bluuebunny/crossref_metadata_2025 huggingface dataset:
A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5'-disrupted myb loci.
Authors: Shen-Ong G L , Lüscher B , Eisenman R N
Published: October 2015

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.
Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice.
Authors: Shanahan C M , Rigby N W , Murray J D , Marshall J T , Townrow C A , Nancarrow C D , Ward K A
Published: October 2015

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.
Lymphoid and mesenchymal tumors in transgenic mice expressing the v-fps protein-tyrosine kinase.
Authors: Yee S P , Mock D , Greer P , Maltby V , Rossant J , Bernstein A , Pawson T
Published: October 2015

src, abl, and fps/fes are prototypes for a family of genes encoding nonreceptor protein-tyrosine kinases. The oncogenic potential of the v-fps protein-tyrosine kinase was investigated by introduction of the gag-fps coding sequence of Fujinami sarcoma virus into the mouse germ line. Transgenic mice with v-fps under the transcriptional control of a 5' human beta-globin promoter (GF) or with both 5' and 3' beta-globin regulatory sequences (GEF) were viable. Unexpectedly, both GF and GEF transgenes were expressed in a wide variety of tissues and induced a spectrum of benign and malignant tumors. These tumors, which included lymphomas, thymomas, fibrosarcomas, angiosarcomas, hemangiomas, and neurofibrosarcomas, developed with various frequencies after latent periods of 2 to 12 months. The majority of lymphoid neoplasms appeared to be of T-cell origin and were monoclonal, as judged by rearrangements of the T-cell receptor beta or immunoglobulin genes. Some tissues that expressed the v-fps oncogene, such as heart, brain, lung, and testes, developed no malignant tumors. The v-fps protein-tyrosine kinase therefore has a broad but not unrestricted range of oncogenic activity in cells of lymphoid and mesenchymal origin. The incomplete penetrance of the neoplastic phenotype and the monoclonality of lymphoid tumors suggest that tumor formation in v-fps mice requires genetic or epigenetic events in addition to expression of the P130gag-fps protein-tyrosine kinase.
Complex regulation of transforming growth factor beta 1, beta 2, and beta 3 mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factors beta 1 and beta 2.
Authors: Bascom C C , Wolfshohl J R , Coffey R J , Madisen L , Webb N R , Purchio A R , Derynck R , Moses H L
Published: October 2015

Regulation of transforming growth factor beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 mRNAs in murine fibroblasts and keratinocytes by TGF beta 1 and TGF beta 2 was studied. In quiescent AKR-2B fibroblasts, in which TGF beta induces delayed stimulation of DNA synthesis, TGF beta 1 autoregulation of TGF beta 1 expression was observed as early as 1 h, with maximal induction (25-fold) after 6 to 12 h. Increased expression of TGF beta 1 mRNA was accompanied by increased TGF beta protein production into conditioned medium of AKR-2B cells. Neither TGF beta 2 nor TGF beta 3 mRNA, however, was significantly induced, but both were apparently down regulated at later times by TGF beta 1. Protein synthesis was not required for autoinduction of TGF beta 1 mRNA in AKR-2B cells. Nuclear run-on analyses and dactinomycin experiments indicated that autoregulation of TGF beta 1 expression is complex, involving both increased transcription and message stabilization. In contrast to TGF beta 1, TGF beta 2 treatment of quiescent AKR-2B cells increased expression of TGF beta 1, TGF beta 2, and TGF beta 3 mRNAs, but with different kinetics. Autoinduction of TGF beta 2 mRNA occurred rapidly with maximal induction at 1 to 3 h, enhanced TGF beta 3 mRNA levels were observed after 3 h, and increased expression of TGF beta 1 occurred later, with maximal mRNA levels obtained after 12 to 24 h. Nuclear run-on analyses indicated that TGF beta 2 regulation of TGF beta 2 and TGF beta 3 mRNA levels is transcriptional, while TGF beta 2 induction of TGF beta 1 expression most likely involves both transcriptional and posttranscriptional controls. In BALB/MK mouse keratinocytes, minimal autoinduction of TGF beta 1 occurred at only the 12- and 24-h time points and protein synthesis was required for this autoinduction. The results of this study provide an example in which TGF beta 1 and TGF beta 2 elicit different responses and demonstrate that expression of TGF beta 1, and TGF beta 3 are regulated differently. The physiological relevance of TGF beta 1 autoinduction in the context of wound healing is discussed.
Expression of REX-1, a gene containing zinc finger motifs, is rapidly reduced by retinoic acid in F9 teratocarcinoma cells.
Authors: Hosler B A , LaRosa G J , Grippo J F , Gudas L J
Published: October 2015

In the presence of retinoic acid (RA), cultured F9 murine teratocarcinoma stem cells differentiate into nontumorigenic cells resembling the extraembryonic endoderm of the early mouse embryo. By differential hybridization screening of an F9 cell cDNA library, we isolated a 1,745-nucleotide cDNA for a gene, REX-1 (for reduced expression), whose steady-state mRNA level began to decline in F9 cells in monolayer culture within 12 h after the addition of RA. By 48 to 96 h after RA treatment of F9 cells in monolayer culture, the REX-1 steady-state mRNA level was more than sevenfold lower than the level in undifferentiated F9 stem cells. The REX-1 mRNA decrease did not result from the reduction in cell growth rate associated with the differentiation process, since the REX-1 mRNA level did not decline in F9 cells that were partially growth arrested after 48 h of isoleucine deprivation. The RA-associated REX-1 mRNA decrease resulted primarily from a reduction in the transcription rate of the REX-1 gene in the presence of RA. In contrast to results in F9 cells, we have been unable thus far to detect REX-1 mRNA in day 7.5 to 12.5 mouse embryo RNA samples or in the P19 teratocarcinoma stem cell line. The putative REX-1 protein identified by DNA sequence analysis contains four repeats of the zinc finger nucleic acid-binding motif and a potential acidic activator domain, suggesting that REX-1 encodes a regulatory protein. The REX-1 gene is not identical to the previously reported murine genes that encode zinc finger-containing proteins.
In vitro analysis of the pea chloroplast 16S rRNA gene promoter.
Authors: Sun E , Wu B W , Tewari K K
Published: October 2015

A cloned pea chloroplast 16S rRNA gene promoter has been characterized in detail by use of a homologous in vitro transcription system that contains a highly purified chloroplast RNA polymerase. The in vivo and in vitro 16S rRNA transcriptional start site has been identified to be a T on the plus strand, 158 bases upstream of the mature 5' end of the gene. BAL 31 deletions of the 16S rRNA leader region demonstrated that the bases between -66 to +30 relative to the transcriptional start site (+1) are necessary for specific 16S transcription. Disruption of canonical TTGACA or TATAAT elements within this region caused complete transcriptional inactivation and prevented protein binding. The topological requirement for 16S transcription was examined by using a construct that synthesized a transcript from the 16S promoter and released it from a pea plastid putative terminator sequence. This minigene was relaxed in vitro with a topoisomerase I from pea chloroplast. It was shown that the 16S promoter was most active when the minigene plasmid was supercoiled.
Tyrosine kinase oncogenes abrogate interleukin-3 dependence of murine myeloid cells through signaling pathways involving c-myc: conditional regulation of c-myc transcription by temperature-sensitive v-abl.
Authors: Cleveland J L , Dean M , Rosenberg N , Wang J Y , Rapp U R
Published: October 2015

Retroviral expression vectors carrying the tyrosine kinase oncogenes abl, fms, src, and trk abrogate the requirements of murine myeloid FDC-P1 cells for interleukin-3 (IL-3). Factor-independent clones constitutively express c-myc in the absence of IL-3, whereas in parental cultures c-myc transcription requires the presence of the ligand. To directly test the effect of a tyrosine kinase oncogene on c-myc expression, retroviral constructs containing three different temperature-sensitive mutants of v-abl were introduced into myeloid IL-3-dependent FDC-P1 and 32D cells. At the permissive temperature, clones expressing temperature-sensitive abl behaved like wild-type abl-containing cells in their growth properties and expressed c-myc constitutively. Temperature shift experiments demonstrated that both IL-3 abrogation and the regulation of c-myc expression correlated with the presence of functional v-abl. Induction of c-myc expression by reactivation of temperature-sensitive v-abl mimicked c-myc induction by IL-3 in that it did not require protein synthesis and occurred at the level of transcription, with effects on both initiation and a transcription elongation block. However, v-abl-regulated FDC-P1 cell growth differed from IL-3-regulated growth in that c-fos and junB, which are normally induced by IL-3, were not induced by activation of v-abl.
Transfection of a human gene that corrects the Lec1 glycosylation defect: evidence for transfer of the structural gene for N-acetylglucosaminyltransferase I.
Authors: Kumar R , Stanley P
Published: October 2015

Chinese hamster ovary (CHO) glycosylation mutants provide an approach to cloning mammalian glycosyltransferases by transfection and gene rescue. In this paper, complementation of the lec1 CHO mutation by human DNA is described. Lec1 transfectants expressed human N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and possessed common human DNA fragments. Cloning of GlcNAc-TI should therefore be possible.
Location of signal sequences for membrane insertion of the Na+,K+-ATPase alpha subunit.
Authors: Homareda H , Kawakami K , Nagano K , Matsui H
Published: October 2015

To study the membrane insertion of the Na+,K+-ATPase (EC 3.6.1.37) alpha subunit with six to eight transmembrane segments, mRNAs encoding the entire alpha subunit and its four different domains were prepared and translated in rabbit reticulocyte lysate with rough microsomal membranes. On the basis of the resistance of the membrane-inserted products to alkali extraction and the failure to insert the translation products into N-ethylmaleimide-treated membranes, it is suggested that at least two signal sequences are contained within the four transmembrane segments from the amino terminus of the alpha subunit.
Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro.
Authors: Roberts J D , Preston B D , Johnston L A , Soni A , Loeb L A , Kunkel T A
Published: October 2015

We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.
The C proteins of HeLa 40S nuclear ribonucleoprotein particles exist as anisotropic tetramers of (C1)3 C2.
Authors: Barnett S F , Friedman D L , LeStourgeon W M
Published: October 2015

The C proteins (C1 and C2) of HeLa 40S heterogeneous nuclear ribonucleoprotein particles copurify under native conditions as a stable complex with a fixed molar protein ratio (S.F. Barnett, W.M. LeStourgeon, and D.L. Friedman, J. Biochem. Biophys. Methods 16:87-97, 1988). Gel filtration chromatography and velocity sedimentation analyses of these complexes revealed a large Stokes radius (6.2 nm) and a sedimentation coefficient of 5.8S. On the basis of these values and a partial specific volume of 0.70 cm3/g based on the amino acid composition, the molecular weight of the complex was calculated to be 135,500. This corresponds well to 129,056, the sequence-determined molecular weight of a (C1)3C2 tetramer. Reversible chemical cross-linking with dithiobis(succinimidyl propionate) and analysis of cross-linked and cleaved complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that the C proteins exist as tetramers, most or all of which are composed of (C1)3C2. The tetramer is stable in a wide range of NaCl concentrations (0.09 to 2.0 M) and is not dissociated by 0.5% sodium deoxycholate. This stability is not the result of disulfide bonds or interactions with divalent cations. The hydrodynamic properties of highly purified C-protein tetramers are the same for C-protein complexes released from intact particles with RNase or high salt. These findings support previous studies indicating that the core particle protein stoichiometry of 40S heterogeneous nuclear ribonucleoproteins is N(3A1-3A2-1B1-1B2-3C1-1C2), where N = 3 to 4, and demonstrate that the C-protein tetramer is a fundamental structural element in these RNA-packaging complexes. The presence of at least three tetramers per 40S monoparticle, together with the highly anisotropic nature of the tetramer, suggesting that one-third of the 700-nucleotide pre-mRNA moiety packaged in monoparticles is associated through a sequence-independent mechanism with the C protein.
Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element.
Authors: Anachkova B , Hamlin J L
Published: October 2015

To study initiation of DNA replication in mammalian chromosomes, we have established a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains approximately 1,000 copies of the early replicating dihydrofolate reductase (DHFR) domain. We have previously shown that DNA replication in the prevalent 243-kilobase (kb) amplicon type in this cell line initiates somewhere within a 28-kb region located downstream from the DHFR gene. In an attempt to localize the origin of replication with more precision, we blocked the progress of replication forks emanating from origins at the beginning of the S phase by the introduction of trioxsalen cross-links at 1- to 5-kb intervals in the parental double-stranded DNA. The small DNA fragments synthesized under these conditions (which should be centered around replication origins) were then used as hybridization probes on digests of cosmids and plasmids from the DHFR domain. These studies suggested that in cells synchronized by this regimen, DNA replication initiates at two separate sites within the previously defined 28-kb replication initiation locus, in general agreement with results described in the accompanying paper (T.-H. Leu and J. L. Hamlin, Mol. Cell. Biol. 9:523-531, 1989). One of these sites contains a repeated DNA sequence element that is found at or near many other initiation sites in the genome, since it was also highly enriched in the early replicating DNA isolated from cross-linked CHO cells that contain only two copies of the DHFR domain.
Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules.
Authors: Porter S E , Champoux J J
Published: October 2015

Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.
Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.
Authors: Stanton V P , Nichols D W , Laudano A P , Cooper G M
Published: October 2015

Activation of transforming potential of the cellular raf gene has uniformly been associated with the deletion of amino-terminal coding sequences. In order to determine whether 5' truncation alone could activate cellular raf, we constructed 21 human c-raf-1 cDNAs with variable BAL 31-generated deletions distal to a Moloney murine sarcoma virus long terminal repeat and a consensus translation initiation sequence. The deletions ranged from 136 to 1,399 nucleotides of coding sequence and shortened the 648-amino-acid raf protein by 44 to 465 amino acids. The full-length c-raf-1 cDNA was nontransforming upon transfection of NIH 3T3 cells, as were four mutants with deletions of 142 or fewer amino acids. Seven of nine mutants with deletions of 154 to 273 amino acids induced transformation with efficiencies ranging from 0.25 to 70 foci per micrograms of DNA. Mutants with deletions of 303 to 324 amino acids displayed high transforming activities (comparable with that of v-raf), with a peak activity of 2,400 foci per microgram of DNA when 305 amino acids were deleted. Deletions of greater than 383 amino acids, extending into the raf kinase domain, lacked transforming activity. Northern (RNA) blotting and immunoprecipitation assays indicated that transfected NIH cells expressed raf RNAs and proteins of the expected sizes. Thus, 5' truncation alone can activate raf transforming potential, with a sharp peak of activation around amino acid 300. Analysis of three raf genes previously detected by transfection of tumor DNAs indicated that these genes were activated by recombination in raf intron 7 and encoded fusion proteins containing amino-terminal non-raf sequences. The extend of deletion of raf sequences in these recombinant genes corresponded to BAL 31 mutants which did not display high transforming activity, suggesting that the fused non-raf coding sequences may also contribute to biological activity.
Characterization of mutant mitochondrial plasmids of Neurospora spp. that have incorporated tRNAs by reverse transcription.
Authors: Akins R A , Kelley R L , Lambowitz A M
Published: October 2015

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) whose nucleotide sequences and genetic organization suggest relationships to mitochondrial introns and retroelements. We have characterized nine suppressive mutants of these plasmids that outcompete mitochondrial DNA and lead to impaired growth. All nine suppressive plasmids contain small insertions, corresponding to or including a mitochondrial tRNA (tRNATrp, tRNAGly, or tRNAVal) or a tRNA-like sequence. The insertions are located at the position corresponding to the 5' end of the major plasmid transcript or 24 nucleotides downstream near a cognate of the sequence at the major 5' RNA end. The structure of the suppressive plasmids suggests that the tRNAs were inserted via an RNA intermediate. The 3' end of the wild-type plasmid transcript can itself be folded into a secondary structure which has tRNA-like characteristics, similar to the tRNA-like structures at the 3' ends of plant viral RNAs. This structure may play a role in replication of the plasmids by reverse transcription. Major transcripts of the suppressive plasmids begin at the 5' end of the inserted mitochondrial tRNA sequence and are present in 25- to 100-fold-higher concentrations than are transcripts of wild-type plasmids. Mapping of 5' RNA ends within the inserted mtDNA sequences identifies a short consensus sequence (PuNPuAG) which is present at the 5' ends of a subset of mitochondrial tRNA genes. This sequence, together with sequences immediately upstream in the plasmids, forms a longer consensus sequence, which is similar to sequences at transcription initiation sites in Neurospora mitochondrial DNA. The suppressive behavior of the plasmids is likely to be directly related to the insertion of tRNAs leading to overproduction of plasmid transcripts.
Levels of RNA from a family of putative serine protease genes are reduced in Drosophila melanogaster dunce mutants and are regulated by cyclic AMP.
Authors: Yun Y , Davis R L
Published: October 2015

We have isolated several genes expressed at abnormal levels in the memory mutant, dunce (dnc), of Drosophila melanogaster. These mutants have an elevated cyclic AMP (cAMP) content due to a mutation in the structural gene for cAMP phosphodiesterase, so the isolated genes are potentially ones regulated by cAMP. Here, we describe the characterization of a genomic clone and corresponding cDNA clones which contain sequences that are underexpressed in dnc mutants. Sequence analysis of portions of the genomic clone and representative cDNAs revealed the presence of two uninterrupted and complete open reading frames (SER1 and SER2) and part of a third (SER3). The predicted amino acid sequences of all of these were found to be homologous to the serine protease family of enzymes. The genomic clone was localized to the polytene chromosome region 99C-D, although genome-blotting experiments indicated the existence of several other genes related to the cloned serine protease-like genes. Hybridization experiments with probes representing each of the three sequenced genes showed that only the SER1-related genes were differentially expressed in dnc mutants. The putative serine protease genes were abundantly expressed in the larval gut, suggesting a major function in digestion. Feeding normal flies cAMP, isobutylmethylxanthine, or forskolin resulted in a decreased RNA level of the SER1-related genes. Thus, RNA levels of this serine protease gene family are negatively regulated by cAMP.
The bcl-2 candidate proto-oncogene product is a 24-kilodalton integral-membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14;18) translocation.
Authors: Chen-Levy Z , Nourse J , Cleary M L
Published: October 2015

We have identified a 24-kilodalton protein that is the product of the human bcl-2 gene, implicated as an oncogene because of its presence at the site of t(14;18) translocation breakpoints. The Bcl-2 protein was detected by specific, highly sensitive rabbit antibodies and was shown to be present in a number of human lymphoid cell lines and tissues, as well as in mouse B cells transfected with a bcl-2 cDNA construct. Characterization of the Bcl-2 protein demonstrated that it has a lipophilic nature and is associated with membrane structures, probably by means of its hydrophobic carboxy-terminal membrane-spanning domain. In t(14;18)-carrying cell lines, the protein is predominantly localized to the perinuclear endoplasmic reticulum, with a minor fraction in the plasma membrane. These properties, together with the observations that Bcl-2 does not have a characteristic signal peptide and is not glycosylated, suggest that it is an integral-membrane protein that spans the bilayer at its C-terminal hydrophobic region but is exposed only at the cytoplasmic surface. The relative abundance of the Bcl-2 protein in various human lymphoid cell lines correlated with transcription of the bcl-2 gene. The protein was abundant in all t(14;18)-carrying cell lines and lymphomas and was also found at lower levels in pre-B-cell lines and nonmalignant lymphoid tissues that do not carry t(14;18) translocations. These results suggest that the Bcl-2 protein is functional in normal B lymphocytes and that a quantitative difference in its expression may play a role in the pathogenesis of lymphomas carrying the t(14;18) translocation.
Octamer transcription factors 1 and 2 each bind to two different functional elements in the immunoglobulin heavy-chain promoter.
Authors: Poellinger L , Roeder R G
Published: October 2015

Immunoglobulin heavy-chain genes contain two conserved sequence elements 5' to the site of transcription initiation: the octamer ATGCAAAT and the heptamer CTCATGA. Both of these elements are required for normal cell-specific promoter function. The present study demonstrates that both the ubiquitous and lymphoid-cell-specific octamer transcription factors (OTF-1 and OTF-2, respectively) interact specifically with each of the two conserved sequence elements, forming either homo- or heterodimeric complexes. This was surprising, since the heptamer and octamer sequence motifs bear no obvious similarity to each other. Binding of either factor to the octamer element occurred independently. However, OTF interaction with the heptamer sequence appeared to require the presence of an intact octamer motif and occurred with a spacing of either 2 or 14 base pairs between the two elements, suggesting coordinate binding resulting from protein-protein interactions. The degeneracy in sequences recognized by the OTFs may be important in widening the range over which gene expression can be modulated and in establishing cell type specificity.
Structure, chromosome location, and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos.
Authors: Chavrier P , Janssen-Timmen U , Mattéi M G , Zerial M , Bravo R , Charnay P
Published: October 2015

We have analyzed the structure and the regulation of Krox-20, a mouse zinc finger-encoding gene which is transiently activated following serum stimulation of quiescent fibroblast cells in culture. The gene is localized on chromosome 10, band B5, in the mouse, and the homologous human gene also maps to chromosome 10 (region q21.1 to q22.1). Alternative splicing of the 5'-most intron of the Krox-20 gene gives rise to mRNAs encoding putative zinc finger proteins with different N termini. The first exon contains a sequence element with strong similarity to the c-fos proto-oncogene serum response element (SRE). This element can functionally substitute for the c-fos SRE, and it binds the same nuclear protein. It is probably responsible for the serum induction of Krox-20, possibly in combination with a weaker SRE located in the 5'-flanking region of the gene. Our findings suggest that c-fos, Krox-20, and a number of immediate-early serum response genes are coregulated and that the SRE and its cognate protein are essential components of this regulatory pathway.
Dual functions of a cis-acting element within the rat prolactin gene promoter.
Authors: Barron E A , Cao Z , Schneider B G , Kraig E , Carrillo A J , Sharp Z D
Published: October 2015

Within the promoter region of the rat prolactin gene lies a TA-rich imperfect palindrome. The possible functions of the 18-base-pair symmetrical sequence were investigated by using an in vitro transcription system. Prolactin templates with and without the palindrome were transcriptionally assayed in both pituitary and nonpituitary extracts. Our results indicated that the palindromic sequence has at least two functions in the regulation of prolactin transcription.
Individual protein-binding domains of the insulin gene enhancer positively activate beta-cell-specific transcription.
Authors: Karlsson O , Walker M D , Rutter W J , Edlund T
Published: October 2015

A beta-cell-specific enhancer is located in the 5'-flanking DNA of the rat insulin 1 gene. Two homologous 8-base-pair sequences in the enhancer (IEB1 and IEB2) significantly stimulated transcription from a heterologous promoter (two- to fourfold) in a cell-specific fashion. When the elements were combined or duplicated, more than 50% of the activity of the intact enhancer was obtained. These two cis-acting elements appear to play a dominant role in the positive control of beta-cell-specific transcription of the insulin gene.
Structural organization of upstream exons and distribution of transcription start sites in the chicken c-myb gene.
Authors: Hahn S L , Hahn M , Hayward W S
Published: October 2015

We mapped and sequenced three upstream exons of the chicken c-myb gene and the regions flanking the first coding exon. We found multiple potential binding sites for transcription factors in the 5'-noncoding region, a T-rich stretch of 78 base pairs (bp) (68% T) in the first intron, and four fairly long open reading frames in the antisense direction of the first coding exon and its flanking regions. Three major transcription start sites, contained within a single 11-bp region, were identified by S1 nuclease analysis and primer extension. A sequence comparison of the avian and murine c-myb genes revealed a highly conserved sequence of 124 bp in the 5'-noncoding region. Its location between the putative transcription factor binding sites and the major transcription start sites suggests that it may play an important regulatory role in c-myb expression.
Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells.
Authors: Fornace A J , Zmudzka B , Hollander M C , Wilson S H
Published: October 2015

Only a few of the genes involved in DNA repair in mammalian cells have been isolated, and induction of a DNA repair gene in response to DNA damage has not yet been established. DNA polymerase beta (beta-polymerase) appears to have a synthetic role in DNA repair after certain types of DNA damage. Here we show that the level of beta-polymerase mRNA is increased in CHO cells after treatment with several DNA-damaging agents.
Transgenic mice carrying the mouse mammary tumor virus ras fusion gene: distinct effects in various tissues.
Authors: Tremblay P J , Pothier F , Hoang T , Tremblay G , Brownstein S , Liszauer A , Jolicoeur P
Published: October 2015

Transgenic mice carrying the v-Ha-ras oncogene under the control of the mouse mammary tumor virus long terminal repeat were produced. These mice exhibit several phenotypes: mammary tumors, bilateral hyperplasia of the harderian lacrimal gland, primary bronchio-alveolar lung adenocarcinoma, and splenomegaly. High levels of the transgene RNA were detected in mammary, harderian, and lung tumors. Accumulation of cells of the myeloid lineages was found in enlarged spleens. This phenotype may represent an indirect effect of v-Ha-ras expression on myeloid progenitors. Our data illustrate the cell-specific effects of v-Ha-ras.
Transforming growth factor alpha: an aromatic side chain at position 38 is essential for biological activity.
Authors: Lazar E , Vicenzi E , Van Obberghen-Schilling E , Wolff B , Dalton S , Watanabe S , Sporn M B
Published: October 2015

Site-directed mutagenesis has been performed in the human transforming growth factor alpha gene. When tyrosine 38 is mutated into phenylalanine or tryptophane, biological activity is retained. In contrast, other alterations between cysteine 34 and cysteine 43 and disruption of disulfide bonds 8 to 21 and 34 to 43 resulted in loss of activities. The presence of an aromatic side chain at position 38 of transforming growth factor alpha seems to be essential for its activity.
Isolation of human sequences that replicate autonomously in human cells.
Authors: Krysan P J , Haase S B , Calos M P
Published: October 2015

We have isolated a heterogeneous collection of human genomic sequences which replicate autonomously when introduced into human cells. The novel strategy for the isolation of these sequences involved cloning random human DNA fragments into a defective Epstein-Barr virus vector. This vector alone was unable to replicate in human cells, but appeared to provide for the nuclear retention of linked DNA. The human sequences persist in a long-term replication assay (greater than 2 months) in the presence of the viral nuclear retention sequences. Using a short-term (4-day) assay, we showed that the human sequences are able to replicate in the absence of all viral sequences. The plasmids bearing human sequences were shown to replicate based on the persistence of MboI-sensitive plasmid DNA in the long-term assay and the appearance of DpnI-resistant DNA in the short-term assay. The human sequences were shown to be responsible for the replication activity and may represent authentic human origins of replication.
Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila.
Authors: Yao M C , Yao C H
Published: October 2015

The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T. thermophila.
Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs.
Authors: Mottram J , Perry K L , Lizardi P M , Lührmann R , Agabian N , Nelson R G
Published: October 2015

Trypanosomes use trans splicing to place a common 39-nucleotide spliced-leader sequence on the 5' ends of all of their mRNAs. To identify likely participants in this reaction, we used antiserum directed against the characteristic U RNA 2,2,7-trimethylguanosine (TMG) cap to immunoprecipitate six candidate U RNAs from total trypanosome RNA. Genomic Southern analysis using oligonucleotide probes constructed from partial RNA sequence indicated that the four largest RNAs (A through D) are encoded by single-copy genes that are not closely linked to one another. We have cloned and sequenced these genes, mapped the 5' ends of the encoded RNAs, and identified three of the RNAs as the trypanosome U2, U4, and U6 analogs by virtue of their sequences and structural homologies with the corresponding metazoan U RNAs. The fourth RNA, RNA B (144 nucleotides), was not sufficiently similar to known U RNAs to allow us to propose an identify. Surprisingly, none of these U RNAs contained the consensus Sm antigen-binding site, a feature totally conserved among several classes of U RNAs, including U2 and U4. Similarly, the sequence of the U2 RNA region shown to be involved in pre-mRNA branchpoint recognition in yeast, and exactly conserved in metazoan U2 RNAs, was totally divergent in trypanosomes. Like all other U6 RNAs, trypanosome U6 did not contain a TMG cap and was immunoprecipitated from deproteinized RNA by anti-TMG antibody because of its association with the TMG-capped U4 RNA. These two RNAs contained extensive regions of sequence complementarity which phylogenetically support the secondary-structure model proposed by D. A. Brow and C. Guthrie (Nature [London] 334:213-218, 1988) for the organization of the analogous yeast U4-U6 complex.
Identification of members of the P-glycoprotein multigene family.
Authors: Ng W F , Sarangi F , Zastawny R L , Veinot-Drebot L , Ling V
Published: October 2015

Overproduction of P-glycoprotein is intimately associated with multidrug resistance. This protein appears to be encoded by a multigene family. Thus, differential expression of different members of this family may contribute to the complexity of the multidrug resistance phenotype. Three lambda genomic clones isolated from a hamster genomic library represent different members of the hamster P-glycoprotein gene family. Using a highly conserved exon probe, we found that the hamster P-glycoprotein gene family consists of three genes. We also found that the P-glycoprotein gene family consists of three genes in mice but has only two genes in humans and rhesus monkeys. The hamster P-glycoprotein genes have similar exon-intron organizations within the 3' region encoding the cytoplasmic domains. We propose that the hamster P-glycoprotein gene family arose from gene duplication. The hamster pgp1 and pgp2 genes appear to be more closely related to each other than either gene is to the pgp3 gene. We speculate that the hamster pgp1 and pgp2 genes arose from a recent gene duplication event and that primates did not undergo this duplication and therefore contain only two P-glycoprotein genes.
Variation of tandem repeats in the developmentally regulated procyclic acidic repetitive proteins of Trypanosoma brucei.
Authors: Mowatt M R , Wisdom G S , Clayton C E
Published: October 2015

The procyclic acidic repetitive proteins (PARPs) of Trypanosoma brucei are developmentally regulated surface proteins encoded by a family of polymorphic genes. We have determined the complete nucleotide sequence of a novel member of the PARP gene family and investigated its expression. The amino acid sequence deduced from the parpA alpha gene showed a marked conservation of both the amino- and carboxy-terminal regions compared with other PARPs but revealed the substitution of a pentapeptide for the dipeptide repeating unit that is characteristic of all other PARPs. Northern hybridization analysis indicated that expression of the parpA alpha gene, like that of other members of this gene family, is confined to the procyclic stage of the T. brucei life cycle. This result implies coordinate regulation of the unlinked genetic loci that encode PARPs.
The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues.
Authors: Croop J M , Raymond M , Haber D , Devault A , Arceci R J , Gros P , Housman D E
Published: October 2015

The gene responsible for multidrug resistance (mdr), which encodes the P-glycoprotein, is a member of a multigene family. We have identified distinct mdr gene transcripts encoded by three separate mdr genes in the mouse. Expression levels of each mdr gene are dramatically different in various mouse tissues. Specific mdr RNA transcripts of approximately 4.5, 5, and 6 kilobases have been detected. Each of the mdr genes has a specific RNA transcript pattern. These results should be considered in relation to understanding the normal physiological function of the mdr multigene family.
Interacting proteins identified by genetic interactions: a missense mutation in alpha-tubulin fails to complement alleles of the testis-specific beta-tubulin gene of Drosophila melanogaster.
Authors: Hays T S , Deuring R , Robertson B , Prout M , Fuller M T
Published: October 2015

In this paper we demonstrate that failure to complement between mutations at separate loci can be used to identify genes that encode interacting structural proteins. A mutation (nc33) identified because it failed to complement mutant alleles of the gene encoding the testis-specific beta 2-tubulin of Drosophila melanogaster (B2t) did not map to the B2t locus. We show that this second-site noncomplementing mutation is a missense mutation in alpha-tubulin that results in substitution of methionine in place of valine at amino acid 177. Because alpha- and beta-tubulin form a heterodimer, our results suggest that the genetic interaction, failure to complement, is based on the structural interaction between the protein products of the two genes. Although the nc33 mutation failed to complement a null allele of B2t (B2tn), a deletion of the alpha-tubulin gene to which nc33 mapped complemented B2tn. Thus, the failure to complement appears to require the presence of the altered alpha-tubulin encoded by the nc33 allele, which may act as a structural poison when incorporated into either the tubulin heterodimer or microtubules.
Expression of replication-dependent histone genes in avian spermatids involves an alternate pathway of mRNA 3'-end formation.
Authors: Challoner P B , Moss S B , Groudine M
Published: October 2015

In somatic cells the expression of replication-dependent histone genes is coupled to the S phase of the cell cycle. However, we have found a number of novel H2a, H2b, and H3 poly(A)+ RNA species in avian haploid round spermatids. The spermatid-specific H2a and H2b 0.8-kilobase RNAs are transcribed from a subset of the replication-dependent H2a and H2b gene families. Two cDNAs derived from the spermatid-specific H2b transcripts were isolated and sequenced. The structures of these cDNAs reveal that the spermatid-specific RNAs are identical to the 0.5-kilobase poly(A)- H2b mRNAs expressed in proliferating somatic cells, except for the addition of poly(A) at the 3' ends. The site of poly(A) addition in the spermatid-specific RNAs is located 26 to 28 nucleotides 3' of the poly(A)- H2b mRNA terminus. Thus, the hairpin structures and purine-rich elements required for the U7 small nuclear ribonucleoprotein-mediated cleavage reaction that generates the 3' ends of poly(A)- H2b mRNAs are not utilized in spermatids and are retained in the poly(A)+ H2b RNAs.
An internal regulatory element controls troponin I gene expression.
Authors: Yutzey K E , Kline R L , Konieczny S F
Published: October 2015

During skeletal myogenesis, approximately 20 contractile proteins and related gene products temporally accumulate as the cells fuse to form multinucleated muscle fibers. In most instances, the contractile protein genes are regulated transcriptionally, which suggests that a common molecular mechanism may coordinate the expression of this diverse and evolutionarily unrelated gene set. Recent studies have examined the muscle-specific cis-acting elements associated with numerous contractile protein genes. All of the identified regulatory elements are positioned in the 5'-flanking regions, usually within 1,500 base pairs of the transcription start site. Surprisingly, a DNA consensus sequence that is common to each contractile protein gene has not been identified. In contrast to the results of these earlier studies, we have found that the 5'-flanking region of the quail troponin I (TnI) gene is not sufficient to permit the normal myofiber transcriptional activation of the gene. Instead, the TnI gene utilizes a unique internal regulatory element that is responsible for the correct myofiber-specific expression pattern associated with the TnI gene. This is the first example in which a contractile protein gene has been shown to rely primarily on an internal regulatory element to elicit transcriptional activation during myogenesis. The diversity of regulatory elements associated with the contractile protein genes suggests that the temporal expression of the genes may involve individual cis-trans regulatory components specific for each gene.