Visualization of an inverted terminal repetition in vaccinia virus DNA.

An inverted terminal repetition was observed in DNA molecules extracted from vaccinia virus. The repeated sequence was visualized by (i) nicking the hairpin loops present of the ends of vaccinia virus DNA, (ii) separating the strands of DNA by alkali denaturation, (iii) allowing the single strands to self-anneal, and (iv) examining the DNA with an electron microscope. Single-stranded circular molecules, each of which contained a duplex projection (3.54 +/- 0.12 micron) representing the terminal repetition, readily formed. Similar size projections were also seen in heteroduplex structures formed by crosshybridization of the separated strands of the two terminal HindIII restriction fragments. Based on contour length measurements and the electrophoretic mobility of the isolated inverted terminal repetition, a molecular weight of approximately 6.9 X 10(6), equivalent to about 10,500 nucleotide base pairs, was estimated. Evidence was obtained from DNA-RNA hybridization studies that the terminal repetition is transcribed.     

Endonuclease recognition sites mapped on Zea mays chloroplast DNA

The closed-circular DNA molecules of 85 × 10⁶ daltons from Zea mays chloroplasts were isolated, digested with the restriction endonucleases Sal I, Bam I, and EcoRI, and the resulting fragments sized by agarose gel electrophoresis. A map of maize chloroplast DNA showing the relative location of all the Sal I recognition sequences and many of the Bam I and EcoRI recognition sites was determined. A DNA sequence representing approximately 15% of the Zea mays chloroplast genome is repeated. The two copies of this sequence are in an inverted orientation with respect to one another and are separated by a nonhomologous sequence representing approximately 10% of the genome length. The inverted repeats contain the genes for chloroplast ribosomal RNAs.     

Nucleotide sequence at the termini of the DNA of Bacillus subtilis phage phi 29

Phage phi 29 DNA cannot be phosphorylated with polynucleotide kinase and [gamma-32P]ATP because of the presence of a viral protein covalently linked to the 5' termini. The 5' ends can, however, be made susceptible to phosphorylation by treatment with alkali and alkaline phosphatase. Restriction fragments Hpa II C and Hpa II F, corresponding to the right and left ends of phi 29 DNA, respectively, were labeled at the 5' ends with polynucleotide kinase and [gamma-32P]ATP or at the 3' ends with terminal transferase and [alpha-32P]ATP or [alpha-32P]cordycepin 5'-triphosphate. After a secondary cleavage of the labeled fragments, the sequence of the first 150-180 nucleotides at the termini of phi 29 DNA was determined by the method of Maxam and Gilbert. The ends of phi 29 DNA are flush, and a six-nucleotides-long inverted terminal repetition was found. The functional implications of the sequences determined are discussed.     

Specific Cleavage of Simian Virus 40 DNA by Restriction Endonuclease of Hemophilus Influenzae

A bacterial restriction endonuclease has been used to produce specific fragments of SV40 DNA. Digestion of DNA from plaque-purified stocks of SV40 with the restriction endonuclease from Hemophilus influenzae gave 11 fragments resolvable by polyacrylamide gel electrophoresis, eight of which were equimolar with the original DNA. The fragments ranged from about 6.5 × 10⁵ to 7.4 × 10⁴ daltons, as determined by electron microscopy, DNA content, or electrophoretic mobility.     

Arrangement of sequences in the inverted terminal repetition of adenovirus 18 DNA.

In contrast to the single-stranded circular molecules produced with denatured DNA from other adenoviruses, there was associated with nearly all circular molecules of adenovirus type 18 a visible, duplex projection. These projections had a mean contour length of 0.31 +/- 0.12 mum, equivalent to approximately 3% of genome length. Individual projections ranged in size from 0.1 to 2 mum. Alkaline sucrose gradient purification of single-stranded molecules did not affect formation of these projections, and treatment of a preparation of circular molecules with Neurospora crassa single-strand specific nucleases yielded 0.34 +/- 0.09 mum duplex fragments. Single-stranded circles did not form if a limited number of nucleotides were removed from the 3' ends of native molecules by Escherichia coli exonuclease III digestion prior to denaturation and annealing. In addition, preformed single-stranded circles could be converted to linear molecules by similar treatment. Based on the formation of specific heteroduplex structures when preparations of native DNA were denatured and reannealed and the absence of branches on linear, single-stranded molecules, we conclude that projections are generated by unusually long, inverted terminal repetitions. The repetitious sequences occur in place of rather than in addition to regular sequences. These data provide direct, visual evidence for the arrangement of bases in the inverted terminal repetition of adenovirus DNA.     

Sequence-specific ligation of DNA using RecA protein

A method is described that allows the sequence-specific ligation of DNA. The method is based on the ability of RecA protein from Escherichia coli to selectively pair oligonucleotides to their homologous sequences at the ends of fragments of duplex DNA. These three-stranded complexes were protected from the action of DNA polymerase. When treated with DNA polymerase, unprotected duplex fragments were converted to fragments with blunt ends, whereas protected fragments retained their cohesive ends. By using conditions that greatly favored ligation of cohesive ends, a second DNA fragment could be selectively ligated to a previously protected fragment of DNA. When this second DNA was a vector, selected fragments were preferentially cloned. The method had sufficient power to be used for the isolation of single-copy genes directly from yeast or human genomic DNA, and potentially could allow the isolation of much longer fragments with greater fidelity than obtainable by using PCR.     

Molecular genetics of vaccinia virus: demonstration of marker rescue.

Two genomic variants of vaccinia virus isolated from serially propagated stocks were used to demonstrate marker rescue. The smaller (S variant) virus contains a 6.3 megadalton (MDal) deletion of unique DNA sequences present in the 123-MDal larger (L variant) virus. The deletion was mapped at 6.85 MDal from the left terminus of the genome, just outside of the inverted terminal repetition. Rescue of the unique deleted DNA sequences by infectious S variant virus was obtained in CV-1 cells by using the calcium orthophosphate precipitation technique of intact or restriction endonuclease-treated L-variant DNA. Restriction fragments that overlapped the deletion allowed marker rescue, but restriction of the L-variant DNA within the unique deleted sequences gave negative results. Restriction endonuclease analysis of the DNA obtained from twice-plaque-purified recombinant virus derived from the rescue of overlap donor fragments gave a restriction pattern identical to that of L-variant virus, indicating that the donor DNA was inserted into the rescuing virus by double recombination. No amplification of the unique sequences was observed from intact L-variant DNA in the absence of infectious S-variant virus, suggesting that deproteinized vaccinia DNA is noninfectious and that the donor DNA was neither integrated into the host DNA nor present as an episomal structure. By using 1 microgram of intact L-variant DNA per CV-1 monolayer in a 6-cm Petri dish, approximately 1--5% of the plaques contained the L-variant genotype, and the dose--response curve was essentially linear from 0.1 to 2 microgram of DNA.     

Instability and reiteration of DNA sequences within the vaccinia virus genome.

The sequence arrangement within the nontranscribed portion of the inverted terminal repetition of the vaccinia virus genome exists in quasi-stable and unstable forms that are not distinguishable on the basis of viral infectivity. The unstable forms, which composed about 20% of a serially passaged stock of virus, were recognized by terminal heterogeneity on restriction endonuclease analysis. Instead of a single terminal fragment from each end of the genome, an array of eight or more fragments differing in size by 1650-base-pair increments was detected. This feature was not eliminated by repeated plaque purification, indicating that the population of DNA molecules with various numbers of reiterations can rapidly evolve from the DNA of a single virus particle. However, at each successive round of plaque purification, about 20% of the unstable isolates revert back to the more stable form. Stable forms are characterized by the presence of a set of 13-17 tandem 70-base-pair repeats on each side of a 435-base-pair intervening sequence near both ends of the genome. In contrast, the unstable forms possess sets of tandem repeats and intervening sequences that alternate many times in series. The transition between the two genomic forms and the evolution of the unstable form appear to be mediated by recombinational events.     

Chromosome ends in Drosophila without telomeric DNA sequences.

We have recovered terminal chromosome deletions of the X chromosome of Drosophila [Df(1)RT; RT = receding tips] that break in various positions of the yellow gene (y) region and delete all distal DNA sequences. Terminal DNA fragments are heterogeneous in length. Molecular cloning and sequencing of the terminal DNA fragments revealed that the broken ends of the deleted chromosomes do not carry any telomeric DNA sequences, yet the broken chromatids do not fuse to one another. Moreover, we confirmed by sequence analysis of 49 independently cloned terminal DNA fragments from two RT lines collected at different times that they lose DNA sequences from their distal ends at a rate of 70-75 base pairs per fly generation. We calculate that the rate of loss from these ends is consistent with the removal of an octanucleotide RNA primer at each round of DNA replication in the germ line.     

A cosmid for selecting genes by complementation in Aspergillus nidulans: Selection of the developmentally regulated yA locus

We constructed a 9.9-kilobase cloning vector, designated pKBY2, for isolating genes by complementation of mutations in Aspergillus nidulans. pKBY2 contains the bacteriophage λ cos site, to permit in vitro assembly of phage particles; a bacterial origin of replication and genes for resistance to ampicillin and chloramphenicol, to permit propagation in Escherichia coli; the A. nidulans trpC⁺ gene, to permit selection in Aspergillus; and a unique BamHI restriction site, to permit insertion of DNA fragments produced by digestion with restriction endonucleases BamHI, BglII, Mbo I, or Sau3A. We used this cosmid to form a quasirandom recombinant DNA library containing 35- to 40-kilobase DNA fragments from a wild-type strain of A. nidulans. DNA from this library transformed a yellow-spored (yA^-) pabaA^-trpC^-Aspergillus strain (FGSC237) to trpC⁺ at frequencies of approximately 10 transformants per μg of DNA. Three of approximately 1000 trpC⁺pabaA^- colonies obtained were putative yA⁺ transformants, because they produced wild-type (green) spores. DNA from each of the green-spored transformants contained pKBY2 sequences and DNA from two transformants transduced E. coli to ampicillin resistance following treatment in vitro with a λ packaging extract. The cosmids recovered in E. coli had similar restriction patterns and both yielded trpC⁺ transformants of A. nidulans FGSC237, 85% of which produced green spores. Several lines of evidence indicate that the recovered cosmids contain a wild-type copy of the yA gene.     

Evidence for reduced copying fidelity of DNA polymerases α, δ, and ε from Novikoff hepatoma cells

To investigate whether or not DNA polymerases α, δ, and ε from tumor cells have acquired properties that might be responsible for mutations found in tumor development, we investigated copying fidelities of DNA polymerases α, δ, and ε from the highly malignant Novikoff hepatoma cells and compared them to the corresponding enzymes from normal rat liver. DNA polymerases were purified more than 300-fold by three chromatographic steps. Copying fidelity was studied using steady-state kinetics and an 18-mer oligonucleotide primed with a 12-mer (13-mer for extension experiments) as DNA primer-template. Three experimental approaches were chosen: i) extension of DNA primers with mismatched 3’-OH ends opposite dGMP, ii) DNA insertion of nucleotides opposite m⁶G in the template and iii) extension of DNA primers with mismatched 3’-OH ends opposite m⁶G. i) Extension of DNA primers with mismatched 3’-OH ends opposite dGMP. DNA primer templates containing G:T and G:A mispairs at the 3’-OH position of the primer were easily extended by DNA polymerases α, δ and ε from both normal rat liver and Novikoff hepatoma cells. The G:G mismatch was elongated with low efficiency. Notably, DNA polymerase α from Novikoff hepatoma cells extended G:A and G:G mismatches significantly faster than the enzyme from normal cells. ii) Insertion of nucleotides opposite m ⁶ G. DNA polymerases α, δ, and ε from normal rat liver preferably catalyzed incorporation of dAMP opposite m⁶G at dNTP concentrations <100 μM. When dNTP concentrations were raised to ≥100 μM, dCMP (DNA polymerases δ and ε) and dTMP (DNA polymerase α) were also incorporated. The same insertion characteristics were found for the enzymes from Novikoff cells, however, insertion efficiencies of dAMP and dCMP were significantly higher for polymerases δ and ε. iii) Extension of primers with mismatched 3’-OH ends opposite m ⁶ G. Only m⁶G:dAMP and m⁶G:dCMP mismatches were extended by DNA polymerases α, δ and ε from both sources. No differences in extension efficiency were observed between the enzymes from normal and hepatoma cells. Taken together, our results suggest that DNA polymerases α, δ, and ε from Novikoff cells catalyzed incorporation of the wrong nucleotides more readily and extended mismatches more easily. These results may provide a rationale why numerous mutations accumulate during tumor development.     

Translesion DNA polymerases are required for spontaneous deletion formation in Salmonella typhimurium

How spontaneous deletions form in bacteria is still a partly unresolved problem. Here, we show that deletion formation in Salmonella typhimurium requires the presence of functional translesion polymerases. First, in wild-type bacteria, removal of the known translesion DNA polymerases, PolII (polB), PolIV (dinB), PolV (umuDC), and SamAB (samAB), resulted in a 10-fold decrease in the deletion rate, indicating that 90% of all spontaneous deletions require these polymerases for their formation. Second, overexpression of these polymerases by derepression of the DNA damage-inducible LexA regulon caused a 25-fold increase in deletion rate that depended on the presence of functional translesion polymerases. Third, overexpression of the polymerases PolII and PolIV from a plasmid increased the deletion rate 12- to 30-fold, respectively. Last, in a recBC⁻ mutant where dsDNA ends are stabilized due to the lack of the end-processing nuclease RecBC, the deletion rate was increased 20-fold. This increase depended on the translesion polymerases. In lexA(def) mutant cells with constitutive SOS expression, a 10-fold increase in DNA breaks was observed. Inactivation of all 4 translesion polymerases in the lexA(def) mutant reduced the deletion rate 250-fold without any concomitant reduction in the amount of DNA breaks. Mutational inactivation of 3 endonucleases under LexA control reduced the number of DNA breaks to the wild-type level in a lexA(def) mutant with a concomitant 50-fold reduction in deletion rate. These findings suggest that the translesion polymerases are not involved in forming the DNA breaks, but that they require them to stimulate deletion formation.     

Sequence arrangement of a highly methylated satellite DNA of a plant, Scilla: A tandemly repeated inverted repeat

G+C-rich satellite DNA, representing about 19% of total nuclear DNA, was isolated from various tissues of the monocotyledonous plant, Scilla siberica, by using Ag⁺-Cs₂SO₄ gradient techniques. This satellite DNA had an unusually high melting point and a high methylcytosine (m⁵C) content (≈25% of total bases; m⁵C/cytosine ratio ≈1.5) and was localized, by in situ hybridization, in the heterochromatin regions of the chromosomes. Digestion with restriction endonuclease Hae III yielded a series of fragments ranging from 35 to several hundred nucleotide pairs. The major fragments, I-IV (35, 50, 59, and 69, nucleotide pairs, respectively), were isolated, and their nucleotide sequences were determined. The dominant fragment I was a highly symmetrical molecule, with a basically palindromic arrangement. This sequence represented the basic unit of Scilla satellite DNA and was tandemly repeated many times, with some base substitutions and multiple successive insertions of the tetranucleotide G-T-C-C. The dinucleotide CpG was the commonest nearest-neighbor sequence. Thin layer chromatography, DNA sequence analysis, and gas chromatography combined with mass spectrometry showed the high m⁵C content (m⁵C/Cyt = 2.2 and 2.8, respectively, for fragments II and III). Identical cleavage fragments were found in satellite DNAs from two other species of this genus (S. amoena and S. ingridae), which suggests that this constitutively methylated sequence is evolutionarily stable. The sequence arrangement of this plant satellite DNA is compared with those reported for several animal satellite DNAs.     

Multiple rounds of adenovirus DNA synthesis in vitro

Adenovirus (Ad) type 2 DNA synthesis can be initiated in the presence of a soluble extract of uninfected HeLa cell nuclei, a 25-60% saturated ammonium sulfate fraction of infected cytoplasm and viral DNA covalently linked to a 5′-terminal protein (Ad DNA-prot). As the purification, from either the nuclei or cytoplasm, of factors active in DNA replication proceeded, various nonreplicative reactions which incorporate labeled deoxynucleotides were uncovered. In order to distinguish replicative from repair reactions, an assay was developed in which the Ad DNA-prot was digested with Xba I, all of the fragments so produced were used (without separation) in a replication reaction, and the products were assayed by electrophoresis on neutral agarose gels. In replicative reactions, most of the radioactivity was incorporated into the terminal fragments, with the internal fragments remaining unlabeled. Infected cytoplasm contains a “discrimination” function in addition to specific factors for Ad DNA replication. The discrimination factors inhibit the nonspecific nucleotide incorporation by uninfected HeLa nuclear extracts on Ad DNA-prot. The specific replicative incorporation into the terminal Ad DNA-prot fragments has also allowed an independent assay for reinitiation of progeny molecules synthesized in vitro. After the first round of replication, the 5′ strand of the progeny duplex from each end is labeled. These same labeled strands will be displaced during the second round of replication and appear in new bands which have been shown to be the single-strand equivalents of the terminal fragments. Thus, at least two rounds of Ad DNA synthesis can initiate at each terminus in vitro. The appearance of displaced single strands requires DNA replication because the addition of dideoxycytidine triphosphate after the first round of synthesis prevents the displacement reaction. Both the progeny single- and double-stranded DNA appear to be linked to protein.     

Chromosomal arm replacement generates a high level of intraspecific polymorphism in the terminal inverted repeats of the linear chromosomal DNA of Streptomyces ambofaciens

The chromosomal DNA of the bacteria Streptomyces ambofaciens DSM40697 is an 8-Mb linear molecule that ends in terminal inverted repeats (TIRs) of 210 kb. The sequences of the TIRs are highly variable between the different linear replicons of Streptomyces (plasmids or chromosomes). Two spontaneous mutant strains harboring TIRs of 480 and 850 kb were isolated. The TIR polymorphism seen is a result of the deletion of one chromosomal end and its replacement by 480 or 850 kb of sequence identical to the end of the undeleted chromosomal arm. Analysis of the wild-type sequences involved in these rearrangements revealed that a recombination event took place between the two copies of a duplicated DNA sequence. Each copy was mapped to one chromosomal arm, outside of the TIR, and encoded a putative alternative sigma factor. The two ORFs, designated hasR and hasL, were found to be 99% similar at the nucleotide level. The sequence of the chimeric regions generated by the recombination showed that the chromosomal structure of the mutant strains resulted from homologous recombination events between the two copies. We suggest that this mechanism of chromosomal arm replacement contributes to the rapid evolutionary diversification of the sequences of the TIR in Streptomyces.     

Construction of plasmids carrying the cI gene of bacteriophage lambda.

By techniques of recombination in vitro, we have constructed a plasmid bearing the repressor gene (cI) of bacteriophage lambda fused to the promoter of the lac operon. Strains carrying this plasmid overproduce lambda repressor. This functional cI gene was reconstituted by joining DNA fragments bearing different parts of that gene. Flush end fusion techniques, involving no sequence overlap, were necessary for the construction; in certain cases, the abutting of the DNA molecules bearing ends generated by different restriction endonucleases creates a sequence at the junction which is recognized by one of the restriction endonucleases.     

Site-specific inversion sequence of the herpes simplex virus genome: Domain and structural features

The genome of herpes simplex virus-1 consists of two covalently linked components, L and S, that invert relative to each other. The L and S components consist of unique DNA sequences bracketed by inverted repeats. The inverted repeats of the L component are designated ab and b′ a′ and those of the S component are designated a′ c′ and ca. The number of a sequences at the termini and at the L-S component junction varies from one to several copies. Insertion into the middle of the L component of a DNA fragment consisting of 156 base pairs (bp) of the b sequence, an entire a sequence of 501 bp, and 618 bp of the c sequence created a new site through which additional inversions in the genome occurred. Comparison of the nucleotide sequences of DNA fragments containing one and two a sequences defined the domain of the a sequence. The single a sequence consists of two 20-bp direct repeats (designated as DR1) bracketing a region that contains 19 tandem direct repeats of a 12-bp sequence (DR2) adjacent to three direct repeats of a 37-bp sequence (DR4), in addition to short stretches of unique sequences. The fragment with two tandem a sequences contained three copies of DR1—i.e., the intervening DR1 was shared by the two a sequences. Furthermore, one a sequence contained 22 copies of DR2 and two copies of DR4 whereas the second a sequence contained 19 copies of DR2 and two copies of DR4. These observations suggest that (i) amplification of the number of terminal and internal a sequences is the consequence of intramolecular or intermolecular recombination through DR1, (ii) the number of copies of DR2 and DR4 within the a sequence is not fixed and may vary as a consequence of unequal crossing over or slippage, and (iii) inversion results from intramolecular recombination between terminal and inverted a sequences.     

Adenovirus-2 DNA Contains an Inverted Terminal Repetition

Denaturation and renaturation of the adenovirus-2 chromosome (a duplex rod) generates single-stranded circles of unit length. These circles can be opened into linear DNA molecules by digestion with exonuclease III, indicating that hydrogen bonding between the two ends of an adenovirus strand is responsible for maintaining the rod in a circular state.The formation of adenovirus single-stranded circles, and their sensitivity to exonuclease III, indicate that the mature adenovirus-2 DNA molecule contains an inverted terminal repetition. That is, the base sequence at one end of the molecule is inverted and appears again at the other end of the molecule. This is the first example of such a structure, and its function is unknown.