bluuebunny/crossref_metadata_2025 huggingface dataset:
Tx1: a transposable element from Xenopus laevis with some unusual properties.
Authors: Garrett J E , Carroll D
Published: September 2015

A family of transposable genetic elements in the genome of the frog, Xenopus laevis, is described. They are designated Tx1. Transposability of the elements was deduced by characterization of a chromosomal locus which is polymorphic for the presence or absence of a Tx1 element. Nucleotide sequence analysis suggested that Tx1 elements show target site specificity, as they are inserted at the pentanucleotide TTTAA in all four cases that were examined. The elements appear to have 19-base-pair (bp) inverted terminal repeats, and they are flanked by 4-bp target duplications (TTAA), although the possibility that they do not create target site duplications is discussed. Tx1 elements have several unusual characteristics: the central portion of each element is comprised of a variable number of two types of 393-bp repeating units; the rightmost 1,000 bp of the element contains separate regions potentially capable of forming bends, left-handed Z-form DNA, and alternative stem-loop structures. Comparisons among single frogs suggest that germ line transposition is relatively infrequent and that variations in numbers of internal repeats accumulate quite slowly at any locus.
Multiple growth-associated nuclear proteins immunoprecipitated by antisera raised against human c-myc peptide antigens.
Authors: Persson H , Gray H E , Godeau F , Braunhut S , Bellvé A R
Published: September 2015

Different antisera raised against various regions of the human c-myc protein were used to identify four human c-myc proteins with apparent molecular masses in sodium dodecyl sulfate-polyacrylamide gels ranging from 64 to 68 kilodaltons (phosphoproteins pp64 and pp67 and nonphosphorylated proteins p65 and p68). pp64 and p65 were the major detectable c-myc proteins, and pp67 and p68 were minor but specific components of the immunoprecipitates. The c-myc proteins were all localized in the cell nucleus. Accumulation of [35S]methionine-labeled p65 was observed after pulse-labeling and chase, suggesting that the stable p65 c-myc protein is generated posttranslationally from short-lived precursors. pp64, pp67, and p68 possessed short half-lives and may therefore be precursors of the stable p65. Confirmation of the nuclear localization of the human c-myc proteins was obtained by immunofluorescent staining. The human c-myc proteins were revealed as a pattern of punctate nuclear staining with, particularly for p65, nucleolar enhancement that left an unstained annulus surrounding the nucleolus.
Localization of a novel v-erbB-related gene, c-erbB-2, on human chromosome 17 and its amplification in a gastric cancer cell line.
Authors: Fukushige S , Matsubara K , Yoshida M , Sasaki M , Suzuki T , Semba K , Toyoshima K , Yamamoto T
Published: September 2015

The c-erbB-2 gene is a v-erbB-related proto-oncogene which is distinct from the gene encoding the epidermal growth factor receptor. By using two independent methods, hybridization of both sorted chromosomes and metaphase spreads with cloned c-erbB-2 DNA, we mapped the c-erbB-2 locus on human chromosome 17 at q21, a specific breakpoint observed in a translocation associated with acute promyelocytic leukemia. Furthermore, we observed amplification and elevated expression of the c-erbB-2 gene in the MKN-7 gastric cancer cell line. These data suggest possible involvement of the c-erbB-2 gene in human cancer.
Regulation of immunoglobulin D synthesis in murine neonatal B lymphocytes.
Authors: Yuan D
Published: September 2015

We analyzed the regulatory basis for the lower expression of immunoglobulin D (IgD) in lymphocytes from neonatal mice of various ages. The results indicate that the relative transcriptional rate of RNA for delta chains is similar to adult levels even in cells which have not started to express IgD. These results suggest that very early after the initiation of mu gene transcription, a defined fraction of polymerases is programmed to progress through the termination site to the delta exons regardless of the developmental stage of the cell. Similar results were obtained from adult CBA/N mice whose spleens contain a large fraction of cells expressing low levels of IgD. On the other hand, the relative steady state level of mRNA in neonatal lymphocytes is approximately half of that in adults, suggesting that there may be differences in the processing or stability of the nascent transcript. In addition, measurements of the in vivo translation rate show that an inefficient delta polypeptide chain processing machinery in neonatal lymphocytes is also an important factor contributing to the reduced expression of IgD.
Differential elimination of rDNA genes in bobbed mutants of Drosophila melanogaster.
Authors: Terracol R , Prud'homme N
Published: September 2015

In Drosophila melanogaster, the multiply repeated genes encoding 18S and 28S rRNA are located on the X and Y chromosomes. A large percentage of these repeats are interrupted in the 28S region by insertions of two types. We compared the restriction patterns from a subcloned wild-type Oregon R strain to those of spontaneous and ethyl methanesulfonate-induced bobbed mutants. Bobbed mutations were found to be deficiencies that modified the organization of the rDNA locus. Genes without insertions were deleted about twice as often as genes with type I insertions. Type II insertion genes were not decreased in number, except in the mutant having the most bobbed phenotype. Reversion to wild type was associated with an increase in gene copy number, affecting exclusively genes without insertions. One hypothesis which explains these results is the partial clustering of genes by type. The initial deletion could then be due either to an unequal crossover or to loss of material without exchange. Some of our findings indicated that deletion may be associated with an amplification phenomenon, the magnitude of which would be dependent on the amount of clustering of specific gene types at the locus.
A specific DNA sequence controls termination of transcription in the gastrin gene.
Authors: Sato K , Ito R , Baek K H , Agarwal K
Published: September 2015

We located and characterized a downstream transcriptional regulatory element in the human gastrin gene by transferring the gastrin gene 3' fragment, from which the polyadenylation signal sequence was deleted, into the shuttle vector pSCAT10 at a site located immediately downstream from the chloramphenicol acetyltransferase (CAT) gene and upstream from the simian virus 40 polyadenylation region. Study of CAT RNA derived from the hybrid plasmids, indicated regulation of transcription on the gastrin gene fragment. Analysis of deletion mutants generated from the 5' region of the fragment by CAT assay and by S1 nuclease mapping of mRNAs indicated the possible involvement of an oligothymidylate-rich sequence in transcription regulation. Mapping of gastrin gene RNA 3' ends to the 5' side proximal to the oligothymidylate-rich sequence clearly demonstrated that this sequence is a transcriptional terminator element. This unique sequence, interspersed with one or two adenines, which also functions in an orientation-dependent manner, is located 192 nucleotides downstream from the gastrin gene polyadenylation site, and serves as a transcriptional termination signal.
Antipeptide antiserum identifies a widely distributed cellular tyrosine kinase related to but distinct from the c-fps/fes-encoded protein.
Authors: Feldman R A , Tam J P , Hanafusa H
Published: September 2015

We raised antibodies directed against a synthetic peptide representing an amino acid sequence of the conserved kinase domain of the transforming protein of Fujinami sarcoma virus (FSV) (P140). The antiserum obtained specifically recognized FSV-P140 and its cellular homolog and in addition, it recognized a new cellular protein of 94,000 daltons (NCP94) in avian and mammalian cells. NCP94 was found to be associated with a cyclic nucleotide-independent protein kinase activity that was specific for tyrosine residues. Although NCP94 and FSV-P140 share antigenic determinants, NCP94 is not a cellular homolog of FSV-P140: NCP94 and the previously identified c-fps/fes product were different in their tryptic fingerprints and in their tissue specificities. Thus, the function of NCP94 in normal cells is probably different than that of the c-fps/fes product. NCP94 was expressed in every tissue and cell line that was examined. In chickens, NCP94 levels were highest during embryonic development and NCP94 expression was high in gizzard, brain, and spleen throughout embryonic and adult life. The universal expression of NCP94 suggests that this protein may be involved in an essential function of normal cells. NCP94 may be a new cellular tyrosine kinase of the src gene family.
Transfer of cloned human class I major histocompatibility complex genes into HLA mutant human lymphoblastoid cells.
Authors: Shimizu Y , Koller B , Geraghty D , Orr H , Shaw S , Kavathas P , DeMars R
Published: September 2015

Three new kinds of recombinant DNA constructs were used to transfer cloned human class I HLA genes (A2 and B8) into unique HLA mutant lymphoblastoid cells: pHeBo(x): a class I gene, "x," in plasmid vector pHeBo, which contains a hygromycin resistance gene and Epstein-Barr virus oriP element that sustains extrachromosomal replication; pHPT(x): gene x in a vector with a hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene; pHPTe(x): gene x in a vector with the HPRT gene and oriP element. Cell surface class I antigen expression was strong in transferents made with class I-deficient lymphoblastoid cell line mutants .144 (A-null), .53 (B-null), and .184 (A-null, B-null). Transferents expressing HLA-A2 were recognized specifically by HLA-A2-specific cytotoxic T lymphocytes. When introduced on either of the vectors with the Epstein-Barr virus oriP element, the class I gene replicated extrachromosomally and was lost at rates of 0.2 to 0.3 per cell division. When introduced with vector pHPT (lacking Epstein-Barr virus oriP), the B8 gene was inserted at different chromosomal locations. Introduction of the HLA-B8 gene failed to restore antigen expression by HLA-B-null mutant .174, providing evidence that, unlike mutants exemplified by .53, .144, and .184, some HLA antigen loss mutants are deficient in a trans-acting function needed for class I antigen expression. Of more general interest, the results obtained with HLA class I genes in vectors that replicate extrachromosomally suggest ways of relating genic expression to chromatin structure and function and of attempting to clone functional human centromeres.
Chromatin structure and developmental expression of the human alpha-globin cluster.
Authors: Yagi M , Gelinas R , Elder J T , Peretz M , Papayannopoulou T , Stamatoyannopoulos G , Groudine M
Published: September 2015

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.
Electron microscopic study of Saccharomyces cerevisiae rDNA chromatin replication.
Authors: Saffer L D , Miller O L
Published: September 2015

An electron microscopic study was made of the replication of rDNA chromatin of Saccharomyces cerevisiae. Two different methods were used to synchronize cells. cdc7-1 cells were raised to a restrictive temperature, whereas A364a cells were blocked with mating factor. Replication bubbles typically opened in the nontranscribed spacers of rDNA repeats in both cell types. The mean position of the center of these bubbles corresponds closely to a position where an autonomously replicating sequence previously has been mapped in an rDNA repeat. Clusters of replication bubbles containing up to four bubbles spaced one to three genes apart were seen opening in early S phase.
Expression and localization of Drosophila melanogaster hsp70 cognate proteins.
Authors: Palter K B , Watanabe M , Stinson L , Mahowald A P , Craig E A
Published: September 2015

Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants with the major heat shock proteins hsp70 and hsp68. While two of the proteins are major proteins at all developmental stages, one heat shock cognate protein, hsc70, is especially enriched in embryos. hsc70 is shown to be the product of a previously identified gene, Hsc4. We have examined the levels of hsp70-related proteins in adult flies and larvae during heat shock and recovery. At maximal induction in vivo, hsp70 and hsp68 never reach the basal levels of the major heat shock cognate proteins. Monoclonal antibodies to hsc70 have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated around the nucleus.
Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.
Authors: Jat P S , Cepko C L , Mulligan R C , Sharp P A
Published: September 2015

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.
Human argininosuccinate synthetase minigenes are subject to arginine-mediated repression but not to trans induction.
Authors: Boyce F M , Anderson G M , Rusk C D , Freytag S O
Published: September 2015

The human argininosuccinate synthetase locus is subject to metabolite-mediated repression by arginine in some cultured cell lines. To gain insight into the mechanism underlying this regulation, chloramphenicol acetyltransferase (CAT) minigenes under the transcriptional control of the human argininosuccinate synthetase promoter were constructed and tested for regulation. When the minigenes were introduced into RPMI 2650 cells, a human cell line that shows sixfold regulation of the argininosuccinate synthetase gene, CAT expression was repressed three- to fivefold when arginine was present in the culture medium. A minigene containing only 149 base pairs of 5'-flanking sequence was expressed at similar levels and regulated to the same degree as one having approximately 3 kilobases of 5'-flanking sequence. Therefore, the cis-acting sequences required for the arginine-mediated repression are likely to be located within the region of the transcription initiation site. The arginine-mediated repression of the CAT minigenes was not observed in canavanine-resistant variants of RPMI 2650 cells, and therefore they showed the appropriate cell-type specificity. Cultured cells having 200-fold-increased levels of argininosuccinate synthetase can be selected by growth in medium containing the arginine analog canavanine. It was previously demonstrated that the increased expression of argininosuccinate synthetase in canavanine-resistant human lymphoblasts was due to a trans-acting mechanism. To gain further support for a trans-acting mechanism, we tested our CAT minigenes for the trans induction in canavanine-resistant variants of RPMI 2650 cells. Transfection of the CAT minigenes into RPMI 2650 cells and canavanine-resistant variants of this cell line yielded no difference in transient CAT expression. Furthermore, cloned canavanine-resistant variant cells having integrated copies of the CAT minigenes expressed CAT at similar levels as compared to the parental cell lines. Since these cell lines do exhibit arginine-mediated repression of CAT but not trans induction, these data indicate that the argine-mediated repression is a regulatory event that occurs independently of the trans induction.
Diverse long terminal repeats are associated with murine retroviruslike (VL30) elements.
Authors: Itin A , Keshet E
Published: September 2015

The VL30 family is a retroviruslike gene family with no apparent nucleic acid homology to any known retrovirus. Over 100 copies of VL30 DNA elements are dispersed throughout the mouse genome. Sequence analysis of the VL30 long terminal repeat (LTR) units showed that, whereas the LTR units of a given VL30 DNA element were almost identical, the LTR units associated with distinct members of the family were very different from one another. Comparison of the LTR sequences possessed by two particular VL30 DNA elements revealed a pattern of extensively homologous DNA segments adjacent to only distantly related DNA sequences. With the aid of sub-LTR probes, it was shown that a certain LTR is composed of both U5 sequences that are abundantly present in all species of the genus Mus and a U3 region detected only in Mus musculus. In addition, we isolated a VL30 DNA element in which the LTR units were replaced by the LTR units of an apparently novel retroviruslike family. These findings suggest that recombinations have played a role in generating the diverse population of VL30-associated LTRs.
Biochemical and functional analyses of a secreted H-2Ld molecule.
Authors: Murre C , Parker K C , Reiss C S , Driller J P , Wiley D C , Burakoff S J , Seidman J G
Published: September 2015

A truncated H-2Ld gene was constructed by deleting the transmembrane and cytoplasmic exons. The truncated H-2Ld gene was introduced into mouse L cells using the thymidine kinase gene as a selectable marker. Transformants were isolated and screened for the presence of truncated H-2Ld antigen. The truncated H-2Ld gene product was present in both the cytoplasm and culture medium, but not on the cell surface. The truncated H-2Ld antigen was stable in culture medium for at least 9 h and was secreted into the medium at a rate similar to the kinetics with which complete H-2 antigens reach the cell surface. Transformants expressing the truncated H-2Ld molecule were not recognized by cytotoxic T lymphocytes specific for the H-2Ld antigen.
Rescue of chromosomal T-antigen sequences onto extrachromosomally replicating, defective simian virus 40 DNA by homologous recombination.
Authors: Subramani S
Published: September 2015

Recombination between chromosomal and extrachromosomal DNA sequences was analyzed by investigation of the recombinational rescue of a 1,018-base-pair (bp) segment of the T-antigen gene of simian virus 40 from the chromosome of monkey COS cells to two different, extrachromosomally replicating, simian virus 40 DNA molecules lacking this 1,018-bp sequence. The ratio of rescued to unrecombined virus was as high as 10(-3). The rescued molecules, detected optimally 5 to 9 days after transfection of COS cells, had completely recovered the 1,018-bp DNA segment from the chromosome. The recombination event is proposed to occur either by double reciprocal recombination or by gene conversion between the chromosomal T-antigen gene and the extrachromosomal molecules missing the 1,018-bp sequence.
An anomalous Ty1 structure attributed to an error in reverse transcription.
Authors: Errede B , Company M , Swanstrom R
Published: September 2015

We have determined the nucleotide sequence of both delta elements of a Ty1 transposon inserted near the CYC7 gene in the Saccharomyces cerevisiae CYC7-H2 mutant. The upstream delta element in this Ty1 has an unusual inverted repeat structure that may have been formed by an error during reverse transcription.
Regulated expression of a murine class I gene in transgenic mice.
Authors: Bieberich C , Scangos G , Tanaka K , Jay G
Published: September 2015

The major histocompatibility complex class I genes play an essential role in the immune presentation of aberrant cells. To gain further insight into the regulation of the expression of these class I genes and to better define the functions of their protein products, we made use of the technique of gene transfer into the germ line of inbred mice. With the use of locus-specific DNA probes, we observed that a transgenic class I gene was expressed in a tissue-dependent fashion analogous to that of an endogenous class I gene. In addition, the level of expression of the transgenic gene was substantially higher that that of the endogenous gene. The availability of transgenic mice properly expressing a foreign murine class I gene provides a unique system to further define the role of the class I antigens in the maturation of the immune response and in determining the malignant and metastatic phenotypes of tumor cells.
Structure of the c-Ki-ras gene in a rat fibrosarcoma induced by 1,8-dinitropyrene.
Authors: Tahira T , Hayashi K , Ochiai M , Tsuchida N , Nagao M , Sugimura T
Published: September 2015

Restriction enzyme maps were made of the region around exons 1 and 2 of activated c-Ki-ras of a fibrosarcoma (1,8-DNP2) induced in a rat by 1,8-dinitropyrene. Nucleotide sequence analysis revealed that activated c-Ki-ras shows a G----T transversion in codon 12 and consequently encodes cysteine instead of glycine in normal rat c-Ki-ras.
The Saccharomyces cerevisiae chromosome III left telomere has a type X, but not a type Y', ARS region.
Authors: Button L L , Astell C R
Published: September 2015

A yeast Saccharomyces cerevisiae telomeric region was isolated by chromosome walking from HML alpha, the most distal known gene on the chromosome III left (IIIL) end. The terminal heterodisperse 3.3-kilobase (kb) SalI fragment on chromosome IIIL, 8.6 kb distal to HML alpha, was cloned in a circular vector to generate a telomeric probe. Southern hybridization and DNA sequencing analyses indicated that 0.6 kb (+/- 200 base pairs) of 5'-C1-3A-3' simple tandem repeat sequence, adjacent to a 1.2-kb type X ARS region, constitutes the telomere on the chromosome IIIL end, and no type Y' ARS region homologies exist between HML alpha and the IIIL terminus.
Polarity of DNA replication through the avian alpha-globin locus.
Authors: James C D , Leffak M
Published: September 2015

Toward understanding the controls affecting eucaryotic chromosome replication, we used a runoff replication assay to investigate whether the activity of a gene is related to its use of an upstream or downstream replication origin. When in vivo-initiated DNA polymerases are allowed to complete replication in vitro in the presence of bromodeoxyuridine triphosphate the density label is preferentially incorporated into origin-distal regions of DNA. Isopycnic centrifugation and blot hybridization analysis of the relative bromodeoxyuridine triphosphate incorporation into fragments spanning the chicken alpha-globin locus indicate that this region is replicated from an upstream origin both in chicken lymphocytes and in erythrocytes. Thus the replication polarity of these genes does not change as a function of transcriptional activity, consistent with earlier suggestions that DNA replication in the transcriptional direction may be a necessary but not sufficient condition for gene expression.
Characterization of the human p53 gene.
Authors: Lamb P , Crawford L
Published: September 2015

Cosmid and lambda clones containing the human p53 gene were isolated and characterized in detail. The gene is 20 kilobases (kb) long and has 11 exons, the first and second exons being separated by an intron of 10 kb. Restriction fragments upstream of sequences known to be within the first identified exon were tested for promoter activity by cloning them in front of the chloramphenicol acetyltransferase gene and transfecting the resulting constructs into HeLa cells. A 0.35-kb DNA fragment was identified that had promoter activity. Results of primer extension experiments indicated that the mRNA cap site falls within this fragment, as expected. Analysis of the sequence upstream of the presumptive cap site indicated that the human p53 promoter may be of an unusual type.
Expression of hepatitis B viral core region in mammalian cells.
Authors: Roossinck M J , Jameel S , Loukin S H , Siddiqui A
Published: September 2015

We studied the expression of the core region of the hepatitis B virus genome in mammalian cells with recombinant plasmid vectors. Stably transformed rat fibroblast cell lines were established by transfection with vectors containing subgenomic and genome-length hepatitis B virus DNA, followed by G418 selection. The RNA transcripts directed by the core region were characterized by Northern blot hybridization and S1 nuclease mapping. Using the chloramphenicol acetyltransferase gene expression system, the promoter activity located upstream of the core open reading frame was confirmed. The synthesis of core and e polypeptides was studied with a commercial radioimmunoassay. These studies show that partial deletion of the precore sequences abolished secretion of the e antigen, but there was pronounced synthesis of the core antigen in transfected cells.
Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation.
Authors: Whitfield C , Abraham I , Ascherman D , Gottesman M M
Published: September 2015

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.
Restriction endonuclease activity induced by PBCV-1 virus infection of a Chlorella-like green alga.
Authors: Xia Y N , Burbank D E , Uher L , Rabussay D , Van Etten J L
Published: September 2015

An enzyme was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1 which exhibits type II restriction endonuclease activity. The enzyme recognized the sequence GATC and cleaved DNA 5' to the G. Methylation of deoxyadenosine in the GATC sequence inhibited enzyme activity. In vitro the enzyme cleaved host Chlorella nuclear DNA but not viral DNA because host DNA contains GATC and PBCV-1 DNA contains GmATC sequences. PBCV-1 DNA is probably methylated in vivo by the PBCV-1-induced methyltransferase described elsewhere (Y. Xia and J. L. Van Etten, Mol. Cell. Biol. 6:1440-1445). Restriction endonuclease activity was first detected 30 to 60 min after viral infection; the appearance of enzyme activity required de novo protein synthesis, and the enzyme is probably virus encoded. Appearance of enzyme activity coincided with the onset of host DNA degradation after PBCV-1 infection. We propose that the PBCV-1-induced restriction endonuclease participates in host DNA degradation and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.
Hepatitis B surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide.
Authors: Eble B E , Lingappa V R , Ganem D
Published: September 2015

Hepatitis B surface antigen (HBsAg), the major coat protein of hepatitis B virus, is also secreted from cells as a subviral particle, without concomitant cleavage of N-terminal amino acid sequences. We examined this unusual export process in a cell-free system and showed that the initial product of HBsAg biosynthesis is an integral transmembrane protein, with most or all of its C-terminal half on the lumenal side of the endoplasmic reticulum membrane. To study the nature of its topogenic signals, we synthesized fusion proteins between HBsAg and the nonsecreted protein alpha-globin. Fusion proteins in which approximately 100 amino acids of globin preceded all HBsAg sequences were successfully translocated in vitro; the same domain as in the wild-type HBsAg was transported into the vesicle lumen. Fusions in which the entire globin domain was C terminal were able to translocate both the C-terminal region of HBsAg and its attached globin domain. Thus, uncleaved signal sequences in p24s function to direct portions of the molecule across the membrane and are able to perform this function even when positioned in an internal protein domain.
Tissue-specific expression of the mouse dioxin-inducible P(1)450 and P(3)450 genes: differential transcriptional activation and mRNA stability in liver and extrahepatic tissues.
Authors: Kimura S , Gonzalez F J , Nebert D W
Published: September 2015

Expression of the P(1)450 and P(3)450 genes was examined in liver and five extrahepatic tissues of mice after they were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene. All six tissues were shown to have increased P(1)450 and P(3)450 mRNA concentrations after treatment with these inducers. P(3)450 mRNA induction was more sensitive than P(1)450 mRNA induction to small doses of TCDD in liver, kidney, and lung. When transcription run-on assays were compared with mRNA prevalence, control P(3)450 mRNA in liver, kidney, and lung was shown to be 20 to 30 times more stable than control P(1)450 mRNA. After TCDD treatment the increases in mRNA concentrations did not necessarily parallel the increases in transcriptional rate. Thus, the inducer appeared to enhance mRNA stability in some instances. This was evident for liver P(1)450 mRNA, in which an 8-fold rise in transcription was associated with a 27-fold increase in mRNA content, and for kidney P(3)450 mRNA, in which a 2-fold rise in transcription was accompanied by a 12-fold increase in mRNA content. In the kidney and lung of control and TCDD-treated mice, transcriptional rates of the P(3)450 gene were at least 10-fold less than those of the P(1)450 gene. These data indicate that even though both genes are controlled by the same receptor, striking tissue-specific differences in transcription and mRNA stabilization affect the final mRNA concentrations.
4.5S RNA is encoded by hundreds of tandemly linked genes, has a short half-life, and is hydrogen bonded in vivo to poly(A)-terminated RNAs in the cytoplasm of cultured mouse cells.
Authors: Schoeniger L O , Jelinek W R
Published: September 2015

4.5S RNA is a group of RNAs 90 to 94 nucleotides long (length polymorphism due to a varying number of UMP residues at the 3' end) that form hydrogen bonds with poly(A)-terminated RNAs isolated from mouse, hamster, or rat cells (W. R. Jelinek and L. Leinwand, Cell 15:205-214, 1978; F. Harada, N. Kato, and H.-O. Hoshino, Nucleic Acids Res. 7:909-917, 1979). We have cloned a gene that encodes the 4.5S RNA. It is repeated 850 (sigma = 54) times per haploid mouse genome and 690 (sigma = 59) times per haploid rat genome. Most, if not all, of the repeats in both species are arrayed in tandem. The repeat unit is 4,245 base pairs long in mouse DNA (the complete base sequence of one repeat unit is presented) and approximately 5,300 base pairs in rat DNA. This accounts for approximately 3 X 10(6) base pairs of genomic DNA in each species, or 0.1% of the genome. Cultured murine erythroleukemia cells contain 13,000 molecules per cell of the 4.5S RNA, which can be labeled to equilibrium in 90 min by [3H]uridine added to the culture medium. The 4.5S RNA, therefore, has a short half-life. The 4.5S RNA can be cross-linked in vivo by 4'-aminomethyl-4,5',8-trimethylpsoralen to murine erythroleukemia cell poly(A)-terminated cytoplasmic RNA contained in ribonucleoprotein particles.
Anomalous placement of introns in a member of the intermediate filament multigene family: an evolutionary conundrum.
Authors: Lewis S A , Cowan N J
Published: September 2015

The origin of introns and their role (if any) in gene expression, in the evolution of the genome, and in the generation of new expressed sequences are issues that are understood poorly, if at all. Multigene families provide a favorable opportunity for examining the evolutionary history of introns because it is possible to identify changes in intron placement and content since the divergence of family members from a common ancestral sequence. Here we report the complete sequence of the gene encoding the 68-kilodalton (kDa) neurofilament protein; the gene is a member of the intermediate filament multigene family that diverged over 600 million years ago. Five other members of this family (desmin, vimentin, glial fibrillary acidic protein, and type I and type II keratins) are encoded by genes with six or more introns at homologous positions. To our surprise, the number and placement of introns in the 68-kDa neurofilament protein gene were completely anomalous, with only three introns, none of which corresponded in position to introns in any characterized intermediate filament gene. This finding was all the more unexpected because comparative amino acid sequence data suggest a closer relationship of the 68-kDa neurofilament protein to desmin, vimentin, and glial fibrillary acidic protein than between any of these three proteins and the keratins. It appears likely that an mRNA-mediated transposition event was involved in the evolution of the 68-kDa neurofilament protein gene and that subsequent events led to the acquisition of at least two of the three introns present in the contemporary sequence.
Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.
Authors: Esteban R , Wickner R B
Published: September 2015

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).
Bidirectional transcription from a solo long terminal repeat of the retrotransposon TED: symmetrical RNA start sites.
Authors: Friesen P D , Rice W C , Miller D W , Miller L K
Published: September 2015

A single copy of the retrotransposon TED was found integrated within the DNA genome of the insect baculovirus, Autographa californica nuclear polyhedrosis virus. After excision of the element from the viral genome, a single long terminal repeat (LTR) remained behind. We have examined the effect of this solo TED LTR on the local pattern of viral transcription. Most prominent was the transcription of two sets of abundant RNAs; both originated within the LTR but extended in opposite directions into flanking viral genes. By promoting symmetric transcription of adjacent genes, the solo LTR has the capacity to activate or repress gene expression in two directions. Primer extension analysis demonstrated that the divergent LTR transcripts were initiated near the same point within a 22-base-pair sequence having hyphenated twofold symmetry. Analogous symmetries at the initiation sites of other retrotransposon LTRs, including copia and Ty, suggested that these sequences serve to establish the precise start for transcription.
Differential accumulation of plant defense gene transcripts in a compatible and an incompatible plant-pathogen interaction.
Authors: Bell J N , Ryder T B , Wingate V P , Bailey J A , Lamb C J
Published: September 2015

Phenylalanine ammonia-lyase and chalcone synthase catalyze the first reaction of phenylpropanoid biosynthesis and the first reaction of a branch pathway specific for flavonoid-isoflavonoid biosynthesis, respectively. These enzymes are key control elements in the synthesis of kievitone, phaseollin, and related isoflavonoid-derived phytoalexins. RNA blot hybridization with 32P-labeled cDNA sequences was used to demonstrate marked accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs in excision-wounded hypocotyls of Phaseolus vulgaris L. (dwarf French bean) and during race-cultivar-specific interactions between hypocotyls of P. vulgaris and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant), early concomitant accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs, localized mainly but not entirely in tissue adjacent to the site of infection, was observed prior to the onset of phytoalexin accumulation and expression of localized, hypersensitive resistance. In contrast, in a compatible interaction (host susceptible) there was no early accumulation of these transcripts; instead, there was a delayed widespread response associated with phytoalexin accumulation during attempted lesion limitation. Two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides synthesized in vitro by translation of isolated polysomal RNA demonstrated stimulation of the synthesis of characteristic sets of phenylalanine ammonia-lyase and chalcone synthase isopolypeptides in directly infected tissue and distant, hitherto uninfected tissue in both compatible and incompatible interactions. Our data show that specific accumulation of plant defense gene transcripts is a key early component in the sequence of events leading to expression of defense responses in wounded tissue and in infected tissue during race-cultivar-specific interactions and that an elicitation signal is transmitted intercellularly in response to infection.
Domain structure of the simian virus 40 core origin of replication.
Authors: Deb S , DeLucia A L , Baur C P , Koff A , Tegtmeyer P
Published: September 2015

The simian virus 40 core origin of replication consists of nucleotides 5211 through 31. These 64 base pairs contain three functional domains with strict sequence requirements and two spacer regions with relaxed sequence specificity but precise positional constraints. The early domain extends for 10 contiguous base pairs between nucleotides 5211 and 5220. A 9-base pair spacer from sequences 5221 through 5229 separates the early domain from the 23-base pair central palindrome that directs the binding of T antigen. The late end of the core between nucleotides 12 and 31 also contains spacer and sequence-specific functions that are not yet completely mapped. We propose that the sequence-specific domains are interaction sites for viral and cellular proteins, determinants of DNA conformation, or both. The spacers would position these signals at required distances and rotations relative to one another.
Isolation of the oncogene and epidermal growth factor-induced transin gene: complex control in rat fibroblasts.
Authors: Matrisian L M , Leroy P , Ruhlmann C , Gesnel M C , Breathnach R
Published: September 2015

Various oncogenes or epidermal growth factor (EGF) induce transcription of a 1.9-kilobase RNA (transin RNA) in rat fibroblasts. The induction by EGF can be blocked by cycloheximide. Thus the response of the transin gene to EGF appears to require de novo protein synthesis. Transin RNA induction is specific to EGF, as neither insulin, platelet-derived growth factor, fibroblast growth factor, nor transforming growth factor beta could elicit the same response. However, transforming growth factor beta could block the EGF induction of transin RNA. Whereas the calcium ionophore A23187 and the tumor promoter TPA, either alone or administered together, did not increase transin RNA levels, TPA could synergise with a serum factor to effect such an increase. Dibutyryl cyclic AMP also induced transin RNA. Treatment of cells with the microfilament-disrupting agent cytochalasin B, but not the microtubule-disrupting agent colcemid, resulted in an increase in transin RNA levels, suggesting a role for the cytoskeleton in control of transin gene expression. The transin RNA does not contain repeated sequences and appears to be encoded by a single-copy gene. The protein sequence encoded by the last four exons of the transin gene shows some homology to two regions of the heme-binding protein hemopexin.
Gamma interferon and 5-azacytidine cause transcriptional elevation of class I major histocompatibility complex gene expression in K562 leukemia cells in the absence of differentiation.
Authors: Chen E , Karr R W , Frost J P , Gonwa T A , Ginder G D
Published: September 2015

We studied the effects of gamma interferon (IFN-gamma) on HLA class I gene expression, differentiation, and proliferative capacity of K562 human leukemia cells. In the uninduced state, K562 cells show little or no class I gene expression but actively express the erythroid-specific gamma-globin gene as well as genes associated with cell proliferation, including the transferrin receptor, c-myc, and alpha-actin genes At both the surface protein and mRNA levels, IFN-gamma induces class I and beta 2-microglobulin gene expression, but does not alter the expression of the gamma-globin, transferrin receptor, c-myc, or alpha-actin genes. A 10-fold maximal induction of both class I surface protein and mRNA occurs at 48 h and is reversible upon withdrawal of IFN-gamma from the culture medium. In vitro nuclear run-on transcription assays were performed to directly establish that IFN-gamma exerts an early effect at the level of transcription, with maximal transcription rates occurring within 4 h. The difference between the time course of transcription induction and that of mRNA accumulation suggests that the regulation of class I gene expression in this human leukemic cell line also involves posttranscriptional mechanisms. Measurements of cell proliferation rates and cell cycle distribution, as well as the reversibility of the effects of IFN-gamma, demonstrate that the selective induction of class I genes in these cells occurs in the absence of differentiation.
ADR1-mediated regulation of ADH2 requires an inverted repeat sequence.
Authors: Shuster J , Yu J , Cox D , Chan R V , Smith M , Young E
Published: September 2015

DNA sequence analysis of wild-type and mutant ADH2 loci suggested that two unusual features 5' of the promoter, a 22-base-pair perfect dyad sequence and a (dA)20 tract, were important for regulation of this gene (D. W. Russell, M. Smith, D. Cox, V. M. Williamson, and E. T. Young, Nature [London] 304:652-654, 1983). Oligonucleotide-directed mutagenesis was used to construct ADH2 genes lacking the 22-base-pair dyad or the (dA)20 tract (V.-L. Chan and M. Smith, Nucleic Acids Res. 12:2407-2419, 1984). These mutant genes and other ADH2 deletions constructed by BAL 31 endonuclease digestion were studied after replacing the wild-type chromosomal locus with the altered alleles by the technique of gene transplacement (T. L. Orr-Weaver, J. W. Szostak, and R. S. Rothstein, Proc. Natl. Acad. Sci. USA 78:6354-6358, 1981), using canavanine resistance as the selectable marker. Deletions lacking the dyad failed to derepress normally and did not respond to mutations at the ADR1 locus, which encodes a protein necessary to activate ADH2. Deletions of the (dA)20 tract did not have a detectable phenotype. A small deletion located just 3' to the (dA)20 tract (between positions -164 and -146) had a low amount of ADR1-dependent transcription during repressed growth conditions, indicating that the regulatory protein encoded by ADR1 is present in a potentially active form during repression and that alterations of a DNA sequence in the promoter region can unmask its latent activity.
Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells.
Authors: Kleinberger T , Etkin S , Lavi S
Published: September 2015

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.