bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant transforming growth factor type beta 3: biological activities and receptor-binding properties in isolated bone cells.
Authors: ten Dijke P , Iwata K K , Goddard C , Pieler C , Canalis E , McCarthy T L , Centrella M
Published: October 2015

We have recently cloned the cDNA for transforming growth factor type beta 3 (TGF-beta 3), a new member of the TGF-beta gene family. We examined the biological effects of recombinant TGF-beta 3 protein in osteoblast-enriched bone cell cultures. In this report we demonstrate that TGF-beta 3 is a potent regulator of functions associated with bone formation, i.e., mitogenesis, collagen synthesis, and alkaline phosphatase activity. In a direct comparison between TGF-beta 3 and TGF-beta 1, TGF-beta 3 appeared to be three- to fivefold more potent than TGF-beta 1. Our cross-linking experiments with iodinated TGF-beta showed that in osteoblast-enriched bone cell cultures, both TGF-beta 3 and TGF-beta 1 associated with the same three cell surface binding sites. Scatchard analysis of receptor competition studies indicated the presence of high-affinity binding sites for TGF-beta 3 in the picomolar range. TGF-beta 3 showed an approximately fourfold-higher apparent affinity than TGF-beta 1 in overall binding.
Glucose-induced hyperaccumulation of cyclic AMP and defective glucose repression in yeast strains with reduced activity of cyclic AMP-dependent protein kinase.
Authors: Mbonyi K , van Aelst L , Argüelles J C , Jans A W , Thevelein J M
Published: October 2015

Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-mediated cyclic AMP (cAMP) signal that induces a protein phosphorylation cascade. In yeast mutants (tpk1w1, tpk2w1, and tpk3w1) containing reduced activity of cAMP-dependent protein kinase, fermentable sugars, as opposed to nonfermentable carbon sources, induced a permanent hyperaccumulation of cAMP. This finding confirms previous conclusions that fermentable sugars are specific stimulators of cAMP synthesis in yeast cells. Despite the huge cAMP levels present in these mutants, deletion of the gene (BCY1) coding for the regulatory subunit of cAMP-dependent protein kinase severely reduced hyperaccumulation of cAMP. Glucose-induced hyperaccumulation of cAMP was also observed in exponential-phase glucose-grown cells of the tpklw1 and tpk2w1 strains but not the tpk3w1 strain even though addition of glucose to glucose-repressed wild-type cells did not induce a cAMP signal. Investigation of mitochondrial respiration by in vivo 31P nuclear magnetic resonance spectroscopy showed the tpk1w1 and tpk2w1 strains, to be defective in glucose repression. These results are consistent with the idea that the signal transmission pathway from glucose to adenyl cyclase contains a glucose-repressible protein. They also show that a certain level of cAMP-dependent protein phosphorylation is required for glucose repression. Investigation of the glucose-induced cAMP signal and glucose-induced activation of trehalase in derepressed cells of strains containing only one of the wild-type TPK genes indicates that the transient nature of the cAMP signal is due to feedback inhibition by cAMP-dependent protein kinase.
Synthesis of functional mRNA in mammalian cells by bacteriophage T3 RNA polymerase.
Authors: Zhou Y , Giordano T J , Durbin R K , McAllister W T
Published: October 2015

We found that the 5' nontranslated leader sequence from encephalomyocarditis virus (EMCV) allowed transcripts that were synthesized by the T3 RNA polymerase in mammalian cells to be translated in a cap-independent fashion. Stable mouse cell lines that carry the T3 RNA polymerase gene expressed the chloramphenicol acetyltransferase (CAT) gene under the control of a phage promoter when the CAT gene was fused to the EMCV leader and introduced into the cells by transient DNA uptake. The level of gene expression in such cells was similar to or greater than that observed with a conventional transient expression vector that is dependent on transcription by the host RNA polymerase II. Expression of the EMCV-CAT fusion gene was stimulated by cotransfection of the cells with a gene that encodes the poliovirus protease 2A protein (which inhibits cap-dependent translation), demonstrating that the EMCV-CAT fusion gene was expressed in a cap-independent fashion. Introduction of both the T3 RNA polymerase gene and the EMCV-CAT fusion gene into a variety of cultured mammalian cell lines (HeLa, BSC40, Ltk-, NIH 3T3, and C127) demonstrated that the T3-EMCV expression system functions in a broad range of cell types.
Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis.
Authors: Banta L M , Vida T A , Herman P K , Emr S D
Published: October 2015

vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.
The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae.
Authors: Schultz J , Marshall-Carlson L , Carlson M
Published: October 2015

The SSN6 protein functions as a negative regulator of a variety of genes in Saccharomyces cerevisiae and is required for normal growth, mating, and sporulation. It is a member of a family defined by a repeated amino acid sequence, the TPR (tetratricopeptide repeat) motif. Here, we have used specific antibody to identify and characterize the SSN6 protein. Both SSN6 and a bifunctional SSN6-beta-galactosidase fusion protein were localized in the nucleus by immunofluorescence staining. The N-terminal one-third of the protein containing the TPR units was identified as the region that is important for SSN6 function. Analysis of four nonsense alleles, isolated as intragenic suppressors of an ssn6::URA3 insertion, revealed that polypeptides truncated after TPR unit 7 provide SSN6 function. Deletion analysis suggested that TPR units are required but that 4 of the 10 TPR units are sufficient. In addition, deletion studies indicated that three very long, homogeneous tracts of polyglutamine and poly(glutamine-alanine) are dispensable. Previous genetic evidence suggested the SSN6 protein as a possible target of the SNF1 protein kinase. Here, we show that the C terminus of SSN6 is phosphorylated in vivo and that the SNF1 kinase is not responsible for most of the phosphorylation. Finally, SSN6 has a modest effect on the maintenance of minichromosomes.
Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.
Authors: Burgess W H , Dionne C A , Kaplow J , Mudd R , Friesel R , Zilberstein A , Schlessinger J , Jaye M
Published: October 2015

Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma.
A single amino acid change in CUP2 alters its mode of DNA binding.
Authors: Buchman C , Skroch P , Dixon W , Tullius T D , Karin M
Published: October 2015

CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene. In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc). Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc. Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc. A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2. This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein. Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.
Upstream elements repress premature expression of an Aspergillus developmental regulatory gene.
Authors: Adams T H , Timberlake W E
Published: October 2015

The Aspergillus nidulans abaA gene regulates intermediate steps in asexual reproductive development and is itself developmentally regulated. An 822-base-pair DNA fragment from the abaA 5'-flanking region is sufficient to drive developmentally appropriate expression of the Escherichia coli lacZ gene. Deletion analysis showed that this fragment contains elements that repress transcription in vegetative cells and immature conidiophores and that activate transcription later during development. A 45-base-pair region encompassing the major and minor abaA transcription initiation sites contains directly repeated sequences related to the mammalian initiator (Inr) element (S. T. Smale and D. Baltimore, Cell 57:103-113, 1989). This element or sequences in the untranslated leader were sufficient for correct transcription initiation and for measurable developmental induction. Similar elements were present at or near the initiation sites of other developmentally regulated genes. We propose that the temporal and spatial specificity of expression of these genes results from modulation of the activity of Inr elements.
The first pre-rRNA-processing event occurs in a large complex: analysis by gel retardation, sedimentation, and UV cross-linking.
Authors: Kass S , Sollner-Webb B
Published: October 2015

The first processing event that mouse pre-rRNA undergoes occurs within the external transcribed spacer and is efficiently reproduced in vitro. Analysis with nondenaturing polyacrylamide gels revealed the formation of heparin-resistant complexes of retarded electrophoretic mobility on the substrate rRNA. The specificity of these complexes was demonstrated by their elimination due to competition with processing-competent, but not with processing-incompetent, rRNAs. Furthermore, complex formation, like the processing cleavage, required only 28 nucleotides of rRNA sequence adjacent to the processing site but was stimulated by additional downstream conserved sequences. These processing complexes formed in a time-dependent manner, and once assembled, they were stable to challenge by competitor rRNA and remained on the processed rRNA. Their sedimentation coefficient was approximately 20S. UV cross-linking studies with 4-thiouridine-substituted rRNA have identified six polypeptides, 52 to 250 kilodaltons, that are specifically bound to the rRNA processing substrate.
Domains of human c-myc protein required for autosuppression and cooperation with ras oncogenes are overlapping.
Authors: Penn L J , Brooks M W , Laufer E M , Littlewood T D , Morgenstern J P , Evan G I , Lee W M , Land H
Published: October 2015

Amino acids 106 to 143 and 354 to 433 of the human c-myc protein (439 amino acids) were shown to be required for the protein to suppress c-myc gene transcription and were found to exactly overlap with those necessary for c-myc to cooperate with ras oncogenes in the transformation of rat embryo fibroblasts. The essential carboxyl-terminal region harbors structural motifs (a basic region, a helix-loop-helix motif, and a "leucine zipper"), which, in other proteins, can mediate dimerization and sequence-specific DNA binding.
A short, highly repetitive element in intron -1 of the human c-Ha-ras gene acts as a block to transcriptional readthrough by a viral promoter.
Authors: Lowndes N F , Bushel P , Mendelsohn L , Wu J , Yen M Y , Allan M
Published: October 2015

We have identified a short, highly repetitive element within intron -1 of the human c-Ha-ras gene. This element was found to be transcribed in both orientations and to be homologous to heterogeneous nonpolyadenylated transcripts. The repetitive element blocked transcriptional readthrough from a strong upstream viral promoter but allowed unimpaired readthrough from the c-Has-ras promoter. We suggest that it may serve to prevent excessive transcription into the coding region of the gene under such circumstances as viral insertion.
trans activation of human alcohol dehydrogenase gene expression in hepatoma cells by C/EBP molecules bound in a novel arrangement just 5' and 3' to the TATA box.
Authors: Stewart M J , Shean M L , Duester G
Published: October 2015

A promoter sequence between nucleotide -51 and nucleotide -10 in the human alcohol dehydrogenase gene ADH2 has been shown to bind the transcription factor CCAAT/enhancer-binding protein (C/EBP). A series of 5'-end deletions of the ADH2 promoter was cotransfected with a C/EBP expression plasmid in a human hepatoma cell line, and trans activation by C/EBP was seen when at least 171 base pairs of 5'-flanking DNA was present. Mutations in the ADH2 promoter indicate that the mechanism of C/EBP trans activation involves two binding sites, one located just upstream of the TATA box and one located in an unusual location between the TATA box and the transcription start point.
Mechanisms of nonhomologous recombination in mammalian cells.
Authors: Roth D B , Porter T N , Wilson J H
Published: September 2015

The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed.
Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum.
Authors: Firtel R A , Silan C , Ward T E , Howard P , Metz B A , Nellen W , Jacobson A
Published: September 2015

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.
Characterization of the human c-fms gene product and its expression in cells of the monocyte-macrophage lineage.
Authors: Woolford J , Rothwell V , Rohrschneider L
Published: September 2015

The McDonough strain of feline sarcoma virus contains an oncogene called v-fms whose ultimate protein product (gp140v-fms) resembles a cell surface growth factor receptor. To identify and characterize the protein product of the proto-oncogene c-fms, antisera were prepared to the viral fms sequences and used to detect specific cross-reacting sequences in human choriocarcinoma cells (BeWo) known to express c-fms mRNA. Both tumor-bearing rat sera and a rabbit antiserum prepared to a segment of v-fms expressed in Escherichia coli detected a 140-kilodalton (kDa) glycoprotein in the BeWo cells. Tryptic fingerprint analysis of [35S]methionine-labeled proteins indicated that the viral fms proteins and the 140-kDa BeWo cell protein were highly related. This 140-kDa glycoprotein contained an associated tyrosine kinase activity in vitro and was labeled principally on serine after 32Pi metabolic labeling. These results suggest that the 140-kDa protein in BeWo cells is the protein product of the human c-fms proto-oncogene. This conclusion is supported by the finding that a similar protein is detectable only in other human cells that express c-fms mRNA. These other human cells include adherent monocytes and the cell line ML-1, which can be induced to differentiate along the monocyte-macrophage pathway. This is in agreement with current thought that the c-fms proto-oncogene product functions as the CSF-1 receptor specific to this pathway.
Bacterial beta-galactosidase as a marker of Rous sarcoma virus gene expression and replication.
Authors: Norton P A , Coffin J M
Published: September 2015

We have developed a convenient and sensitive assay of eucaryotic gene expression which uses the Escherichia coli lacZ gene product, beta-galactosidase, as a nonselectable marker. This system has been applied to the analysis of Rous sarcoma virus replication and gene expression. Avian cells were transfected with plasmids encoding in-frame gene fusions of the N-terminal portion of the gag gene to a 'lacZ gene, which requires both transcriptional and translational initiation signals; these were supplied by the virus long terminal repeat and leader region. Readily detectable quantities of beta-galactosidase were synthesized in transfected cells; it was demonstrated that the levels of enzyme activity induced in such cultures increased linearly with the input DNA concentration and also correlated with mRNA levels. By using a Rous sarcoma virus-derived vector containing the src gene and a related virus as a helper, it was shown that lac sequences were compatible with all phases of the virus life cycle. gag-lacZ fusion proteins were immunoprecipitable from cultures which stably expressed lacZ as well as src. Virus rescued from stably transfected cultures resulted in continued lac and src expression in recipient cells. One particular construction was efficiently transmitted as virus, although it lacked sequences thought to be important for encapsidation of RNA into virions. The data presented here demonstrate the use of lacZ as a marker of retrovirus gene expression and replication.
Transcription of herpes simplex virus tk sequences under the control of wild-type and mutant human RNA polymerase I promoters.
Authors: Smale S T , Tjian R
Published: September 2015

We studied RNA polymerase I transcription in cells transfected with a plasmid, prHuTK, containing the herpes simplex virus tk gene fused to a human rRNA promoter. Primer extension analysis of tk RNA isolated from COS cells transfected with prHuTK reveals that transcription from the RNA polymerase I promoter is highly efficient and initiates at the same position used for the synthesis of endogenous rRNA in HeLa cells. The RNA products derived from prHuTK are distinguishable from normal RNA polymerase II transcripts of tk in that they are not polyadenylated, are extremely unstable, and are found predominantly in the nucleus. Moreover, the transcription observed is resistant to 300 micrograms of alpha-amanitin per ml. These results strongly suggest that prHuTK transcription is under the control of the human rRNA promoter and RNA polymerase I. To further characterize the activity of the human rDNA promoter in vivo, a series of 5' and 3' deletion mutants was tested in this transfection assay. The deletion analysis indicates that a core region of ca. 40 base pairs overlapping the initiation site is critical for transcription. In addition, a region between nucleotides -234 and -131 upstream from the core sequence serves to modulate the efficiency of transcription. Insertion into prHuTK of additional ribosomal nontranscribed spacer DNA or the simian virus 40 enhancer element has no apparent effect on the promoter activity. Surprisingly, RNA polymerase II transcripts synthesized at low levels from two start sites within the core control element of the wild-type RNA polymerase I promoter are activated upon deletion of upstream RNA polymerase I promoter sequences. However, these RNA polymerase II transcripts are not expressed from the endogenous rRNA promoter.
Rapid induction of selective transcription by auxins.
Authors: Hagen G , Guilfoyle T J
Published: September 2015

Nuclei isolated from excised soybean plumules that were treated with 2,4-dichlorophenoxyacetic acid (2,4-D) were active in transcription of four auxin-regulated genes or DNA sequences, which have been described previously (G. Hagen, A. Kleinschmidt, and T. Guilfoyle, Planta 162:147-153, 1984). The rates of transcription of the auxin-responsive sequences were 10- to 100-fold greater with nuclei isolated from auxin-treated plumules than with those from untreated plumules. The transcriptional response was also observed with hypocotyls of intact soybean seedlings and hypocotyl sections, as well as with green bean and mung bean plumules that were treated with 2,4-D. Other auxins, including 2,4,5-trichlorophenoxyacetic acid, alpha-naphthaleneacetic acid, and indole-3-acetic acid, also induced the transcriptional response. Increased transcription rates were observed within 5 min after application of auxins to excised plumules, and half-maximal to maximal transcription rates were achieved by 15 min after application of auxins. As little as 10(-7) to 10(-8) M 2,4-D induced a transcriptional response, but maximal transcription rates were achieved at 10(-3) M 2,4-D. Brief treatment with the protein synthesis inhibitor cycloheximide did not inhibit the induction of transcription by auxins. These results clearly demonstrated that auxin-regulated gene expression is under rapid transcriptional control.
Direct identification of palmitic acid as the lipid attached to p21ras.
Authors: Buss J E , Sefton B M
Published: September 2015

p21v-H-ras, the transforming protein of Harvey murine sarcoma virus, contains a covalently attached lipid. Using thin-layer chromatography, we identified the acyl group as the 16-carbon saturated fatty acid palmitic acid. No myristic acid was detected in fatty acids released from in vivo-labeled p21v-H-ras. The p21v-K-ras protein encoded by Kirsten sarcoma virus was also palmitylated. The processing and acylation of p21v-K-ras however differed from that of p21v-H-ras. Three forms of [3H]palmitic acid-labeled p21ras proteins were detected in Kirsten sarcoma virus-transformed cells. This contrasted with Harvey sarcoma virus, in which two forms of p21v-H-ras contained palmitic acid. Analysis by partial proteolysis of p21v-H-ras labeled with [3H]palmitic acid suggested that all of the lipid found in intact p21v-H-ras was located in the C-terminal region. On sodium dodecyl sulfate-polyacrylamide gels, p21v-H-ras labeled with [3H]palmitic acid migrated slightly ahead of the majority of p21v-H-ras. Of the mature forms of p21v-H-ras, apparently only a subpopulation contains palmitic acid.
Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae.
Authors: Yeh E , Carbon J , Bloom K
Published: September 2015

We used DNA fragments from the centromere regions of yeast (Saccharomyces cerevisiae) chromosomes III and XI to examine the transcriptional activity within this chromosomal domain. DNA transcripts were found 200 to 300 base pairs from the 250-base-pair centromere core and lie within an ordered chromatin array. No transcripts were detected from the functional centromere region. We examined the cellular function of one of these tightly centromere-linked transcripts. (CEN11)L, by disrupting the coding sequences in vivo and analyzing the phenotype of the mutant yeast cell. Diploids heterozygous for the (CEN11)L disruption sporulated at wild-type levels, and the absence of the (CEN11)L gene product had no effect on the viability or mitotic growth of haploid cells. Diploids homozygous for the (CEN11)L disruption were unable to sporulate when induced by the appropriate nutritional cues. The mutant cells were competent for intragenic recombination and appeared to be blocked at the mononucleate stage. The temporal ordering of (CEN11)L function with respect to the sporulation mutant spo13 suggests that the (CEN11)L gene product may be required at both the first and second meiotic cell divisions. This new sporulation gene has been termed SPO15.
5'-flanking sequence required for regulated expression of a muscle-specific Drosophila melanogaster actin gene.
Authors: Geyer P K , Fyrberg E A
Published: September 2015

We have functionally tested derivatives of a muscle-specific Drosophila melanogaster actin gene in which 5'-flanking sequences have been deleted or rearranged. From our results we conclude that approximately 1,000 nucleotides of 5'-flanking sequence are required for wild-type levels of mRNA accumulation during flight muscle development. Derivatives having 875 or 865 nucleotides of upstream sequence could be expressed normally, but were prone to influence by flanking foreign DNA sequences. Derivatives retaining 600 or fewer nucleotides of flanking DNA did not direct detectable levels of mRNA accumulation. The sequence residing between -919 and -640 could be inverted and yet retain normal function. Deletion of this sequence reduced mRNA accumulation markedly, but did not affect its spatial localization, suggesting that elements which confer tissue specificity reside close to the point of transcription initiation.
Circumsporozoite protein of Plasmodium berghei: gene cloning and identification of the immunodominant epitopes.
Authors: Eichinger D J , Arnot D E , Tam J P , Nussenzweig V , Enea V
Published: September 2015

The gene encoding the circumsporozoite (CS) protein of the rodent malaria parasite Plasmodium berghei was cloned and characterized. A cDNA library made from P. berghei sporozoite RNA was screened with a monoclonal antibody for expression of CS protein epitopes. The resulting cDNA clone was used to isolate the CS protein gene from a lambda library containing parasite blood-stage DNA. The CS protein gene contains a central region encoding two types of tandemly repeated amino acid units, flanked by nonrepeated regions encoding amino- and carboxy-terminal signal and anchorlike sequences, respectively. One of the central repeated amino acid unit types contains the immunodominant epitopes.
The additional guanylate at the 5' terminus of Escherichia coli tRNAHis is the result of unusual processing by RNase P.
Authors: Orellana O , Cooley L , Söll D
Published: September 2015

In eucaryotes the 5'-terminal guanylate moiety of mature tRNAHis is added posttranscriptionally. To determine whether the same mechanism occurs in procaryotes, we processed in vitro-derived Escherichia coli tRNAHis precursors to mature tRNA, either in E. coli extracts or by using pure M1-RNA, the catalytic component of RNase P. The results show that the extra guanylate at the 5' end of mature E. coli tRNAHis is encoded in the gene and is found in tRNA as the result of an unusual cleavage by RNase P.
Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae.
Authors: Patterson M , Sclafani R A , Fangman W L , Rosamond J
Published: September 2015

The product of the CDC7 gene of Saccharomyces cerevisiae appears to have multiple roles in cellular physiology. It is required for the initiation of mitotic DNA synthesis. While it is not required for the initiation of meiotic DNA replication, it is necessary for genetic recombination during meiosis and for the formation of ascospores. It has also been implicated in an error-prone DNA repair pathway. Plasmids capable of complementing temperature-sensitive cdc7 mutations were isolated from libraries of yeast genomic DNA in the multicopy plasmid vectors YRp7 and YEp24. The complementing activity was localized within a 3.0-kilobase genomic DNA fragment. Genetic studies that included integration of the genomic insert at or near the CDC7 locus and marker rescue of four cdc7 alleles proved that the cloned fragment contains the yeast chromosomal CDC7 gene. The RNA transcript of CDC7 is about 1,700 nucleotides. Analysis of the nucleotide sequence of a 2.1-kilobase region of the cloned fragment revealed the presence of an open reading frame of 1,521 nucleotides that is presumed to encode the CDC7 protein. Depending on which of two possible ATG codons initiates translation, the calculated size of the CDC7 protein is 58.2 or 56 kilodaltons. Comparison of the predicted amino acid sequence of the CDC7 gene product with other known protein sequences suggests that CDC7 encodes a protein kinase.
Overexpression and amplification of five genes in a multidrug-resistant Chinese hamster ovary cell line.
Authors: Van der Bliek A M , Van der Velde-Koerts T , Ling V , Borst P
Published: September 2015

Multidrug-resistant cells are cross-resistant to a wide range of unrelated drugs, many of which are used in cancer chemotherapy. We constructed a cDNA library from RNA of the multidrug-resistant Chinese hamster ovary cell line CHRC5. By differential screening we isolated cDNAs derived from mRNAs that are overexpressed in this cell line. The cDNAs could be grouped in five classes on the basis of transcript lengths detected in RNA blots. We infer that each class codes for a separate protein. The corresponding genes are amplified 10 or 30 times in CHRC5 DNA, providing an explanation for the constitutive overexpression found in this cell line. Despite differential amplification, the genes may be linked in one large amplicon as indicated by the hybridization analysis of large fragments of CHRC5 DNA separated by pulsed field gradient gel electrophoresis. Therefore, some of these genes might be fortuitously coamplified and not contribute functionally to the resistant phenotype. It is also possible, however, that genes involved in drug resistance are clustered. One of our clones cross-hybridized with the recently described cDNA pCHP1 (J. R. Riordan, K. Deuchars, N. Kartner, N. Alon, J. Trent, and V. Ling, Nature [London] 316:817-819, 1985) encoding part of the 170-kilodalton P-glycoprotein, a protein which is frequently overproduced in multidrug-resistant cells. The nature of the four other genes is still unknown. Sequences of four of the five classes of cDNAs are conserved in mouse and human DNA.
Bidirectional activity of the rat insulin II 5'-flanking region in transgenic mice.
Authors: Efrat S , Hanahan D
Published: September 2015

We have identified a new transcription initiation site in the 5'-flanking regulatory region of the rat insulin II gene. This site is located on the opposite strand with respect to the insulin gene promoter, upstream of the insulin gene transcriptional enhancer. The cell-specific activity of this reverse promoter element is demonstrated in two lineages of transgenic mice, in which it directs expression of simian virus 40 T antigen specifically to the beta cells of the endocrine pancreas, resulting in development of pancreatic tumors. Analysis of RNA from the tumor cells demonstrates bidirectional transcription from the insulin regulatory region of the transgene. These data raise the possibility that bidirectional activity is a quality of the regulatory region of the insulin gene in its natural genomic context.
Interaction of GAL4 and GAL80 gene regulatory proteins in vitro.
Authors: Lue N F , Chasman D I , Buchman A R , Kornberg R D
Published: September 2015

The GAL80 protein of Saccharomyces cerevisiae, synthesized in vitro, bound tightly to GAL4 protein and to a GAL4 protein-upstream activation sequence DNA complex, as shown by (i) coimmunoprecipitation of GAL4 and GAL80 proteins with anti-GAL4 antiserum, (ii) an electrophoretic mobility shift of a GAL4 protein-upstream activation sequence DNA complex upon the addition of GAL80 protein, and (iii) GAL4-dependent binding of GAL80 protein to upstream activation sequence DNA immobilized on Sepharose beads. Anti-GAL4 antisera were raised against a GAL4-URA3 fusion protein, which could be purified to homogeneity in a single step with the use of an affinity chromatographic procedure for the URA3 gene product.
Promoter and enhancer elements from the rat elastase I gene function independently of each other and of heterologous enhancers.
Authors: Ornitz D M , Hammer R E , Davison B L , Brinster R L , Palmiter R D
Published: September 2015

An elastase-human growth hormone (hGH) fusion gene containing 205 base pairs of elastase 5' flanking region is expressed exclusively in pancreatic acinar cells of transgenic mice. This paper shows that the promoter region (-72 to +8) and the enhancer (-205 to -73) function independently of each other. The elastase enhancer can activate the heterologous mouse metallothionein gene and the hGH gene promoters; conversely, enhancers from the thymocyte-specific murine leukemia virus MCF13 and the metal regulatory elements from the metallothionein gene can activate the elastase promoter in a variety of cell types. Combinations of immunoglobulin and elastase enhancers with a heterologous promoter and the hGH gene result in expression in all of the tissues predicted by the sum of each enhancer acting alone. Thus these enhancer elements act independently of each other, suggesting that they do not have silencing activity in cells in which they are normally inactive.
Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules.
Authors: Lee B A , Maher D W , Hannink M , Donoghue D J
Published: September 2015

The v-vis gene encodes p28sis, the transforming protein of simian sarcoma virus. This gene resulted from a fusion of the env gene of simian sarcoma-associated virus and the woolly monkey gene for the B chain of platelet-derived growth factor (PDGF). Previous work has shown that the v-sis gene product undergoes signal sequence cleavage, glycosylation, dimerization, and proteolytic processing to yield a secreted form of the protein. It transport across the endoplasmic reticulum is blocked by the introduction of a charged amino acid residue within the signal sequence, the protein does not dimerize, is not secreted, and is no longer transforming as assayed by focus-forming ability in NIH 3T3 cells. Instead, this mutant protein localizes to the nucleus as demonstrated by both indirect immunofluorescence and cell fractionation. Using a series of deletion mutations, we delimited an amino acid sequence within this protein which is responsible for nuclear localization. This region is completely conserved in the predicted human c-sis protein, although it lies outside of regions required for transformation by the v-sis gene product. This nuclear transport signal is contained within amino acid residues 237 to 255, RVTIRTVRVRRPPKGKHRK. An amino acid sequence containing these residues is capable of directing cytoplasmic v-sis mutant proteins to the nucleus. This sequence is also capable of directing less efficient nuclear transport of a normally cytoplasmic protein, pyruvate kinase. Pulse-chase experiments indicate that the half-lives of nuclear and cytoplasmic v-sis mutant proteins are approximately 35 min. Using the heat-inducible hsp70 promoter from Drosophila melanogaster, we showed that the nuclear v-sis protein accumulates in the nucleus within 30 min of induction. The identification of a nuclear transport signal in the v-sis gene product raises interesting questions regarding the possibility of some function for PDGF or PDGF-related molecules in the nucleus.
Identification of the gene for the yeast ribonucleotide reductase small subunit and its inducibility by methyl methanesulfonate.
Authors: Hurd H K , Roberts C W , Roberts J W
Published: September 2015

We have identified, cloned, and sequenced the gene for the small subunit of ribonucleotide diphosphate reductase of Saccharomyces cerevisiae. The protein and its transcript are induced about 10-fold by the alkylating agent methyl methanesulfonate, a result which suggests that the gene is induced by DNA damage.
Genetic and biochemical characterization of clathrin-deficient Saccharomyces cerevisiae.
Authors: Payne G S , Hasson T B , Hasson M S , Schekman R
Published: September 2015

Clathrin is important but not essential for yeast cell growth and protein secretion. Diploid Saccharomyces cerevisiae cells heterozygous for a clathrin heavy-chain gene (CHC1) disruption give rise to viable, slow-growing, clathrin heavy-chain-deficient meiotic progeny (G. Payne and R. Schekman, Science 230:1009-1014, 1985). The possibility that extragenic suppressors account for growth of clathrin-deficient cells was examined by deletion of CHC1 from haploid cell genomes by single-step gene transplacement and independently by introduction of a centromere plasmid carrying the complete CHC1 gene into diploid cells before eviction of a chromosomal CHC1 locus and subsequent tetrad analysis. Both approaches yielded clathrin-deficient haploid strains. In mutants missing at least 95% of the CHC1 coding domain, transcripts related to CHC1 were not detected. The time course of invertase modification and secretion was measured to assess secretory pathway functions in the viable clathrin-deficient cells. Core-glycosylated invertase was converted to the mature, highly glycosylated form at equivalent rates in mutant and wild-type cells. Export of mature invertase from mutant cells was delayed but not prevented. Abnormal vacuoles, accumulated vesicles, and Golgi body-derived structures were visualized in mutant cells by electron microscopy. We conclude that extragenic suppressors do not account for the viability of clathrin-deficient cells and, furthermore, that many standard laboratory strains can sustain a CHC1 disruption. Clathrin does not appear to mediate protein transfer from the endoplasmic reticulum to the Golgi body but may function at a later stage of the secretory pathway.
Identification of protein products encoded by the proto-oncogene int-1.
Authors: Brown A M , Papkoff J , Fung Y K , Shackleford G M , Varmus H E
Published: September 2015

The proto-oncogene int-1 is activated by adjacent insertions of proviral DNA in mouse mammary tumor virus-induced tumors and has transforming activity in certain mammary epithelial cell lines. The gene is normally expressed in the central nervous system of mid-gestational embryos and in the adult testis. We raised antibodies against synthetic int-1 peptides and used these to identify protein products of the gene in cells transfected or infected with retroviral vectors expressing int-1. Four protein species of 36,000, 38,000, 40,000, and 42,000 Mr were immunoprecipitated by antibodies against two different int-1 peptides and were not present in control cells. Partial degradation with V8 protease showed the four species to be structurally related to each other and to int-1 polypeptide synthesized in vitro. Treatment of the cells with tunicamycin prevented the appearance of all but the 36,000-Mr species, suggesting that the slower-migrating forms are glycosylated derivatives. The unglycosylated 36,000-Mr species migrated faster in polyacrylamide gels than the in vitro translation product of int-1 and has probably undergone cleavage of an amino-terminal signal peptide.
Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.
Authors: Trudel M , Magram J , Bruckner L , Costantini F
Published: September 2015

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.
Alternative splicing of chicken fibronectin in embryos and in normal and transformed cells.
Authors: Norton P A , Hynes R O
Published: September 2015

To study the alternative splicing of fibronectin during embryogenesis and oncogenic transformation, we isolated cDNA clones of chicken fibronectin. The partial amino acid sequence deduced from sequencing of these clones showed that, overall, chicken fibronectin is approximately 80% identical with mammalian fibronectins. However, two of the three known regions of alternative splicing differed from this average. The V region was significantly more divergent, and RNA from embryonic chicken liver showed a pattern of V exon splicing which was distinct from that seen in human or rat fibronectins. In contrast, the EIIIB segment was very highly conserved (96%). As in mammals, this segment and another (EIIIA) were alternatively spliced in a cell-type-specific fashion. EIIIA+ and EIIIB+ species were almost absent in liver but predominated in total embryo RNA at all times from 2.5 to 11 days postfertilization. We also examined the possible contributions of fibronectin splicing and integrin receptor expression to the loss of fibronectin on oncogenic transformation. We detected little change in fibronectin splicing, other than a slight increase in representation of EIIIB+ species in fibroblasts after transformation by Rous sarcoma virus. It was also established that the overall reduction in fibronectin mRNA level observed after transformation was not accompanied by a decrease in integrin mRNA levels, indicating that fibronectin and integrin receptors are not coordinately regulated at the transcriptional level.
Repair of UV-induced lesions in Xenopus laevis oocytes.
Authors: Legerski R J , Penkala J E , Peterson C A , Wright D A
Published: September 2015

We characterized a DNA repair system in frog oocytes by comicroinjection of UV-irradiated pBR322 DNA and radiolabeled nucleotides. Repair synthesis was monitored by incorporation of label into recovered pBR322 DNA and by a novel method in which the removal of UV photoproducts was determined from the shift of DNA topoisomers that occurs during gel electrophoresis upon repair of these lesions. We investigated the effects of several drugs in the oocyte system and found that although novobiocin, an inhibitor of topoisomerase II, was an effective inhibitor of repair, VM-26, another inhibitor of topoisomerase II, was not. In addition, the topoisomerase I inhibitor camptothecin had no effect on repair in this system. Finally, circular DNA (either supercoiled or nicked circular) was repaired at least 50 times more rapidly than linear DNA.
Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.
Authors: Bartolomei M S , Corden J L
Published: September 2015

RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene.