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A restriction endonuclease has been partially purified from Haemophilus influenzae Rf232 containing the genetically determined system of restriction and modification of DNA. The enzyme requires ATP for the degradation of transfecting phage DNA.
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Cleavage maps of bacteriophage BF23 DNA have been constructed for the restriction endonucleases SalI (3 fragments), BamHI (5 fragments), EcoRI, (8 fragments), BalI (13 fragments), and HpaI (49 fragments, 32 of which have been ordered). The maps were determined by (i) analysis of deletion mutants, (ii) digestion with two endonucleases, (iii) digestion of isolated fragments with a second enzyme, (iv) analysis of partial digests, and (v) digestion after treatment with lambda exonuclease.
Analysis of the infected cell polypeptides and the DNA restriction profiles of 31 HSV-1 isolates from the trigeminal, superior cervical and vagus ganglia from 17 individuals (12 U.S.A., 2 Japanese, 3 Norwegian) could be classified as 15 different virus strains. With the exception of the three Norwegian isolates which gave identical profiles, virus isolates from the ganglia of different individuals could all be distinguished from one another. In contrast virus isolates from the trigeminal, superior cervical and vagus ganglia of the same individual, or virus isolates from the left and right ganglia of the same individual or multiple isolates from different explants of a single ganglion were indistinguishable. In conclusion, a single virus strain infects each individual initially and virus descended from this event subsequently infects and becomes latent in different cells of the same ganglion as well as in different ganglia.
Normal human metaphase chromosomes isolated from fibroblasts were resolved into 14 peaks based on total Hoechst 33258 fluorescence and sorted with the fluorescence-activated cell sorter. The chromosomal DNA was extracted and characterized by EcoRI analysis. As expected, analysis of the peak containing chromosomes 16 and 18 detected the alpha-globin genes and of the peak containing chromosomes 9, 10, 11, and 12 detected the beta-, gamma-, and delta-globin genes. Translocations were then used to localize further the beta-, gamma-, and delta-globin genes. The first translocation t(11;22)(q25;q11), which moved nearly all of chromosome 11 to a different peak, confirmed that the beta-, gamma-, and delta-globin genes are on this chromosome. The second, t(4;11)(q25;q13), which moved the distal portion of the long arm of chromosome 11 to a new peak, showed that the genes are not in this segment. The third, t(X;11)(q11;p13), moved the distal region of the short arm of chromosome 11 to a peak which now contained the beta-, gamma-, and delta-globin genes. Therefore, the beta-, gamma-, and delta-globin genes residue on the distal portion of the chromosome 11 short arm including bands p13, p14, and p15. This sorting method may be used generally to assign other genes to chromosomal segments of the entire chromosome complement.
phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).
The action of two restriction endonucleases on polyoma virus DNA has been examined and the sites at which they cleave the DNA located. One of the enzymes, KpnI from Klebsiella pneumoniae OK8, cleaves polyoma DNA twice at about 11-6 and 59-2% from the EcoRI site. The other enzyme, PstI from Providencia stuartii 164, cleaves polyoma DNA five times at about 14-8, 16-5, 32-6, 50-3 and 80-0% from the EcoRI site. Some of the cleavages produced by these enzymes alone, or in conjunction with other endonucleases, may be of use in the isolation of regions of particular interest from the virus DNA.
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Extracts from Agrobacterium tumefaciens strain ID135 contain three enzymes that have been characterized and partially purified. The first enzyme, a DNA topoisomerase, appeared to relax only negatively twisted DNA. The second enzyme, Atu I, a type II restriction endonuclease, generated the identical DNA digestion pattern as EcoRII when several DNAs were used. The third enzyme, endonuclease A, showed a preference for superhelical DNAs as substrates. When plasmid pCK135DNA, obtained from the virulent strain IDI135 of A. tumefaciens, or plant DNA was exposed to the three enzymes, changes in DNA patterns were observed due to either conformational changes or digestion of the DNAs. These enzymes may function in vivo in the processing and incorporation of bacterial DNA in plant cells.
Rat epitheloid lung cells were transformed with various preparations of SV40 dna using the Ca2+-precipitation technique. The amount of SV40 genetic information integrated into transformed clones was evaluated by DNA-DNA renaturation kinetics. The growth properties on plastic and in soft-agar were examined, as well as the ability to induce tumors in syngeneic new-born animals or in adult nude mice. One particular transformed line, which had received the Hpa II/BamH I A (59 per cent) fragment, was found to contain about 3 integrated copies of this fragment per cell and no significant amount of the Hpa II/BamH I B (41 per cent) fragment. This line which grew to high saturation densities and efficiently formed clones in low serum on plastic, produced tumors in both syngeneic rats and nude mice. Thus the Hpa II/BamH I A fragment, which mainly includes early viral information, was sufficient to impart these properties to rat epitheloid lung cells.
The five satellite DNAs of Drosophila melanogaster have been isolated by the combined use of different equilibrium density gradients and hydrolyzed by seven different restriction enzymes; Hae III, Hind II + Hind III, Hinf, Hpa II, EcoR I and EcoR II. The 1.705 satellite is not hydrolyzed by any of the enzymes tested. Hae III is the only restriction enzyme that cuts the 1.672 and 1.686 satellites. The cleavage products from either of these reactions has a heterogeneous size distribution. Part of the 1.688 satellite is cut by Hae III and by Hinf into three discrete fragments with M.W. that are multiples of 2.3 X 10(5) daltons (approximately 350 base pairs). In addition, two minor bands are detected in the 1.688-Hinf products. The mole ratios of the trimer, dimer and monomer are: 1:6.30 : 63.6 for 1.688-Hae III and 1 : 22.0 : 403 for 1.688-Hinf. Circular mitochondrial DNA (rho = 1.680) is cut into discrete fragments by all of the enzymes tested and molecular weights of these fragments have been determined.
In Staphylococcus aureus transduction of different tetracycline and chloramphenicol plasmids with a group I/III modification was possible to group I and III strains. Group II strains, containing a restriction endonuclease, had a restriction both for the phage and the plasmids: two restriction-deficient group II strains were good acceptors for these plasmids.
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Escherichia coli RNA polymerase has been shown to bind to a limited number of Hind and Hae III restriction enzyme fragments. On S13 replicative form DNA there are three major binding sites, and the locations correlate with promoter sites at the beginning of genes a and B and a site overlapping gene D and the beginning of gene E. Two less definite binding sites have been localized, one in gene F and one at the gene G-H junction. In phiX174 replicative form DNA, five sites, each with apparently similar binding properties, have been located, four of which correspond exactly to binding sites in S13. One site, at the beginning of the B gene, could not be assigned to exactly the same location found in S13. This was due in part to differences in the restriction cleavage maps in the area of the DNA and possibly to the higher background of nonspecific binding in the phiX174 experiments. The location of two of the phiX174 sites at the beginning of genes A and D-E corresponds very well with transcription data, but the site at the start of the B gene indicates the promoter site is closer to the initiation sequence of the B protein than was previously suggested on the basis of transcription data.
Circular dichroic spectra of T7 RNA polymerase show minima at 222 nm ([theta]m=-7.9 X 10(3) deg cm2/dmol) and 208 nm ([theta]m =-7.55 X 10(3) deg cm2/dmol) and a maximum at 193 nm ([theta]m = 1.2 X 10(4) deg cm2/dmol). The small mean residue ellipticity above 200 nm indicates that the secondary structure contains approximately 12% alpha helix. The secondary structure is unaltered by high salt, glycerol, -SH reagents, nitration of tyrosyl residues, and chelating agents. Binding of the native enzyme to [32P]T7 DNA has been measured by the retention of the protein-[32P]DNA complexes on nitrocellulose filters. At 37degrees T7 RNA polymerase binds to its promoters in the absence of NTP's. Binding and catalytic activity are both abolished at 0degree. Binding of the initiating [gamma-32P]GTP can also be detected by the filter binding assay. Native T7 RNA polymerase is inactivated by reaction with 1 mol of 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) or 1 mol of [14C]iodoacetamide. The latter reaction is blocked by Nbs2 suggesting that a single -SH group is required for activity. Alkylation of the -SH group does not alter binding of the enzyme to the DNA template, but modifies the binding of GTP to the enzyme. Nitration of approximately4 surface tyrosyl residues of the protein prevents binding to T7 DNA. The restriction endonuclease, Hpa II, cuts T7 DNA into approximately40 fragments and reduces total RNA synthesis by T7 RNA polymerase by 70%. Fragmentation of the DNA template by Hpa II does not alter the rate of RNA chain initiation by T7 polymerase, and restriction fragments accounting for approximately25% of the T7 DNA still bind tightly to the enzyme. Thus the T7 RNA polymerase promoters remain intact on the restriction fragments. Gel electrophoresis of the transcription products, using restriction fragments as templates, show that of the seven in vitro transcripts produced by T7 RNA polymerase from whole T7 DNA, only the smallest (representing the last 1.5% of the genome) is transcribed from Hpa II fragments. The remaining transcripts are replaced by six new and much shorter mRNA's. The DNA fragments containing the promoters for these mRNA's have been removed from the fragment mix by binding them to the enzyme and retaining the complexes on nitrocellulose filters.
On pre-infection of the host Escherichia coli B with u.v.-inactivated T3 or T7 phage able to express their early genes (like 0.3), B-specific modification of superinfecting, successfully multiplying viruses does not take place. The ocr gene function (gene 0.3) of T3 and T7 not only prevents host-specific DNA restriction but also modification, probably by inhibiting the same late step in the interaction between the restriction enzyme and DNA.
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A restriction enzyme analysis was performed on satellite DNA components, isolated, as described in the preceding paper, from the bovine genome by a combination of Cs2SO4/BAMD and Cs2SO4/Ag+ density gradient centrifugation. Such an analysis has led to the unambiguous identification of eight satellite DNA components and to new information on their repeat units; this indicates that identical repeat lengths are shared by them, a fact strongly suggesting a common origin.