EcoRI Cleavage Sites Research Articles

Cloning of the marmoset herpesvirus thymidine kinase gene and analyses of the boundaries of the coding region

In order to delimit the approximate boundaries of the marmoset herpesvirus (MarHV) thymidine kinase (TK) gene, HindIII and BamHI digests of MarHV DNA were cloned in plasmid pBR322. Several recombinant plasmids which transformed E. coli K12 strain RR1 to ampicillin resistance were isolated. The MarHV DNA inserts in these plasmids accounted for about half of the MarHV genome. One of the plasmids, pMAR4, contained a 9.1-kbp fragment of MarHV DNA ( HindIII-G), transformed LM(TK −) cells to TK +, and hybridized to the BamHI-I fragment of MarHV DNA, which had previously been shown to have TK-transforming activity. pMAR4 DNA had little or no homology to the 2-kbp PuvII fragment of HSV-1 DNA, which contains the HSV-1 TK gene. Cleavage with PvuII, SacI, SmaI, and KpnI inactivated the TK-transforming activity of pMAR4, but cleavage with HindIII, PstI, EcoRI, XhoI, XbaI, and BamHI did not. Deletion mutants pMAR401 and pMAR420, which lacked the 2.6-kbp KpnI and the 2.75-kbp EcoRI fragments, respectively, of pMAR4, lost transforming activity, whereas pMAR410, which lacked a 2.9kbp XhoI fragment of pMAR4 did not. Recombinant plasmid pMAR430, which contained a 3-kbp PstI fragment of pMAR4, also transformed LM(TK −) cells to TK +. The results strongly suggest that the coding region of the MarHV TK gene was within a 2.4-kbp pMAR4 sequence extending from the PstI (0.33 kbp) to the EcoRI (2.7 kbp) cleavage sites.

Characterization of AKR murine leukemia virus sequences in AKR mouse substrains and structure of integrated recombinant genomes in tumor tissues.

A specific cDNA probe of AKR murine leukemia virus (AKR-MLV) was prepared to detect AKR-MLV sequences in normal and tumor tissues in a variety of AKR mouse substrains. AKR strains contained up to six endogenous AKR-MLV genomes. All substrains tested had one AKR-MLV locus in common, and closely related substrains had several proviruses integrated in an identical site. Virus-induced tumors in the AKR/FuRdA and AKR/JS strains showed a reintegration pattern of AKR-MLV sequences unique for the individual animal, suggesting a monoclonal origin for the outgrown tumors. An analysis of tumor DNAs from the AKR/FuRdA and AKR/JS substrains with restriction enzymes cleaving within the proviral genome revealed a new EcoRI restriction site and BamHI restriction site not present in normal tissues. The positions of these sites corresponded both with cleavage sites of EcoRI and BamHI in integrated Moloney recombinants and with the structure of isolated AKR mink cell focus-forming viruses. All tumors analyzed to data contain nearly identical integrated recombinant genomes, suggesting a causal relationship between the formation of recombinants and the leukemogenic process.

Physical and functional mapping of Tn2603, a transposon encoding ampicillin, streptomycin, sulfonamide, and mercury resistance.

A map of cleavage sites for restriction endonuclease EcoRI, BamHI, HindIII, and SalI on Tn2603, a transposon encoding resistance to ampicillin, streptomycin, sulfonamide, and mercury, was constructed by an analysis of restriction cleavage patterns of plasmid pMK1.::Tn2603 and its deletion derivative. By cloning the fragments generated from pMK1.::Tn2603 with these restriction endonucleases to a pACYC184 plasmid vehicle, the regions necessary for expression of resistance were located on the restriction cleavage map of Tn2603. Ampicillin, streptomycin, and sulfonamide-resistance genes were mapped in a cluster on the region between the center and the right and the mercury-resistance gene was located to the left of the map. The final functional map of Tn2603 was compared with those of Tn4 and Tn21 and the evolutional relationships between them were discussed.

A map of specific cleavage sites and tRNA genes in the chloroplast genome of Euglena gracilis bacillaris

A map showing locations of 22 of the 30 endonuclease EcoRI cleavage sites and 54 additional sites for eight other restriction endonucleases is presented. The regions of chloroplast DNA that hybridize with chloroplast tRNA are also shown.

Physical map of tupaia adenovirus DNA by cleavage with restriction endonucleases and partial denaturation.

The double-stranded DNA of an adenovirus of tupaia (TAV) which has a mol. wt. of 21.5 X 10(6) was analysed as follows: the cleavage sites of restriction endonucleases BamHI, EcoRI, KpnI, SmaI, BstEII, ClaI and HpaI were determined by complete, partial and double digestion followed by gel electrophoretic separation of the resulting fragments. Terminal HpaI fragments were determined by hydrolysing the intact DNA with exonuclease III before restriction enzyme cleavage. Partial denaturation mapping of uncleaved DNA and EcoRI fragments revealed the cleavage sites of EcoRI as well as A + T-rich regions at both termini of the genome.

Bacteriophage phi 1 as a gene-cloning vector in Bacillus subtilis.

We attempted to use Bacillus subtilis phage phi 1 as a gene-cloning vector since the phi 1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A phi 1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, phi 1E1 and phi 1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant phi 1E2 delta 1 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage rho 11 DNA, that phi 1E2 delta 1 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten phi 1 clones isolated from independent transfectants and found that six of them carried rho 11 DNA fragments inserted at either of the two EcoRI-cleavage sites. Some of the hybrid phage DNAs were found to be cleaved with BamHI and HaeIII endonucleases and the rho 11 DNA portion, whereas the parental phi 1E2 delta 1 DNA was insensitive to any of these enzymes. These hybrid phages would therefore be useful vectors for cloning foreign DNA fragments generated by cleavage with BamHI or HaeIII endonucleases.

Physical and functional mapping of RP4-TOL plasmid recombinants: analysis of insertion and deletion mutants.

Cleavage sites for the restriction endonucleases XhoI, BamHI, HindIII, and EcoRI were mapped on the pTN2 plasmid, a recombinant of TOL and RP4, which specifies the toluene-degrading enzymes in the same way as the wild-type TOL plasmid. The pTN2 plasmid, purified from a strain of Escherichia coli, contained the entire length of the RP4 plasmid (about 54 kilobase pairs [kb]) and the TOL segment (about 56 kb). The TOL segment is inserted at about 12 and 5 kb away from the EcoRI and BamHI cleavage sites of RP4, respectively. Cleavage sites for XhoI, BamHI, HindIII, and EcoRI were also mapped on an insertion mutant, pTN1, and two deletion mutants, pTN81 and pTN9. Analysis of pTN81 and pTN9 allowed estimation of the region of the gene cluster for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, as well as the region required for toluate oxygenase activity. Induction of TOL enzymes directed by pTN1 suggested the location and orientation of transcription of the gene cluster for catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase. Analysis of strains carrying both pTN9 and a xylR mutant of the TOL plasmid demonstrated that xylR+ is trans dominant over xylR.

In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals.

We report the construction and use of a series of plasmid vectors suitable for the detection and cloning of translational control signals and 5' coding sequences of exogenously derived genes. In these plasmids, the first eight codons of the amino-terminal end of the lactose operon beta-galactosidase gene, lacZ, were removed, and unique BamHI, EcoRI, and SmaI (XmaI) endonuclease cleavage sites were incorporated adjacent to the eighth codon of lacZ. Introduction of deoxyribonucleic acid fragments containing appropriate regulatory signals and 5' coding sequences into such lac fusion plasmids led to the production of hybrid proteins consisting of the carboxyl-terminal segment of a beta-galactosidase remnant plus a peptide fragment that contained the amino-terminal amino acids encoded by the exogenous deoxyribonucleic acid sequence. These hybrid peptides retained beta-galactosidase enzymatic activity and yielded a Lac+ phenotype. Such hybrid proteins are useful for purifying peptide sequences encoded by exogenous deoxyribonucleic acid fragments and for studies relating the structure and function of specific peptide segments.

Characterisation of complete type II insertions in cloned segments of ribosomal DNA from Drosophila melanogaster

We have isolated cloned segments of ribosomal DNA that have EcoRI restrictable (type II) insertions in their 28 S genes. The type II insertions in these plasmids are homologous sequences and have three characteristic cleavage sites for EcoRI. One of these clones is unusual in that it has undergone a deletion of part of the 28 S gene at or near the site of the type II insertion. A second is unusual in that, in addition to the type II insertion in the rDNA, the transcribed spacer sequences are interrupted by an unidentified sequence. This sequence differs in its arrangement of restriction sites from the sequence that interrupts the transcribed spacer of cDm207 (Glover, 1977). The type II sequences in all these clones share homology with the unusually long ‘insertion’ that interrupts the 28 S gene of cDm207. We have re-examined the nature of the additional sequences linked to the type II sequences of cDm207 and find them to be related to type I rDNA insertion sequences.

Epstein-Barr virus (B95-8) DNA VII: molecular cloning and detailed mapping.

Two of the Sal I fragments and all of the internal BamHI fragments (with the exception of BamHI c, a 0.6 x 10(6) dalton fragment) of Epstein-Barr virus (EBV) DNA have been cloned in pBR322. The termini and other parts of the DNA (including the EcoRI fragment which contains BamHI c) have been cloned as EcoRI fragments in bacteriophage Charon 4A. The cloned DNAs have been used to derive a complete map of the BamHI fragments of EBV DNA and to align the BamHI, EcoRI, HindIII, and SalI cleavage sites in EBV DNA.

A virus in Beechey ground squirrels that is related to hepatitis B virus of humans.

A virus given the name ground squirrel hepatitis virus (or GSHV), with many of the unique characteristics of human hepatitis B virus (HBV), has been found in Beechey ground squirrels in northern California. Common features include virus morphology, viral DNA size and structure, a virion DNA polymerase that repairs a single-stranded region in the viral DNA, crossreacting viral antigens, and persistent infection with viral antigen continuously in the blood. Although similar, GSHV and HBV Are not identical. The ground squirrel virion has a slightly greater diameter, the viral surface antigens crossreact only partially and, thus, are not identical, and GSHV DNA has two restriction endonuclease EcoRI cleavage sites in contrast to the single site in HBV DNA. Thus, GSHV is a member of the virus group that includes HBV and the virus recently found in woodchucks in the eastern United States and named woodchuck hepatitis virus. It is not yet known how closely the ground squirrel and woodchuck viruses are related.

A physical gene map of the bacteriophage P22 late region: Genetic analysis of cloned fragments of P22 DNA

A physical gene map of the late region of the P22 chromosome has been constructed by genetic analysis of restriction enzyme fragments of P22 DNA cloned in a plasmid vector. Cleavage sites for restriction endonucleases SalI, SstI, SmaI, Xhoi, and BglI were mapped on P22 DNA to provide physical reference points in addition to the EcoRI, HindIII, and BamHI cleavage sites previously mapped. Restriction enzymes KpnI, BglII, and Xbal were found to have no cleavage sites on P22 DNA. Fragments of P22 DNA produced by cleavage with EcoRI, BamHI, or EcoRI plus BamHI were cloned in Escherichia coli using the plasmid vector pBR322, and the resulting recombinant plasmids were introduced into Salmonella typhimurium. The genes present on a cloned fragment were identified by the ability of the hybrid plasmid to complement or recombine with P22 amber mutations in known genes when mutant phage were used to infect S. typhimurium strains carrying the recombinant plasmids. These experiments place all phage genes required for P22 head morphogenesis except gene 3 on the physical map between coordinates 0.000 and 0.318. The coding capacity of this interval is in close agreement with the molecular weights of the proteins assigned to it. The single gene for the P22 base plate protein is placed between coordinates 0.376 and 0.420 on the physical map. These results also show that distances on the recombination frequency map are significantly distorted relative to the physical gene map of the late region. The recombination frequency map is expanded in the region of the physical gene map where terminally redundant ends of the circularly permuted mature chromosomes fall.

Genetic analysis of the cloned genome of phage Mu

Randomly sheared fragments of Mu cts62 DNA have been cloned into the EcoRI cleavage site of plasmid p0P203(UV-5)-3, a derivative of pMB9 containing the lactose operator promoter, using poly (dA · dT) tailing with terminal transferase. The extent of Mu DNA carried by the recombinant plasmids was determined genetically by crosses with known markers in essential genes of Mu in rec + backgrounds. Plasmids which rescued essential genes were transformed to rec − backgrounds to check for expression of the Mu genes. Plasmids were found which rescued and complemented 23 of the 24 essential genes of Mu. Many of these plasmids carried overlapping segments of the Mu genome. The plasmid library was also screened for clones containing the nonessential genes gin and mom. Clones carrying gene A were not found in the initial screening, however, several clones which were resistant to infection by Mu due to expression of the Mu repressor were able to rescue and complement a D108 cts-Mu Ats hybrid phage at 43°.

The 5' terminus of the precursor ribosomal RNA of Saccharomyces cerevisiae.

The 5' terminus of Saccharomyces cereviasiae 35S pre rRNA was mapped on the rDNA using two methods: 1) Suitable restriction endonuclease fragments were hybridized to total high molecular weight RNA and extended with reverse transcriptase to the 5' end of the RNA template. 2) Other restriction fragments spanning the 5' terminus of 35S pre rRNA and radioactively labeled at their ends were hybridized to high molecular weight RNA and the non hybridized nucleic acids were digested with S1 nuclease. On the basis of these experiments, the 5' terminus of 35S pre rRNA was placed approximately 670 nucleotides upstream from the 17S rRNA coding region. The exact position was determined by reverse transcription as above, but in the presence of dideoxyribonucleoside triphosphates, which served as a way of sequencing the 5' terminal region. 35S pre rRNA synthesis is initiated at a site in EcoRI restriction fragment B which is 48 nucleotides upstream from the EcoRI cleavage site in the coding strand.

Viable deletion mutant of human papovavirus BK that induces insulinomas in hamsters

A plaque morphology mutant (pm-522) of human papovavirus BK, which was rescued from a human papovavirus BK-induced hamster pineocytoma, was characterized and compared with a cloned wild-type virus (wt-501). Mutant pm-522 formed turbid plaques and grew more slowly than wt-501 in human embryonic kidney (HEK) cells. The immunofluorescence assay revealed that more HEK cells underwent abortive infection with pm-522 than with wt-501. Whereas wt-501 induced brain tumors and osteosarcomas, but no insulinomas, in hamsters, pm-522 induced brain tumors and insulinomas. The DNA of pm-522 was found by electrophoresis and electron microscopy to have a deletion (85 +/- 15 base pairs) and an insertion (40 +/- 10 base pairs) between map coordinates 0.708 and 0.725 from the endonuclease EcoRI cleavage site. These results demonstrate the presence of a viable deletion human papovarivus BK mutant capable of inducing insulinomas in hamsters.

A restriction map of cauliflower mosaic virus DNA (strain PV 147). Mapping of the cleavage sites of HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III.

The virion-extracted DNA (Mr5 x 10(6)) of cauliflower mosaic virus (CaMV) has three single-stranded interruptions. The mapping of this DNA using eleven restriction endonucleases (HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III) is reported here. The existence of the three single-stranded breaks complicates the identification and the molecular weight determination of fragments produced by HpaII, HindIII and HindII + III. Indeed the electrophoretic mobility of some fragments in which a single-stranded discontinuity is located is modified, and the fluorescence of ethidium bromide complexed with these fragments is reduced as compared to that observed for the other fragments existing in a molar ratio. These drawbacks were overcome by performing experiments of nick-translation of CaMV DNA with Escherichia coli DNA polymerase I. FRom the data it follows that the CaMV DNA molecule bears bears 1 site for HhaI and SacI, 2 for AvaI and PvuII, 3 for PstI, 4 for XbaI, 5 for EcoRI, 6 for Bg/II and HincII, 11 for HpaII and 15 for HindII + III. The corresponding fragments have all been ordered and precisely located providing a suitable map for further investigations connected with the study of the fine structure and the function of the CaMV genome.

Transposon A-generated mutations in the mercuric resistance genes of plasmid R100-1

A series of 23 transposon 801(Tn801)-induced mutations of plasmid R100-1 from mercuric salts resistance to sensitivity was studied. Although Tn801 transposed frequently into the mer region of the plasmid, fine structural analysis showed that the site of insertion within mer varied. About one-half of the Tn801 insertion events also caused a deletion of greater than 1 megadalton. Genetic and restriction endonuclease EcoRI and BamHI analysis of the mutant plasmid deoxyribonucleic acid elucidated the organization of the mer operon and suggested the existence of a trans-acting regulatory factor governing resistance to mercuric salts. Tn801 insertions leading to mercuric sensitivity occurred in the restriction endonuclease fragments EcoRI-H and EcoRI-I. Regulatory mutations leading to a 50-fold-reduced synthesis of mercuric reductase enzyme occurred in two complementation classes thought to represent the gene for a trans-acting inducer molecule and a cis-acting operator-promoter sequence. Mutations leading to total loss of the enzyme mercuric reductase occurred on both the EcoRI-H and EcoRI-I fragments, showing that the structural gene for this enzyme (merA) bridges the EcoRI cleavage site separating the segments. Hypersensitivity to mercuric salts resulted when Tn801 insertion occurred in the reductase gene in the operatordistal portion of the operon. Hypersensitive cells inducibly bound three to five times more Hg2+ at low concentrations than did sensitive (plasmidless) cells. This finding led to the proposal that another gene (merT) controls uptake of Hg2+ by the cells. Transcription of the operon was deduced to start in the EcoRI-H fragment and to move into the EcoRI-I fragment of the plasmid genome.

Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Cleavage of the group-A streptococcal macrolide, lincosamide, and streptogramin B (MLS) resistance plasmid pSM19035 yields 2 fragments [13 and 4 megadaltons (MD)] with EcoRI, and 15 fragments with HindIII, 12 of which are 6 pairs of identical fragments derived from the inverted repeats that comprise about 80% of the pSM19035 genome. The large EcoRI fragment was isolated, ligated, and used to transform the Challis strain of Streptococcus sanguis to erythromycin resistance. Plasmids (pDB101, pDB102, and pDB103) isolated from three different transformants had lower molecular masses than the original large EcoRI fragment. HindIII digestion of these molecules and subsequent analysis of fragment radioactivity distributions indicated the loss of plasmid segments of various sizes. The deletions, all of which occurred in the palindrome, did not affect the level and the inducible nature of pSM19035-determined antibiotic resistance. Only pDB101 retained the unique EcoRI cleavage site. The results of this analysis allowed the construction of an EcoRI and HindIII cleavage-site map of pSM19035 and promise to simplify future studies of genetic functions specified by streptococcal MLS resistance plasmids.

Cleavage sites of restriction endonucleases EcoRI, BamHI and HindIII on chicken embryo rDNA

two types of repeats have been found, one of them bearing an insertion of varying length within the 28 S rRNA gene [4-81. Experimental data from the cleavage sites of different restriction enzymes on ribosomal DNA in higher eukaryotes, like mouse [9,10] or man [9], have indi- cated, that the relative length heterogeneity of the repeat unit for these organisms is not so extensive. On the other hand, a minor heterogeneity of restric- tion fragments was detected, when the DNA was isolated from different individuals [9]. Ribosomal genes of chicken are present in -200-240 copies/ haploid genome [ 111. By EcoRI cleavage and analysis of resultant fragments it was suggested that they are also arranged in tandem repeats [ 121. Here we describe the cleavage sites of restriction enzymes HindIII, EcoRI and BamHI and the size of the repeat unit. 2.

Restriction endonuclease HindIII cleavage site map of bacteriophage P22

The 14 HindIII cleavage sites on P22 DNA have been mapped. HindIII cleavage sites were located relative to EcoRI sites by determining the molecular weights and map order of fragments produced by HindIII, or HindIII and EcoRI digestion. Molecular weights were estimated from the electrophoretic mobility of fragments. The HindIII fragment order was established by HindIII cleavage of segments of the P22 genome obtained as isolated EcoRI fragments or as overlapping genetic substitutions in bacteriophage λ chromosomes. The resulting HindIII/ EcoRI cleavage site map defines physical markers in all regions of the P22 genome and defines the locations of a number of P22 genes on this physical map of the P22 chromosome. Three HindIII sites and two HpaI sites have been mapped in immI, one of two P22 gene clusters controlling lysogeny. Two of these HindIII sites lie within the structural gene ant specifying one of the regulatory proteins of the immI region. Assignment of the ant gene to specific HindIII fragments utilized the insertion element Tnl, which was shown to contain no HindIII cleavage sites.