bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron microscopic heteroduplex mapping identifies regions of the engrailed locus that are conserved between Drosophila melanogaster and Drosophila virilis.
Authors: Kassis J A , Wong M L , O'Farrell P H
Published: September 2015

Physical localization of mutations in the engrailed (en) gene suggested that at least 70 kilobases (kb) of genomic sequences contribute to the normal function of this gene. Molecular characterization has suggested that en function is encoded in a small, 4.5-kb primary transcript. To identify functional regions within the 70 kb of the en locus of D. melanogaster, we identified sequences conserved in the D. virilis genome (estimated divergence time, 60 million years). Based on homology to D. melanogaster, we isolated en DNA from a D. virilis genomic library. Electron microscopic heteroduplex analysis indicated that in 70 kb there is 20 kb of conserved DNA in 33 different regions dispersed throughout the en locus, including two which encode parts of the major embryonic transcript. The conserved regions are in the same linear order and are spaced by similar lengths of nonconserved sequences in the D. virilis and D. melanogaster DNAs. What functional constraints have enforced conservation of sequences throughout the entire 70 kb and protected the region from divergence of size and arrangement? Our working hypothesis is that sequences necessary for the complex spatial and temporal pattern of en expression are dispersed throughout the 70-kb en locus and that selection for proper regulation restricts evolutionary divergence.
Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.
Authors: Evan G I , Lewis G K , Ramsay G , Bishop J M
Published: September 2015

Six monoclonal antibodies have been isolated from mice immunized with synthetic peptide immunogens whose sequences are derived from that of the human c-myc gene product. Five of these antibodies precipitate p62c-myc from human cells, and three of these five also recognize the mouse c-myc gene product. None of the antibodies sees the chicken p110gag-myc protein. All six antibodies recognize immunoblotted p62c-myc. These reagents also provide the basis for an immunoblotting assay by which to quantitate p62c-myc in cells.
Structure of the rat alpha 1-acid glycoprotein gene.
Authors: Liao Y C , Taylor J M , Vannice J L , Clawson G A , Smuckler E A
Published: September 2015

The complete nucleotide sequence of the rat alpha 1-acid glycoprotein gene has been determined from an isolated lambda recombinant bacteriophage. Southern blot analysis and DNA sequencing indicate that there is only one gene per genome; it contains six exons and is located within a 3,200-base-pair fragment starting from a TATA box and extending to the polyadenylation signal AATAAA. Transcription starts 37 base pairs upstream from the beginning of the translation codon ATG. The TATA box (TATAAA) lies 26 base pairs upstream from this site. The gene contains several potential glucocorticoid receptor-binding sites, both inside and outside the structural gene.
Isolation of antibodies for phosphotyrosine by immunization with a v-abl oncogene-encoded protein.
Authors: Wang J Y
Published: September 2015

Immunization of rabbits with a tyrosine-phosphorylated v-abl protein resulted in the production of antibodies for the v-abl protein and for phosphotyrosine. The antiphosphotyrosine antibodies could be purified by affinity chromatography with O-phosphotyramine coupled to Sepharose. These antibodies detected a variety of tyrosine-phosphorylated proteins, including receptors for peptide growth factors. The usefulness of these antibodies was demonstrated by the detection of previously unidentified tyrosine-phosphorylated proteins in v-src-, v-abl-, and v-erbB-transformed cell lines.
Developmental expression of rat transforming growth factor-alpha mRNA.
Authors: Lee D C , Rochford R , Todaro G J , Villarreal L P
Published: September 2015

Expression of the gene encoding transforming growth factor-alpha (TGF alpha) was examined in developing rat embryos by using a cloned TGF alpha cDNA as a hybridization probe. Northern blot analysis of RNA isolated from whole fetuses revealed that TGF alpha mRNA was present at relatively high levels in 8- through 10-day-old embryos and then declined to the low or undetectable level, which is characteristic of adult tissues before birth. The level of TGF alpha mRNA present during early gestation was similar to that present in retrovirus-transformed cells in culture, suggesting that TGF alpha expression is not highly localized in the embryo. These observations are consistent with the hypothesis that TGF alpha plays a role in development, possibly as a fetal growth factor.
Characterization of a retrovirus shuttle vector capable of either proviral integration or extrachromosomal replication in mouse cells.
Authors: Berger S A , Bernstein A
Published: September 2015

A retrovirus shuttle vector is described that contains the dominant selectable neo gene which confers resistance to kanamycin in bacteria and to the drug G418 in animal cells. The bacterial supF gene and the origins of DNA replication from polyomavirus and the ColE1 replicon also have been included in this vector. Infection of normal rodent cells results in single-copy proviral integration, whereas infection of mouse (MOP) cells expressing polyoma large T antigen results in extrachromosomal replication of the DNA form of the virus. The copy number of the extrachromosomal circles in MOP cells varies from 0 to 100 copies per cell. G418-resistant MOP cells lose their drug-resistant phenotype after passage under nonselective conditions, suggesting that maintenance of the extrachromosomal circles is unstable. The extrachromosomal form of the virus can be recovered as plasmids in Escherichia coli. Two-thirds of the circles analyzed were found to be structurally intact. The others have undergone rearrangements including deletions and insertions. The bacterial supF gene was found to be intact in the majority of recovered plasmids. The data presented here suggest that these retroviruses should be useful as gene transfer vectors for animal cells in culture or in vivo.
Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression.
Authors: Crawford B D , Enger M D , Griffith B B , Griffith J K , Hanners J L , Longmire J L , Munk A C , Stallings R L , Tesmer J G , Walters R A
Published: September 2015

We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.
Reconstitution of functional mRNA-protein complexes in a rabbit reticulocyte cell-free translation system.
Authors: Greenberg J R , Carroll E
Published: September 2015

A variety of evidence suggests that the cytoplasmic mRNA-associated proteins of eucaryotic cells are derived from the cytoplasm and function there, most likely in protein synthesis or some related process. Furthermore, the evidence suggests that protein-free mRNA added to a cell-free translation system should become associated with a set of proteins similar to those associated with mRNA in native polyribosomes. To test this hypothesis, we added deproteinized rabbit reticulocyte mRNA to a homologous cell-free translation system made dependent on exogenous mRNA by treatment with micrococcal nuclease. The resulting reconstituted complexes were irradiated with UV light to cross-link the proteins to mRNA, and the proteins were analyzed by gel electrophoresis. The proteins associated with polyribosomal mRNA in the reconstituted complexes were indistinguishable from those associated with polyribosomal mRNA in intact reticulocytes. Furthermore, reticulocyte mRNA-associated proteins were very similar to those of cultured mammalian cells. The composition of the complexes varied with the translational state of the mRNA; that is, certain proteins present in polyribosomal mRNA-protein complexes were absent or reduced in amount in 40S to 80S complexes and in complexes formed in the absence of translation. However, other proteins, including a 78-kilodalton protein associated with polyadenylate, were present irrespective of translational state, or else they were preferentially associated with untranslated mRNA. These findings are in agreement with previous data suggesting that proteins associated with cytoplasmic mRNA are derived from the cytoplasm and that they function in translation or some other cytoplasmic process, rather than transcription, RNA processing, or transport from the nucleus to the cytoplasm.
Stage-specific patterns of collagen gene expression during development of Caenorhabditis elegans.
Authors: Cox G N , Hirsh D
Published: September 2015

Collagens are the major protein components of the Caenorhabditis elegans cuticle and are encoded by a large family of 40 to 150 closely related but nonidentical genes. We have determined temporal patterns of mRNA accumulation for a large number of collagen genes by screening recombinant phages and plasmids containing cloned collagen genes under high stringency conditions with 32P-labeled cDNA preparations specific for eggs or three postembryonic molts. We find that collagen mRNA levels are regulated both temporally and quantitatively during C. elegans development. Most genes studied exhibit one of four patterns of mRNA accumulation which correlate with changes in cuticle morphology and collagen protein composition during development. Our results suggest that, in general, there is a progressive activation of new collagen genes during normal development.
Repeat arrays in cellular DNA related to the Epstein-Barr virus IR3 repeat.
Authors: Heller M , Flemington E , Kieff E , Deininger P
Published: September 2015

We isolated clones and determined the sequence of portions of mouse and human cellular DNA which cross-hybridize strongly with the IR3 repetitive region of Epstein-Barr virus. The sequences were found to be tandem arrays of a simple sequence based on the triplet GGA, very similar to the IR3 repeat. The cellular repeats have distinct differences from the viral repeat region, however, and their sequences do not appear capable of being translated into a purely glycine-plus-alanine protein domain like the portion of the Epstein-Barr nuclear antigen coded by IR3. Although the relationship between IR3 and the cellular repeats is left unclear, the cellular repeats have many interesting features. The tandem arrays are about 1 to several kilobases long, much shorter than satellite tandem repeats and larger than other interspersed, tandem repeats. Each of the repeats is a distinct variation, perhaps diverged from a common sequence, (GGA)n. This family is present in the genomes of all species tested and appears to be a ubiquitous feature of all higher eucaryotic genomes.
Regulation of creatine kinase induction in differentiating mouse myoblasts.
Authors: Chamberlain J S , Jaynes J B , Hauschka S D
Published: September 2015

The regulation of creatine kinase (CK) induction during muscle differentiation was analyzed with MM14 mouse myoblasts. These cells withdraw from the cell cycle and commit to terminal differentiation when fed with mitogen-depleted medium. Myoblasts contained trace amounts of an isozyme of brain CK (designated BB-CK), but differentiation was accompanied by the induction of two other isozymes of muscle and brain CKs (designated MM-CK and MB-CK). Increased CK activity was detectable within 6 h of mitogen removal, 3 h after the first cells committed to differentiation and 6 h before fusion began. By 48 h, MM-CK activity increased more than 400-fold, MB-CK activity increased more than 150-fold, and BB-CK activity increased more than 10-fold. Antibodies prepared against purified mouse MM-CK cross-reacted with muscle and brain CKs (designated M-CK and B-CK, respectively) from a variety of species and were used to demonstrate that the increase in enzymatic activity was paralleled by an increase in the protein itself. CK antibodies were also used to aid in identifying cDNA clones to M-CK. cDNA sequences which corresponded to protein-coding regions cross-hybridized with B-CK mRNA; however, a subclone containing the 3'-nontranslated region was unique and was used to quantitate M-CK mRNA levels during myoblast differentiation. M-CK mRNA was not detectable in myoblasts, but within 5 to 6 h of mitogen withdrawal (6 to 7 h before fusion begins) it accumulated to about 30 molecules per cell. By 24 h, myotubes contained approximately 1,100 molecules per nucleus of M-CK mRNA.
Cloning and sequencing of a c-myc oncogene in a Burkitt's lymphoma cell line that is translocated to a germ line alpha switch region.
Authors: Showe L C , Ballantine M , Nishikura K , Erikson J , Kaji H , Croce C M
Published: September 2015

We have cloned and sequenced the translocated c-myc gene from the Burkitt's lymphoma CA46 cell line that carries a reciprocal translocation between chromosomes 8 and 14. The breakpoint lies within the first intron of c-myc, so that the first noncoding exon of the gene remains on the 8q- chromosome. The second and third coding exons are translocated to the 14q+ chromosome into the switch region of C-alpha 1. The orientation of the c-myc gene with relationship to alpha 1 is 5' to 5', with directions of transcription in opposite orientation. DNA sequencing studies predict five changes in the amino acid sequence of the myc protein, two of which occur in a region within the second exon which is highly conserved in evolution. Southern blotting data indicate that the first exon of c-myc is rearranged 3' to 3' with the pseudo-epsilon gene. Because CA46 cells contain two rearranged mu genes, the translocation must have occurred after immunoglobulin rearrangement. The position of the breakpoint in CA46 occurs within a 20-base-pair region of the first intron of c-myc to which breakpoints have been mapped for two additional B-cell lymphomas with the t(8;14) translocation, ST486 and the Manca cell line. The region of the heavy chain locus to which c-myc has translocated is different in each case. Comparisons have been made of the levels of transcripts of the translocated c-myc gene in ST486 and CA46, where the gene is not associated with the heavy chain enhancer, with its expression in the Manca cell, in which it is. The c-myc gene is transcribed at similar levels in all three cases.
Structure and transcription of a telomeric surface antigen gene of Trypanosoma brucei.
Authors: Bernards A , Kooter J M , Borst P
Published: September 2015

The gene encoding variant surface glycoprotein 221 in Trypanosoma brucei is located adjacent to a chromosome end and can be activated with or without a concomitant gene duplication. To test whether transcription initiates within the cloned segment of the 221 gene, we analyzed nascent and stable transcripts. We show here that the 221 coding region and 8.5 kilobases of adjacent upstream DNA are transcribed into nascent RNA at a similar rate when gene 221 is activated without duplication. Since only part of this transcribed upstream segment is transferred with the coding region to another telomere upon duplicative activation of gene 221, we infer that initiation of variant surface glycoprotein gene transcription occurs outside the gene segment that moves into an expression site by gene conversion. Our analysis shows that part of the variant surface glycoprotein 221 transcription unit consists of an unusual 3.5-kilobase tandem array of ca. 50 repeat segments and that a rearrangement in this array accompanies the nonduplicative activation of gene 221. A variant surface glycoprotein pseudogene is located within the transcription unit of gene 221, and we discuss models that account for this unusual situation.
A complex control region of the mouse rRNA gene directs accurate initiation by RNA polymerase I.
Authors: Miller K G , Tower J , Sollner-Webb B
Published: September 2015

To determine the size and location of the mouse rDNA promoter, we constructed systematic series of deletion mutants approaching the initiation site from the 5' and 3' directions. These templates were transcribed in vitro under various conditions with S-100 and whole-cell extracts. Surprisingly, the size of the rDNA region that determines the level of transcription differed markedly, depending on the reaction conditions. In both kinds of cell extracts, the apparent 5' border of the promoter was at residue ca. -27 under optimal transcription conditions, but as reaction conditions became less favorable, the 5' border moved progressively out to residues -35, -39, and -45. The complete promoter, however, extends considerably further, for under other nonoptimal conditions, we observed major effects of promoter domains extending in the 5' direction to positions ca. -100 and -140. In contrast, the apparent 3' border of the mouse rDNA promoter was at residue ca. +9 under all conditions examined. We also show that the subcloned rDNA region from -39 to +9 contains sufficient information to initiate accurately and that the region between +2 and +9 can influence the specificity of initiation. These data indicate that, although the polymerase I transcription factors recognize and accurately initiate with only the sequences downstream of residue -40, sequences extending out to residue -140 greatly favor the initiation reaction; presumably, this entire region is involved in rRNA transcription in vivo.
Cloned mouse DNA fragments can replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro.
Authors: Ariga H , Tsuchihashi Z , Naruto M , Yamada M
Published: September 2015

Mouse liver DNA was cut out with BamHI and cloned into YIp5, which contained the URA3 gene of Saccharomyces cerevisiae in pBR322. Of the several plasmids isolated, two plasmids, pMU65 and pMU111, could transform S. cerevisiae from the URA- to the URA+ phenotype and could replicate autonomously within the transformant, indicating that mouse DNA fragments present in pMU65 or pMU111 contain autonomously replicating sequences (ARS) for replication in S. cerevisiae. Furthermore, to determine the correlation between ARS function in yeast cells and that in much higher organisms, we tried to challenge these plasmids with the simian virus 40 (SV40) DNA replication system. Of the two plasmids tested, the EcoRI-BglII region of pMU65 could be hybridized with a chemically synthesized 13-nucleotide fragment corresponding to the origin region of SV40 DNA. Both pMU65 (the EcoRI-BglII region cloned in pBR322) and its subclone pMU65EB could replicate semiconservatively, and initiation of DNA replication started from the EcoRI-BglII region when the replicating activity of these plasmids was tested in the in vitro SV40 DNA replication system we have established before. Furthermore, pMU65 and pMU65EB could replicate autonomously within monkey Cos cells which produce SV40 T antigen constitutively. These results show that a 2.5-kilobase fragment of the EcoRI-BglII region in pMU65 contains the ARS needed for replication in the SV40 DNA replication system.
Abundant nuclear ribonucleoprotein form of CAD RNA.
Authors: Sperling R , Sperling J , Levine A D , Spann P , Stark G R , Kornberg R D
Published: September 2015

Transcripts of the CAD gene in Syrian hamster cells are as abundant in the nucleus as in the cytoplasm. This was shown by in situ hybridization of whole cells and by solution and blot hybridization of subcellular fractions. Similar results were obtained both for wild-type cells and for a mutant containing amplified CAD genes in which the level of CAD RNA is 150-fold greater. CAD nuclear RNA is indistinguishable from mature mRNA by gel electrophoresis and blot hybridization. Discrete higher-molecular-weight precursors are undetectable, although the persistence of a short length of intervening sequence in the otherwise fully processed RNA is not excluded. CAD RNA is released from nuclei by sonication in physiological conditions in a ribonucleoprotein form that sediments as a broad peak at about 200S in a sucrose gradient. CAD sequences extracted from nuclei by treatment with EDTA and RNase are found in the 30S particles previously described.
Characterization and purification of lupus antigen La, and RNA-binding protein.
Authors: Francoeur A M , Chan E K , Garrels J I , Mathews M B
Published: September 2015

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.
Cell cycle control of the human HSP70 gene: implications for the role of a cellular E1A-like function.
Authors: Kao H T , Capasso O , Heintz N , Nevins J R
Published: September 2015

The gene encoding the human 70-kilodalton heat shock protein (HSP70) is subject to activation by the adenovirus E1A gene product and appears to be regulated in the absence of heat shock by a cellular activity similar to E1A. Given the relation of E1A to alteration of growth control, we have investigated the expression of the HSP70 gene during the cell cycle. Assay of mRNA levels after release from a thymidine-aphidicolin block revealed a 20-fold increase in mRNA abundance, reaching a peak level in the post-S-phase period. Upon reaching this peak level, the abundance of the mRNA then declined as the cells entered the next cycle. Control of the abundance of the mRNA during the cell cycle appeared to be primarily at the level of transcription as measured in nuclear runoff assays. Very similar results were obtained by analyzing the expression of the HSP70 gene in the adenovirus-transformed 293 cell line. Furthermore, the E1A gene was also found to be cell cycle regulated; the activation and peak level of the E1A mRNA occurred at an earlier time than those of the heat shock mRNA, consistent with, but not proof of, the hypothesis that E1A is responsible for the cell cycle control of the HSP70 expression. We therefore suggest that the E1A-like cellular activity may govern certain aspects of cell cycle transcription.
Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression.
Authors: Veldman G M , Lupton S , Kamen R
Published: September 2015

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.
Control of globin gene transcription by erythropoietin in erythroblasts from friend virus-infected mice.
Authors: Bondurant M C , Lind R N , Koury M J , Ferguson M E
Published: September 2015

Splenic erythroblasts of mice infected with the anemia-inducing strain of Friend virus can be isolated in large numbers with less than 5% contamination with other cell types. In short-term culture, the isolated cells will initiate globin synthesis and undergo other aspects of terminal differentiation only if erythropoietin (EP) is added to the medium. An early effect of the hormone on these cells is stimulation of total RNA synthesis. EP also causes initiation of transcription of the beta-globin genes after a lag period of 4 to 6 h. By 6 h, the transcription rate of beta-globin RNA is enhanced threefold, and by 12 h, it is nearly maximal at ca. 20 times the level of control cells which received no EP. Transcription rates of alpha and beta-globin genes are approximately equal to each other throughout the period of terminal differentiation. In the splenic erythroblasts, the chromatin structure in the vicinity of the beta-major globin gene was analyzed with two nucleases during these transcription rate changes. No S1 nuclease-hypersensitive site is detectable near the gene. The beta-major gene is quite sensitive to DNase I in comparison with the albumin gene; however, the level of sensitivity is the same before EP addition as it is during maximal gene transcription after EP addition. Also, a hypersensitive site near the 5' cap site of the beta-major gene is quantitatively equivalent both before and after EP addition. Analysis of cytosine methylation at two sites upstream from the gene showed no changes upon induction of beta-globin gene transcription by EP. Thus, the initiation of beta-globin transcription by EP appears to be at some step after chromatin structural alteration such as synthesis, release, or activation of a specific transcription initiation factor.
UV stimulation of DNA-mediated transformation of human cells.
Authors: van Duin M , Westerveld A , Hoeijmakers J H
Published: September 2015

Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells (complementation groups A and F), which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. No stimulation was found after damaging vector DNA by treatment with DNase or gamma rays. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome.
Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating.
Authors: Kurjan J
Published: September 2015

The role of alpha-factor structural genes MF alpha 1 and MF alpha 2 in alpha-factor production and mating has been investigated by the construction of mf alpha 1 and mf alpha 2 mutations that totally eliminate gene function. An mf alpha 1 mutant in which the entire coding region is deleted shows a considerable decrease in alpha-factor production and a 75% decrease in mating. Mutations in mf alpha 2 have little or no effect on alpha-factor production or mating. The mf alpha 1 mf alpha 2 double mutants are completely defective in mating and alpha-factor production. These results indicate that at least one alpha-factor structural gene product is required for mating in MAT alpha cells, that MF alpha 1 is responsible for the majority of alpha-factor production, and that MF alpha 1 and MF alpha 2 are the only active alpha-factor genes.
Isolation and characterization of genes differentially expressed during conidiation of Neurospora crassa.
Authors: Berlin V , Yanofsky C
Published: September 2015

A Neurospora crassa genomic DNA library was screened with a cDNA probe enriched in sequences expressed in conidiating cultures. Clones were isolated that preferentially hybridized to this probe versus a second cDNA probe complementary to polyadenylated RNA isolated from mycelia. Twelve clones contained unique sequences that hybridized to 22 transcripts, 19 of which accumulated preferentially in conidiating cultures. Eight transcripts were present in higher levels in conidiating cultures than in mycelia. Eleven transcripts were detected only in conidiating cultures and first appeared at different times during the asexual cycle. We mapped genomic sequences homologous to the 11 clones by conventional crosses using restriction fragment-length polymorphisms as genetic markers. The sequences homologous to genes expressed preferentially in conidiating cultures are distributed on six of the seven chromosomes. Clones that map to the same chromosome are linked. No recombination occurred between genomic sequences homologous to three clones, suggesting that the genes contained in these clones may constitute a gene cluster.
Characterization and developmental expression of a Drosophila ras oncogene.
Authors: Mozer B , Marlor R , Parkhurst S , Corces V
Published: September 2015

We cloned a Drosophila melanogaster ras gene (Dmras64B) on the basis of its homology to the ras oncogen from Harvey murine sarcoma virus. This gene mapped at chromosomal position 64B on the left arm of the third chromosome. Sequencing of Dmras64B revealed extensive amino acid homology with the proteins encoded by the human and Saccharomyces cerevisiae ras genes. The coding region of the Drosophila gene is interrupted by two introns located in different positions with respect to its human counterpart. Dmras64B encodes three different RNAs (1.6, 2.1, and 2.6 kilobases long) that are constantly expressed throughout the development of the fly.
Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.
Authors: Alwine J C
Published: September 2015

The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.
Gene transfer method for transient gene expression, stable transformation, and cotransformation of suspension cell cultures.
Authors: Gopal T V
Published: September 2015

A new method was developed to study transient gene expression, stable transformation, and cotransformation in suspension cells, such as mouse myeloma and erythroleukemia cells. This method involves attachment of cells to a concanavalin A-coated tissue culture dish, treatment of cells with DEAE-dextran to adsorb plasmid DNA to the attached cells, and finally treatment with a 40% solution of polyethylene glycol to facilitate the uptake of DNA by the cells. Plasmids pSV2cat and pSV2neo were used as markers to optimize the conditions for transient gene expression and stable transformation, respectively, of mouse myeloma and erythroleukemia cells. This method was successfully used to obtain cotransformants of mouse myeloma cells.
The simian virus 40 sequences between 0.169 and 0.423 map units are not essential to immortalize early-passage rat embryo cells.
Authors: Sompayrac L , Danna K J
Published: September 2015

F8dl is a simian virus 40 early-region deletion mutant that lacks the sequences between 0.169 and 0.423 map units. We show that cloned F8dl DNA immortalized early-passage Fisher rat embryo cells with an efficiency that was about 20% of that of cloned wild-type simian virus 40 DNA. In contrast, we detected no immortalized colonies when we transfected the cells with DNA of five other early-region deletion mutants that do not make stable truncated forms of T antigen. Since all five of these mutants have intact early- and late-region control sequences, we conclude that these control sequences are not sufficient for immortalization. Three of the mutants that did not immortalize did make a normal small t antigen, suggesting that the expression of this protein alone is not sufficient for immortalization of early-passage Fisher rat embryo cells.
Cloning, sequencing, and functional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis.
Authors: Weller S K , Spadaro A , Schaffer J E , Murray A W , Maxam A M , Schaffer P A
Published: September 2015

The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of oriS located within the repeated sequences flanking the short unique arm (US), and one copy of oriL located within the long unique arm (UL). Precise localization and characterization of oriL have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to oriS. In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.
Ubiquitin is a heat shock protein in chicken embryo fibroblasts.
Authors: Bond U , Schlesinger M J
Published: September 2015

Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.
Specific regulation of transcription of the discoidin gene family in Dictyostelium discoideum.
Authors: Alexander S , Shinnick T M
Published: September 2015

Dictyostelium discoideum strains that carry the dis mutations fail to express the family of developmentally regulated discoidin lectin genes during morphogenesis. We show here that this absence of discoidin lectin expression is due to the failure to transcribe the discoidin genes. Furthermore, the dis mutations appear to affect only discoidin expression and not the expression of other proteins during development, as assessed by a two-dimensional gel analysis of pulse-labeled proteins and by the accumulation of developmentally regulated enzymes. The dis mutations appear to define trans-acting regulatory loci, the products of which act at the transcriptional level to control specifically the developmental expression of the discoidin gene family.
Calcium ionophore A23187 induces expression of glucose-regulated genes and their heterologous fusion genes.
Authors: Resendez E , Attenello J W , Grafsky A , Chang C S , Lee A S
Published: September 2015

Using two cDNA clones which encode hamster genes specifically induced by glucose starvation, we demonstrated that an 8- and 30-fold increase, respectively, in the transcription rates of these genes was coordinately effected by calcium ionophore A23187 treatment, resulting in a similar increase in the steady-state levels of their mRNAs. This response was observed within several hours of ionophore treatment in several mammalian cell types and appeared to be specifically mediated by A23187 but not by other ionophores in general. To define the regulatory sequence which mediates this Ca2+-induced response, we showed by gene transfection techniques that the 5' flanking sequence of a rat glucose-regulated gene contained the region for induction by A23187. The system reported here offers attractive features for the study of specific gene regulation by Ca2+.
Interrelationships of protein and DNA syntheses during replication of mammalian cells.
Authors: Sariban E , Wu R S , Erickson L C , Bonner W M
Published: September 2015

During the replication of chromatin, the syntheses of the histone protein and DNA components are closely coordinated but not totally linked. The interrelationships of total protein synthesis, histone protein synthesis, DNA synthesis, and mRNA levels have been investigated in Chinese hamster ovary cells subjected to several different types of inhibitors in several different temporal combinations. The results from these studies and results reported elsewhere can be brought together into a consistent framework which combines the idea of autoregulation of histone biosynthesis as originally proposed by W. B. Butler and G. C. Mueller (Biochim. Biophys. Acta 294:481-496, 1973] with the presence of basal histone synthesis and the effects of protein synthesis on DNA synthesis. The proposed framework obviates the difficulties of Butler and Mueller's model and may have wider application in understanding the control of cell growth.
Structure of a murine alpha interferon pseudogene with a repetitive R-type sequence in the 3' flanking region.
Authors: Le Roscouet D , Vodjdani G , Lemaigre-Dubreuil Y , Tovey M G , Latta M , Doly J
Published: September 2015

A murine alpha interferon pseudogene was identified in a mouse genomic library. The nucleotide sequence revealed several in-phase termination codons within the gene and repetitive oligonucleotides in the flanking regions. The nucleotide sequences and the amino acids of the peptide signal sequences were compared with known human alpha interferon genes and the pseudogene.
Structural and evolutionary relationships among five members of the human gamma-crystallin gene family.
Authors: Meakin S O , Breitman M L , Tsui L C
Published: September 2015

We have characterized five human gamma-crystallin genes isolated from a genomic phage library. DNA sequencing of four of the genes revealed that two of them predict polypeptides of 174 residues showing 71% homology in their amino acid sequence; the other two correspond to closely related pseudogenes which contain the same in-frame termination codon at identical positions in the coding sequence. Two of the genes and one of the pseudogenes are oriented in a head-to-tail fashion clustered within 22.5 kilobases. All three contain a TATA box 60 to 80 base pairs upstream of the initiation codon and a highly conserved segment of 44 base pairs in length immediately preceding the TATA box. The two genes and the two pseudogenes are similar in structure: each contains a small 5' exon encoding three amino acids followed by two larger exons that correspond exactly to the two similar structural domains of the polypeptide. The first intron varies from 100 to 110 base pairs, and the second intron ranges from 1 to several kilobases, rendering an overall gene size of 1.7 to 4.5 kilobases. At least one of the two pseudogenes appears to have been functional before inactivation, suggesting that their identical mutation was generated by gene conversion.
Collagen expression in embryonic chicken chondrocytes treated with phorbol myristate acetate.
Authors: Finer M H , Gerstenfeld L C , Young D , Doty P , Boedtker H
Published: September 2015

Growth of embryonic chicken sternal chondrocytes in the presence of phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, resulted in a dramatic morphological change from spherical floating cells to adherent fibroblastic cells. This morphological change was accompanied by a quantitative switch from synthesis of cartilage-specific type II procollagen to type I procollagen. Type II procollagen mRNA levels decreased 10-fold in PMA-treated cells. Activation of type I collagen genes led to the accumulation of type I procollagen mRNA levels comparable to those of type II mRNA in these cells. However, only type I procollagen mRNA was translated. In addition to gene activation, unprocessed pro alpha 1(I) transcripts present at low levels in control chondrocytes were processed to mature mRNA species. Redifferentiation of PMA-treated chondrocytes was possible if cells were removed from PMA after the morphological change and cessation of type II procollagen synthesis but before detectable amounts of type I procollagen were synthesized. Production of type I collagen thus marks a late phase of chondrocyte "dedifferentiation" from which reversion is no longer possible. Redifferentiated cell populations contained 24-fold more pro alpha 1(II) collagen mRNA than pro alpha 1(I) collagen mRNA, but the rates of procollagen synthesis were comparable. This suggests that the PMA-mediated dedifferentiation of chondrocytes as well as their redifferentiation is under both transcriptional and posttranscriptional regulation.
Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody.
Authors: Hattori S , Ulsh L S , Halliday K , Shih T Y
Published: September 2015

The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli. We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant. The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase. Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 X 10(-8)M for GTP and 1.0 X 10(-8)M for GDP. The binding site can be specifically labeled with a [3H]GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP. To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities. We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule. These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature [London] 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function.
Structure of the promoter of the Dictyostelium discoideum prespore EB4 gene.
Authors: Barklis E , Pontius B , Barfield K , Lodish H F
Published: September 2015

EB4 is one of several cloned cDNAs that is expressed as mRNA only after the aggregation stage of Dictyostelium discoideum differentiation and exclusively in prespore and spore cells (E. Barklis and H. F. Lodish, Cell 32:1139-1148, 1983). We have isolated the unique genome fragment corresponding to the 5' portion of the EB4 message and the EB4 promoter. The EB4 transcript has an unusually long, G + C-rich, 5' noncoding region, but initiates at several start sites within a region of DNA that is 96% A + T. The sequence GTGGTGG, along with slight variations, occurs several times in the promoter. We have used the EB4 promoter to drive the transcription of an EB4/beta-galactosidase fusion transcript in yeast cells. Although the cap sites of the fused transcript in yeast cells are located in the region where multiple EB4 transcripts are initiated in Dictyostelium, the unregulated expression of the fusion transcript in yeast does not mimic the normal regulated pattern of EB4 mRNA expression in D. discoideum.