bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.
Authors: Stringer J R
Published: September 2015

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.
Locus encoding a family of small heat shock genes in Caenorhabditis elegans: two genes duplicated to form a 3.8-kilobase inverted repeat.
Authors: Russnak R H , Candido E P
Published: September 2015

The genes coding for hsp 16-48, previously identified by cDNA cloning, and for another 16-kilodalton heat shock protein designated hsp16-1 were characterized by DNA sequencing. The two genes were arranged in a head-to-head orientation. Both the coding and flanking regions were located within a 1.9-kilobase module which was duplicated exactly to form a 3.8-kilobase inverted repeat structure. The inverted repeat structure ended in an unusual guanine-plus-cytosine-rich sequence 24 nucleotides in length. The identity of the two modules at the nucleotide sequence level implies that the duplication event may have occurred recently. Alternatively, gene conversion between the two modules could also maintain homology of the two gene pairs. The small heat shock genes of Caenorhabditis elegans contained TATA boxes and heat-inducible promoters, the latter agreeing closely with the Drosophila melanogaster consensus sequence described by Pelham (Cell 30:517-528, 1982). Unlike the homologous D. melanogaster genes, each of these C. elegans genes contained a short intron, the position of which has been conserved in a related murine alpha-crystallin gene. The intron separated variable and conserved regions within the amino acid sequences of the encoded heat shock polypeptides.
Expression of a human U1 RNA gene introduced into mouse cells via bovine papillomavirus DNA vectors.
Authors: Schenborn E T , Lund E , Mitchen J L , Dahlberg J E
Published: September 2015

We introduced a gene for human U1 small nuclear RNA, HU1-1, into mouse C127 cells via bovine papillomavirus (BPV) vectors. After transfection, up to 15% of the total U1 RNA in transformed cells was encoded by the introduced human genes. High levels of expression of the human gene were observed when the recombinant viral DNAs were maintained either as plasmids or after integration into high-molecular-weight DNA. As few as 400 and 35 base pairs of 5' and 3' flanking region sequences, respectively, were sufficient for transcription of human U1 RNA, and no increase in the level of expression was observed with HU1-1 DNA containing several kilobases of flanking region sequences. Several of the transformed cell lines contained the recombinant BPV DNA apparently integrated into the host genome. Integration or rearrangement or both of the U1-BPV DNA was promoted when the HU1-1 gene was positioned at the BamHI site downstream of the BPV transforming region. At least two variants of the U1-BPV DNAs were able to cause morphological transformation of cells despite the fact that these DNAs lacked a BPV transcriptional enhancer element.
Nucleotide sequence analysis and enhancer function of long terminal repeats associated with an endogenous African green monkey retroviral DNA.
Authors: Kessel M , Khan A S
Published: September 2015

The nucleotide sequence and enhancer activity of the long terminal repeats (LTRs) associated with a cloned endogenous African green monkey (AGM) retroviral DNA designated as lambda-AGM-1 was studied. A unique feature of the endogenous AGM proviral LTRs was the presence of multiple copies of two types of directly repeating units in the U3 region: 16 8-base-pair (bp) repeats were present in the 5' LTR and 12 were present in the 3' LTR which were bound by a 6-bp perfect direct repeat; tandem duplication of a 32-bp sequence resulted in 3.5 copies in the 5' LTR and 2.5 copies in the 3' LTR. Nucleotide sequence homology was seen between the 8-bp direct repeats located in the AGM proviral LTRs and a 10-bp repeat unit of the deca-satellite present in AGM cellular DNA. The 32-bp repeats of the AGM proviral LTRs contained sequences which were related to the SV40 21-bp repeats and to the "core" of the SV40 72-bp enhancer element. Furthermore, the AGM provirus was distinct from known infectious retroviruses due to the presence of a primer-binding sequence complementary to the 3' terminus of mammalian tRNAGly. Functional analysis of the 3' LTR present in lambda-AGM-1 DNA by chloramphenicol acetyltransferase assay demonstrated enhancer activity associated with the 32-bp direct repeats. Sequences outside the 32-bp unit were necessary for full activator function, suggesting the presence of multiple enhancer domains in the AGM provirus.
Structure and developmental regulation of a wheat gene encoding the major chlorophyll a/b-binding polypeptide.
Authors: Lamppa G K , Morelli G , Chua N H
Published: September 2015

A genomic clone for a major chlorophyll a/b-binding polypeptide of the light-harvesting complex has been sequenced from wheat. This gene, whAB1.6, encodes a 70-nucleotide 5'-nontranslated spacer, a 34-amino-acid NH2-terminal extension, i.e., the transit peptide, and a mature coding protein of 232 amino acid residues. The exact molecular weight of the precursor polypeptide is 28,560. The transit peptide is basic and is rich in serines. No intervening sequences are found in this gene. The transcription start site of the whAB1.6 gene occurs at AAAC as determined by S1 nuclease analysis. Putative regulatory sequences occur upstream of the gene at -25 (TTTAAATA) and at -72 (CCAACCA). Northern blots show a single RNA species estimated to be 1,100 nucleotides. Heterogeneity of the RNA population is demonstrated in S1 nuclease analyses with a 5'-end-labeled fragment that extends 191 nucleotides into the mature protein coding sequence. At least seven different transcripts can be recognized. The highest levels of RNA transcribed from the whAB1.6 gene are found in the basal segments of the wheat leaf, whereas other chlorophyll a/b-binding transcripts in the cell show a different pattern of abundance. As a control, we show that roots do not contain chlorophyll a/b-binding RNA. The most abundant RNA species shows an interrupted homology with the whAB1.6 gene at the start of the mature protein coding sequence; another species shows homology beginning at the start of the transit peptide and does not include the nontranslated region. Chlorophyll a/b-binding polypeptides accumulate toward the tip of the leaf as shown by Western blot analysis of total thylakoid proteins.
H-2Ld antigen encoded by a recombinant retrovirus genome is expressed on the surface of infected cells.
Authors: Weis J H , Reiss C S , Mulligan R C , Burakoff S J , Seidman J G
Published: September 2015

A recombinant murine retrovirus was constructed which contains, within its genome, a truncated version of the gene encoding the murine H-2Ld major histocompatibility antigen. The H-2Ld gene, which was inserted 3' of the env splice acceptor site in the recombinant retrovirus MSV-neo, lacked the 5' promoter and TATA sequences and the 3' transcription termination and polyadenylate addition sites of the normal H-2Ld gene. Transfection of the MSV-neo/H-2Ld plasmid (pLTV-11) into Y-2 cells resulted in the production of the transmissible recombinant retrovirus LTV-11. Cells infected with LTV-11 virus were resistant to the eucaryotic antibiotic G418 and expressed H-2Ld on the cell surface. These infected cells contained a viral RNA species which possessed both the H-2Ld and the neomycin resistance gene sequences but did not contain significant levels of the smaller H-2Ld-specific mRNA. The H-2Ld antigen expressed on the surface of infected cells functioned as a target for cytolytic T cells specific for the H-2Ld antigen.
trans Activation of the simian virus 40 late transcription unit by T-antigen.
Authors: Brady J , Khoury G
Published: September 2015

We have investigated the role of simian virus 40 (SV40) T-antigen in the induction of late gene expression independent of its function in amplifying templates through DNA replication. Northern blot and S1 nuclease analyses showed that stimulation occurred at the transcriptional level. At least two template elements, the T-antigen-binding sites and the 72-base-pair repeats, appeared to be important for this induction. Using template mutants, we demonstrated that deletions within T-antigen-binding site II decreased T-antigen-mediated late gene expression approximately 10- to 20-fold. In addition, multiple point mutations within a single retained copy of the SV40 72-base-pair repeat decreased T-antigen-mediated late gene expression. Using in vivo competition studies, we demonstrated that competitor DNA fragments containing the SV40 control region (nucleotides 5171 through 272) quantitatively decreased SV40 late gene expression in COS-1 cells. In contrast, competition with a plasmid containing SV40 nucleotides 1 through 294 (which removes all of T-antigen-binding site I and half of site II) was much less efficient. Finally, we demonstrated that in vivo competition experiments employing competitor fragments distal to the T-antigen-binding sites within the late template region (SV40 nucleotides 180 through 2533) resulted in superinduction of late gene expression in COS-1 cells. This finding suggests that negative factors such as repressors or attenuators may modulate late SV40 gene expression before induction. Our results are consistent with a model in which induction of late gene expression involves an interaction of the SV40 origin region with DNA-binding proteins, one of which may be T-antigen. Activation of the SV40 late transcription unit may involve induction of the SV40 enhancer or removal of a repressor-like protein or both.
Altered beta-actin gene expression in phorbol myristate acetate-treated chondrocytes and fibroblasts.
Authors: Gerstenfeld L C , Finer M H , Boedtker H
Published: September 2015

Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, was shown to have opposite effects on the cellular morphology and steady-state levels of beta-actin mRNA in embryonic chicken muscle fibroblasts and sternal chondrocytes. When fibroblasts were treated with PMA, they formed foci of densely packed cells, ceased to adhere to culture plates, and had significantly reduced levels of beta-actin mRNA and protein. Conversely, when treated with PMA, floating chondrocytes attached to culture dishes, spread out, and began to accumulate high levels of beta-actin mRNA and proteins. In the sternal chondrocytes the stimulation of the beta-actin mRNA production was accompanied by increased steady-state levels of fibronectin mRNAs and protein. These alterations were concomitant with a fivefold reduction in type II collagen mRNA and a cessation in its protein production. After fibronectin and actin mRNAs and proteins reached their maximal levels, type I collagen mRNA and protein synthesis were turned on. Removal of PMA resulted in reduced beta-actin mRNA levels in chondrocytes and in a further alteration in the cell morphology. These observed correlations between changes in cell adhesion and morphology and beta-actin expression suggest that the effect of PMA on cell shape and adhesion may result in changes in the microfilament organization of the cytoskeleton which ultimately lead to changes in the extracellular matrix produced by the cells.
Accurate and efficient transcription of human c-myc genes injected into Xenopus laevis oocytes.
Authors: Nishikura K , Goldflam S , Vuocolo G A
Published: September 2015

We investigated the expression of the cloned human c-myc gene in Xenopus laevis oocytes microinjected with different recombinants. We found that microinjected plasmid DNA carrying an intact human c-myc gene directs efficient and faithful transcription from its own two promoters in X. laevis oocytes. This active transcription was unaffected by the presence of previously identified enhancing elements such as simian virus 72-base pair repeats or mouse immunoglobulin heavy-chain gene enhancer sequences in the construct in cis. This suggests that all necessary DNA sequences for accurate and faithful transcription recognized by the transcription machinery of the frog oocyte are self-contained. In addition, we have found that human c-myc transcripts synthesized in oocytes are properly polyadenylated at either one of two sites and also that the transcripts are spliced correctly but with low efficiency.
Incorporation of a charged amino acid into the membrane-spanning domain blocks cell surface transport but not membrane anchoring of a viral glycoprotein.
Authors: Adams G A , Rose J K
Published: September 2015

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G protein) consists of a continuous stretch of 20 uncharged and mostly hydrophobic amino acids. We examined the effects of two mutations which change the amino acid sequence in this domain. These mutations were generated by oligonucleotide-directed mutagenesis of a cDNA clone encoding the G protein, and the altered G proteins were then expressed in animal cells. Replacement of an isoleucine residue in the center of this domain with a strongly polar but uncharged amino acid (glutamine) had no effect on membrane anchoring or transport of the protein to the cell surface. Replacement of this same isoleucine residue with a charged amino acid (arginine) generated a G protein that still spanned intracellular membranes but was not transported efficiently to the cell surface. The protein accumulated in the Golgi region in about 50% of the cells, and about 20% of the cells had detectable protein levels in a punctate pattern on the cell surface. In the remaining cells the protein accumulated in a vesicular pattern throughout the cytoplasm. Models which might explain the abnormal behavior of this protein are discussed.
Induction of cellular thymidine kinase occurs at the mRNA level.
Authors: Stuart P , Ito M , Stewart C , Conrad S E
Published: September 2015

The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- cells with human chromosomal DNA, and genomic libraries were made in lambda Charon 30 from a second-round TK+ transformant. When the library was screened with a human Alu probe, seven overlapping lambda clones from the human TK locus were obtained. None of the seven contained a functional TK gene as judged by transfection analysis, but several combinations of clones gave rise to TK+ colonies when cotransfected into TK- cells. A functional cDNA clone encoding the human TK gene has also been isolated. Using this cDNA clone as a probe in restriction enzyme/blot hybridization analyses, we have mapped the coding sequences and direction of transcription of the gene. We have also used a single-copy subclone from within the coding region to monitor steady-state levels of TK mRNA in serum-stimulated and simian virus 40-infected simian CV1 tissue culture cells. Our results indicate that the previously reported increase in TK enzyme levels seen after either treatment is paralleled by an equivalent increase in the steady-state levels of TK mRNA. In the case of simian virus 40-infected cells, the induction was delayed by 8 to 12 h, which is the length of time after infection required for early viral protein synthesis. In both cases, induction of TK mRNA coincides with the onset of DNA synthesis, but virally infected cells ultimately accumulate more TK mRNA than do serum-stimulated cells.
Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover.
Authors: Warner J R , Mitra G , Schwindinger W F , Studeny M , Fried H M
Published: September 2015

The rate of accumulation of each ribosomal protein is carefully regulated by the yeast cell to provide the equimolar ratio necessary for the assembly of the ribosome. The mechanisms responsible for this regulation have been examined by introducing into the yeast cell extra copies of seven individual ribosomal protein genes carried on autonomously replicating plasmids. In each case studied the plasmid-borne gene was transcribed to the same degree as the genomic gene. Nevertheless, the cell maintained a balanced accumulation of ribosomal proteins, using a variety of methods other than transcription. (i) Several ribosomal proteins were synthesized in substantial excess. However, the excess ribosomal protein was rapidly degraded. (ii) The excess mRNA for two of the ribosomal protein genes was translated inefficiently. We provide evidence that this was due to inefficient initiation of translation. (iii) The transcripts derived from two of the ribosomal protein genes were spliced inefficiently, leading to an accumulation of precursor RNA. We present a model which proposes the autogenous regulation of mRNA splicing as a eucaryotic parallel of the autogenous regulation of mRNA translation in procaryotes. Finally, the accumulation of each ribosomal protein was regulated independently. In no instance did the presence of excess copies of the gene for one ribosomal protein affect the synthesis of another ribosomal protein.
High-resolution mapping of S1- and DNase I-hypersensitive sites in chromatin.
Authors: Weintraub H
Published: September 2015

A new and easy technique for accurately mapping DNase I- and S1 nuclease-hypersensitive sites is described. The technique is a modification of primer extension and S1 nuclease methods conventionally used to map RNA ends.
Expression of ras cellular oncogenes during development of Drosophila melanogaster.
Authors: Lev Z , Kimchie Z , Hessel R , Segev O
Published: September 2015

The transcription patterns of three v-Ha-ras-related cellular oncogenes in Drosophila melanogaster were studied. Each gene coded for at least two distinct transcripts. The larger transcript of each gene was expressed at a similar abundance during the entire life cycle of fruit flies, whereas the shorter transcripts were much more abundant in embryonic stages than at later stages.
Gene disruption by transformation in Neurospora crassa.
Authors: Paietta J V , Marzluf G A
Published: September 2015

To establish conditions which might permit deliberate gene disruptions in Neurospora crassa, we studied transformation with linear DNA fragments. The transformation frequency observed was increased about twofold in comparison with that obtained with circular plasmid DNA. However, only a low proportion, approximately 10%, of the integration events occurred at the homologous site, whereas most integrations of transforming DNA took place in nonhomologous regions. It was also found that multiple integration events frequently occurred in individual transformants. A plasmid, designated pJP12, was constructed that contains the N. crassa am+ gene interrupted by insertion into its coding region of a DNA segment carrying a functional Neurospora qa-2+ gene. A fragment of Neurospora DNA that contains this am qa-2+ construction was obtained from plasmid pJP12 and used to transform an am+ qa-2 strain in an attempt to disrupt the resident am+ gene. After the initial qa-2+ transformants were converted to homokaryons by appropriate crosses, 10 independent transformants with an am mutant phenotype were found among 117 examined. Each of these qa-2+ am transformants showed the loss of a hybridization band in Southern blots of genomic DNA that corresponded to the normal am+ gene and the presence of a new hybridization band, consistent with an alteration in the am+ region.
Eucaryotic transcription complexes are specifically associated in large sedimentable structures: rapid isolation of polymerase I, II, and III transcription factors.
Authors: Culotta V C , Wides R J , Sollner-Webb B
Published: September 2015

RNA synthesis in eucaryotes takes place on template molecules that are activated by stably associating with limiting transcription factors. In this paper we demonstrate that such stable transcription complexes can be specifically sedimented from in vitro transcription reaction mixtures by mild centrifugation. This occurs with stable complexes of genes transcribed by all three classes of eucaryotic RNA polymerase and with S-100 as well as whole-cell extracts. However, the transcriptional capacity of the isolated complex differs for the three polymerase classes. The pelleted ribosomal DNA (polymerase I) complex contains all the factors necessary for transcription, each purified 25- to 50-fold, whereas the pelleted adenovirus major late promoter (polymerase II) complex lacks a factor that remains in the supernatant. In the case of 5S DNA (polymerase III), a necessary factor associates slowly with the sedimentable complex. Notably, the interactions responsible for this rapid sedimentation are specific for DNA molecules in stable complexes, suggesting that the in vitro sedimentable complex mirrors the in vivo structural organization of active genes.
Reconstruction of appropriate tubulin and actin gene regulation after transient transfection of cloned beta-tubulin and beta-actin genes.
Authors: Lau J T , Pittenger M F , Cleveland D W
Published: September 2015

Most animal cells rapidly depress the synthesis of new alpha- and beta-tubulin polypeptides in response to microtubule inhibitors that increase the pool of depolymerized subunits. This apparent autoregulatory control of tubulin synthesis is achieved through the modulation of tubulin mRNA levels. To begin to analyze the molecular mechanism responsible for such regulation, we have introduced exogenous beta-tubulin gene sequences into cultured mouse cells by DEAE-dextran-mediated DNA transfection. We find that the heterologous tubulin genes are expressed and that their RNA transcripts are accurately processed to mature mRNAs. Moreover, after drug-induced microtubule depolymerization, the expression of unintegrated tubulin gene sequences is regulated coordinately with the endogenous mouse alpha- and beta-tubulin RNA transcripts. Such regulation appears to be specific for transfected tubulin genes, since similar down-regulation is not observed in a contransfected beta-actin gene. Curiously, in response to microtubule depolymerization, the amount of RNA transcripts from a transfected beta-actin gene increases twofold, which qualitatively and quantitatively parallels that seen by the RNAs encoded by the endogenous actin genes. Thus, the transient DNA transfection approach may permit the unambiguous elucidation of regulatory sequences involved in establishing the proper level of expression of these two important cytoskeletal gene families.
Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.
Authors: Waters S H , Distel R J , Hecht N B
Published: September 2015

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.
Effect of ARS1 mutations on chromosome stability in Saccharomyces cerevisiae.
Authors: Srienc F , Bailey J E , Campbell J L
Published: September 2015

We have used a set of deletion mutations in the ARS1 element of Saccharomyces cerevisiae to measure their effect on chromosome stability. This work establishes the previously proposed existence of three domains in ARS1. Domain C, which we have previously inferred, but not proved, to be a part of ARS1, is now established. In addition, we show that increasingly large deletions of the domain have increasingly large effects, which was not realized before. Furthermore, we have provided the first positive evidence for the central importance of a 14-base-pair core sequence containing the ARS consensus element by showing that it has the ability to act as a replicator on a plasmid containing no other ARS1 flanking sequence. The method of analyzing plasmid stability used in our study employs a novel and sensitive flow cytometry assay for beta-galactosidase. We discuss ways in which flow cytometry, based on this assay, could be generalized beyond its particular application in this work to studying other aspects of the cell biology of yeast and higher cells. The actual flow cytometry method will be described in detail elsewhere.
Characterization of the functional gene and several processed pseudogenes in the human triosephosphate isomerase gene family.
Authors: Brown J R , Daar I O , Krug J R , Maquat L E
Published: September 2015

The functional gene and three intronless pseudogenes for human triosephosphate isomerase were isolated from a recombinant DNA library and characterized in detail. The functional gene spans 3.5 kilobase pairs and is split into seven exons. Its promoter contains putative TATA and CCAAT boxes and is extremely rich in G and C residues (76%). The pseudogenes share a high degree of homology with the functional gene but contain mutations that preclude the synthesis of an active triosephosphate isomerase enzyme. Sequence divergence calculations indicate that these pseudogenes arose approximately 18 million years ago. We present evidence that there is a single functional gene in the human triosephosphate isomerase gene family.
Neoplastic transformation of normal and carcinogen-induced preneoplastic Syrian hamster embryo cells by the v-src oncogene.
Authors: Gilmer T M , Annab L A , Oshimura M , Barrett J C
Published: September 2015

The ability of cloned Rous sarcoma virus (RSV) DNA encoding the v-src oncogene to neoplastically transform normal, diploid Syrian hamster embryo (SHE) cells was examined. Transfection of RSV DNA into early passage SHE cells resulted in a low but significant number of tumors when treated cells were injected into nude mice. Tumors formed with a low frequency (two tumors out of ten sites injected) and only after a long latency period (14 weeks). In contrast to the normal SHE cells, several different carcinogen-induced preneoplastic immortal SHE cell lines were highly susceptible to transformation by the v-src oncogene to the neoplastic phenotype. Tumors formed with high efficiency and a short latency period (less than 3 weeks). Further studies were performed to determine the basis for the inefficient transformation of the normal SHE cells. NeoR clones isolated after cotransfection of SHE cells with pSV2-neo and RSV DNAs were neither morphologically altered nor immortal and did not contain detectable levels of the v-src gene product. These results suggest that neoplastic transformation by v-src DNA in the normal cells is initially suppressed. However, cells from a v-src-induced tumor expressed v-src RNA, and antibody to v-src protein precipitated from the tumor cells a 60,000-molecular-weight protein which displayed protein kinase activity. Karyotypic analyses confirmed that the tumor was derived from Syrian hamster cells and suggested that it was clonal in nature. These results indicate that the v-src oncogene was primarily responsible for neoplastic transformation of SHE cells. In contrast to the results with the v-src oncogene, our previous studies showed that v-Ha-ras oncogene alone is unable to induce neoplastic transformation of SHE cells. Furthermore, the v-myc oncogene was able to compliment v-Ha-ras to neoplastically transform SHE cells, while cotransfection with v-src plus v-myc did not increase the incidence of tumors.
Construction of a cDNA to the hamster CAD gene and its application toward defining the domain for aspartate transcarbamylase.
Authors: Shigesada K , Stark G R , Maley J A , Niswander L A , Davidson J N
Published: September 2015

cDNA complementary to hamster mRNA encoding the CAD protein, a multifunctional protein which carries the first three enzymes of pyrimidine biosynthesis, was constructed. The longest of these recombinants (pCAD142) covers 82% of the 7.9-kilobase mRNA. Portions of the cDNA were excised and replaced by a lac promoter-operator-initiation codon segment. The resultant plasmids were transfected into an Escherichia coli mutant defective in aspartate transcarbamylase, the second enzyme of the pathway. Complementation of the bacterial defect was observed with as little as 2.2 kilobases of cDNA sequence, corresponding to the 3' region of the mRNA. DNA sequencing in this region of the hamster cDNA reveals stretches which are highly homologous to the E. coli gene for the catalytic subunit of aspartate transcarbamylase; other stretches show no homology. The highly conserved regions probably reflect areas of protein structure critical to catalysis, while the nonconserved regions may reflect differences between the quaternary structures of E. coli and mammalian aspartate transcarbamylases, one such difference being that the bacterial enzyme in its native form is allosterically regulated and the mammalian enzyme is not.
Erbb is linked to the alpha-globin locus on mouse chromosome 11.
Authors: Silver J , Whitney J B , Kozak C , Hollis G , Kirsch I
Published: September 2015

A fragment of the human gene for c-erb-B was used to map homologous sequences in mice. Analysis of somatic cell hybrids and recombinant inbred and congenic mouse strains indicated that this gene, designated Erbb, is closely linked to the gene for alpha-globin on mouse chromosome 11. Several genes controlling hematopoietic differentiation map to mouse chromosome 11.
Linear simian virus 40 DNA fragments exhibit a propensity for rolling-circle replication.
Authors: Deichaite I , Laver-Rudich Z , Dorsett D , Winocour E
Published: September 2015

A linear simian virus 40 origin-containing DNA fragment replicated in monkey COS cells, generating tandemly repeated (head-to-tail) structures. Electron microscopy revealed circle-and-tail configurations characteristic of rolling-circle replication intermediates. Circularization of the same DNA before transfection led to a theta type of replication which generated supercoiled DNA molecules.
Large-scale production of polyoma middle T antigen by using genetically engineered tumors.
Authors: Kaplan D R , Bockus B , Roberts T M , Bolen J , Israel M , Schaffhausen B S
Published: September 2015

A recombinant plasmid containing a metallothionein promoter-polyoma middle T cDNA fusion was constructed and used to transfect NIH 3T3 cells. Transformed cells expressing middle T were injected into nude mice. Within 3 weeks, each mouse produced tumors containing middle T equivalent to that in 250 to 1,000 100-mm dishes of polyomavirus-infected cells. This middle T, partially purified by immunoaffinity chromatography, retained activity as measured by its ability to be phosphorylated in vitro. The combined approach of fusing strong promoters to genes of interest and utilizing nude mice to grow large quantities of cells expressing the gene provides a quick, inexpensive alternative to other expression systems.
Activation of Ha-ras p21 by substitution, deletion, and insertion mutations.
Authors: Chipperfield R G , Jones S S , Lo K M , Weinberg R A
Published: September 2015

The transforming activity of naturally arising ras oncogenes results from point mutations that affect residue 12 or 61 of the encoded 21-kilodalton protein (p21). By use of site-directed mutagenesis, we showed that deletions and insertions of amino acid residues in the region of residue 12 are also effective in conferring oncogenic activity on p21. Common to these various alterations is the disruption that they create in this domain of the protein, which we propose results in the inactivation of a normal function of the protein.
Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.
Authors: Crouse G F , Leys E J , McEwan R N , Frayne E G , Kellems R E
Published: September 2015

The use of murine dihydrofolate reductase (dhfr) gene amplification mutants enabled us to identify important structural and functional features of the dhfr promoter region. We found another transcription unit, at least 14 kilobases in size, which initiates within 130 base pairs of the major dhfr transcript and is transcribed divergently. The 5' ends of both transcripts were analyzed and found to have multiple initiation sites. The major dhfr transcript and the divergent transcript appear to share the same promoter region; the longer transcripts of the dhfr gene overlap with the divergent transcripts and use a different promoter region. The divergent transcript appears to code for a protein; an homologous sequence to its first exon is found in the corresponding location near the human dhfr gene.
Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation.
Authors: Keller J M , Alwine J C
Published: September 2015

The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.
Control functions of adenovirus transformation region E1A gene products in rat and human cells.
Authors: Bellett A J , Li P , David E T , Mackey E J , Braithwaite A W , Cutt J R
Published: September 2015

Altered control of the rat cell cycle induced by adenovirus requires expression of transformation region E1A, but not of E1B, E2A, E2B, or late genes. We show here that neither E3 nor E4 is required, so the effect results directly from an E1A product. Mutants with defects in the 289-amino-acid (aa) E1A product had little or no effect on the rat cell cycle even at 1,000 IU per cell. A mutant (pm975) lacking the 243-aa E1A product altered cell cycle progression, but less efficiently than did wild-type virus. The 289-aa E1A protein is therefore essential for cell cycle effects; the 243-aa protein is also necessary for the full effect but cannot act alone. Mutants with altered 289-aa E1A proteins showed different extents of leak expression of viral early region E2A as the multiplicity was increased; each leaked more in human than in rat cells. dl312, with no E1A products, failed to produce E2A mRNA or protein at 1,000 IU per cell in rat cells but did so in some experiments in human cells. There appears to be a very strict dependence of viral early gene expression on E1A in rat cells, whereas dependence on E1A is more relaxed in HeLa cells, perhaps due to a cellular E1A-like function. Altered cell cycle control is more dependent on E1A function than is early viral gene expression.
Expression of human beta-globin genes in transgenic mice: effects of a flanking metallothionein-human growth hormone fusion gene.
Authors: Townes T M , Chen H Y , Lingrel J B , Palmiter R D , Brinster R L
Published: September 2015

In an attempt to place a human beta-globin gene in an open chromatin domain regardless of its site of integration in the mouse genome, we microinjected into fertilized mouse eggs a construct in which the human beta-globin gene and a mouse metallothionein-human growth hormone fusion gene were juxtaposed and oriented in opposite directions. Mice that developed from injected eggs and that grew larger than normal were analyzed for human beta-globin mRNA. The globin genes were not expressed in erythroid tissue but were expressed with the same tissue specificity as metallothionein-human growth hormone. These results suggest that sequences which control metallothionein-human growth hormone gene expression are capable of stimulating the expression of a flanking gene in an orientation-independent and tissue-specific manner. As a control for this experiment, we deleted the metallothionein-human growth hormone transcription unit and noted that the human beta-globin gene then was expressed at high levels with erythroid tissue specificity.
Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.
Authors: Michaeli T , Prives C
Published: September 2015

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.
Reproducible and variable genomic rearrangements occur in the developing somatic nucleus of the ciliate Tetrahymena thermophila.
Authors: Howard E A , Blackburn E H
Published: September 2015

We analyzed the extent, reproducibility, and developmental control of genomic rearrangements in the somatic macronucleus of the ciliate Tetrahymena thermophila. To exclude differences caused by genetic polymorphisms, we constructed whole-genome homozygotes, and we compared the homozygous progeny derived from single macronuclear differentiation events. This strategy enabled us to identify a novel form of variable rearrangement and to confirm previous findings that rearranged sequences occur at a high frequency in the Tetrahymena genome. Rearrangements studied here were deletions of both unique and interchromosomally dispersed repetitive DNA sequences involving DNA rejoining of internal, nontelomeric regions of macronuclear DNAs. We showed that although rearrangements of some sequence classes are reproducible among independently developed macronuclei, other specific sequence classes are variably rearranged in macronuclear development. The variable somatic genomes so produced may be the source of phenotypically variant cell lines.
Induction of acquired thermotolerance in Tetrahymena thermophila: effects of protein synthesis inhibitors.
Authors: Hallberg R L , Kraus K W , Hallberg E M
Published: September 2015

When Tetrahymena thermophila cells growing at 30 degrees C are shifted to either 40 or 43 degrees C, the kinetics and extent of induction of heat shock mRNAs in both cases are virtually indistinguishable. However, the cells shifted to 40 degrees C show a typical induction of heat shock protein (HSP) synthesis and survive indefinitely (100% after 24 h), whereas those at 43 degrees C show an abortive synthesis of HSPs and die (less than 0.01% survivors) within 1 h. Cells treated at 30 degrees C with the drugs cycloheximide or emetine, at concentrations which are initially inhibitory to protein synthesis and cell growth but from which cells can eventually recover and resume growth, are after this recovery able to survive a direct shift from 30 to 43 degrees C (ca. 70% survival after 1 h). This induction of thermotolerance by these drugs is as efficient in providing thermoprotection to cells as is a prior sublethal heat treatment which elicits the synthesis of HSPs. However, during the period when drug-treated cells recover their protein synthesis ability and simultaneously acquire the ability to subsequently survive a shift to 43 degrees C, none of the major HSPs are synthesized. The ability to survive a 1-h, 43 degrees C heat treatment, therefore, does not absolutely require the prior synthesis of HSPs. But, as extended survival at 43 degrees Celsius depends absolutely on the ability of cells to continually synthesize HSPs, it appears that a prior heat shock as well as the recovery from protein synthesis inhibition elicits a change in the protein synthetic machinery which allows the translation of HSP mRNAs at what would otherwise be a nonpermissive temperature for protein synthesis.
Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids.
Authors: Kern F G , Dailey L , Basilico C
Published: September 2015

In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression.
RNA-mediated gene duplication: the rat preproinsulin I gene is a functional retroposon.
Authors: Soares M B , Schon E , Henderson A , Karathanasis S K , Cate R , Zeitlin S , Chirgwin J , Efstratiadis A
Published: September 2015

Rats and mice have two, equally expressed, nonallelic genes encoding preproinsulin (genes I and II). Cytological hybridization with metaphase chromosomes indicated that both genes reside on rat chromosome I but are approximately 100,000 kilobases apart. In mice the two genes reside on two different chromosomes. DNA sequence comparisons of the gene-flanking regions in rats and mice indicated that the preproinsulin gene I has lost one of the two introns present in gene II, is flanked by a long (41-base) direct repeat, and has a remnant of a polydeoxyadenylate acid tract preceding the downstream direct repeat. These structural features indicated that gene I was generated by an RNA-mediated duplication-transposition event involving a transcript of gene II which was initiated upstream from the normal capping site. Sequence divergence analysis indicated that the pair of the original gene and its retroposed, but functional, counterpart (which appeared about 35 million years ago) is maintained by strong negative selection operating primarily on the segments encoding the chains of the mature hormone, whereas the segments encoding the parts of the polypeptide that are eliminated during processing and also the introns and the flanking regions are evolving neutrally.