bluuebunny/crossref_metadata_2025 huggingface dataset:
RNA turnover in Trypanosoma brucei.
Authors: Ehlers B , Czichos J , Overath P
Published: September 2015

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procyclic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27 degrees C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, beta-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of beta-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (t1/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t1/2, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37 degrees Celsius caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while beta-tubulin mRNA was stabilized 2- to 3-fold and mRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.
Kinds of mutations formed when a shuttle vector containing adducts of benzo[a]pyrene-7,8-diol-9,10-epoxide replicates in COS7 cells.
Authors: Yang J L , Maher V M , McCormick J J
Published: September 2015

We have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of the (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) replicates in the monkey kidney cell line COS7. The target for detecting mutations was the 200-base pair gene for a tyrosine suppressor tRNA (supF), inserted at the EcoRI site in shuttle vector p3AC (Sarkar et al., Mol. Cell. Biol. 4:2227-2230, 1984). When introduced by transformation, a functioning supF gene in progeny plasmid recovered from COS7 cells allows suppression of a lacZ amber mutation in the indicator Escherichia coli host. Treatment of p3AC with BPDE caused a linear increase in the number of BPDE residues bound per plasmid. Untreated plasmids and plasmids containing 6.6 BPDE residues were transfected into COS7 cells, and the progeny were assayed for mutations in the supF gene. The frequency of mutants generated during replication of the BPDE-treated plasmids was not higher than that from untreated plasmids, but the two populations differed markedly in the kinds of mutations they contained. Gel electrophoresis analysis of the size alterations of 77 mutant plasmids obtained with untreated DNA and 45 obtained with BPDE-treated DNA showed that the majority of the mutant progeny of untreated plasmids exhibited gross alterations, principally large deletions. In contrast, the majority of the mutants generated during replication of the BPDE-treated plasmids contained only minor alterations, principally point mutations. Sequence analysis of progeny of untreated plasmids containing putative point mutations showed insertions and deletions of bases and a broad spectrum of base substitutions; in those from BPDE-treated plasmids, all base substitutions involved guanosine . cystosine pairs.
Episomal maintenance of a bovine papilloma virus vector in transgenic mice.
Authors: Elbrecht A , DeMayo F J , Tsai M J , O'Malley B W
Published: September 2015

We have used a bovine papillomavirus-based vector to generate transgenic mice. Transgenic mice result from either pronuclear or cytoplasmic injections of the vector into fertilized eggs. Of 30 mice generated by microinjection, 27 (90%) contained the vector in its episomal form, at less than one copy per cell. This represents a highly efficient means of gene transfer in which the transgene is in a controlled genetic environment.
Target sequences for cis-acting regulation within the dual promoter of the human c-myc gene.
Authors: Lipp M , Schilling R , Wiest S , Laux G , Bornkamm G W
Published: September 2015

Recombinant plasmids of the human c-myc promoter-leader region and the bacterial chloramphenicol acetyltransferase (cat) gene were constructed. After transfection into different rodent and human cells, the 862-base-pair (bp) PvuII fragment carrying both c-myc promoters and 350 bp of the untranslated leader conferred 1/15 to 1/30 of the CAT activity mediated by the simian virus 40 promoter. The presence of additional sequences upstream of the PvuII fragment had an overall negative effect on c-myc promoter activity detectable by titration analysis with small amounts of transfected plasmid DNA. The analysis of numerous deletion constructs in the c-myc promoter-leader region as well as S1 mapping experiments demonstrated that the high CAT activity depended largely on the presence of the second promoter. By cotransfection of c-myc-cat constructs with plasmids carrying different parts of the c-myc promoter locus, targets for positively acting cellular factors were identified. Two positive regulatory elements were mapped within the 862-bp PvuII fragment. One was localized within the 248-bp PvuII-SmaI fragment -101 to -349 bp upstream of the first cap site and the other within the 142-pb XhoI-NaeI fragment of the first exon, comprising positions -95 to +47 relative to the second cap site. We conclude that the dual promotor of the human c-myc gene represents a strong eucaryotic promotor regulated by cooperation of positively and negatively acting cellular transcription factors.
Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification.
Authors: Meinkoth J , Killary A M , Fournier R E , Wahl G M
Published: September 2015

We analyzed the amplification of the CAD gene in independently isolated N-(phosphonacetyl)-L-aspartate-resistant clones derived from single parental clones in two mouse cell lines. We report for the first time that the CAD gene is amplified unstably in mouse cells, that the degree of instability varies greatly between clones, and that minute chromosomes and highly unstable chromosomelike structures contain the amplified sequences. These data are most consistent with the idea that the amplified unit in each clone consists of different flanking DNA and that such differences engender amplified sequences with unequal stability. We also introduced the mouse chromosome containing the CAD gene into hamster cells by microcell-mediated chromosome transfer to determine whether the propensity for unstable extrachromosomal amplification of the mouse CAD gene would prevail in the hamster cell nuclear environment. We report that the mouse CAD gene was amplified stably in expanded chromosomal regions in each of seven hybrids that were analyzed. This observation is consistent with the idea that the nuclear environment influences whether mutants containing intra- or extrachromosomally amplified sequences will be isolated.
Expression of the c-myc oncogene under control of an immunoglobulin enhancer in E mu-myc transgenic mice.
Authors: Alexander W S , Schrader J W , Adams J M
Published: September 2015

Transgenic mice bearing a cellular myc oncogene coupled to the immunoglobulin heavy-chain enhancer (E mu) exhibit perturbed B-lymphocyte development and succumb to B lymphoid tumors. To investigate how the enhancer has affected myc expression, we analyzed the structure and abundance of myc transcripts in tissues of prelymphomatous mice and in the lymphomas. Expression of the E mu-myc transgene appeared to be confined largely to B lymphoid cells, being dominant in bone marrow, spleen, and lymph nodes, with no detectable expression in T cells or other hematopoietic lineages examined. The myc transcripts initiated very predominantly at the normal myc promoters, although use of the more upstream myc promoter was accentuated and an enhancer-associated promoter may be used infrequently. The level of E mu-myc transcripts in the preneoplastic lymphoid tissues and in the E mu-myc tumors was not markedly higher than myc RNA levels in proliferating normal lymphocytes. Thus, enforced expression of structurally normal myc transcripts at only a modestly elevated level has profound biological consequences. The absence of detectable endogenous c-myc RNA in any tumor, or in preneoplastic bone marrow, supports a negative feedback model for normal c-myc regulation.
DNA sequence analysis of spontaneous mutations at the aprt locus of hamster cells.
Authors: Nalbantoglu J , Phear G , Meuth M
Published: September 2015

To determine the nature of spontaneous mutational events in cellular genes in hamster cells, mutant adenine phosphoribosyltransferase (aprt) genes were cloned and the regions to which we mapped alterations were sequenced. A variety of nucleotide changes were found to occur in the 12 mutant genes analyzed. Most mutations were simple base-pair substitutions-transitions (both G X C----A X T and A X T----G X C) and transversions. The only multiple mutation was a simple transition next to a single-base-pair insertion. Of the 12 mutations, 4 were more complex, involving small deletions or duplications. Two of these were similar to previously described deletions in that they occurred between short direct sequence repeats. No hot spots were detected. Three independent mutations were characterized at one restriction endonuclease site, although no other mutations were detected in the nucleotides surrounding this site in other mutant strains. At a functional level, sequence changes were either in exons (resulting in missense and, in one instance, nonsense mutations) or at splicing sites.
Signal processing, glycosylation, and secretion of mutant hemagglutinins of a human influenza virus by Saccharomyces cerevisiae.
Authors: Abdul Jabbar M , Nayak D P
Published: September 2015

We investigated the nature of signal recognition, transport, and secretion of mutant hemagglutinins (HAs) of a human influenza virus by the yeast Saccharomyces cerevisiae. The cDNA sequences encoding variant forms of influenza HA were expressed in S. cerevisiae. The HA polypeptides (HA500 and HA325) that were synthesized with their N-terminal signal peptides were correctly targeted to the membrane compartment where they were glycosylated. In contrast, the HA polypeptides (HA484 and HA308) lacking the signal peptide were expressed in the cytoplasm and did not undergo any glycosidic modification, demonstrating the importance of the heterologous signal sequence in the early steps of translocation in S. cerevisiae. The analysis of the N-terminal amino acid sequence of HA500 and HA325 polypeptides demonstrated the correct cleavage of the signal peptide, indicating the structural compatibility of a heterologous signal peptide for efficient recognition and processing by the yeast translocation machinery. The membrane-sequestered and glycosylated HA polypeptides were relatively stable in S. cerevisiae compared with the signal-minus, nonglycosylated HA molecules. Although both the anchor-minus HA (HA500) and HA1 (HA325) polypeptides were targeted efficiently to the membrane, their glycosylation and transport patterns were shown to be different. During pulse-chase, the HA500 remained cell-associated with no detectable secretion into the extracellular medium, whereas the HA325 secreted into the medium. Furthermore, only the cell-associated and secreted forms of HA325 and not HA500 appeared to have undergone hyperglycosylation with the extensive addition of high-molecular-weight outer-chain mannans. Possible reasons for the observed phenotypic behavior of these two mutant HAs are discussed.
Glucocorticoid-regulated compartmentalization of cell surface-associated glycoproteins in rat hepatoma cells: evidence for an independent response that requires receptor function and de novo RNA synthesis.
Authors: Haffar O K , Vallerga A K , Marenda S A , Witchel H J , Firestone G L
Published: September 2015

The role of glucocorticoid hormones in the compartmentalization of cell surface-associated mouse mammary tumor virus (MMTV) glycoproteins was examined in M1.54, a cloned line of MMTV-infected rat hepatoma tissue culture cells. The expression of cellular [2-3H]mannose-labeled and cell surface 125I-labeled MMTV glycoproteins was examined throughout a time course of exposure to dexamethasone, a synthetic glucocorticoid. Posttranslational localization of cell surface MMTV glycoproteins required 6 h of exposure to hormone and occurred approximately 4 h after their initial production in an intracellular fraction. This regulated localization to the cell surface correlated with glucocorticoid receptor occupancy and was inhibited by exposure to RU 38486, a powerful antagonist of glucocorticoid-mediated responses. Cell surface immunoprecipitation demonstrated that actinomycin D, an inhibitor of de novo RNA synthesis, prevented regulated expression of cell surface viral glycoproteins, suggesting that newly synthesized cellular components mediate this process. The localization of cell surface MMTV glycoproteins appeared normal in a transcriptional variant (CR1) that produces basal levels of MMTV RNA and glycoprotein precursors in the presence of dexamethasone. Thus, regulated compartmentalization of viral glycoproteins is not an obligate consequence of a critical precursor concentration. Taken together, our results suggest that posttranslational trafficking of cell surface-destined MMTV glycoproteins resulted from an independent glucocorticoid hormone response that required receptor function and de novo RNA synthesis.
Functional simian virus 40 T antigen is expressed in hybrid cells having finite proliferative potential.
Authors: Pereira-Smith O M , Smith J R
Published: September 2015

Simian virus 40-transformed human cells fused with other independently derived simian virus 40-transformed cells and tumor-derived cells containing activated H-ras and N-ras oncogenes yielded hybrids capable of indefinite division. Fusions with various other immortal cells yielded hybrids that had limited division potential. T antigen expressed in limited-division hybrids was functional for the induction of cellular DNA synthesis.
Restriction of P-element insertions at the Notch locus of Drosophila melanogaster.
Authors: Kelley M R , Kidd S , Berg R L , Young M W
Published: September 2015

P elements move about the Drosophila melanogaster genome in a nonrandom fashion, preferring some chromosomal targets for insertion over others (J. C. J. Eeken, F. H. Sobels, V. Hyland, and A. P. Schalet, Mutat. Res. 150:261-275, 1985; W. R. Engels, Annu. Rev. Genet. 17:315-344, 1983; M. D. Golubovsky, Y. N. Ivanov, and M. M. Green, Proc. Natl. Acad. Sci. USA 74:2973-2975, 1977; M. J. Simmons and J. K. Lim, Proc. Natl. Acad. Sci. USA 77:6042-6046, 1980). Some of this specificity may be due to recognition of a particular DNA sequence in the target DNA; derivatives of an 8-base-pair consensus sequence are occupied by these transposable elements at many different chromosomal locations (K. O'Hare and G. M. Rubin, Cell 34:25-36, 1983). An additional level of specificity of P-element insertions is described in this paper. Of 14 mutations induced in the complex locus Notch by hybrid dysgenesis, 13 involved P-element insertions at or near the transcription start site of the gene. This clustering was not seen in other transposable element-induced mutations of Notch. DNA sequences homologous to the previously described consensus target for P-element insertion are not preferentially located in this region of the locus. The choice of a chromosomal site for integration appears to be based on more subtle variations in chromosome structure that are probably associated with activation or expression of the target gene.
Context-dependent gene expression: cis-acting negative effects of specific procaryotic plasmid sequences on eucaryotic genes.
Authors: Peterson D O , Beifuss K K , Morley K L
Published: September 2015

A sequence element within pBR322 DNA mediates a cis-acting negative effect on expression from eucaryotic genes in transient expression assays. The negative element overlaps with sequences that inhibit DNA replication, but its effect is observed in the absence of detectable replication of transfected DNA.
Expression and characterization of transforming growth factor alpha precursor protein in transfected mammalian cells.
Authors: Gentry L E , Twardzik D R , Lim G J , Ranchalis J E , Lee D C
Published: September 2015

Analysis of a cDNA clone derived from retrovirus-transformed rat fibroblasts has recently suggested that the mature 50-amino-acid form of transforming growth factor alpha (TGF alpha) is derived from a 159-amino-acid transmembrane precursor by proteolytic cleavage. To understand the processing of the TGF alpha precursor molecule in more detail, we have expressed this protein in baby hamster kidney (BHK) fibroblasts under control of the metal-ion-inducible metallothionein promoter and characterized the expressed precursor with site-specific antipeptide antibodies. One of the BHK transfectants, termed 5:2, expressed the TGF alpha mRNA in a cadmium- and zinc-inducible manner. The TGF alpha precursor protein was detected by immunoprecipitation analysis of radiolabeled cell cultures. In the induced 5:2 cells, a polypeptide of Mr 13,000 to 17,000 was readily identified by peptide antisera made to three different regions of the TGF alpha precursor protein. No such protein species were observed in BHK cells treated with cadmium and zinc or in uninduced 5:2 cells. However, two cell lines known to produce TGF alpha naturally, Leydig testicular tumor cells and Snyder-Theilan feline sarcoma virus-transformed Fisher rat embryo fibroblasts, possessed detectable levels of immunologically related Mr 13,000 to 17,000 proteins. Cell fractionation studies indicate that the Mr 13,000 to 17,000 species expressed in induced 5:2 cells is membrane associated, consistent with predictions based on the cDNA sequence of the TGF alpha precursor. Media conditioned by induced 5:2 cells contained epidermal growth factor receptor-competing activity, which, upon size fractionation, was similar in size to the mature processed form of TGF alpha. These data show that these nontransformed BHK cells possess the ability to process the TGF alpha precursor molecule into its native form.
Repair of single-stranded DNA nicks, gaps, and loops in mammalian cells.
Authors: Ayares D , Ganea D , Chekuri L , Campbell C R , Kucherlapati R
Published: September 2015

We studied the ability of mammalian cells to repair single-stranded nicks, gaps, and loops in DNA duplexes. Heteroduplexes prepared from derivatives of the shuttle vector pSV2neo were introduced into monkey COS cells. After replication, the plasmids were recovered and used to transform Escherichia coli. Plasmid DNA from the recovered colonies was tested for repair at each of six different sites. We observed that mammalian cells are capable of repairing single-stranded gaps and free single-stranded ends most efficiently. Regions containing twin loops were recognized, and one of the loops was excised. Portions of the molecules containing small single loops were also repaired. Markers which were 58 nucleotides apart were corepaired with nearly 100% efficiency, while markers which were 1,000 nucleotides or more apart were never corepaired. The mechanisms involved in heteroduplex repair in mammalian cells seem to be similar to those involved in repairing DNA lesions caused by physical and chemical agents.
Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin.
Authors: Carroll S M , Gaudray P , De Rose M L , Emery J F , Meinkoth J L , Nakkim E , Subler M , Von Hoff D D , Wahl G M
Published: September 2015

In a previous study (G. M. Wahl, B. Robert de Saint Vincent, and M. L. De Rose, Nature (London) 307:516-520, 1984), we used gene transfer of a CAD cosmid to demonstrate that gene position profoundly affects amplification frequency. One transformant, T5, amplified the donated CAD genes at a frequency at least 100-fold higher than did the other transformants analyzed. The CAD genes in T5 and two drug-resistant derivatives were chromosomally located. In this report, we show that a subclone of T5 gives rise to an extrachromosomal molecule (CAD episome) containing the donated CAD genes. Gel electrophoresis indicated that the CAD episome is approximately 250 to 300 kilobase pairs, and a variety of methods showed that it is a covalently closed circle. We show that the CAD episome replicates semiconservatively and approximately once per cell cycle. Since the CAD cosmid, which comprises most of the CAD episome, does not replicate autonomously when transfected into cells, our results indicate that either the process which generated the episome resulted in a cellular origin of DNA replication being linked to the CAD sequences or specific rearrangements within the episome generated a functional origin. The implications of these results for mechanisms of gene amplification and the genesis of minute chromosomes are discussed.
Naturally occurring methylation inhibitor: DNA hypomethylation and hemoglobin synthesis in human K562 cells.
Authors: Lyon S B , Buonocore L , Miller M
Published: September 2015

A naturally occurring methylation inhibitor isolated from rabbit liver and named methinin inhibits a number of methyltransferases. Methinin is a low-molecular-weight compound (1,400) that has an active amine group. This compound inhibits the DNA methyltransferase of human erythroleukemia cells (K562) in vitro. When the K562 cells were grown in medium containing methinin, fetal hemoglobin was produced. Small but detectable amounts of adult hemoglobin were also produced. Methinin was not toxic to these cells. The overall rate of genomic DNA methylation was reduced by 60% in cells grown in medium containing methinin. Southern blots of genomic DNA from methinin-treated cells and untreated cells hybridized to a 32P-labeled globin gene probe showed that one site in the globin gene region was hypomethylated. Methinin is a naturally occurring compound which inhibits DNA methylation both in vitro and in vivo.
Molecular cloning and characterization of an activated human c-raf-1 gene.
Authors: Fukui M , Yamamoto T , Kawai S , Mitsunobu F , Toyoshima K
Published: September 2015

Results of previous studies have shown that a raf-related transforming DNA sequence is present in NIH 3T3 transformants that are derived from GL-5-JCK human glioblastoma DNA transfection. The transforming DNA was molecularly cloned by using cosmid vector pJB8 to determine its structure and origin. Analyses of selected clones revealed that the transforming DNA consisted of three portions of human DNA sequences, with the 3' half of the c-raf-1 gene as its middle portion. This raf region was about 20 kilobases long and contained exons 8 to 17 and the poly(A) addition site. RNA blot analysis showed that the raf-related transforming DNA was transcribed into 5.3-, 4.8-, and 2.5-kilobase mRNAs; the 2.5-kilobase transcript was thought to be the major transcript. Immunoprecipitation analyses revealed that a 44-kilodalton raf-related protein was specifically expressed in the NIH 3T3 transformants. The raf-related transforming DNA was considered to be activated when its amino-terminal sequence was truncated and the DNA was coupled with a foreign promoter sequence. On hybridization analysis of the original GL-5-JCK glioblastoma DNA, no rearrangement of c-raf-1 was detectable in the tumor DNA. The rearrangement of c-raf-1 may have occurred during transfection or may have been present in a small population of the original tumor cells as a result of tumor progression.
Identification and characterization of cDNA clones encoding two homologous proteins that are part of the asialoglycoprotein receptor.
Authors: McPhaul M , Berg P
Published: September 2015

The asialoglycoprotein receptor (ASGP-R) from rat liver contains the following three distinct protein species when it is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: RHL1 (42 kilodaltons), RHL2 (49 kilodaltons), and RHL3 (54 kilodaltons). In this paper we describe the isolation of cDNA clones encoding RHL1 and RHL2 from a cDNA library constructed from rat liver mRNA. A comparison of the predicted coding sequence for RHL2 with that for RHL1 showed that these sequences are highly homologous. The library also contained numerous cDNA clones for both RHL1 and RHL2 that were derived from unspliced precursor mRNAs. Differential splicing at the 5' end of the RHL1 transcript was inferred from the finding that two different types of RHL1 cDNA were identified, each having a different 5' terminus.
Conservation of promoter sequence but not complex intron splicing pattern in human and hamster genes for 3-hydroxy-3-methylglutaryl coenzyme A reductase.
Authors: Luskey K L
Published: September 2015

Regulation of the expression of 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase is a critical step in controlling cholesterol synthesis. Previous studies in cultured Chinese hamster ovary cells have shown that HMG-CoA reductase is transcribed from a cholesterol-regulated promoter to yield a heterogeneous collection of mRNAs with 5' untranslated regions of 68 to 670 nucleotides in length. Synthesis of these molecules is initiated at multiple sites, and multiple donor sites are used to excise an intron in the 5' untranslated region. In the current paper, I report that human HMG-CoA reductase gene resembles the Chinese hamster gene in having multiple sites of transcription initiation that are subject to suppression by cholesterol. The human gene differs from the hamster gene in that a single donor splice site is used to excise the intron in the 5' untranslated region. All of the resulting RNAs have short 5' untranslated regions of 68 to 100 nucleotides. This difference in the splicing pattern of the first intron is species specific and not a peculiarity of cultured cells in that HMG-CoA reductase mRNAs from Syrian hamster livers resemble those of the cultured Chinese hamster ovary cells. Comparison of the DNA sequences of the HMG-CoA reductase promoters from three different species--humans, Syrian hamsters, and Chinese hamsters--shows a highly conserved region of 179 nucleotides that extends from 220 to 42 nucleotides upstream of the transcription initiation sites. This region is 88% identical between the human and Chinese hamster promoter. When fused to the coding region of the Escherichia coli chloramphenicol acetyltransferase gene, this highly conserved region of the reductase gene directs the cholesterol-regulated expression of chloramphenicol acetyltransferase in transfected hamster cells, further indicating the interspecies conservation of the regulatory elements.
A 12-base-pair sequence is an essential element of the ribosomal gene terminator in Xenopus laevis.
Authors: Labhart P , Reeder R H
Published: September 2015

rRNA transcription in Xenopus laevis terminates near a 7-base-pair (bp) conserved sequence (T3 box) located 200 bp upstream of the site of transcription initiation for the adjacent gene promoter. We present evidence here that a 12-bp element containing the T3 box is an essential part of the terminator. Using an oocyte injection assay, we found that the 12-bp element (but not the T3 box alone) severely reduced the amount of RNA detectable at sites downstream from itself and that the T3 box within the 12-bp element was required to specify the formation of correct 3' ends. This requirement for the 12-bp element was also seen in pulse-label experiments by using a homogenate of oocyte nuclei, but the present data did not allow us to determine the exact mechanism by which the 12-bp element acts. Removal of the T3 region from its normal location allowed a significant amount of readthrough transcripts to accumulate, indicating that additional sequences may be required for complete terminator function.
Distinct replication-independent and -dependent phases of histone gene expression during the Physarum cell cycle.
Authors: Carrino J J , Kueng V , Braun R , Laffler T G
Published: September 2015

During the S phase of the cell cycle, histone gene expression and DNA replication are tightly coupled. In mitotically synchronous plasmodia of the myxomycete Physarum polycephalum, which has no G1 phase, histone mRNA synthesis begins in mid-G2 phase. Although histone gene transcription is activated in the absence of significant DNA synthesis, our data demonstrate that histone gene expression became tightly coupled to DNA replication once the S phase began. There was a transition from the replication-independent phase to the replication-dependent phase of histone gene expression. During the first phase, histone mRNA synthesis appears to be under direct cell cycle control; it was not coupled to DNA replication. This allowed a pool of histone mRNA to accumulate in late G2 phase, in anticipation of future demand. The second phase began at the end of mitosis, when the S phase began, and expression became homeostatically coupled to DNA replication. This homeostatic control required continuing protein synthesis, since cycloheximide uncoupled transcription from DNA synthesis. Nuclear run-on assays suggest that in P. polycephalum this coupling occurs at the level of transcription. While histone gene transcription appears to be directly switched on in mid-G2 phase and off at the end of the S phase by cell cycle regulators, only during the S phase was the level of transcription balanced with the rate of DNA synthesis.
DNA sequences homologous to the Drosophila opa repeat are present in murine mRNAs that are differentially expressed in fetuses and adult tissues.
Authors: Duboule D , Haenlin M , Galliot B , Mohier E
Published: September 2015

A mouse embryonic cDNA containing two opa-like (CAX)n repeats was isolated on the basis of its cross-hybridization with a Drosophila K10 cDNA. Such repeated sequences were present in different murine mRNAs, some of which were specifically expressed during fetal life or in different adult tissues. This suggests that, as already described for Drosophila, opa-like sequences are parts of proteins involved in ontogenic or cell-type-specific functions in vertebrates. However, unlike Drosophila, such repeated sequences were not found within the murine homeo-boxes containing genes of the Hox-1 complex.
Amplification of the neu (c-erbB-2) oncogene in human mammmary tumors is relatively frequent and is often accompanied by amplification of the linked c-erbA oncogene.
Authors: van de Vijver M , van de Bersselaar R , Devilee P , Cornelisse C , Peterse J , Nusse R
Published: September 2015

We investigated alterations in the structure and expression of oncogenes in mammary tumors and mammary tumor-derived cell lines. In 16 of 95 samples, we detected amplification of the human neu oncogene, also known as c-erB-2, accompanied by overexpression in the tumors from which intact RNA could be isolated. In 10 of these DNAs, the linked oncogene c-erbA was also amplified, whereas another gene on human chromosome 17, p53, was present in normal copy numbers. Overexpression of c-erbA could not be detected in the tumors analyzed. The relatively high frequency of neu amplification points to a functional role in human breast cancer. Coamplification of the c-erbA oncogene could contribute to this disease as well but is most likely fortuitous.
Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells.
Authors: Brash D E , Reddel R R , Quanrud M , Yang K , Farrell M P , Harris C C
Published: September 2015

Strontium ion formed DNA-phosphate precipitates analogous to those formed by calcium but lacking the lethal and differentiation-inducing effects of calcium on many epithelial cell types in primary culture. Human primary bronchial epithelial cells were transiently and stably transfected by using strontium phosphate; the frequency of stable transformation with a plasmid carrying the simian virus 40 large-T-antigen gene was greater than 10(-4).
Relatively stable population of c-myc RNA that lacks long poly(A).
Authors: Swartwout S G , Preisler H , Guan W D , Kinniburgh A J
Published: September 2015

We examined the turnover of c-myc RNA in the human promyelocytic cell line HL-60. In whole-cell RNA from rapidly growing cells we observed two major size classes of c-myc RNA, 2.4 and 2.2 kilobases (kb). When HL-60 cells were treated with actinomycin D for 30 min to inhibit transcription, the 2.4-kb c-myc RNA population was rapidly degraded, while the 2.2-kb c-myc RNA was degraded much more slowly. S1 nuclease transcript mapping and promoter-specific probes were utilized to show that both the stable 2.2-kb and the labile 2.4-kb c-myc RNA populations have 5' ends at the second promoter site (P2) and 3' ends at the second poly(A) addition site. To examine further possible structural differences between these two RNA populations, we fractionated RNA on an oligo(dT)-cellulose column to separate RNAs that contained long poly(A) tails from those which did not. We found that the labile 2.4-kb c-myc RNA population bound to oligo(dT)-cellulose, while the more stable 2.2-kb c-myc RNA population did not. Preliminary estimates of their half-lives (t1/2) showed that the poly(A)+ c-myc RNA had a t1/2 of 12 min, while the c-myc RNA that did not bind to oligo(dT)-cellulose had a t1/2 of greater than 1 h. Several other cell types contain both poly(A)+ and nonpoly(A)+ c-myc RNAs including HeLa cells, normal human bone marrow cells, and normal mouse fetal liver cells. In agreement with the results in HL-60 cell, HeLa cell poly(A)+ c-myc RNA was more labile than c-myc RNA that lacked poly(A). The stable, nonpoly(A)+ c-myc RNA population may be important in the posttranscriptional regulation of c-myc expression.
Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene.
Authors: Kemper B , Jackson P D , Felsenfeld G
Published: September 2015

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.
Control of proliferin gene expression in serum-stimulated mouse cells.
Authors: Linzer D I , Wilder E L
Published: September 2015

The serum-inducible expression of proliferin genes in BALB/c 3T3 cells was found to be dependent on both protein synthesis and an extended presence of serum in the medium. Even though no mature proliferin mRNA was detected in serum-starved cells, transcription of the proliferin genes occurred in these resting-cell cultures, indicating that posttranscriptional events may be important for regulating proliferin mRNA levels. These results suggest that protein synthesis after serum stimulation of quiescent mouse fibroblasts is required for posttranscriptional processing or stabilization of proliferin RNA. Proliferin RNA levels were found to be heterogeneous among serum-stimulated cells analyzed by in situ hybridization. This heterogeneity is probably due to asynchrony in the population and may point to a correlation between the time of proliferin expression and the time of entry of a cell into S phase.
Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes.
Authors: Giorda R , Ennis H L
Published: September 2015

A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.
Differential early viral gene expression in two stages of human papillomavirus type 16 DNA-induced malignant transformation.
Authors: Yasumoto S , Doniger J , DiPaolo J A
Published: September 2015

Human papillomavirus (HPV) type 16 DNA induces progressive transformation in NIH 3T3 cells. Two types of cell lines, PM3T3G0 and PM3T3Fo, were isolated by G418 or focus selection, respectively, after transfection of cells by a recombinant HPV 16 DNA carrying the neo gene. These cell lines exhibited distinct phenotypes compared with controls. Saturation densities of PM3T3G0 and PM3T3Fo lines were two- to three- and five- to sevenfold greater than that of control NIH 3T3 cells, respectively. Neither cell type required high serum for growth, in contrast to NIH 3T3 cells. PM3T3G0 lines were premalignant, whereas PM3T3Fo lines manifested tumorigenicity within 2 weeks. Subpopulations of three PM3T3G0 lines underwent progressive transformation as reflected by focus formation. Analysis of HPV 16-specific mRNA species demonstrated that high levels of early and late gene expression were detected in premalignant PM3T3G0 lines, whereas relatively low quantities of selected gene messages were expressed in malignant transformants. Thus, high levels of viral gene expression are not crucial for malignant transformation.
Flounder antifreeze protein synthesis under heat shock control in transgenic Drosophila melanogaster.
Authors: Rancourt D E , Walker V K , Davies P L
Published: September 2015

The gene coding for the most abundant antifreeze protein (AFP) in the winter flounder was placed downstream of the Drosophila melanogaster hsp70 promoter and introduced into the D. melanogaster germ line by P-element-mediated transformation. In each of six transgenic strains tested, heat shock treatment induced the expression of two major AFP gene transcripts and one minor one. All three transcripts were spliced despite the lack of an obvious D. melanogaster internal intron-splicing sequence. The variation in transcript length was caused by selection of different polyadenylation sites. Western blots showed the presence of immunoreactive AFP in hemolymph from heat-shocked transformants. The immunoreactive material had a molecular weight of 6,200, which is consistent with the loss of the signal sequence from the primary translation product and the retention of the pro sequence. Thus, all the signals for flounder pre-mRNA and preprotein processing were recognized in D. melanogaster.
Interferon-induced revertants of ras-transformed cells: resistance to transformation by specific oncogenes and retransformation by 5-azacytidine.
Authors: Samid D , Flessate D M , Friedman R M
Published: September 2015

Prolonged alpha/beta interferon (IFN-alpha/beta) treatment of NIH 3T3 cells transformed by a long terminal repeat-activated Ha-ras proto-oncogene resulted in revertants that maintained a nontransformed phenotype long after IFN treatment had been discontinued. Cloned persistent revertants (PRs) produced large amounts of the ras-encoded p21 and were refractile to transformation by EJras DNA and by transforming retroviruses which carried the v-Ha-ras, v-Ki-ras, v-abl, or v-fes oncogene. Transient treatment either in vitro or in vivo with cytidine analogs that alter gene expression by inhibiting DNA methylation resulted in transformation of PR, but not of NIH 3T3, cells. The PR retransformants reverted again with IFN, suggesting that DNA methylation is involved in IFN-induced persistent reversion.
Modification of fos proteins: phosphorylation of c-fos, but not v-fos, is stimulated by 12-tetradecanoyl-phorbol-13-acetate and serum.
Authors: Barber J R , Verma I M
Published: September 2015

We have investigated the covalent modification of the proteins encoded by the murine fos proto-oncogene (c-fos) and that of the corresponding gene product of FBJ murine osteosarcoma virus (v-fos). Both proteins are posttranslationally processed in the cell, resulting in forms with lower electrophoretic mobilities than that of the initial translation product on sodium dodecyl sulfate-polyacrylamide gels. Treatment with alkaline phosphatase indicates that most, if not all, of this electrophoretic shift is due to phosphoesterification of both proteins. These phosphoryl groups stoichiometrically modify the v-fos and c-fos proteins on serine residues and turn over rapidly in vivo in the presence of protein kinase inhibitors (half-life, less than 15 min). Direct quantitative comparison of steady-state labeling studies with L-[35S]methionine and [32P]phosphate reveals that the c-fos protein is four- to fivefold more highly phosphorylated than the v-fos protein is. Comparison of tryptic fragments from [32P]phosphate-labeled proteins indicates that although the two proteins have several tryptic phosphopeptides in common, the c-fos protein contains unique major tryptic phosphopeptides that the v-fos protein lacks. These unique sites of c-fos phosphorylation have been tentatively localized to the carboxy-terminal 20 amino acid residues of the protein. Phosphorylation of the c-fos protein, but not the v-fos protein, can be stimulated at least fivefold in vivo by the addition of either 12-tetradecanoyl-phorbol-13-acetate or serum. This increase in the steady-state degree of phosphorylation of c-fos appears to be independent of protein kinase C since phosphorylation is Ca2+ and diacylglycerol independent. The possible role of phosphorylation of these proteins in cellular transformation is discussed.
12-O-tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5'-flanking region.
Authors: Angel P , Baumann I , Stein B , Delius H , Rahmsdorf H J , Herrlich P
Published: September 2015

Genomic clones coding for human fibroblast collagenase were isolated. By constructing and transfecting mutants with 5' and 3' deletion mutations of the 5' control region of the gene into human or murine cells, we delimited a 32-base-pair sequence at positions -73 to -42 which is required for the induction of transcription by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. The DNA element behaves as a 12-O-tetradecanoyl-phorbol-13-acetate-inducible enhancer: it mediates the stimulation of transcription to the heterologous herpes simplex virus thymidine kinase promoter and acts in a position- and orientation-independent manner. Differences in enhancer efficiency in different cell lines are interpreted to indicate differences in the activity of a trans-acting factor.
Homologous recombination between coinjected DNA sequences peaks in early to mid-S phase.
Authors: Wong E A , Capecchi M R
Published: September 2015

We have examined the effect of cell cycle position on homologous recombination between plasmid molecules coinjected into synchronized rat fibroblasts. Recombination activity was found to be low in G1 and to rise 10- to 15-fold, peaking in early to mid-S phase.
Individual mouse VL30 elements transferred to rat cells by viral pseudotypes retain their responsiveness to activators of protein kinase C.
Authors: Rodland K D , Brown A M , Magun B E
Published: September 2015

By exploiting the retroviral characteristics of mouse VL30 elements, we transferred individual copies of VL30 to Rat-1 cells by infection. Analysis of clonal isolates containing single VL30 elements integrated into the Rat-1 genome indicates that responsiveness to activators of protein kinase C is an inherent property of at least some of the VL30 sequences.
Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.
Authors: Simon J R , Moore P D
Published: September 2015

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.
Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function.
Authors: Deschenes R J , Broach J R
Published: September 2015

Two proteins in the yeast Saccharomyces cerevisiae that are encoded by the genes RAS1 and RAS2 are structurally and functionally homologous to proteins of the mammalian ras oncogene family. We examined the role of fatty acylation in the maturation of yeast RAS2 protein by creating mutants in the putative palmitate addition site located at the carboxyl terminus of the protein. Two mutations, Cys-318 to an opal termination codon and Cys-319 to Ser-319, were created in vitro and substituted in the chromosome in place of the normal RAS2 allele. These changes resulted in a failure of RAS2 protein to be acylated with palmitate and a failure of RAS2 protein to be localized to a membrane fraction. The mutations yielded a Ras2- phenotype with respect to the ability of the resultant mutants to grow on nonfermentable carbon sources and to complement ras1- mutants. However, overexpression of the ras2Ser-319 product yielded a Ras+ phenotype without a corresponding association of the mutant protein with the membrane fraction. We conclude that the presence of a fatty acyl moiety is important for localizing RAS2 protein to the membrane where it is active but that the fatty acyl group is not an absolute requirement of RAS2 protein function.