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Bme 361 I, a new site-specific type II deoxyribonuclease, was purified from Bacillus megaterium 361 by chromatography on phosphocellulose P 11 and hydroxylapatite. The enzyme recognizes and cleaves the nucleotide sequence 5'-GG decreases CC-3' in double-strand DNA. Thus it is a true isoschizomer of deoxyribonucleases Hae III and BspR I.
The viral and host factors involved in herpes simplex virus (HSV) recombination are little understood. To identify features of the process, recombination in HSV-1 and HSV-2 has been studied by analysing the segregation of unselected markers in the form of restriction endonuclease (RE) sites. By confining parental interactions to only one strain of virus of each serotype, restrictions imposed by non-homology are overcome and differential growth phenotypes can be discounted. The analysis of unselected and selected recombinants using RE sites in conjunction with temperature-sensitive mutations is consistent with (i) HSV being highly recombinogenic, (ii) parental and progeny molecules taking part in the process, (iii) the four genomic isomers participating in recombination, (iv) genome alignment being part of the recombination process and (v) cellular factors in conjunction with genome homology influencing the efficiency of recombination.
Mitochondrial DNA (mtDNA) polymorphism was studied in 20 mongrel dogs using 14 restriction enzymes. The polymorphism was observed in the cleavage patterns of Apa I, EcoR I, EcoR V, Hinc II and Sty I. Three morphs using EcoR I and Apa I and two morphs using EcoR V, Hinc II and Sty I were found. However, no polymorphism was detected in the cleavage patterns of BamH I, Bgl II, Hae II, Hind III, Pst I, Sca I, Stu I and Xba I. The examined dogs were classified in seven types using five restriction endonucleases which recognized six nucleotide sequences. The value of nucleotide diversity was estimated to be 0.0055. A phylogenetic tree constructed by genetic distances among seven restriction types showed at least two clusters of mtDNA.
For many years, the resolution of disputed paternity cases by genetic means relied on laboratory blood tests of red cell antigens and red cell and serum protein electrophoresis. These systems are generally characterized as lacking polymorphism and having low powers of exclusion, therefore it was necessary to use a panel of up to 23 marker systems. Over the past five years, a variety of DNA restriction fragment length polymorphism (RFLP) systems have been developed that characterize individuals at the DNA level. In this paper we describe the implementation of a Hae III RFLP system utilized in our laboratory for the resolution of disputed parentage cases as well as forensic casework. Since the analysis of highly polymorphic VNTR loci has proven to be the most powerful method to date for the determination of biological relatedness the utilization of the conventional methodologies should be completely replaced.
Fingerprint patterns were generated from strains of Neisseria meningitidis by digestion of chromosomal DNA samples with 'rare-site' restriction endonucleases and resolution of the resultant fragments by pulsed field gel electrophoresis (PFGE). The potential of this technique for the rapid establishment of the clonal relationships between different isolates of the meningococcus was investigated. The fingerprint patterns from various serogroup A strains, previously assigned to clonal subgroups on the basis of their electrophoretic types (ETs), were compared. Fingerprints generated with the endonucleases SfiI, SpeI and NheI each gave distinctive patterns for the clonal subgroups I-IV of serogroup A. Further, the endonucleases SpeI and, particularly, NheI were capable of resolving differences between various subgroup III strains isolated at different times and geographical locations. Strains isolated during the 'new wave' pandemic, which was associated with the Haj, from Europe, America, and Africa, had a characteristic fingerprint pattern and appeared to be distinct from 'old wave' pandemic strains. The PFGE technique is a relatively rapid and sensitive method for establishing clonal relationships among epidemic strains of N. meningitidis.
Previously we described the B-Z junctions produced in oligomers containing (5meCG)4 segments in the presence of 5.0 M NaCl or 50 uM Co(NH3)6+3 [Sheardy, R.D. & Winkle, S.A., Biochemistry 28, 720-725 (1989); Winkle, S.A., Aloyo, M.C., Morales, N., Zambrano, T.Y. & Sheardy, R.D., Biochemistry 30, 10601-10606 (1991)]. The circular dichroism spectra of an analogous unmethylated oligomer containing (CG)4, termed BZ-IV, in 5.0 M NaCl and in 50 uM Co(NH3)+3 suggest, however, that this oligomer does not form a B-Z hybrid. BZ-IV possesses Hha I sites (CGCG) in the (CG)4 segment and an Mbo I site (GATC) at the terminus of the (CG)4 segment. BZ-IV is equally digestible in the presence and absence of cobalt hexamine by Hha I, further indicating that the structure of BZ-IV is fully B-like under these conditions. The Mbo I cleavage site at the juncture between the (CG)4 segment and the adjacent random segment displays enhanced cleavage by both Mbo I and its isoschizomer Sau3AI in the presence of cobalt hexamine. In addition, exonuclease III digestion of BZ-IV is inhibited at this juncture. Actinomycin inhibits Mbo I activity in the presence of cobalt hexamine but not in the absence. Together, these results suggest that enzymes recognize the interfaces of (CG)n and adjacent random sequences as altered substrates even in the absence of a B-Z junction formation.
The influence of BrdU substitution of DNA in Chinese hamster cells on the frequencies of chromosomal aberrations induced by three restriction endonucleases which recognize thymine-rich sequences in DNA has been studied. The restriction enzymes chosen were Eco RI (recognition site G/AATTC), Sca I (AGT/ACT), and Dra I (TTT/AAA). A restriction enzyme that does not have thymine in the recognition sequence, Hae III (GG/CC), was also tried. These enzymes were introduced into cells by electroporation after two cell cycles of BrdU substitution and the aberration yields compared with that observed in non-substituted cells. Our results seem to indicate that the BrdU-substituted chromatin becomes resistant to the chromosome-breaking activity of the restriction enzymes recognizing thymine-rich DNA sequences. These observations are compared with the patterns of cutting of isolated DNA as shown by agarose gel electrophoresis.
Seventeen Mexican Trypanosoma cruzi stocks and five South American reference strains were analyzed by Hind III restriction fragment length polymorphisms associated with ribosomal RNA gene spacers and by HinfI digestion patterns of total DNA. Our findings demonstrate the occurrence of genetic heterogeneity within these stocks. Hierarchic and non-hierarchic clustering of these molecular characters allowed the formation of groups that correlate with the geographic origin of the stocks. The HinfI digestion pattern permitted the identification of DNA fragments from the kinetoplast maxi-circles, and therefore represents a simple and convenient method for conducting epidemiologic surveys in laboratories in developing countries.
In the budding yeast, S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (+/- 300 bp). In contrast, in mammalian cells and Drosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just like S. cerevisiae origins. Because the fission yeast, Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than does S. cerevisiae, we thought that S. pombe replication origins might prove to resemble--and thus be helpful models for--animal cell origins. An attempt to test this possibility using 2D gel techniques resulted in identification of a replication origin near the ura4 gene on chromosome III of S. pombe. The 2D gel patterns produced by this S. pombe origin indeed resemble the patterns produced by animal cell origins and show that the S. pombe origin cannot be precisely located. The data suggest an initiation zone of 3-5 kbp. Some aspects of the 2D gel patterns detected at the S. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.
DNA from unrelated individuals (Caucasians; n = 200-271) from Switzerland were digested with Hae III and successively hybridized to the DNA probes D1S7, D2S44, D4S139, D10S28, D14S13, D17S26 and D17S79. An allele frequency distribution was determined for each locus. Furthermore, from the same individuals (n = 200) after amplification of DNA the allele and genotype frequencies at the HLA-DQ alpha locus were determined. The allele frequency distribution in Swiss Caucasians is statistically similar to American Caucasian population samples. In criminal cases a DNA-profile derived from four single-locus probes always leads to a very high value of discrimination and in paternity testing the probability of paternity always exceeds 99.9% regardless to the reference population used for biostatistical evaluation. Therefore for use in forensic analysis and paternity testing pooled caucasian databases might be used for the determination of the frequency of occurrence of a DNA-profile.
The mobility shift assay was used to study the competition of the minor groove binder distamycin A with either an Antennapedia homeodomain (Antp HD) peptide or derivatives of a fushi tarazu homeodomain (ftz HD) peptide for their AT-rich DNA binding site. The results show that distamycin and the homeodomain peptides compete under the conditions: (i) preincubation of DNA with distamycin and subsequent addition of HD peptide; (ii) simultaneous incubation of DNA with distamycin and HD peptide; and (iii) preincubation of DNA with HD peptide and subsequent addition of distamycin. There is also competition when using a peptide which lacks the N-terminal arm of ftz HD that is involved in contacts in the minor groove. It is proposed that the protein's binding affinity is diminished by distamycin-induced conformational changes of the DNA. The feasibility of the propagation of conformational changes upon binding in the minor groove is also shown for the inhibition of restriction endonucleases differing in the AT content of their recognition site and of their flanking DNA sequences. Thus, it is demonstrated that minor groove binders can compete with the binding of proteins in the major groove, providing an experimental indication for the influence of biological activities exerted by DNA ligands binding in the minor groove.
The fragile-X syndrome of mental retardation is associated with an expansion in the number of CGG repeats present in the FMR1 gene. The repeat region is within sequences characteristic of a CpG island. Methylation of CpG dinucleotides that are 5' to the CGG repeat has been shown to occur on the inactive X chromosome of normal females and on the X chromosome of affected fragile-X males, and is correlated with silencing of the FMR1 gene. The methylation status of CpG sites 3' to the repeat and within the repeat itself has not previously been reported. We have used two methylation-sensitive restriction enzymes, AciI and Fnu4HI, to further characterize the methylation pattern of the FMR1 CpG island in normal individuals and in those carrying fragile-X mutations. Our results indicate that: (i) CpG dinucleotides on the 3' side of the CGG repeat are part of the CpG island that is methylated during inactivation of a normal X chromosome in females; (ii) the CGG repeats are also part of the CpG island and are extensively methylated as a result of normal X-chromosome inactivation; (iii) similar to normal males, unaffected fragile-X males with small CGG expansions are unmethylated in the CpG island; for affected males, the patterns of methylation are similar to those of a normal, inactive X chromosome; (iv) in contrast to the partial methylation observed for certain sites in lymphocyte DNA, complete methylation was observed in DNA from cell lines containing either a normal inactive X chromosome or a fragile-X chromosome from an affected male.(ABSTRACT TRUNCATED AT 250 WORDS)
We have purified three chromatographically distinct human enzyme activities from HeLa cells, that are capable of converting bleomycin-treated DNA into a substrate for E. coli DNA polymerase I. The bleomycin-treated DNA substrate used in this study has been characterized via a 32P-postlabeling assay and shown to contain strand breaks with 3'-phosphoglycolate termini as greater than 95% of the detectable dose-dependent lesions. The purified HeLa cell enzymes were shown to be capable of removing 3'-phosphoglycolates from this substrate. Also 3'-phosphoglycolate removal and nucleotide incorporation were enzyme dependent. In addition, all three Hela cell enzymes have been determined to possess Class II AP endonuclease activity. The enzymes lack 3'----5' exonuclease activity and are, therefore, dissimilar to exonuclease III--an E. coli enzyme that can remove 3'-phosphoglycolate.
Transcription of the phage Mu com/mom operon is trans-activated by another phage gene product, C, a site-specific DNA binding protein. To gain insight into the mechanism by which C activates transcription, we carried out footprinting analyses of Escherichia coli RNA polymerase (= RNAP) binding to various com-lacZ fusion plasmids. KMnO4-sensitive sites (diagnostic of the melted regions in open-complexes) and DNase I-sensitive sites were located by primer-extension analysis. The results are summarized as follows: (i) in vivo, in the absence of C, RNAP bound in the wild-type (wt) promoter region at a site designated P2; in vitro DNase I-footprinting showed that P2 extends from -74 to -24 with respect to transcription initiation. This overlaps a known strong C-binding site (at -35 to -54). RNAP bound at P2 appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (ii) In contrast, when C was present in vivo, RNAP bound in the wt promoter region at a different site, designated P1, located downstream and partially overlapping P2. RNAP bound at P1 also appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (iii) Two C-independent mutants, which initiate transcription at the same position as the wt, were also analyzed. In vivo, in the absence of C, RNAP bound mutant tin7 (contains a T to G substitution at -14) predominantly at P1; in vitro DNase I-footprinting showed that P1 extends from -56 to +21. With mutant tin6 (a 63 base-pair deletion removing P2, as well as part of P1 and the C-binding site from -35 to -54), RNAP bound to P1 independent of C. We conclude that P1 is the 'functional' RNAP binding site for mom-transcription initiation, and that C activates transcription by promoting binding at P1, while blocking binding at P2.
We have developed a new PCR-RFLP method for HLA-DQB1 typing. This method was easy to follow, requiring only one DQB1 generic amplification and 5 endonucleases to assign 14 out of 15 HLA-DQB1 alleles. In addition, we determined that by using one generic amplification and two enzymes (Sau96 I and Hae III) it was possible to type the generic specificities: DQw2, DQw4, DQw5, DQw6, and DQw7, DQw8-9, providing a practical alternative for serological HLA-DQ generic typing. We also performed a side-by-side correlation with a PCR-SSO typing method and found an almost 100% concordance between the methods. The limitations of these methods were: 1) the PCR-RFLP method did not allow the differentiation between the HLA-DQB1*0602 and *0603 alleles; 2) the PCR-SSO method gave crosshybridization signals in the detection of *0302 or *0303 alleles. Our results suggested that both methods, PCR-RFLP and PCR-SSO, are useful alternatives for HLA-DQB1 typing.
Two-dimensional gel electrophoretic replicon mapping techniques were used to identify all functional DNA replication origins and termini in a 26.5-kbp stretch in the left arm of yeast chromosome III. Only one origin was detected; it coincided with an ARS element (ARS306), as have all previously mapped yeast origins. A replication termination region was identified in a 4.3-kbp stretch at the telomere-proximal end of the investigated region, between the origin identified in this paper and the neighboring, previously mapped, ARS305-associated origin (previously called the A6C origin). Termination does not occur at a specific site; instead, it appears to be the consequence of replication forks converging in a stretch of DNA of at least 4.3 kbp.
Significant DNA polymorphisms have been reported in the beta-globin gene cluster of epsilon-G gamma-A gamma-psi beta-delta-beta-gene region, in normal (Hb AA) individuals and in patients with sickle cell anaemia (SCA). Investigations of the extent of the DNA polymorphisms in the beta A- and beta S-globin gene cluster using Hind III, Hinc II, Ava II, Xmn I, and Hpa I, revealed several associations with mild SCA. The correlation of the presence (+) or absence (-) of the restriction endonuclease site to clinical severity in patients homozygous for beta S-gene showed that the mild form of SCA was associated mainly (> 90%) with the Xmn I polymorphic site 5' to G gamma, and to a lesser extent with Hinc II polymorphic site 5' to epsilon and in the psi beta-gene, with Hind III polymorphic site in G gamma and Hpa I polymorphic site 3' to the beta-globin gene, while in the severe form of SCA these polymorphic sites were absent in most patients. The polymorphism in the beta-globin gene cluster was significantly related to the expression of the beta S-gene and clinical severity of SCA.
A rapid detection method was developed for DNA polymorphisms in the human lipoprotein lipase (LPL) gene. The examined polymorphisms include an A-C transversion in the 5'-region of intron 3, a T-G transversion that occurs within a Hind III site of intron 8, and the previously described C-T transition that causes a Pvu II polymorphism in intron 6. Gene fragments encompassing each polymorphic site were amplified by the polymerase chain reaction (PCR) and digested with an appropriate restriction enzyme whose recognition site was either naturally affected by the polymorphism or artificially created with a mismatched PCR-primer. According to the digestion profiles, genotypes were unambiguously distinguished. With this method, respective allelic frequencies were determined for 50 or 70 normal subjects. The procedure will facilitate LPL genotyping in the large population.