All Publications
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Biotechnology. A prudent path forward for genomic engineering and germline gene modification.
Science
2015; 348 (6230): 36-38
View details for DOI 10.1126/science.aab1028
View details for PubMedID 25791083
View details for PubMedCentralID PMC4394183
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The Dual-Use Conundrum
SCIENCE
2012; 337 (6100): 1273-1273
View details for DOI 10.1126/science.1229789
View details for Web of Science ID 000308705000001
View details for PubMedID 22984033
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High-throughput method for analyzing methylation of CpGs in targeted genomic regions
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (28): 12587-12592
Abstract
A unique microarray-based method for determining the extent of DNA methylation has been developed. It relies on a selective enrichment of the regions to be assayed by target amplification by capture and ligation (mTACL). The assay is quantitatively accurate, relatively precise, and lends itself to high-throughput determination using nanogram amounts of DNA. The measurements using mTACLs are highly reproducible and in excellent agreement with those obtained by sequencing (r = 0.94). In the present work, the methylation status of >145,000 CpGs from 5,472 promoters in 221 samples was measured. The methylation levels of nearby CpGs are correlated, but the correlation falls off dramatically over several hundred base pairs. In some instances, nearby CpGs have very different levels of methylation. Comparison of normal and tumor samples indicates that in tumors, the promoter regions of genes involved in differentiation and signaling are preferentially hypermethylated, whereas those of housekeeping genes remain hypomethylated. mTACL is a platform for profiling the state of methylation of a large number of CpG in many samples in a cost-effective fashion, and is capable of scaling to much larger numbers of CpGs than those collected here.
View details for DOI 10.1073/pnas.1005173107
View details for Web of Science ID 000279843200036
View details for PubMedID 20616066
View details for PubMedCentralID PMC2906552
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Personal Reflections on the Origins and Emergence of Recombinant DNA Technology
GENETICS
2010; 184 (1): 9-17
Abstract
The emergence of recombinant DNA technology occurred via the appropriation of known tools and procedures in novel ways that had broad applications for analyzing and modifying gene structure and organization of complex genomes. Although revolutionary in their impact, the tools and procedures per se were not revolutionary. Rather, the novel ways in which they were applied was what transformed biology.
View details for DOI 10.1534/genetics.109.112144
View details for Web of Science ID 000281784000003
View details for PubMedID 20061565
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Moments of discovery
ANNUAL REVIEW OF BIOCHEMISTRY
2008; 77: 14-44
View details for DOI 10.1146/annurev.biochem.76.051605.153715
View details for Web of Science ID 000257596800002
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Retrospective - Arthur Kornberg (1918-2007)
SCIENCE
2007; 318 (5856): 1564-1564
View details for DOI 10.1126/science.1152989
View details for Web of Science ID 000251421700025
View details for PubMedID 18063778
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Origins of the Human Genome Project: Why sequence the human genome when 96% of it is junk?
AMERICAN JOURNAL OF HUMAN GENETICS
2006; 79 (4): 603-605
View details for Web of Science ID 000240855900001
View details for PubMedID 16960796
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A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (5): 1412-1417
Abstract
A striking feature of the human genome is the dearth of CpG dinucleotides (CpGs) interrupted occasionally by CpG islands (CGIs), regions with relatively high content of the dinucleotide. CGIs are generally associated with promoters; genes, whose promoters are especially rich in CpG sequences, tend to be expressed in most tissues. However, all working definitions of what constitutes a CGI rely on ad hoc thresholds. Here we adopt a direct and comprehensive survey to identify the locations of all CpGs in the human genome and find that promoters segregate naturally into two classes by CpG content. Seventy-two percent of promoters belong to the class with high CpG content (HCG), and 28% are in the class whose CpG content is characteristic of the overall genome (low CpG content). The enrichment of CpGs in the HCG class is symmetric and peaks around the core promoter. The broad-based expression of the HCG promoters is not a consequence of a correlation with CpG content because within the HCG class the breadth of expression is independent of the CpG content. The overall depletion of CpGs throughout the genome is thought to be a consequence of the methylation of some germ-line CpGs and their susceptibility to mutation. A comparison of the frequencies of inferred deamination mutations at CpG and GpC dinucleotides in the two classes of promoters using SNPs in human-chimpanzee sequence alignments shows that CpGs mutate at a lower frequency in the HCG promoters, suggesting that CpGs in the HCG class are hypomethylated in the germ line.
View details for DOI 10.1073/pnas.0510310103
View details for Web of Science ID 000235094300047
View details for PubMedID 16432200
View details for PubMedCentralID PMC1345710
- Brilliant Science, Dark Politics, Uncertain Law Jurimetrics 2006; 46
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Reflections on the Lasker Prize for basic biomedical research
JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION
2005; 294 (11): 1419-1420
View details for Web of Science ID 000231987800022
View details for PubMedID 16174707
- George Beadle: From Genes to Proteins Nature Reviews Genetics 2005
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Timeline - George Beadle: from genes to proteins
NATURE REVIEWS GENETICS
2004; 5 (12): 949-954
Abstract
George W. Beadle's life spanned much of the period during which genetics changed from an abstract to a molecular science. Beadle himself catalysed the transition from classical to molecular genetics when, together with Edward Tatum, he discovered that each gene is linked to the production of a protein. This article traces his life from a modest farm to the centre of biology and a principal role in the development of the scientific enterprise.
View details for DOI 10.1038/nrg1494
View details for Web of Science ID 000225416800016
View details for PubMedID 15573126
- Aslilomar and Recombinant DNA www.nobel.se/chemistry/articles 2004
- A Centennial: George W. Beadle, 1903-1989 Genetics 2004; 166: 1-10
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Moments of discovery: My favorite experiments
JOURNAL OF BIOLOGICAL CHEMISTRY
2003; 278 (42): 40417-40424
View details for DOI 10.1074/jbc.X300004200
View details for Web of Science ID 000185847200001
View details for PubMedID 12893814
- George Beadle: An Uncommon Farmer 2003
- Moments of Discovery: My Favorite Experiments J.Biol.Chem. 2003; 278: 40417-24
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Asilomar and recombinant DNA
SCIENTIST
2002; 16 (6): 19-19
View details for Web of Science ID 000175030200016
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Reflections on Asilomar 2 at Asilomar 3 - twenty-five years later
PERSPECTIVES IN BIOLOGY AND MEDICINE
2001; 44 (2): 183-185
View details for Web of Science ID 000168491200004
View details for PubMedID 11370151
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Rad51 uses one mechanism to drive DNA strand exchange in both directions
JOURNAL OF BIOLOGICAL CHEMISTRY
2000; 275 (6): 3970-3976
Abstract
The Rad51 protein of Saccharomyces cerevisiae, like its bacterial counterpart RecA, promotes strand exchange between circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in vitro. However, the two proteins differ in the requirement for initiating joint molecules and in the polarity of branch migration. Whereas RecA initiates joint molecules from any type of ends on the dsDNA and branch migration proceeds exclusively in the 5'- to 3'-direction with respect to the single strand DNA substrate, initiation mediated by Rad51 requires a complementary 3' or 5' overhanging end of the linear dsDNA and branch migration proceeds in either direction. Here we report that the rates of Rad51-mediated branch migration in either the 5'- to 3'- or 3'- to 5'-directions are affected to the same extent by temperature and MgCl(2). Furthermore, branch migration in both directions is equally impeded by insertions of non-homologous sequences in the dsDNA, inserts of 6 base pairs or more being completely inhibitory. We have also found that the preference of strand exchange in the 5'- to 3'-direction does not change if RPA is replaced by Escherichia coli SSB or T4 gene 32 proteins, suggesting that the preference for the direction of strand exchange is intrinsic to Rad51. Based on these results, we conclude that Rad51-promoted branch migration in either direction occurs fundamentally by the same mechanism, quite probably by stabilizing successively formed heteroduplex base pair.
View details for Web of Science ID 000085288800036
View details for PubMedID 10660552
- Scientists and the Public: An Ambivalent Partnership Useful Knowledge, Amer. Phil. Soc. 1999: 71-78
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Inspired choices
SCIENCE
1998; 282 (5390): 873-874
View details for Web of Science ID 000076727300017
View details for PubMedID 9841431
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Branch migration during Rad51-promoted strand exchange proceeds in either direction
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1998; 95 (18): 10477-10481
Abstract
The Saccharomyces cerevisiae Rad51 protein is important for genetic recombination and repair of DNA double-strand breaks in vivo and can promote strand exchange between linear double-stranded DNA and circular single-stranded DNA in vitro. However, unlike Escherichia coli RecA, Rad51 requires an overhanging complementary 3' or 5' end to initiate strand exchange; given that fact, we previously surmised that the fully exchanged molecules resulted from branch migration in either direction depending on which type of end initiated the joint molecule. Our present experiments confirm that branch migration proceeds in either direction, the polarity depending on whether a 3' or 5' end initiates the joint molecules. Furthermore, heteroduplex DNA is formed rapidly, first at the overhanging end of the linear double-stranded DNA's complementary strand and then more slowly by progressive lengthening of the heteroduplex region until strand exchange is complete. Although joint molecule formation occurs equally efficiently when initiated with a 3' or 5' overhanging end, branch migration proceeds more rapidly when it is initiated by an overhanging 3' end, i.e., in the 5' to 3' direction with respect to the single-stranded DNA.
View details for Web of Science ID 000075730500026
View details for PubMedID 9724728
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Understanding our genes: A legacy for the next millennium
PROCEEDINGS OF THE AMERICAN PHILOSOPHICAL SOCIETY
1998; 142 (3): 416-424
View details for Web of Science ID 000076402100009
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Interaction of Rad51 with ATP and Mg2+ induces a conformational change in Rad51
BIOCHEMISTRY
1998; 37 (34): 11932-11939
Abstract
The presumptive first step in the Rad51-promoted formation of joint molecules is binding of the protein to ssDNA in the presence of ATP and Mg2+. In this paper, we report that Rad51's ability to bind DNA is rapidly inactivated when incubated at 30-37 degrees C but is stabilized by the presence of ATP and Mg2+. Although unable to promote binding to DNA, ATP-gamma-S also prevents inactivation of Rad51 at 37 degrees C. AMP-P-N-P lacks this property, while ADP protects partially but only at 5-10 times higher concentrations than ATP. These observations correlate with the dissociation constant of those nucleotides for Rad51 determined by equilibrium dialysis. Rad51 binds ATP and ATP-gamma-S with a 1:1 stoichiometry and Kds of 21 and 19 microM, respectively. The presence of DNA significantly increases the affinity of Rad51 for ATP, while DNA has a smaller effect on the affinity of ATP-gamma-S. Competition binding studies show that ADP and AMP-P-N-P bind with a 5- and 55-fold lower affinity, respectively, than ATP. The CD spectrum of Rad51 with negative double minima at around 210 and 222 nm is characteristic of an alpha-helical protein. Upon binding ATP and Mg2+, the CD spectrum is altered in the regions 194-208 and 208-235 nm, changes that are indicative of a more structured state; this change does not occur with Rad51 that has been inactivated at 37 degrees C. We surmise that the active conformation is more resistant to inactivation at elevated temperature. Our data suggest that one of the roles of ATP and Mg2+ in Rad51-mediated strand exchange is to induce the proper protein structure for binding the two DNA substrates.
View details for Web of Science ID 000075793100025
View details for PubMedID 9718317
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Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells
MOLECULAR AND CELLULAR BIOLOGY
1998; 18 (7): 4070-4078
Abstract
To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.
View details for Web of Science ID 000074380100045
View details for PubMedID 9632791
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Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA
MOLECULAR AND CELLULAR BIOLOGY
1998; 18 (7): 4400-4406
Abstract
The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication. A Saccharomyces cerevisiae strain carrying the rfa1-44 allele displays a number of impaired recombination and repair phenotypes, all of which are suppressible by overexpression of RAD52. We demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placing RFA1 and RAD52 in the same genetic pathway. Furthermore, two-hybrid analysis indicates the existence of interactions between Rad52 and all three subunits of RPA. The nature of this Rad52-RPA interaction was further explored by using two different mutant alleles of rad52. Both mutations lie in the amino terminus of Rad52, a region previously defined as being responsible for its DNA binding ability (U. H. Mortenson, C. Beudixen, I. Sunjeuaric, and R. Rothstein, Proc. Natl. Acad. Sci. USA 93:10729-10734, 1996). The yeast two-hybrid system was used to monitor the protein-protein interactions of the mutant Rad52 proteins. Both of the mutant proteins are capable of self-interaction but are unable to interact with Rad51. The mutant proteins also lack the ability to interact with the large subunit of RPA, Rfa1. Interestingly, they retain their ability to interact with the medium-sized subunit, Rfa2. Given the location of the mutations in the DNA binding domain of Rad52, a model incorporating the role of DNA in the protein-protein interactions involved in the repair of DNA double-strand breaks is presented.
View details for Web of Science ID 000074380100078
View details for PubMedID 9632824
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Binding of Rad51p to DNA - Interaction of Rad5lp with single- and double-stranded DNA
JOURNAL OF BIOLOGICAL CHEMISTRY
1998; 273 (11): 6177-6182
Abstract
Like RecA, Saccharomyces cerevisiae Rad51p promotes strand exchange between circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA). We have investigated several parameters characteristic of the interaction of Rad51p with ssDNA and dsDNA, particularly the effects of the nucleotide cofactors ATP and ADP and the analogs adenosine 5'-O-(thiotriphosphate) (ATPgammaS) and adenylyl-imidodiphosphate (AMP-PNP). Rad51p binding to both 1-N6-ethenoadenosine and 3-N4-ethenocytidine ssDNA (epsilonDNA) and dsDNA requires the presence of Mg2+ and ATP; no binding occurs in the presence of ADP, AMP-PNP, or ATPgammaS. Binding of Rad51p to dsDNA also requires ATP; ADP is ineffective, whereas ATPgammaS and AMP-PNP are considerably less able to promote binding and only at elevated concentrations of Rad51p. ATP binding, not ATP hydrolysis, is required for Rad51p binding to DNA. The Kd values for ATP for promoting binding of Rad51p to ssDNA and dsDNA are 1 and 3 microM, respectively. Rad51p binding occurs with a stoichiometry of one monomer of Rad51p per approximately 6.3 nucleotides of epsilonDNA and approximately 3.3 base pairs of dsDNA. Once formed, Rad51p. ssDNA complexes are stable so long as sufficient ATP levels are maintained. ATP hydrolysis causes dissociation of Rad51p from DNA. Moreover, the preformed complex is stable in the presence of a 10-fold excess of ADP or AMP-PNP over ATP. ATPgammaS, however, in the same -fold excess over ATP causes dissociation of the Rad51p. ssDNA complex.
View details for Web of Science ID 000072488500031
View details for PubMedID 9497339
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Characterization of strand exchange activity of yeast Rad51 protein
MOLECULAR AND CELLULAR BIOLOGY
1997; 17 (9): 5359-5368
Abstract
The Saccharomyces cerevisiae RAD51 gene product takes part in genetic recombination and repair of DNA double strand breaks. Rad51, like Escherichia coli RecA, catalyzes strand exchange between homologous circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in the presence of ATP and ssDNA-binding protein. The formation of joint molecules between circular ssDNA and linear dsDNA is initiated at either the 5' or the 3' overhanging end of the complementary strand; joint molecules are formed only if the length of the overhanging end is more than 1 nucleotide. Linear dsDNAs with recessed complementary or blunt ends are not utilized. The polarity of strand exchange depends upon which end is used to initiate the formation of joint molecules. Joint molecules formed via the 5' end are processed by branch migration in the 3'-to-5' direction with respect to ssDNA, and joint molecules formed with a 3' end are processed in the opposite direction.
View details for Web of Science ID A1997XR72400046
View details for PubMedID 9271413
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Saccharomyces cerevisiae mutants defective in plasmid chromosome recombination
MOLECULAR & GENERAL GENETICS
1996; 252 (5): 530-538
Abstract
We have studied the recombinational repair of a double-strand break (DSB) in a plasmid-borne ade2::HO-site by an intact ade2 allele following the induction of a galactose-inducible GAL-HO gene. If GAL-HO expression is not attenuated by the presence of a low level of glucose in the galactose medium, deleterious effects are observed. Our comparison of the effects of several rad mutations on the relative efficiencies of DSB repair at both the ade2::HO-site and at the chromosomal MAT locus indicate that the two processes share common functions. Not surprisingly, most of the recombination-defective mutants found using our assay are alleles of genes in the RAD52 epistasis group. The recombination and repair deficiencies vary among the different mutant groups and also among mutants within a group. In general, there is a correlation between the extents of the recombination and repair defects. Our screen also turned up a novel rfa1 allele with a pronounced deficiency in DSB repair and recombination and a srs2 mutation which causes only a mild defect.
View details for Web of Science ID A1996VQ51400005
View details for PubMedID 8914514
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The HIV Nef protein associates with protein kinase C theta
JOURNAL OF BIOLOGICAL CHEMISTRY
1996; 271 (28): 16753-16757
Abstract
Expression of the human immunodeficiency virus (HIV) Nef protein has been linked to both decreased cell surface expression of CD4 and an impairment of signal transduction. The recently reported association of Nef with an unidentified serine kinase provides a clue as to how Nef might exert its effects. Considering the key role of protein kinase C (PKC) in T cell activation, we investigated the possibility that Nef interacts with PKC. Our results, using two approaches for detecting interactions between Nef and PKC isozymes in Jurkat cells, show that Nef interacts preferentially with thetaPKC. The interaction of Nef and thetaPKC is independent of calcium, enhanced by phospholipid activators of PKC and not affected by a PKC pseudosubstrate peptide. Phorbol 12-myristate 13-acetate and phytohemagglutinin stimulation of Jurkat cells expressing Nef fails to produce the usual translocation of thetaPKC from the cytosol to the particulate fraction; translocation of betaPKC and epsilonPKC was unaffected. Indeed, there appears to be a net loss of thetaPKC in Nef-expressing cells following stimulation. The loss of thetaPKC, which may be a result of inhibition of its binding to RACKs due to Nef binding, could contribute to the various impairments of T cell function associated with HIV infection and Nef expression.
View details for PubMedID 8663223
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Review of Kornberg's the Golden Helix
NEW ENGLAND JOURNAL OF MEDICINE
1996; 334 (15): 994-995
View details for Web of Science ID A1996UD59600031
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THE RECOMBINANT-DNA CONTROVERSY - 20 YEARS LATER
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1995; 92 (20): 9011-9013
View details for Web of Science ID A1995RX20000001
View details for PubMedID 7568062
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Co-chairman's remarks: reverse genetics: directed modification of DNA for functional analysis.
Gene
1993; 135 (1-2): 261-264
Abstract
The classic paradigm for identifying the genetic basis of a particular organism's properties proceeds from the phenotype to the gene, and thence to the molecular structures of the corresponding DNA, RNA and protein. 'Positional cloning' of disease genes and the molecular characterization of the responsible mutations (inappropriately referred to as reverse genetics, initially) exemplifies this approach. Now, the ability to clone, modify and test the biological activities of DNA segments provides a new approach, referred to as 'reverse genetics'. This paradigm begins with a segment of DNA whose molecular structure is known, and proceeds to explore the gene's contribution to the organism's phenotype; thus, the experimental path is from the gene as a nucleotide sequence to the corresponding phenotypic characteristic. Such a strategy follows from the ability to modify these sequences in highly directed and nearly unlimited ways, and to assess the phenotypic relevance of such alterations either in vitro, in cultured cells, or even in whole organisms. This approach permits the full panoply of molecular techniques to be used for creating uniquely altered structures and obviates the reliance on chance events as the source of mutations. As a consequence, the range of questions that can be studied is greatly expanded, and the information that is obtained is all the richer.
View details for PubMedID 8276267
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RAPID ASSEMBLY AND DISASSEMBLY OF COMPLEMENTARY-DNA STRANDS THROUGH AN EQUILIBRIUM INTERMEDIATE STATE MEDIATED BY A1 HNRNP PROTEIN
JOURNAL OF BIOLOGICAL CHEMISTRY
1992; 267 (20): 13815-13818
Abstract
A1 hnRNP protein, which rapidly renatures complementary strands of nucleic acids in vitro, affects both the equilibrium and kinetic properties of the reaction (single-stranded DNA in equilibrium with double-stranded DNA). A1 lowers the melting transition of duplex DNA. However, at temperatures above this new Tm, both single- and double-stranded DNAs are present at equilibrium and are rapidly interconverting. Although the ratio of single and double strands under these conditions is a function of both the A1 protein and complementary DNA strand concentrations, it is not strongly affected by further increases in temperature. These surprising results demonstrate that A1 does not act as a simple catalyst in promoting renaturation and indicate how A1 and other proteins could act to speed the turnover of intermediate complexes in important biological processes.
View details for Web of Science ID A1992JD32500007
View details for PubMedID 1629182
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HOMOLOGOUS RECOMBINATION OF COPACKAGED RETROVIRUS RNAS DURING REVERSE TRANSCRIPTION
JOURNAL OF VIROLOGY
1992; 66 (4): 2378-2388
Abstract
According to prevailing models, the high frequency of recombination in retroviruses occurs during reverse transcription of two genetically different genomes copackaged into virion particles. This view has been tested in our studies of the mechanism of recombination within homologous sequences of two retroviral genomes during a single round of virus replication and in the absence of helper virus. The recombination substrates were Moloney murine leukemia virus-based vectors, each of which contains an altered defective neomycin gene (neo) under the transcriptional control of the 5' long terminal repeat; the 3' sequences of each construct contain either the Moloney murine leukemia virus or simian virus 40 large-T polyadenylation sequence. One neo gene contained a linker insertion mutation at the 5' end (neo minus), and the other contained a deletion and linker insertion at the 3' end (neo delta 3). Each of the mutant neo constructs was introduced into the packaging helper cell line psi 2 by sequential cotransfection, and individual psi 2 double transformants were selected. Supernatant fluids from the cloned psi 2 double transformants were used to infect NIH 3T3 cells, and recombinant neo+ proviruses were detected by their ability to confer G418 resistance during infection of NIH 3T3 cells. Our results show that (i) recombination between a homologous sequence of about 560 bp occurred with a frequency of about 10(-4) per virus replication cycle; (ii) recombination occurred only after the viral RNAs had been packaged into particles, i.e., recombination between the two vector DNAs or between viral RNAs prior to packaging was not detected; and (iii) copackaging of two different genomic RNAs as a heterodimer is a prerequisite for recombination. Furthermore, our results indicate that recombination can occur during the DNA negative-strand synthesis of reverse transcription.
View details for Web of Science ID A1992HJ50400065
View details for PubMedID 1372369
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RAPID RENATURATION OF COMPLEMENTARY-DNA STRANDS MEDIATED BY CATIONIC DETERGENTS - A ROLE FOR HIGH-PROBABILITY BINDING DOMAINS IN ENHANCING THE KINETICS OF MOLECULAR ASSEMBLY PROCESSES
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1991; 88 (18): 8237-8241
Abstract
The rate of renaturation for complementary DNA strands can be enhanced greater than 10(4)-fold by the addition of simple cationic detergents, and the reaction is qualitatively and quantitatively very similar to that found with purified heterogeneous nuclear ribonucleoprotein A1 protein. Under optimal conditions, renaturation rates are greater than 2000-fold faster than reactions run in 1 M NaCl at 68 degrees C. The reaction is second-order with respect to DNA concentration, and reaction rates approach or equal the rate with which complementary strands are expected to encounter each other in solution. Renaturation can even be observed well above the expected melting temperature of the duplex DNA, demonstrating that some cationic detergents have DNA double-helix-stabilizing properties. The reaction is also extremely rapid in the presence of up to a 10(6)-fold excess of noncomplementary sequences, establishing that renaturation is specific and relatively independent of heterologous DNA. This finding also implies that up to several thousand potential target sequences can be sampled per strand per second. Such reagents may be useful for procedures that require rapid nucleic acid renaturation, and these results suggest ways to identify and design other compounds that increase the kinetics of association reactions. Moreover, this work provides further support for a model relating the existence of flexible, weakly interacting, repeating domains to their function in rapid molecular assembly processes in vitro and in vivo.
View details for Web of Science ID A1991GF10100070
View details for PubMedID 1896475
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EXPRESSION OF THE TYPE-1 HUMAN-IMMUNODEFICIENCY-VIRUS NEF PROTEIN IN T-CELLS PREVENTS ANTIGEN RECEPTOR-MEDIATED INDUCTION OF INTERLEUKIN-2 MESSENGER-RNA
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1991; 88 (12): 5326-5330
Abstract
Stable transformants of the Jurkat T-cell line have been obtained that express either of two distinct forms of the type 1 human immunodeficiency virus nef gene: the nef-1-encoded protein (Nef-1) contains alanine, glycine, and valine at positions 15, 29, and 33, respectively; the protein specified by nef-2 (Nef-2) has threonine, arginine, and alanine at the corresponding positions. When Jurkat cells or their Nef-2-expressing transformants are treated with phorbol 12-myristate 13-acetate (PMA) plus either phytohemagglutinin (PHA) or antibodies against CD3 epsilon, T-cell receptor beta chain, or CD2, there is a prompt increase in interleukin 2 (IL-2) mRNA and intracellular calcium and in the IL-2 receptor alpha chain on the cell surface. Although cells expressing Nef-1 also induce calcium mobilization and the production of IL-2 receptor alpha chain, the formation of IL-2 mRNA is blocked in response to these stimuli. Moreover, Nef-1-expressing cells transfected with a plasmid in which the IL-2 promoter is fused to the chloramphenicol acetyltransferase (CAT) gene fail to induce CAT following treatment with PMA and PHA. By contrast, the parental and Nef-2-containing cells induce CAT normally. Nef-1-expressing cells can produce IL-2 mRNA in response to a combination of PMA and ionomycin, although much less efficiently than the parental Jurkat cells or Nef-2-expressing cells. These findings, and others described herein, suggest that the virally encoded Nef protein interferes with a signal emanating from the T-cell receptor complex that induces IL-2 gene transcription.
View details for Web of Science ID A1991FR44800054
View details for PubMedID 2052609
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REVERSE GENETICS - ITS ORIGINS AND PROSPECTS
BIO-TECHNOLOGY
1991; 9 (4): 342-344
View details for Web of Science ID A1991FE66700010
View details for PubMedID 1367005
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The human genome initiative. All our collective ingenuity will be needed.
FASEB journal
1991; 5 (1): 75-?
View details for PubMedID 1991590
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REPAIR OF DELETIONS AND DOUBLE-STRAND GAPS BY HOMOLOGOUS RECOMBINATION IN A MAMMALIAN INVITRO SYSTEM
MOLECULAR AND CELLULAR BIOLOGY
1991; 11 (1): 445-457
Abstract
We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed.
View details for Web of Science ID A1991ER08100048
View details for PubMedID 1986239
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TRANSDUCTION OF CELLULAR NEO MESSENGER-RNA BY RETROVIRUS-MEDIATED RECOMBINATION
JOURNAL OF VIROLOGY
1990; 64 (12): 5783-5796
Abstract
Transduction of cellular oncogenes by retroviruses is thought to be a multistep process, involving transcriptional activation of a cellular gene by upstream proviral integration and joining of cellular DNA to retroviral transcriptional signals, followed by copackaging and recombination with a helper virus genome during reverse transcription. To examine the molecular mechanism of the reverse transcriptase-mediated recombination, we introduced into mouse fibroblast cells a variety of constructs in which the neo selectable marker was joined to flanking retroviruslike or cell-like sequences. After superinfection and copackaging with a replication-competent Mo-MuLVsupF virus, the formation of recombinant neo transducing viruses was assessed in a second round of virus infection by the ability to confer G418 resistance to infected cells. Our results showed that recombinant neo proviruses were generated from neo RNA containing either a 5' or 3' retroviral end, implying that one recombination event with helper virus RNA was sufficient to incorporate the neo gene into proviral DNA. Recombination occurred with an apparent frequency of 10(-4) to 10(-5) per replication cycle in the absence of homology between the two recombining partners. This frequency, however, increased at least 100-fold if homology was provided at the site of recombination. Our results support the hypothesis that neo-transducing viruses arise via reverse transcriptase-mediated recombination of RNA rather than by recombination proceeding through DNA intermediates. Unexpectedly, removal of the retroviral packaging site psi reduced the number of neo recombinants only slightly. Our data indicated that although RNAs lacking the psi site are poorly packaged into virions, those RNAs that are included in the virions undergo frequent recombination, even if there is no selection for recombination. Many of the neo recombinants formed with the psi- constructs had undergone additional recombinations and often incorporated the psi site from the helper RNA.
View details for Web of Science ID A1990EJ20200011
View details for PubMedID 1700824
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RENATURATION OF COMPLEMENTARY-DNA STRANDS MEDIATED BY PURIFIED MAMMALIAN HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN A1-PROTEIN - IMPLICATIONS FOR A MECHANISM FOR RAPID MOLECULAR ASSEMBLY
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1990; 87 (21): 8403-8407
Abstract
Purified heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein, which is found in vivo associated with heterogeneous nuclear RNA (hnRNA), promotes the rapid renaturation of nucleic acid strands. Maximal renaturation activity requires the glycine-rich carboxyl-terminal one-third of the protein, although the amino-terminal two-thirds also has activity. The A1-mediated reaction is second-order with respect to complementary DNA concentration, and the renaturation rate constant at 37 degrees C with A1 is about 3000-fold greater than in the absence of the protein. At 60 degrees C, the A1-mediated renaturation rate is even faster, and is about 300-fold greater than protein-free reactions carried out at 68 degrees C in 1 M NaCl. Provided that sufficient A1 protein is present to coat all strands in solution, the presence of nonhomologous, single-stranded DNA does not significantly inhibit the reaction. Moreover, renaturation of short strands to their complement contained in very long strands is nearly as efficient as between two short strands. These results indicate that A1 may be useful for procedures that rely on nucleic acid renaturation. We propose that A1 promotes rapid renaturation primarily by reducing the entropic barrier of bimolecular strand association through relatively transient interactions between A1-coated strands. Such interactions, mediated by flexible repeating domains, may act generally to increase the association kinetics of highly specific molecular assemblies in processes such as RNA maturation, transcription, translation, and transport.
View details for Web of Science ID A1990EG22000045
View details for PubMedID 2236048
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GENE TARGETING AT THE HUMAN CD4 LOCUS BY EPITOPE ADDITION
GENES & DEVELOPMENT
1990; 4 (2): 157-166
Abstract
Homologous recombination at the CD4 locus in a human T-cell line has been achieved by an approach called epitope addition. The endogenous CD4 gene provided transcription, translation, and leader sequences to a crippled introduced Thy-1 gene, resulting in the expression of murine Thy-1 epitopes on the surface of the human cells. Thy-1+ cells were selected using the Fluorescence Activated Cell Sorter (FACS). An estimated 700-fold enrichment for homologous versus nonhomologous integration events was obtained, such that 70% of cells scoring positive for Thy-1 were derived from gene targeting. Three of the Thy-1+ cell lines expressed protein only from the targeted allele; thus, these cells were functionally CD4-.
View details for Web of Science ID A1990CN76300001
View details for PubMedID 1692556
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HOMOLOGOUS INTEGRATION IN MAMMALIAN-CELLS WITHOUT TARGET GENE SELECTION
GENES & DEVELOPMENT
1988; 2 (11): 1353-1363
Abstract
Homologous integrations into a nonselectable target locus have been highly enriched for following DNA transfections into mammalian cells. The target gene, the SV40 early region in COS1 cells, provides transcription signals to activate a defective selectable marker, the gpt gene. We find that nearly half of the selected clones have integrated the gpt gene at the homologous sequence in the COS1 genome. This is an estimated 100-fold enrichment for homologous events compared with transfections in which the gpt gene is transcriptionally active. As shown for yeast integration events, a double-strand break at a position of homology between the transfected DNA and the genomic target is necessary to achieve a high frequency of homologous integrations. Furthermore, the arrangement of sequences at the integration site includes a repair of the double-strand gap, which was present on the transfected DNA, suggesting that similarities exist between yeast and mammalian integrations. The experimental design, in which a defective marker is activated following a homologous integration, may have general applications for gene targeting in mammalian cells.
View details for Web of Science ID A1988Q936200001
View details for PubMedID 2850258
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COMPARISON OF INTRON-DEPENDENT AND INTRON-INDEPENDENT GENE-EXPRESSION
MOLECULAR AND CELLULAR BIOLOGY
1988; 8 (10): 4395-4405
Abstract
Recombinant simian virus 40 viruses carrying rabbit beta-globin cDNA failed to express the beta-globin sequence unless an intron was included in the transcription unit. The addition of either beta-globin IVS1 or IVS2 caused a 400-fold increase in RNA production. Stable beta-globin RNA production required sequences in IVS2 that were very close to the splice sites and that coincided with those needed for mRNA splicing. In addition to the recombinant viruses, intron-dependent expression was observed with both replicating and nonreplicating plasmid vectors in short-term transfections of cultured animal cells. Unlike transcriptional enhancer elements, IVS2 failed to increase stable RNA production when it was placed downstream of the polyadenylation site. Using a plasmid vector system to survey different inserted sequences for their dependence on introns for expression, we found that the presence of IVS2 stimulated the expression of these sequences 2- to 500-fold. Sequences from the transcribed region of the herpes simplex virus thymidine kinase gene, a gene that lacks an intervening sequence, permitted substantial intron-independent expression (greater than 100-fold increase) in the plasmid vector system.
View details for Web of Science ID A1988Q294000048
View details for PubMedID 3185553
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CONSERVATION OF SHORT PATCHES OF AMINO-ACID SEQUENCE AMONGST PROTEINS WITH A COMMON FUNCTION BUT EVOLUTIONARILY DISTINCT ORIGINS - IMPLICATIONS FOR CLONING GENES AND FOR STRUCTURE-FUNCTION ANALYSIS
NUCLEIC ACIDS RESEARCH
1988; 16 (18): 9017-9026
Abstract
Small patches of identical amino acid sequences commonly occur in proteins that have the same function but are derived from evolutionarily distant organisms. Reverse translation of such patches into degenerate pools of oligonucleotides provide useful hybridization probes for cloning the gene for the corresponding protein from other organisms. Since the conserved patches of identical amino acid sequence are probably important for the protein's biological function, they are preferred targets for reverse genetic studies aimed at defining structure-function relationships.
View details for Web of Science ID A1988Q146000017
View details for PubMedID 2845364
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EPSTEIN-BARR VIRUS SHUTTLE VECTOR FOR STABLE EPISOMAL REPLICATION OF CDNA EXPRESSION LIBRARIES IN HUMAN-CELLS
MOLECULAR AND CELLULAR BIOLOGY
1988; 8 (7): 2837-2847
Abstract
Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.
View details for Web of Science ID A1988P009800021
View details for PubMedID 2841588
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CLONING AND CHARACTERIZATION OF A CDNA-ENCODING HUMAN GALACTOSE-1-PHOSPHATE URIDYL TRANSFERASE
MOLECULAR BIOLOGY & MEDICINE
1988; 5 (2): 107-122
Abstract
We report the cloning and characterization of a cDNA that encodes a functional human galactose-1-phosphate uridyl transferase (GALT). The cDNA is 1400 bases in length and encodes a 43,000 Mr protein. The cloning strategy involved the identification of short peptide sequences conserved between the homologous enzymes from Escherichia coli and yeast, and the construction of oligonucleotide pools corresponding to the conserved patches. These patches of conserved amino acids tend to be conserved in humans as well.
View details for Web of Science ID A1988N716900005
View details for PubMedID 2840550
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DNA CROSS-LINKED BY CISPLATIN - A NEW PROBE FOR THE DNA-REPAIR DEFECT IN XERODERMA-PIGMENTOSUM
MOLECULAR BIOLOGY & MEDICINE
1987; 4 (5): 277-290
Abstract
Xeroderma pigmentosum (XP) is an inherited disease characterized by the defective repair of DNA damaged by ultraviolet radiation and a number of chemicals. In this paper, plasmid DNA carrying a marker gene is cross-linked in vitro by the antitumor drug cisplatin and successfully introduced into tissue culture cells by both calcium phosphate coprecipitation and electroporation. Transient expression of the marker gene is greatly decreased in XP cells compared to wild-type. As few as seven lesions will inactivate the marker gene in XP cells. Furthermore, the biochemical defect must include an impaired capacity for repair of cisplatin-DNA intrastrand cross-links. Since the host cell itself is not exposed to chemical modification, a cisplatin cross-linked plasmid shuttle vector can be used as a specific probe for the DNA repair capacity of cultured cells. Paradoxically, when cisplatin cross-linked plasmid carrying the selectable marker neo is introduced into cells, there is an increase in the number of stable neo+ transformants in both XP and wild-type cells. Thus, cisplatin damage appears to stimulate the integration of transfected DNA into the host chromosome by a mechanism that is independent of the defective repair pathway in XP.
View details for Web of Science ID A1987K947300003
View details for PubMedID 3695939
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IDENTIFICATION AND CHARACTERIZATION OF CDNA CLONES ENCODING 2 HOMOLOGOUS PROTEINS THAT ARE PART OF THE ASIALOGLYCOPROTEIN RECEPTOR
MOLECULAR AND CELLULAR BIOLOGY
1987; 7 (5): 1841-1847
Abstract
The asialoglycoprotein receptor (ASGP-R) from rat liver contains the following three distinct protein species when it is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: RHL1 (42 kilodaltons), RHL2 (49 kilodaltons), and RHL3 (54 kilodaltons). In this paper we describe the isolation of cDNA clones encoding RHL1 and RHL2 from a cDNA library constructed from rat liver mRNA. A comparison of the predicted coding sequence for RHL2 with that for RHL1 showed that these sequences are highly homologous. The library also contained numerous cDNA clones for both RHL1 and RHL2 that were derived from unspliced precursor mRNAs. Differential splicing at the 5' end of the RHL1 transcript was inferred from the finding that two different types of RHL1 cDNA were identified, each having a different 5' terminus.
View details for Web of Science ID A1987H060600031
View details for PubMedID 3600647
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AIDS AGREEMENT
NATURE
1987; 326 (6111): 326-326
View details for Web of Science ID A1987G578100019
View details for PubMedID 3645305
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ELECTROPORATION FOR THE EFFICIENT TRANSFECTION OF MAMMALIAN-CELLS WITH DNA
NUCLEIC ACIDS RESEARCH
1987; 15 (3): 1311-1326
Abstract
A simple and reproducible procedure for the introduction of DNA into mammalian cells by electroporation is described. The parameters involving the cells, the DNA, and the electric field are investigated. The procedure has been applied to a broad range of animal cells. It is capable of transforming more than 1% of the viable cells to the stable expression of a selectable marker.
View details for Web of Science ID A1987F976100032
View details for PubMedID 3029703
View details for PubMedCentralID PMC340526
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FORMATION OF FUNCTIONAL ASIALOGLYCOPROTEIN RECEPTOR AFTER TRANSFECTION WITH CDNAS ENCODING THE RECEPTOR PROTEINS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1986; 83 (23): 8863-8867
Abstract
The rat asialoglycoprotein receptor (ASGP-R) has been expressed in cultured rat hepatoma cells (HTC cells) after transfection with cloned cDNAs. Fluorescence-activated cell sorting of transfected cells was used to identify the functional cDNA clones and to isolate cells expressing the ASGP-R. Simultaneous or sequential transfections with two cloned cDNAs that encode related but distinctive polypeptide chains were needed to obtain ASGP-R activity; transfection with either cDNA alone failed to produce detectable ASGP-R. The affinity of transduced ASGP-R for asialo orosomucoid is less than that of the native rat ASGP-R, and the number of surface receptors in clones expressing ASGP-R is about one-fifth that found on rat hepatocytes.
View details for Web of Science ID A1986F147100010
View details for PubMedID 3466162
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ISOLATION AND CHARACTERIZATION OF EXPRESSIBLE CDNA CLONES ENCODING THE M1 AND M2 SUBUNITS OF MOUSE RIBONUCLEOTIDE REDUCTASE
MOLECULAR AND CELLULAR BIOLOGY
1986; 6 (10): 3433-3442
Abstract
Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2, which are differentially regulated during the cell cycle. We have isolated expressible cDNA clones of both subunits from an Okayama-Berg cDNA library made with mRNA from hydroxyurea-resistant, M2 protein-overproducing mouse TA3 cells. Expression of M2 protein could be demonstrated by electron paramagnetic resonance spectroscopy after transfection of COS-7 monkey cells with the plasmid. Electrophoresis and blot analyses of the parent and hydroxyurea-resistant TA3 mRNA revealed two M2 transcripts, a major one of 2.1 kilobases and a minor one of about 1.6 kilobases. Restriction endonuclease mapping of the corresponding cDNAs indicated that the two mRNAs differed only in the length of the 3' untranslated ends. By contrast, there was only one mRNA corresponding to the M1 protein, and its mobility corresponded to about 3.1 kilobases. The hydroxyurea-resistant TA3 cells contained a 50- to 100-fold excess of the M2 mRNAs over that of the parent cells and a 10-fold excess of the M1 mRNA. However, a Southern blot analysis of the corresponding genomic DNA sequences showed that the M2 gene was amplified fivefold but the M1 gene was still single copy. The complete nucleotide sequence of the 2,111-base-pair-long M2 cDNA revealed an open reading frame coding for 390 amino acids, which corresponds to a molecular weight of 45,100. The mouse M2 protein sequence was quite homologous to the equivalent protein in the clam Spisula solidissima, while the homology to the smaller subunits of Epstein-Barr virus, herpes simplex virus type 2, and Escherichia coli ribonucleotide reductases were less pronounced.
View details for Web of Science ID A1986E227200017
View details for PubMedID 3025593
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EFFECT OF UPSTREAM READING FRAMES ON TRANSLATION EFFICIENCY IN SIMIAN VIRUS-40 RECOMBINANTS
MOLECULAR AND CELLULAR BIOLOGY
1986; 6 (7): 2704-2711
Abstract
In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.
View details for Web of Science ID A1986C935600047
View details for PubMedID 3023946
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TERMINATION-REINITIATION OCCURS IN THE TRANSLATION OF MAMMALIAN-CELL MESSENGER-RNAS
MOLECULAR AND CELLULAR BIOLOGY
1986; 6 (7): 2695-2703
Abstract
Many examples of internal translation initiation in eucaryotes have accumulated in recent years. In many cases terminators of upstream reading frames precede the internal initiation site, suggesting that translational reinitiation may be a mechanism for initiation at internal AUGs. To test this idea, a series of recombinants was constructed in the mammalian expression vector pSV2. Each contained a dicistronic transcription unit comprising the coding sequence for mouse dihydrofolate reductase (DHFR) followed by the gene for xanthine-guanine phosphoribosyl transferase (XGPRT) from Escherichia coli. Various versions of this pSV2dhfr-gpt recombinant plasmid altered the location at which the DHFR reading frame was terminated relative to the XGPRT initiation codon and demonstrated that this is a critical factor for the expression of XGPRT activity in transfected Cos-1 cells. Thus, when the DHFR frame terminated upstream or a very short distance downstream of the XGPRT initiator AUG, substantial levels of XGPRT activity were observed. When the DHFR frame terminated 50 nucleotides beyond the XGPRT initiator, activity was reduced about twofold. However, when the DHFR and XGPRT sequences were fused in-frame so that ribosomes which initiated at the DHFR AUG did not terminate until they encountered the XGPRT terminator, production of XGPRT activity was abolished. This dependence of internal translation initiation on the position of terminators of the upstream reading frame is consistent with the hypothesis that mammalian ribosomes are capable of translational reinitiation.
View details for Web of Science ID A1986C935600046
View details for PubMedID 3023945
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IMMORTALIZATION OF XERODERMA PIGMENTOSUM-CELLS BY SIMIAN VIRUS-40 DNA HAVING A DEFECTIVE ORIGIN OF DNA-REPLICATION
SOMATIC CELL AND MOLECULAR GENETICS
1986; 12 (1): 13-20
Abstract
A simian virus 40 (SV40) DNA fragment, encompassing the whole early region and having a defective origin of DNA replication, has been used to transform human fibroblast cells derived from two xeroderma pigmentosum (XP) patients. Two of the SV40-transformed XP cell lines, belonging to complementation group C, had acquired the characteristic of indefinite life-span in culture. These XP cell lines synthesize T antigen as shown by immunofluorescence and retain the high sensitivity to UV irradiation. Detailed karyotype analysis shows very few chromosomal changes, while the transfecting SV40 DNA is integrated into cellular DNA sequences. These are the first immortalized XP cell lines derived from complementation group C. In view of the extreme difficulty in obtaining immortalized human fibroblasts, we suggest a possible advantage of replication defective SV40 DNA molecules for immortalizing human fibroblast cells of any source.
View details for Web of Science ID A1986A002700002
View details for PubMedID 3003928
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RAPID ASSAY FOR DETECTION OF ESCHERICHIA-COLI XANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE ACTIVITY IN TRANSDUCED CELLS
NUCLEIC ACIDS RESEARCH
1985; 13 (8): 2921-2930
Abstract
Cultured mammalian cells transduced with the Escherichia coli gene, Ecogpt, synthesize the bacterial enzyme xanthine-guanine phosphoribosyl transferase (XGPT) (1). This paper describes a method for measuring XGPT activity in crude cell extracts by following the conversion of 14C-xanthine (X) to 14C-xanthine monophosphate (XMP) and 14C-xanthosine (XR) by thin layer chromatography. The method is rapid, easy to use, sensitive and linear over a wide range of XGPT activity and has been useful for detecting XGPT in cells that were transiently transfected or stably transformed with Ecogpt. During our studies, we have found that a human cell line (XP20S) converts xanthine to XMP. This activity is probably catalyzed by a variant hypoxanthine-guanine phosphoribosyltransferase (HGPT) since the low activity is readily inhibited by hypoxanthine. A low level of conversion of X to XMP may explain why some cell lines are not killed in a medium containing mycophenolic acid and X.
View details for Web of Science ID A1985AGN0300018
View details for PubMedID 3889850
View details for PubMedCentralID PMC341204
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TRANSCRIPTION FROM A PLANT GENE PROMOTER IN ANIMAL-CELLS
NUCLEIC ACIDS RESEARCH
1985; 13 (22): 7945-7957
Abstract
The promoter segment of a plant gene (maize alcohol dehydrogenase 1 (Adh 1)) has been fused to two bacterial reporter genes, Ecogpt (1) and neo (2), in pSV2-derived vectors and introduced into cultured mammalian cells by DNA transfection. The pAdh1-gpt plasmids transformed the recipient cells for resistance to mycophenolic acid plus xanthine (3) and the analogous pAdh1-neo plasmid transformed cells to G418 resistance (2). S1 analysis of transient transfections of CV1 cells with various derivatives of pAdh1-gpt confirmed that production of gpt mRNA is initiated at the Adh1 promoter at and near the same site used in transcription of the intact Adh1 gene in maize. Moreover, expression of the Adh1 promoter was increased 10-20 fold if the SV40 early region enhancer sequence was included in the same molecule.
View details for Web of Science ID A1985AVD0300003
View details for PubMedID 2866489
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BACTERIOPHAGE-LAMBDA VECTOR FOR TRANSDUCING A CDNA CLONE LIBRARY INTO MAMMALIAN-CELLS
MOLECULAR AND CELLULAR BIOLOGY
1985; 5 (5): 1136-1142
Abstract
We have developed a bacteriophage lambda vector (lambda NMT) that permits efficient transduction of mammalian cells with a cDNA clone library constructed with the pcD expression vector (H. Okayama and P. Berg, Mol. Cell. Biol. 3:280-289, 1983). The phage vector contains a bacterial gene (neo) fused to the simian virus 40 early-region promoter and RNA processing signals, providing a dominant-acting selectable marker for mammalian transformation. The phage DNA can accommodate pcD-cDNA recombinants with cDNA of up to about 9 kilobases without impairing the ability of the phage DNA to be packaged in vitro and propagated in vivo. Transfecting cells with the lambda NMT-pcD-cDNA recombinant phage yielded G418-resistant clones at high frequency (approximately 10(-2]. Cells that also acquired a particular cDNA segment could be detected among the G418-resistant transformants by a second selection or by a variety of screening protocols. Reconstitution experiments indicated that the vector could transduce 1 in 10(6) cells for a particular phenotype if the corresponding cDNA was present as 1 functional cDNA clone per 10(5) clones in the cDNA library. This expectation was confirmed by obtaining two hypoxanthine-guanine phosphoribosyltransferase (HPRT)-positive transductants after transfecting 10(7) HPRT-deficient mouse L cells with a simian virus 40-transformed human fibroblast cDNA library incorporated into the lambda NMT phage vector. These transductants contained the human HPRT cDNA sequences and expressed active human HPRT.
View details for Web of Science ID A1985AGF2000029
View details for PubMedID 3158804
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RAD3 GENE OF SACCHAROMYCES-CEREVISIAE - NUCLEOTIDE-SEQUENCE OF WILD-TYPE AND MUTANT ALLELES, TRANSCRIPT MAPPING, AND ASPECTS OF GENE-REGULATION
MOLECULAR AND CELLULAR BIOLOGY
1985; 5 (1): 17-26
Abstract
We determined the complete nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae. The coding region of the gene contained 2,334 base pairs that could encode a protein with a calculated molecular weight of 89,796. Analysis of RAD3 mRNA by Northern blots and by S1 nuclease mapping indicated that the transcript was approximately 2.5 kilobases and did not contain intervening sequences. Fusions between the RAD3 gene and the lac'Z gene of Escherichia coli were constructed and used to demonstrate that the RAD3 gene was not inducible by DNA damage caused by UV radiation or 4-nitroquinoline-1-oxide. Two UV-sensitive chromosomal mutant alleles of RAD3, rad3-1 and rad3-2, were rescued by gap repair of a centromeric plasmid, and their sequences were determined. The rad3-1 mutation changed a glutamic acid to lysine, and the rad3-2 mutation changed a glycine to arginine. Previous studies have shown that disruption of the RAD3 gene results in loss of an essential function and is associated with inviability of haploid cells. In the present experiments, plasmids carrying the rad3-1 and rad3-2 mutations were introduced into haploid cells containing a disrupted RAD3 gene. These plasmids expressed the essential function of RAD3 but not its DNA repair function. A 74-base-pair deletion at the 3' end of the RAD3 coding region or a fusion of this deletion to the E. coli lac'Z gene did not affect either function of RAD3.
View details for Web of Science ID A1985TZ25000003
View details for PubMedID 3885009
View details for PubMedCentralID PMC366672
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NUCLEOTIDE-SEQUENCE OF 3-HYDROXY-3-METHYL-GLUTARYL COENZYME-A REDUCTASE, A GLYCOPROTEIN OF ENDOPLASMIC-RETICULUM
NATURE
1984; 308 (5960): 613-617
Abstract
The nucleotide sequence of a 4.8-kilobase mRNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase, the endoplasmic reticulum enzyme that controls cholesterol biosynthesis, shows that it is a protein of 887 amino acids (molecular weight 97,092) which contains three potential sites for asparagine-linked glycosylation. The reductase is a transmembrane glycoprotein, but in contrast to many other transmembrane glycoproteins, it lacks a cleavable or hydrophobic NH2-terminal signal sequence.
View details for Web of Science ID A1984SM02100053
View details for PubMedID 6546784
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UNUSUAL REGULATION OF SIMIAN VIRUS-40 EARLY-REGION TRANSCRIPTION IN GENOMES CONTAINING 2 ORIGINS OF DNA-REPLICATION
MOLECULAR AND CELLULAR BIOLOGY
1984; 4 (9): 1915-1928
Abstract
As part of our efforts to create multifunctional vectors for the transduction of animal cells, a set of simian virus 40 recombinants were constructed which contain an inverted duplication of the region including the origin of viral DNA replication (ori) and the early-region promoter. The unusual aspects of the structure of these recombinant genomes revealed several unexpected features of their function. In particular, transcription from the early-region promoters on these recombinants occurred primarily after the start of DNA replication, and, in that sense, these promoters behaved as if they were late-region promoters. This behavior results from the fact that these genomes contain multiple ori segments, and, therefore, they replicate earlier and faster than wild-type virus DNA, thereby causing a precocious shift in the initiation of early-region transcription from sites downstream of ori to sites located upstream of ori. The abnormal expression from multiple ori genomes is consistent with our present notions regarding the replication-dependent shift in early-region transcriptional start sites (Buchman et al., Mol. Cell. Biol. 4:1900-1914). Since our experiments demonstrate that RNAs initiated upstream of ori contribute to T-antigen formation late in infection, we suggest that the shift in early-region transcription starts modulates large T-antigen production in concert with viral DNA replication.
View details for Web of Science ID A1984TG62000031
View details for PubMedID 6092947
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COMPLEX REGULATION OF SIMIAN VIRUS-40 EARLY-REGION TRANSCRIPTION FROM DIFFERENT OVERLAPPING PROMOTERS
MOLECULAR AND CELLULAR BIOLOGY
1984; 4 (9): 1900-1914
Abstract
During simian virus 40 lytic infection there is a shift in initiation sites used to transcribe the early region, which encodes large T and small t antigens. Early in infection, transcription is initiated almost exclusively from sites that are downstream of the origin of DNA replication, whereas transcripts produced later are initiated mainly from sites on the upstream side. We have used mutant virus and specially constructed plasmid DNAs to investigate the factors regulating this transcriptional shift. In our studies simian virus 40 large T antigen appears to mediate the shift in transcription in two ways: first, T antigen represses transcription at the downstream sites late in infection by binding to the region where these RNAs are initiated; second, T antigen promotes transcription from sites on the upstream side by its ability to initiate replication or amplification, or both, of the template DNA. In addition, transcription from the downstream sites is heavily dependent on enhancer sequences located in the 72-base-pair repeat region, whereas transcription from the upstream sites late in infection does not require enhancer sequences. Thus, different overlapping promoters regulate simian virus 40 early-region expression in a manner that apparently coordinates the production of large T antigen with the increase in viral DNA.
View details for Web of Science ID A1984TG62000030
View details for PubMedID 6092946
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HOMOLOGOUS RECOMBINATION BETWEEN DEFECTIVE NEO GENES IN MOUSE 3T6 CELLS
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY
1984; 49: 171-181
View details for Web of Science ID A1984AEJ8000019
View details for PubMedID 6597755
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HOMOLOGOUS AND NONHOMOLOGOUS RECOMBINATION IN MONKEY CELLS
MOLECULAR AND CELLULAR BIOLOGY
1983; 3 (6): 1040-1052
Abstract
Though recombinational events are important for the proper functioning of most cells, little is known about the frequency and mechanisms of recombination in mammalian cells. We have used simian virus 40 (SV40)-pBR322 hybrid plasmids constructed in vitro as substrates to detect and quantitate intramolecular homologous and nonhomologous recombination events in cultured monkey cells. Excision of wild-type or defective SV40 DNAs by recombination from these plasmids was scored by the viral plaque assay, in either the absence or the presence of DNA from a temperature-sensitive helper virus. Several independent products of homologous and nonhomologous recombination have been isolated and characterized at the DNA sequence level. We find that neither DNA replication of the recombination substrate nor SV40 large T antigen is essential for either homologous or nonhomologous recombination involving viral or pBR322 sequences.
View details for Web of Science ID A1983QS67700009
View details for PubMedID 6308421
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Transcription in vivo from SV40 early promoter deletion mutants without repression by large T antigen.
Journal of molecular and applied genetics
1983; 2 (1): 127-135
Abstract
Transfection of monkey cells with recombinant plasmids containing a beta-globin coding region fused to wild-type or deleted simian virus 40 (SV40) early region promoters has allowed an analysis of the transcriptional activity of these promoters in the absence of repression by large T antigen. We find that deletion of the TATA sequence does not reduce the transcriptional effectiveness of the promoter; however, the 5' ends of the transcripts are heterogeneous rather than being restricted to the usual sites. The short GC-rich repeat sequences between nucleotides 37 and 107 and the tandemly repeated 72-bp segment between nucleotides 107 and 250 are each essential for promoter function. The GC-rich repeats are functionally redundant and probably interchangeable, since several subsets of two or three of the GC-rich segments are sufficient. One copy of the 72-bp sequence is sufficient to permit transcription. Moreover, the 72-bp repeat sequences function normally even if they are inverted relative to their normal orientation.
View details for PubMedID 6842119
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A CDNA CLONING VECTOR THAT PERMITS EXPRESSION OF CDNA INSERTS IN MAMMALIAN-CELLS
MOLECULAR AND CELLULAR BIOLOGY
1983; 3 (2): 280-289
Abstract
This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).
View details for Web of Science ID A1983QB11000016
View details for PubMedID 6300662
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ISOLATION AND CHARACTERIZATION OF A FULL-LENGTH EXPRESSIBLE CDNA FOR HUMAN HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
1983; 80 (2): 477-481
Abstract
We have cloned a full-length 1.6-kilobase cDNA of a human mRNA coding for hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) into a simian virus 40-based expression vector and have determined its full nucleotide sequence. The inferred amino acid sequence agrees with a partial amino acid sequence determined for authentic human HPRT protein. Transfection of HPRT-deficient mouse LA9 cells with the purified plasmid leads to the expression of human HPRT enzyme activity in cells stably transfected and selected for enzyme activity in hypoxanthine/aminopterin/thymidine medium.
View details for Web of Science ID A1983PZ62700034
View details for PubMedID 6300847
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SIMIAN VIRUS-40 EARLY-REGION AND LATE-REGION PROMOTER FUNCTIONS ARE ENHANCED BY THE 72-BASE-PAIR REPEAT INSERTED AT DISTANT LOCATIONS AND INVERTED ORIENTATIONS
MOLECULAR AND CELLULAR BIOLOGY
1983; 3 (6): 991-999
Abstract
Tandemly repeated 72-base-pair (bp) segments located between nucleotides 107 and 250 of the simian virus 40 genome are essential for early region transcription. The functional requirement for the 72-bp repeat was supplied even when that segment was translocated to several locations distant from, and in different orientation, relative to, the promoter. Regardless of the position of the 72-bp enhancer segment, transcription was initiated at the same locations as with the normal promoter. Translocation of the 72-bp repeat segment to other sites in the genome resulted in the appearance of DNase I hypersensitivity at that site in the intranuclear viral minichromosomes. One of the translocations which did not produce enhancement of early- and late-region expression also failed to create a DNase I-hypersensitive site at the translocated 72-bp segment.
View details for Web of Science ID A1983QS67700004
View details for PubMedID 6308429
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HIGH-EFFICIENCY CLONING OF FULL-LENGTH CDNA
MOLECULAR AND CELLULAR BIOLOGY
1982; 2 (2): 161-170
Abstract
A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 10(5) plasmid-cDNA recombinants obtained per microgram of rabbit reticulocyte mRNA, about 10% contained a complete alpha- of beta-globin mRNA sequence, and at least 30 to 50%, but very likely more, contained the entire globin coding regions. We attribute the high efficiency of cloning full- or nearly full-length cDNA to (i) the fact that the plasmid DNA vector itself serves as the primer for first- and second-strand cDNA synthesis, (ii) the lack of any nuclease treatment of the products, and (iii) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA's over those with truncated cDNA's.
View details for Web of Science ID A1982NA76500008
View details for PubMedID 6287227
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Deletion mapping of DNA regions required for SV40 early region promoter function in vivo.
Journal of molecular and applied genetics
1982; 1 (5): 457-481
Abstract
The SV40 early region promoter, previously localized to the DNA segment bounded by the HpaII and HindIII restriction sites (nucleotides 346 and 5171), was further defined by construction of an extensive set of deletions within this region and measurement of their effects on (a) viral DNA replication, (b) virus multiplication and the ability to complement early and late mutations, (c) transformation of rat cells, (d) large T antigen formation, and (e) the location of the 5' ends of early mRNAs. One set of mutations is represented by deletions that begin at the HpaII site and extend unidirectionally for varying lengths toward the BglI site at ori. A second set of mutants contains deletions that start at ori and extend unidirectionally for varying lengths towards the HpaII site. A third set of mutants, with deletions or duplications of various lengths and boundaries, lie between the HpaII and BglI sites. Our studies indicate the following. (a) Ori, the sequence needed for initiating SV40 DNA replication, extends from the sequences needed for initiating SV40 DNA replication, extends from the sequences needed to bind T antigen to the palindrome in site II to nucleotide 34, the late region edge of the AT block. Flanking sequences adjacent to the AT block facilitate DNA replication. (b) The SV40 early region promoter comprises two functionally distinct nucleotide sequence elements. One is flanked by nucleotides 5231 and 107, and contains the RNA initiation sites at nucleotides 5231-5237, a positioning element resembling the TATAAATA consensus sequence about 20-25 nucleotides upstream, and an RNA polymerase II recognition sequence contributed by short GC-rich sequences clustered between nucleotides 35 and 107; we refer to this as the RNA polymerase II interaction site. The second distinct sequence element is contained within each of two 72-bp segments located between nucleotides 107 and 250; the behavior of this element suggests that it may influence the accessibility of RNA polymerase II for the interaction site or the efficiency of RNA chain initiation. Large T antigen binding sites I, II, and III overlap with the putative RNA polymerase II interaction site; since large T antigen does not prevent elongation of RNA transcripts initiated upstream, T antigen probably represses early region expression by preventing RNA polymerase II binding to the promoter.
View details for PubMedID 6296253
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SIMIAN VIRUS-40 RABBIT BETA-GLOBIN RECOMBINANTS LACKING LATE MESSENGER-RNA SPLICE SITES EXPRESS CYTOPLASMIC RNAS WITH ALTERED STRUCTURES
JOURNAL OF VIROLOGY
1982; 42 (1): 262-274
Abstract
Deletions were introduced at exon-intron boundaries in the late region of a simian virus 40-beta-globin cDNA recombinant to study the role of splicing in the formation of simian virus 40 late cytoplasmic RNAs. The recombinant was used as a wild type because it allowed characterization of mutant RNAs expressed from defective genomes in the presence of comparable RNAs contributed by the coinfecting helper virus. Removal of a 17-base pair segment at map position 0.76, which included a portion of the leader sequence implicated in the splicing of the major 16S mRNA, prevented expression of 16S-type mRNA. The same mutant accumulated cytoplasmic 19S-type mRNA, but the assortment of the 5' ends of these mRNAs differed from the assortment of the wild-type counterparts. Another mutant that lacks nucleotide sequences implicated in the splicing of the major 16S mRNA and one of the principal 19S-type RNAs accumulated a 16S-type mRNA with a previously undetected leader splice, and assortment of 19S mRNAs with new or normally underrepresented splices, and even a species of unspliced cytoplasmic 19S mRNA.
View details for Web of Science ID A1982NK39300030
View details for PubMedID 6283144
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REGULATED EXPRESSION OF HUMAN INTERFERON BETA-1 GENE AFTER TRANSDUCTION INTO CULTURED MOUSE AND RABBIT-CELLS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
1982; 79 (17): 5166-5170
Abstract
The human interferon beta 1 gene has been inserted into simian virus 40 hybrid plasmid vectors carrying the bacterial phosphotransferase gene (neo), and introduced into cultured mammalian cells by DNA transfection. A majority of the transformants resistant to the antibiotic G418 were capable of synthesizing and secreting biologically active human interferon. The neo/interferon transformants contain several copies of the transfecting DNA integrated into cellular DNA sequences. In most transformants the production of human interferon and its mRNA is induced by the addition of poly(rI) X poly(rC); by contrast, the level of neo mRNA is not increased under the same conditions. The 5' end of the human interferon mRNA produced after induction was indistinguishable from the interferon mRNA induced in human fibroblasts. This indicates that information enabling human beta 1 interferon gene to be induced by poly(rI) X poly(rC) is localized to sequences within, or 5'-proximal to, the coding sequence.
View details for Web of Science ID A1982PE86500011
View details for PubMedID 6957856
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DISSECTIONS AND RECONSTRUCTIONS OF GENES AND CHROMOSOMES
BIOSCIENCE REPORTS
1981; 1 (4): 269-287
View details for Web of Science ID A1981MM78700001
View details for PubMedID 6271279
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SELECTION FOR ANIMAL-CELLS THAT EXPRESS THE ESCHERICHIA-COLI GENE CODING FOR XANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
1981; 78 (4): 2072-2076
Abstract
Cultured monkey (TC7) and mouse (3T6) cells synthesize an Excherichia coli enzyme, xanthine-guanine phosphoribosyltransferase (XGPRT; 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase, EC 2.4.2.22), after transfection with DNA vectors carrying the corresponding bacterial gene, Ecogpt. In contrast to mammalian cells, which do not efficiently use xanthine for purine nucleotide synthesis, cells that produce E. coli XGPRT can synthesize GMP from xanthine via XMP. After transfection with vector-Ecogpt DNAs, surviving cells producing XGPRT can be selectively grown with xanthine as the sole precursor for guanine nucleotide formation in a medium containing inhibitors (aminopterin and mycophenolic acid) that block de novo purine nucleotide synthesis. Cells transformed for Ecogpt arise with a frequency of 10(-4) to 10(-5); they appear to be genetically stable in as much as there is no discernible decrease in XGPRT formation or loss on their ability to grow in selective medium after propagation in nonselective medium. Although several of the vector-gpt DNAs can replicate in monkey and mouse cells, none of the transformants contain autonomously replicating vector-gpt DNA. Rather, the gpt transformants contain one to five copies of the transfecting DNA associated with, and most probably integrated into, cellular DNA sequences. In several transformants, vector-coded gene products for which there was no selection are also synthesized. This suggests that recombinant DNAs containing Ecogpt as a selective marker can be useful for cotransformation of nonselectable genes.
View details for Web of Science ID A1981LP04000009
View details for PubMedID 7017722
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DISSECTIONS AND RECONSTRUCTIONS OF GENES AND CHROMOSOMES
SCIENCE
1981; 213 (4505): 296-303
View details for Web of Science ID A1981LW75000010
View details for PubMedID 6264595
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GLUCOCORTICOIDS REGULATE EXPRESSION OF DIHYDROFOLATE-REDUCTASE CDNA IN MOUSE MAMMARY-TUMOR VIRUS CHIMAERIC PLASMIDS
NATURE
1981; 294 (5838): 228-232
Abstract
Fusions between the mouse mammary tumour virus long terminal repeat and a mouse dihydrofolate reductase cDNA have been constructed in a SV40 vector. When these plasmids are transferred into recipient cells, the production of dihydrofolate reductase is regulated by glucocorticoid hormones. These results define a hormonally responsive region of the viral genome.
View details for Web of Science ID A1981MQ49400029
View details for PubMedID 6272123
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EXPRESSION OF THE MOUSE DIHYDROFOLATE-REDUCTASE COMPLEMENTARY DEOXYRIBONUCLEIC-ACID IN SIMIAN-VIRUS 40 VECTORS
MOLECULAR AND CELLULAR BIOLOGY
1981; 1 (9): 854-864
Abstract
A mouse complementary deoxyribonucleic acid segment coding for the enzyme dihydrofolate reductase has been cloned in two general classes of vectors containing simian virus 40 deoxyribonucleic acid: (i) those that can be propagated as virions in permissive cells and (ii) those that can be introduced into and maintained stably in various mammalian cells. Both types of vectors express the mouse dihydrofolate reductase by using signals supplied by simian virus 40 deoxyribonucleic acid sequences. Moreover, plasmid vectors carrying the complementary deoxyribonucleic acid segment can complement Chinese hamster ovary cells lacking dihydrofolate reductase.
View details for Web of Science ID A1981ME71700009
View details for PubMedID 9279398
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FACTORS GOVERNING THE EXPRESSION OF A BACTERIAL GENE IN MAMMALIAN-CELLS
MOLECULAR AND CELLULAR BIOLOGY
1981; 1 (5): 449-459
Abstract
Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.
View details for Web of Science ID A1981LV34100007
View details for PubMedID 6100966
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Introduction of liposome-encapsulated SV40 DNA into cells.
journal of biological chemistry
1980; 255 (21): 10431-10435
Abstract
DNA, isolated from Simian virus 40 (SV40), has been encapsulated in large (0.4-micrometer diameter) unilamellar phospholipid vesicles. The procedure used for liposome preparation encapsulated the SV40 DNA at high efficiency (30 to 50% entrapment) and did not alter the physical or biological properties of the DNA molecules. The biological activity of the liposome-entrapped viral DNA was determined by plaque assays on a permissive monkey cell line. The infectivity of liposome-entrapped SV40 DNA was enhanced at least 100-fold over that of free naked DNA. Importantly, the infectivity of vesicle-entrapped DNA was resistant to DNase digestion, dependent on the amount of DNA encapsulated per vesicle and on the vesicle lipid composition. Liposomes composed of phosphatidylserine were the most efficient for delivery of DNA to cells (1.8 x 10(3) plaque-forming units/micrograms of DNA). Following the incubation of DNA-containing liposomes with cells, their infectivity could be enhanced an additional 10- to 200-fold by exposing the cells to high concentrations of polyethylene glycol or glycerol. Under these conditions the infectivity of liposome-encapsulated SV40 DNA (3 x 10(5) plaque-forming units/microgram) was comparable with values reported using the calcium phosphate method. In addition to providing a sensitive assay for monitoring and optimizing the delivery of vesicle contents to cells, the liposome-mediated delivery of nucleic acids may have potential for increasing the efficiency of DNA delivery to cells and for extending the number of cell types which can be transformed or transfected.
View details for PubMedID 6253474
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INTRODUCTION OF LIPOSOME-ENCAPSULATED SV40 DNA INTO CELLS
JOURNAL OF BIOLOGICAL CHEMISTRY
1980; 255 (21): 431-435
View details for Web of Science ID A1980KP68200068
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A MODEL FOR THE MECHANISM OF SPLICE FORMATION - RNA BRIDGING SEQUENCES COMPLEMENTARY TO THE SPLICE JUNCTION CAN APPOSE THE RNA EXONS
PROC AUST BIOCHEMICAL SOC. 1980: 84–84
View details for Web of Science ID A1980KD61300195
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EXPRESSION OF A BACTERIAL GENE IN MAMMALIAN-CELLS
SCIENCE
1980; 209 (4463): 1422-1427
Abstract
Transfection of cultured monkey kidney cells with recombinant DNA constructed with a cloned Escherichia coli gene that codes for xanthine-guanine phosphoribosyltransferase and several different SV40 DNA-based vectors, results in the synthesis of readily measurable quantities of the bacterial enzyme. Moreover, the physiological defect in purine nucleotide synthesis characteristic of human Lesch-Nyhan cells can be overcome by the introduction of the bacterial gene into these cells.
View details for Web of Science ID A1980KG02000020
View details for PubMedID 6251549
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A 3RD SPLICE SITE IN SV40 EARLY MESSENGER-RNA
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY
1979; 44: 55-62
View details for Web of Science ID A1979KQ93600007
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NUCLEOTIDE-SEQUENCE DELETIONS WITHIN THE CODING REGION FOR SMALL-T-ANTIGEN OF SIMIAN VIRUS-40
JOURNAL OF VIROLOGY
1979; 30 (3): 674-682
Abstract
Simian virus 40 early mutants with deletions mapping in the 0.53-0.60 region have been sequenced by the Maxam and Gilbert approach. All these deletions effect the small-t gene. The size of the shortened small-t-related polypeptides produced by several of the mutants has been compared with the molecular weight as deduced from the nucleotide sequence. There was good agreement for the mutants dl890, dl891, and dl2102. For dl2121 and dl2122 the small-t-related protein was considerably larger than expected. It is possible to explain this result on the basis of the nucleotide sequence: the normal splicing event of the small-t mRNA still occurs, but as the deletion shifts the reading frame, translation of the small-t-related polypeptide continues beyond the small-t splice, but in a different reading frame than large-T. Mutants dl883, dl884, and dl2112 have lost one of the small-t splicing boundaries, and no (or minute amonts of) small-t-related protein has been observed in mutant-infected cells. The possible relationship between splicing and transport of polyadenylic acid-containing mRNA from the nucleus to the cytoplasm in vertebrae cells is discussed.
View details for Web of Science ID A1979GX88800005
View details for PubMedID 225536
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MUTATIONAL ALTERATIONS WITHIN THE SIMIAN-VIRUS 40 LEADER SEGMENT GENERATE ALTERED 16S-MESSENGER RNA AND 19S-MESSENGER RNA
JOURNAL OF VIROLOGY
1979; 29 (1): 209-219
Abstract
We have analyzed the structure of the late cytoplasmic RNAs made after infection with wild-type simian virus 40 and a set of viable mutants, four of which have deletions and one an insertion within the nucleotide sequence specifying the leader segment of the 16S and 19S mRNA's. The principal findings are: (i) simian virus 40 16S and 19S mRNA's made during infections with wild-type virnds and possibly in the nucleotide sequence comprising the "leader" segments. (II) "Spliced" 16S and 19S mRNA's are made during infections with each of the mutants although, in some cases, the ratio of 19S to 16S mRNA species is reduced. (iii) The deletion or insertion of nucleotides within the DNA segment defined by map position 0.70 to 0.75 causes striking alterations in the types of leader structures in the late mRNAs. (iv) Many of the late RNA leader segments produced after infection with the mutants appear to be multiply spliced, i.e., instead of the major 200- to 205-nucleotide-long leader segment present in wild-type 16S mRNA, the RNAs produced by several of the deletion mutants have leaders with whort discontiguous segments.
View details for Web of Science ID A1979GD34500024
View details for PubMedID 219218
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CONSTRUCTION, PROPAGATION AND EXPRESSION OF SIMIAN-VIRUS 40 RECOMBINANT GENOMES CONTAINING THE ESCHERICHIA-COLI GENE FOR THYMIDINE KINASE AND A SACCHAROMYCES-CEREVISAE GENE FOR TYROSINE TRANSFER-RNA
JOURNAL OF MOLECULAR BIOLOGY
1979; 133 (3): 359-383
View details for Web of Science ID A1979HR56100004
View details for PubMedID 231664
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SYNTHESIS OF RABBIT BETA-GLOBIN IN CULTURED MONKEY KIDNEY CELLS FOLLOWING INFECTION WITH A SV40 BETA-GLOBIN RECOMBINANT GENOME
NATURE
1979; 277 (5692): 108-114
Abstract
Rabbit beta-globin complementary DNA (cDNA) has been inserted into SV40 DNA in place of the gene coding for the virus' major capsid protein, VP1. The recombinant genome, SVGT5-RabetaG, multiplies efficiently in CV1 monkey kidney cell cultures and is transcribed to yield cytoplasmic, polyadenylated hybrid mRNAs containing the beta-globin coding sequence. Cells propagating SVGT5-RabetaG produce substantial quantities of rabbit beta-globin polypeptide.
View details for Web of Science ID A1979GD60900030
View details for PubMedID 215915
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PHYSICAL AND GENETIC ORGANIZATION OF A VIRAL GENOME
CRC CRITICAL REVIEWS IN BIOCHEMISTRY
1979; 7 (1): 75-82
Abstract
Mutants of SV40 with deletions of a few to several thousand base pairs have been constructed in vitro and cloned in cultured monkey cells. The location and size of these deletions has been determined by restriction endonuclease mapping and electron microscopic and enzymatic analysis of DNA heteroduplex molecules. Analysis of the phenotype of these deletion mutants permits us to specify the locations of the known SV40 genes, in particular, the novel organization of SV40s two early genes that are required for oncogenesis.
View details for Web of Science ID A1979HW46600005
View details for PubMedID 227644
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SIMIAN VIRUS-40 MUTANTS WITH DELETIONS AT THE 3' END OF THE EARLY REGION ARE DEFECTIVE IN ADENOVIRUS HELPER FUNCTION
JOURNAL OF VIROLOGY
1979; 30 (3): 683-691
Abstract
Coinfection of monkey cells with simian virus 40 (SV40) and adenovirus type 2 (Ad2) increased the Ad2 yield 1,000-fold over that obtained by Ad2 infection alone of monkey cells (A. S. Rabson, G. T. O'Conor, I. K. Berezesky, and F. J. Paul, Proc. Soc. Exp. Biol. Med. 116:187-190, 1964). The ability of viable mutants of SV40 that contain deletions at various sites in the viral DNA to enhance Ad2 growth in monkey cells was examined. Only those mutants with deletions near the 3' end of the early region were deficient in providing this helper function. Mutants dl1265, lacking 39 base pairs at map position 0.18, and dl1263, lacking 33 base pairs at map position 0.20 (H. van Heuverswyn, C. Cole, P. Berg, and W. Fiers, J. Virol. 30:936-941, 1979), were approximately 4 and 30% as effective as wild-type SV40, respectively. The extent of enhancement of Ad2 yield depended on the multiplicity of infection by SV40, but not by Ad2 (at a multiplicity of infection of
View details for Web of Science ID A1979GX88800006
View details for PubMedID 225537
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EVALUATION METHODOLOGY IN ORGANIZATION-DEVELOPMENT - ANALYSIS AND CRITIQUE
JOURNAL OF APPLIED BEHAVIORAL SCIENCE
1978; 14 (2): 151-173
View details for Web of Science ID A1978FA48500002
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ORGANIZATION AND EXPRESSION OF EARLY GENES OF SIMIAN-VIRUS 40
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1978; 75 (1): 117-121
Abstract
The early region of simian virus 40 codes for at least two immunologically related polypeptides: large-T and small-t, with apparent molecular weights of 90,000-100,000 and 15,000-20,000, respectively. Because small-t shares methionine-containing tryptic peptides with large-T, the two polypeptides are probably coded, in part, by a common nucleotide sequence. To locate the coding sequences for large-T and small-t in the DNA, the production of these proteins was examined after infection of CV-1 cells with wild-type and deletion mutants of simian virus 40. We found that a deletion at the distal portion of the early region alters the structure of large-T but not of small-t; but deletions within the region between map coordinates 0.59 and 0.55 result in an alteration or absence of small-t and a normal large-T. These findings have been rationalized by a model that proposes the existence of two early mRNAs, one coding for large-T and the other for small-t. Both mRNAs span virtually the entire early region; but the mRNA coding for large-T lacks the nucleotide sequence between map coordinates 0.59 and 0.54. We suggest that small-t is translated from the larger of the two mRNAs, beginning at or near its 5' end and terminating at a termination codon at about map coordinate 0.54. Larger-T, on the other hand, is translated from the shorter mRNA, beginning at the same initiator codon, and, because of the deletion of the terminator codon at 0.54, translation proceeds to the terminator codon at or near map position 0.18.
View details for Web of Science ID A1978EM12800027
View details for PubMedID 203926
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EXCISION OF DNA SEGMENTS INTRODUCED INTO CLONING VECTORS BY POLY(DA.DT) JOINING METHOD
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1978; 75 (4): 1763-1767
Abstract
A method is described for excising cloned DNA segments that have been inserted into their vectors by poly(dA-dT) joins. The recombinant DNA is cleaved within the vector DNA portion by one or more restriction endonucleases to generate a linear DNA molecule with the insert DNA sequence flanked by the poly(dA-dT) joins. After denaturation, the single strands "snap back" because of the intrastrand poly(dA) and poly(dT) sequences to form circular structures with "tails" of vector DNA. The vector portion of the DNA is then digested by Escherichia coli exonuclease VII, while the insert portion remains resistant to attack. The resistant strands are annealed and purified by electrophoresis in agarose. The insert DNA segment free of contaminating vector sequences can be used as a hybridization probe and for insertion into a new vector since suitable cohesive termini are generated from the retained poly(dA) and poly(dT) tails by an appropriate exonuclease.
View details for Web of Science ID A1978EY18300035
View details for PubMedID 347445
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ALTERING SPECIFICITY OF RESTRICTION ENDONUCLEASE - EFFECT OF REPLACING MG2+ WITH MN2+
BIOCHEMISTRY
1978; 17 (1): 131-138
Abstract
In the presence of 100 mM Tris buffer (pH 7.5) and 1-10 mM Mg2+ EcoRI endonuclease cleaves DNA at a specific nucleotide sequence and in a characteristic way: -GAATTC-. But if Mg2+ is replaced by Mn2+, the specificity of the cleavage is relaxed and cleavages occur at many other sites; moreover, there appears to be a hierarchy of cleavage rates at the pseudo-EcoRI restriction sites. For example, SV40 DNA is cleaved only once in the usual digestion conditions, but with Mn2+ more than ten cleavages are made; the five most rapidly cleaved SV40 DNA map locations are 0/1.0 larger than 0.93 larger than 0.33 approximately equal to 0.42 larger than 0.29 approximately equal to 0.40 larger than 0.25. Mn2+ also alters the restriction specificity of HindIII but not HpaII endonuclease.
View details for Web of Science ID A1978EG71400019
View details for PubMedID 201281
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NEW REGION OF SIMIAN VIRUS-40 GENOME REQUIRED FOR EFFICIENT VIRAL TRANSFORMATION
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1978; 75 (5): 2473-2477
Abstract
Viable mutants of simian virus 40 with deletions in three regions of the virus genome (map coordinates 0.21-0.17, 0.59-0.54, and 0.67-0.74) have been tested for their ability to transform rat fibroblasts to anchorage independence. Only those mutants whose deletions occur between 0.59 and 0.55 in the proximal part of the early region are defective in transforming ability. The most severely defective of these transform with less than one-hundredth the efficiency of wild type. They retain their defect when tested in Chinese hamster lung cells and when infection is initiated with viral DNA instead of intact virions. Complementation for transformation can be observed between these transformation-defective deletions and a simian virus 40 temperature-sensitive A mutant.
View details for Web of Science ID A1978FB94300091
View details for PubMedID 209467
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DOES SIMIAN-VIRUS 40 DNA INTEGRATE INTO CELLULAR DNA DURING PRODUCTIVE INFECTION
JOURNAL OF VIROLOGY
1978; 28 (2): 475-489
Abstract
Late after infection of permissive monkey cells by simian virus 40 (SV40), large amounts of SV40 DNA (30,000 to 220,000 viral genome equivalents per cell) can be isolated with the high-molecular-weight fraction of cellular DNA. Hirai and Defendi (J. Virol.9:705-707, 1972) and Hölzel and Sokol (J. Mol. Biol. 84:423-444, 1974) suggested that this SV40 DNA is covalently integrated into the cellular DNA. However, our data indicate that the high-molecular-weight viral DNA is composed of tandem, "head-to-tail" repeats of SV40 DNA and that very little, if any, of this viral DNA is covalently joined to the cellular DNA. This was deduced from the following experimental findings. The size of the SV40 DNA associated with the high-molecular-weight cellular DNA fraction is greater than 45 kilobases, based on its electrophoretic mobility in agarose gels. In this form the SV40 DNA did not produce heteroduplex structures with a marker viral DNA (an SV40 genome with a characteristic deletion and duplication). After the high-molecular-weight DNA was digested with EcoRI or HpaII endonucleases, enzymes which cleave SV40 DNA once, more than 95% of the SV40 DNA migrated as unit-length linear molecules and, after hybridization with the marker viral DNA, the expected heteroduplex structures were easily detected. Digestion of the high-molecular-weight DNA fraction with restriction endonucleases that cleave cellular, but not SV40. DNA did not alter the electrophoretic mobility of the polymeric SV40 DNA, nor did it give rise to molecules that form heteroduplex structures with the marker viral DNA. Polymeric SV40 DNA molecules produced after coinfection by two physically distinguishable SV40 genomes contain only a single type of genome, suggesting that they arise by replication rather than by recombination. The polymeric form of SV40 DNA is highly infectious for CV-1P monolayers (6.5 X 10(4) PFU per microgram of SV40 DNA), yielding virtually exclusively normal, covalently closed circular, monomer-length DNA. Quite clearly these cells have an efficient mechanism for generating monomeric viral DNA from the SV40 DNA polymers.
View details for Web of Science ID A1978FV38300008
View details for PubMedID 214574
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CHARACTERIZATION OF COMPONENTS RELEASED BY ALKALI DISRUPTION OF SIMIAN VIRUS-40
JOURNAL OF VIROLOGY
1977; 21 (3): 1079-1084
Abstract
Treatment of simian virus 40 (SV40) particles at pH 9.8 in the presence of 1 mM dithiothreitol for 5 min at 37 degrees C disrupted the virions into a 60S DNA-protein complex and DNA-free 7S protein particles. The DNA-protein complex contained approximately equal amounts of DNA and protein, and appeared by electron microscopy to be relaxed circular structures with an average of 21 beads joined by short, thin bridges. The major protein components in the complex were host cell histones, but SV40-specific proteins VP3 and VP2 were also present. The 7S protein particles were almost exclusively VP1 and, in negatively stained samples, resembled the capsomer structures of intact virions.
View details for Web of Science ID A1977CZ24600029
View details for PubMedID 15134
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PHYSICAL AND GENETIC CHARACTERIZATION OF DELETION MUTANTS OF SIMIAN VIRUS-40 CONSTRUCTED INVITRO
JOURNAL OF VIROLOGY
1977; 24 (1): 277-294
Abstract
Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with Hae II or Bam HI endonuclease or partial cleavage with either Hae III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage lambda 5' -exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the Hae II endonuclease site (map location 0.83), define a new genetic complementation group, E, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the D complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the EcoRI endonuclease site (0/1.0) and eleven with deletions at the BamHI endonuclease site (0.15) fall into the B/C complementation group. Six additional mutants with deletions at the BamHI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the B/C group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the E, D, and B/C genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members of the A complementation group. Thus, the A gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene A function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed.
View details for Web of Science ID A1977DV96800027
View details for PubMedID 198579
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GENETIC ENGINEERING - CHALLENGE AND RESPONSIBILITY
AMBIO
1977; 6 (5): 253-261
View details for Web of Science ID A1977EC66000002
View details for PubMedID 11662815
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LABELING DEOXYRIBONUCLEIC-ACID TO HIGH SPECIFIC ACTIVITY INVITRO BY NICK TRANSLATION WITH DNA-POLYMERASE I
JOURNAL OF MOLECULAR BIOLOGY
1977; 113 (1): 237-251
View details for Web of Science ID A1977DM10700015
View details for PubMedID 881736
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HYBRIDIZATION INSITU OF SV40 PLAQUES - DETECTION OF RECOMBINANT SV40 VIRUS CARRYING SPECIFIC SEQUENCES OF NONVIRAL DNA
SCIENCE
1977; 196 (4286): 183-185
Abstract
The detection and recovery of SV40 genomes containing foreign DNA sequences can be facilitated, and the risk of accidental dispersal reduced, by in situ hybridization and radioautography.
View details for Web of Science ID A1977DA10600020
View details for PubMedID 191907
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STRUCTURE AND FORMATION OF CIRCULAR DIMERS OF SIMIAN VIRUS-40 DNA
JOURNAL OF VIROLOGY
1977; 24 (1): 295-302
Abstract
Most of the viral DNA extracted from simian virus 40 (SV40)-infected African green monkey kidney cells consists of circular molecules about 5.3 kilobases in contour length. However, about 1% of the viral DNA was found to occur as closed circular dimers that appeared to be formed, preferentially, late in infection. The monomeric units of dimers were organized in a head-to-tail, tandem arrangement; moreover, the monomeric units were not defective; i.e., they lacked deletions or other rearrangements. After infections with dimer DNA, nondefective monomers were formed. These findings suggest that dimers are not intermediates in the production of defective SV40 genomes. The majority of the dimers formed in mixed infections with two mutants were homodimers, but about 5% of the circular dimers were heterodimers and must have arisen by intermolecular recombination.
View details for Web of Science ID A1977DV96800028
View details for PubMedID 198580
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RECOMBINANT DNA RESEARCH CAN BE SAFE
TRENDS IN BIOCHEMICAL SCIENCES
1977; 2 (2): N26-N27
View details for Web of Science ID A1977CU64500001
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ISOLATION AND PROPAGATION OF A SEGMENT OF SIMIAN-VIRUS 40 GENOME CONTAINING ORIGIN OF DNA-REPLICATION
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1976; 73 (5): 1513-1517
Abstract
Heteroduplex DNA molecules formed from two DNAs that differ from each other by a deletion can be cleaved at the mismatched region (a deletion loop) with the single-strand-specific S1 endonuclease. A heteroduplex DNA molecule, constructed from the DNA of simian virus 40 (SV40) mutant with a deletion of the map region 0.54-0.55 and the DNA of a second SV40 mutant having a deletion of the map segment 0.70-0.73, is cleaved twice with S1 endonuclease. One of the products is a DNA fragment of about 0.13 the length of SV40 DNA which contains the origin of SV40 DNA replication (0.67 on the SV40 DNA map).
View details for Web of Science ID A1976BS77400033
View details for PubMedID 179089
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CONSTRUCTION OF HYBRID VIRUSES CONTAINING SV40 AND GAMMA PHAGE DNA SEGMENTS AND THEIR PROPAGATION IN CULTURED MONKEY CELLS
CELL
1976; 9 (4): 695-705
Abstract
This paper describes the successful construction and propagation of a transducing animal virus. A segment of DNA approximately 2 kilobases (kb) in length was removed from the late region of the SV40 genome by sequential cleavages with Hpa II and Bam HI endonucleases (at 0.735 and 0.13, respectively, on the SV40 DNA map). A segment of about 1.5 kb of lambda phage containing ORI (the origin of lambda DNA replication), the two structural genes CII and cro, and four transcriptional promoters, was inserted into the late region of SV40 by the poly(dA:dT) joining procedure. The resulting hybrid DNAs were cloned and propagated as virions in CV-1 monkey kidney cells by mixed infections at 41 degrees C with tsA58, an early mutant of SV40. The location, size, and orientation of the inserted lambda DNA segment was verified by restriction endonuclease digestions and by heteroduplex analysis. Clones with each of the two possible orientations of the lambda DNA segment were isolated. CV-1 cells infected with lambda-SV40 hybrid virus contain little or no lambda-specific RNA or proteins, even though the hybrid virus replicates nearly as well as the helper virus.
View details for Web of Science ID A1976CV58900007
View details for PubMedID 189942
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CONSTRUCTION AND ANALYSIS OF VIABLE DELETION MUTANTS OF SIMIAN VIRUS-40
JOURNAL OF VIROLOGY
1976; 18 (2): 664-671
Abstract
Viable mutants of simian virus 40 (SV40), with deletions ranging in size from 15 to 200 base pairs, have been obtained by infecting CV-1P cells with circularly permuted linear SV40 DNA. The linear DNA was produced by cleavage of closed circular DNA with DNase I in the presence of Mn2+, followed, in some cases, by mild digestion with lambda 5'-exonuclease. The SV40 map location and the size of each deletion were determined by using the S1 nuclease mapping procedure (Shenk et al., 1975) and the change in size of fragments produced by Hind II + III endonuclease cleavage. Deletions in at least three regions of the SV40 chromosome have slight or no effect on the rate or yield of viral multiplication and on vira-induced cellular transformation. These regions are located at the following coordinates on the SV40 physical map: 0.17 to 0.18; 0.54 to 0.59; and 0.68 to 0.74.
View details for Web of Science ID A1976BP99900032
View details for PubMedID 178902
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CHARACTERIZATION OF ALKALINE DISRUPTION OF SV40 VIRIONS
FEDERATION AMER SOC EXP BIOL. 1976: 1688–88
View details for Web of Science ID A1976BR40301679
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SUMMARY STATEMENT OF ASILOMAR CONFERENCE ON RECOMBINANT DNA-MOLECULES
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1975; 72 (6): 1981-1984
View details for Web of Science ID A1975AG70300001
View details for PubMedID 806076
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SYNTHESIS OF SUPERHELICAL SIMIAN VIRUS-40 DEOXYRIBONUCLEIC-ACID IN CELL LYSATES
JOURNAL OF BIOLOGICAL CHEMISTRY
1975; 250 (11): 4340-4347
Abstract
In vivo-labeled SV40 replicating DNA molecules can be converted into covalently closed superhelical SV40 DNA (SV40(I) using a lysate of sv40-infected monkey cells containing intact nuclei. Replication in vitro occurred at one-third the in vivo rate for 30 min at 30 degrees. After 1 hour of incubation, about 54% of the replicating molecules had been converted to SV40(I), 5% to nicked, circular molecules (SV40(II), 5% to covalently closed dimers; the remainder failed to complete replication although 75% of the prelabeled daughter strands had been elongated to one-genome length. Density labeling in vitro showed that all replicating molecules had participated during DNA synthesis in vitro. Velocity and equilibrium sedimentation analysis of pulse-chased and labeled DNA using radioactive and density labels suggested that SV40 DNA synthesis in vitro was a continuation of normal ongoing DNA synthesis. Initiation of new rounds of SV40 DNA replication was not detectable.
View details for Web of Science ID A1975AE57800044
View details for PubMedID 165197
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Isolation and characterization of individual clones of simian virus 40 mutants containing deletions duplications and insertions in their DNA.
Cold Spring Harbor symposia on quantitative biology
1975; 39: 69-84
View details for PubMedID 169098
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Mapping of mutational alterations in DNA with S1 nuclease: the location of deletions, insertions and temperature-sensitive mutations in SV40.
Cold Spring Harbor symposia on quantitative biology
1975; 39: 61-67
View details for PubMedID 169094
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BIOCHEMICAL METHOD FOR MAPPING MUTATIONAL ALTERATIONS IN DNA WITH S1 NUCLEASE - LOCATION OF DELETIONS AND TEMPERATURE-SENSITIVE MUTATIONS IN SIMIAN VIRUS 40
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1975; 72 (3): 989-993
Abstract
S1 nuclease (EC 3.1.4.X), a single-strand-specific nuclease, can be used to accurately map the location of mutational alterations in simian virus 40 (SV40) DNA. Deletions of between 32 and 190 base pairs, which are at or below the limit of detectability by conventional electron microscopic analysis of heteroduplex DNAs, have been located in this way. To map a deletion, a mixture of unit length, linear DNA, prepared from the SV40 deletion mutant and its wild-type parent, are denatured and reannealed to form heteroduplexes. S1 nuclease can cut such heteroduplexes at the nonbase-paired region to produce fragments whose lengths correspond to the position of the deletion. Similarly, specific fragments are produced when S1 nuclease cleaves a heteroduplex formed from the DNAs of SV40 temperature-sensitive mutants and either their revertants or wild-type parents. Thus, the positions of the nonhomology between these DNAs can be determined.
View details for Web of Science ID A1975W171200048
View details for PubMedID 165498
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ASILOMAR CONFERENCE ON RECOMBINANT DNA-MOLECULES
SCIENCE
1975; 188 (4192): 991-994
View details for Web of Science ID A1975AD09200007
View details for PubMedID 1056638
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BIOCHEMICAL PROCEDURE FOR PRODUCTION OF SMALL DELETIONS IN SIMIAN VIRUS-40 DNA
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1975; 72 (4): 1392-1396
Abstract
A simple biochemical procedure for producing small deletions (15 to 50 base pairs) at virtually any location in simian virus 40 DNA has been developed. The steps involved are: cleavage of the closed-circular DNA to produce a linear structure followed by 5'-exonuclease digestion to expose a short single-stranded segment at each 3' end of the molecule. Mutants containing deletions at the site of the cleavage are obtained by infecting permissive monkey kidney cells with the exonuclease-treated DNA in the presence or absence of a helper DNA (depending upon whether or not the site of cleavage and therefore the deletion occurred in a gene required for vegetative multiplication). In this paper viable mutants with deletions at the HpaII endonuclease cleavage site (0.735 map position) and defective trans-complementable mutants with deletions at the EcoRI endonuclease cleavage site (0/1.0 map position) were isolated.
View details for Web of Science ID A1975AB51800038
View details for PubMedID 165505
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CONSTRUCTION INVITRO OF MUTANTS OF SIMIAN VIRUS 40 - INSERTION OF A POLY(DA-DT) SEGMENT AT HEMOPHILUS-PARAINFLUENZA II RESTRICTION ENDONUCLEASE CLEAVAGE SITE
JOURNAL OF MOLECULAR BIOLOGY
1975; 98 (1): 1-15
View details for Web of Science ID A1975AV68400001
View details for PubMedID 172641
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ELECTRON-MICROSCOPE LOCALIZATION OF A PROTEIN BOUND NEAR ORIGIN OF SIMIAN VIRUS 40 DNA-REPLICATION
JOURNAL OF VIROLOGY
1975; 15 (1): 167-172
Abstract
A salt-stable complex of protein and viral DNA obtained from Simian virus 40 (SV40)-infected monkey cells or mature SV40 virions has a novel structure. When viewed by high resolution electron microscopy, the circular SV40 DNA molecule has bound to it one to three globular protein "knobs". Using ecoRI and hpaII restriction endonucleases, each of which can cleave SV40 DNA once at a known location (10, 11, 12, 14), the bound protein can be localized at 0.7 plus or minis 0.05 on the SV40 DNA physical map (SV40 fractional length, clockwise from the ecoRI endonuclease-cleavage site).
View details for Web of Science ID A1975V345100023
View details for PubMedID 163341
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Letter: Potential biohazards of recombinant DNA molecules.
Science
1974; 185 (4148): 303
View details for PubMedID 4600381
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MAPPING OF MUTATIONAL ALTERATIONS IN DNA WITH S1 NUCLEASE - LOCATION OF DELETIONS, INSERTIONS AND TEMPERATURE-SENSITIVE MUTATIONS IN SV40
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY
1974; 39: 61-67
View details for Web of Science ID A1974AB46100009
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VIABLE DELETION MUTANTS OF SIMIAN VIRUS 40 - SELECTIVE ISOLATION BY MEANS OF A RESTRICTION ENDONUCLEASE FROM HEMOPHILUS-PARAINFLUENZAE
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1974; 71 (12): 4879-4883
Abstract
Resistance of simian virus 40 (SV40) DNA to cleavage by Hemophilus parainfluenzae II (HpaII) restriction endonuclease has been used as a positive, in vitro selection for mutants lacking the one HpaII endonuclease-cleavage site of wild-type SV40 DNA. Each of 10 viable mutants isolated by this procedure multiplies significantly more slowly than wild-type virus and contains a small deletion (80 to 190 base pairs in size) of the region of the genome that includes the HpaII endonuclease-recognition sequence. These well-defined mutants, having a selective disadvantage for growth, would not have been readily obtained by conventional methods used to screen for viral mutants. Therefore, in certain circumstances, restriction endonucleases are effective reagents for the selection of new classes of mutants. Because these small deletions can be visualized in heteroduplexes, these mutants provide internal markers for mapping other alterations or features of the simian virus 40 genome.
View details for Web of Science ID A1974V197000052
View details for PubMedID 4373732
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ISOLATION AND CHARACTERIZATION OF INDIVIDUAL CLONES OF SIMIAN VIRUS 40 MUTANTS CONTAINING DELETIONS, DUPLICATIONS AND INSERTIONS IN THEIR DNA
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY
1974; 39: 69-84
View details for Web of Science ID A1974AB46100010
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DEFECTIVE SIMIAN VIRUS 40 GENOMES - ISOLATION AND GROWTH OF INDIVIDUAL CLONES
VIROLOGY
1974; 62 (1): 112-124
View details for Web of Science ID A1974U800900010
View details for PubMedID 4370797
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GLYCYL TRANSFER RIBONUCLEIC-ACID SYNTHETASE FROM ESCHERICHIA-COLI - PURIFICATION, PROPERTIES, AND SUBSTRATE BINDING
BIOCHEMISTRY
1974; 13 (7): 1338-1348
View details for Web of Science ID A1974S551200006
View details for PubMedID 4594761
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SINGLE MUTATIONAL MODIFICATION OF A TRYPTOPHAN-SPECIFIC TRANSFER-RNA PERMITS AMINOACYLATION BY GLUTAMINE AND TRANSLATION OF CODON UAG
JOURNAL OF MOLECULAR BIOLOGY
1974; 86 (2): 245-260
View details for Web of Science ID A1974T618600007
View details for PubMedID 4606150
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CONVENIENT MICRO METHOD FOR QUANTITATION OF CLOSED CIRCULAR DEOXYRIBONUCLEIC-ACID
BIOCHEMISTRY
1974; 13 (11): 2410-2413
View details for Web of Science ID A1974T032800027
View details for PubMedID 4364778
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MAPPING OF SIMIAN VIRUS 40 EARLY FUNCTIONS ON VIRAL CHROMOSOME
JOURNAL OF VIROLOGY
1973; 12 (3): 653-658
Abstract
The simian virus 40 (SV40) DNA segment in the nondefective adenovirus 2-SV40 hybrid, Ad2(+)ND(4), is colinear with the segment between 0.11 and 0.59 SV40 fractional length from the site at which the R(1) restriction endonuclease cleaves SV40 DNA. This specifies the region of the SV40 DNA molecule which induces the early SV40 antigens: U antigen, tumor specific transplantation antigen, and T antigen. A variant of Ad2(+)ND(4), called Ad2(+)ND(4del), was found which has a deletion of the DNA segment between 0.50 and 0.57 SV40 fractional length from the R(1) endonuclease cleavage point.
View details for Web of Science ID A1973Q499000028
View details for PubMedID 4355862
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CLEAVAGE OF CIRCULAR, SUPERHELICAL SIMIAN VIRUS-40 DNA TO A LINEAR DUPLEX BY S1 NUCLEASE
JOURNAL OF VIROLOGY
1973; 12 (6): 1303-1313
Abstract
S(1) nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S(1) nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S(1) nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR(I) restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.
View details for Web of Science ID A1973R192500014
View details for PubMedID 4357509
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DUPLICATION OF STRUCTURAL GENE FOR GLYCYL-TRANSFER RNA SYNTHETASE IN ESCHERICHIA-COLI
JOURNAL OF MOLECULAR BIOLOGY
1971; 58 (2): 595-?
View details for Web of Science ID A1971J588800016
View details for PubMedID 4933418
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GLYCYL-TRANSFER RNA SYNTHETASE - AN OLIGOMERIC PROTEIN CONTAINING DISSIMILAR SUBUNITS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1970; 67 (4): 1967-?
Abstract
Glycyl-tRNA synthetase from Escherichia coli is a tetramer containing two small (33,000 daltons) and two large (80,000 daltons) subunits. The subunits can be separated and then reassembled to form active enzyme. Genetically altered glycyl-tRNA synthetases are modified in either the large or the small subunit. Wild type activity can be reconstituted in vitro by mixing extracts containing enzymes with modifications in different subunits.
View details for Web of Science ID A1970I017400050
View details for PubMedID 4923123
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ISOLATION AND PARTIAL CHARACTERIZATION OF ESCHERICHIA-COLI MUTANTS WITH ALTERED GLYCYL TRANSFER RIBONUCLEIC ACID SYNTHETASES
JOURNAL OF BACTERIOLOGY
1970; 102 (1): 193-?
Abstract
Isolates with mutations in glyS, the structural gene for glycyl-transfer ribonucleic acid (tRNA) synthetase (GRS) in Escherichia coli, are frequently found among glycine auxotrophs. Extracts of glyS mutants have altered GRS activities. The mutants grow with normal growth rates in minimal media when high levels of glycine are provided. No other metabolite of a variety tested is capable of restoring normal growth. The glyS mutants fail to make ribonucleic acid (RNA) when depleted of exogenous glycine in strains which are RC(str) but do so when the cells are RC(rel). In contrast, biosynthetic mutants which are unable to synthesize glycine (glyA mutants) do not make RNA when deprived of glycine even if they are RC(rel); in this case, RNA is synthesized upon glycine deprivation only when the nucleic acid precursors made from glycine are provided in the medium. The level of serine transhydroxymethylase is unaltered in extracts of any of the glyS mutants, even though the level of charged tRNA(Gly) is at least 20-fold lower than that found in a prototrophic parent; this indicates that, if there is control over the synthesis of serine transhydroxymethylase, it is not modified by reduced levels of charging of the major species of tRNA(Gly).
View details for Web of Science ID A1970F807100026
View details for PubMedID 4908671
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CHARACTERIZATION OF ALTERED FORMS OF GLYCYL TRANSFER RIBONUCLEIC ACID SYNTHETASE AND EFFECTS OF SUCH ALTERATIONS ON AMINOACYL TRANSFER RIBONUCLEIC ACID SYNTHESIS IN-VIVO
JOURNAL OF BACTERIOLOGY
1970; 102 (1): 204-?
Abstract
The glycyl transfer ribonucleic acid (tRNA) synthetase (GRS) activities of several Escherichia coli glyS mutants have been partially characterized; the K(m) for glycine and the apparent V(max) of several of the altered GRS differ significantly from the parental GRS. Paradoxically, some of the altered forms exhibit more activity in vitro than the GRS from a prototrophic strain (GRS(L)); several parameters of these activities have been studied in an attempt to resolve this problem. The amount of acylated tRNA(Gly) in vivo was examined to assess the GRS activities inside the cells. During exponential growth in media containing glycine, moderate amounts of acylated tRNA(Gly) occur in the glyS mutants; glycine deprivation leads to a dramatic drop in the amount of acylated tRNA(Gly). An alternative measure of the in vivo activities of the altered enzymes is the efficiency of suppression of the trpA36 locus by su(36) (+); glyS mutants grown with added glycine exhibit one-third to one-fourth the suppression efficiency of the prototrophic glyS(H) parent, presumably because they are less efficient, even in the presence of high levels of glycine, in charging the tRNA(Gly) species which functions as the translational suppressor.
View details for Web of Science ID A1970F807100027
View details for PubMedID 4908672
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RECESSIVE LETHALS - A NEW CLASS OF NONSENSE SUPPRESSORS IN ESCHERICHIA COLI
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1969; 63 (2): 392-?
Abstract
Two new nonsense suppressors in Escherichia coli were found in partial diploids carrying F'14 and were shown to be on the episome. These suppressors can exist only in cells which also contain the su(-) allele, i.e., su(+)/su(-) heterozygotes. Presumably the mutations cause an alteration of an essential cellular component, the complete loss of which is lethal. Su7, an amber suppressor, has an efficiency of 76 per cent and su8, an ochre suppressor, an efficiency of 4 per cent.
View details for Web of Science ID A1969D965700027
View details for PubMedID 4895537
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RECESSIVE LETHAL NONSENSE SUPPRESSOR IN ESCHERICHIA COLI WHICH INSERTS GLUTAMINE
NATURE
1969; 223 (5213): 1340-?
View details for Web of Science ID A1969E204200017
View details for PubMedID 4897374
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[A new example of anti-platelet antibody Ko].
Vox sanguinis
1963; 8 (3): 341-347
View details for PubMedID 5872213
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[A new example of anti-platelet antibody Ko].
Vox sanguinis
1963; 8: 341-347
View details for PubMedID 14025260
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UN NOUVEL EXEMPLE DANTICORPS ANTI-PLAQUETTAIRE KO
VOX SANGUINIS
1963; 8 (3): 341-?
View details for Web of Science ID A19633725C00005