Methylation of somatic vs germ cell DNAs analyzed by restriction endonuclease digestions.

Bacterial restriction endonucleases containing the dinucleotide CpG in their cleavage sequences were used to compare the methylation patterns of primarily repeated DNA sequences in (1) bovine somatic cell native DNAs vs bovine sperm cell native DNA and (2) native vs renatured bovine liver and sperm cell DNAs. The restriction patterns of sperm native DNA differ markedly from those of somatic cell native DNAs when using Hpa II, Hha I, and Ava I but not when using the enzymes Eco RI and Msp I. Digestion patterns of germ cell renatured DNA differed significantly from those of germ cell native DNA when using Hpa II but not when using Msp I or Eco RI. The results may not be due to artifacts of renaturation of the DNAs. The results are consistent with the concept that germ cell DNA may be strand asymmetrically hemimethylated. The data also suggest that methylation of the 5'-cytosine in the sequence CCGG renders this site insensitive to cleavage by Msp I.     

Methylation of satellite sequences in mouse spermatogenic and somatic DNAs.

The distribution of 5-methyl cytosine (5-MeC) residues in a highly repetitive sequence, mouse major satellite, was examined in germinal versus somatic DNAs by digestion with the methylation sensitive isoschizomers Msp I and Hpa II and Southern blot analysis, using a cloned satellite probe. DNA from liver, brain, and a mouse fibroblast cell line, C3H 10T1/2, yielded a multimeric hybridization pattern after digestion with Msp I (and control Eco RI) but were resistant to digestion with Hpa II, reflecting a high level of methylation of the satellite sequences. In contrast, DNA from mature sperm was undermethylated at these same sequences as indicated by the ability of Hpa II to generate a multimeric pattern. DNAs from purified populations of testis cells in different stages of spermatogenesis were examined to determine when during germ cell differentiation the undermethylation was established. As early as in primitive type A, type A, and type B spermatogonia, an undermethylation of satellite sequences was observed. This suggest that this highly specific undermethylation of germ cell satellite DNA occurs very early in the germ cell lineage, prior to entry into meiosis.     

On the possibility of localizing in situ Mus musculus and Drosophila virilus satellite DNAs by Alu I and Eco RI restriction endonucleases

Mus musculus and Drosophila virilus metaphase chromosomes have been treated with Alu I or Eco RI restriction endonucleases and, to ascertain possible selective digestion, subsequently stained with the DNA-specific dye ethidium bromide. The correlation between our findings and previously known cytological and biochemical data allowed us to show that Alu I is an enzyme capable of cytologically detecting repetitive DNAs, while Eco RI is unable to do so. This would be a consequence of the fact that Alu I extensively digests and extracts all chromosomal DNA except that localized in those regions where the presence of satellite DNAs has previously demonstrated. Eco RI, on the contrary, is not capable of doing so and its activity seems to be obstructed by alcohol:acid fixation procedures.     

DNA determinants important in sequence recognition by Eco RI endonuclease

Alkylation interference and protection methods (Siebenlist, U., and Gilbert, W., (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 122-126) have been utilized to deduce potential DNA contacts involved in specific complex formation between Eco RI endonuclease and its recognition sequence. The endonuclease protected the N7 position (major groove) of the dG and the N3 position (minor groove) of both dA residues within the Eco RI sequence against alkylation by dimethylsulfate, d(GpApApTpTpC), suggesting the presence of poly-peptide in both grooves in the vicinity of affected nitrogens. Results of methylation interference analysis suggest that the N7 of the Eco RI site dG and the N3 of the central dA, d(GpApApTpTpC), are utilized as contacts by the enzyme. The failure to observe interference upon methylation of the 5'-penultimate dA within the sequence implies that the endonuclease does not bond to the N3 of this residue, despite the fact that it is protected against alkylation by the protein. Ethylation interference patterns suggest four major phosphate contacts between endonuclease and each DNA strand. Two of these phosphates are 5'-external to the Eco RI sequence, d(pNpGpApApTpTpC), suggesting involvement of outside phosphates in electrostatic interactions. Moreover, alkylation protection and interference effects on the two DNA strands display perfect 2-fold symmetry. Thus, the endonuclease interacts with a minimum of 10 nucleotide pairs to yield a DNA-protein complex characterized by elements of symmetry. In contrast, specific alkylation effects were not observed in comparable experiments with the endonuclease and a DNA which had been previously methylated by the Eco RI modification enzyme.     

The integration sites of endogenous and exogenous Moloney murine leukemia virus

Specific cDNA probes of Moloney and AKR murine leukemia viruses have been prepared to characterize the proviral integration sites of these viruses in the genomes of Balb/Mo and Balb/c mice. The genetically transmitted Moloney provirus of Balb/Mo mice was detected in a characteristic Eco RI DNA fragment of 16 x 10(6) daltons. No fragment of this size was detected in tissue DNAs from Balb/c mice infected as newborns with Moloney virus. We conclude that a viral integration site, occupied in preimplantation mouse embryos, is not necessarily occupied when virus infects cells in post-natal animals. Balb/Mo and Balb/c mice do carry the AkR structural gene in an Eco RI DNA fragment of 12 x 10(6) daltons. Further restriction analysis of this fragment indicated that both mouse lines carry one AKR-type provirus. Leukemogenesis in Balb/Mo and newborn infected Balb/c mice is accompanied by reintegration of Moloney viral sequences in new chromosomal sites of tumor tissues. Part of the reintegrated Moloney viral sequences are of subgenomic size. The AKR viral sequences, however, are not found in new sites. Further restriction analysis revealed that the development of Moloney virus-induced leukemia in Balb/Mo mice does not lead to detectable structural alteration of the genetically transmitted Moloney and AKR structural genes. Possible mechanisms of the reintegration process are also discussed.     

Preparation and properties of insolubilized restriction endonucleases.

Type II restriction endonucleases Bam HI and Eco RI were covalently coupled to Sepharose. These insolubilized enzymes generated fragment patterns for several viral DNAs identical to those produced by the respective free enzymes. Conditions for optimal activity were similar for both bound and unbound forms of the enzymes. Insolubilization improved thermal stability of Bam HI and Eco RI. The bound enzyme can be recovered from reaction mixtures and reused several times. Upon storage at 4 degrees C, coupled endonucleases remained stable for several months.     

The ability of bacterial DNA methyltransferases to use methylcobalamine as a cofactor in DNA methylation reactions

The ability of bacterial DNA methyltransferases Alu I, Cfr I, Cfr 6, Cfr 10, Eco RI, Eco RII, Msp I, Mva I, Pvu I, Pvu II, and Sau 3A to use methylcobalamine and methylmethionine as cofactors of DNA methylation in vitro has been investigated. These bacterial DNA methyltransferases used methylcobalamine, but not methylmethionine for DNA methylation.     

Isolation of deletion and substitution mutants of adenovirus type 5

The infectivity of adenovirus type 5 DNA can be increased to about 5 x 10³ plaque-forming units per μg DNA if the DNA is isolated as a DNA-protein complex. Utilizing this improved infectivity, a method was developed for the selection of mutants lacking restriction endonuclease cleavage sites. The procedure involves three steps. First, the DNA-protein complex is cleaved with a restriction endonuclease. The Eco RI restriction endonuclease was used here. It cleaves adenovirus type 5 DNA to produce three fragments: fragment A (1–76 map units), fragment C (76–83 map units) and fragment B (10–83 map units). Second, the mixture of fragments is rejoined by incubating with DNA ligase, and, third, the modified DNA is used to infect cells in a DNA plaque assay. Mutants were obtained which lacked the endonuclease cleavage site at 0.83 map units. Such mutant DNAs were selected by this procedure because they were cleaved by the Eco RI endonuclease to produce only two fragments: a normal A fragment and a fused B/C fragment. These two fragments could be rejoined to produce a viable DNA molecule as a result of a bimolecular reaction with one ligation event; this exerted a strong selection for such molecules since a trimolecular reaction (keeping the C fragment in its proper orientation) and two ligation events were required to regenerate a wild-type molecule. The alterations resulting in the loss of the Eco RI endonuclease cleavage site at 0.83 map units include both deletion and substitution mutations. The inserted sequences in the substitution mutations are cellular in origin.     

Preservation of Xenopus laevis rDNA-containing plasmid, pXlr101A, injected into the fertilized egg of Xenopus laevis

A circular rDNA-containing recombinant plasmid, pXlr101A, and its vector pBR322 were microinjected into the cytoplasm of fertilized Xenopus laevis eggs. The DNAs extracted from injected embryos at various stages of development were analyzed by hybridization with 32P-labeled pBR322 as the probe. Neither pXlr101A nor pBR322 were replicated, but they were maintained until the tailbud stage, disappearing during the muscular response stage. When pXlr101A-injected embryos were raised until the 2-week-old tadpole stage, sequences homologous to pBR322 were detectable in two Eco RI fragments of the pXlr101A-injected tadpole DNA. The sizes of the Eco RI fragments suggested that the plasmid sequences were preserved most probably in the chromosome-integrated form.     

Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid.

A composite plasmid has been constructed in vitro from colicin E1 factor (mass of 4.2 megadaltons [Md]) and nontransmissible resistance factor RSF 1010 (mass, 5.5. Md) deoxyribonucleic acids (DNAs) by the sequential action of Escherichia coli endonuclease (RI (Eco RI) and T4 phage DNA ligase on the covalently closed circular forms of the constituents. The composite plasmid was selected and amplified in vivo by sequential transformation of E. coli C600 with the ligated mixture and selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-cesium chloride solution. Treatment of the composite plasmid with Eco RI yielded two fragments with mobilities corresponding to the linear forms of the parental plasmids, whereas Serratia marscesens endonuclease R (SmaR), which introduces a single scission in the colicin E1 factor but not in RSF 1010, convErted the composite plasmid to a single linear molecule (mass, 9.7 Md). Sequential degradation of colicin E1 factor with Sma R and Eco RI produced two fragments with masses of 3.5 and 0.7 Md; sequential degradation of RSF 1010 produced only one fragment (due to the cleavage with Eco RI), and sequential degradation of the composite plasmid produced the expected three fragments--an RSF 1010 Eco RI linear and the two expected products from the colicin E1 factor moiety. The composite plasmid conferred on the host cell resistance to streptomycin, sulfonamides, and colicin E1, but colicin E1 itself was not synthesized. In contrast, colicin E1 was synthesized by cells containing simultaneously both colicin E1 factor and RSF 1010 as separate entities. In the presence of chloramphenicol, the composite plasmid continued to replicate for 6 h. whereas replication of RSF 1010 and chromosomal DNA stopped within 2 h. Continued replication in the presence of chloramphenicol suggests that the replicator of the colicin E1 factor is functional in the composite plasmid.     

Transformation by subgenomic fragments of Rous sarcoma virus DNA

Subgenomic fragments of Rous sarcoma virus (RSV) DNA, generated by Eco RI digestion of DNA of RSV-infected chicken cells, induced transformation of NIH/3T3 mouse cells with efficiencies that were 100–1000 fold lower than the efficiency of transformation by intact RSV DNA. Analysis of the DNAs of NIH cells transformed by Eco RI-digested RSV DNA indicated that these cells contained no more than 2 × 10⁶ daltons of RSV DNA, and did not contain sequences from the 5′ terminus of RSV RNA which are included in the leader sequence of subgenomic src mRNA of RSV-infected cells. The product of the RSV src gene (pp60^src), however, was produced in apparently similar quantities by NIH cells transformed by Eco RI fragments of RSV DNA and by intact RSV DNA. Thus expression of the src gene of RSV in NIH cells transformed by subgenomic fragments of RSV DNA did not require the terminal sequences of the RSV genome, which appear to be involved in synthesis and processing of src mRNA in RSV-infected cells. DNAs of NIH cells transformed by Eco RI-digested RSV DNA were found to induce transformation in secondary transfection assays with efficiencies that were similar to the efficiency of transformation by intact RSV DNA. These results suggest that transformation by subgenomic fragments of RSV DNA may be a consequence of integration of src gene-containing DNA fragments in the vicinity of a promoter site in the recipient cell genome, leading to efficient expression of the RSV src gene.     

Four new type I restriction enzymes identified in Escherichia coli clinical isolates

Using a plasmid transformation method and the RM search computer program, four type I restriction enzymes with new recognition sites and two isoschizomers (EcoBI and Eco377I) were identified in a collection of clinical Escherichia coli isolates. These new enzymes were designated Eco394I, Eco826I, Eco851I and Eco912I. Their recognition sequences were determined to be GAC(5N)RTAAY, GCA(6N)CTGA, GTCA(6N)TGAY and CAC(5N)TGGC, respectively. A methylation sensitivity assay, using various synthetic oligonucleotides, was used to identify the adenines that prevent cleavage when methylated (underlined). These results suggest that type I enzymes are abundant in E.coli and many other bacteria, as has been inferred from bacterial genome sequencing projects.     

Translation in Escherichia coli mini-cells containing hamster mitochondrial DNA-Co1E1 - Ampr recombinant plasmids.

Hamster mitochondrial DNA is cleaved into two fragments (4.2 and 11.4 kilobase pairs of DNA (kb)) by the restriction enzyme, Eco RI. Recombinant DNA molecules formed in vitro between an Escherichia coli plasmid, Co1E1 - Ampr, and Eco RI-digested hamster mitochondrial DNA were transformed into E. coli K12. The translation products of the parent plasmid, Co1E1 - Ampr, and recombinant plasmid DNAs containing (i) the 4.2 kb mitochondrial DNA fragment and (ii) the 11.4 kb fragment were characterized on sodium dodecyl sulfate-polyacrylamide gels using bacterial mini-cell lysates. The Co1E1 - Ampr plasmid specifies at least six polypeptides whose structural genes comprise 56% of the plasmid DNA. Insertion of hamster mitochondrial DNA at the Eco RI site of the plasmid alters the relative rate of synthesis of these six polypeptides and induces the occurrence of a new band on sodium dodecyl sulfate-polyacrylamide gels which is probably not specified by the inserted mitochondrial DNA sequences.     

The methylation status of DNA derived from potato plants recovered from slow growth

The in vitro conservation of potato using tissue culture medium supplemented with the growth retardant mannitol causes morphological changes in the propagated material. These culture conditions seem to have an affect on the DNA extracted from the regenerated plants, when it is digested by the methylation sensitive restriction enzymes Hpa II/Msp I and Eco RII/Bst NI, compared to the control material. In most of these plants, there appears to be preferential methylation of nuclear domains that contain Eco RII/Bst NI recognition sites in contrast to those that contain Hpa II/Msp I sites. The refractory nature of the isolated DNA to these restriction enzymes was attributed to hypermethylation of genomic DNA and the ribosomal RNA genes. These findings indicate that methylation of DNA sequences may be an adaptive response to conditions of high osmotic stress. The importance of these results for the conservation of potato germplasm and international exchange is discussed.     

Limited accessibility of DNA methylation sites for bacterial methylases M. Eco RII and M.Eco dam in chromatin at different levels of organization

The bacterial methylases M. Eco RII and M. Eco dam can methylate DNA in rat liver chromatin to form the 5-methylcytosine (m5C) and N6-methyladenine (m6A) residues, respectively. The CH3-accepting capacity of DNA in chromatin (mono- and dinucleosomes, mono- and dinucleomers) is 15 - 30 times less than that of free total DNA in rat liver. Such a low level of DNA methylation in chromatin in vitro suggests that the accessibility and recognition of methylation sites by DNA-methylases are decreased in comparison with free DNA both in the core-particle DNA and in the internucleosomal DNA. The degree of DNA methylation in chromatin particles depends on the ionic strength and Mg2+; when the former is decreased from 0.515 down to 0.176, the DNA methylation by both enzymes is increased 2-fold. An addition of Mg2+ (1 - 2 mM) decreases the CH3-accepting capacity of nucleomeric DNA, that of nucleosomal DNA remains unchanged. Thus, the accessibility of DNA for methylases is variable depending on the conformational changes of chromatin. The values of the m6A to m5C ratio for free and nucleosomal DNAs formed by methylation with a methylation of nucleomeric DNA, i. e. 1.01, 0.92 and 0.51, respectively. As Mg/4 concentration rises, the m6A/m5C ratio for nucleosomal and nucleomeric DNA is increased. It seems therefore that at different levels of organization and upon certain conformation changes the number and, probably, the nature of exposed DNA methylation sites in chromatin are different. Bacterial DNA-methylases can be used as an effective probe for a fine analysis of chromatin ultrastructure, in particular at its different functional states.     

The ovalbumin split gene: molecular cloning of Eco RI fragments "c" and "d".

The Eco RI fragments "c" and "d" of the ovalbumin gene (1, 2) have been isolated by molecular cloning. Restriction enzyme mapping and electron microscopy have confirmed that the two fragments contain the same ovalbumin mRNA coding sequences. These sequences are split into two regions which have been mapped in fragments "c" and "d". There is no evidence that the ovalbumin mRNA sequences contained in these fragments could be further interrupted. Our results confirm that the presence of Eco RI fragment "d" in some chickens is due to the existence of an allelic variant of the ovalbumin gene which contains an additional Eco RI site within the region corresponding to Eco RI fragment "c". This additional Eco RI site appears to be the main difference between the two alleles. Finally, our results provide a direct demonstration that most of the ovalbumin mRNA sequences are encoded for by Eco RI fragments "a", "b" and "c".     

Recent amplification of an alpha satellite DNA in humans.

A repeat sequence 682 base pairs (bp) long produced by cleavage of human DNA with Xba I restriction enzyme is composed of four tandemly arranged subunits with lengths of 171, 170, 171, and 170 bp each. The sequence organization of the 682 bp Xba I repeat bears a striking resemblance to other complex satellite DNAs of primates, including the Eco RI human alpha satellite family which also occurs as a 170 bp repeat. The Eco RI tetramer and the 682 bp Xba I repeat show a sequence divergence of 21%. The 682 bp Xba I repeat sequence is restricted to humans and is only distantly related to the previously reported 340 bp Xba human repeated DNA sequence. These finding are consistent with the concept of occasional amplifications of members or groups of members of alpha satellite DNA during human evolution. Amplifications apparently occurred after humans, apes and gibbons diverged from Old World monkeys (Eco RI satellite), after humans and apes diverged from gibbons (340 bp Xba I satellite) and after humans diverged from the great apes (682 bp Xba I satellite).     

Structural organization and evolution of the plastid genome of Vaucheria sessilis (Xanthophyceae)

The plastid DNAs of 18 Vaucheria sessilis strains from various habitats in western Europe were digested with the restriction endonucleases Eco RI, Sal I, Bam HI and Pvu II. Their restriction patterns showed variable fragment divergencies. Two main groups of plastid genomes were recognized, which were substantiated by morphological features. The differences among the restriction patterns could be attributed to the loss or appearance of restriction sites and to minor size variations caused by deletions/insertions. The Sal I and Bam HI restriction sites which together discriminate six different plastid genomes were mapped on the circular molecule of 124 kilobase paris (kbp). The plastid genomes of several Vaucheria sessilis strains were shown to exist in two inversion isomers caused by intramolecular recombination within the inverted repeat segments.