bluuebunny/crossref_metadata_2025 huggingface dataset:
Yeast CBP1 mRNA 3' end formation is regulated during the induction of mitochondrial function.
Authors: Mayer S A , Dieckmann C L
Published: October 2015

Alternative mRNA processing is one mechanism for generating two or more polypeptides from a single gene. While many mammalian genes contain multiple mRNA 3' cleavage and polyadenylation signals that change the coding sequence of the mature mRNA when used at different developmental stages or in different tissues, only one yeast gene has been identified with this capacity. The Saccharomyces cerevisiae nuclear gene CPB1 encodes a mitochondrial protein that is required for cytochrome b mRNA stability. This 66-kDa protein is encoded by a 2.2-kb mRNA transcribed from CPB1. Previously we showed that a second 1.2-kb transcript is initiated at the CBP1 promoter but has a 3' end near the middle of the coding sequence. Furthermore, it was shown that the ratio of the steady-state level of 2.2-kb CBP1 message to 1.2-kb message decreases 10-fold during the induction of mitochondrial function, while the combined levels of both messages remain constant. Having proposed that regulation of 3' end formation dictates the amount of each CBP1 transcript, we now show that a 146-bp fragment from the middle of CBP1 is sufficient to direct carbon source-regulated production of two transcripts when inserted into the yeast URA3 gene. This fragment contains seven polyadenylation sites for the wild-type 1.2-kb mRNA, as mapped by sequence analysis of CBP1 cDNA clones. Deletion mutations upstream of the polyadenylation sites abolished formation of the 1.2-kb transcript, whereas deletion of three of the sites only led to a reduction in abundance of the 1.2-kb mRNA. Our results indicate that regulation of the abundance of both CBP1 transcripts is controlled by elements in a short segment of the gene that directs 3' end formation of the 1.2-kb transcript, a unique case in yeast cells.
trans-Activation of a globin promoter in nonerythroid cells.
Authors: Evans T , Felsenfeld G
Published: October 2015

We show that expression in fibroblasts of a single cDNA, encoding the erythroid DNA-binding protein Eryf1 (GF-1, NF-E1), very efficiently activates transcription of a chicken alpha-globin promoter, trans-Activation in these cells occurred when Eryf1 bound to a single site within a minimal globin promoter. In contrast, efficient activation in erythroid cells required multiple Eryf1 binding sites. Our results indicate that mechanisms exist that are capable of modulating the trans-acting capabilities of Eryf1 in a cell-specific manner, without affecting DNA binding. The response of the minimal globin promoter to Eryf1 in fibroblasts was at least as great as for optimal constructions in erythroid cells. Therefore, the assay provides a very simple and sensitive system with which to study gene activation by a tissue-specific factor.
The core proteins A2 and B1 exist as (A2)3B1 tetramers in 40S nuclear ribonucleoprotein particles.
Authors: Barnett S F , Theiry T A , LeStourgeon W M
Published: October 2015

The six "core" proteins of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) package 700-nucleotide lengths of pre-mRNA into a repeating array of regular particles. We have previously shown that the C proteins exist as anisotropic tetramers of (C1)3C2 in 40S hnRNP particles and that each particle probably contains three such tetramers. We report here that proteins A2 and B1 also exist in monoparticles as (A2)3B1 tetramers and that each monoparticle contains at least three such tetramers. Proteins A2 and B1 dissociate from isolated monoparticles as a stable tetramer upon nuclease digestion. In low-salt gradients, the tetramers sediment at 6.8S, which is consistent with a mass of 145 kDa. In 200 mM salt, the concentration which dissociates these proteins from RNA, only 4.2S dimers exist in solution. Tetramers of (A2)3B1 possess the ability to package multiples of 700 nucleotides of RNA in vitro into an array of regular, 22.5-nm 43S particles. Unlike the in vitro assembly of intact 40S hnRNP, the (A2)3B1 tetramers assemble by means of a highly cooperative process. These findings indicate that the (A2)3B1 tetramers play a major role in hnRNP assembly and they further support the contention that 40S monoparticles are regular structures composed of three copies of three different tetramers, i.e., 3[(A1)3B2, (A2)3B1, (C1)3C2].
Colony-stimulating factor 1 expression is down-regulated during the adipocyte differentiation of H-1/A marrow stromal cells and induced by cachectin/tumor necrosis factor.
Authors: Umezawa A , Tachibana K , Harigaya K , Kusakari S , Kato S , Watanabe Y , Takano T
Published: October 2015

We isolated clonal sublines of the established mouse marrow stromal cell line, H-1. These clonal sublines underwent differentiation into adipocytes in various degrees. One subline, H-1/A, underwent adipocyte differentiation after confluence, while another subline, H-1/D, did not differentiate. In H-1/A cells, the 4.5- and 2.5-kb major mRNA species of colony-stimulating factor 1 (CSF-1) were expressed before differentiation and were down-regulated at a posttranscriptional level during the differentiation of H-1/A cells. The down-regulation of the CSF-1 gene was not a result of arrested cellular growth, because no down-regulation was detected in the nondifferentiating sister line, H-1/D. This down-regulation appeared to be an early event in differentiation. Cachectin/tumor necrosis factor transiently induced the expression of CSF-1 and inhibited the differentiation of H-1/A cells into adipocytes. This induced expression of CSF-1 was due to an increased rate of transcription.
Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization.
Authors: Dang C V , Barrett J , Villa-Garcia M , Resar L M , Kato G J , Fearon E R
Published: October 2015

The physiological significance of in vitro leucine zipper interactions was studied by the use of two strategies which detect specific protein-protein interactions in mammalian cells. Fusion genes were constructed which produce chimeric proteins containing leucine zipper domains from several proteins fused either to the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein or to the transcriptional activation domain of the herpes simplex virus VP16 protein. Previous studies in mammalian cells have demonstrated that a single chimeric polypeptide containing these two domains will activate transcription of a reporter gene present downstream of the GAL4 DNA-binding site. Similarly, if the GAL4 DNA-binding domain of a chimeric protein could be complexed through leucine zipper interactions with the VP16 activation domain of another chimeric protein, then transcriptional activation of the reporter gene would be detected. Using this strategy for detecting leucine zipper interactions, we observed homo-oligomerization between leucine zipper domains of the yeast protein GCN4 and hetero-oligomerization between leucine zipper regions from the mammalian transcriptional regulating proteins c-Jun and c-Fos. In contrast, homo-oligomerization of the leucine zipper domain from c-Myc was not detectable in cells. The inability of the c-Myc leucine zipper to homo-oligomerize strongly in cells was confirmed independently. The second strategy to detect leucine zipper interactions takes advantage of the observation that the addition of nuclear localization sequences to a cytoplasmic protein will allow the cytoplasmic protein to be transported to and retained in the nucleus. Chimeric genes encoding proteins with sequences from a cytoplasmic protein fused either to the GCN4 or c-Myc leucine zipper domains were constructed. Experiments with the c-Myc chimeric protein failed to demonstrate transport of the cytoplasmic marker protein to the nucleus in cells expressing the wild-type c-Myc protein. In contrast, the cytoplasmic marker was translocated into the nucleus when the GCN4 leucine zippers were present on both the cytoplasmic marker and a nuclear protein, presumably as a result of leucine zipper interaction. These results suggest that c-Myc function requires hetero-oligomerization to an as yet undefined factor.
The ubiquitous transcription factor Oct-1 and the liver-specific factor HNF-1 are both required to activate transcription of a hepatitis B virus promoter.
Authors: Zhou D X , Yen T S
Published: October 2015

The liver-specific transcription factor HNF-1 activates transcription of several mammalian hepatocyte-specific genes. The hepatitis B virus preS1 promoter shows hepatocyte specificity, which has been ascribed to binding of HNF-1 to a cognate DNA sequence upstream of the TATA box. We show here that there is an adjacent site that binds the ubiquitous transcription factor Oct-1. Both the Oct-1 and HNF-1 sites are necessary for liver-specific transcription of the preS1 promoter, but neither site alone activates transcription. The Oct-1 site is also necessary for activation of the preS1 promoter in HeLa cells, expressing transfected HNF-1. Our results show that while Oct-1 is not restricted to hepatocytes, it nevertheless can play a critical role in the expression of a liver-specific gene.
RNA polymerase II elongation complexes paused after the synthesis of 15- or 35-base transcripts have different structures.
Authors: Linn S C , Luse D S
Published: October 2015

We have purified specific RNA polymerase II elongation intermediates initiated at the adenovirus type 2 major late promoter and paused either 15 or 35 to 36 bases downstream of the transcription initiation site. Transcription was arrested at these two sites by combining modification of the promoter sequence with limitation of appropriate nucleotide concentrations in the in vitro reaction. The resultant complexes were remarkably stable and could be purified away from free DNA and contaminating protein-DNA complexes, without loss of activity, by the use of sucrose gradient sedimentation and low-ionic-strength polyacrylamide gel electrophoresis. The complexes were characterized by both DNase I and o-phenanthroline-copper ion nuclease protection assays. The DNase I footprints revealed that the structures of the 15- and 35- to 36-nucleotide transcription complexes differed from those previously reported for an adenovirus type 2 major late preinitiation complex and a subsequent intermediate formed upon addition of ATP. Furthermore, the 35- to 36-nucleotide complex protected a significantly smaller portion of the template than the 15-nucleotide species and migrated at a slightly higher rate in polyacrylamide gels. These observations suggest that changes in structural organization may continue to occur in transcription complexes which are already committed to elongation.
The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.
Authors: Buss J E , Quilliam L A , Kato K , Casey P J , Solski P A , Wong G , Clark R , McCormick F , Bokoch G M , Der C J
Published: October 2015

Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.
Interaction of a lens cell transcription factor with the proximal domain of the mouse gamma F-crystallin promoter.
Authors: Liu Q R , Tini M , Tsui L C , Breitman M L
Published: October 2015

The elements regulating lens-specific expression of the mouse gamma F-crystallin gene were examined. Here we show that mouse gamma F-crystallin sequences -67 to +45 contain a low basal level of lens-specific promoter activity and that sequences -67 to -25, which are highly conserved among different gamma-crystallin genes, are able to function as a strong transcriptional activator when duplicated and placed upstream of the TATA box. We also show that nuclear factors from lens and nonlens cells are able to form different complexes with sequences centered at -46 to -36 and demonstrate that binding of the factor from lens cells correlates with lens-specific promoter activity of the mouse gamma F-crystallin gene.
Identification of a cis-acting sequence required for germ line-specific splicing of the P element ORF2-ORF3 intron.
Authors: Chain A C , Zollman S , Tseng J C , Laski F A
Published: October 2015

P element transposition in Drosophila melanogaster is limited to the germ line because the third intron (the ORF2-ORF3 intron) of the P element transcript is spliced only in germ line cells. We describe a systematic search for P element sequences that are required to regulate the splicing of the ORF2-ORF3 intron. We have identified three adjacent mutations that abolish the germ line specificity and allow splicing of this intron in all tissues. These mutations define a 20-base regulatory region located in the exon, 12 to 31 bases from the 5' splice site. Our data show that this cis-acting regulatory sequence is required to inhibit the splicing of the ORF2-ORF3 intron in somatic cells.
Characterization of the Epstein-Barr virus-inducible gene encoding the human leukocyte adhesion and activation antigen BLAST-1 (CD48).
Authors: Fisher R C , Thorley-Lawson D A
Published: October 2015

BLAST-1 (CD48) (previously referred to as BCM-1 by the Human Gene Nomenclature Committee) is an early-activation-associated membrane glycoprotein expressed on the surface of human leukocytes and induced to a high level following infection of B cells by the Epstein-Barr virus. It is a member of the immunoglobulin superfamily, mediates cell adhesion, and has significant sequence homology to two other adhesion molecules, CD2 and LFA3. Here we report the isolation and characterization of the BLAST-1 gene. The gene is at least 28.6 kb in length, is split into 4 exons, and contains a restriction fragment-length polymorphism. The overall genomic organization is consistent with other members of the immunoglobulin superfamily, in which extracellular immunoglobulinlike domains are encoded by discrete exons. Transcription is initiated at a series of major and minor sites in both normal and tumor-derived lymphoid cells. Appropriately located TATA and CCAAT box sequences were not detected. These characteristics have also been demonstrated for the recently described B-cell-specific genes B29 and CD20. The expression of these genes in B cells may involve the use of multiple promoters and novel transcription initiator-binding proteins. A 1.58-kb genomic DNA fragment, consisting of the 5'-flanking region located immediately upstream of the ATG initiation codon, was able to drive the expression of a reporter gene in an orientation-dependent and tissue-restricted manner.
Replication-competent human mitochondrial DNA lacking the heavy-strand promoter region.
Authors: Moraes C T , Andreetta F , Bonilla E , Shanske S , DiMauro S , Schon E A
Published: October 2015

We identified two patients with progressive external ophthalmoplegia, a mitochondrial disease, who harbored a population of partially deleted mitochondrial DNA (mtDNA) with unusual properties. These molecules were deleted from mtDNA positions 548 to 4,442 and encompassed not only rRNA sequences but the heavy-strand promoter region as well. A 13-bp direct repeat was found flanking the breakpoint precisely, with the repeat at positions 535 to 547 located within the binding site for mitochondrial transcription factor 1 (mtTF1). This is the second mtDNA deletion involving a 13-bp direct repeat reported but is at least 10 times less frequent in the patient population than the former one. In situ hybridization studies showed that transcripts under the control of the light-strand promoter were abundant in muscle fibers with abnormal proliferation of mitochondria, while transcripts directed by the heavy-strand promoter, whether of genes residing inside or outside the deleted region, were not. The efficient transcription from the light-strand promoter implies that the major heavy-and light-strand promoters, although physically close, are functionally independent, confirming previous in vitro studies.
Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis.
Authors: Duester G , Shean M L , McBride M S , Stewart M J
Published: October 2015

Retinoic acid regulation of one member of the human class I alcohol dehydrogenase (ADH) gene family was demonstrated, suggesting that the retinol dehydrogenase function of ADH may play a regulatory role in the biosynthetic pathway for retinoic acid. Promoter activity of human ADH3, but not ADH1 or ADH2, was shown to be activated by retinoic acid in transient transfection assays of Hep3B human hepatoma cells. Deletion mapping experiments identified a region in the ADH3 promoter located between -328 and -272 bp which confers retinoic acid activation. This region was also demonstrated to confer retinoic acid responsiveness on the ADH1 and ADH2 genes in heterologous promoter fusions. Within a 34-bp stretch, the ADH3 retinoic acid response element (RARE) contains two TGACC motifs and one TGAAC motif, both of which exist in RAREs controlling other genes. A block mutation of the TGACC sequence located at -289 to -285 bp eliminated the retinoic acid response. As assayed by gel shift DNA binding studies, the RARE region (-328 to -272 bp) of ADH3 bound the human retinoic acid receptor beta (RAR beta) and was competed for by DNA containing a RARE present in the gene encoding RAR beta. Since ADH catalyzes the conversion of retinol to retinal, which can be further converted to retinoic acid by aldehyde dehydrogenase, these results suggest that retinoic acid activation of ADH3 constitutes a positive feedback loop regulating retinoic acid synthesis.
Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.
Authors: Pepin M C , Barden N
Published: October 2015

Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.
A gene required for RNase P activity in Candida (Torulopsis) glabrata mitochondria codes for a 227-nucleotide RNA with homology to bacterial RNase P RNA.
Authors: Shu H H , Wise C A , Clark-Walker G D , Martin N C
Published: October 2015

We have mapped a gene in the mitochondrial DNA of Candida (Torulopsis) glabrata and shown that it is required for 5' end maturation of mitochondrial tRNAs. It is located between the tRNAfMet and tRNAPro genes, the same tRNA genes that flank the mitochondrial RNase P RNA gene in the yeast Saccharomyces cerevisiae. The gene is extremely AT rich and codes for AU-rich RNAs that display some sequence homology with the mitochondrial RNase P RNA from S. cerevisiae, including two regions of striking sequence homology between the mitochondrial RNAs and the bacterial RNase P RNAs. RNase P activity that is sensitive to micrococcal nuclease has been detected in mitochondrial extracts of C. glabrata. An RNA of 227 nucleotides that is one of the RNAs encoded by the gene that we mapped cofractionated with this mitochondrial RNase P activity on glycerol gradients. The nuclease sensitivity of the activity, the cofractionation of the RNA with activity, and the homology of the RNA with known RNase P RNAs lead us to propose that the 227-nucleotide RNA is the RNA subunit of the C. glabrata mitochondrial RNase P enzyme.
Meiotic induction by Xenopus cyclin B is accelerated by coexpression with mosXe.
Authors: Freeman R S , Ballantyne S M , Donoghue D J
Published: October 2015

We have investigated the relationship between Xenopus laevis c-mos (mosXe) and the cyclin B component of maturation-promoting factor. Microinjection of Xenopus oocytes with in vitro-synthesized RNAs encoding Xenopus cyclin B1 or cyclin B2 induces the progression of meiosis, characterized by germinal vesicle breakdown (GVBD). By preinjecting oocytes with a mosXe-specific antisense oligonucleotide, we show that GVBD induced by cyclin B does not require expression of the mosXe protein. GVBD induced by cyclin B proceeds significantly faster than GVBD induced by progesterone or MosXe. However, coinjection of RNAs encoding cyclin B1 or cyclin B2 with mosXe RNA results in a 2.5- to 3-fold acceleration in GVBD relative to that induced by cyclin B alone. This acceleration of GVBD does not correlate with changes in the level of cyclin B1 and cyclin B2 phosphorylation.
Tst-1, a member of the POU domain gene family, binds the promoter of the gene encoding the cell surface adhesion molecule P0.
Authors: He X , Gerrero R , Simmons D M , Park R E , Lin C J , Swanson L W , Rosenfeld M G
Published: October 2015

Tst-1, a member of the POU domain gene family, is expressed in specific neurons and in myelinating glia in the mammalian nervous system. Bacterially expressed Tst-1 binds specifically to the promoter of the gene encoding myelin protein P0, a Schwann cell surface adhesion molecule. In cotransfection assays, Tst-1 can specifically repress the P0 promoter. The N-terminal part of Tst-1 protein is highly glycine- and alanine-rich, a structural feature shared by the helix-loop-helix protein TFEB.
Substitution of foreign protein sequences into a chimeric toxin composed of transforming growth factor alpha and Pseudomonas exotoxin.
Authors: Debinski W , Siegall C B , Fitzgerald D , Pastan I
Published: October 2015

TGF alpha-PE40 is a chimeric toxin made by replacing domain Ia of Pseudomonas exotoxin (PE) with transforming growth factor alpha (TGF alpha). We have now replaced a portion of domain Ib of PE with different polypeptides or an extra domain III of PE in transforming growth factor alpha-PE40 and maintained cell killing. Thus, TGF alpha-PE40 can be used to transport foreign protein sequences into the cytosol of cells.
Induction of a cyclic AMP-responsive gene in living cells requires the nuclear factor CREB.
Authors: Meinkoth J L , Montminy M R , Fink J S , Feramisco J R
Published: October 2015

We constructed cell lines containing various enhancer elements cloned upstream from a marker gene. By microinjection of specific antibodies directed against transcriptional activator proteins into these cell lines, we have developed a functional assay for factors which regulate the activity of target promoters. Here we show that microinjection of a highly specific antibody to the cyclic AMP enhancer element-binding (CREB) protein diminishes gene expression in response to cyclic AMP in living fibroblasts.
DNase I-hypersensitive sites and transcription factor-binding motifs within the mouse E beta meiotic recombination hot spot.
Authors: Shenkar R , Shen M H , Arnheim N
Published: October 2015

The second intron of the E beta gene in the mouse major histocompatibility complex is the site of a meiotic recombination hot spot. We detected two DNase I-hypersensitive sites in this intron in meiotic cells isolated from mouse testes. One site appears to be constitutive and is found in other tissues regardless of whether or not they express the E beta gene. Near this hypersensitive site are potential binding motifs for H2TF1/KBF1, NF kappa B, and octamer transcription factors. Gel retardation studies with mouse lymphoma cell nuclear extracts confirmed that each of these motifs is capable of binding protein. The binding of transcription factors may contribute to the enhancement of recombination potential by altering chromatin structure and increasing the accessibility of the DNA to the recombination machinery.
Recognition of U1 and U2 small nuclear RNAs can be altered by a 5-amino-acid segment in the U2 small nuclear ribonucleoprotein particle (snRNP) B" protein and through interactions with U2 snRNP-A' protein.
Authors: Bentley R C , Keene J D
Published: October 2015

We have investigated the sequence elements influencing RNA recognition in two closely related small nuclear ribonucleoprotein particle (snRNP) proteins, U1 snRNP-A and U2 snRNP-B". A 5-amino-acid segment in the RNA-binding domain of the U2 snRNP-B" protein was found to confer U2 RNA recognition when substituted into the corresponding position in the U1 snRNP-A protein. In addition, B", but not A, was found to require the U2 snRNP-A' protein as an accessory factor for high-affinity binding to U2 RNA. The pentamer segment in B" that conferred U2 RNA recognition was not sufficient to allow the A' enhancement of U2 RNA binding by B", thus implicating other sequences in this protein-protein interaction. Sequence elements involved in these interactions have been localized to variable loops of the RNA-binding domain as determined by nuclear magnetic resonance spectroscopy (D. Hoffman, C.C. Query, B. Golden, S.W. White, and J.D. Keene, Proc. Natl. Acad. Sci. USA, in press). These findings suggest a role for accessory proteins in the formation of RNP complexes and pinpoint amino acid sequences that affect the specificity of RNA recognition in two members of a large family of proteins involved in RNA processing.
Control of protein phosphatase 2A by simian virus 40 small-t antigen.
Authors: Yang S I , Lickteig R L , Estes R , Rundell K , Walter G , Mumby M C
Published: October 2015

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity. Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Small-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone H1 as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.
Effects of controlled RAD52 expression on repair and recombination in Saccharomyces cerevisiae.
Authors: Dornfeld K J , Livingston D M
Published: October 2015

We have examined the effects of RAD52 overexpression on methyl methanesulfonate (MMS) sensitivity and spontaneous mitotic recombination rates. Cells expressing a 10-fold excess of RAD52 mRNA from the ENO1 promoter are no more resistant to MMS than are wild-type cells. Similarly, under the same conditions, the rate of mitotic recombination within a reporter plasmid does not exceed that measured in wild-type cells. This high level of expression is capable of correcting the defects of rad52 mutant cells in carrying out repair and recombination. From these observations, we conclude that wild-type amounts of Rad52 are not rate limiting for repair of MMS-induced lesions or plasmid recombination. By placing RAD52 under the control of the inducible GAL1 promoter, we find that induction results in a 12-fold increase in the fraction of recombinants within 4 h. After this time, the fraction increases less rapidly. When RAD52 expression is quickly repressed during induction, the amount of RAD52 mRNA decreases rapidly and no nascent recombinants are formed. This result suggests a short active half-life for the protein product. Induction of RAD52 in G1-arrested mutant cells also causes a rapid increase in recombinants, suggesting that replication is not necessary for plasmid recombination.
A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems.
Authors: Gröning K , Palfi Z , Gupta S , Cross M , Wolff T , Bindereif A
Published: October 2015

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.
Accurate transcription of a plant mitochondrial gene in vitro.
Authors: Hanic-Joyce P J , Gray M W
Published: October 2015

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.
The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.
Authors: Fazioli F , Kim U H , Rhee S G , Molloy C J , Segatto O , Di Fiore P P
Published: October 2015

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.
Activation of the silent endogenous cholesterol-7-alpha-hydroxylase gene in rat hepatoma cells: a new complementation group having resistance to 25-hydroxycholesterol.
Authors: Leighton J K , Dueland S , Straka M S , Trawick J , Davis R A
Published: October 2015

The oxysterol 25-hydroxycholesterol acts both as a regulatory sterol determining the expression of genes governed by sterol regulatory elements and as a substrate for 7-alpha-hydroxylase, the first and rate-limiting enzyme in the bile acid synthetic pathway. Most wild-type nonhepatic cells are killed by the cytotoxic action of 25-hydroxycholesterol. In contrast, liver cells, which express 7-alpha-hydroxylase activity, are resistant to killing by 25-hydroxycholesterol. We examined the possibility that selection for resistance to 25-hydroxycholesterol might lead to the derivation of a cell line expressing 7-alpha-hydroxylase. A rat hepatoma cell line (7-alpha-hydroxylase minus) was transfected with human DNA and screened for resistance to 25-hydroxycholesterol. Although parental hepatoma cells were all killed within a week, a 25-hydroxycholesterol-resistant cell line (L35 cells) which showed stable expression of 7-alpha-hydroxylase activity and mRNA was obtained. These cells exhibited normal inhibition of cholesterol biosynthesis by 25-hydroxycholesterol. Blocking 7-alpha-hydroxylase activity with ketoconazole also blocked the resistance of L35 cells to 25-hydroxycholesterol. Isolation of microsomes from these cells showed levels of 7-alpha-hydroxylase activity (22.9 pmol/min/mg of protein) that were comparable to the activity (33.2 pmol/min/mg) of microsomes isolated from the livers of rats killed during the high point of the diurnal cycle. Parental cells had no detectable activity. These data show a new complementation group for 25-hydroxycholesterol resistance: expression of 7-alpha-hydroxylase. Dexamethasone increased both the activity and the cellular content of mRNA coding for 7-alpha-hydroxylase. Since dactinomycin blocked the ability of dexamethasone to induce mRNA, active transcription is required. Southern analysis of genomic DNA showed that L35 cells contain the rat (endogenous) gene but not the human gene. Furthermore, the RNA expressed by L35 cells is similar in size to rat RNA and is distinct from the human form of 7-alpha-hydroxylase. The combined data indicate that L35 cells are resistant to 25-hydroxycholesterol because they express 7-alpha-hydroxylase. The mechanism responsible involves activation of the endogenous (silent) gene of the parental rat hepatoma cell.
Simian virus 40 large-T antigen expresses a biological activity complementary to the p300-associated transforming function of the adenovirus E1A gene products.
Authors: Yaciuk P , Carter M C , Pipas J M , Moran E
Published: October 2015

In this report we present evidence that simian virus 40 T antigen encodes a biological activity that is functionally equivalent to the transforming activity lost by deletion of the E1A p300-binding region. T-antigen constructs from which the pRb-binding region has been deleted are virtually unable to induce foci of transformed cells in a ras cooperation assay in primary baby rat kidney cells. Nevertheless, such a construct can cooperate with an E1A N-terminal deletion mutant, itself devoid of transforming activity, to induce foci in this assay. The heterologous trans-cooperating activity observed between E1A and T-antigen deletion products is as efficient as trans cooperation between mutants expressing individual E1A domains. The cooperating function can be impaired by a deletion near the N terminus of T antigen. Such a deletion impairs neither the p53-binding function nor the activity of the pRb-binding region.
Organization and chromosomal localization of the human platelet-derived endothelial cell growth factor gene.
Authors: Hagiwara K , Stenman G , Honda H , Sahlin P , Andersson A , Miyazono K , Heldin C H , Ishikawa F , Takaku F
Published: October 2015

Human platelet-derived endothelial cell growth factor (hPD-ECGF) is a novel angiogenic factor which stimulates endothelial cell growth in vitro and promotes angiogenesis in vivo. We report here the cloning and sequencing of the gene for hPD-ECGF and its flanking regions. This gene is composed of 10 exons dispersed over a 4.3-kb region. Its promoter lacks a TATA box and a CCAAT box, structures characteristic of eukaryotic promoters. Instead, six copies of potential Sp1-binding sites (GGGCGG or CCGCCC) were clustered just upstream of the transcription start sites. Southern blot analysis using genomic DNAs from several vertebrates suggested that the gene for PD-ECGF is conserved phylogenetically among vertebrates. The gene for hPD-ECGF was localized to chromosome 22 by analysis of a panel of human-rodent somatic cell hybrid lines.
In vitro studies of the binding of the ARGR proteins to the ARG5,6 promoter.
Authors: Dubois E , Messenguy F
Published: October 2015

ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae. We show here that they are also required in vitro to observe a protein-DNA complex with the promoter of the ARG5,6 gene. The specific binding of ARGR proteins in vitro is stimulated by arginine. Antibodies raised against a synthetic MCM1 polypeptide retard the migration of ARGR-DNA complex on gel mobility shift assays. This result suggests that MCM1 could be an additional regulatory element of arginine metabolism.
A family of genes related to a new expression site-associated gene in Trypanosoma equiperdum.
Authors: Florent I C , Raibaud A , Eisen H
Published: October 2015

Two genes, belonging to a new expression site-associated gene family of six to eight members in Trypanosoma equiperdum and Trypanosoma brucei, have been cloned from a T. equiperdum variant. One of them, called ESAG-9c, is contained in the 1.78-C expression site and is found just upstream of the 5' barren region. The other one, called ESAG-9u, is unique in the family, is not telomere linked, and apparently is not expression site related. A 2-kb poly(A)+ mRNA is detected with probes for this ESAG-9 family in all T. equiperdum variants examined. By using polymerase chain reaction and restriction fragment length polymorphism techniques, it has been possible to distinguish between ESAG-9c and ESAG-9u and to show that ESAG-9c is transcribed in an expression site-specific manner. However, ESAG-9u (or another gene in the family having identical characteristics) is transcribed in all variants, regardless of the expression site used by these variants. Thus, this ESAG-9 family contains at least one gene that is under expression site control but might have other genes that are not. The function of these ESAG-9 genes is unknown. Transcripts homologous to ESAG-9 were detected in T. brucei bloodstream forms but not in procyclics.
Identification of DNA regions required for mitotic and meiotic functions within the centromere of Schizosaccharomyces pombe chromosome I.
Authors: Hahnenberger K M , Carbon J , Clarke L
Published: October 2015

We have determined the structural organization and functional roles of centromere-specific DNA sequence repeats in cen1, the centromere region from chromosome I of the fission yeast Schizosaccharomyces pombe. cen1 is composed of various classes of repeated sequences designated K', K"(dgl), L, and B', arranged in a 34-kb inverted repeat surrounding a 4- to 5-kb nonhomologous central core. Artificial chromosomes containing various portions of the cen1 region were constructed and assayed for mitotic and meiotic centromere function in S. pombe. Deleting K' and L from the distal portion of one arm of the inverted repeat had no effect on mitotic centromere function but resulted in greatly increased precocious sister chromatid separation in the first meiotic division. A centromere completely lacking K' and L, but containing the central core, one copy of B' and K" in one arm, and approximately 2.5 kb of the core-proximal portion of B' in the other arm, was also fully functional mitotically but again did not maintain sister chromatid attachment in meiosis I. However, deletion of K" from this minichromosome resulted in complete loss of centromere function. Thus, one copy of at least a portion of the K" (dgl) repeat is absolutely required but is not sufficient for S. pombe centromere function. The long centromeric inverted-repeat region must be relatively intact to maintain sister chromatid attachment in meiosis I.
Transcripts from amplified sequences of an inherited homogeneously staining region in chromosome 1 of the house mouse (Mus musculus).
Authors: Eckert W A , Plass C , Weith A , Traut W , Winking H
Published: October 2015

Several populations of the house mouse, Mus musculus, are polymorphic for the presence or absence of an inherited homogeneously staining region (HSR) in chromosome 1. The HSR consists of highly amplified DNA sequences, present in low copy numbers in the HSR- genome. A cloned HSR-derived genomic sequence detected transcripts of about 1.3 and 4.5 kb on blots of poly(A)+ RNA from liver of HSR+ mice but not from that of HSR- mice. A cDNA library was established from RNA of HSR+ mice and screened with the HSR-derived genomic clone. Positive clones were isolated and shown to be complementary to the 1.3-kb RNA species and to amplified DNA sequences in the HSR+ genome. The combined sequence of four overlapping cloned cDNAs is 959 nucleotides long and includes an open reading frame encoding a putative protein of 208 amino acids. The pertinent gene is unidentified. No homologous sequence is stored in the EMBL data base. A stretch of 109 nucleotides at the 3' end of the 1.3-kb RNA homology region in the same genomic fragment, as indicated by hybridization data and sequence motifs resembling promoter elements. Thus, our data suggest that at least two genes or gene families are encoded in the HSR.
In vitro genetic transfer of protein synthesis and respiration defects to mitochondrial DNA-less cells with myopathy-patient mitochondria.
Authors: Chomyn A , Meola G , Bresolin N , Lai S T , Scarlato G , Attardi G
Published: October 2015

A severe mitochondrial protein synthesis defect in myoblasts from a patient with mitochondrial myopathy was transferred with myoblast mitochondria into two genetically unrelated mitochondrial DNA (mtDNA)-less human cell lines, pointing to an mtDNA alteration as being responsible and sufficient for causing the disease. The transfer of the defect correlated with marked deficiencies in respiration and cytochrome c oxidase activity of the transformants and the presence in their mitochondria of mtDNA carrying a tRNA(Lys) mutation. Furthermore, apparently complete segregation of the defective genotype and phenotype was observed in the transformants derived from the heterogeneous proband myoblast population, suggesting that the mtDNA heteroplasmy in this population was to a large extent intercellular. The present work thus establishes a direct link between mtDNA alteration and a biochemical defect.
Characterization of a telomere-binding protein from Physarum polycephalum.
Authors: Coren J S , Epstein E M , Vogt V M
Published: October 2015

We have partially purified a nuclear protein (PPT) from Physarum polycephalum that binds to the extrachromosomal ribosomal DNA telomeres of this acellular slime mold. Binding is specific for the (T2AG3)n telomere repeats, as evidenced by nitrocellulose filter binding assays, by gel mobility shift assays with both DNA fragments and double-stranded oligonucleotides, and by DNase I footprinting. PPT is remarkably heat stable, showing undiminished binding activity after incubation at 90 degrees C. It sediments at 1.2S, corresponding to a molecular weight of about 10,000 (for a globular protein), and its binding activity is undiminished by incubation with RNase, suggesting that it is not a ribonucleoprotein. We hypothesize that PPT plays a structural role in telomeres, perhaps preventing nucleolytic degradation or promoting telomere extension by a telomere-specific terminal transferase.