==================================BSR45================================== 45. Site-specific DNA integration is topic of interest. Word searches: site-specific integration; integration with sequences or sequence; resolvase; virus with integration (or retrovirus); DNA with integration; bacteriophage P1 and lox or recombination; recombination with site or sequence; rec A or rec BC. 1 UI - 87080325 AU - Minter SJ ; Clore GM ; Gronenborn AM ; Davies RW TI - Cooperative DNA binding by lambda integration protein--a key component of specificity. AB - Quantitative analysis of nitrocellulose filter binding data by the method of Clore, Gronenborn and Davies [(1982) J. Mol. Biol. 155, 447-466] has been used to show that lambda integration protein (Int) exhibits cooperativity in binding to specific recognition sites within the attachment site region (lambda attP) of bacteriophage lambda DNA. Optimal values of the equilibrium constant obtained were 3.0(+/- 1.0) X 10(10) M-1 for the P' site using a model of three sites with equal affinity and 1.9(+/- 0.4) X 10(10) M-1 for the P1 site on a two-site model. The value of the cooperativity parameter alpha is 172(+106)(-66) in all cases. The occurrence of a consensus recognition sequence is necessary but not sufficient for strong binding; cooperative interaction between Int molecules binding to adjacent members of an array of binding sites is also essential. The occurrence of binding site arrays distinguishes lambda attP very clearly from other DNA sequences containing single recognition sites by chance. MH - Allosteric Site ; Binding, Competitive ; Collodion ; DNA/*METABOLISM ; DNA Nucleotidyltransferases/*METABOLISM ; DNA Restriction Enzymes ; Filtration ; Models, Chemical ; Protein Binding ; Proteins/*METABOLISM ; Viral Proteins/*METABOLISM SO - Eur J Biochem 1986 Dec 15;161(3):727-31 2 UI - 87112709 AU - Gardner JF ; Nash HA TI - Role of Escherichia coli IHF protein in lambda site-specific recombination. A mutational analysis of binding sites. AB - The phage lambda attachment site, attP, contains three binding sites for an Escherichia coli protein, IHF, that is needed for efficient integrative recombination. We have used synthetic oligodeoxyribonucleotides to direct multiple base changes at each of these three sites. Alteration by two base-pairs of the consensus sequence for the leftmost binding site specifically interferes with IHF binding to that site and modestly depresses recombination in vitro. For each of the three binding sites, alteration of the consensus sequence by four base-pairs strongly depresses recombination in vitro, indicating that all three sites are important for attP function. The mutated attP sites are also depressed for recombination in vivo but some of the mutants are less affected than they are in vitro. The disparity between effects in vivo and in vitro for some mutants but not others suggests that the three binding sites are not functionally equivalent and that at some sites additional E. coli factors may replace or assist IHF. The non-equivalence of the three IHF sites is also indicated by the behavior of prophage attachment sites carrying mutations in the binding sites. MH - Attachment Sites (Microbiology) ; Bacterial Proteins/*GENETICS ; Binding Sites ; DNA, Viral ; Escherichia Coli/GENETICS ; Mutation ; Phage Lambda/ *GENETICS ; *Recombination, Genetic ; Support, U.S. Gov't, Non-P.H.S. SO - J Mol Biol 1986 Sep 20;191(2):181-9 3 UI - 87089704 AU - Hoffman-Liebermann B ; Liebermann D ; Troutt A ; Kedes LH ; Cohen SN TI - Human homologs of TU transposon sequences: polypurine/polypyrimidine sequence elements that can alter DNA conformation in vitro and in vivo. AB - We previously have shown that homologs of the outer domain segment of the inverted repeat termini (IVR-OD) of the sea urchin TU transposons are conserved among multiple eucaryotic species, including humans. We report here that two cloned human DNA IVR-OD homologs, Hut2 and Hut17, consist of a series of tandem repeats of the trimer AGG/TCC, forming segments (313 and 221 base pairs in length, respectively) of polypurine/polypyrimidine (pPu/pPy or "Puppy:) asymmetry in the two DNA strands; these are punctuated at certain sites with variant trimers, which are different for the two clones. Sequences homologous to the Hut2 pPu/pPy tract exist at multiple sites in the DNA of a wide variety of eucaryotes. Hybridization of human DNA with a Hut2 probe or with a previously described chicken DNA pPu/pPy sequence indicates that pPu/pPy sequences can be grouped into families distinguishable by the extent of their homology with each probe at different hybridization stringencies. Moreover, particular pPu/pPy tracts show species-specific differences in their distribution. Both the Hut2 and Hut17 pPu/pPy tracts are cleaved by S1 nuclease when tested on supercoiled plasmids. Most if not all of the 313-base-pair Hut2 pPu/pPy tract is also sensitive to S1 in its native location in HeLa cell chromatin, indicating that the sequence contains conformational information that can be expressed in vivo. This view is supported by evidence that exogenously derived Hut2 pPu/pPy tracts introduced into mouse L cells and integrated in chromatin can assume an S1-sensitive conformation. MH - Animal ; Base Sequence ; Cloning, Molecular ; DNA/*GENETICS ; *DNA Insertion Elements ; DNA Restriction Enzymes ; Human ; *Nucleic Acid Conformation ; Nucleic Acid Hybridization ; Phage Lambda/GENETICS ; Plasmids ; Purines ; Pyrimidines ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. ; Transfection SO - Mol Cell Biol 1986 Nov;6(11):3632-42 4 UI - 87086779 AU - Reynolds AE ; Mahadevan S ; LeGrice SF ; Wright A TI - Enhancement of bacterial gene expression by insertion elements or by mutation in a CAP-cAMP binding site. AB - The regulatory region (bglR) of the cryptic bgl operon was characterized by DNA sequence analysis and transcription mapping. Bgl(-)-specific transcription was found to occur in both the wild-type Bgl- and mutant Bgl+ cells. However, the steady-state level of bgl RNA was much higher in the Bgl+ mutant than in the wild-type. Activation of the bgl operon by insertion sequence-mediated bglR mutations or point mutations in bglR is therefore the result of increased transcription. The ethylmethane sulfonate-induced point mutations in bglR are alterations in a single base in the cAMP binding protein (CAP) binding site, leading to a stronger binding of the CAP-cAMP complex. The IS1 and IS5-mediated bglR mutations analyzed show that the insertion sequences can activate the bgl operon by integration 78 to 125 base-pairs upstream from the transcription initiation site. The role of the insertion sequences in activation of the bgl operon is discussed. MH - Adenosine Cyclic Monophosphate/METABOLISM ; Base Sequence ; Carrier Proteins/*GENETICS/METABOLISM ; *DNA Insertion Elements ; DNA, Bacterial ; *Enhancer Elements (Genetics) ; Escherichia Coli/GENETICS/METABOLISM ; *Gene Expression Regulation ; *Genes, Regulator ; *Mutation ; Plasmids ; Support, Non-U.S. Gov't ; Transcription, Genetic SO - J Mol Biol 1986 Sep 5;191(1):85-95 5 UI - 87064642 AU - Anderson RA ; Eliason SL TI - Recombination of homologous DNA fragments transfected into mammalian cells occurs predominantly by terminal pairing. AB - The mechanism by which double-strand cleavages stimulate the joining of plasmid DNA fragments introduced into cultured mammalian cells was investigated by cotransfecting pairs of plasmids encoding deletion mutations in a dominant selectable gene into LMtk- cells. Plasmid recombination substrates were produced by creating deletions of different sizes within the neo coding region of the pSV2neo plasmid. Complementing pairs of deleted plasmid DNAs were linearized at specific unique sites before cotransfection into mouse LMtk- cells by the calcium phosphate precipitation method. Cleaving one donor plasmid produced a 4- to 10-fold stimulation in the production of colonies able to survive in medium containing G-418. The linearization of the second plasmid further increased the efficiency by another factor of 6 to 15 when the cut was made on the opposite side of the homology, approximately equidistant from the center of the overlap. Fifty-seven individual G-418-resistant colonies representing the products of individual crosses were isolated, and the genomic DNAs containing the presumably integrated, functional recombinant neo genes were analyzed on Southern blots. A band consistent with the exchange of markers flanking the neo gene was present in 90% of the DNAs examined. In only one case was the pattern indicative of either a double crossover or a gene conversion event. These results support the idea that homologous extrachromosomal DNA fragments are joined through annealing of overlapping single-stranded ends. This DNA-joining phenomenon may represent the activity of cellular DNA repair enzymes; its relationship to genetic recombination occurring at the chromosomal level remains to be determined. MH - Adenine Phosphoribosyltransferase/GENETICS ; Animal ; DNA Restriction Enzymes ; DNA, Recombinant/*METABOLISM ; *Genes, Structural ; L Cells/ ENZYMOLOGY ; Mice ; Mutation ; *Plasmids ; Sequence Homology, Nucleic Acid ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; Thymidine Kinase/GENETICS SO - Mol Cell Biol 1986 Sep;6(9):3246-52 6 UI - 87064575 AU - Bourgaux-Ramoisy D ; Gendron D ; Chartrand P ; Bourgaux P TI - An excision event that may depend on patchy homology for site specificity. AB - In mouse cells transformed by a mutant polyomavirus genome, recombination between integrated viral DNA and flanking cellular DNA resulted in the excision of two readily amplifiable chimeras, designated RmI and RmII. The crossing-over that generated RmII was unique in that it involved a simple cellular sequence in which the triplet 5'-CTG-3' was repeated many times. We show that the sequence across the junction resulting from excision was identical in several molecules of RmII, as if the cross-over generating this junction always involved exactly the same two sites on the viral and cellular DNA. We also show that the cellular site mapped where the replacement of a G by an A in one of many successive 5'-CTG-3' triplets generated a homology of five nucleotides (5'-CTACT-3') with the viral site. Oligonucleotides on both sides of these sites are probably involved in matching the two DNAs prior to recombination. MH - Animal ; Chimera ; DNA/ANALYSIS ; DNA Restriction Enzymes/METABOLISM ; DNA, Viral/ANALYSIS ; Mice ; Mutation ; Polyomaviruses/*GENETICS ; Recombination, Genetic ; Structure-Activity Relationship ; Support, Non-U.S. Gov't SO - Mol Cell Biol 1986 Jul;6(7):2727-30 USER: 7 UI - 87064339 AU - Murnane JP TI - Inducible gene expression by DNA rearrangements in human cells. AB - A permanent human cell line, cell line LM205, was established by transforming primary human fibroblasts with a plasmid containing both simian virus 40 sequences with a defective origin of replication and a G418 resistance gene (neo) that lacked a eucaryotic transcriptional promoter. G418-resistant cells appeared spontaneously in clonal populations of LM205 cells at a frequency of approximately 10(-5) cell per cell plated in the presence of 400 micrograms of G418 per ml. G418 resistance was stable and correlated with the appearance of neo-specific RNA. Characterization of the neo gene in the G418-sensitive parental cell line by both a Southern blot analysis and a restriction map analysis of cloned sequences demonstrated that there was a stable integration site containing a single neo coding sequence. A Southern blot analysis of five G418-resistant subclones indicated that there were heterogeneous DNA rearrangements in the region of the neo gene that were unique in each subclone. Restriction mapping of a fragment containing the neo gene isolated from one of the resistant subclones demonstrated that the rearrangement was a tandem duplication that resulted in the relocation of the simian virus 40 bidirectional transcriptional promoter 5' to the neo gene. Tandem duplication was also consistent with the Southern blot polymorphisms observed in the other resistant subclones, suggesting that there were heterogeneous sites of recombination with respect to both the neo gene and the simian virus 40 promoter. Although these rearrangements resulted in an increase in neo gene copy number per cell, amplification showed no correlation quantitatively with the large increase in neo-specific RNA in these cells. Therefore, G418-resistant colony formation in cell line LM205 provides a method for studying both the mechanisms involved in this type of recombination and the factors influencing its frequency. MH - Cell Line ; Cell Transformation, Viral ; Cloning, Molecular ; DNA/ *GENETICS ; DNA Restriction Enzymes ; *Genes ; Human ; Plasmids ; RNA/ GENETICS/ISOLATION & PURIFICATION ; Skin ; Support, U.S. Gov't, Non-P.H.S. ; SV40 Virus/GENETICS ; *Transcription, Genetic ; Transfection SO - Mol Cell Biol 1986 Feb;6(2):549-58 8 UI - 87060955 AU - Leong JM ; Nunes-D:uby SE ; Oser AB ; Lesser CF ; Youderian P ; Susskind MM ; Landy A TI - Structural and regulatory divergence among site-specific recombination genes of lambdoid phage. AB - The lambdoid bacteriophage phi 80 and P22 have site-specific recombination systems similar to that of lambda. Each of the three phage has a different insertion specificity, but structural analysis of their attachment sites suggests that the three recombination pathways share similar features. In this study, we have identified and sequenced the int and xis genes of phi 80 and P22. phi 80 int and xis were identified using a plasmid recombination assay in vivo, and the P22 genes were mapped using Tn1 insertion mutations. In all three phage, the site-specific recombination genes are located directly adjacent to the phage attachment site. Interestingly, the transcriptional orientation of the phi 80 int gene is opposite to that of lambda and P22 int, resulting in convergent transcription of phi 80 int and xis. Because of its transcriptional orientation, phi 80 int cannot be expressed by the major leftward promoter, PL, and the regulatory strategy of phi 80 integration and excision must differ significantly from that of lambda. The deduced amino acid sequences of the recombination proteins of the three systems show surprisingly little homology. Sequences homologous to the lambda PI promoter are more conserved than the protein-coding sequences. Nevertheless, the Int proteins are locally related in the C-terminal sequences, particularly for a stretch of some 25 amino acid residues that lie approximately 50 residues from the C terminus. The Xis proteins can be aligned at their N termini. MH - Amino Acid Sequence ; Attachment Sites (Microbiology) ; Bacteriophages/ *GENETICS ; Base Sequence ; DNA, Viral ; *Genes, Viral ; Promoter Regions (Genetics) ; *Recombination, Genetic ; Sequence Homology, Nucleic Acid ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. ; Viral Proteins/GENETICS SO - J Mol Biol 1986 Jun 20;189(4):603-16 9 UI - 87057043 AU - Thompson JF ; Waechter-Brulla D ; Gumport RI ; Gardner JF ; Moitoso de Vargas L ; Landy A TI - Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory implications. AB - The manner in which integration host factor (IHF) regulates lambda site-specific recombination has been analyzed by examining the behavior of both wild-type and mutant DNAs in integrative and excisive recombination as well as in protein binding. While integrative recombination of an attP with two base changes in the H1 site required 8-fold more IHF than did wild type, binding to this site was lowered at least 500-fold, suggestive of cooperative interactions. A mutant attP with nine base changes did not integrate at all in vitro, with the defect being less severe in vivo. IHF inhibition of excisive recombination was relieved by both mutations in vitro and in vivo. These results imply that occupancy of the H1 site is critical for determining the direction of recombination. It is proposed that IHF inhibition of excision provides a monitor of the strength of the induction stimulus and the nutritional state of the cell; this would allow the prophage to excise selectively in conditions which favor successful completion of the lytic cycle. MH - Bacterial Proteins/GENETICS/*METABOLISM ; Binding Sites ; DNA, Viral/ GENETICS/*METABOLISM ; Escherichia Coli/GENETICS/*METABOLISM ; *Lysogeny ; Phage Lambda/GENETICS/*PHYSIOLOGY ; Protein Binding ; *Recombination, Genetic ; Support, U.S. Gov't, P.H.S. SO - J Bacteriol 1986 Dec;168(3):1343-51 10 UI - 87053881 AU - Naylor LH ; Lilley DM ; van de Sande JH TI - Stress-induced cruciform formation in a cloned d(CATG)10 sequence. AB - The synthetic alternating purine-pyrimidine sequence, d(CATG)10.d(CATG)10, has been cloned into a 2.079-kb pBR322-derived plasmid (pLN1) and its conformation studied under torsional stress. The resultant plasmid, pLNc40, is hypersensitive to cleavage by the single strand-specific nucleases, S1 nuclease and Bal31 nuclease, and to modification by the single strand-selective reagent, osmium tetroxide. The S1-hypersensitive site of this plasmid predominates over those previously mapped in pBR322. Site-specific cleavage of pLNc40 with the resolvase T4 endonuclease VII demonstrates that this alternating purine-pyrimidine tract selectively forms a cruciform structure when stably integrated into a negatively supercoiled plasmid. Quantitative measurements of the twist change (-4.3 +/- 0.2) and free energy of formation (16.2 +/- 0.5 kcal/mol) of this cruciform have been made from two-dimensional gel electrophoresis experiments, and correspond well with the predicted values of cruciform formation for this sequence. We conclude that cruciform extrusion versus the B-Z transition is the favoured conformation of this insert under torsional stress. MH - Base Sequence ; *Cloning, Molecular ; DNA Restriction Enzymes ; DNA, Recombinant ; DNA, Superhelical ; *Nucleic Acid Conformation ; *Oligodeoxyribonucleotides/GENETICS ; Plasmids ; Support, Non-U.S. Gov't ; Thermodynamics SO - EMBO J 1986 Sep;5(9):2407-13 11 UI - 87036910 AU - Herman SA ; Coffin JM TI - Differential transcription from the long terminal repeats of integrated avian leukosis virus DNA. AB - In avian leukosis virus-induced lymphoma and erythroblastosis, the expression of the proto-oncogenes c-myc and c-erbB is activated by downstream or readthrough transcripts initiated within integrated proviral DNA. To determine the relative abundance of viral RNAs extending into the downstream cellular sequences independently of the effects that may be exerted by specific sites of proviral integration, we examined the RNA of infected avian fibroblasts. Using a nuclease protection strategy to detect downstream, readthrough, and normal viral RNAs and to distinguish them from each other, we found that transcripts initiated within the 3' long terminal repeat, i.e., downstream transcripts, were undetectable in infected fibroblasts and could not have amounted to more than 1 to 2% of the total viral RNA. However, readthrough RNAs, which are transcripts initiated within the 5' long terminal repeat and extended beyond the viral polyadenylation site into the downstream cellular DNA, were present at relatively high levels, making up approximately 15% of the total viral RNA. MH - Animal ; Avian Leukosis/FAMILIAL & GENETIC/MICROBIOLOGY ; Avian Leukosis Viruses/*GENETICS ; Cells, Cultured ; Chick Embryo ; DNA, Viral/GENETICS ; Lymphoma/FAMILIAL & GENETIC/MICROBIOLOGY ; Proto-Oncogenes ; Recombination, Genetic ; Repetitive Sequences, Nucleic Acid ; RNA, Viral/ ANALYSIS/GENETICS ; Support, U.S. Gov't, P.H.S. ; *Transcription, Genetic SO - J Virol 1986 Nov;60(2):497-505 12 UI - 87028239 AU - Yanofsky MF ; Porter SG ; Young C ; Albright LM ; Gordon MP ; Nester EW TI - The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. AB - Tumor formation by Agrobacterium tumefaciens involves the transfer and integration of a defined segment (T-DNA) of tumor-inducing (Ti) plasmid DNA into the plant nuclear genome. A set of plasmid genes outside the T-DNA, the vir genes, are thought to mediate the transfer process. We report here that the virD operon encodes a site-specific endonuclease that cleaves at a unique site within each of the 24 bp direct repeats that flank the T-DNA. The endonuclease function was further localized to the 5' end of this operon by demonstrating that cleavage does not occur in virD mutant strains of Agrobacterium and that the 5' end of the virD operon is sufficient to direct cleavage in E. coli. Analysis of nucleotide sequence and protein data indicate that two proteins of 16.2 and 47.4 kd are involved. MH - Agrobacterium/*GENETICS ; Amino Acid Sequence ; Base Sequence ; DNA, Viral ; Endonucleases/*GENETICS ; Escherichia Coli ; Mutation ; *Operon ; Plant Tumors ; Plasmids ; Ribonucleases/METABOLISM ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. SO - Cell 1986 Nov 7;47(3):471-7 13 UI - 86304548 AU - Berg CA ; Gall JG TI - Microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs. AB - Telomeres are essential structures that stabilize the ends of eukaryotic chromosomes and allow complete replication of linear DNA molecules. We examined the structure and replication of telomeres by observing the fate of the linear extrachromosomal rDNA of Tetrahymena after injection into unfertilized Xenopus eggs. The rDNA replicated efficiently as a linear extrachromosomal molecule, increasing in mass 30-50-fold by 15-20 h after injection. In addition, the molecules increased in length by addition of up to several kilobases of DNA to their termini. Sequence analysis demonstrated that the added DNA bore no resemblance to known telomeres. The junction between the rDNA and added DNA was apparently random, indicating that the addition reaction did not involve a site-specific recombination or integration event. Surprisingly, Southern blot analysis showed that the added DNA did not derive from Xenopus DNA, but rather from co-purifying and therefore co-injected Tetrahymena DNA. The nonspecific ligation of random DNA fragments to the rDNA termini suggests that microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs. MH - Animal ; Base Sequence ; DNA Replication ; DNA, Recombinant/*METABOLISM ; DNA, Ribosomal/*GENETICS/METABOLISM ; Extrachromosomal Inheritance ; Female ; Microinjections ; Oocytes ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Tetrahymena/*GENETICS ; Xenopus laevis/*METABOLISM SO - J Cell Biol 1986 Sep;103(3):691-8 14 UI - 86289722 AU - DeVries PJ ; Davidson RL ; Clough DW TI - Site specificity of DNA methylation and expression of herpes simplex thymidine kinase gene. AB - Analysis of the methylation pattern of a single-copy herpes simplex virus thymidine kinase gene integrated into the genome of mouse L cells revealed that hypomethylation of five specific AvaI sites correlates with expression of the TK gene in all of the cell lines tested. Of these specific sites, one lies 5' to the coding region, one 3' to the coding region, and three lie within the coding region of the thymidine kinase gene. Analysis of methylation at a variety of other sites using other methylation-sensitive endonucleases revealed considerable variation in the methylation patterns, apparently unrelated to gene expression and subject to variation with time in culture. MH - Animal ; Chromosome Mapping ; DNA Restriction Enzymes ; DNA, Viral/ *GENETICS/METABOLISM ; *Genes, Viral ; Herpesvirus Hominis/ENZYMOLOGY/ *GENETICS ; L Cells/*MICROBIOLOGY ; Methylation ; Mice ; Support, U.S. Gov't, P.H.S. ; Thymidine Kinase/*GENETICS SO - Somatic Cell Mol Genet 1986 Jul;12(4):385-93 15 UI - 86287281 AU - Lee CY ; Iandolo JJ TI - Integration of staphylococcal phage L54a occurs by site-specific recombination: structural analysis of the attachment sites. AB - Lysogenization by staphylococcal phage L54a induces the loss of lipase (glycerol ester hydrolase) activity in its host Staphylococcus aureus. The attachment site of the bacterial chromosome (attB) for the phage is at the 3' end of the lipase gene, geh. The DNA fragment containing the attB (base pairs 2620-2637 inclusive) site has been sequenced. We have also cloned and determined the nucleotide sequence of the DNA fragments containing the other three attachment sites--i.e., the attP locus on the circularly permuted phage genome and the attL and attR loci at the left and right ends of the prophage in the lysogenized strain. These results reveal that an 18-base-pair core sequence is common to all four att sites. These data indicate that the crossover point must exist within the core sequence and, further, that integration is site- and orientation-specific. We also localized the viral recombinase gene to a 2.1-kilobase DNA segment extending rightward to the attP site. This region was found to be essential for integration of plasmids containing the attP site. MH - Base Sequence ; Chromosome Mapping ; DNA Nucleotidyltransferases/GENETICS ; DNA, Bacterial/GENETICS ; DNA, Viral/GENETICS ; Genes, Viral ; Lipase/ GENETICS ; *Lysogeny ; *Recombination, Genetic ; Staphylococcal Phages/ *GENETICS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. SO - Proc Natl Acad Sci USA 1986 Aug;83(15):5474-8 16 UI - 86283308 AU - Merregaert J ; Michiels L ; van der Rauwelaert E ; Lommel M ; Gol-Winkler R ; Janowski M TI - Oncogene involvement in radiation- and virus-induced mouse osteosarcomas. AB - Internal irradiation of mice using bone seeking radionuclides results in the activation of endogenous retroviruses and in the subsequent development of bone tumors. Genomic DNA from an osteosarcoma cell line, derived from an 90Sr-induced bone tumor, was cotransfected with the plasmid pSV2-neo into NIH/3T3 cells and G418-resistant transfectants gave rise to colonies in soft agar. Southern blot analysis of these first cycle transformants revealed the presence of extra copies of c-ras. We have analysed the arrangement of ecotropic murine leukemia proviral sequences in seven 90Sr-induced bone tumors and one osteosarcoma cell line of CF1-mice. Integration of ecotropic and/or ecotropic recombinant proviruses seems to be involved in rearrangements of 3' provirus cellular junction fragments occurring in all tumor DNAs analysed, but no indication for site-specific integration was found. We also determined the primary structure of FBR-MuSV, a transforming retrovirus able to induce bone tumors in newborn mice. FBR-MuSV contains sequences from all four exons of the murine c-fos gene, but lacks sequences encoding the first 24 and the last 98 amino acids of the c-fos gene product. The coding region of FBR-MuSV has also undergone two small in frame deletions. Thus, the v-fosFBR-MuSV retains 236 amino acids of the 380 amino acids of the murine c-fos product. In FBR-MuSV-transformed cells two fos-containing mRNAs have been detected: a 3.3-kb full-size genomic RNA and a 2.2-kb subgenomic mRNA as revealed by both fos- and MuLV-hybridization probes. MH - Animal ; Cell Line ; Chromosome Mapping ; DNA Restriction Enzymes/ DIAGNOSTIC USE ; DNA, Neoplasm/GENETICS ; Gene Expression Regulation ; Mice ; Mouse Sarcoma Viruses/*GENETICS ; Neoplasms, Radiation-Induced/ *FAMILIAL & GENETIC ; *Oncogenes ; Sarcoma, Osteogenic/ETIOLOGY/*FAMILIAL & GENETIC ; Strontium Radioisotopes/TOXICITY ; Support, Non-U.S. Gov't SO - Leuk Res 1986;10(7):915-21 17 UI - 86283301 AU - Janowski M ; Merregaert J ; Reddy P TI - Retroviruses in radiation-induced lymphomas. AB - The nucleotide sequence of RadLV/VL3 (T+L+), the thymotropic and leukemogenic entity of the in-vitro propagated radiation leukemia virus complex (RadLV/VL3), is that of a recombinant retrovirus. The gag, pol and most of the env gene are very similar to the homologous regions of Akv MuLV. The 3' end of the env gene and the LTR appear to have derived from a xenotropic MuLV. However, the LTR has acquired a feature shared by other lymphomagenic MuLVs. This feature consists in sequence rearrangements resulting in the generation of presumed enhancer elements. RadLV/VL3(T+L+)-specific proviral sequences were found adjacent to the c-myc gene in several virus-induced thymic lymphomas of the rat, suggesting that the enhancer elements might play a role in lymphomagenesis. However, we found that the presence of a provirus at a specific DNA site can lead to an in-vitro growth advantage and to clonal cell selection independently of a lymphomagenic process. We conclude from this observation that clonal appearance of an integrated provirus in cultured radiogenic lymphoma cells does not necessarily reflect a viral induction of radiation-induced leukemogenesis. MH - Animal ; DNA, Viral/GENETICS ; Gene Expression Regulation ; Genes, Viral ; Leukemia, Radiation-Induced/FAMILIAL & GENETIC/*MICROBIOLOGY ; Linkage (Genetics) ; Lymphoma/FAMILIAL & GENETIC/*MICROBIOLOGY ; Mice ; Oncogenes ; Recombination, Genetic ; Retroviridae/GENETICS/*PATHOGENICITY ; Support, Non-U.S. Gov't SO - Leuk Res 1986;10(7):833-42 18 UI - 86250597 AU - Hagblom P ; Korch C ; Jonsson AB ; Normark S TI - Intragenic variation by site-specific recombination in the cryptic plasmid of Neisseria gonorrhoeae. AB - Cryptic plasmid DNA of Neisseria gonorrhoeae was found integrated into the gonococcal chromosome in both plasmid-bearing strains and plasmid-free strains. At several chromosomal locations only segments of the plasmid were found. However, in at least two strains an intact copy of the plasmid seemed to be present with the joints between the plasmid and the chromosomal DNA being located within the cppB gene of the cryptic plasmid. The cppB gene was shown to undergo a sequence-specific intragenic deletion. The deletion removed 54 base pairs, representing 18 amino acids, and did not affect the reading frame. It is proposed that the cryptic plasmid integrates into the chromosome and other gonococcal plasmids within this site-specific deletion region. Models for the site-specific recombination are presented. MH - Amino Acid Sequence ; Bacterial Proteins/GENETICS ; Base Sequence ; Chromosome Deletion ; Chromosomes, Bacterial ; DNA, Bacterial/GENETICS ; Genes, Structural ; Models, Genetic ; Neisseria Gonorrhoeae/*GENETICS ; *Plasmids ; *Recombination, Genetic ; Support, Non-U.S. Gov't SO - J Bacteriol 1986 Jul;167(1):231-7 19 UI - 86248254 AU - Rynditch AV ; Yatsula BA ; Hlo:z:anek I ; Dost:alov:a V ; Mach O TI - Virus-specific nucleotide sequences in duck cells transformed by chicken and duck-adapted Rous sarcoma virus. AB - Adaptation of PR-RSV-C on duck cells results in successful and efficient replication of the adapted virus in duck cells. The adapted variant, daPR-RSV-C, was compared with the parental chicken-cell derived PR-RSV-C. No differences in the efficiency of integration and in the number of integrated proviral copies in duck cells were found. However, the structure of proviral DNA of the adapted virus was different. Whereas EcoRI and HindIII digestion showed no differences between chicken-cell derived PR-RSV-C and the daPR-RSV-C, a new restriction site was found for BamHI endonuclease, which is probably located at the 3' end of the env gene. MH - Adaptation, Physiological ; Animal ; Avian Sarcoma Viruses/*GENETICS/ PHYSIOLOGY ; Base Sequence ; Cells, Cultured ; Chickens ; Ducks ; DNA Restriction Enzymes ; DNA, Viral/*GENETICS ; Nucleic Acid Hybridization ; *Transformation, Genetic ; Virus Replication SO - Folia Biol (Praha) 1986;32(2):145-53 20 UI - 86223823 AU - Omer CA ; Cohen SN TI - Structural analysis of plasmid and chromosomal loci involved in site-specific excision and integration of the SLP1 element of Streptomyces coelicolor. AB - SLP1int (integrated [int] form of Streptomyces lividans plasmid 1 [SLP1]) is a Streptomyces coelicolor A3(2) transmissible sequence capable of autonomous replication as well as site-specific integration into and excision from the S. coelicolor chromosome. We report here that the plasmid and chromosomal loci involved in the integration of SLP1 and the two loci at which the recombination occurs during excision all share at least 111 base pairs of a 112-base-pair DNA sequence. Recombinational cross-over during integration or excision occurred nonrandomly within the common att sequence at or near a 25-base-pair inverted repeat. We suggest that chromosomally integrated plasmidogenic segments such as SLP1int may be involved in the acquisition and structural organization of genes encoding the diverse metabolic capabilities observed in different streptomycetes. MH - Base Sequence ; *Chromosome Mapping ; DNA, Bacterial/ANALYSIS ; Endonucleases/METABOLISM ; *Nucleic Acid Conformation ; *Plasmids ; Recombination, Genetic ; Repetitive Sequences, Nucleic Acid ; Streptomyces/*GENETICS ; Substrate Specificity ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. SO - J Bacteriol 1986 Jun;166(3):999-1006 21 UI - 86089316 AU - Colicelli J ; Goff SP TI - Isolation of a recombinant murine leukemia virus utilizing a new primer tRNA. AB - We have previously described the construction of a mutant of Moloney murine leukemia virus bearing a deletion at the normal site of integration of the viral DNA. We have now recovered a revertant of the virus after abortive infection of mouse cells and have determined the structure of the new virus. The revertant is a recombinant virus containing a 500-base-pair patch of new sequences derived from the mouse genome. The integration site was perfectly restored to the wild-type sequence, although the patch of DNA was overall only 80% homologous to Moloney murine leukemia virus. Surprisingly, the tRNA primer binding site was no longer homologous to the usual proline tRNAs, but was a perfect match for glutamine tRNA. This result suggests that the Moloney murine leukemia virus reverse transcriptase is not specific to one tRNA, but can utilize different tRNAs to prime the synthesis of viral DNA. Comparisons with published reports allowed the identification of sequences that are 94% homologous to the patch sequence, present in one of the endogenous retroviral sequences of the mouse. No replication-competent members of this family, utilizing the glutamine tRNA primer, have been previously isolated. MH - Animal ; Base Sequence ; Binding Sites ; DNA, Viral/BIOSYNTHESIS ; Mice/ GENETICS ; Moloney Leukemia Virus/GENETICS/*ISOLATION & PURIFICATION ; Recombination, Genetic ; Retroviridae Proteins/GENETICS ; Reverse Transcriptase/METABOLISM ; RNA/PHYSIOLOGY ; RNA, Transfer, Amino Acyl/ PHYSIOLOGY ; RNA, Viral/PHYSIOLOGY ; Sequence Homology, Nucleic Acid ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Viral Proteins/ METABOLISM ; *Virus Replication SO - J Virol 1986 Jan;57(1):37-45 22 UI - 86120304 AU - Stone TW ; Potter KN TI - Methylation blockage and other improvements to a comprehensive DNA analysis program. AB - A comprehensive DNA analysis computer program was described in the second special issue of Nucleic Acids Research on the applications of computers to research on nucleic acids by Stone and Potter (1). Criteria used in designing the program were user friendliness, ability to handle large DNA sequences, low storage requirement, migratability to other computers and comprehensive analysis capability. The program has been used extensively in an industrial-research environment. This paper talks about improvements to that program. These improvements include testing for methylation blockage of restriction enzyme recognition sites, homology analysis, RNA folding analysis, integration of a large DNA database (GenBank), a site specific mutagenesis analysis, a protein database and protein searching programs. The original design of the DNA analysis program using a command executive from which any analytical programs can be called, has proven to be extremely versatile in integrating both developed and outside programs to the file management system employed. MH - Base Sequence ; *Computers ; DNA/*ANALYSIS ; Information Systems ; Methylation ; Mutation ; *Software SO - Nucleic Acids Res 1986 Jan 10;14(1):255-64 23 UI - 86200459 AU - Grandgenett DP ; Vora AC ; Swanstrom R ; Olsen JC TI - Nuclease mechanism of the avian retrovirus pp32 endonuclease. AB - In vivo, the inferred circular retrovirus DNA precursor to the provirus contains two long terminal repeats (LTRs) in tandem. We studied the site-specific nicking of supercoiled DNA that contains tandem copies of avian retrovirus LTR DNA in vitro by using purified avian myeloblastosis virus pp32 endonuclease, Mg2+, and viral DNA substrates containing different LTR circle junction sequences. The results confirmed our previous observation that the pp32 protein generates two nicks, one in either viral DNA strand, each 2 nucleotides from the circle junction site. The specificity of nicking by pp32 was unchanged over an eight-fold range of protein concentration and with different avian retrovirus LTR circle junction substrates. These data are consistent with models which propose a role for the endonuclease in removal of two nucleotides from the LTR termini on integration of viral DNA in vivo. MH - Avian Leukosis Viruses/*ENZYMOLOGY ; DNA, Viral/ANALYSIS ; Endonucleases/ *PHARMACOLOGY ; Manganese/PHARMACOLOGY ; Myeloblastosis Virus, Avian/ *ENZYMOLOGY ; Phosphoproteins/*PHARMACOLOGY ; Repetitive Sequences, Nucleic Acid ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. SO - J Virol 1986 Jun;58(3):970-4 24 UI - 86168046 AU - Sargent MG ; Bennett MF TI - Identification of a specific membrane-particle-associated DNA sequence in Bacillus subtilis. AB - After the Bacillus subtilis nucleoid was dissected with restriction endonucleases, a specific DNA sequence from the purA region was isolated in a particulate form that probably originated from the cell membrane. Precise definition of the binding region within this sequence was achieved by a novel procedure based on a previously reported observation that additional copies of the binding region, introduced into the chromosome using an integrative plasmid, were also predominantly particle bound. Subsections of the original plasmid insertion were cloned into the integrative plasmid and introduced into B. subtilis, in which they became tandemly reiterated under appropriate selective conditions. HaeIII sites in the vector, flanking each insertion, were used to excise the latter for subsequent tests of particle association. Examination of 10 strains containing subsections of the original 5.2-kilobase-pair region showed that the binding region was confined to 283 base pairs. This was confirmed by dissection in vitro of a larger, isolated, particle-bound sequence. The nucleotide sequence of a 1,300-base-pair region that contained this site was determined. The entire region had a notably high A + T content and was deficient in open reading frames for transcription. MH - Bacillus Subtilis/*GENETICS ; Base Sequence ; Binding Sites ; Cell Membrane/ANALYSIS ; Cloning, Molecular ; DNA Insertion Elements ; DNA, Bacterial/*ANALYSIS SO - J Bacteriol 1986 Apr;166(1):38-43 25 UI - 86164249 AU - Garcia M ; Wellinger R ; Vessaz A ; Diggelmann H TI - A new site of integration for mouse mammary tumor virus proviral DNA common to BALB/cf(C3H) mammary and kidney adenocarcinomas. AB - The BALB/cf/Cd substrain of mice, developed by inbreeding and selection, shows a 70% incidence of spontaneous kidney adenocarcinomas. Initially foster-nursed on C3H mothers, these mice no longer produce mammary tumors, but there is evidence that mouse mammary tumor virus (MMTV) is involved in the formation of these renal carcinomas. We identified a chromosomal region called int-41, representing a locus interrupted by the integration of an exogenous MMTV provirus in a BALB/c mammary tumor. We found a DNA rearrangement and transcriptional activation in the domain specified by the int-41 probe in one primary kidney adenocarcinoma. A 5.2-kb int-41 specific mRNA was detected in the kidney tumor cell line established from a transplantable renal adenocarcinoma. This mRNA hybridized with MMTV and int-41 specific probes suggesting that it is a hybrid molecule composed of MMTV-LTR sequences covalently linked to host cell RNA. This mRNA was strongly stimulated by the presence of glucocorticoid hormone in the culture medium. Our data are compatible with the hypothesis that the int-41 chromosomal domain is involved in the neoplastic transformation of epithelial cells from different organs. MH - Adenocarcinoma/*FAMILIAL & GENETIC/MICROBIOLOGY ; Animal ; Cell Line ; Chimera ; Cloning, Molecular ; DNA Restriction Enzymes ; DNA, Neoplasm/ *GENETICS ; DNA, Viral/*GENETICS ; Kidney Neoplasms/*FAMILIAL & GENETIC/ MICROBIOLOGY ; Mammary Cancer Virus/*GENETICS ; Mammary Neoplasms, Experimental/*FAMILIAL & GENETIC/MICROBIOLOGY ; Mice ; Mice, Inbred Strains ; Nucleic Acid Hybridization ; Support, Non-U.S. Gov't ; Transcription, Genetic ; Translation, Genetic SO - EMBO J 1986 Jan;5(1):127-34 26 UI - 86144098 AU - Ellis DM ; Dean DH TI - Location of the Bacillus subtilis temperate bacteriophage phi 105 attP attachment site. AB - Chromosomal DNAs of lysogens of phi 105 and phi 105 DI:1t were digested with restriction enzymes EcoRI and HpaI and were probed with nick-translated mature phi 105 DNA. Altered bacteriophage-specific bands in the lysogens were detected, indicating that the phage integrates into the host chromosome at a single site, probably via a Campbell-type circular intermediate. The phage attachment site is centrally located in the phage genome and lies between the phage immunity region and the nonessential deletable region of phi 105. MH - Bacillus Subtilis/*GENETICS ; Bacteriophages/*GENETICS ; Chromosome Mapping ; DNA, Bacterial/GENETICS ; DNA, Viral/*GENETICS ; Genes, Viral ; Lysogeny ; Recombination, Genetic SO - J Virol 1986 Apr;58(1):223-4 27 UI - 86120364 AU - Di Nocera PP ; Graziani F ; Lavorgna G TI - Genomic and structural organization of Drosophila melanogaster G elements. AB - The properties and the genomic organization of G elements, a moderately repeated DNA family of D. melanogaster, are reported. G elements lack terminal repeats, generate target site duplications at the point of insertion and exhibit at one end a stretch of A residues of variable length. In a large number of recombinant clones analyzed G elements occur in tandem arrays, interspersed with specific ribosomal DNA (rDNA) segments. This arrangement results from the insertion of members of the G family within the nontranscribed spacer (NTS) of rDNA units. Similarity of the site of integration of G elements to that of ribosomal DNA insertions suggests that distinct DNA sequences might have been inserted into rDNA through a partly common pathway. MH - Animal ; Base Sequence ; *Cloning, Molecular ; Drosophila Melanogaster/ *GENETICS ; DNA/*ISOLATION & PURIFICATION ; DNA Restriction Enzymes ; DNA, Ribosomal/ISOLATION & PURIFICATION ; Nucleic Acid Hybridization ; Repetitive Sequences, Nucleic Acid ; Support, Non-U.S. Gov't SO - Nucleic Acids Res 1986 Jan 24;14(2):675-91 28 UI - 86106220 AU - Thomas KR ; Folger KR ; Capecchi MR TI - High frequency targeting of genes to specific sites in the mammalian genome. AB - We corrected a defective gene residing in the chromosome of a mammalian cell by injecting into the nucleus copies of the same gene carrying a different mutation. We determined how the number, the arrangement, and the chromosomal position of the integrated gene, as well as the number of injected molecules influence the gene-targeting frequency. Recombination between the newly introduced DNA and its chromosomal homolog occurred at a frequency of 1 in 10(3) cells receiving DNA. Correction events were mediated by either double reciprocal recombination or gene conversion. This resulted in sequences in the genome being replaced by sequences of the introduced DNA or, in separate experiments, sequences in the incoming DNA being replaced by chromosomal sequences. Both point mutations and deletion mutations were corrected; however, the nature of the mutation carried by the respective sequence influenced whether the integrated or injected sequence was corrected. MH - Alleles ; Animal ; Base Sequence ; Cell Line ; Chromosome Deletion ; Drug Resistance ; DNA Restriction Enzymes ; DNA, Recombinant ; Mice ; Models, Genetic ; *Mutation ; Neomycin/PHARMACOLOGY ; Plasmids ; *Recombination, Genetic ; Transfection SO - Cell 1986 Feb 14;44(3):419-28 29 UI - 86098685 AU - Gray DA ; McGrath CM ; Jones RF ; Morris VL TI - A common mouse mammary tumor virus integration site in chemically induced precancerous mammary hyperplasias. AB - Mammary carcinomas can be induced by chemical and hormonal as well as viral carcinogens. Irrespective of the class of inducer, these tumors develop in discrete stages, of which alveolar hyperplasia is one of the earliest identifiable. Since carcinogenesis by the mammary tumor virus is now thought to involve proviral activation of adjacent cell genes at specific loci, we sought to determine if a similar mechanism also played a role in chemical and hormonal carcinogenesis and if its role was stage specific. Three high-tumor-incidence BALB/c hyperplastic alveolar nodule outgrowths of two different etiologies were found to have exogenous mouse mammary tumor virus proviruses integrated at the same site in the genome. This common site of integration is not within the bounds of the int-1 and int-2 loci into which proviruses detected at these loci are clustered in MMTV-induced mammary tumors. All three HANs are commonly impaired in end-point differentiation. We propose that mouse mammary tumor virus integration at this site is responsible for a specific abnormality in differentiation associated with the preneoplastic phenotype. MH - Animal ; Base Sequence ; Breast Diseases/*CHEMICALLY INDUCED/MICROBIOLOGY/ PATHOLOGY ; Caseins/BIOSYNTHESIS ; Cell Line ; DNA/ANALYSIS ; DNA Restriction Enzymes ; DNA, Viral/ANALYSIS ; Female ; Gene Expression Regulation ; Hormones/TOXICITY ; Hyperplasia ; Mammary Cancer Virus/ *ISOLATION & PURIFICATION/PHYSIOLOGY ; Mice ; Mice, Inbred BALB C ; Precancerous Conditions/*CHEMICALLY INDUCED/MICROBIOLOGY/PATHOLOGY ; Recombination, Genetic ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; 9,10-Dimethyl-1,2-Benzanthracene/TOXICITY SO - Virology 1986 Jan 30;148(2):360-8 30 UI - 86089311 AU - Moreau-Gachelin F ; D'Auriol L ; Robert-Lezenes J ; Galibert F ; Tambourin P ; Tavitian A TI - Analysis of integrated proviral DNA sequences with an octadecanucleotide probe designed for specific identification of spleen focus-forming virus in the mouse genome. AB - The Friend spleen focus-forming virus (SFFV) is an envelope gene recombinant between the ecotropic Friend murine leukemia virus and the endogenous xenotropic mink cell focus-forming retroviral sequences. We synthesized an octadecanucleotide complementary to the 3' end of the SFFV env gene designed for discriminating the SFFV proviruses from the xenotropic mink cell focus-forming virus and ecotropic exogenous or endogenous viral sequences. Under appropriate hybridization conditions this probe allowed the identification, in addition to few endogenous DNA fragments, of multiple SFFV proviruses integrated in the genome of Friend malignant cells. Therefore this probe should be of interest in further characterizing the SFFV integration sites and possibly the SFFV ancestor gene. MH - Animal ; Base Sequence ; DNA, Neoplasm/ANALYSIS ; DNA, Viral/*ANALYSIS ; Friend Virus/GENETICS ; Genetic Marker ; Leukemia, Experimental/FAMILIAL & GENETIC/*MICROBIOLOGY ; Mice/GENETICS/MICROBIOLOGY ; Mink Cell Focus-Inducing Viruses/GENETICS ; Mouse Leukemia Viruses/*ISOLATION & PURIFICATION ; Nucleic Acid Hybridization ; Recombination, Genetic ; Spleen Focus-Forming Viruses/GENETICS/*ISOLATION & PURIFICATION ; Support, Non-U.S. Gov't ; Viral Envelope Proteins/GENETICS SO - J Virol 1986 Jan;57(1):349-52 31 UI - 86085687 AU - Waldman AS ; Fitzmaurice WP ; Scocca JJ TI - Integration of the bacteriophage HP1c1 genome into the Haemophilus influenzae Rd chromosome in the lysogenic state. AB - Restriction fragments hybridizing to phage HP1c1 DNA were identified in digests of DNA from lysogenic strains of Haemophilus influenzae. The results showed that the cohesive ends of the mature phage DNA were joined in lysogens and that the phage genome was covalently linked to the host DNA, indicating that lysogeny involves recombination between specific sites on the phage and host chromosomes. The site on the phage chromosome at which this recombination occurred was between 110 and 750 base pairs of the left end on the mature phage genome. MH - Bacteriophages/*GENETICS ; *Chromosomes, Bacterial ; DNA, Viral ; *Genes, Viral ; Haemophilus Influenzae/*GENETICS ; *Lysogeny ; Support, U.S. Gov't, P.H.S. SO - J Bacteriol 1986 Jan;165(1):297-300