypeptide of the variant AFP. Administration of glucocorticoids to RLA209-15 cells grown at 33 degrees C stimulated synthesis of both the fetal and variant AFPs, but the levels of the 2.2-kb AFP mRNA were preferentially increased. RLA209-15 cells contained two glucocorticoid receptor mRNAs of 6.8 and 4.5 kb. The glucocorticoid-mediated maturation described above was blocked by the antiglucocorticoid RU486.
A 29-nucleotide DNA segment containing an evolutionarily conserved motif is required in cis for cell-type-restricted repression of the chicken alpha-smooth muscle actin gene core promoter.
Authors: Carroll S L , Bergsma D J , Schwartz R J
Published: September 2015

A series of 5' deletion mutations of the upstream flanking sequences of the chicken alpha-smooth muscle (aortic) actin gene was prepared and inserted into the chloramphenicol acetyltransferase expression vector pSV0CAT. Deletion recombinants were transfected into fibroblasts, which actively express the alpha-smooth muscle actin gene, and primary myoblast cultures, which accumulate much lower quantities of alpha-smooth muscle actin mRNAs. The first 122 nucleotides of 5'-flanking DNA were found to contain a "core" promoter capable of accurately directing high levels of transcription in both fibroblasts and myotubes. The activity of this core promoter is modulated in fibroblasts by a "governor" element(s) located at least in part between nucleotides -257 and -123. This region contains sequences potentially conserved between mammalian and avian alpha-smooth muscle actin genes as well as one of a pair of 16-base-pair inverted CCAAT box-associated repeats which are conserved among all chordate muscle actin genes examined to date. A smaller DNA segment (-151 to -123) containing these upstream CCAAT box-associated repeats was sufficient to suppress expression of the core promoter in muscle cultures, suggesting that the upstream CCAAT box-associated repeats play a negative role in the alpha-smooth muscle actin gene promoter.
Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma 3.
Authors: Schiff L A , Nibert M L , Co M S , Brown E G , Fields B N
Published: September 2015

By atomic absorption analysis, we determined that the reovirus outer capsid protein sigma 3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, we localized the zinc- and dsRNA-binding activities of sigma 3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxy-terminal fragment. By these techniques, new zinc- and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor IIIA-like zinc-binding site within sigma 3. We suggest that the zinc- and dsRNA-binding activities of sigma 3 may be important for its proposed regulatory effects on viral and host cell transcription and translation.
Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors.
Authors: Takahashi M , Nishizawa M , Negishi K , Hanaoka F , Yamada M A , Hayatsu H
Published: September 2015

To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.
Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells.
Authors: Evans M J , Scarpulla R C
Published: September 2015

To investigate the transcriptional control of nuclear-encoded respiratory genes in mammals, we have performed a deletional analysis of cis-acting regulatory sequences in the rat somatic cytochrome c gene. Three major regions are required for maximal expression of the transfected gene in kidney cell lines CV-1 and COS-1. One of these, region III (+71 to +115 from the transcription initiation site), is an unusual intragenic controlling element found in the 5' end of the first intron, while the other two, region I (-191 to -165) and region II (-139 to -84), define the upstream promoter. Region II contains two consensus CCAAT boxes and mediates a constitutive level of expression in both cell lines. In contrast, regions I and III are both required for the increased promoter activity observed in COS-1 cells compared with promoter activity observed in CV-1 cells, and the regions function individually as competitors with the full promoter for trans-acting factors or complexes. Region III contains a perfect octanucleotide homology with region I in addition to a consensus Sp1-transcription-factor-binding site. Promoter stimulation in COS-1 cells can be duplicated in CV-1 cells by cotransfecting with a T-antigen-producing vector, but purified T antigen does not bind anywhere in the cytochrome c promoter. A control promoter from the mouse metallothionein I gene is similarly activated in T-antigen-producing cells only in the presence of zinc, which activates its upstream regulatory sites. We conclude that T antigen stimulates these cellular promoters through the activation or induction of cellular factors or complexes that mediate their effects through promoter-specific regulatory elements. Cytochrome c promoter regions activated in this system may play a physiological role in controlling gene expression.
Complex regulation of heat shock- and glucose-responsive genes in human cells.
Authors: Watowich S S , Morimoto R I
Published: September 2015

We have isolated a human genomic clone that encodes the glucose-responsive protein GRP78 and have used this cloned gene probe, together with a cloned HSP70 gene, to study the expression of both stress-induced genes in response to inhibitors of cellular metabolism. On the basis of the effects of this group of chemicals on GRP78 and HSP70 expression, we have identified three classes of stress gene inducers. The first class induces GRP78 expression and includes inhibitors of glycoprotein processing. The second class results in coordinate activation of both GRP78 and HSP70 synthesis and includes amino acid analogs and heavy metals. Chemicals in the third class coordinately induce GRP78 and repress HSP70 expression; this class includes the calcium ionophore A23187 and the glucose analog 2-deoxyglucose. Whereas induction of GRP78 or HSP70 expression is primarily due to transcriptional activation, chemicals that repress HSP70 expression act through posttranscriptional regulation. These results reveal that the regulation of GRP78 and HSP70 expression is complex and may be dependent on the specificity and magnitude of physiological damage.
Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum.
Authors: Rubin E J , Gill D M , Boquet P , Popoff M R
Published: September 2015

Exoenzyme C3 from Clostridium botulinum types C and D specifically ADP-ribosylated a 21-kilodalton cellular protein, p21.bot. Guanyl nucleotides protected the substrate against denaturation, which implies that p21.bot is a G protein. When introduced into the interior of cells, purified exoenzyme C3 ADP-ribosylated intracellular p21.bot and changed its function. NIH 3T3, PC12, and other cells rapidly underwent temporary morphological alterations that were in certain respects similar to those seen after microinjection of cloned ras proteins. When injected into Xenopus oocytes, C3 induced migration of germinal vesicles and potentiated the cholera toxin-sensitive augmentation of germinal vesicle breakdown by progesterone, also as caused by ras proteins. Nevertheless, p21.bot was immunologically distinct from p21ras.
6;7 chromosomal translocation in spontaneously arising rat immunocytomas: evidence for c-myc breakpoint clustering and correlation between isotypic expression and the c-myc target.
Authors: Pear W S , Wahlström G , Nelson S F , Axelson H , Szeles A , Wiener F , Bazin H , Klein G , Sümegi J
Published: September 2015

Our previous studies have shown that spontaneously arising immunocytomas in the LOU/Ws1 strain of rats contain a t(6;7) chromosomal translocation in all seven tumors studied (F. M. Babonits, J. Spira, G. Klein, and H. Bazin, Int. J. Cancer 29:431-437, 1982). We have also shown that the c-myc is located on chromosome 7 (J. Sümegi, J. Spira, H. Bazin, J. Szpirer, G. Levan, and G. Klein, Nature (London) 306:497-499, 1983) and the immunoglobulin H cluster on chromosome 6 (W.S. Pear, G. Wahlström, J. Szpirer, G. Levan, G. Klein, and J. Sümegi, Immunogenetics 23:393-395, 1986). We now report a detailed cytogenetic and molecular analysis of nine additional rat immunocytomas. The t(6;7) chromosomal translocation is found in all tumors. Mapping of the c-myc breakpoints showed that in 10 of 14 tumors, the c-myc breakpoints are clustered in a 1.5-kilobase region upstream of exon 1. In contrast with sporadic Burkitt's lymphoma and mouse plasmacytoma, only 1 of 14 tumors contains the c-myc breakpoints in either exon 1 or intron 1. Analysis of the sequences juxtaposed to the c-myc show that immunoglobulin H switch regions are the targets in at least five tumors and that there is a strong correlation between the secreted immunoglobulin and the c-myc target. Unlike sporadic Burkitt's lymphoma and mouse plasmacytoma, at least two rat immunocytomas show recombination of the c-myc with sequences distinct from immunoglobulin switch regions.
The cap of both miniexon-derived RNA and mRNA of trypanosomes is 7-methylguanosine.
Authors: Sutton R E , Boothroyd J C
Published: September 2015

Most, if not all, trypanosome mRNAs have the same 35-base sequence at their 5' terminus which is derived from a short RNA (medRNA) probably by the process of trans-splicing. It is of interest, evolutionarily and mechanistically, to determine the chemical structure of the 5' terminus of the precursor (medRNA) and product (mRNA). We demonstrate here that the cap structure of both is most probably 7-methylguanosine in a 5',5' triphosphate linkage, consistent with a precursor/product relationship.
B-cell control region at the 5' end of a major histocompatibility complex class II gene: sequences and factors.
Authors: Dorn A , Fehling H J , Koch W , Le Meur M , Gerlinger P , Benoist C , Mathis D
Published: September 2015

Transcription of major histocompatibility complex class II genes is elaborately regulated. Mouse class II genes are transcribed primarily in B cells, peripheral macrophages and interdigitating cells, and thymic cortical and medullary cells. In this study, we began to identify the DNA sequences and protein factors that control expression of a class II gene in B cells, addressing in particular how closely they resemble those that regulate immunoglobulin gene expression. We describe a region upstream of the E alpha gene that is crucial for its transcription in the B cells of transgenic mice but is less important in cultured B-cell lines. The sequence of this region reveals several familiar motifs, including a second X-Y pair reminiscent of that residing in the promoter-proximal region of all class II genes, a B motif strikingly homologous to that associated with the immunoglobulin kappa gene enhancer, several Ephrussi motifs, and a Pu box-like sequence very similar to that implicated in simian virus 40 and lymphotrophic papovavirus expression in B cells. Careful study of the proteins that bind specifically to these different motifs prompts us to suggest that major histocompatibility complex class II and immunoglobulin genes rely on quite different factors to achieve B-cell-specific expression.
Sequence and structural requirements of a herpes simplex viral DNA replication origin.
Authors: Lockshon D , Galloway D A
Published: September 2015

Herpes simplex virus (HSV) types 1 and 2 contain two classes of origins of DNA replication, oriS and oriL, which are closely related. A series of plasmids was constructed which contained specifically altered versions of the HSV type 2 oriS replication origin. Their ability to replicate in an in vivo replicon assay allowed a core origin of 75 base pairs (bp) to be defined. It included both arms of a 56-bp palindrome and from 13 to 20 bp of sequence leftward of the palindrome. The AT-rich sequence at the center of the palindrome was essential. Sequences on either side of the core origin enhanced replication. When additional copies of the -AT-dinucleotide were introduced progressively into the center of the palindrome, an oscillating effect on origin function was observed. These and other data implicate a linear rather than a cruciform conformation of the oriS palindrome in the initiation of HSV replication.
Identification of the human c-myc protein nuclear translocation signal.
Authors: Dang C V , Lee W M
Published: September 2015

We identified and characterized two regions of the human c-myc protein that target proteins into the nucleus. Using mutant c-myc proteins and proteins that fuse portions of c-myc to chicken muscle pyruvate kinase, we found that residues 320 to 328 (PAAKRVKLD; peptide M1) induced complete nuclear localization, and their removal from c-myc resulted in mutant proteins that distributed in both the nucleus and cytoplasm but retained rat embryo cell cotransforming activity. Residues 364 to 374 (RQRRNELKRSP; peptide M2) induced only partial nuclear targeting, and their removal from c-myc resulted in mutant proteins that remained nuclear but were cotransformationally inactive. We conjugated synthetic peptides containing M1 or M2 to human serum albumin and microinjected the conjugate into the cytoplasm of Vero cells. The peptide containing M1 caused rapid and complete nuclear accumulation, whereas that containing M2 caused slower and only partial nuclear localization. Thus, M1 functions as the nuclear localization signal of c-myc, and M2 serves some other and essential function.
Interaction of nuclear proteins with muscle-specific regulatory sequences of the human cardiac alpha-actin promoter.
Authors: Gustafson T A , Miwa T , Boxer L M , Kedes L
Published: September 2015

The human cardiac alpha-actin promoter is involved in the muscle-specific transcriptional regulation of the gene. In this study, we utilized gel mobility shift, methylation interference, and DNase I protection assays to examine protein factor interaction with the promoter in vitro. All assays demonstrated specific interaction of nuclear factors with a region of the promoter encompassed by nucleotides -93 to -113 base pairs from the transcriptional start site. This region contains a CC(A + T-rich)6GG element, termed a CArG box, which has previously been implicated in the muscle-specific transcriptional regulation of the gene by functional assays. Although the gene is only expressed in muscle cells, identical binding activity was present in nuclear extracts of all cell types examined, including those of muscle (C2, L8, and L6 cells) and nonmuscle (HeLa, NIH 3T3, HuT12, and L cells) origin. Furthermore, methylation interference assays showed that identical nucleotides interacted with factors isolated from C2 and HeLa cells. Competition studies showed that the CArG-binding factor, designated as CBF, also interacts with the c-fos serum responsive element, which contains a CArG element, but not with the simian virus 40 enhancer and early promoter. Thus, a region of the human cardiac alpha-actin promoter known to be functionally involved in muscle-specific regulation of the gene appears to interact in vitro, and in an identical manner, with a factor(s) which is neither muscle nor gene specific, suggesting a more complex mode of regulation than previously envisioned.
Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes.
Authors: Shimamura A , Tremethick D , Worcel A
Published: September 2015

We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.