bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and nucleotide sequence analysis of the Saccharomyces cerevisiae RAD1 gene.
Authors: Yang E , Friedberg E C
Published: September 2015

We have screened a yeast genomic library for complementation of the UV sensitivity of mutants defective in the RAD1 gene and isolated a plasmid designated pNF1000 with an 8.9-kilobase insert. This multicopy plasmid quantitatively complemented the UV sensitivity of two rad1 mutants tested but did not affect the UV resistance of other rad mutants. The location of the UV resistance function in pNF1000 was determined by deletion analysis, and an internal fragment of the putative RAD1 gene was integrated into the genome of a RAD1 strain. Genetic analysis of several integrants showed that integration occurred at the chromosomal RAD1 site, demonstrating that the internal fragment was derived from the RAD1 gene. A 3.88-kilobase region of pNF1000 was sequenced and showed the presence of a small open reading frame 243 nucleotides long that is apparently unrelated to RAD1, as well as a 2,916-nucleotide larger open reading frame presumed to encode RAD1 protein. Depending on which of two possible ATG codons initiates translation, the size of the RAD1 protein is calculated at 110 or 97 kilodaltons.
Cloning of DNA corresponding to rare transcripts of rat brain: evidence of transcriptional and post-transcriptional control and of the existence of nonpolyadenylated transcripts.
Authors: Brilliant M H , Sueoka N , Chikaraishi D M
Published: September 2015

To examine the expression of genes encoding rare transcripts in the rat brain, we have characterized genomic DNA clones corresponding to this class. In brain cells, as in all cell types, rare transcripts constitute the majority of different sequences transcribed. Moreover, when compared with other tissues or cultured cells, brain tissue may be expected to have an even larger set of rare transcripts, some of which could be restricted to subpopulations of neural cells. We have identified seven clones whose transcripts are nonabundant, averaging less than three copies per cell. Clone rg13 (rat genomic 13) RNA was detected only in the brain, whereas RNA of a second clone, rg40, was also detected in the brain and in a melanoma. Transcripts of rg13 were found in cerebellum, cerebral cortex, and regions underlying the cortex, whereas rg40 transcripts were not detected in the cerebellum. Transcripts of both rg13 and rg40 were found in pelleted polysomal RNA. RNA of another clone, rg34, was found in the brain, liver, and kidney but was found in pelleted polysomal RNA only in the brain, suggesting that its expression may be post-transcriptionally controlled. The remaining four clones represent rare transcripts that are common to the brain, liver, and kidney; rg18 RNA is restricted to the nucleus, whereas rg3, rg26, and rg36 transcripts are found in the cytoplasm of all three tissues. Transcripts of the brain-specific clone, rg13, and the commonly expressed clone, rg3, are nonpolyadenylated, presumably belonging to the high-complexity, nonpolyadenylated class of transcripts in the mammalian brain.
Translational efficiency of cMyc mRNA in Burkitt lymphoma cells.
Authors: Nilsen T W , Maroney P A
Published: September 2015

The translational efficiency of cMyc mRNAs was assessed in a variety of cell lines: HeLa cells and Epstein-Barr virus-transformed lymphocytes, both of which contain only the germ line cMyc allele; Daudi, a Burkitt cell line containing a translocated cMyc gene with no apparent alteration; and P3HR-1, a Burkitt line in which the 5' end of the translocated cMyc gene has been altered by the chromosomal translocation. Translational efficiency was inferred by measuring the number of ribosomes associated with the cMyc mRNA, using a procedure by which individual polysomal fractions were analyzed by blot hybridization. Since polysome size is a function of the length of the translated sequence as well as the rate of initiation of protein synthesis, we also determined the number of ribosomes associated with a control mRNA (alpha tubulin) which codes for a protein of similar size to cMyc. We found that the cMyc mRNA was associated with a number of ribosomes comparable to that associated with alpha tubulin mRNA in all the cell lines tested.
High spontaneous mutation frequency in shuttle vector sequences recovered from mammalian cellular DNA.
Authors: Ashman C R , Davidson R L
Published: September 2015

The recombinant shuttle vector pSV2gpt was introduced into V79 Chinese hamster cells, and stable transformants expressing the Escherichia coli gpt gene were selected. Two transformants carrying tandem duplications of the plasmid at a single site were identified and fused to simian COS-1 cells. Plasmid DNA recovered from the heterokaryons was used to transform a Gpt- derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. The high frequency of Gpt- plasmids (ca. 1%) was similar to that observed when plasmid was recovered from COS-1 cells which had been transfected with pSV2gpt. Most of the mutant plasmids had rearrangements in the region containing the gpt gene.
Cloning and expression of a cDNA that comprises part of the gene coding for a spore coat protein of Dictyostelium discoideum.
Authors: Dowds B C , Loomis W F
Published: September 2015

The three major spore coat proteins of Dictyostelium discoideum are developmentally regulated, cell-type-specific proteins. They are packaged in prespore vesicles and then secreted to form the outer layer of spore coats. We have isolated a cDNA clone from the gene coding for one of these proteins, SP96, a glycoprotein of 96,000 daltons. We screened the cDNA bank by the method of hybrid select translation followed by immunoprecipitation of the translation products with SP96-specific polyclonal antiserum. We found that the gene was first transcribed into stable mRNA a few hours before the time of detection of SP96 synthesis and that the mRNA, like the protein, accumulated specifically in prespore cells and spores. SP96 constituted the same proportion of newly synthesized protein as the proportion of its message in polyadenylated RNA. SP96 appeared to be encoded by a single gene as judged by Southern blot analysis of digested genomic DNA hybridized to the cDNA clone.
Processed pseudogenes for rat cytochrome c are preferentially derived from one of three alternate mRNAs.
Authors: Scarpulla R C
Published: September 2015

Three cytochrome c mRNAs (1,400, 1,100 and 700 nucleotides) are colinear with RC4, a gene that has introns and correctly encodes cytochrome c. A comparison of RC4 to six nonallelic clones isolated from the rat cytochrome c multigene family demonstrates that all three mRNAs are represented in the genome as processed pseudogenes. Four of the six pseudogenes are derived from the 1,100-nucleotide mRNA, and genomic hybridizations further establish that nearly all of the 30 or so gene family members are also genomic copies of this mRNA despite the equimolar ratio of the three messages in rat tissues. Thus, the surprising multiplicity of cytochrome c sequences in the rat genome is mainly accounted for by the selective use of the 1,100-nucleotide mRNA for the formation of processed pseudogenes. In contrast to 700- and 1,400-nucleotide species which are polyadenylated downstream from AAGUAAA and AAUUAAA, respectively, the 1,100-nucleotide mRNA uses the ubiquitous AAUAAA and also displays a unique stem and loop structure (delta G = -59.4 kJ) centered 37 base pairs upstream from this sequence.
Specific transcripts are elevated in Saccharomyces cerevisiae in response to DNA damage.
Authors: McClanahan T , McEntee K
Published: September 2015

Differential hybridization has been used to identify genes in Saccharomyces cerevisiae displaying increased transcript levels after treatment of cells with UV irradiation or with the mutagen/carcinogen 4-nitroquinoline-1-oxide (NQO). We describe the isolation and characterization of four DNA damage responsive genes obtained from screening ca. 9,000 yeast genomic clones. Two of these clones, lambda 78A and pBR178C, contain repetitive elements in the yeast genome as shown by Southern hybridization analysis. Although the genomic hybridization pattern is distinct for each of these two clones, both of these sequences hybridize to large polyadenylated transcripts ca. 5 kilobases in length. Two other DNA damage responsive sequences, pBRA2 and pBR3016B, are single-copy genes and hybridize to 0.5- and 3.2-kilobase transcripts, respectively. Kinetic analysis of the 0.5-kilobase transcript homologous to pBRA2 indicates that the level of this RNA increases more than 15-fold within 20 min after exposure to 4-nitroquinoline-1-oxide. Moreover, the level of this transcript is significantly elevated in cells containing the rad52-1 mutation which are deficient in DNA strand break repair and gene conversion. These results provide some of the first evidence that DNA damage stimulates transcription of specific genes in eucaryotic cells.
Expression of the amplified domain in human neuroblastoma cells.
Authors: Michitsch R W , Montgomery K T , Melera P W
Published: September 2015

Screening of a partial cDNA library prepared from the human neuroblastoma cell line BE(2)-C with genomic DNA probes containing sequences representative of the amplified domain of that cell line allowed us to identify cloned transcripts from an active gene within the domain. The gene BE(2)-C-59 is amplified ca. 150-fold and encodes a 3.0- and a 1.5-kilobase RNA transcript, both of which are overproduced in BE(2)-C cells. A survey of a large variety of human tumor cell types indicated that this gene is amplified to varying degrees in all neuroblastoma cell lines and a retinoblastoma cell line that exhibit obvious cytological manifestations of DNA sequence amplification, i.e., homogeneously staining regions and double-minute chromosomes. The BE(2)-C-59 gene is not amplified, however, in other nonrelated tumor types, even those containing amplified DNA. Although the functional significance of this specific gene amplification in neuroblastoma cells remains unknown, an indication that it may relate to the malignant phenotype of these cells follows from the remainder of our data which show that the amplified BE(2)-C-59 gene shares partial homology with both the second and third exons, but not the first exon, of the human c-myc oncogene.
Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step.
Authors: Barnes G , Hansen W J , Holcomb C L , Rine J
Published: September 2015

Asparagine-linked glycosylation is a form of covalent modification that distinguishes proteins that are either membrane bound or are in cellular compartments topologically outside of the cell from those proteins that remain soluble in the cytoplasm. This type of glycosylation occurs stepwise, with core oligosaccharide added in the endoplasmic reticulum and subsequent modifications occurring in the golgi. We used tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharide, to select for mutants that are resistant to this antibiotic. Genetic, biochemical, and physiological experiments led to the following conclusions. The synthesis of N-linked oligosaccharide is an essential function in cells. In contrast to mammalian cells, yeast cells do not transport tunicamycin by a glucosamine transport function. We identified a gene, ALG7, that is probably the structural gene for UDP-N-acetylglucosamine-1-P transferase, the enzyme inhibited by tunicamycin. Dominant mutations in this gene result in increased activity of the transferase and loss of the ability of the cell to sporulate. In addition, we identified another gene, TUN1, in which recessive mutations result in resistance to tunicamycin. The ALG7 and TUN1 genes both map on chromosome VII.
Isolation and characterization of the RNA2, RNA3, and RNA11 genes of Saccharomyces cerevisiae.
Authors: Last R L , Stavenhagen J B , Woolford J L
Published: September 2015

Temperature-sensitive mutations in the genes RNA2 through RNA11 cause accumulation of intervening sequence containing precursor mRNAs in Saccharomyces cerevisiae. Three different plasmids have been isolated which complement both the temperature-sensitive lethality and precursor mRNA accumulation when introduced into rna2, rna3, and rna11 mutant strains. The yeast sequences on these plasmids have been shown by Southern transfer hybridization and genetic mapping to be derived from the RNA2, RNA3, and RNA11 genomic loci. Part of the RNA2 gene is homologous to more than one region of the yeast genome, whereas the RNA3 and RNA11 genes are single copy. RNAs homologous to these loci have been identified by RNA transfer hybridization, and the specific RNAs which are associated with the Rna+ phenotype have been mapped. This was done by a combination of transcript mapping, subcloning, and in vitro mutagenesis. The transcripts are found to be enriched in polyadenylated RNA and are of very low abundance (0.01-0.001% polyadenylated RNA).
Immunological and genetic characterization of 2-deoxygalactose-resistant, galactokinase-deficient mutants of Chinese hamster cells: evidence for structural mutations at the galK locus.
Authors: Talbot B , de Souza C A , Banville D , Thirion J P
Published: September 2015

Ten independent mutants resistant to 2-deoxygalactose and without any detectable galactokinase activity (null-galactokinase mutations) were isolated from mutagenized Chinese hamster somatic cells. They were analyzed for the presence of serologically cross-reacting material (CRM) with antiserum generated against highly purified Chinese hamster galactokinase. All 10 mutants contain cross-reacting material (i.e., were CRM+), indicating that all the mutations affect the correct expression of a product of the galactokinase structural gene. Complementation analysis among them shows that the 10 mutations fall in one functional genetic unit.
Post-transcriptional regulation of the abundance of mRNAs encoding alpha-tubulin and a 94,000-dalton protein in teratocarcinoma-derived stem cells versus differentiated cells.
Authors: Howe C C , Lugg D K , Overton G C
Published: September 2015

Changes in the expression of the genes encoding alpha-tubulin and a 94,000-dalton protein (p94) specified by a cDNA clone, p4-30, were examined in a differentiated teratocarcinoma-derived parietal endoderm cell line, PYS-2, and an undifferentiated teratocarcinoma stem cell line, F9. Relative to other proteins or mRNA species, the synthesis rate of the alpha-tubulins and of p94, as well as the levels of their corresponding cytoplasmic mRNAs, were lower in PYS-2 than in F9 cells. The decrease was greater for the relative abundance of cytoplasmic alpha-tubulin mRNA than for p94 mRNA. Similarly, induction of differentiation of F9 cells by simultaneous exposure to retinoic acid (RA) and dibutyryl cyclic AMP resulted in reduced relative levels of the cytoplasmic mRNAs for these proteins. The reduction in abundance of the two RNA species was not due to a decrease in growth rate since the differentiated cells, PYS-2, RA-treated F9, and RA plus dibutyryl cyclic AMP-treated F9 cells, grew at a rate similar to that of undifferentiated F9 cells. However, induction of differentiation of F9 cells by treatment with RA alone did not cause down-regulation of the two RNA species. The relative levels of total cellular RNA encoding alpha-tubulin and p94 in PYS-2 cells were also lower than those in F9 cells to an extent comparable to the decrease in the cytoplasmic RNAs. Since the apparent relative rates of RNA transcription were similar in both cell types, we conclude that the reduction in relative levels of the alpha-tubulin and p94 RNAs in the cell depends largely on the relative stability of the two RNAs and not on the relative rates of transcription. The faster disappearance of the two RNA species relative to other cellular RNAs from actinomycin D-treated PYS-2 compared with F9 cells is consistent with this interpretation.
Characterization of the expressed gene and several processed pseudogenes for the mouse ribosomal protein L30 gene family.
Authors: Wiedemann L M , Perry R P
Published: September 2015

Five cloned genes encoding the mouse ribosomal protein L30 were isolated from a recombinant DNA library and characterized by restriction mapping and nucleotide sequence analysis. Only one of these genes has introns and is expressed; the others are inactive processed pseudogenes. The expressed gene consists of five exons and four introns spanning 2,723 nucleotides. Transcripts of this gene are processed into the mature L30 mRNA by pathways that exhibit both constraints and flexibility with regard to the order of intron excision. The L30 mRNA which is 457 to 468 nucleotides in length excluding the polyadenylic acid tail, exhibits some microheterogeneity at its 3' end and encodes a basic protein of 115 amino acids. The 5' portion of the rpL30 gene has some novel features which are remarkably similar to the previously characterized mouse rpL32 gene. These include homologous sequences in the -60 to -340 region, the absence of a good TATA consensus sequence, and the presence of a palindromic pyrimidine sequence that spans the cap site.
Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.
Authors: Brewer B J , Chlebowicz-Sledziewska E , Fangman W L
Published: September 2015

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.
Erythroleukemia (K562) cells contain a functional beta-globin gene.
Authors: Donovan-Peluso M , Young K , Dobkin C , Bank A
Published: September 2015

K562 cells are human erythroid cells that synthesize embryonic and fetal globins but not adult beta-globin. A cloned beta-globin gene was isolated from K562 cells and transfected into HeLa cells. The RNA transcripts produced were comparable in both amount and size to those obtained with a normal beta-globin gene.
The two embryonic U1 small nuclear RNAs of Xenopus laevis are encoded by a major family of tandemly repeated genes.
Authors: Lund E , Dahlberg J E , Forbes D J
Published: September 2015

We have identified a large family of U1 RNA genes in Xenopus laevis that encodes two distinct species of U1 RNA. These genes are expressed primarily at the onset of transcription in the 4,000-cell embryo (D. J. Forbes, M. W. Kirschner, D. Caput, J. E. Dahlberg, and E. Lund, Cell 38:681-689, 1984). The two types of embryonic U1 RNA genes are interspersed and are organized in large tandem arrays. The basic 1.9-kilobase repeating unit contains a single copy of each of the embryonic genes and is reiterated ca. 500-fold per haploid genome. This repetitive U1 DNA accounts for more than 90% of all U1 DNA in X. laevis. In addition to this major family, there exist several minor families of dispersed U1 RNA genes, which presumably encode the oocyte and somatic species of X. laevis U1 RNA. Although the embryonic genes are normally inactive in stage VI oocytes, they are expressed when cloned copies are injected into oocyte nuclei.
Characterization of genomic poly(dT-dG).poly(dC-dA) sequences: structure, organization, and conformation.
Authors: Hamada H , Petrino M G , Kakunaga T , Seidman M , Stollar B D
Published: September 2015

Hybridization studies suggest the abundant presence of poly(dT-dG).poly(dC-dA) (TG-element), a potential Z-DNA sequence, in eucaryotic genomes. We have isolated and characterized TG-elements from different locations in the human genome: from randomly isolated clones, associated with the actin gene family, and linked to another repeated element. The results indicate that the following features are typical of these TG-elements: the elements consist of 20 to 60 base pairs of (dT-dG)n.(dC-dA)n, the sequences characterized in our study were not flanked by direct or inverted repeats, the sequences are interspersed rather than in satellite blocks, the elements are not usually associated with other repeated elements, and some of the elements are found near coding sequences or in introns. Studies on the conformation of a genomic TG-element in a supercoiled plasmid indicate several distinct properties of the TG-element: it is in the Z-form only at low ionic strength, S1 nuclease recognizes its Z-form with a marked preference for one of the B-Z junctions, and the sensitive region extends for 20 base pairs near the B-Z junction. In contrast to the result with the supercoiled plasmid, S1 nuclease failed to recognize the TG-element in minichromosomes.
Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster.
Authors: Burns D K , Stark B C , Macklin M D , Chooi W Y
Published: September 2015

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.
A deletion that includes the signal peptidase cleavage site impairs processing, glycosylation, and secretion of cell surface yeast acid phosphatase.
Authors: Haguenauer-Tsapis R , Hinnen A
Published: September 2015

We transformed Saccharomyces cerevisiae with a high-copy-number plasmid carrying either the wild-type gene coding for a repressible cell surface acid phosphatase or two modified genes whose products lack a 13- or 14-amino-acid segment spanning or immediately adjacent to the signal peptidase cleavage site. The wild-type gene product underwent proteolytic cleavage of the signal peptide, core glycosylation, and outer chain glycosylation. The deletion spanning the signal peptidase cleavage site led to an unprocessed protein. This modified protein exhibited core glycosylation, whereas its outer chain glycosylation was severely inhibited. Secretion of the deleted protein was impaired, and active enzyme accumulated within the cell. The deletion immediately adjacent to the signal peptidase cleavage site exhibited only a small decrease in the efficiency of processing and had no effect on the efficiency of secretion.
Noncoordinate histone synthesis in heat-shocked Drosophila cells is regulated at multiple levels.
Authors: Farrell-Towt J , Sanders M M
Published: September 2015

Transferring Drosophila tissue culture cells from 25 to 37 degrees C (heat shock) causes histone protein synthesis to become noncoordinate. To determine the level at which this is controlled, the synthesis, degradation, and translation of individual histone mRNAs was studied under both heat shock and control conditions. The increased synthesis of histone H2b protein during heat shock appears to be controlled primarily at the level of translation. During heat shock, H2b mRNA is transcribed at about the same level as in the control. However, H2b mRNA is more stable under heat shock than under control conditions and is predominantly found in polysomes. The reduction in synthesis of H2a, H3, and H4 protein during heat shock appears to be controlled at both the transcriptional and translational levels. Although transcription of H2a, H3, and H4 mRNAs is reduced during heat shock, like H2b mRNA, they are more stable. However, unlike H2b mRNA, these mRNAs are not predominantly associated with polysomes during heat shock. Regulation of H1 synthesis during heat shock is completely different from that of the other histones. During heat shock, H1 mRNA is not transcribed, and unlike all of the other Drosophila mRNAs studied to date, its mRNA is not stable in heat-shocked cells. Results from in vitro translation studies support the conclusion that noncoordinate synthesis of the core histone proteins during heat shock is controlled at the level of translation.
Myristic acid is attached to the transforming protein of Rous sarcoma virus during or immediately after synthesis and is present in both soluble and membrane-bound forms of the protein.
Authors: Buss J E , Kamps M P , Sefton B M
Published: September 2015

Myristic acid, a minor component of cellular fatty acids, has been shown previously to be covalently bound to most molecules of p60src, the transforming protein of Rous sarcoma virus. We have now determined at what time during the life cycle of p60src, and where within the cell, this lipid becomes attached to the protein. p60src was found to acquire myristic acid at only one time, during or immediately after its synthesis. p60src is known to be synthesized on free polysomes and appears at the cytoplasmic face of the plasma membrane after a lag of 10 min. The addition of myristic acid to p60src therefore precedes the binding of the protein to the plasma membrane. The lipid attached to p60src is a permanent, metabolically stable part of the protein; we found no evidence for turnover of the myristyl moiety. However, we did find myristate attached to various soluble forms of p60src and to a large number of cytosolic cellular proteins as well. This demonstrates that the attachment of myristic acid to a protein is not in itself sufficient to convert a soluble protein into a membrane-bound protein.
Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells.
Authors: Voronova A F , Buss J E , Patschinsky T , Hunter T , Sefton B M
Published: September 2015

The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid.
Regulation of human histone gene expression during the HeLa cell cycle requires protein synthesis.
Authors: Sive H L , Heintz N , Roeder R G
Published: September 2015

We have examined the effects of protein synthesis inhibition on histone gene expression during the HeLa cell cycle. Histone mRNAs, which normally are rapidly degraded in the absence of DNA synthesis, persist and increase in concentration when translation is inhibited before DNA replication is halted. This is not a function of polysomal shielding of these mRNAs from active degradation mechanisms since inhibitors of translation initiation alone effect stabilization and induction. The superinduction of histone mRNAs by protein synthesis inhibition is effective at the G1/S border, and in the S-phase and non-S-phase periods of the cell cycle. However, the relative increase in histone mRNA is greater when cells not synthesizing DNA are treated with a protein synthesis inhibitor than when S-phase cells are so treated. Non-histone mRNAs examined are not superinduced by translation inhibition. Transcription rates from both histone and non-histone genes increase after protein synthesis inhibition. Although the decrease in histone gene transcription associated with DNA synthesis inhibition is prevented and reversed by protein synthesis inhibition, we have no evidence that histone gene-specific transcriptional regulation is dependent on protein synthesis. Transcriptional increases may contribute to the superinduction effect but cannot explain its differential extent during the cell cycle, since these increases are similar when replicating or nonreplicating cells are treated with a protein synthesis inhibitor. We believe that changes in histone mRNA stability can account for much of the differential superinduction effect. Our results indicate a requirement for continuing protein synthesis in the cell cycle regulation of histone mRNAs.
Primary structure of the RAD52 gene in Saccharomyces cerevisiae.
Authors: Adzuma K , Ogawa T , Ogawa H
Published: September 2015

The RAD52 gene of Saccharomyces cerevisiae, which is involved in genetic recombination and DNA repair, was cloned by transformation of rad52-1 mutant cells to methyl methanesulfonate resistance with BamHI fragments of Rad+ genomic DNA inserted into the Escherichia coli-S. cerevisiae shuttle vector YRp7. A plasmid carrying a 2.0-kilobase BamHI fragment was found to partially complement methyl methanesulfonate sensitivity of the rad52-1 mutant. By using this fragment as a hybridization probe, a plasmid that fully complemented the methyl methanesulfonate sensitivity of the mutant was isolated, which carries a 3.3-kilobase SalI fragment containing most of the 2.0-kilobase BamHI fragment. Analysis of the nucleotide sequence of the SalI fragment revealed the presence of a large open reading frame of 1,512 nucleotides. The rad52-1 mutant DNA has a single-base change in this reading frame, which leads to an amino acid substitution. Analysis of mRNA synthesized in yeast by the S1 mapping technique disclosed possible transcription initiation and termination points of the RAD52 gene and suggested formation of the gene product without splicing of the transcript.
HeLa cell beta-tubulin gene transcription is stimulated by adenovirus 5 in parallel with viral early genes by an E1a-dependent mechanism.
Authors: Stein R , Ziff E B
Published: September 2015

We report that the rate of transcription of cellular beta-tubulin genes increases during the early phase of adenovirus infection of HeLa cells, with kinetics very similar to those routinely found for viral genes. This activation depends upon adenovirus early region E1a, which encodes products that activate early virus transcription. To compare the responses of viral and cellular genes to E1a, we infected HeLa cells with dl312, a transcriptionally inactive deletion mutant that lacks a functional E1a gene. We then superinfected the cells with a helper virus, dl327, which encodes active E1a products, and measured changes in the rates of transcription of various cell and viral genes. Early region E3 of dl312 was activated 0 to 6 h postinfection and then repressed at 8 h postinfection, thus reproducing the two-step kinetics characteristic of a wild-type infection. Synthesis of beta-tubulin nuclear RNA was also transiently induced two- to six-fold, rising and falling in a manner similar to E3 transcription. An increase in helper virus multiplicity gave an increase in beta-tubulin stimulation, but dl312 alone, even at a high multiplicity of infection, gave no induction, confirming the requirement for E1a. beta-Actin nuclear RNA was actively synthesized before infection, but it was not further stimulated by the virus. Cellular beta-globin gene transcription was not stimulated by the virus, although transcription of a transfected beta-globin plasmid was induced by the virus or from a cotransfected E1a expression plasmid. We conclude that adenovirus 5 can stimulate beta-tubulin gene transcription. We discuss the significance for the viral life cycle of viral stimulation of cell genes and consider possible mechanisms in the light of the results obtained with beta-actin and beta-tubulin.
Alternative splicing of a Drosophila tropomyosin gene generates muscle tropomyosin isoforms with different carboxy-terminal ends.
Authors: Basi G S , Boardman M , Storti R V
Published: September 2015

The muscle tropomyosin I (mTm I) gene from Drosophila melanogaster has been analyzed and shown to express a complex transcription unit consisting of two sets of tissue-specific mRNAs. A 1.3- and 1.6-kilobase set of mRNAs is expressed during myogenesis in embryos, and in myogenic cell cultures. The mRNAs encode a 34,000-dalton muscle tropomyosin isoform. The same mTm I gene expresses a different set of 1.7- and 1.9-kilobase mRNAs in thoracic flight muscle tissue of the adult. The thorax RNAs encode a new tropomyosin isoform resolved on two-dimensional gels. The structure of the gene has been determined, and we show that the embryonic and thoracic mRNAs are generated by alternative splicing. The alternate exon splicing patterns determine a different 27 amino acids at the carboxy-terminal end of the two tropomyosin isoforms. These results show that the carboxy-terminal domain of tropomyosin is highly regulated in determining tropomyosin function. The results also show that contractile protein isoforms can be generated by single as well as multiple genes.
Cell cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae.
Authors: Storms R K , Ord R W , Greenwood M T , Mirdamadi B , Chu F K , Belfort M
Published: September 2015

Synchronous populations of Saccharomyces cerevisiae cells, generated by two independent methods, have been used to show that thymidylate synthase, in contrast to the vast majority of cellular proteins thus far examined, fluctuates periodically during the S. cerevisiae cell cycle. The enzyme, as assayed by two different methods, accumulated during S period and peaked in mid to late S phase, and then its level dropped. These observations suggest that both periodic synthesis and the instability of the enzyme contribute to the activity profile seen during the cell cycle. Accumulation of thymidylate synthase is determined at the level of its transcript, with synthase-specific mRNA levels increasing at least 10-fold to peak near the beginning of S period and then falling dramatically to basal levels after the onset of DNA synthesis. This mRNA peak coincided with the time during the cell cycle when thymidylate synthase levels were increasing maximally and immediately preceded the peak of DNA synthesis, for which the enzyme provides precursor dTMP.
Nuclear envelope localization of an adenovirus tumor antigen maintains the integrity of cellular DNA.
Authors: White E , Blose S H , Stillman B W
Published: September 2015

The adenovirus early-region 1B 19,000-molecular-weight tumor antigen is required for oncogenic transformation of cells by adenovirus. We have demonstrated that this tumor antigen is located in the nuclear envelope of infected and transformed cells and that a fraction of the protein within the nuclear envelope is associated with the nuclear lamina. During cell division in the transformed cells, the nuclear envelope containing the tumor antigen dissociates at metaphase and then reforms around the separated daughter chromosomes at telophase. Adenovirus mutants carrying lesions in the gene encoding this tumor antigen cause degradation of host cell chromosomal DNA, and in these mutants, the intracellular localization of the 19,000-dalton protein is altered. These results demonstrate that components of the nuclear envelope function in the organization of chromatin in infected and transformed cells and that a virus-encoded protein plays a critical role in this process.
Correct temperature induction and developmental regulation of a cloned heat shock gene transformed into the Drosophila germ line.
Authors: Hoffman E P , Corces V G
Published: September 2015

We have constructed a size variant of the Drosophila hsp28 gene by deleting 207 base pairs of the protein coding region, beginning 33 base pairs downstream of the ATG protein initiation codon. After transformation of Drosophila melanogaster rosy (ry506) flies with this altered gene, using the P transposable element system, it was found that the transformed gene was regulated correctly both after temperature elevation and during the development of the flies. Levels of the variant mRNA were as high as those of the endogenous hsp28 during all patterns of expression, and the variant mRNA appeared in all cases to be processed correctly and to be as stable as the endogenous mRNA. Nevertheless, the chromosomal locus of the transformed gene did not puff after heat shock, suggesting that normal transcription of the gene does not require puffing of the locus. The deleted hsp28 gene retained the reading frame of the endogenous one, and a protein of the expected molecular weight of 18,500 was made after heat shock at levels comparable to those of the endogenous hsp28.
Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.
Authors: Mercer W E , Avignolo C , Baserga R
Published: September 2015

Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.
Saccharomyces cerevisiae RAD2 gene: isolation, subcloning, and partial characterization.
Authors: Naumovski L , Friedberg E C
Published: September 2015

A plasmid (pNF2000) containing a 9.7-kilobase pair DNA insert that complements the UV sensitivity of rad2-1, rad2-2, and rad2-4 mutants of Saccharomyces cerevisiae has been isolated from a yeast genomic library. Genetic analysis of strains derived by transformation of rad2 mutants with an integrating plasmid containing a 9.3-kilobase pair fragment from pNF2000 shows that the fragment integrates exclusively at the chromosomal rad2 gene. We therefore conclude that this plasmid contains the RAD2 gene. The 9.3-kilobase pair fragment was partially digested with Sau3A and cloned into a multicopy yeast vector designed for easy retrieval of Sau3A inserts. The smallest subclone that retains the RAD2 gene is 4.5 kilobase pairs. This fragment was partially digested with Sau3A and cloned into an integrating plasmid. These plasmids were isolated and integrated into a heterozygous rad2/RAD2 strain. Plasmids containing internal fragments of the RAD2 gene were identified because they yielded UV-sensitive transformants due to disruption of the RAD2 gene. Sporulation of diploids transformed with integrating plasmids containing internal fragments of RAD2 gave rise to four viable haploids per tetrad, indicating that unlike the RAD3 gene of S. cerevisiae, the RAD2 gene is not essential for the viability of haploid cells under normal growth conditions. Measurements of the RNA transcript by RNA-DNA hybridization with the internal fragment as the probe indicate a size of approximately 3.2 kilobases.
Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.
Authors: Storrie B , Sachdeva M , Viers V S
Published: September 2015

We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.
Genetic mapping of Ty elements in Saccharomyces cerevisiae.
Authors: Klein H L , Petes T D
Published: September 2015

We used transformation to insert a selectable marker at various sites in the Saccharomyces cerevisiae genome occupied by the transposable element Ty. The vector CV9 contains the LEU2+ gene and a portion of the repeated element Ty1-17. Transformation with this plasmid resulted in integration of the vector via a reciprocal exchange using homology at the LEU2 locus or at the various Ty elements that are dispersed throughout the S. cerevisiae genome. These transformants were used to map genetically sites of several Ty elements. The 24 transformants recovered at Ty sites define 19 distinct loci. Seven of these were placed on the genetic map. Two classes of Ty elements were identified in these experiments: a Ty1-17 class and Ty elements different from Ty1-17. Statistical analysis of the number of transformants at each class of Ty elements shows that there is preferential integration of the CV9 plasmid into the Ty1-17 class.
Isolation of the Drosophila melanogaster dunce chromosomal region and recombinational mapping of dunce sequences with restriction site polymorphisms as genetic markers.
Authors: Davis R L , Davidson N
Published: September 2015

Using the method of chromosomal walking, we have isolated a contiguous region of the Drosophila melanogaster X chromosome which corresponds to salivary gland chromosome bands 3C12 to 3D4. This five-band region contains approximately 100 kilobases of DNA, including those sequences comprising dunce, a gene which functions in memory and cyclic nucleotide metabolism. Genome blots of DNA from flies carrying several different chromosomal aberrations with breakpoints in the region have been probed with the isolated clones to map the breakpoints on the cloned DNA and to delimit dunce sequences. This has localized dunce to a 50-kilobase region. In addition, we have searched this 50-kilobase region for restriction site polymorphisms between X chromosomes from different Drosophila strains by genome blotting experiments, and we have followed the segregation of detected polymorphisms and dunce alleles after meiotic recombination. The data map one dunce mutation between two polymorphisms located 10 to 12 kilobases apart.
Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector.
Authors: Pennock G D , Shoemaker C , Miller L K
Published: September 2015

The N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), has been fused to DNA encoding Escherichia coli beta-galactosidase. The fused gene was inserted into the AcNPV DNA genome by cotransfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA. Recombinant viruses were selected as blue plaques in the presence of a beta-galactosidase indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Studies of one such virus, L1GP-gal3, indicated that the synthesis of beta-galactosidase is temporally controlled beginning late (20 h) in infection after the release of infectious virus particles from the cell. By 48 h postinfection, a remarkably high level of expression is achieved. On the basis of these results, AcNPV should be a useful vector for the stable propagation and expression of passenger genes in a lepidopteran cell background. A generalized transplacement vector that facilitates the construction and selection of recombinant viruses carrying passenger genes under their own promoter control has also been developed.