Single EcoRI Site Research Articles

Construction of an M13 histidine-transducing phage: A single-stranded cloning vehicle with one EcoRI site

In order to create a ready source of single-stranded DNA for DNA sequence determination by the dideoxy chain-termination method, the promoter-proximal part of the histidine operon, the hisOGD region of Salmonella typhimurium, was cloned onto the single-stranded phage M13. Both orientations of the his DNA were cloned to supply DNA template for sequencing of each strand. Insertion was achieved at an HaeIII site in the intergenic region (IR) of M13, and a single EcoRI site was purposely regenerated at one boundary of the his DNA insert. Infected colonies, not plaques, were selected using the hisD gene as a selective marker. The single RI site and the hisD marker for auxotrophic selection represent improvements on the wild type M13 as a single-stranded vector for cloning other DNA.

Heterodupkxes of φ174 and G4 DNAs: orientation to genetic map and comparison with predictions from nucleotide sequences

Heteroduplexes between the viral DNA of phiX174 and DNA from the replicative form (RF) of phage G4 were examined by electron microscopy. The single Eco RI site of G4-RF was utilized as a physical marker by preparing the heteroduplexes from the denatured, linear DNA obtained by restricting G4-RF with Eco RI endonuclease. Restriction fragments of phiX were used in a separate series of heteroduplexes to align the heteroduplex map and the G4 Eco RI site with the similar genetic maps of the two phages. The positions of the branch migrating junctions of recombinant phiX-G4 figure-8s, previously located only with respect to the G4-Eco RI site, have now been located with high proability within the gene A region of the two genomes. The degree of mismatch between the known nucleotide sequences of phi X and G4 accounts for positions of all of the regions of single-strandedness in the observed heteroduplexes, but unexplained discrepancies were also found.

Ribosomal DNA in Drosophila melanogaster: I. Isolation and characterization of cloned fragments

Fragments of rDNA‡ from Drosophila melanogaster produced by the restriction endonuclease EcoRI were cloned in the form of recombinant plasmids in Escheriehia coli. Maps were prepared showing the location of the coding regions and of several restriction endonuclease sites. Most rDNA repeats have a single EcoRI site in the 18 S gene region. Thus, 19 of 24 recombinant clones contained a full repeat of rDNA. Ten repeats with continuous 28 S genes and repeats containing insertions in the 28 S gene of 0.5, 1 and 5 kb were isolated. The 0.5 and 1 kb insertion sequences are homologous to segments of the 5 kb insertions; because of this homology they are grouped together and identified as type 1 insertions. Four recombinant clones contain an rDNA fragment that corresponds to only a portion of a repeating unit. In these fragments the 28 S gene is interrupted by a sequence which had been cleaved by EcoRI. The interrupting sequences in these clones are not homologous to any portion of type 1 insertions and are therefore classified as type 2. In one of the above clones the 28 S gene is interrupted at an unusual position; such a structure is rare or absent in genomic rDNA from the fly. Another unusual rDNA fragment was isolated as a recombinant molecule. In this fragment the entire 18 S gene and portions of the spacer regions surrounding it are missing from one repeat. A molecule with the same structure has been found in uncloned genomic rDNA by electron microscopic examination of RNA/DNA hybrids.

Construction and characterization of new cloning vehicles III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR325. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Ap r) and tetracycline (Tc r) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Ap r and Tc r genes from pBR322 and the cloramphenicol resistance gene (Cm r) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cm r gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selecion of EcoRI, BamHI, HindIII, PstI, HincII, SalI, ( XamI), SmaI, ( XmaI)), BglII and DpnII restriction generated DNA molecules.

Regulated expression by readthrough translation from a plasmid-encoded beta-galactosidase

We have characterized expression of beta-galactosidase from a plasmid cloning vehicle, pBGP120, which carries most of the lacZ gene and contains a single EcoRI site near the end of lacZ. In addition, we have examined expression of heterologous DNA inserted at the position of the EcoRI site. The EcoRI site was shown to be within the sequence coding for beta-galactosidase and its precise location and phase were deduced. Insertion of heterologous EcoRI-generated DNA fragments altered the molecular weight of the plasmid-encoded beta-galactosidase polypeptide. Those insertions that were in the correct phase were expressed at a high level as a fused protein. The different forms of beta-galactosidase polypeptides produced by various hybrid plasmids were all stable proteins. The level of expression of the plasmid-encoded beta-galactosidase was several times higher than maximal expression of chromosome-encoded beta-galactosidase, suggesting that expression is proportional to gene copy number. The expression of the plasmid lacZ gene was controlled by cyclic AMP. When grown in a cya strain (DG74), expression was dependent on exogenous cyclic AMP. Although in normal strains there was insufficient lac repressor to inactivate all copies of the plasmid, repressor regulation was restored when the plasmid was grown in a strain (M96) that overproduces the lac repressor.

572 THE MOLECULAR ANATOMY OF A HUMAN GENE

The ability of bacterial restriction endonuclease to recognize specific senuences in DNA makes them a potent tool for dissection of the human nenome. The organization of ribosomal nenes in normal and neoplastic human tissues has been studied by the restriction of purified DNA. Franments of DNA produced by the restriction endonucleases EcoRI and HindIII were separated accordinn to size by agarose gel electronhoresis. DNA franments which contained ribosomal genes were identified by hybridization to 125I-labelled human ribosomal RNA and autoradiography. Analysis of these studies showed that EcoRI cleaves ribosomal DNA from human spleen into fragments of three sizes (molecular weights 12, 5, and 4.2 × 106). Digestion with HindIII yields fragments of two sizes (molecular weights 9.3, and 8.4 × 106). These data permit the construction of a single map for the human ribosomal gene of 17 × 106 daltons. A similar analysis of human DNA from several fibroblast and tumor cell lines shows variation only at a single EcoRI site. Despite marked alterations in karyotype, the organization of ribosomal nenes has been strongly conserved in the neoplastic tissues so far examined.

Physical map of the origin of defective DNA in herpes simplex virus type 1 DNA

The origin of defective DNA (dDNA) of the Patton strain of herpes simplex virus type 1 (HSV-1) was physically mapped with BamHI in the parental DNA. The dDNA obtained from virus passaged at high multiplicities of infection was resistant to cleavage with HindIII, whereas digestion with EcoRI yielded a cluster of fragments 5.4 to 5.7 megadaltons (Mdal) in size. Cleavage with BamHI gave a cluster of fragments 2.6 to 3.2 Mdal in size, plus two homogeneous, comigrating 1-Mdal fragments. One of the latter fragments contained the single EcoRI site approximately 65 base pairs from one end. Hybridization of in vitro labeled dDNA probe to EcoRI, HindIII, BamHI, and Hpa I digests of nondefective HSV-1 DNA demonstrated that, in addition to the S-region terminal repeat, only one end of the S region was involved in the generation of this class of dDNA. Thus, the dDNA probe did not hybridize to either the S region 3.0-Mdal HindIIIN fragment or a 3.0-Mdal BamHI fragment of the adjacent 8.7-Mdal HindIIIG fragment, but did hybridize to four BamHI fragments of HindIII G (approximately 5.7 Mdal). The cluster of 2.6- to 3.2-Mdal fragments obtained with BamHI digestion of dDNA appears to represent a novel junction between the termination of dDNA adjacent to the 3.0-Mdal BamHI fragment in HindIII G and the 2.0- to 2.3-Mdal BamHI fragment terminal in HSV-1 DNA.

Chromosomal transfer promoted by the promiscuous plasmid RP4

We have studied the properties of the recombinant plasmid RP4λ att. This plasmid possesses the EcoRI-generated fragment of phage λ containing the genes att- int- xis (srIλ2–3) inserted into the single EcoRI site of the promiscuous plasmid RP4. The insertion of this λ fragment has no detectable effect on normal plasmid functions. However, it confers the ability to promote low-frequency polarized chromosomal transfer by int-promoted integration into the host λ attachment site attλ. We have succeeded in isolating an Hfr derivative which has the plasmid stably integrated at attλ. The Hfr derivative is unusual in having both an integrated and an autonomous RP4λ att stably coexisting in the same cell.

Recombinant plasmids capable to replication in B. subtilis and E. coli.

The plasmid pBC16 (4.25 kbases), originally isolated from Bacillus cereus, determines tetracycline resistance and can be transformed into competent cells of B. subtilis. A miniplasmid of pBC16 (pBC16-1), 2,7 kb) which has lost an EcoRI fragment of pBC16 retains the replication functions and the tetracycline resistance. This plasmid which carries only one EcoRI site has been joined in vitro to pBS1, a cryptic plasmid previously isolated from B. subtilis and shown to carry also a single EcoRI site (Bernhard et al., 1978). The recombinant plasmid is unstable and dissociates into the plasmid pBS161 (8.2 kb) and the smaller plasmid pBS162 (2.1 kb). Plasmid pBS161 retains the tetracycline resistance. It possesses a single EcoRI site and 6 HindIII sites. The largest HindIII fragment of pBS161 carries the tetracycline resistance gene and the replication function. After circularization in vitro of this fragment a new plasmid, pBS161-1 is generated, which can be used as a HindIII and EcoRI cloning vector in Bacillus subtilis. Hybrid plasmids consisting of the E. coli plasmids pBR322, pWL7 or pAC184 and different HindIII fragments of pBS161 were constructed in vitro. Hybrids containing together with the E. coli plasmid the largest HindIII fragment of pBS161 can replicate in E. coli and B. subtilis. In E. coli only the replicon of the E. coli plasmid part is functioning whereas in B. subtilis replication of the hybrid plasmid is under the control of the Bacillus replicon. The tetracycline resistance of the B. subtilis plasmid is expressed in E. coli, but several antibiotic resistances of the E. coli plasmids (ampicillin, kanamycin and chloramphenicol) are not expressed in B. subtilis. The hybrid plasmids seem to be more unstable in B. subtilis than in E. coli.

Mapping of the ribosomal RNA genes on spinach chloroplast DNA.

Spinach chloroplast ribosomal RNAs have been hybridized to restriction endonuclease fragments of spinach chloroplast DNA. All three RNA species (23S, 16S and 5S) hybridized to a single large fragment when the DNA was digested with either Sall or Pstl. Hybridization of 23S RNA to fragments produced by Smal yielded two radioactive bands which corresponded to the bi-molar 2.5 X 10(6) and 1.15 X 10(6) Mr fragments. 16S RNA also hybridized to two, bi-molar Smal fragments (3.4 X 10(6) and 2.5 X 10(6) Mr) and 5S RNA hybridized to the 1.15 X 10(6) Mr bi-molar Smal fragment. The 23S RNA and 16S RNA cistrons were each also shown to contain a single EcoRI site. From the data it was possible to conclude that the ribosomal RNA genes are located on the inverted repeat region of the spinach chloroplast DNA restriction map [1,2], that the sequence of the cistrons is 16S - 23S - 5S and that the size of the spacer between the 16S and 23S RNA cistrons is approximately 0.90 X 10(6) Mr.

Location of the origin-terminus of the viral strand of the duplex replicative form of bacteriophage G4 DNA

One of the products of bacteriophage G4 DNA replication late in the infectious process is an open-circular, duplex replicative form DNA, RFII. These molecules contain a single discontinuity located at a specific site in the viral strand. Limited enzymatic repair of such RFII molecules with 32P-labeled deoxyribonucleoside triphosphates specifically labels restriction fragments HpaII A, HaeIII Z 2, Hind(II and III) A and Hind(II and III) D 2 and places the 3′OH terminus of the viral strand at a point approximately half-way round the genome from the single EcoRI site. These results taken together with the in vitro localization of the origin of the complementary strand at a point close to the EcoRI site (Zechel et al., 1975) suggest that G4 replicates by a mechanism involving distinct and widely separated origins of the individual strands (e.g., a displacement-loop mechanism).

Detection and characterization of naturally occuring plasmids in Bacillus subtilis

Among 83 B. subtilis strains screened, eight were shown to have a significant amount of their DNA in the form of covalently closed circular molecules. One of these strains (1264) carries two such molecular species (plasmids pGY31 and pGY32). A group of four strains (14A1, X35, X36, X43) each carry one plasmid (pGY5 to pGY8). These four plasmids were found to be very similar to each other. Strain 2588 carries a large plasmid (pGY1). The satellite DNA's of the remaining two strains (11A1 and 13F3) are very heterogeneous. The plasmids were isolated and characterized as regards sedimentation velocity, copy number per chromosome equivalent and EcoRI cleavage pattern. The sedimentation coefficient of pGY1 suggested a molecular weight of the order of 40 Md. This plasmid is present in no more than two copies per chromosome. It possesses many EcoRI sites. Plasmids pGY5 to pGY8 each have two EcoRI sites. Cleavage pattern and sedimentation coefficient are the same in all four of them. Their estimated M.W. is 4.9 Md. pGY7 of this class was studied in more detail. Its average copy number is 20. pGY31 and pGY32 both have a single EcoRI site. Their M.W.'s are respectively 7.6 and 3.6 Md and the minimal copy numbers are 7 and 15. Contour lengths were determined for pGY7 (=2.94 μ), pGY31 (=4.46 μ) and pGY32 (=1.84 μ). No phenotype associated with any of the above plasmids has so far been detected. Among the eight strains harboring plasmids only one (2588, plasmid pGY1) was found to be genetically homologous to strain 168, by transformation. Strains 1264 and X36 are indistinguishable from 168 in physiological tests.

The nucleotide sequence surrounding the origin of DNA replication of Col E1

The DNA of Col E1 replicates from a unique origin located at a distance of 17-19% of the genome length from the single Eco RI clevage site. The nucleotide sequence about this site has been determined by a combination of RNA and DNA sequencing techniques. The principal features of the sequence are two palindromes, one of which resembles a palindrome located in the intercistronic region of 0X174. The sequence also contains stretches of purine and pyrimidine clusters of the following compositions: pAT5G, pC2T5G, pGT5G. The origin sequence demonstrates that initiation of DNA replication takes place in an intercistronic region of Col E1DNA, although the possibility that this region makes small polypeptides 30-40 residues long cannot be strictly eliminated at this time.

A plasmid cloning vehicle allowing regulated expression of eukaryotic DNA in bacteria.

We have constructed a plasmid cloning vehicle in which transcription of inserted heterologous DNA fragments can be regulated by a defined bacterial operator and promoter. The lambda plac 5 EcoRIDNA fragment containing the operator, promoter, and beta-galactosidase gene of the lactose operon was linked to the ColE1 derivative plasmid pSF2124, creating a plasmid designated pBGP100, pBGP100 contains one EcoRI site at the lac DNA/pSF2124 DNA junction and another at the lambda DAN/pSF2124 DNA junction. We deleted the latter EcoRI site to generate a plasmid (pBGP120) retaining a single EcoRI site at the lac DNA/nSF2124 DNA junction. To determine whether DNA introduced at the EcoRI site of pBGP120 was expressed under lactose control, we inserted the EcoRI fragment containing 28S ribosomal DNA of Xenopus laevis, creating the hybrid plasmid pBGP123. RNA-DNA hybridization of pulse-labeled RNA from cells containing pBGP123 showed that induction of the lac operon increases the percentage of labeled RNA complementary to Xenopus 28S DNA about 9-fold. This vehicle may be of use for production of eukaryotic gene products in bacteria.

Molecular Vehicle Properties of the Broad Host Range Plasmid RK2

The pladmid RK2 is stably maintained in a broad range of gram-negative bacteria. The RK2 DNA has a single Eco RI restriction site. The insertion of a DNA fragment into this site does not interfere with either plasmid maintenance or self-transmissibility. Because RK2 has a broad host range, it should be useful for the construction in vitro of hybrid plasmid molecules capable of being established by conjugal transfer or transformation into many genera of gram-negative organisms.

The generation of a ColE1-Apr cloning vehicle which allows detection of inserted DNA.

A 3.2 Mdal sequence of DNA, TnA, which contains the ampicillin (Ap) resistance determinant has been translocated from an R plasmid to the plasmid ColE1. A total of 12 isolates were studied. There are at least 8 sites in ColE1 at which TnA has inserted. Insertion at five of these has resulted in a Col-phenotype. One ColE1-Apr plasmid, RSF2124, was examined further and its replication properties are found to be similar to that of the parent plasmid. RSF2124 appears to be a useful plasmid vehicle for the molecular cloning of DNA from diverse prokaryotic sources: it codes for readily detectable Ap resistance and contains a single EcoRI site in a gene affecting colicin biosynthesis so that it is unable to produce colicin upon ligation to other DNA.