Propagation of a segment of bacteriophage lamda-DNA in monkey cells after covalent linkage to a defective simian virus 40 genome.

A 520 base pair DNA segment was excised from the bacteriophage lamda-genome by cleavage with the bacterial restriction endonuclease, endo R. Hindll. This segment was covalently joined in vitro to an 880 base pair simian virus 40 (SV40) DNA segment which contains the initation site for SV40 DNA replication. The latter segment was derived from the genome of a defective reiteration mutant of SV40 also by endo R. Hindlll cleavage. When the recombinant molecule, together with wild-type SV40 DNA as helper, was introduced into monkey cells by DNA infection, replication of the lamda-DNA sequences was observed, and hybrid genomes were encapsidated into progeny SV40 virions. The structure of the lamda-DNA segment after serial passage in monkey cells was examined by use of restriction endonucleases and electron microscopic heteroduplex analysis.     

Transformation with specific fragments of adenovirus DNAs. II. Analysis of the viral DNA sequences present in cells transformed with a 7% fragment of adenovirus 5 DNA

Five clones of rat kidney cells transformed by a small restriction endonuclease fragment of adenovirus 5 (Ad5) DNA (fragment HsuI G, which represents the left terminal 7% of the adenovirus genome) were analyzed with respect to the viral DNA sequences present in the cellular DNAs. In these analyses, the kinetics of renaturation of 32P-labeled specific fragments of Ad5 DNA was measured in the presence of a large amount of DNA extracted either from each of the transformed cell lines or from untransformed cells. The fragments were produced by digestion of 32P-labeled adenovirus 5 DNA with endo R.HsuI, or by digestion of 32P-labeled fragment HsuI G of adeno 5 DNA with endo R.HpaI. All five transformed lines were found to contain DNA sequences homologous to 75--80% of Ad5 fragment HsuI G only. Clones II and V contained approximately 48 copies per quantity of diploid cell DNA, clone VI about 35 copies, clone IV 22 copies and clone III 5--10 copies. These results indicate that a viral DNA segment as small as 5.5% of the Ad5 genome, contains sufficient information for the maintenance of transformation.     

Cleavage map of bacteriophage T4 cytosine-containing DNA by sequence-specific endonucleases SalI and KpnI.

Cytosine-containing T4 DNA from endoII- endoIV- dCTPase- alc2 phage grown in a sup+ rB- mB- host is cleaved by endo R.EcoRI and endo R.HindIII to greater than 40 fragments and by endo R.SalI and endo R.KpnI to 8 and 6 fragments, respectively. The latter two fragment sets have been correlated to each other to produce a cleavage map of the genome. The sum of the molecular weights of the fragments calculated from electrophoretic mobility in agarose gels yields a genome molecular weight for cytosine-containing T4 DNA of 105 x 10(6).     

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40². For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · HindIII) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hind digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to HindIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond HindIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · HindIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.     

Complementary action of restriction enzymes endo R-DpnI and Endo R-DpnII on bacteriophage f1 DNA

Bacteriophage f1 duplex DNA was isolated from Escherichia coli strains containing different DNA methylases and assayed for its sensitivity to endonucleolytic cleavage by the enzymes endo R · DpnI and endo R · DpnII. The former enzyme is specific for methylated DNA, the latter for unmethylated DNA (Lacks &; Greenberg, 1975). The E. coli dam methylase was found to be responsible for making f1 resistant to endo R · DpnII and sensitive to endo R · DpnI. Endo R · DpnI cleaved f 1 DNA from dam⁺ cells at four sites. Additional methylation by enzymes other than the dam methylase gave no further cleavage. Endo R · DpnII cleaved f1 DNA from dam^? cells also at four sites to give restriction fragments identical to those obtained with endo R · DpnI cleavage. Thus, the two enzymes are complementary in that they recognize and cleave within the same DNA sequence, one if the DNA is methylated, the other if it is unmethylated. DNA duplexes containing one methylated strand (dam ⁺) and one unmethylated strand (dam^?) were prepared in vitro. These methylated hybrids were refractory to endonucleolytic cleavage by both endo R · DpnI and endo R · DpnII. Neither enzyme, therefore, appears to make even a single strand break at a methylated/unmethylated hybrid site.     

The generation and analysis of clones containing bacteriophage T4 DNA fragments

Cytosine-containing DNA of bacteriophage T4 was digested with three restriction endonucleases: endo R · EcoRI, endo R · HindIII and endo R · PstI, and each digestion ligated with a cloning vector to generate three independent collections of T4 DNA-containing clones. The T4 clones were screened for their T4 genetic content by recombinational analysis using amber mutants of T4. Complementation of T4 amber mutant growth and labeling of proteins in vivo provided evidence of expression of specific (g30, g39, g44 and g46) cloned T4 genes.     

Effects of 2''-deoxy-2''-azidocytidine on polyoma virus DNA replication: evidence for rolling circle-type mechanism.

Rolling circle-type molecules were found in polyoma virus-infected cells after inhibition of DNA synthesis with 2'-deoxy-2'-azidocytidine. The circular DNA molecules were always relaxed and of polyoma length. Most of the attached tails were less than two times the length of the polyoma genome, but tails with a length of up to 4.75 times the genome were also found. After cleavage of the total pool of replicating molecules with either endo R.EcoRI or endo R.BamI, Y-shaped molecules with replicated portions of various lengths were generated from rolling circle-type molecules. Moreover, after cleavage, Y-shaped molecules with three unequal arms were found, which could be explained as derived from the tail in rolling circle-type molecules starting from the normal origin, i.e., 29% from the endo R.EcoRI cleavage site. Rolling circle-type molecules were also found during a normal, noninhibited infection cycle. In such cells, a relatively higher frequency of rolling circle-type molecules was observed late during infection. Compared with control cultures, cultures inhibited with 2'-deoxy-2'-azidocytidine showed a greater amount of rolling circle-type molecules relative to normal replicative intermediates. 2'-Deoxy-2'-azidocytidine has previously been shown to inhibit the initiation of new rounds of replication; thus, the result obtained here indicates that a rolling circle-type mechanism is independent of the reinitiation of DNA synthesis.     

Complementary strand-specific sequences from unique fragments of adenovirus type 2 DNA for hybridization-mapping experiments

Separation of the complementary strands of adenovirus type 2 DNA by poly(U,G)-CsCl density gradient centrifugation permitted studies of Ad2³ DNA renaturation with independently variable concentrations of each complementary strand. Single-stranded DNA was isolated by hydroxylapatite chromatography following exhaustive incubation under such conditions, and was found to selectively represent sequences of the complement present in excess during the incubation. This result was exploited in a general method for isolation of complementary strand-specific sequences of radioactively labeled Ad2 DNA or restriction enzyme fragments of Ad2 DNA. Liquid phase saturation-hybridization experiments were carried out with labeled DNA representing each complementary strand of the six endo R.EcoRI cleavage fragments of Ad2 DNA and unlabeled messenger RNA prepared from HeLa cells late after productive infections with Ad2. The results were combined with the known endo R.EcoRI cleavage map of Ad2 DNA to construct a low-resolution map showing physically separated regions, on both complementary strands of Ad2 DNA, represented in mRNA late after infection.     

Protection of particular cleavage sites of restriction endonucleases by distamycin A and actinomycin D.

It is shown here that distamycin A and actinomycin D can protect the recognition sites of endo R.EcoRI, EcoRII, HindII, HindIII, HpaI and HpaII from the attack of these restriction endonucleases. At proper distamycin concentrations only two endo R.EcoRI sites of phage lambda DNA are available for the restriction enzyme--sRI1 and sRI4. This phenomenon results in the appearance of larger DNA fragments comprising several consecutive fragments of endo R.EcoRI complete cleavage. The distamycin fragments isolated from the agarose gels can be subsequently cleaved by endo R.EcoRI with the yield of the fragments of complete digestion. We have compared the effect of distamycin A and actinomycin D on a number of restriction endonucleases having different nucleotide sequences in the recognition sites and established that antibiotic action depends on the nucleotide sequences of the recognition sites and their closest environment     

Identification of a telomeric DNA sequence in Trypanosoma brucei

A simple repetitive DNA sequence in the nuclear genome of Trypanosoma brucei, consisting of tandem repeats of the hexanucleotide 5' CCCTAA 3', was identified as being telomeric by several criteria. This sequence was specifically labeled with T. brucei genomic DNA as the template for in vitro nick translation by DNA polymerase I, and was present in Bal 31 nuclease sensitive, genomic restriction fragments of the large sizes expected in this organism for at least some telomeric regions. The same repeated sequence was found in six other flagellates tested. A segment of DNA from T. brucei including this telomeric sequence was cloned in pBR322 and characterized. The cloned segment contained a sequence highly homologous to the 3' ends of several variant surface glycoprotein mRNAs, upstream of the tandemly repeated hexanucleotide sequence.     

Use of repetitive DNA for diagnosis of chromosomal rearrangements

Summary We have used two repeated DNA fragments (3.4 and 2.1 kb) released from Y chromosome DNA by digestion with the restriction endonuclease Hae III to analyze potential Y chromosome/autosome translocations. Two female patients were studied who each had an abnormal chromosome 22 with extra quinacrine fluorescent material on the short arm. The origin of the 22p+ chromosomes was uncertain after standard cytologic examinations. Analysis of one patient's DNA with the Y-specific repeated DNA probes revealed the presence of both the 3.4 and 2.1 kb Y-specific fragments. Thus, in this patient, the additional material was from the Y chromosome. Analysis of the second patient's DNA for Y-specific repeated DNA was negative, indicating that the extra chromosomal segment was not from the long arm of the Y chromosome. These two cases demonstrate that repeated DNA can distinguish between similar appearing aberrant chromosomes and may be useful in karyotypic and prenatal diagnosis.     

Initiation and termination of DNA transfer during conjugation of IncI1 plasmid R64: roles of two sets of inverted repeat sequences within oriT in termination of R64 transfer

Intercellular transfer of plasmid DNA during bacterial conjugation initiates and terminates at a specific origin of transfer, oriT. We have investigated the oriT structure of conjugative plasmid R64 with regard to the initiation and termination of DNA transfer. Using recombinant plasmids containing two tandemly repeated R64 oriT sequences with or without mutations, the subregions required for initiation and termination were determined by examining conjugation-mediated deletion between the repeated oriTs. The oriT subregion required for initiation was found to be identical to the 44-bp oriT core sequence consisting of two units, the conserved nick region sequence and the 17-bp repeat A sequence, that are recognized by R64 relaxosome proteins NikB and NikA, respectively. In contrast, the nick region sequence and two sets of inverted repeat sequences within the 92-bp minimal oriT sequence were required for efficient termination. Mutant repeat A sequences lacking NikA-binding ability were found to be sufficient for termination, suggesting that the inverted repeat structures are involved in the termination process. A duplication of the DNA segment between the repeated oriTs was also found after mobilization of the plasmid carrying initiation-deficient but termination-proficient oriT and initiation-proficient but termination-deficient oriT, suggesting that the 3' terminus of the transferred strand is elongated by rolling-circle-DNA synthesis.     

Direct sequencing of deleted mitochondrial DNA in myopathic patients

To investigate the mechanism of mitochondrial DNA deletion in human diseases, we amplified the deleted mitochondrial DNA of five patients with mitochondrial myopathy by using the polymerase chain reaction, and directly sequenced the crossover regions of the deleted mitochondrial DNA without cloning. In Patient 1, a 7-bp directly repeated sequence of 5'-ATCCCCA-3' was found at the boundaries of deleted segment spanning 7,039 bp between the ATPase 6 and the cytochrome b genes. In Patients 2, 3, and 4, a 13-bp sequence of 5'-ACCTCCCTCACCA-3' was found in the boundaries of deleted segment spanning 4,977 bp between the ATPase 8 and the ND5 genes. In Patient 5, a 3-bp sequence of 5'-CCT-3' was found in the boundaries of deleted segment spanning 3,717 bp between the ATPase 6 and the ND5 genes. Similar directly repeated sequences may contribute to mitochondrial DNA deletions in human degenerative diseases.     

Specific association of repetitive DNA sequences with major histocompatibility genes.

The DNA sequence organization of a 17.8-kilobase segment of porcine DNA, containing a functional major histocompatibility (MHC) gene, has been studied. The DNA flanking the MHC gene contains at least 10 distinct repetitive DNA sequence elements, each of which occurs only once within the 17.8-kilobase DNA segment. Their reiteration frequencies in the genome range from 10(2) to 10(4). The genomic organization of seven of these sequence elements has been examined; all are interspersed with other, unrelated DNA sequences. These seven repeated sequences are not generally associated in the genome. However, they appear to be nonrandomly linked in MHC-associated regions of the genome: at least two additional DNA segments containing MHC-homologous DNA also contain sequences homologous to DNA fragments bearing the seven different repeats. Of the seven sequences, four can be detected in splenic total RNA. These results suggest that these repeated elements are specifically associated with the MHC locus.     

Conformational flexibility in DNA structure and its implications in understanding the organization of DNA in chromatin.

X-ray crystallographic studies of drug-nucleic acid crystalline complexes have suggested that DNA first bends or 'kinks' before accepting an intercalative drug or dye. This flexibility in DNA structure is made possible by altering the normal C2' endo deoxyribose sugar puckering in B DNA to a mixed sugar puckering pattern of the type C3' endo (3'-5') C2' endo and partially unstacking base pairs. A kinking scheme such as this would require minimal sterochemical rearrangement and would also involve small energies. This has prompted us to ask more generally if a conformational change such as this could be used by proteins in their interactions with DNA. Here we describe an interesting superhelical DNA structure formed by kinking DNA every ten base pairs. This structure may be used in the organization of DNA within the nucleosome structure in chromatin.     

Purification and characterization of HeLa endonuclease R. A G-specific mammalian endonuclease

We previously reported a double-stranded endonuclease from HeLa cells, endonuclease R (endo R), which specifically cleaves duplex DNA at sites rich in G.C base pairs. In this report we describe the purification of endo R to near homogeneity by conventional and affinity chromatography. The molecular mass of the active form of endo R is approximately 115-125 kDa. SDS-gel electrophoresis reveals a major protein species of 100 kDa. The enzyme requires Mg2+ as a cofactor and is equally active on closed circular and linear duplex DNA substrates that contain G-rich sequences. A 50% reduction in cleavage activity is observed with Ca2+ ions and no double-stranded cleavage occurs with Zn2+. Use of Mn2+ causes an altered specificity at low concentrations of enzyme or divalent metal ion and nonspecific degradation of the substrate at higher concentrations. Endo R is strongly inhibited by sodium or potassium chloride and exhibits a wide pH optimum of 6.0-9.0. The pI of the enzyme is between 6.5 and 7.0. A 2-fold stimulation is observed with the addition of dGTP or dATP but specific cleavage is inhibited by ATP at an equivalent concentration. Cleavage activity is competitively inhibited 10-fold more efficiently by single-stranded poly(dG)12 than by other DNA competitors. The ends of endo R cleavage products contain 5'-phosphate and 3'-hydroxyl groups, and a significant portion of these products were substrates for T4 DNA ligase. Endo R appears to be a previously uncharacterized mammalian endonuclease.     

Restriction and modification in B. subtilis

Summary The content of 5-methylcytosine (5MC) and 6-methyladenine (6MA) in modified and nonmodified DNAs from B. subtilis and B. subtilis phage SPP1 were determined. Nonmodified SPP1 · O DNA contains about 15 5MC residues/molecule. Each modified SPP1 ·R DNA molecule carries 190 modification specific methyl groups. This number is sufficient to account for modification of the 80 restriction sites in SPP1 DNA (Bron and Murray, 1975) against endo R · Bsu R, assuming each modified site contains two 5MC residues. Resistance of SP01 DNA against endo R · Bsu R restriction both in vivo and in vitro is probably not due to methylation of endo R·Bsu R recognition sites.     

Highly repeated DNA of the baboon: organization of sequences homologous to highly repeated DNA of the African green monkey.

In the African green monkey genome, 20% of the total DNA consists of a highly reiterated DNA sequence that occurs largely in long tandem arrays of a repeat unit that is 172 base-pairs in length. The DNA of the baboon contains sequences homologous to this repeat unit. However, in the baboon genome, these sequences comprise roughly 6% of the total DNA and alternate in a regular fashion with a DNA segment that may be distantly related to the monkey repeat unit. The sequences in the baboon that are homologous to the monkey repeat unit are contained within a 340 base-pair repeat unit of the highly repeated DNA fraction of the baboon. The extent of nucleotide divergence of the homologous repeated sequences between the two species is estimated to be about 10%.     

In vitro packaging of a lambda Dam vector containing EcoRI DNA fragments of Escherichia coli and phage P1.

In this report we describe a coliphage lambda vector system for cloning endo R. EcoRI DNA fragments. This system differs significantly from those previously described in two ways. First, restricted and ligated DNA is encapsidated in vitro. Second, with increasing lambda DNA size in the range 78 to 100% that of wild-type, the efficiency of DNA encapsidation into infectious phage particles markedly increases. For lambda wild-type DNA the efficiency of in vitro packaging (10(6) to 10(7) plaques produced per microgram of added DNA) is equal to, or better than, the standard CaCl2 transfection method. The use of a Dam mutation to facilitate recognition of size classes of inserted fragments is described. Using this vector and in vitro packaging, several E. coli and phage P1 and R.EcoRI fragments were cloned.