bluuebunny/crossref_metadata_2025 huggingface dataset:
Yeast STE7, STE11, and STE12 genes are required for expression of cell-type-specific genes.
Authors: Fields S , Chaleff D T , Sprague G F
Published: September 2015

Cell type specialization in yeast haploids involves the mutually exclusive expression of one of two sets of genes, the a-specific and alpha-specific genes. We demonstrated that the products of the STE7, STE11, and STE12 genes were required for the expression of both gene sets. RNA levels transcribed from these gene sets were significantly decreased but not abolished in haploids containing a null mutation in the STE7, STE11, or STE12 gene. Transcript levels from the a- and alpha-specific gene sets were not further reduced in strains harboring mutations in all three STE genes, suggesting that STE7, STE11, and STE12 are required for the same aspect of transcription. We further showed that the requirement for these products was not the same for each member of a particular gene set. However, for any given a- or alpha-specific gene, the effect on RNA levels of any of the three ste mutations was similar.
The human c-fps/fes gene product expressed ectopically in rat fibroblasts is nontransforming and has restrained protein-tyrosine kinase activity.
Authors: Greer P A , Meckling-Hansen K , Pawson T
Published: September 2015

A 13-kilobase EcoRI genomic restriction fragment containing the human c-fps/fes proto-oncogene locus was expressed transiently in Cos-1 monkey cells and stably in Rat-2 fibroblasts. In both cases, human c-fps/fes directed synthesis of a 92-kilodalton protein-tyrosine kinase (p92c-fes) indistinguishable from a tyrosine kinase previously identified with anti-fps antiserum which is specifically expressed in human myeloid cells. Transfected Rat-2 cells containing approximately 50-fold more human p92c-fes than is found in human leukemic cells remained morphologically normal and failed to grow in soft agar. Synthesis of p92c-fes in this phenotypically normal line exceeded that of the P130gag-fps oncoprotein in a v-fps-transformed Rat-2 line. Despite this elevated expression, human p92c-fes induced no substantial increase in cellular phosphotyrosine and was not itself phosphorylated on tyrosine. In contrast, p92c-fes immunoprecipitated from these Rat-2 cells or expressed as an enzymatically active fragment in Escherichia coli from a c-fps/fes cDNA catalyzed tyrosine phosphorylation with an activity similar to that of v-fps/fes polypeptides. Thus, p92c-fes is not transforming when ectopically overexpressed in Rat-2 fibroblasts. This lack of transforming activity correlates with a restriction imposed on the kinase activity of the normal c-fps/fes product in vivo which is apparently lifted for v-fps/fes oncoproteins, suggesting that regulatory interactions within the host cell modify fps/fes protein function and normally restrain its oncogenic potential.
Different cis-acting DNA elements control expression of the human apolipoprotein AI gene in different cell types.
Authors: Sastry K N , Seedorf U , Karathanasis S K
Published: September 2015

In mammals, the gene coding for apolipoprotein AI (apoAI), a protein of the plasma lipid transport system, is expressed only in the liver and the intestine. A series of plasmids containing various lengths of sequences flanking the 5' end of the human apoAI gene were constructed and assayed for transient expression after introduction into cultured human hepatoma (HepG2), colon carcinoma (Caco-2), and epithelial (HeLa) cells. The results showed that while most of these constructs are expressed in HepG2 and Caco-2 cells, none of them is expressed in HeLa cells. In addition, the results indicated that a DNA segment located between nucleotides -256 and -41 upstream from the transcription start site of this gene is necessary and sufficient for maximal levels of expression in HepG2 but not in Caco-2 cells, while a DNA segment located between nucleotides -2052 and -192 is required for maximal levels of expression in Caco-2 cells. Moreover, it was shown that the -256 to -41 DNA segment functions as a hepatoma cell-specific transcriptional enhancer with both homologous and heterologous promoters. These results indicate that different cis- and possibly trans-acting factors are involved in the establishment and subsequent regulation of expression of the apoAI gene in the mammalian liver and intestine.
DNA-binding properties of the Drosophila melanogaster zeste gene product.
Authors: Mansukhani A , Crickmore A , Sherwood P W , Goldberg M L
Published: September 2015

The ability of the zeste moiety of beta-galactosidase-zeste fusion proteins synthesized in Escherichia coli to bind specific DNA sequences was examined. Such fusion proteins recognize a region of the white locus upstream of the start of transcription; this region has previously been shown to be required for genetic interaction between the zeste and white loci. Another strong binding site was localized to a region between 50 and 205 nucleotides before the start of the Ubx transcriptional unit; expression of the bithorax complex is also known to be influenced by the zeste locus. Weaker binding sites were also seen in the vicinity of the bxd and Sgs-4 genes, but it is currently unclear whether these binding sites play a role in transvection effects. The DNA-binding activity of the zeste protein is restricted to a domain of approximately 90 amino acids near the N terminus. This domain does not appear to contain homeobox or zinc finger motifs found in other DNA-binding proteins. The DNA-binding domain is not disrupted by any currently characterized zeste mutations.
The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing.
Authors: Wieczorek D F , Smith C W , Nadal-Ginard B
Published: September 2015

Tropomyosin (TM), a ubiquitous protein, is a component of the contractile apparatus of all cells. In nonmuscle cells, it is found in stress fibers, while in sarcomeric and nonsarcomeric muscle, it is a component of the thin filament. Several different TM isoforms specific for nonmuscle cells and different types of muscle cell have been described. As for other contractile proteins, it was assumed that smooth, striated, and nonmuscle isoforms were each encoded by different sets of genes. Through the use of S1 nuclease mapping, RNA blots, and 5' extension analyses, we showed that the rat alpha-TM gene, whose expression was until now considered to be restricted to muscle cells, generates many different tissue-specific isoforms. The promoter of the gene appears to be very similar to other housekeeping promoters in both its pattern of utilization, being active in most cell types, and its lack of any canonical sequence elements. The rat alpha-TM gene is split into at least 13 exons, 7 of which are alternatively spliced in a tissue-specific manner. This gene arrangement, which also includes two different 3' ends, generates a minimum of six different mRNAs each with the capacity to code for a different protein. These distinct TM isoforms are expressed specifically in nonmuscle and smooth and striated (cardiac and skeletal) muscle cells. The tissue-specific expression and developmental regulation of these isoforms is, therefore, produced by alternative mRNA processing. Moreover, structural and sequence comparisons among TM genes from different phyla suggest that alternative splicing is evolutionarily a very old event that played an important role in gene evolution and might have appeared concomitantly with or even before constitutive splicing.
Identification of negative and positive regulatory elements associated with a class I major histocompatibility complex gene.
Authors: Ehrlich R , Maguire J E , Singer D S
Published: September 2015

Regulatory DNA sequence elements were functionally identified in the 5'-flanking region of a gene, PD1, which encodes a porcine classical transplantation antigen. Both a positive regulatory element and a novel negative regulatory DNA element were mapped within 1.1 kilobases upstream of exon 1. The negative regulatory element reduced the activity of both the homologous PD1 promoter and a heterologous simian virus 40 promoter. In vivo competition experiments indicated that the functions of the PD1 positive and negative regulatory elements are mediated by distinct cellular trans-acting factors. The PD1 positive regulatory element interacted with cellular factors in common with those binding to the simian virus 40 enhancer. Finally, the negative regulatory element required the presence of a positive regulatory element to function. This interaction between positive and negative regulatory elements represents a novel mechanism for regulating gene expression.
Effects of single-base substitutions within the Acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I.
Authors: Kownin P , Bateman E , Paule M R
Published: September 2015

Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decreased specific RNA synthesis in vitro. These were within the region which is protected from DNase I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.
Expression of a Drosophila melanogaster acetylcholine receptor-related gene in the central nervous system.
Authors: Wadsworth S C , Rosenthal L S , Kammermeyer K L , Potter M B , Nelson D J
Published: September 2015

We isolated Drosophila melanogaster genomic sequences with nucleotide and amino acid sequence homology to subunits of vertebrate acetylcholine receptor by hybridization with a Torpedo acetylcholine receptor subunit cDNA probe. Five introns are present in the portion of the Drosophila gene encoding the unprocessed protein and are positionally conserved relative to the human acetylcholine receptor alpha-subunit gene. The Drosophila genomic clone hybridized to salivary gland polytene chromosome 3L within region 64B and was termed AChR64B. A 3-kilobase poly(A)-containing transcript complementary to the AChR64B clone was readily detectable by RNA blot hybridizations during midembryogenesis, during metamorphosis, and in newly enclosed adults. AChR64B transcripts were localized to the cellular regions of the central nervous system during embryonic, larval, pupal, and adult stages of development. During metamorphosis, a temporal relationship between the morphogenesis of the optic lobe and expression of AChR64B transcripts was observed.
Dictyostelium discoideum myosin: isolation and characterization of cDNAs encoding the essential light chain.
Authors: Chisholm R L , Rushforth A M , Pollenz R S , Kuczmarski E R , Tafuri S R
Published: September 2015

We used an antibody specific for Dictyostelium discoideum myosin to screen a lambda gt11 cDNA expression library to obtain cDNA clones which encode the Dictyostelium essential myosin light chain (EMLC). The amino acid sequence predicted from the sequence of the cDNA clone showed 31.5% identity with the amino acid sequence of the chicken EMLC. Comparisons of the Dictyostelium EMLC, a nonmuscle cell type, with EMLC sequences from similar MLCs of skeletal- and smooth-muscle origin, showed distinct regions of homology. Much of the observed homology was localized to regions corresponding to consensus Ca2+-binding of E-F hand domains. Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the EMLC. Examination of the pattern of EMLC mRNA expression showed that a significant increase in EMLC message levels occurred during the first few hours of development, coinciding with increased actin expression and immediately preceding the period of maximal chemotactic activity.
The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro.
Authors: Furdon P J , Kole R
Published: September 2015

We have shown previously that truncation of the human beta-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit beta-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.
Sequence-specific interactions between a cellular DNA-binding protein and the simian virus 40 origin of DNA replication.
Authors: Traut W , Fanning E
Published: September 2015

The core origin of simian virus 40 (SV40) DNA replication is composed of a 64-base-pair sequence encompassing T-antigen-binding site II and adjacent sequences on either side. A 7-base-pair sequence to the early side of T-antigen-binding site II which is conserved among the papovavirus genomes SV40, BK, JC, and SA12 was recently shown to be part of a 10-base-pair sequence required for origin activity (S. Deb, A.L. DeLucia, C.-P. Baur, A. Koff, and P. Tegtmeyer, Mol. Cell. Biol. 6:1663-1670, 1986), but its functional role was not defined. In the present report, we have used gel retention assays to identify a monkey cell factor that interacts specifically with double-stranded DNA carrying this sequence and also binds to single-stranded DNA. DNA-protein complexes formed with extracts from primate cells are more abundant and display electrophoretic mobilities distinct from those formed with rodent cell extracts. The binding activity of the factor on mutant templates is correlated with the replication activity of the origin. The results suggest that the monkey cell factor may be involved in SV40 DNA replication.
Comparison of the structure and cell cycle expression of mRNAs encoded by two histone H3-H4 loci in Saccharomyces cerevisiae.
Authors: Cross S L , Smith M M
Published: September 2015

The haploid genome of Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 gene pairs, termed the copy I and copy II loci. The structures of the mRNA transcripts from each of these four genes were examined by nuclease protection and primer extension mapping. For each gene, several species of mRNAs were identified that differed in the lengths of their 5' and 3' untranslated regions. The cell cycle accumulation pattern of the H3 and H4 mRNAs was determined in cells from early-exponential-growth cultures fractionated by centrifugal elutriation. The RNA transcripts from all four genes were regulated with the cell division cycle, and transcripts from the nonallelic gene copies showed tight temporal coordination. Cell cycle regulation did not depend on selection of a particular histone mRNA transcript since the ratio of the multiple species from each gene remained the same across the division cycle. Quantitative measurements showed significant differences in the amounts of mRNA expressed from the two nonallelic gene sets. The mRNAs from the copy II H3 and H4 genes were five to seven times more abundant than the mRNAs from the copy I genes. There was no dosage compensation in the steady-state levels of mRNA when either set of genes was deleted. In particular, there was no increase in the amount of copy I H3 or H4 transcripts in cells in which the high-abundance copy II genes were deleted.
Gene conversion associated with site-specific recombination in yeast plasmid pSR1.
Authors: Matsuzaki H , Araki H , Oshima Y
Published: September 2015

A circular DNA plasmid, pSR1, isolated from Zygosaccharomyces rouxii has a pair of inverted repeats consisting of completely homologous 959-base pair (bp) sequences. Intramolecular recombination occurs frequently at the inverted repeats in cells of Saccharomyces cerevisiae, as well as in Z. rouxii, and is catalyzed by a protein encoded by the R gene of its own genome. The recombination is, however, independent of the RAD52 gene of the host genome. A site for initiation of the intramolecular recombination in the S. cerevisiae host was delimited into, at most, a 58-bp region in the inverted repeats by using mutant plasmids created by linker insertion. The 58-bp region contains a pair with 14-bp dyad symmetry separated by a 3-bp spacer sequence. The recombination initiated at this site was accompanied by a high frequency of gene conversion (3 to 50% of the plasmid clones examined). Heterogeneity created by the linker insertion or by a deletion (at most 153 bp so far tested) at any place on the inverted repeats was converted to a homologous combination by the gene conversion, even in the rad52-1 mutant host. A mechanism implying branch migration coupled with DNA replication is discussed.
Introduction of new genetic material into human myeloid leukemic blast stem cells by retroviral infection.
Authors: Smith L J , Benchimol S
Published: September 2015

An amphotropic retroviral vector containing the bacterial neomycin phosphotransferase gene (neo) was used to infect blast cells from patients with acute myeloblastic leukemia. The infected cells acquired a G418-resistant phenotype that was stable as measured in a clonogenic assay and in long-term suspension culture. Thus, gene transfer into stem cells was accomplished by this procedure. This approach for manipulating gene expression in blast stem cells provides a means to assess the roles of a variety of genes in self-renewal, differentiation, and leukemogenesis.
Tumor promoter and epidermal growth factor stimulate phosphorylation of the c-erbB-2 gene product in MKN-7 human adenocarcinoma cells.
Authors: Akiyama T , Saito T , Ogawara H , Toyoshima K , Yamamoto T
Published: September 2015

Treatment of human adenocarcinoma MKN-7 cells with epidermal growth factor (EGF) or phorbol tetradecanoate acetate (TPA) stimulated phosphorylation of the c-erbB-2 gene product. EGF induced a rapid increase in phosphotyrosine followed by relatively gradual increases in phosphoserine and phosphothreonine. On the other hand, the TPA-induced increase in phosphorylations occurred exclusively on serine and threonine residues. Tryptic phosphopeptide mapping analysis suggested that treatments with EGF and TPA induced phosphorylation of many common sites in the c-erbB-2 gene product. However, in contrast to TPA, EGF increased the phosphorylation of the c-erbB-2 protein in cells whose protein kinase C had been down-regulated by long-term pretreatment with TPA, suggesting that EGF and TPA induce phosphorylation by different mechanisms. Since the c-erbB-2 gene product did not show detectable EGF-binding activity, phosphorylation of tyrosine of the c-erbB-2 gene product might be catalyzed directly by the EGF receptor kinase that was activated by EGF.
A cell-specific enhancer of the mouse alpha 1-antitrypsin gene has multiple functional regions and corresponding protein-binding sites.
Authors: Grayson D R , Costa R H , Xanthopoulos K G , Darnell J E
Published: September 2015

We have previously described the isolation and characterization of genomic clones corresponding to the mouse alpha 1-antitrypsin gene (Krauter et al., DNA 5:29-36, 1986). In this report, we have analyzed the DNA sequences upstream of the RNA start site that direct hepatoma cell-specific expression of this gene when incorporated into recombinant plasmids. The 160 nucleotides 5' to the cap site direct low-level expression in hepatoma cells, and sequences between -520 and -160 bp upstream of the RNA start site functioned as a cell-specific enhancer of expression both with the alpha 1-antitrypsin promoter and when combined with a functional beta-globin promoter. Within the enhancer region, three binding sites for proteins present in hepatoma nuclear extracts were identified. The location of each site was positioned, using both methylation protection and methylation interference experiments. Each protein-binding site correlated with a functionally important region necessary for full enhancer activity. These experiments demonstrated a complex arrangement of regulatory elements comprising the alpha 1-antitrypsin enhancer. Significant qualitative differences exist between the findings presented here and the cis-acting elements operative in regulating expression of the human alpha 1-antitrypsin gene (Ciliberto et al., Cell 41:531-540, 1985; De Simone et al., EMBO J. 6:2759-2766, 1987).
Cloning of the RNA8 gene of Saccharomyces cerevisiae, detection of the RNA8 protein, and demonstration that it is essential for nuclear pre-mRNA splicing.
Authors: Jackson S P , Lossky M , Beggs J D
Published: September 2015

Strains of Saccharomyces cerevisiae that bear the temperature-sensitive mutation rna8-1 are defective in nuclear pre-mRNA splicing at the restrictive temperature (36 degrees C), suggesting that the RNA8 gene encodes a component of the splicing machinery. The RNA8 gene was cloned by complementation of the temperature-sensitive growth defect of an rna8-1 mutant strain. Integrative transformation and gene disruption experiments confirmed the identity of the cloned DNA and demonstrated that the RNA8 gene encodes an essential function. The RNA8 gene was shown to be represented once per S. cerevisiae haploid genome and to encode a low-abundance transcript of approximately 7.4 kilobases. By using antisera raised against beta-galactosidase-RNA8 fusion proteins, the RNA8 gene product was identified in S. cerevisiae cell extracts as a low-abundance protein of approximately 260 kilodaltons. Immunodepletion of the RNA8 protein specifically abolished the activity of S. cerevisiae in vitro splicing extracts, confirming that RNA8 plays an essential role in splicing.
Functional analysis of the long terminal repeats of intracisternal A-particle genes: sequences within the U3 region determine both the efficiency and direction of promoter activity.
Authors: Christy R J , Huang R C
Published: September 2015

The transcriptional activity of five intracisternal A-particle (IAP) long terminal repeats (LTRs) in mouse embryonal carcinoma PCC3-A/1 cells and in Ltk- cells was determined. We tested the promoter activity of the LTRs by coupling them to the reporter gene chloramphenicol acetyltransferase (CAT) or guanosine-xanthine phosphoribosyltransferase (gpt). Each LTR was tested for promoter function in both the sense (5' to 3') and antisense (3' to 5') orientation preceding the reporter gene. The transcriptional activity of individual IAP gene LTRs varied considerably, and the LTR from IAP81 possessed promoter activity in both directions. The bidirectional activity of the IAP81 LTR confirmed by monitoring Ecogpt expression in stably transfected Ltk- cells, with the initiation sites for sense and antisense transcription being localized to within the IAP81 LTR by S1 nuclease mapping. Deletions of LTR81 show that for normal 5'-to-3' gene transcription (sense direction), the 3'U3/R region determines the basal level of transcription, whereas sequences within the 5'U3 region enhance transcription four- to fivefold. Deletion mapping for antisense transcription indicates that a 64-base-pair region (nucleotides 47 to 110) within the U3 region is essential for activity. These data indicate that the U3 region contains all the regulatory elements for bidirectional transcription in IAP LTRs.
Characterization of Gmhsp26-A, a stress gene encoding a divergent heat shock protein of soybean: heavy-metal-induced inhibition of intron processing.
Authors: Czarnecka E , Nagao R T , Key J L , Gurley W B
Published: September 2015

We determined the DNA sequence and mapped the corresponding transcripts of a genomic clone containing the Gmhsp26-A gene of soybean. This gene is homologous to the previously characterized cDNA clone pCE54 (E. Czarnecka, L. Edelman, F. Schöffl, and J. L. Key, Plant Mol. Biol. 3:45-58, 1984) and is expressed in response to a wide variety of physiological stresses including heat shock (HS). S1 nuclease mapping of transcripts and a comparison of the cDNA sequence with the genomic sequence indicated the presence of a soybean seedlings with either CdCl2 or CuSO4. Analysis of the 5' termini of transcripts indicated the presence of one major and at least two minor start sites. In each case, initiation occurred 27 to 30 base pairs downstream from a TATA-like motif, and thus each initiation site appears to be promoted by the activity of a separate subpromoter. The three subpromoters are all associated with sequences showing low homology to the HS consensus element of Drosophila melanogaster HS genes and are differentially induced in response to various stresses. Within the carboxyl-terminal half of the protein, hydropathy analysis of the deduced amino acid sequence indicated a high degree of relatedness to the small HS proteins. A comparison of the primary amino acid sequence of hsp26-A with sequences of the small HS proteins suggested that this stress protein is highly diverged and may therefore be specialized for stress adaptation in soybean.
Genomic organization and sequence of the Gus-s alpha allele of the murine beta-glucuronidase gene.
Authors: Funkenstein B , Leary S L , Stein J C , Catterall J F
Published: September 2015

The Gus-s alpha allele of the mouse beta-glucuronidase gene exhibits a high degree of inducibility by androgens due to its linkage with the Gus-r alpha regulatory locus. We isolated Gus-s alpha on a 28-kilobase pair fragment of mouse chromosome 5 and found that it contains 12 exons and 11 intervening sequences spanning 14 kilobase pairs of this genomic segment. The mRNA cap site was identified by ribonuclease protection and primer extension analyses which revealed an unusually short 5' noncoding sequence of 12 nucleotides. Proximal regulatory sequences in the 5'-flanking DNA and the complete sequence of the Gus-s alpha mRNA transcript were also determined. Comparison of the amino acid sequence determined from the Gus-s alpha nucleotide sequence with that of human beta-glucuronidase indicated that the two human mRNA species differ due to alternate splicing of an exon homologous to exon 6 of the mouse gene.
Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.
Authors: Lazar E , Watanabe S , Dalton S , Sporn M B
Published: September 2015

To study the relationship between the primary structure of transforming growth factor alpha (TGF-alpha) and some of its functional properties (competition with epidermal growth factor (EGF) for binding to the EGF receptor and induction of anchorage-independent growth), we introduced single amino acid mutations into the sequence for the fully processed, 50-amino-acid human TGF-alpha. The wild-type and mutant proteins were expressed in a vector by using a yeast alpha mating pheromone promoter. Mutations of two amino acids that are conserved in the family of the EGF-like peptides and are located in the carboxy-terminal part of TGF-alpha resulted in different biological effects. When aspartic acid 47 was mutated to alanine or asparagine, biological activity was retained; in contrast, substitutions of this residue with serine or glutamic acid generated mutants with reduced binding and colony-forming capacities. When leucine 48 was mutated to alanine, a complete loss of binding and colony-forming abilities resulted; mutation of leucine 48 to isoleucine or methionine resulted in very low activities. Our data suggest that these two adjacent conserved amino acids in positions 47 and 48 play different roles in defining the structure and/or biological activity of TGF-alpha and that the carboxy terminus of TGF-alpha is involved in interactions with cellular TGF-alpha receptors. The side chain of leucine 48 appears to be crucial either indirectly in determining the biologically active conformation of TGF-alpha or directly in the molecular recognition of TGF-alpha by its receptor.
Developmental aspects of uridine addition within mitochondrial transcripts of Trypanosoma brucei.
Authors: Feagin J E , Stuart K
Published: September 2015

The mitochondrial respiratory system is absent in slender bloodstream forms of Trypanosoma brucei, incomplete in stumpy bloodstream forms, and complete in procyclic (insect) forms. The steady-state abundance of transcripts of some mitochondrially encoded components of the respiratory system correlates with its differential expression in different life cycle stages. Recently, it was reported that uridines which are not encoded in the genome are added to cytochrome b and cytochrome oxidase II transcripts. We now report that the (U)+ transcripts of both genes are found in procyclic forms and to some degree in stumpy forms but are absent in slender forms. The uridine additions to cytochrome oxidase II correct a frameshift in the gene and presumably allow production of a full-length protein, whereas those added to cytochrome b create an in-frame AUG which extends the N terminus of the predicted protein by 20 amino acids. The stage specificity of uridine additions to these transcripts thus reflects the life cycle stage during which the protein products would be used. Transcripts of MURF2, a gene of unknown function, have additional uridines in both slender and procyclic forms which create two in-frame AUGs. MURF2 transcripts additionally differ from the DNA sequence in ways which cannot be explained by uridine addition alone, implying that other processes alter these transcripts.
Alternate trans splicing in Trypanosoma equiperdum: implications for splice site selection.
Authors: Layden R E , Eisen H
Published: September 2015

We examined the structures of the 5' ends of mRNAs encoding variant surface glycoprotein 78 (VSG-78) and VSG-1(78) in Trypanosoma equiperdum. Several mRNA species were found for each gene, and all contained the 35-base miniexon (or spliced leader) sequence attached at different positions on their 5' ends. Thus, the generation of multiple messages for each VSG occurred by attachment of the miniexon at one of several 3' splice acceptor sites. The frequency with which individual splice sites were used varied from less than 1 to 95% of the RNA produced from a particular gene. We propose that the miniexon RNA and RNA from the VSG genes may interact via base pairing and that this in part specifies the use of particular acceptor sites. Sequences complementary to the miniexon primary transcript, termed the "med-comp site," were found in both genes and in several published sequences. Splice sites were most often used if they were the first site 3' of the med-comp site and contained a high pyrimidine content in the bases preceding the AG acceptor signal.
The structural gene for a phosphorus-repressible phosphate permease in Neurospora crassa can complement a mutation in positive regulatory gene nuc-1.
Authors: Mann B J , Akins R A , Lambowitz A M , Metzenberg R L
Published: September 2015

van+, a gene encoding a phosphorus-repressible phosphate permease, was isolated by its ability to complement nuc-1, a positive regulatory locus that normally regulates van+ expression. This was unexpected because the nuc-1 host already contained a resident van+ gene. Plasmids carrying van+ complemented a nuc-2 mutation as well. Probing of RNA from untransformed wild-type (nuc-1+) and constitutive (nuc-1c) strains by van+ probes indicated that levels of the van+ transcript were subject to control by nuc-1+. Probing of the same RNAs with a cosmid clone, containing approximately 15 kilobases of upstream and downstream DNA, revealed no other detectable phosphorus-regulated transcripts within this 40-kilobase region of the chromosome.
Two-dimensional gel electrophoretic method for mapping DNA replicons.
Authors: Nawotka K A , Huberman J A
Published: September 2015

We describe in detail a method which allows determination of the directions of replication fork movement through segments of DNA for which cloned probes are available. The method uses two-dimensional neutral-alkaline agarose gel electrophoresis followed by hybridization with short probe sequences. The nascent strands of replicating molecules form an arc separated from parental and nonreplicating strands. The closer a probe is to its replication origin or to the origin-proximal end of its restriction fragment, the shorter the nascent strands that are detected by the probe. The use of multiple probes allows determination of directions of replication fork movement, as well as locations of origins and termini. In this study, we used simian virus 40 as a model to demonstrate the feasibility of the method, and we discuss its applicability to other systems.
Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus.
Authors: Macnab J C , Adams R L , Rinaldi A , Orr A , Clark L
Published: September 2015

Infection of rat embryo cells with herpes simplex virus type 2 caused undermethylation of host cell DNA synthesized during infection. DNA made prior to infection was not demethylated, but some of its degradation products, including methyl dCMP, were incorporated into viral DNA. The use of mutant virus showed that some viral DNA synthesis appears to be required for the inhibition of methylation. Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.
Molecular analysis of hybrid dysgenesis-induced derivatives of a P-element allele at the vg locus.
Authors: Williams J A , Pappu S S , Bell J B
Published: September 2015

Secondary and tertiary derivatives of a P-element insertion allele at the vestigial (vg) locus were induced by hybrid dysgenesis. The derivatives were characterized by Southern analyses and, in four cases, by DNA sequencing. The alterations found were P-element internal deletions, deletions of the insert and/or adjacent vg region DNA, or novel insertions of P-element sequences into existing P-element inserts. The relatively high frequency of secondary insertions into P-element sequences observed herein is unusual, since secondary insertions have seldom been recovered in other dysgenic screens. The effects of the alleles on vg expression were determined. The results are consistent with a model in which the insertions disrupt vg gene expression by transcriptional interference.
Replication from a proximal simian virus 40 origin is severely inhibited by multiple reiterations of the 72-base-pair repeat enhancer sequence.
Authors: Kumar R , Yoon K P , Subramanian K N
Published: September 2015

In a previous study in our laboratory, the effect of the reiteration frequency of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer on transcription from the proximal SV40 early promoter was investigated (R. Kumar, T. A. Firak, C. T. Schroll, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 83:3199-3203, 1986). Increasing the enhancer copy number to four increased transcription proportionately; further increments in enhancer copy number reversed this effect, resulting in a decrease in the transcriptional activation. In the present study, the effect of enhancer reiteration on the replication efficiency of plasmids containing the SV40 origin of replication was investigated in transient replication assays in vivo in COS-1 monkey kidney cells producing the SV40 large tumor antigen required for replication. A plasmid containing the SV40 core origin and three copies of the replication-activating, G+C-rich 21-bp repeat promoter element replicated efficiently. Plasmids containing multiple copies of the 72-bp repeat enhancer cloned in head-to-tail linkage adjacent to the 21-bp repeat and the core origin replicated less efficiently; the decrease in replication efficiency could be correlated with the number of copies of the 72-bp repeat; replication was severely curtailed when 10 or more copies of the 72-bp repeat were present. Replication was not significantly inhibited by an increase in the number of copies of the 21-bp repeat to 15 or by the presence of three copies of a 360-bp pBR322 sequence in the immediate vicinity. Multiple copies of the 72-bp enhancer in cis were unable to inhibit replication from a second SV40 origin of replication situated 2 kilobase pairs away from the enhancer reiteration. Replication of four different test plasmids was not inhibited in trans by cotransfection of an excess of a potential competitor plasmid containing a 24-copy reiteration of the 72-bp enhancer. These results indicate that multiple tandem reiterations of the 72-bp enhancer inhibit replication only when they are present in cis adjacent to the origin of replication. Possible explanations for this inhibitory effect, such as an unfavorable local chromatin structure induced by the multimeric enhancer region or reduced or improper communications between factors bound to the multimeric region and the adjacent replication origin, are discussed.
Opsin expression in the rat retina is developmentally regulated by transcriptional activation.
Authors: Treisman J E , Morabito M A , Barnstable C J
Published: September 2015

The gene for rhodopsin, the primary light sensor of the visual system, is specifically expressed in the rod photoreceptor cells of the retina. We show here that in the rat, opsin RNA first accumulates to detectable levels at postnatal day 2 (PN2) and that nascent transcripts can be detected at PN1; this is the time when peak numbers of photoreceptor cells are generated by the final division of their neuroepithelial precursors. Accumulated opsin RNA then increases to reach the adult level, 0.06% of total retinal RNA, at about PN10. The transcription rate of the opsin gene increases to a similar extent over the same time course between PN3 and adulthood, suggesting that transcriptional activation is responsible for the increase in opsin expression. We used the antibody RET-P1 to show that rhodopsin protein is also detectable at PN2 and that the number of cells expressing the protein increases with time in a central-to-peripheral gradient in the retina. This increase in the number of differentiating photoreceptors in the tissue appears to account for much of the increase in opsin gene transcription and RNA accumulation. In situ hybridization to opsin RNA shows that it is restricted to the photoreceptor layer from the time it can first be detected, at PN7. Later in development, when RET-P1 staining shifts to the photoreceptor outer segments, opsin RNA becomes localized to the inner segments, suggesting that the distributions of opsin protein and RNA are related.
mRNA structures influencing translation in the yeast Saccharomyces cerevisiae.
Authors: Baim S B , Sherman F
Published: September 2015

The mRNA sequence and structures that modify and are required for translation of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae were investigated with sets of CYC1 alleles having alterations in the 5' leader region. Measurements of levels of CYC1 mRNA and iso-1-cytochrome c in strains having single copies of altered alleles with nested deletions led to the conclusion that there is no specific sequence adjacent to the AUG initiator codon required for efficient translation. However, the nucleotides preceding the AUG initiator codon at positions -1 and -3 slightly modified the efficiency of translation to an order of preference similar to that found in higher cells. In contrast to large effects observed in higher eucaryotes, the magnitude of this AUG context effect in S. cerevisiae was only two- to threefold. Furthermore, introduction of hairpin structures in the vicinity of the AUG initiator codon inhibited translation, with the degree of inhibition related to the stability and proximity of the hairpin. These results with S. cerevisiae and published findings on other organisms suggest that translation in S. cerevisiae is more sensitive to secondary structures than is translation in higher eucaryotes.
Enforced expression of the c-myc oncogene inhibits cell differentiation by precluding entry into a distinct predifferentiation state in G0/G1.
Authors: Freytag S O
Published: September 2015

A broad base of data has implicated a role for the c-myc proto-oncogene in the control of the cell cycle and cell differentiation. To further define the role of myc in these processes, I examined the effect of enforced myc expression on several events that are thought to be important steps leading to the terminally differentiated state: (i) the ability to arrest growth in G0/G1, (ii) the ability to replicate the genome upon initiation of the differentiation program, and (iii) the ability to lose responsiveness to mitogens and withdraw from the cell cycle. 3T3-L1 preadipocyte cell lines expressing various levels of myc mRNA were established by transfection with a recombinant myc gene under the transcriptional control of the Rous sarcoma virus (RSV) promoter. Cells that expressed high constitutive levels of pRSVmyc mRNA arrested in G0/G1 at densities similar to those of normal cells at confluence. Upon initiation of the differentiation program, such cells traversed the cell cycle with kinetics similar to those of normal cells and subsequently arrested in G0/G1. Thus, enforced expression of myc had no effect on the ability of cells to arrest growth in G0/G1 or to replicate the genome upon initiation of the differentiation program. Cells were then tested for their ability to reenter the cell cycle upon exposure to high concentrations of serum and for their capacity to differentiate. In contrast to normal cells, cells expressing high constitutive levels of myc RNA reentered the cell cycle when challenged with 30% serum and failed to terminally differentiate. The block to differentiation could be reversed by high expression of myc antisense RNA, showing that the induced block was specifically due to enforced expression of pRSVmyc. These findings indicate that 3T3-L1 preadipocytes enter a specific state in G0/G1 after treatment with differentiation inducers, into which cells expressing high constitutive levels of myc RNA are precluded from entering. I propose that myc acts as a molecular switch and directs cells to a pathway that can lead to continued proliferation or to terminal differentiation.
Differential protein binding in lymphocytes to a sequence in the enhancer of the mouse retrovirus SL3-3.
Authors: Thornell A , Hallberg B , Grundström T
Published: September 2015

An electrophoretic mobility shift assay was used to characterize interactions of nuclear proteins with a DNA segment in the enhancer element of the leukemogenic murine retrovirus SL3-3. Mutation of a DNA sequence of the 5'-TGTGG-3' type decreased transcription in vivo specifically in T-lymphocyte cell lines. Extracts of nuclei from different T-lymphocyte cell lines or cells from lymphoid organs resulted in much higher amounts of complexes in vitro with this DNA sequence than did extracts from other cell lines or organs tested. Differences were also found in the sets of complexes obtained with extracts from the different types of cells. The DNA sequence specificities of the different SL3-3 enhancer factor 1 (SEF1) protein complexes were found to be distinct from those of several other previously identified DNA motifs of the TGTGG type because of differences in several nucleotides critical for binding and because these other DNA motifs could not compete with the identified DNA sequence for binding of SEF1. Limited treatment with several different proteases cleaved the SEF1 proteins such that their DNA-binding domain(s) remained and created complexes with decreased and nondistinguishable electrophoretic mobility shifts and with new properties. These results indicate that the SEF1 proteins have a structure with a flexible and relatively vulnerable hinge region linking a DNA-binding domain(s) to a more variable domain(s) with other functions. We suggest that the binding of SEF1 is an essential factor for the T-cell tropism of SL3-3 and the ability of this virus to cause T-cell lymphomas.
Mapping functional regions of transcription factor TFIIIA.
Authors: Vrana K E , Churchill M E , Tullius T D , Brown D D
Published: September 2015

Functional deletion mutants of the trans-acting factor TFIIIA, truncated at both ends of the molecule, have been expressed by in vitro transcription of a cDNA clone and subsequent cell-free translation of the synthetic mRNAs. A region of TFIIIA 19 amino acids or less, near the carboxyl terminus, is critical for maximal transcription and lies outside the DNA-binding domain. The elongated protein can be aligned over the internal control region (ICR) of the Xenopus 5S RNA gene with its carboxyl terminus oriented toward the 5' end of the gene and its amino terminus oriented toward the 3' end of the gene. The nine "zinc fingers" and the linkers that separate them comprise 80% of the protein mass and correspond to the DNA-binding domain of TFIIIA. The zinc fingers near the amino terminus of the protein contribute more to the overall binding energy of the protein to the ICR than do the zinc fingers near the carboxyl end. The most striking feature of TFIIIA is its modular structure. This is demonstrated by the fact that each zinc finger binds to just one of three short nucleotide sequences within the ICR.
Haploid-specific transcription of protamine-myc and protamine-T-antigen fusion genes in transgenic mice.
Authors: Stewart T A , Hecht N B , Hollingshead P G , Johnson P A , Leong J A , Pitts S L
Published: September 2015

The protamines are small, basic, arginine-rich proteins synthesized postmeiotically in the testes. Analysis of the regulation of synthesis of the protamine mRNA and protein is restricted by the difficulty in culturing and manipulating the cells in which transcription and translation occur. To avoid these problems, we have produced transgenic mice carrying fusion genes in which sequences 5' to the mouse protamine-2 gene have been linked to exons 2 and 3 of the mouse c-myc gene and, separately, to the simian virus 40 (SV40) early region. We show here that the prot.myc gene is correctly regulated; transcription is detected only in the round spermatids. In one family of transgenic mice carrying the 5' protamine-SV40 T-antigen fusion gene, SV40 early-region mRNA accumulated to the highest level in the testes but was also detected in the thymuses, brains, hearts, and preputial glands of the animals. Although we have demonstrated specific transcription of these fusion genes in the round spermatids, we were not able to detect the SV40 T-antigen protein.
Amplification of the structurally and functionally altered epidermal growth factor receptor gene (c-erbB) in human brain tumors.
Authors: Yamazaki H , Fukui Y , Ueyama Y , Tamaoki N , Kawamoto T , Taniguchi S , Shibuya M
Published: September 2015

By using Southern blot analysis, we found that in two cases of human glioblastoma multiforme, cells carried amplified c-erbB genes which bore short deletion mutations within the ligand-binding domain of the epidermal growth factor (EGF) receptor. The products of these mutated c-erbB genes were about 30 kilodalton (kDa) smaller than the normal 170-kDa EGF receptor, and the tumor cell membrane fractions containing the 140-kDa abnormal EGF receptor showed a significant elevation of tyrosine kinase activity without its ligand. In view of the similarity to the activated viral and cellular erbB genes in the avian system, these mutated and overexpressed EGF receptors might play a role in the onset or development of human glioblastoma cells.