A general method to cleave a known DNA sequence at any site.

We describe a new method for obtaining DNA fragments starting at a desired point where there is no recognition sequence for any known restriction endonuclease. A single-stranded DNA containing the fragment of interest is annealed to a synthetic oligonucleotide hybridizing at the 5' end of the required fragment. Then, a partially double-stranded DNA is synthesized using the Klenow fragment of DNA polymerase I in the presence of the four deoxynucleoside triphosphates. The remaining single-stranded regions are removed by digestion with a single-strand nuclease, and the resulting 5' blunt-ended fragment is finally released by digestion with a restriction endonuclease at any site downstream its 3' end. The usefulness of the method was exemplified here by insertion of an epidermal growth factor-like African swine fever virus gene immediately downstream of the ribosome binding site of an expression vector.     

Cloning of a repeat sequence from human DNA which contains a BamHI site

A human 1.5 kilobase BamHI repeated DNA fragment has been cloned from a genomic library and subcloned in pBR322. It is part of the human homogeneous main-band DNA, it has properties similar to those of long interspersed repetitive sequences (LINES), and differs from the families of human repeated DNA already described.     

Mu insertion duplicates a 5 base pair sequence at the host inserted site.

Nucleotide sequences were analyzed across the two ends of lysogenic Mu DNA. These ends were cloned separately in lambdapMu hybrid particles that derived from a single Mu lysogen in the lac Z part of lambdaplac5. The obtained data imply that Mu lysogenization was associated with the duplication of 5 base pairs present in lac DNA at the Mu insertion site. As a result of this duplication, Mu DNA is flanked by two copies of five identical base pairs oriented as direct repeats. A similar conclusion has been obtained independently by other investigators with the use of a different Mu lysogen (D. Kamp and R. Kahmann, personal communication). Thus Mu insertion seems to have a striking similarity to typical IS-mediated insertions that were found to be associated with a short DNA duplication at the target site.     

Anatomy of a preferred target site for the bacterial insertion sequence IS903

Like many transposons the bacterial insertion sequence IS903 was thought to insert randomly. However, using both genetic and statistical approaches, we have derived a target site for IS903 that is used 84% of the time. Computational and genetic analyses of multiple IS903 insertion sites predicted a preferred target consisting of a 21 bp palindromic pattern centered on the 9 bp target duplication generated during transposition. Here we show that targeting can be dissected into four components: the 5 bp flanking sequences, the most important sequences required for site-specific insertion; the 7 bp palindromic core within the target duplication; the dinucleotide pair at the transposon-target junction; and the local DNA context. Finally, using a substrate with multiple target sites we show that a target site is more likely found by a local bind-and-slide model and not by extended DNA tracking.     

Methylation site within a facultatively persistent sequence in the macronucleus of Tetrahymena thermophila.

DNA methylation occurs at the adenines in the somatic macronucleus of Tetrahymena thermophila. We report on a methylation site within a DNA segment showing facultative persistence in the macronucleus. When the site is present, methylation occurs on both strands, although only 50% of the DNA molecules are methylated.     

The extent of DNA sequence required for a functional bacterial attachment site of phage lambda.

We have investigated the extent of DNA sequence required to form a bacterial attachment site (attB) that functions in bacteriophage lambda integration. A DNA fragment carrying attB of Escherichia coli was trimmed, recloned and tested for recombination proficiency. We found that the common core sequence plus the adjoining 4-bp sequences of both the B and B' arms are required for full activity, while plasmids with an even shorter attB sequence retain some capacity to function as attB in vivo. We also found that the nonspecific DNA that is joined to the required attachment site sequence does not significantly influence the rate of the recombination reaction.     

A symmetrical six-base-pair target site sequence determines Tn10 insertion specificity

Transposon Tn10 inserts at many sites in the bacterial chromosome, but preferentially inserts at particular hotspots. We believe we have identified the target DNA signal responsible for this specificity. We have determined the DNA sequences of 11 Tn10 insertion sites and identified a particular 6 base pair (bp) symmetrical consensus sequence (GCTNAGC) common to those sites. The sequences at some sites differ from the consensus sequence but only in limited and well defined ways. The sequences at some sites differ from the consensus sequence than do sequences at other sites, and the consensus sequence and closely related sequences are generally absent from potential target regions where Tn10 is known not to insert. Other aspects of the target DNA can significantly influence the efficiency with which a particular target site sequence is used. The 6 bp consensus sequence is symmetrically located within the 9 bp target DNA sequence that is cleaved and duplicated during Tn10 insertion. This juxtaposition of recognition and cleavage sites plus the symmetry of the perfect consensus sequence suggest that the target DNA may be both recognized and cleaved by the symmetrically disposed subunits of a single protein, as suggested for type II restriction endonucleases. There is plausible homology between the consensus sequence and the very ends of Tn10, compatible with recognition of transposon ends and target DNA by the same protein. The sequences of actual insertion sites deviate from the perfect consensus sequence in a way which suggests that the 6 bp specificity determinant may be recognized through protein-DNA contacts along the major groove of the DNA double helix.     

Functional compromises among pH tolerance, site specificity, and sequence tolerance for a DNA-hydrolyzing deoxyribozyme

We recently reported the identification by in vitro selection of 10MD5, a deoxyribozyme that requires both Mn2+ and Zn2+ to hydrolyze a single-stranded DNA substrate with formation of 5′-phosphate and 3′-hydroxyl termini. DNA cleavage by 10MD5 proceeds with kobs=2.7 h(?1) and rate enhancement of 10(12) over the uncatalyzed P?O hydrolysis reaction. 10MD5 has a very sharp pH optimum near 7.5, with greatly reduced DNA cleavage rate and yield when the pH is changed by only 0.1 unit in either direction. Here we have optimized 10MD5 by reselection (in vitro evolution), leading to variants with broader pH tolerance, which is important for practical DNA cleavage applications. Because of the extensive Watson?Crick complementarity between deoxyribozyme and substrate, the parent 10MD5 is inherently sequence-specific; i.e., it is able to cleave one DNA substrate sequence in preference to other sequences. 10MD5 is also site-specific because only one phosphodiester bond within the DNA substrate is cleaved, although here we show that intentionally creating Watson?Crick mismatches near the cleavage site relaxes the site specificity. Newly evolved 10MD5 variants such as 9NL27 are also sequence-specific. However, the 9NL27 site specificity is relaxed for some substrate sequences even when full Watson?Crick complementarity is maintained, corresponding to a functional compromise between pH tolerance and site specificity. The site specificity of 9NL27 may be restored by expanding its “recognition site” from ATGT (as for 10MD5) to ATGTT or larger, i.e., by considering 9NL27 to have reduced substrate sequence tolerance relative to 10MD5. These findings provide fundamental insights into the interplay among key deoxyribozyme characteristics of tolerance and selectivity, with implications for ongoing development of practical DNA-catalyzed DNA hydrolysis.     

A protein target site in an early replicated human DNA sequence: a highly conserved binding motif

We have previously reported that a human nuclear factor, probably corresponding to the USF/MLTF protein [1,2], is able to bind specifically to a DNA sequence present in DNA replicated at the onset of S-phase [3]. Here we demonstrate that the same factor binds also to several other similar sequences, present in eukaryotic and viral genomes. Mutations or methylation in a CpG dinucleotide, central in the palindromic binding site, completely abolish binding. Furthermore, we present evidence for the existence of at least two other nuclear proteins in human cells with the same DNA binding specificity. The data presented suggest a strong evolutionary conservation, among distantly related organisms, of the binding motif, which is probably the target of a number of nuclear factors that share the same DNA binding specificity albeit in the context of different functions.     

A human dihydrofolate reductase intronless pseudogene with an Alu repetitive sequence: multiple DNA insertions at a single chromosomal site

A dihydrofolate reductase (DHFR) pseudogene, hDHFR-psi 3 has been isolated from a human genomic DNA fragment library. Sequence analysis of this gene revealed a lack of introns and the presence of a tract of nine adenines, 90 bp downstream from the end of the coding sequence. These features suggest that hDHFR-psi 3 was derived from a processed RNA molecule that has been converted into DNA and inserted into a chromosome, analogous to the origin of three intronless human DHFR genes previously described. An interesting feature of hDHFR-psi 3 is the presence of a member of the Alu moderately repetitive DNA sequence family within the DHFR coding region. This Alu element is flanked by a 16 bp directly repeated DNA segment derived from DHFR coding sequences. The Alu element apparently has been inserted into the intronless DHFR pseudogene and thus, there have been two insertions at a single chromosomal locus. The hDHFR-psi 3 contains only the 3' half of the DHFR coding sequence. Immediately upstream from the directly repeated sequence before the Alu element is an adenine-rich tract. The DNA farther upstream is moderately repetitive and is related to neither DHFR nor Alu DNA sequence. Therefore, it seems possible that a third insertion has occurred at the same site further disrupting the hDHFR coding sequences.     

Site-specific DNA splicing: a general procedure for the creation of a restriction site at a predetermined position in a DNA sequence

A method is described for creating any of a wide array of restriction sites at a predetermined position in a known DNA sequence. The method utilizes the exonuclease activity of BAL 31 and a specially designed bifunctional oligodeoxynucleotide linker. The desired restriction site is generated when the linker is ligated to those BAL 31-digested DNA fragments which end with the target sequence. The proper ligation product is then identified by a highly specific hybridization procedure. The method is versatile and specific and is especially useful in the isolation of functional elements of a gene.     

178-Nucleotide sequence surrounding the cos site of bacteriophage lambda DNA.

A nucleotide sequence of 61 nucleotides at the left end and 117 nucleotides at the right end of DNA from bacteriophage lambdacI857Sam7 was determined by the Maxam and Gilbert method. A perfect inverted repeat sequence of 10 nucleotides is near the left end, and one of 15 nucleotides is near the right end. DNA from another closely related lambda strain, lambdacI857prm116Sam7, has about 10% divergence in the sequence of the first 110 nucleotides at the right end and has a 17-member perfect inverted repeat sequence.     

Identification and sequence characterization of a 1.3 Kb EcoRI repeat fragment that harbors a DNA repair site of rat pachytene spermatocytes

Introduction of well-programmed nicks and gaps and the associated DNA repair activity in the genome at the pachytene interval is a characteristic feature of the meiotic prophase in organisms as varied as lilium and mouse. In the present study we have shown that the DNA synthetic activity in rat pachytene spermatocytes is insensitive to aphidicolin, a specific inhibitor of DNA polymerase , and , suggesting DNA -polymerase-mediated repair synthesis in these cells. We have developed a novel approach for the isolation of the DNA repair sites by combining two independent techniques. Following incorporation of BrdUrd into pachytene spermatocytes in the presence of aphidicolin, the repair sites were released as ssDNA fragments by treatment of nuclei with 30 mM NaOH. Subsequently, the BrdUrd containing ssDNA fragments were specifically isolated using polyclonal anti-BrdUrd antibodies. The DNA fragments released were of two size classes, namely 4–7S (major) and 9–12S (minor) and constituted approximately 1.75% of the pachytene genomic DNA. These DNA repair fragments were distinct from Okazaki fragments and other replicative intermediates isolated from rat bone marrow cells as evidenced by (a) their different size distribution and (b) little cross-hybridization. Southern hybridization of restriction enzyme digests of rat genomic DNA with probes made against BrdUrd-ssDNA fragments revealed that although the repair sites were distributed throughout the genome, strong hybridization signals were observed in EcoRI, (1.3 kb and 2.4 kb), BamH1 (9 kb) and HindIII (5 kb) repetetive DNA fragments. The EcoRI 1.3 kb family were cloned into M13 mp19, and a repair positive (1.3 A) and a repair negative (1.3 B) were identified and sequenced. The repair positive clone contained (a) (CA)₂₂ repeat, (b) a (CAGA)₆ repeat and (c) 4 sequences sharing high homology with various hypervariable minisatellite (HVMS) sequences. One of the HVMS sequence contained a GGCAGG motif known to be responsible for germline instability. The repair negative clone had (a) (CA)₆ repeat and (b) a HVMS like sequence without GGCAGG. The significance of these motifs and their relevance to the events of DNA metabolism at pachytene interval have been discussed.     

The sequence selectivity of DNA-targeted 9-aminoacridine cisplatin analogues in a telomere-containing DNA sequence

In this study, the detailed DNA sequence specificity of four acridine Pt complexes was examined and compared with that of cisplatin. The DNA sequence specificity was determined in a telomere-containing DNA sequence using a polymerase stop assay, with a fluorescent primer and an automated capillary DNA sequencer. The Pt compounds included an acridine intercalating moiety that was modified to give a 9-aminoacridine derivative, a 7-methoxy-9-aminoacridine derivative, a 7-fluoro-9-aminoacridine derivative and a 9-ethanolamine-acridine derivative. Compared with cisplatin, the DNA sequence specificity was most altered for the 7-methoxy-9-aminoacridine compound, followed by the 9-aminoacridine derivative, the 7-fluoro-9-aminoacridine compound and the 9-ethanolamine-acridine derivative. The DNA sequence selectivity for the four acridine Pt complexes was shifted away from runs of consecutive guanines towards single guanine bases, especially 5′-GA dinucleotides and sequences that contained 5′-CG. The sequence specificity was examined in telomeric and non-telomeric DNA sequences. Although it was found that telomeric DNA sequences were extensively damaged by the four acridine Pt complexes, there was no extra preference for telomeric sequences.     

Characterization and sequence analysis of a recombination site in the hybrid virus Ad2+ND.

Restriction endonuclease mapping of an adenovirus-simian virus 40 hybrid virus and adenovirus-2 DNA allowed the characterization of fragments of Ad2⁺ND₁² which contain the two junctions between simian virus (SV40) sequences and adenovirus-2 sequences. The corresponding fragments of Ad2⁺⁺ DNA were also characterized. One fragment of Ad2⁺ND₁ containing a recombination site, and the corresponding fragment of Ad2⁺⁺ were analyzed by direct DNA sequence analysis. Comparison of nucleotide sequences in Ad2⁺⁺, Ad2⁺ ND₁, and SV40 DNAs precisely localized those sequences involved in the final recombination event which produced the stable hybrid virus Ad2⁺ND₁. No sequence homology was detected between the two parent DNAs.     

Target site cleavage by the monomeric restriction enzyme BcnI requires translocation to a random DNA sequence and a switch in enzyme orientation

Endonucleases that generate double-strand breaks in DNA often possess two identical subunits related by rotational symmetry, arranged so that the active sites from each subunit act on opposite DNA strands. In contrast to many endonucleases, Type IIP restriction enzyme BcnI, which recognizes the pseudopalindromic sequence 5'-CCSGG-3' (where S stands for C or G) and cuts both DNA strands after the second C, is a monomer and possesses a single catalytic center. We show here that to generate a double-strand break BcnI nicks one DNA strand, switches its orientation on DNA to match the polarity of the second strand and then cuts the phosphodiester bond on the second DNA strand. Surprisingly, we find that an enzyme flip required for the second DNA strand cleavage occurs without an excursion into bulk solution, as the same BcnI molecule acts processively on both DNA strands. We provide evidence that after cleavage of the first DNA strand, BcnI remains associated with the nicked intermediate and relocates to the opposite strand by a short range diffusive hopping on DNA.     

Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site.

The nucleotide sequence of the long terminal repeat (LTR) of three murine retroviral DNAs has been determined. The data indicate that the U5 region (sequences originating from the 5' end of the genome) of various LTRs is more conserved than the U3 region (sequences from the 3' end of the genome). The location and sequence of the control elements such as the 5' cap, "TATA-like" sequences, "CCAAT-box," and presumptive polyadenylic acid addition signal AATAAA in the various LTRs are nearly identical. Some murine retroviral DNAs contain a duplication of sequences within the LTR ranging in size from 58 to 100 base pairs. A variant of molecularly cloned Moloney murine sarcoma virus DNA in which one of the two LTRs integrated into the viral DNA was also analyzed. A 4-base-pair duplication was generated at the site of integration of LTR in the viral DNA. The host-viral junction of two molecularly cloned AKR-murine leukemia virus DNAs (clones 623 and 614) was determined. In the case of AKR-623 DNA, a 3- or 4-base-pair direct repeat of cellular sequences flanking the viral DNA was observed. However, AKR-614 DNA contained a 5-base-pair repeat of cellular sequences. The nucleotide sequence of the preintegration site of AKR-623 DNA revealed that the cellular sequences duplicated during integration are present only once. Finally, a striking homology between the sequences flanking the preintegration site and viral LTRs was observed.     

Nucleotide sequence at the site of single-strand DNA breaks in Pseudomonas aeruginosa bacteriophage phi kF77

It is known that DNA molecules from the phage group phi k specific to Pseudomonas aeruginosa possess single-strand breaks (nicks). The sequences around the nicks in the bacteriophage phi kF77 DNA have been determined by various methods. In addition, an EcoRV-HindIII fragment, containing a nick, was cloned into the plasmid pUC9 and sequenced by Maxam-Gilbert technique. The sequence common for all nicks was CCTAohpCTCCGG.