bluuebunny/crossref_metadata_2025 huggingface dataset:
Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src.
Authors: Coussens P M , Cooper J A , Hunter T , Shalloway D
Published: September 2015

The tyrosine protein kinase activities of pp60c-src and pp60v-src were compared. The activities were qualitatively similar in vitro when the src proteins were bound in an immune complex with monoclonal antibody; both proteins utilized either ATP or GTP as phosphate donors, preferred Mn2+ to Mg2+, and had similar exogenous substrate specificities. The specific activity of pp60c-src was about 10-fold lower than that of pp60v-src for exogenous substrate phosphorylation but was only 1.1- to 2-fold lower than that of pp60v-src for autophosphorylation. Six glycolytic enzymes, including three not previously identified as substrates for pp60src phosphorylation, were phosphorylated by both pp60c-src and pp60v-src. Levels of pp60c-src fourfold higher than the amount of pp60v-src in src-plasmid-transformed cells did not detectably alter the level of phosphotyrosine in cellular proteins, but increasing the expression of pp60c-src another twofold (which induces cells to form foci in monolayer culture (P.J. Johnson, P.M. Coussens, A.V. Danko, and D. Shalloway, Mol. Cell. Biol. 5:1073-1083, 1985) resulted in a threefold increase in the level of cellular protein phosphotyrosine. Immunoprecipitation and analysis of the alkali-stable phosphoproteins by two-dimensional electrophoresis showed that, in contrast to pp60v-src-transformed cells, pp36 and enolase are only weakly phosphorylated in these high-level pp60c-src overexpresser cells. Even allowing for the in vitro differences in specific activities of phosphorylation, these results suggest that the pp60c-src tyrosine protein phosphorylating activity may be restricted relative to that of pp60v-src by additional in vivo mechanisms.
Stimulation of sea urchin H2B histone gene transcription by a chromatin-associated protein fraction depends on gene sequences downstream of the transcription start site.
Authors: Mous J , Stunnenberg H , Georgiev O , Birnstiel M L
Published: September 2015

We isolated a chromosomal protein fraction derived from chromatin of sea urchin embryos which specifically stimulated the expression of the histone H2B gene by a factor of 5- to 10-fold when the complete sea urchin histone gene repeat h22 was injected in Xenopus laevis oocyte nuclei. Gene manipulation experiments revealed the existence of two different target sites in the H2B gene which appear to mediate the response to injection of the stimulatory sea urchin chromatin-associated proteins; both are located downstream of the transcription initiation site. The first sequence element which is shown to be implicated is within, or at least includes, the H2B 5' untranslated leader sequence between nucleotides 11 and 76. The second element resides within an H2B DNA segment located near the 3' end of the gene, extending from 90 base pairs upstream of the mRNA 3' terminus to 140 base pairs in the spacer sequences downstream.
Genetic characterization of human c-rel sequences.
Authors: Brownell E , O'Brien S J , Nash W G , Rice N
Published: September 2015

We isolated and sequenced a human genomic-DNA segment that is homologous to a portion of v-rel, the transforming gene of reticuloendotheliosis virus (strain T). We also localized the human rel sequences to human chromosome 2 by screening a panel of rodent X human somatic-cell hybrids with the newly described human rel segment.
ras gene Amplification and malignant transformation.
Authors: Pulciani S , Santos E , Long L K , Sorrentino V , Barbacid M
Published: September 2015

Morphologic transformation of NIH 3T3 mouse cells occurs upon transfection of these cells with large amounts (greater than or equal to 10 micrograms) of recombinant DNA molecules carrying the normal human H-ras-1 proto-oncogene. We provide experimental evidence indicating that transformation of these NIH 3T3 cells results from the combined effect of multiple copies of the H-ras-1 proto-oncogene rather than from spontaneous mutation of one of the transfected H-ras-1 clones (E. Santos, E.P. Reddy, S. Pulciani, R.J. Feldman, and M. Barbacid, Proc. Natl. Acad. Sci. USA 80:4679-4683, 1983). Levels of H-ras-1 RNA and p21 expression are highly elevated in the NIH 3T3 transformants, and in those cases examined, these levels correlate with the malignant properties of these cells. We have also investigated the presence of amplified ras genes in a variety of human carcinomas. In 75 tumor biopsies, we found amplification of the human K-ras-2 locus in one carcinoma of the lung. These results indicate that ras gene amplification is an alternative pathway by which ras genes may participate in the development of human neoplasia.
Surprising S1-resistant trimolecular hybrids: potential complication in interpretation of S1 mapping analyses.
Authors: Lopata M A , Sollner-Webb B , Cleveland D W
Published: September 2015

Although the technique of S1 mapping is a powerful analytical tool for the analysis of RNA, we now report a surprising complication involving a trimolecular hybrid between two RNA species and a single DNA probe molecule which, if unrecognized, can lead to misleading interpretations. We document that such trimolecular hybrids can be efficiently formed under some hybridization conditions and that the probe DNA sequence at the junction of the two RNA molecules can be remarkably stable to digestion with S1. Trimolecular hybrids can arise in any instance whenever a distal region of an end-labeled DNA probe is homologous to a moderately abundant RNA in the sample to be analyzed. This situation presents a serious, potential complication for a variety of S1 analyses, particularly those in which DNA transfection has been utilized to reintroduce in vitro-engineered genes into cultured animal cells.
Evolution of the dispersed SUC gene family of Saccharomyces by rearrangements of chromosome telomeres.
Authors: Carlson M , Celenza J L , Eng F J
Published: September 2015

The SUC gene family of Saccharomyces contains six structural genes for invertase (SUC1 through SUC5 and SUC7) which are located on different chromosomes. Most yeast strains do not carry all six SUC genes and instead carry natural negative (suc0) alleles at some or all SUC loci. We determined the physical structures of SUC and suc0 loci. Except for SUC2, which is an unusual member of the family, all of the SUC genes are located very close to telomeres and are flanked by homologous sequences. On the centromere-proximal side of the gene, the conserved region contains X sequences, which are sequences found adjacent to telomeres (C. S. M. Chan and B.-K. Tye, Cell 33:563-573, 1983). On the other side of the gene, the homology includes about 4 kilobases of flanking sequence and then extends into a Y' element, which is an element often found distal to the X sequence at telomeres (Chan and Tye, Cell 33:563-573, 1983). Thus, these SUC genes and flanking sequences are embedded in telomere-adjacent sequences. Chromosomes carrying suc0 alleles (except suc20) lack SUC structural genes and portions of the conserved flanking sequences. The results indicate that the dispersal of SUC genes to different chromosomes occurred by rearrangements of chromosome telomeres.
Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.
Authors: Liu H T , Baserga R , Mercer W E
Published: September 2015

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.
Mechanism for differential sensitivity of the chromosome and growth cycles of mammalian cells to the rate of protein synthesis.
Authors: Wu R S , Bonner W M
Published: September 2015

It has been documented widely that when the generation times of eucaryotic cells are lengthened by slowing the rate of protein synthesis, the duration of the chromosome cycle (S, G2, and M phases) remains relatively invariant. Paradoxically, when the growth of exponentially growing cultures of CHO cells is partially inhibited with inhibitors of protein synthesis, the immediate effect is a proportionate reduction in the rate of total protein, histone protein, and DNA synthesis. However, on further investigation it was found that over the next 2 h the rates of histone protein and DNA synthesis recover, in some cases completely to the uninhibited rate, while the synthesis rates of other proteins do not recover. We called this process chromosome cycle compensation. The amount of compensation seen in CHO cell cultures can account quantitatively for the relative invariance in the length of the chromosome cycle (S, G2, and M phases) reported for these cells. The mechanism for this compensation involves a specific increase in the levels of histone mRNAs. An invariant chromosome cycle coupled with a lengthening growth cycle must result in a disproportionate lengthening of the G1 phase. Thus, these results suggest that chromosome cycle invariance may be due more to specific cellular compensation mechanisms rather than to the more usual interpretation involving a rate-limiting step for cell cycle progression in the G1 phase.
Detection of two chromatin proteins which bind specifically to the 5'-flanking region of the rat prolactin gene.
Authors: White B A , Preston G M , Lufkin T C , Bancroft C
Published: September 2015

We employed a protein gel blotting procedure to search for nuclear proteins from rat pituitary cells that bind preferentially to the 5'-flanking region of the rat prolactin gene. By gel blots of chromatin proteins from GH3 rat pituitary tumor cells with a 32P-labeled prolactin genomic clone, we detected two major binding proteins with molecular weights of approximately 44,000 and 48,000, designated NP44 and NP48, respectively. Both NP44 and NP48 are minor chromatin proteins which are extracted at low salt concentrations (0.4 M NaCl) and exhibit a range of slightly acidic isoelectric variants. NP44 and NP48 were detected at similar levels in chromatin extracts of GH3 cells, the prolactin-negative GC cell variant of the GH3 cells, and normal rat pituitary tissue. Considerably lower levels of these two proteins were found in chromatin extracts from rat liver and rat C6 glial cells. NP44 and NP48 exhibit DNA sequence specificity, as evidenced by their strong binding to the upstream flanking region of the prolactin gene, but only very weak binding to plasmid DNA, rat prolactin or growth hormone cDNAs, or upstream flanking regions of two other rat genes. By analyzing subclones of a rat prolactin genomic clone, we established that NP44 and NP48 bind to at least two sites, which are located between 0.4 and 2.0 kilobases (region I) and between 2.0 and 4.8 kilobases (region II) upstream of the transcription initiation site. These findings are discussed in the context of a possible functional association between the strong binding of NP44 and NP48 to the prolactin 5'-flanking region and pituitary-specific expression of the prolactin gene.
Quantitative assessment of actin transcript number in eggs, embryos, and tube feet of the sea star Pisaster ochraceus.
Authors: Kovesdi I , Smith M J
Published: September 2015

Actin coding sequence cDNA probes were used to quantitate the number of transcripts in RNA from eggs, embryos, and tube feet of the sea star Pisaster ochraceus. Transcript concentrations were measured in both total RNA and in poly(A)+ RNA by titration and hybridization kinetic methods. Surprisingly, the actin transcript number in sea star eggs is two orders of magnitude greater than in sea urchin eggs. There are at least 2.9 X 10(5) actin transcripts per sea star egg, 1.2 X 10(5) per 48-h gastrula and 1.9 X 10(5) per 72-h gastrula. The number of actin transcripts per unit mass of extracted tube foot RNA is lower than in developmental stages. The relative abundance and size of actin transcripts was determined by Northern and dot blot analyses using probes containing actin coding DNA or 3'-untranslated-region sequences. The actin transcript in eggs and embryos is 2,300 nucleotides (nt) long and originates from the Cy (cytoplasmic) gene class. In tube feet, the most abundant actin transcript is 2,200 nt long and originates from the M (muscle) gene class. Tube feet also contain, at lower abundance, 2,300-nt transcripts of the Cy gene type expressed in eggs and embryos.
Nuclear compartmentalization of the v-myb oncogene product.
Authors: Boyle W J , Lampert M A , Li A C , Baluda M A
Published: September 2015

Nuclei obtained from chicken leukemic myeloblasts transformed by avian myeloblastosis virus were fractionated into various subnuclear compartments, which were then analyzed by specific immunoprecipitation for the presence of the leukemogenic product, p48v-myb, of the viral oncogene. In cells labeled for 30 or 60 min with L-[35S]methionine and in unlabeled exponentially dividing leukemic cells analyzed by Western blotting, p48v-myb was detected within the nucleoplasm (29 +/- 9% [standard deviation] of the total), chromatin (7 +/- 4%), and lamina-nuclear matrix (64 +/- 9%). Also, in myeloblasts analyzed by immunofluorescence during mitosis, p48v-myb appeared to be dispersed through the cell like the lamina-nuclear matrix complex. Strong attachment to the nuclear matrix-lamina complex suggests that p48v-myb may be involved in DNA replication or transcription or both.
Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression.
Authors: Entian K D , Hilberg F , Opitz H , Mecke D
Published: September 2015

The regulatory hexokinase PII mutants isolated previously (K.-D. Entian and K.-U. Fröhlich, J. Bacteriol. 158:29-35, 1984) were characterized further. These mutants were defective in glucose repression. The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme. Hence, a regulatory domain for glucose repression was postulated. For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics. None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged. The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D. Entian and E. Kopetzki, Eur. J. Biochem. 146:657-662, 1985), was absent from one of these mutants. It is possible that the PIIM modification is directly connected with the triggering of glucose repression. To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function. Unlike hexokinase PII wild-type transformants, glucose repression was not restored in the hexokinase PII mutant transformants. In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PII structural gene.
Differential expression of the human gonadotropin alpha gene in ectopic and eutopic cells.
Authors: Darnell R B , Boime I
Published: September 2015

We have analyzed the regulation of the alpha gonadotropin gene in eutopic placental cells and ectopic tumor cells by constructing a series of plasmid vectors containing alpha genomic 5' flanking DNA placed upstream of the gene encoding the bacterial enzyme chloramphenicol acetyltransferase (CAT). These plasmid DNAs were transfected into a eutopic (JAr) and an ectopic (HeLa) cell line. Both cell types expressed the CAT gene from plasmid constructs containing as much as 1,500 base pairs (bp) and as little as 140 bp of alpha 5' flanking DNA; JAr cells were considerably more efficient than HeLa cells. Ectopic and eutopic cells differed qualitatively in their expression from these alpha-CAT constructs when cells were treated with cAMP or butyrate. Butyrate induced alpha expression in HeLa cells but not in JAr cells, while cAMP induced expression in JAr cells. These results are consistent with and extend previous observations suggesting that there are cell-specific differences in the regulation of alpha gene expression in ectopic and eutopic cells. However, by using deletion constructs of the alpha-CAT gene, we found that the basal expression and cell-specific induction of the alpha gene in ectopic and eutopic cells were dependent on the same 140 bp of alpha 5' flanking DNA. These 140 bp were sequenced and found to contain a 9-bp stretch of DNA homologous with the consensus viral enhancer sequence. Such features of alpha expression common to both ectopic and eutopic cells may be involved in the coordinate expression of the alpha gene and the tumorigenic phenotype observed in each cell type.
Phosphatidylinositol kinase activities in normal and Rous sarcoma virus-transformed cells.
Authors: Sugimoto Y , Erikson R L
Published: September 2015

The phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol kinase activities in the plasma membrane-rich fraction of chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus increased when the cells were shifted from the nonpermissive temperature, 41 degrees C, to the permissive temperature, 35 degrees C. Temperature shift from 35 to 41 degrees C decreased the lipid kinase activities in the membrane vesicles. These changes accompanied the changes observed in pp60v-src protein kinase activity. Thermal inactivation at 41 degrees C did not appreciably reduce PI and PIP kinase activities in membrane vesicles prepared from uninfected or Rous sarcoma virus-transformed cells, whereas pp60v-src protein kinase activity in the membrane vesicles was rapidly inactivated under the same conditions. These data suggest that pp60v-src may indirectly enhance PI and PIP phosphorylation but not directly contribute to this pathway.
Burkitt lymphoma cell line carrying a variant translocation creates new DNA at the breakpoint and violates the hierarchy of immunoglobulin gene rearrangement.
Authors: Denny C T , Hollis G F , Magrath I T , Kirsch I R
Published: September 2015

The Burkitt lymphoma cell line KK124, which contains a reciprocal t(8;22) translocation, was shown to have rearranged in a region 3' to the c-myc proto-oncogene on chromosome 8 and 5' to the lambda constant region on chromosome 22. The breakpoint was cloned and sequenced, revealing that c-myc and a portion of its 3' region abutted a complete lambda variable gene that had undergone V-J recombination. Since this cell line expresses kappa light chain, this lambda rearrangement violates the previously proposed hierarchy of immunoglobulin gene rearrangement. A novel duplication of normal chromosome 8 sequences was also found at the breakpoint. The first exon of c-myc and its flanking sequence from the translocated allele was sequenced and compared with a normal counterpart. Extensive mutation was found within the first exon in contrast to its 3' and 5' flanking regions. S1 nuclease analysis revealed that it was the translocated c-myc being expressed and that there was a promoter shift from P2 to P1. The detailed structural analysis of this cell line provides clues concerning mechanisms of chromosomal translocation and c-myc deregulation in Burkitt lymphomas.
Organization, primary structure, and evolution of histone H2A and H2B genes of the fission yeast Schizosaccharomyces pombe.
Authors: Choe J , Schuster T , Grunstein M
Published: September 2015

The histone H2A and H2B genes of the fission yeast Schizosaccharomyces pombe were cloned and sequenced. Southern blot and sequence analyses showed that, unlike other eucaryotes, Saccharomyces cerevisiae included, S. pombe has unequal numbers of these genes, containing two histone H2A genes (H2A-alpha and -beta) and only one H2B gene (H2B-alpha) per haploid genome. H2A- and H2B-alpha are adjacent to each other and are divergently transcribed. H2A-beta has no other histone gene in close proximity. Preceding both H2A-alpha and -beta is a highly conserved 19-base-pair sequence (5'-CATCAC/AAACCCTAACCCTG-3'). The H2A DNA sequences encode two histone H2A subtypes differing in amino acid sequence (three residues) and size (H2A-alpha, 131 residues; H2A-beta, 130 residues). H2B-alpha codes for a 125-amino-acid protein. Sequence evolution is extensive between S. pombe and S. cerevisiae and displays unique patterns of divergence. Certain N-terminal sequences normally divergent between eucaryotes are conserved between the two yeasts. In contrast, the normally conserved hydrophobic core of H2A is as divergent between the yeasts as between S. pombe and calf.
DNA-mediated transfer and expression of a human DNA repair gene that demethylates O6-methylguanine.
Authors: Ding R , Ghosh K , Eastman A , Bresnick E
Published: September 2015

Human liver DNA was transfected into CHO cells (mex-) along with pSV2gpt and colonies were selected first for resistance to mycophenolic acid and then to chloroethylnitrosourea. Transformants were obtained that contained approximately 10,000 molecules of O6-alkylguanine alkyltransferase (mex+) per cell. Their genome contained at least three copies of the human Alu sequence.
Transformation-defective mutant of avian myeloblastosis virus that is temperature sensitive for production of transforming protein p45v-myb.
Authors: Moscovici M G , Klempnauer K H , Symonds G , Bishop J M , Moscovici C
Published: September 2015

We have characterized a mutant of avian myeloblastosis virus (strain GA907/7) that shows a reduced capacity to transform myelomonocytic cells at the nonpermissive temperature. Myeloblasts transformed by this mutant suffer a substantial decrease in the amount of the transforming protein p45v-myb when shifted from the permissive to the nonpermissive temperature. We presume that the 5- to 10-fold decrease in the amount of p45v-myb causes the loss of the transformed phenotype. The decrease is due to a reduction in the level of v-myb mRNA. Mutant GA907/7 thus provides genetic evidence that p45v-myb is the transforming protein of avian myeloblastosis virus and apparently represents an unusual defect in the production or stability of mRNA.
Major substrate for growth factor-activated protein-tyrosine kinases is a low-abundance protein.
Authors: Cooper J A , Hunter T
Published: September 2015

A scarce, soluble, conserved protein was identified as the nonphosphorylated precursor of two related 42-kilodalton phosphoproteins that contain phosphotyrosine in mitogen-stimulated but not control fibroblasts.
Nucleotide sequence and expression in vitro of cDNA derived from mRNA of int-1, a provirally activated mouse mammary oncogene.
Authors: Fung Y K , Shackleford G M , Brown A M , Sanders G S , Varmus H E
Published: September 2015

The mouse int-1 gene is a putative mammary oncogene discovered as a target for transcriptionally activating proviral insertion mutations in mammary carcinomas induced by the mouse mammary tumor virus in C3H mice. We have isolated molecular clones of full- or nearly full-length cDNA transcribed from int-1 RNA (2.6 kilobases) in a virus-induced mammary tumor. Comparison of the nucleotide sequence of the cDNA clones with that of the int-1 gene (A. van Ooyen and R. Nusse, Cell 39:233-240, 1984) shows the following. The coding region of the int-1 gene is composed of four exons. The splice donor and acceptor sites conform to consensus; however, at least two closely spaced polyadenylation sites are used, and the transcriptional initiation site remains ambiguous. The major open reading frame is preceded by an open frame 10 codons in length. The mRNA encodes a 41-kilodalton protein with several striking features--a strongly hydrophobic amino terminus, a cysteine-rich carboxy terminus, and four potential glycosylation sites. There are no differences in nucleotide sequence between the known exons of the normal and a provirally activated allele. The length of the deduced open reading frame was further confirmed by in vitro translation of RNA transcribed from the cDNA clones with SP6 RNA polymerase.
Recombinant interleukin 2 regulates levels of c-myc mRNA in a cloned murine T lymphocyte.
Authors: Reed J C , Sabath D E , Hoover R G , Prystowsky M B
Published: September 2015

The cellular oncogene c-myc has been implicated in the regulation of growth of normal and neoplastic cells. Recently, it was suggested that c-myc gene expression may control the G0----G1-phase transition in normal lymphocytes that were stimulated to enter the cell cycle by the lectin concanavalin A (ConA). Here we describe the effects of purified recombinant interleukin 2 (rIL2) and of ConA on levels of c-myc mRNA in the noncytolytic murine T-cell clone L2. In contrast to resting (G0) primary cultures of lymphocytes, quiescent L2 cells have a higher RNA content than resting splenocytes and express receptors for interleukin 2 (IL2). Resting L2 cells are therefore best regarded as early G1-phase cells. Purified rIL2 was found to stimulate the rapid accumulation of c-myc mRNA in L2 cells. Levels of c-myc mRNA became maximal within 1 h and declined gradually thereafter. In contrast, ConA induced slower accumulation of c-myc mRNA in L2 cells, with increased levels of c-myc mRNA becoming detectable 4 to 8 h after stimulation. Experiments with the protein synthesis inhibitor cycloheximide demonstrated that the increase in levels of c-myc mRNA that were induced by ConA was a direct effect of this lectin and not secondary to IL2 production. Cyclosporin A, an immunosuppressive agent, markedly reduced the accumulation of c-myc mRNA that was induced by ConA but only slightly diminished the accumulation of c-myc mRNA that was induced by rIL2. Taken together, these data provide evidence that (i) c-myc gene expression can be regulated by at least two distinct pathways in T lymphocytes, only one of which is sensitive to cyclosporine A, and (ii) the accumulation of c-myc mRNA can be induced in T cells by IL2 during the G1 phase of the cell cycle.
Pichia pastoris as a host system for transformations.
Authors: Cregg J M , Barringer K J , Hessler A Y , Madden K R
Published: September 2015

We developed a methylotrophic yeast, Pichia pastoris, as a host for DNA transformations. The system is based on an auxotrophic mutant host of P. pastoris which is defective in histidinol dehydrogenase. As a selectable marker, we isolated and characterized the P. pastoris HIS4 gene. Plasmid vectors which contained either the P. pastoris or the Saccharomyces cerevisiae HIS4 gene transformed the P. pastoris mutant host. DNA transfer was accomplished by a modified version of the spheroplast generation (CaCl2-polyethylene glycol)-fusion procedure developed for S. cerevisiae. In addition, we report the isolation and characterization of P. pastoris DNA fragments with autonomous replication sequence activity. Two fragments, PARS1 and PARS2, when present on plasmids increased transformation frequencies to 10(5)/micrograms and maintained the plasmids as autonomous elements in P. pastoris cells.
Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.
Authors: Chakrabarti S , Brechling K , Moss B
Published: September 2015

We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli beta-galactosidase (beta gal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK- recombinants could be selected by a plaque assay on TK- cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous TK- mutants by the addition of a beta-gal indicator to the agarose overlay. Plaques that expressed beta-gal stained dark blue within several hours at 37 degrees C. Alternatively, TK- selection could be eliminated, and recombinant plaques could be readily identified solely by their blue color. The reverse procedure, in which the starting virus expresses beta-gal (i.e., forms blue plaques) and the desired recombinant has deleted the entire beta-gal gene (i.e., forms white plaques), is another alternative. Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.
Genes for low-molecular-weight heat shock proteins of soybeans: sequence analysis of a multigene family.
Authors: Nagao R T , Czarnecka E , Gurley W B , Schöffl F , Key J L
Published: September 2015

Soybeans, Glycine max, synthesize a family of low-molecular-weight heat shock (HS) proteins in response to HS. The DNA sequences of two genes encoding 17.5- and 17.6-kilodalton HS proteins were determined. Nuclease S1 mapping of the corresponding mRNA indicated multiple start termini at the 5' end and multiple stop termini at the 3' end. These two genes were compared with two other soybean HS genes of similar size. A comparison among the 5' flanking regions encompassing the presumptive HS promoter of the soybean HS-protein genes demonstrated this region to be extremely homologous. Analysis of the DNA sequences in the 5' flanking regions of the soybean genes with the corresponding regions of Drosophila melanogaster HS-protein genes revealed striking similarity between plants and animals in the presumptive promoter structure of thermoinducible genes. Sequences related to the Drosophila HS consensus regulatory element were found 57 to 62 base pairs 5' to the start of transcription in addition to secondary HS consensus elements located further upstream. Comparative analysis of the deduced amino acid sequences of four soybean HS proteins illustrated that these proteins were greater than 90% homologous. Comparison of the amino acid sequence for soybean HS proteins with other organisms showed much lower homology (less than 20%). Hydropathy profiles for Drosophila, Xenopus, Caenorhabditis elegans, and G. max HS proteins showed a similarity of major hydrophilic and hydrophobic regions, which suggests conservation of functional domains for these proteins among widely dispersed organisms.
Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae.
Authors: Abovich N , Gritz L , Tung L , Rosbash M
Published: September 2015

The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.
Coordinate expression of amplified metallothionein I and II genes in cadmium-resistant Chinese hamster cells.
Authors: Griffith J K
Published: September 2015

Recombinant DNA probes complementary to Chinese hamster metallothionein (MT)-1 and MT-2 mRNAs were used to compare MT gene copy numbers, zinc-induced MT mRNA levels, and uninduced MT mRNA levels in cadmium-resistant (Cdr) Chinese hamster ovary cell lines. Quantitative hybridization analyses determined that the MT-1 and MT-2 genes are each present at approximately single-copy levels in the genome of cell line Cdr2C10 and are coordinately amplified approximately 7, 3, and 12 times over the Cdr2C10 value in the genomes of cell lines Cdr20F4, Cdr30F9, and Cdr200T1, respectively. The maximum zinc-induced MT-1 mRNA concentrations in cell lines Cdr20F4, Cdr30F9, and Cdr200T1 were equal to 1, 3, and 15 times that measured in Cdr2C10, respectively. Similarly, the maximum zinc-induced MT-2 mRNA concentrations were equal to 1, 3, and 14 times that measured in Cdr2C10, respectively, and in each instance they were 90 to 150 times greater than their respective concentrations in uninduced cells. Thus, relative MT gene numbers are closely correlated with both zinc-induced and uninduced MT mRNA levels in Cdr2C10, Cdr30F9, and Cdr200T1, but not in Cdr20F4. Each of the latter two lines possesses structurally altered chromosomes whose breakpoints are near the MT locus. Nonetheless, the ratio of the levels of MT-1 to MT-2 mRNAs was constant in each of the four cell lines, including Cdr20F4. These results demonstrate that MT-1 and MT-2 mRNAs are induced coordinately in each Cdr cell line. Therefore, the coordination of the induction of MT-1 and MT-2 mRNA is independent of MT gene amplification, MT gene rearrangement, and the relative inducibilities of amplified MT genes. However, MT mRNA and protein levels each indicate that MT-1 and MT-2 expression is non-coordinate in uninduced cells. Thus, regulation of MT expression may involve two different mechanisms which are differentially operative in induced and uninduced cells.
Effect of dimethyl sulfoxide on human carcinoma cells, inhibition of plasminogen activator synthesis, change in cell morphology, and alteration of response to cholera toxin.
Authors: Ossowski L , Belin D
Published: September 2015

Human carcinoma HEp-3 lost its tumorigenic and metastatic potential upon prolonged culture in vitro. This change was accompanied by a reduced production of plasminogen activator (PA) of the urokinase type (uPA), which is secreted by HEp-3 cells, a change in response to effectors that modulate uPA production, and an alteration of cell morphology. Similar but more rapid changes occurred when malignant HEp-3 cells were exposed to dimethyl sulfoxide (DMSO). uPA activity in the culture medium dropped below 50% of the control level within 6 h after the addition of DMSO and became undetectable after 24 h of treatment. This drop in uPA activity was not caused by an increased production of PA inhibitors. The cell-associated uPA decreased to 25 to 30% of the control level within 6 h of DMSO treatment and remained at this level for at least 96 h; the reduced uPA production was partially accounted for by a rapid decrease in the functional and chemical concentration of uPA mRNA. In contrast, the concentrations of most of the abundant mRNA species did not appear to be significantly affected, and cell growth was only slightly inhibited in the presence of DMSO. Malignant HEp-3 cells treated with DMSO responded to cholera toxin with an enhanced production of uPA, and their morphology became indistinguishable from that of nonmalignant HEp-3 cells grown in vitro for prolonged periods of time. All of the above changes were fully and rapidly reversible. The inhibitory effect of DMSO on PA production appears to be specific for uPA of human cell lines.
Autogenous regulation of the positive regulatory qa-1F gene in Neurospora crassa.
Authors: Patel V B , Giles N H
Published: September 2015

In Neurospora crassa, the qa-1F regulatory gene positively controls transcription of all genes in the quinic acid (qa) gene cluster. qa-1F is transcribed at a low, uninduced level but is subject to strong (50-fold), autogenous regulation as well as to control by the negative regulatory gene, qa-1S, and the inducer quinic acid. Cloned qa-1F DNA sequences hybridize to two related mRNAs of 2.9 and 3.0 kilobases. When wild-type (qa-1F+) cultures are transferred to inducing conditions, qa-1F mRNA increases for 4 h, remains somewhat level, and decreases after 8 to 10 h. That this control is autogenous, i.e., that the qa-1F gene controls the synthesis of its own mRNA, is indicated by the presence of approximately the same low level of qa-1F mRNA in poly(A)+ RNA from noninducible qa-1F- mutant cultures under inducing conditions as that observed in uninduced wild-type cultures. The qa-1S gene also regulates the transcription of qa-1F, since a qa-1S- mutant, whether in noninducing or inducing conditions, contains a level of qa-1F mRNA that corresponds to the low level observed in uninduced wild-type cultures. These results corroborate the hypothesis (M. E. Case and N. H. Giles, Proc. Natl. Acad. Sci. USA 72:553-557, 1975; V. B. Patel, M. Schweizer, C. C. Dykstra, S. R. Kushner, and N. H. Giles, Proc. Natl. Acad. Sci. USA 78:5783-5787, 1981; L. Huiet, Proc. Natl. Acad. Sci. USA 81:1174-1178, 1984) that the qa-1F gene encodes an activator protein and acts positively in controlling transcription of itself and the other qa genes.
DNase I footprinting shows three protected regions in the promoter of the rRNA genes of Xenopus laevis.
Authors: Dunaway M , Reeder R H
Published: September 2015

Extracts prepared from Xenopus laevis oocytes contain a protein(s) which specifically protects three discrete regions of the RNA polymerase I promoter from digestion by DNase I. Protected region I, from nucleotide +15 to nucleotide -10, spans the site of transcription initiation. Protected region II extends from nucleotide -70 to nucleotide -100 relative to initiation, falling within a 42-base-pair sequence which is homologous to the 60/81-base-pair repeated elements which occur outside of the promoter in the spacer. Protected region III is upstream of region II, from nucleotide -120 to nucleotide -140. All three regions correlate with sequences known from deletion studies to be important for promoter function. Deletion mutants which retain either region I or regions II and III together footprint normally. Deletion of region III, however, reduces but does not eliminate footprinting on region II, suggesting either that one protein binds to both regions or that the proteins which bind to these sites interact with each other.
Sequences upstream of c-mos(rat) that block RNA accumulation in mouse cells do not inhibit in vitro transcription.
Authors: van der Hoorn F A , Müller V , Pizer L I
Published: September 2015

An unusual feature of the c-mos oncogene is the lack of expression in mouse tissues. Recombinant plasmids that contain the strong adenovirus late promoter and different amounts of cellular DNA 5' to c-mos(rat) were constructed and tested in transfection and transcription assays. The cellular sequences inhibit RNA accumulation in mouse but not human cells and do not inhibit in vitro transcription of the plasmid DNAs.
Human c-fms proto-oncogene: comparative analysis with an abnormal allele.
Authors: Verbeek J S , Roebroek A J , van den Ouweland A M , Bloemers H P , Van de Ven W J
Published: September 2015

The organization of the human c-fms proto-oncogene has been determined and compared with an abnormal allele. The human v-fms homologous genetic sequences are dispersed discontinuously and colinearly with the viral oncogene over a DNA region of ca. 32 kilobase pairs. The abnormal c-fms locus contains a small deletion in its 3' portion. DNA sequencing analysis indicated that it was 426 base pairs in size and located in close proximity to a putative c-fms exon.
Translocation of oncogene c-sis from chromosome 22 to chromosome 11 in a Ewing sarcoma-derived cell line.
Authors: van Kessel A G , Turc-Carel C , de Klein A , Grosveld G , Lenoir G , Bootsma D
Published: September 2015

Somatic cell hybrids, obtained after fusion of translocation (11;22)-positive Ewing sarcoma cells and Chinese hamster fibroblasts, were assayed for the presence of immunoglobulin C lambda, Philadelphia chromosome breakpoint cluster region, and c-sis oncogene sequences. It was found that c-sis was translocated from chromosome 22 to chromosome 11 in the Ewing sarcoma cells used, indicating that the breakpoint must be proximal to this locus. Moreover, we found that the chromosome 22-linked C lambda and breakpoint cluster region sequences are not translocated. This result confirms an earlier cytogenetic observation that the Ewing sarcoma-associated breakpoint in chromosome 22 is distal to those observed in translocation (8;22)-positive Burkitt lymphoma and in Philadelphia chromosome-positive chronic myeloid leukemia.
Functional analysis of the transcription control region located within the avian retroviral long terminal repeat.
Authors: Cullen B R , Raymond K , Ju G
Published: September 2015

We used several quantitative assays of in vivo transient gene expression to dissect the elements within the Rous sarcoma virus long terminal repeat (LTR) which constitute the retroviral transcription control region. Site-directed deletion mutagenesis was used to locate and define the enhancer and promoter elements within the LTR. In addition, we inserted exogenous DNA fragments into the LTR to examine the effects of position and sequence on the activity of these LTR transcriptional elements. The Rous sarcoma virus enhancer element, which we propose is located entirely within the LTR, was shown to activate both the beta-globin and retroviral LTR promoters when located in cis. We observed a striking correlation between the degree of activation and the distance between the retroviral promoter and enhancer elements. The LTR promoter element mediated the activation effect of the enhancer element, as LTR deletion mutants containing only the enhancer and TATA box region expressed little activity. The promoter region encoded a low but significant level of transcriptional activity even in the absence of an enhancer. Overall LTR transcriptional activity declined sharply with increasing distance between the LTR promoter and initiator elements. These results shed light on both the importance of the spatial arrangement of the sequence elements within this eucaryotic transcription control region and on the functional interrelationship between these elements.
Family of human alpha-interferon-like sequences.
Authors: Feinstein S I , Mory Y , Chernajovsky Y , Maroteaux L , Nir U , Lavie V , Revel M
Published: September 2015

An interferon-alpha-like sequence was isolated from a human genomic library by hybridization with a 15-base oligonucleotide. The sequence also showed homology to alpha-interferon and was most closely related to the leukocyte interferon-M gene fragment. The original isolate cross-hybridized to a family of sequences, 10 of which were isolated as clones. Some of these sequences were located within a few kilobases of alpha-interferon genes, consistent with our assignment of several members of the family to human chromosome 9 which also has the beta 1- and alpha-interferon genes.
gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes.
Authors: Silver S , Roizman B
Published: September 2015

True gamma or gamma 2 genes, unlike alpha, beta, and gamma 1 (beta gamma) genes of herpes simplex virus 1 (HSV-1), stringently require viral DNA synthesis for their expression. We report that gamma 2 genes resident in cells were induced in trans by infection with HSV-1 but that the induction did not require amplification of either the resident gene or the infecting viral genome. Specifically, to test the hypothesis that expression of these genes is amplification dependent, we constructed two sets of gamma 2-thymidine kinase (TK) chimeric genes. The first (pRB3038) consisted of the promoter-regulatory region and a portion of 5'-transcribed noncoding region of the domain of a gamma 2 gene identified by Hall et al. (J. Virol. 43:594-607) in the HSV-1(F) BamHI fragment D' to the 5'-transcribed noncoding and coding regions of the TK gene. The second (pRB3048) contained, in addition, an origin of HSV-1 DNA replication. Cells transfected with either the first or second construct and selected for the TK+ phenotype were then tested for TK induction after superinfection with HSV-1(F) delta 305, containing a deletion in the coding sequences of the TK gene, and viruses containing, in addition, a ts lesion in the alpha 4 regulatory protein (ts502 delta 305) or in the beta 8 major DNA-binding protein (tsHA1 delta 305). The results were as follows: induction by infection with TK- virus of chimeric TK genes with or without an origin of DNA replication was dependent on functional alpha 4 protein but not on viral DNA synthesis; the resident chimeric gene in cells selected for G418 (neomycin) resistance was regulated in the same fashion; the chimeric gene recombined into the viral DNA was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis; the gamma 2-chimeric genes resident in the host and in viral genomes were transcribed from the donor BamHI fragment D' containing the promoter-regulatory domain of the gamma 2 gene. The significance of the differential regulation of gamma 2 genes in the environments of host and viral genomes by viral trans-acting factors is discussed.
Rapid assay for extrachromosomal homologous recombination in monkey cells.
Authors: Rubnitz J , Subramani S
Published: September 2015

Most of the recombination assays based on the regeneration of selectable marker genes after transient infection or stable integration of DNA into mammalian cells are time consuming. We have used plasmids containing two truncated but overlapping segments of the neomycin resistance gene to rapidly quantitate and characterize the time course of extrachromosomal homologous recombination of DNA transfected into monkey COS cells. By transiently infecting cells with these recombination substrates, extracting Hirt DNA after 1 to 4 days, and transforming recombination-deficient Escherichia coli, we have shown that recombination between direct repeats occurs at frequencies of 1 to 4%. We have also used Southern blot analysis to directly characterize the recombination of this DNA in COS cells and to demonstrate that double-strand breaks in the region of homology increase recombination frequencies 10- to 50-fold.