bluuebunny/crossref_metadata_2025 huggingface dataset:
HPR1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene.
Authors: Aguilera A , Klein H L
Published: October 2015

The HPR1 gene has been cloned by complementation of the hyperrecombination phenotype of hpr1-1 strains by using a color assay system. HPR1 is a gene that is in single copy on chromosome IV of Saccharomyces cerevisiae, closely linked to ARO1, and it codes for a putative protein of 752 amino acids (molecular mass, 88 kilodaltons). Computer searches revealed homology (48.8% conserved homology; 24.8% identity) with the S. cerevisiae TOP1 gene in an alpha-helical stretch of 129 amino acids near the carboxy-terminal region of both proteins. The ethyl methanesulfonate-induced hpr1-1 mutation is a single-base change that produces a stop codon at amino acid 559 coding for a protein that lacks the carboxy-terminal TOP1 homologous region. Haploid strains carrying deletions of the HPR1 gene show a slightly reduced mitotic growth rate and extremely high rates of intrachromosomal excision recombination (frequency, 10 to 15%) but have a undetectable effect on rDNA recombination. Double-null mutants hpr1 top1 grow very poorly. We conclude that Hpr1 is a novel eucaryotic protein, mutation of which causes an increase in mitotic intrachromosomal excision recombination, and that it may be functionally related to an activity of the topoisomerase I protein.
c-Ha-ras down regulates the alpha-fetoprotein gene but not the albumin gene in human hepatoma cells.
Authors: Nakao K , Lawless D , Ohe Y , Miyao Y , Nakabayashi H , Kamiya H , Miura K , Ohtsuka E , Tamaoki T
Published: October 2015

We studied the effects of transfection of the normal c-Ha-ras gene, rasGly-12, and its oncogenic mutant, rasVal-12, on expression of the alpha-fetoprotein (AFP) and albumin genes in a human hepatoma cell line, HuH-7. The mutant and, to a lesser extent, the normal ras gene caused reduction of the AFP mRNA but not the albumin mRNA level in transfected HuH-7 cells. Cotransfection experiments with a rasVal-12 expression plasmid and a chloramphenicol acetyltransferase reporter gene fused to AFP regulatory sequences showed that rasVal-12 suppressed the activity of enhancer and promoter regions containing A + T-rich sequences (AT motif). In contrast, rasVal-12 did not affect the promoter activity of the albumin and human hepatitis B virus pre-S1 genes even though these promoters contain homologous A + T-rich elements. ras transfection appeared to induce phosphorylation of nuclear proteins that interact with the AFP AT motif, since gel mobility analysis revealed the formation of slow-moving complexes which was reversed by phosphatase treatment. However, similar changes in complex formation were observed with the albumin and hepatitis B surface antigen pre-S1 promoters. Therefore, this effect alone cannot explain the specific down regulation of the AFP promoter and enhancer activity. ras-mediated suppression of the AFP gene may reflect the process of developmental gene regulation in which AFP gene transcription is controlled by a G-protein-linked signal transduction cascade triggered by external growth stimuli.
A novel cis-acting DNA element required for a high level of inducible expression of the rat P-450c gene.
Authors: Yanagida A , Sogawa K , Yasumoto K I , Fujii-Kuriyama Y
Published: October 2015

A novel cis-acting regulatory element (designated BTE for basic transcription element) was found in the region proximal to the TATA sequence of the P-450c gene by the use of deletion mutations. This DNA element is considered to be involved in the basic transcription of the gene and does not show distinct enhancer activity in itself. Together with the XRE sequence (A. Fujisawa-Sehara, K. Sogawa, M. Yamane, and Y. Fujii-Kuriyama, Nucleic Acids Res. 15:4179-4191, 1987), however, this sequence is required for a high inducible expression of the P-450c gene in response to xenobiotic inducers. The BTE sequence contained the GC box consensus sequence and half of the NF-1-binding consensus or CAT box sequence, but their synthetic oligonucleotides, used as competitors in the gel mobility shift assays, did not compete with the BTE sequence for the binding protein, suggesting that the BTE sequence functions as a different recognition sequence from that for Sp1 or NF-1. Analogous sequences to BTE are found in the region proximal to the TATA sequence of other genes, especially other P-450 genes with different modes of regulation, suggesting that the BTE sequence plays a common regulatory role in basic transcription of genes including a group of the P-450 superfamily. The ubiquitous distribution of nuclear factor(s) binding to this element supports this suggestion.
Multilevel regulation of surface antigen gene expression in Paramecium tetraurelia.
Authors: Gilley D , Rudman B M , Preer J R , Polisky B
Published: October 2015

A family of genes is responsible for production of surface antigenic components of Paramecium tetraurelia. These surface proteins are expressed in a mutually exclusive manner. Individuals rarely display more than one type. However, changes in environmental conditions can cause different surface proteins which replace preexisting types to be expressed. We investigated the nature of regulation of the genes for the A, C, and H surface antigens of P. tetraurelia. A system for in vitro run-on transcription was developed from crude Paramecium extracts and used in this analysis. The genes for surface antigens A and H were controlled at the level of transcription. However, the gene for surface antigen C demonstrated both transcriptional and posttranscriptional control, depending on the serotype being expressed. When animals expressed serotype A, the gene for surface antigen C was not transcribed. However, when animals expressed serotype H, the gene for surface antigen C was actively transcribed and stable surface antigen C mRNA was present in the cells, although surface antigen C was not detectable by serotype testing or by a salt-alcohol extraction method. The kinetics of transformation from serotype H to serotype C were determined by using the in vitro transcription system and monitoring steady-state RNA levels. During the transition, serotype A transcription was detected in run-on transcription experiments, although this RNA did not accumulate. The results indicate that serotype expression is controlled at several levels and that not all serotype genes are controlled in the same manner.
Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols.
Authors: Hoy C A , Lewis E D , Schimke R T
Published: October 2015

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.
mXBP/CRE-BP2 and c-Jun form a complex which binds to the cyclic AMP, but not to the 12-O-tetradecanoylphorbol-13-acetate, response element.
Authors: Ivashkiv L B , Liou H C , Kara C J , Lamph W W , Verma I M , Glimcher L H
Published: October 2015

Proto-oncogene products c-Fos and c-Jun form a complex which binds with high affinity to the 12-O-tetradecanoylphorbol-13-acetate (TPA) response DNA element and which stimulates transcription of phorbol ester- inducible genes. We have previously identified, by screening a lambda gt11 expression library, murine protein mXBP, which binds to a sequence which overlaps the 3' end of the murine class II major histocompatibility complex A alpha gene X box, a conserved transcription element found upstream of all class II genes. Here, we demonstrate that the target sequence for mXBP is a consensus cyclic AMP response element (CRE). mXBP is a member of the leucine zipper family of DNA-binding proteins and has significant homology to oncoproteins c-Fos and c-Jun. The inferred amino acid sequence of mXBP shows near identity to human CRE-BP1, except it does not contain an internal proline-rich domain. Immunoprecipitation and glutaraldehyde cross-linking studies show that mXBP/CRE-BP2 can form a complex with c-Jun. Complex formation is dependent on intact leucine zipper domains in both proteins. mXBP-c-Jun complexes can coexist with c-Fos-c-Jun complexes and can bind with high affinity to CRE, but not to TPA response DNA element, sequences. These results suggest that changes in the expression of mXBP/CRE-BP2, c-Fos, and c-Jun, which alter the ratio of mXBP-c-Jun to c-Fos-c-Jun complexes, would affect the relative expression of cyclic AMP and phorbol ester-responsive genes. This provides support for a combinatorial model of gene regulation, whereby protein-protein interactions which alter the DNA binding specificity of protein complexes can expand the flexibility of cellular transcriptional responses.
The human Pim-1 gene is selectively transcribed in different hemato-lymphoid cell lines in spite of a G + C-rich housekeeping promoter.
Authors: Meeker T C , Loeb J , Ayres M , Sellers W
Published: October 2015

The expression of the Pim-1 proto-oncogene was studied by using the K562, Daudi, and Jurkat cell lines. In K562, Pim-1 mRNA levels were more than 20-fold higher than in Daudi and 50-fold higher than in Jurkat. Nuclear run-on assay data correlated directly with the steady-state mRNA levels, suggesting that the rate of transcription was responsible for the selective expression of this gene. Furthermore, the half-life of Pim-1 mRNA was shown to be 47 min in K562, 71 min in Daudi, and 35 min in Jurkat. This indicated that selective Pim-1 mRNA expression did not depend on posttranscriptional regulation. Therefore, 1.7 kilobases of the Pim-1 promoter was sequenced and studied in detail. The sequence showed that the region from nucleotide -1 to -873 was G + C rich (71%). Study of promoter deletions defined two major functional regions, a proximal element (nucleotide -104 to -1) and a distal element (nucleotide -427 to -336). DNase I protection assays identified binding sites for the Sp1 and AP2 proteins in these elements. A possible new transcription factor binds at position -348 in the distal element. In our study of the 1.7-kilobase Pim-1 promoter, we found no differences between K562 and Jurkat that could explain large differences in transcription. Therefore, the Pim-1 promoter appears to function constitutively, and we conclude that distant elements must regulate the tissue-selective expression of this gene. Although the Pim-1 gene has a G + C-rich housekeeping promoter, expression is carefully regulated at the level of transcription.
mRNAs that mature through trans-splicing in Caenorhabditis elegans have a trimethylguanosine cap at their 5' termini.
Authors: Van Doren K , Hirsh D
Published: October 2015

Approximately 10% of the mRNAs in the nematode Caenorhabditis elegans mature through a trans-splicing mechanism that involves the transfer of a 22-nucleotide spliced leader to the 5' end of the pre-mRNA. The spliced leader RNA exists as a small nuclear ribonucleoprotein particle and has the trimethylguanosine cap that is characteristic of eucaryotic small nuclear RNAs. We found that the trimethylguanosine cap present on the spliced leader RNA was transferred to the pre-mRNA during the trans-splicing reaction. Thereafter, the trimethylguanosine cap was maintained on the mature mRNA. This is the first example of eucaryotic cellular mRNAs possessing a trimethylguanosine cap structure.
Effects of translocations on transcription from PVT.
Authors: Shtivelman E , Bishop J M
Published: October 2015

We have previously described a transcription unit on human chromosome 8, designated as PVT, that is consistently disrupted by the minority forms of translocations [t(2;8) and t(8;22)] in Burkitt's lymphoma. PVT begins 57 kilobase pairs downstream of the proto-oncogene MYC and is more than 200 kilobase pairs in length. In order to explore the pathogenic impact of translocations affecting PVT, we have characterized further the structure and transcription of the locus. In normal cells, PVT is transcribed into a variety of RNAs, the diversity of which remains unexplained. Alleles of PVT affected by translocations give rise to additional RNAs. These RNAs arise from a fusion of the first exon of PVT on chromosome 8 to the constant region of an immunoglobulin light chain on either chromosome 2 or chromosome 22. We have found no evidence that any of the normal or abnormal transcripts of PVT give rise to a protein. Our results suggest that the pathogenic effects of the variant translocations in Burkitt's lymphoma are not executed by a gene situated in a vicinity of the chromosomal breakpoints. Instead, our data leave open the possibility that the effects of the translocations may be mediated by activation of the relatively distant MYC gene.
Genetic exploration of interactive domains in RNA polymerase II subunits.
Authors: Martin C , Okamura S , Young R
Published: October 2015

The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.
Isolation and characterization of eight tumor necrosis factor-induced gene sequences from human fibroblasts.
Authors: Lee T H , Lee G W , Ziff E B , Vilcek J
Published: October 2015

A lambda cDNA library was prepared from polyadenylated RNA isolated from quiescent human diploid FS-4 fibroblasts stimulated with tumor necrosis factor for 3 h. Differential screening was used to isolate cDNA sequences that are stimulated by tumor necrosis factor. Eight distinct tumor necrosis factor-stimulated gene sequences (designated TSG-1, -6, -8, -12, -14, -21, -27, and -37) were partially sequenced and compared with known sequences from GenBank. TSG-1 was identical to the gene for interleukin-8. TSG-8 corresponded to the gene for monocyte chemotactic and activating factor. TSG-21 and -27 were identical to the genes for collagenase and stromelysin, respectively. The other four sequences showed no homologies with known genes. Patterns of induction of mRNAs corresponding to the eight cloned cDNAs by various cytokines, growth factors, and activators of second messenger pathways were analyzed in FS-4 cells.
Identification of a novel neuronal C-SRC exon expressed in human brain.
Authors: Pyper J M , Bolen J B
Published: October 2015

Neuronal cells are known to express at least two different forms of the C-SRC proto-oncogene as a consequence of alternative splicing events which add an 18-nucleotide exon (the NI exon) between C-SRC exons 3 and 4. Here we report that a second neuronal exon of C-SRC is also present between C-SRC exons 3 and 4. This neuronal exon (the NII exon) of C-SRC was isolated from human adult and fetal brain-derived cDNAs and contains 33 nucleotides capable of encoding 11 amino acids (Gln-Thr-Trp-Phe-Thr-Phe-Arg-Trp-Leu-Gln-Arg). The human NI exon was located approximately 390 nucleotides from the end of C-SRC exon 3, whereas the NII exon was approximately 1,000 nucleotides from the beginning of C-SRC exon 4. Analysis of human brain RNA revealed that the NII exon is utilized primarily in conjunction with the NI exon to yield transcripts capable of encoding C-SRC products possessing 17 additional amino acids. These splicing events, which occur between the NI and NII exons, are predicted to alter the sixth amino acid encoded by the NI exon from an arginine to a serine residue, producing a potentially novel phosphorylation site. Analysis of the different C-SRC RNA transcripts revealed that the level of C-SRC RNA containing both NI and NII exons is similar in adult and fetal brain tissue, whereas the level of C-SRC RNA containing only the NI exon or the nonneuronal form of C-SRC RNAs is significantly higher in fetal brain tissues. These results indicate that the expression and splicing pattern of the C-SRC gene are developmentally regulated in the human brain.
Amplification of tandemly repeated origin control sequences confers a replication advantage on rDNA replicons in Tetrahymena thermophila.
Authors: Yu G L , Blackburn E H
Published: October 2015

The macronuclear rRNA genes (rDNA) in the ciliate Tetrahymena thermophila are normally palindromic linear replicons, containing two copies of the replication origin region in inverted orientation. A circular plasmid containing a single Tetrahymena rRNA gene (one half palindrome) joined to a tandem repeat of a 1.9-kilobase (kb) rDNA segment encompassing the rDNA replication origin and known replication control elements was used to transform Tetrahymena macronuclei by microinjection. This plasmid was shown previously to have a replication advantage over the rDNA allele of the recipient cell strain (G.-L. Yu and E. H. Blackburn, Proc. Natl. Acad. Sci. USA 86:8487-8491, 1990). During vegetative cell divisions, the circular and palindromic rDNAs were rapidly replaced by novel, successively longer linear rDNAs that eventually contained up to 30 tandem 1.9-kb repeats, resulting from homologous but unequal crossovers between the 1.9-kb repeats. We present evidence to show that increasing the number of copies of the replication control regions increases the replicative advantage of the rDNA, the first such situation for a cellular nuclear replicon in a eucaryote.
Efficient site-specific cleavage by RNase MRP requires interaction with two evolutionarily conserved mitochondrial RNA sequences.
Authors: Bennett J L , Clayton D A
Published: October 2015

RNase MRP is a site-specific endonuclease that processes primer mitochondrial RNA from the leading-strand origin of mitochondrial DNA replication. Using deletional analysis and saturation mutagenesis, we have determined the substrate requirements for cleavage by mouse mitochondrial RNase MRP. Two regions of sequence homology among vertebrate mitochondrial RNA primers, conserved sequence blocks II and III, were found to be critical for both efficient and accurate cleavage; a third region of sequence homology, conserved sequence block I, was dispensable. Analysis of insertion and deletion mutations within conserved sequence block II demonstrated that the specificity of RNase MRP accommodates the natural sequence heterogeneity of conserved sequence block II in vivo. Heterologous assays with human RNase MRP and mutated mouse mitochondrial RNA substrates indicated that sequences essential for substrate recognition are conserved between mammalian species.
A gene family for acidic ribosomal proteins in Schizosaccharomyces pombe: two essential and two nonessential genes.
Authors: Beltrame M , Bianchi M E
Published: October 2015

We have cloned the genes for small acidic ribosomal proteins (A-proteins) of the fission yeast Schizosaccharomyces pombe. S. pombe contains four transcribed genes for small A-proteins per haploid genome, as is the case for Saccharomyces cerevisiae. In contrast, multicellular eucaryotes contain two transcribed genes per haploid genome. The four proteins of S. pombe, besides sharing a high overall similarity, form two couples of nearly identical sequences. Their corresponding genes have a very conserved structure and are transcribed to a similar level. Surprisingly, of each couple of genes coding for nearly identical proteins, one is essential for cell growth, whereas the other is not. We suggest that the unequal importance of the four small A-proteins for cell survival is related to their physical organization in 60S ribosomal subunits.
SAS1 and SAS2, GTP-binding protein genes in Dictyostelium discoideum with sequence similarities to essential genes in Saccharomyces cerevisiae.
Authors: Saxe S A , Kimmel A R
Published: October 2015

We have identified two novel, very closely related genes, SAS1 and SAS2, from Dictyostelium discoideum. These encode small, approximately 20-kilodaton proteins with amino acid sequences thought to be involved in interaction with guanine nucleotides. The protein sizes, spacings of GTP-binding domains, and carboxyl-terminal sequences suggest their relationship to the ubiquitous ras-type proteins. Their sequences, however, are sufficiently different to indicate that they are not true ras proteins. More extensive sequence identity (approximately 55%) is shared with the YPT1 and SEC4 proteins from Saccharomyces cerevisiae. These yeast proteins are essential for growth and are believed to be involved in intracellular signaling associated with membrane function. SAS1 and SAS2 exhibit distinct patterns of genomic organization and developmentally regulated gene expression. SAS1 contains introns and is associated with a developmentally regulated repetitive element. SAS2 is colinear with its mRNA and does not appear to be closely linked with this repetitive element. Both genes are expressed during growth and throughout development. SAS1 is maximally expressed during cytodifferentiation, when two sizes of SAS1 mRNA are detectable. SAS2 mRNA levels are maximal during culmination. On the basis of the expression patterns of the SAS genes and their relationship to the YPT1 and SEC4 genes, we discuss possible functions of the SAS proteins.
Antibody-induced mitogenicity mediated by a chimeric CD2-c-fms receptor.
Authors: Roussel M F , Transy C , Kato J Y , Reinherz E L , Sherr C J
Published: October 2015

A chimeric receptor composed of the extracellular domain of the human T-cell antigen CD2 (T11) joined to the membrane-spanning segment and the intracellular tyrosine kinase domain of the human colony-stimulating factor 1 receptor (CSF-1R) was expressed in murine NIH 3T3 fibroblasts. Stimulation of these cells with monoclonal antibodies to CD2 induced phosphorylation of the chimeric glycoprotein on tyrosine, receptor downmodulation, and mitogenesis. In contrast, neither human CSF-1R nor the chimeric receptor was able to function in interleukin-2-dependent murine T cells. In fibroblasts, then, CSF-1 per se is not required for activation of the receptor kinase or for a biological response, whereas in T cells, CSF-1R may be unable to engage the downstream signal transduction machinery.
Developmentally regulated interactions of liver nuclear factors with the rat phosphoenolpyruvate carboxykinase promoter.
Authors: Trus M , Benvenisty N , Cohen H , Reshef L
Published: October 2015

A sequential pattern of interactions of trans-acting factors in rat liver with the phosphoenolpyruvate carboxykinase promoter during late development was observed. A liver-enriched factor, possibly AF1, interacted with the promoter in fetal liver, whereas a factor with the characteristics of C/EBP bound the promoter after birth with the onset of the gene expression.
Retroviral and pseudogene insertion sites reveal the lineage of human salivary and pancreatic amylase genes from a single gene during primate evolution.
Authors: Samuelson L C , Wiebauer K , Snow C M , Meisler M H
Published: October 2015

We have analyzed the junction regions of inserted elements within the human amylase gene complex. This complex contains five genes which are expressed at high levels either in the pancreas or in the parotid gland. The proximal 5'-flanking regions of these genes contain two inserted elements. A gamma-actin pseudogene is located at a position 200 base pairs upstream of the first coding exon. All of the amylase genes contain this insert. The subsequent insertion of an endogenous retrovirus interrupted the gamma-actin pseudogene within its 3'-untranslated region. Nucleotide sequence analysis of the inserted elements associated with each of the five human amylase genes has revealed a series of molecular events during the recent history of this gene family. The data indicate that the entire gene family was generated during primate evolution from one ancestral gene copy and that the retroviral insertion activated a cryptic promoter.
Regulation of the chicken embryonic myosin light-chain (L23) gene: existence of a common regulatory element shared by myosin alkali light-chain genes.
Authors: Uetsuki T , Nabeshima Y , Fujisawa-Sehara A , Nabeshima Y
Published: October 2015

The transcriptional regulation of the chicken myosin alkali light-chain (MLC) L23 gene was analyzed. Two different types of cis-regulatory regions were identified: one was a silencerlike region located between 3.7 and 2.7 kilobases upstream of the mRNA initiation site, and the other was essential for the expression of L23 in skeletal muscle cells and was located between 106 and 91 base pairs upstream of the cap site. This 16-base-pair cis-acting element was designated as the MLC box since it is well conserved in various muscle-specific MLC promoter regions. The activity of the MLC box showed tissue specificity. To analyze the relationship between the nucleotide sequence and the activity of the MLC box precisely, mutation analysis was performed. The 16-base-pair sequence was indispensable for the active transcription of L23 gene, and the MLC box could function in either orientation. The inverted sequence of the MLC box was similar to the sequence of the alpha-actin CArG box. By using a gel mobility retardation assay, the nuclear protein(s) that binds to both MLC box and CArG box was detected with nuclear extract prepared from chicken embryonic breast muscle. These observations imply that a common factor regulates the coordinate expression of these contractile proteins in muscle differentiation.
The immunoglobulin heavy-chain enhancer functions as the promoter for I mu sterile transcription.
Authors: Su L K , Kadesch T
Published: October 2015

We demonstrate that the immunoglobulin heavy-chain enhancer functions as the promoter for I mu sterile transcription. The enhancer itself, when placed 5' to the bacterial cat or neo genes, is able to direct transcription by using heterogeneous start sites that are generally the same as those found with bona fide I mu transcripts. In general, promoter activity is dependent on the same sequence motifs important for enhancer activity. However, it appears that a mutation within the conserved octanucleotide ATTTGCAT has a much more severe effect on the promoter activity of the enhancer than the same mutation has on its enhancer activity. This result is consistent with the known role of the octanucleotide as a promoter element, and this is discussed in relation to the biological role of sterile transcription.
Biochemical characterization of the Drosophila dpp protein, a member of the transforming growth factor beta family of growth factors.
Authors: Panganiban G E , Rashka K E , Neitzel M D , Hoffmann F M
Published: October 2015

The decapentaplegic (dpp) gene of Drosophila melanogaster is required for pattern formation in the embryo and for viability of the epithelial cells in the imaginal disks. The dpp protein product predicted from the DNA sequence is similar to members of a family of growth factors that includes transforming growth factor beta (TGF-beta). We have produced polyclonal antibodies to a recombinant dpp protein made in bacteria and used a metallothionein promoter to express a dpp cDNA in Drosophila S2 cells. Similar to other proteins in the TGF-beta family, the dpp protein produced by the Drosophila cells was proteolytically cleaved, and both portions of the protein were secreted from the cells. The amino-terminal 47-kilodalton (kDa) peptide was found in the medium and in the proteins adhering to the plastic petri dish. The carboxy-terminal peptide, the region with sequence similarity to the active ligand portion of TGF-beta, was found extracellularly as a 30-kDa homodimer. Most of the 30-kDa homodimer was in the S2 cell protein adsorbed onto the surface of the plastic dish. The dpp protein could be released into solution by increased salt concentration and nonionic detergent. Under these conditions, the amino-terminal and carboxy-terminal portions of dpp were not associated in a stable complex.
Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase.
Authors: Livi G P , Kmetz P , McHale M M , Cieslinski L B , Sathe G M , Taylor D P , Davis R L , Torphy T J , Balcarek J M
Published: October 2015

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.
Coding and noncoding sequences at the 3' end of yeast histone H2B mRNA confer cell cycle regulation.
Authors: Xu H X , Johnson L , Grunstein M
Published: October 2015

Yeast (Saccharomyces cerevisiae) histone mRNA synthesis is tightly regulated to the S phase of the cell division cycle as a result of both transcriptional and posttranscriptional regulation. We focused on the role of posttranscriptional control in histone H2B1 gene (HTB1) regulation and studied a portion of the HTB1 message required for cell-cycle-specific accumulation. The 3' end of the HTB1 gene containing a 17-amino-acid coding sequence and entire noncoding sequence was fused to the bacterial neomycin phosphotransferase II gene (neo) under control of the GAL1 promoter. The expression of the endogenous and chimeric HTB1 genes was analyzed during the yeast cell cycle. As yeast cells entered a synchronous cell cycle following release from alpha-factor arrest, the level of GAL1-promoter-controlled neo-HTB1 message increased approximately 12-fold during S phase and dropped to basal level when the cells left S phase. This indicates that the 3' end of the HTB1 mRNA is capable of conferring cycle-specific regulation on a heterologous message. Deletion analysis of the 3' end showed that the signal for cell cycle control of HTB1 mRNA includes contiguous coding and noncoding sequences surrounding the stop codon. This differs from the situation in mammalian cells, whose posttranscriptional regulation of histone genes is mediated through a short sequence containing a stem-loop structure near the very terminus of the untranslated 3' end.
Many transcribed regions of the Onchocerca volvulus genome contain the spliced leader sequence of Caenorhabditis elegans.
Authors: Zeng W L , Alarcon C M , Donelson J E
Published: October 2015

Genomic DNAs of the related parasitic nematodes Onchocerca volvulus and Dirofilariae immitis, and a cDNA library of O. volvulus, were examined for the presence of the 22-nucleotide spliced leader (SL) found at the 5' ends of 10 to 15% of the mRNAs in the free-living nematode Caenorhabditis elegans. As in C. elegans, genes for the SL RNA are linked to the repetitive 5S rRNA genes of O. volvulus and D. immitis, but unlike C. elegans, they are in the same orientation as the 5S rRNA genes within the repeat unit. In O. volvulus the SL sequence is also encoded at more than 30 additional genomic locations and occurs at interior sites within many transcripts. Sequence determinations of four different cDNAs of O. volvulus, each containing an internal copy of the SL within a conserved 25mer, and one corresponding genomic DNA clone indicate that this sequence is not trans spliced onto these RNAs, but is encoded within the genes. The RNAs of two of these cDNAs appear to be developmentally regulated, since they occur in adult O. volvulus but were not detected in the infective L3 stage larvae. In contrast, actin mRNAs are present at all developmental stages, and at least one actin mRNA species contains a trans-spliced 5' SL. The internal locations of the SL in various transcripts and its perfect sequence conservation among parasitic and free-living nematodes argues that it serves specific, and perhaps multiple, functions for these organisms.
Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression.
Authors: Wek R C , Ramirez M , Jackson B M , Hinnebusch A G
Published: October 2015

GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast Saccharomyces cerevisiae. GCN2, a translational activator of GCN4 expression, contains a domain homologous to the catalytic subunit of eucaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating GCN4 expression. Elevated GCN2 gene dosage led to derepression of GCN4 under nonstarvation conditions; however, we found that GCN2 mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated posttranslationally in amino acid-starved cells. Three dominant-constitutive GCN2 point mutations were isolated that led to derepressed GCN4 expression under nonstarvation conditions. Two of the GCN2(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which we suggested might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety. Deletions and substitutions in the HisRS-related sequences and in the carboxyl-terminal segment in which one of the GCN2(Con) mutation mapped abolished GCN2 positive regulatory function in vivo without lowering autophosphorylation activity in vitro. These results suggest that sequences flanking the GCN2 protein kinase moiety are positive-acting domains required to increase recognition of physiological substrates or lower the requirement for uncharged tRNA to activate kinase activity under conditions of amino acid starvation.
Activation of the proto-oncogene p60c-src by point mutations in the SH2 domain.
Authors: O'Brien M C , Fukui Y , Hanafusa H
Published: October 2015

To investigate the importance of a conserved region spanning residues 137 to 241 in the noncatalytic domain of p60c-src (SH2 region), we used oligonucleotide-directed mutagenesis to change residues that are highly conserved in this region. Chicken embryo fibroblasts infected with a p60c-src variant containing arginine instead of tryptophan at residue 148 (W148R) appeared more rounded than cells overexpressing a normal c-src gene, and they formed colonies in soft agar. p60c-src variants containing serine instead of arginine at residue 155 (R155S) or isoleucine instead of glycine at residue 170 (G170I) also appeared transformed and were anchorage independent, but to a lesser extent than W148R. Mutation of residue 201 from histidine to leucine (H201L) had no observable effect. The in vitro kinase activity of cells infected with W148R or G170I was elevated twofold. Expression of p60W148R (or, to a lesser extent, of p60G170I) increased the number of proteins phosphorylated on tyrosine in infected cells. All of the mutants were phosphorylated in vivo on Tyr-527, instead of Tyr-416 as observed for p60v-src. Immunoprecipitated p60W148R and p60G170I were found to be associated with a phosphatidylinositol kinase activity, a factor which appears to be necessary for transformation by tyrosine-specific protein kinases. These results show that a single point mutation in the SH2 region of the cellular src gene can activate its transforming potential. This type of activation is in a new category of alterations at the amino terminus that activate but do not cause a shift in phosphorylation at the carboxy terminus.
Multimeric arrays of the yeast retrotransposon Ty.
Authors: Weinstock K G , Mastrangelo M F , Burkett T J , Garfinkel D J , Strathern J N
Published: October 2015

We have identified a novel integrated form of the yeast retrotransposon Ty consisting of multiple elements joined into large arrays. These arrays were first identified among Ty-induced alpha-pheromone-resistant mutants of MATa cells of Saccharomyces cerevisiae which contain Ty insertions at HML alpha that result in the expression of that normally silent cassette. These insertions are multimeric arrays of both the induced genetically marked Ty element and unmarked Ty elements. Structural analysis of the mutations indicated that the arrays include tandem direct repeats of Ty elements separated by only a single long terminal repeat. The Ty-HML junction fragments of one mutant were cloned and shown to contain a 5-base-pair duplication of the target sequence that is characteristic of a Ty transpositional insertion. In addition, the arrays include rearranged Ty elements that do not have normal long terminal repeat junctions. We have also identified multimeric Ty insertions at other chromosomal sites and as insertions that allow expression of a promoterless his3 gene on a plasmid. The results suggest that Ty transposition includes an intermediate that can undergo recombination to produce multimers.
Inhibition of topoisomerase II does not inhibit transcription of RNA polymerase I and II genes.
Authors: Dunaway M
Published: October 2015

Injection of VM-26 (teniposide) into Xenopus oocytes inhibits the activity of topoisomerase II but does not inhibit transcription by RNA polymerases I and II. A specific assay for topoisomerase II, resolution of catenated DNA molecules into product rings, was used to quantitate VM-26 inhibition in vivo. When catenanes were injected without VM-26, about 60% of them were separated into product rings in the first 5 min after injection, and decatenation of the remainder was complete within 15 min. When VM-26 was coinjected, 60% of the catenanes were separated into product rings in the first 5 min after injection, but the remaining 40% were stable over the next 40 min. At 1 h after injection catenanes were no longer detected in the gel analysis, but the increasing numbers of linear product rings indicated that topoisomerase II continued to be inhibited by VM-26. These results suggest that a short lag of approximately 5 min is required for VM-26 to inhibit topoisomerase II and that after this initial period topoisomerase II is inhibited by more than 90%. There was no detectable decrease in transcription of injected rRNA and thymidine kinase (TK) genes or in the activity of the rRNA enhancer when these transcription templates were coinjected with VM-26. The time required for assembly of injected DNA into chromatin doubled in the presence of VM-26.
Mutations in Saccharomyces cerevisiae which confer resistance to several amino acid analogs.
Authors: McCusker J H , Haber J E
Published: October 2015

Four new complementation groups of mutations which confer resistance to several amino acid analogs in Saccharomyces cerevisiae are described. These mutants were isolated on medium containing urea as the nitrogen source, in contrast to previous studies that had used medium containing proline. All four resistance to amino acid analog (raa) complementation groups appear to confer resistance by reducing amino acid analog and amino acid uptake. In some genetic backgrounds, raa leu2 and raa thr4 double mutants are inviable, even on rich medium. The raa4 mutation may affect multiple amino acid transport systems, since raa4 mutants are unable to use proline as a nitrogen source. raa4 is, however, unlinked to a previously described amino acid analog resistance and proline uptake mutant, aap1, or to the general amino acid permease mutant gap1. Both raa4 and gap1 prevent uptake of [3H]leucine in liquid cultures. The raa1, raa2, and raa3 mutants affect only a subset of the amino acid analogs and amino acids affected by raa4. The phenotypes of raa1, -2, and -3 mutants are readily observed on agar plates but are not seen in uptake and incorporation of amino acids measured in liquid media.
Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway.
Authors: de Barros Lopes M , Ho J Y , Reed S I
Published: October 2015

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.
Regulation of protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC-12 cells.
Authors: Lacal J C , Cuadrado A , Jones J E , Trotta R , Burstein D E , Thomson T , Pellicer A
Published: October 2015

Expression of the N-ras oncogene under the control of the glucocorticoid-responsive promoter in the pheochromocytoma cell line UR61, a subline of PC-12 cells, has been used to investigate the differentiation process to neuronal cells triggered by ras oncogenes (I. Guerrero, A. Pellicer, and D. E. Burstein, Biochem. Biophys. Res. Commun. 150:1185-1192, 1988). Using ras-inducible cell lines, we observed that expression of the oncogenic N-ras p21 protein interferes with the ability of phorbol esters to induce downregulation of protein kinase C. This effect was associated with the appearance of immunologically detectable protein kinase C as well as the activity of the enzyme as analyzed either by binding of [3H]phorbol-12,13-dibutyrate in intact cells or by in vitro kinase activity. These results indicate a relationship between ras p21 and protein kinase C in neuronal differentiation in this model system. Comparison to the murine fibroblast system suggests that this relationship may be functional.
Antigen receptor-induced cell cycle arrest in WEHI-231 B lymphoma cells depends on the duration of signaling before the G1 phase restriction point.
Authors: Page D M , DeFranco A L
Published: October 2015

Stimulation of antigen receptors on WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-immunoglobulin M [IgM]) causes irreversible growth arrest. This may be a model for antigen-induced tolerance to self components in the immune system. Antigen receptor stimulation also causes inositol phospholipid hydrolysis, producing diacylglycerol, which activates protein kinase C, and inositol 1,4,5-trisphosphate, which causes release of calcium from intracellular stores. To better understand the nature of the antigen receptor-induced growth arrest of WEHI-231 cells, we have examined the basis for it. WEHI-231 cells in various phases of the cell cycle were isolated by centrifugal elutriation, and their response was evaluated following treatment with either anti-IgM or pharmacologic agents that raise intracellular free calcium levels and activate protein kinase C. Treatment with anti-IgM or the pharmacologic agents did not lengthen the cell cycle. Instead, growth inhibition was solely the result of arrest in the G1 phase. The efficiency of G1 arrest increased with the length of time during which the cells received signaling before reaching the G1 phase arrest point. Maximum efficiency of arrest was achieved after approximately one cell cycle of receptor signaling. These results imply that anti-IgM causes G1 arrest of WEHI-231 cells by slowly affecting components required for S phase progression, rather than by rapidly inhibiting such components or by rapidly activating a suicide mechanism. Antigen receptor stimulation was twice as effective as stimulation via the mimicking reagents phorbol dibutyrate and ionomycin. Thus, although the phosphoinositide second messengers diacylglycerol and calcium probably play roles in mediating the effects of anti-IgM on WEHI-231 cells, other second messengers may also be involved.