Cutting Restriction Enzymes Research Articles

Measurement of DNA Double-Strand Breaks in Mammalian Cells by Pulsed-Field Gel Electrophoresis: A New Approach Using Rarely Cutting Restriction Enzymes

A new method is presented for measuring DNA double-strand breaks (DSBs) which combines the technique of pulsed-field gel electrophoresis (PFGE) with the idea of velocity sedimentation. Purified DNA samples were treated with restriction enzymes, which results in DNA of sizes which can be separated by PFGE. After electrophoresis, unirradiated DNA shows a size distribution (obtained with the help of a specially developed software program) similar to that obtained with the sedimentation technique; with X irradiation, this distribution is shifted to lower molecular weight with increasing dose. The rate of DSB induction was calculated by comparing the curves obtained experimentally with theoretical distributions (based on the assumption that breaks are formed according to Poisson statistics). The method was tested by measuring X-ray-induced DSBs in P3 (derived from human epithelial teratoma) cells. The induction of DSBs was found to be linear with dose and a rate of 5.4 x 10(-3)/Mbp/Gy was obtained.

Construction of a Human MluI YAC Library

We describe a cloning strategy for the construction of a human genomic library in yeast artificial chromosomes (YACs) based on complete digestion of high-molecular-weight DNA with the infrequently cutting restriction enzyme Mlu I. Cloning of MluI fragments in the vector pYAC-RC and one subsequent size fractionation by preparative pulsed-field gel electrophoresis yielded a library with average insert sizes of 600 kb. Ninety-seven percent of the colonies were recombinant. An additional size fractionation of MluI fragments prior to ligation had no significant influence on the size of the YACs. The library currently consists of 5000 clones, which is the equivalent of one human genome. Nineteen percent of the YACs were larger than 1.2 Mb. Since smaller MluI fragments are lost during sizing, we also performed cloning without size fractionation. Only 20% of the colonies were recombinant, probably due to unligated vector fragments that were present during the transformation.

Epidemiologic analysis of sporadic Salmonella typhi isolates and those from outbreaks by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was used to compare and analyze 158 isolates of Salmonella typhi from five well-defined outbreaks of typhoid fever in Malaysia and also isolates involved in sporadic cases of typhoid fever occurring during the same period. Digestion of chromosomal DNAs from these S. typhi isolates with the restriction endonucleases XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3') and then PFGE produced restriction endonuclease analysis (REA) patterns consisting of 11 to 24 DNA fragments ranging in size from 20 to 630 kbp. Analysis of the REA patterns generated by PFGE after digestion with XbaI and SpeI indicated that the S. typhi isolates obtained from sporadic cases of infection were much more heterogeneous (at least 13 different REA patterns were detected; Dice coefficient, between 0.73 and 1.0) than those obtained during outbreaks of typhoid fever. The clonal nature and the close genetic identities of isolates from outbreaks in Alor Setar, Penang, Kota Kinabalu, Johor Bahru, and Kota Bahru were suggested by the fact that only a limited number of REA patterns, which mostly differed by only a single band, were detected (one to four patterns; Dice coefficient, between 0.82 and 1.0), although a different pattern was associated with each of these outbreaks. Comparison of REA patterns with ribotyping for 18 S. typhi isolates involved in sporadic cases of infection showed a good correlation, in that 72% of the isolates were in the same group. There was no clear correlation of phage types with a specific REA pattern. We conclude that PFGE of s. typhi chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for comparing and differentiating S. typhi isolates for epidemiological purposes.

Genomic organization of glycophorin A gene family revealed by yeast artificial chromosomes containing human genomic DNA.

Human glycophorins A, B, and E are members of the glycophorin gene family located at chromosome 4, band q31. These genes were apparently generated by two successive gene duplications, and the following evolutionary pathway was proposed based on the genomic sequences. First, the ancestral gene was duplicated, and one of the duplicated genes evolved directly into the GPA gene. Another duplicated gene acquired a 3' sequence from the precursor genomic segment and was then duplicated to yield glycophorin B and E genes (Onda, M., Kudo, S., Rearden, A., Mattei, M.-G. and Fukuda, M. (1993) Proc. Natl. Acad. Sci. USA 90, 7220-7224). Although the above hypothesis was proposed, it has been difficult to provide solid evidence for this, because no genomic clones studied encompass two members of the gene family. In this study we isolated several yeast artificial chromosome (YAC) clones encoding the entire region or a portion of this gene locus. The physical gene mapping was carried out by testing the presence of specific sequences using a polymerase chain reaction. Moreover, the YAC clones were digested by rare cutting restriction enzymes, and the resultant large DNA fragments were separated by pulse-field electrophoresis. Southern blot analyses of those DNA fragments were carried out using various probes encoding specific portions of glycophorin genes. The results obtained revealed that the distance between glycophorin A and B genes is almost the same as that between glycophorin B and E genes. Moreover, it was shown that no precursor genomic sequence was present in the sequences downstream from the GPB or GPE gene, suggesting that the duplication of glycophorin B/E progenitor gene was made through unequal crossing over homologous recombination. These combined results suggest that two duplication events took place tandemly to produce these three members of the glycophorin gene family.

Determination of Gardnerella vaginalis genome size by pulsed-field gel electrophoresis.

The chromosomal DNA of four strains of Gardnerella vaginalis were digested with rare cutting restriction enzymes and analyzed by pulsed-field gel electrophoresis (PFGE). The four strains studied were two clinical isolates (GVP 004 & GVP 007) and two American Type Culture Collection strains (ATCC 14018 & ATCC 14019). The restriction enzyme SfiI generated two DNA fragments of about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalis genome size of about 1.7 Mb. A similar genome size was calculated utilizing two more GC-rich sequence specific restriction endonucleases, NotI and AscI. When digested with AscI, the chromosomal DNA of all four strains gave rise to 11 to 12 DNA fragments ranging between 0.01 Mb to 0.43 Mb. DNA from the two clinical isolates were digested by NotI (yielding 7 to 9 fragments), while the DNA from the two ATCC strains were resistant to NotI digestion. In contrast the clinical isolates, DNA from the two ATCC strains gave an identical profile for all restriction endonucleases tested. From double digestion experiments, the two SfiI sites could be localized on two AscI fragments. From these PFGE studies, it is concluded that the G. vaginalis genome is a circular DNA that ranges between 1.67 Mb and 1.72 Mb in size.

Long-range physical maps of two loci (Aps-1 and GP79) flanking the root-knot nematode resistance gene (Mi) near the centromere of tomato chromosome 6.

The root knot nematode resistance gene Mi in tomato has been mapped in the pericentromeric region of chromosome 6. With the objective of isolating Mi through a map-based cloning approach, we have previously identified and ordered into a high-resolution genetic linkage map a variety of tightly linked molecular markers. Using pulsed-field gelelectrophoresis and various rarely cutting restriction enzymes in single, double and partial digestions, we now report long-range physical maps of the two closest flanking markers, acid phosphatase-1 (Aps-1) and GP79, which span over 400 and 800 kb, respectively. It is concluded that the physical distance between both markers is larger than predicted on the basis of genetic linkage analysis. Furthermore, two RFLP markers (H3F8 and H4H10) which map genetically to the same locus as Aps-1 do not show physical linkage, indicating severe suppression of recombination in this region of the chromosome. Finally, no evidence was obtained showing the presence of a CpG island near Aps-1.

Studies on the Evolution of Agrobacterium vitis as Based on Genomic Fingerprinting and IS Element Analysis

Summary In order to study evolutionary relationships of the crown gall pathogen on grapevine, Agrobacterium vitis , bacterial genomes were analyzed by genomic fingerprinting. Additionally, A. vitis specific IS elements were used as chromosomal markers to establish genetic relations obtained by restriction analysis. DNA fragments up to 1 Mb in size were produced by digestion of genomic DNA with the rare cutting restriction endonucleases Xba I, Sfi I, and Spe I and separated by pulsed field gel electrophoresis (PFGE). As the result of the macrorestriction analysis of 42 strains six genomic groups with homologous restriction patterns could be identified. Based on similarity coefficients of more than 0.8 A. vitis strains were allocated to one of the six genome types. For every genome type defined, a distinct IS element pattern was found. The synopsis of IS element patterns and genomic fingerprinting allowed the construction of a phylogenetic tree on the evolution of different genome types within A. vitis .

Analysis of rotifer ribosomal gene structure using the polymerase chain reaction (PCR)

We have used the polymerase chain reaction (PCR) to selectively amplify 18 S ribosomal genes in rotifer taxa from major planktonic clades. In each case, we obtained an amplified product of between 1.8 and 2.0 kilobase pairs. We analyzed the PCR products using 6- and 4-base cutting restriction enzymes, comparing fragment mobilities. For example, Brachionus plicatilis (BSL strain) 18S genes have no restriction sites for Hind III or Bam HI and only a single site for Eco RI (all 6-base cutters). The 4-base cutter Msp I, on the other hand, has at least 4 enzymatic sites, producing fragments between approximately 110 and 460 base pairs in length. Results of this type can be used to differentiate among species and species groups within the Rotifera and can be used as the basis for construction of a broad molecular phylogeny of the group.

High‐resolution mapping of genetic loci of Anabaena PCC 7120 required for photosynthesis and nitrogen fixation

A physical map of the Anabaena genome permitted the localization of its genes to chromosomal fragments generated by rarely cutting restriction endonucleases and separated by pulsed-field gel electrophoresis. We introduce a novel means of mapping more precisely to c. 20 kb by use of rare restriction sites within vectors bearing cloned sequences that undergo homologous recombination with the genome. We thereby localize and orient genes encoding principal photosynthetic pigments. The relative spacing of loci within a single restriction fragment was determined with even higher resolution, as illustrated for genes required for heterocyst development and nitrogen fixation that were marked with transposons. Small, newly visualized restriction fragments of the chromosome were also mapped.

RFLP analysis of highly polymorphic loci in barley

Barley middle-repeat sequences were screened for their ability to discriminate 51 barley commercial varieties. Two hordein clones, a clone encoding a leaf-specific thionin, a desiccation induced cDNA clone, a clone coding for 5S-rRNA and one corresponding to ubiquitin genes were tested. A very sensitive RFLP technique including four cutter restriction enzymes and denaturing 4% polyacrylamide gels were used to evidence the highest level of polymorphism.The RFLP data were analyzed by computer. Some probe/enzyme combinations were able to differentiate a large number of the cultivars tested, whereas three probe/enzyme combinations succeeded in identifying all the varieties. The use of this RFLP method can thus be suggested for cultivar identification in barley.

Typing method for N2‐fixing bacteria based on PCR‐F — application to the characterization of Frankia strains

DNA sequences of an intergenic spacer (IGS) and parts of genes in the nif cluster were amplified by the polymerase chain reaction (PCR) using two primers derived from nifD- and nifK-conserved sequences. The PCR products were cleaved by ten 4-base cutting restriction enzymes and the restriction patterns were used as fingerprints to type Frankia strains. The feasability of this PCR-RFLP method for typing Frankia strains was investigated on Frankia reference strains belonging mainly to the Elaeagnaceae infectivity group but also on new Frankia isolates and on other N2-fixing microorganisms. By modulating the stringency of the amplifications, we showed the method allowed to target either Frankia strains or the whole N2-fixing microbial community. DNA digestion patterns were used to estimate the sequence divergence between the Frankia nifD-K fragment. The estimated relationships deduced from these genotypic data correlated well with established Frankia taxonomic schemes.

Rapid discrimination of Echinococcus species and strains using a polymerase chain reaction-based RFLP method

Echinococcus species and genetically distinct strains of Echinococcus granulosus can be rapidly and reliably identified using a polymerase chain reaction (PCR)-linked restriction fragment length polymorphism (RFLP) method which surveys the sequence of a rapidly evolving region of the ribosomal DNA (rDNA) unit. Internal transcribed spacer 1 (ITS1) of the rDNA repeat was amplified from various isolates and the product was digested with one of a number of 4-base cutting restriction enzymes. Characteristic patterns were produced when samples within various species and strain groups were analysed. This method offers an objective, simple, highly sensitive and rapid approach for the discrimination of Echinococcus isolates and for study of other parasite complexes.

Large DNA restriction fragment polymorphism in the Mycobacterium avium-M. intracellulare complex: a potential epidemiologic tool

Mycobacterium avium-M. intracellulare complex (MAI) isolates were studied by comparing the large restriction fragment (LRF) patterns produced by digesting their DNAs with infrequently cutting restriction endonucleases and separating the resultant large fragments by pulsed-field gel electrophoresis. Four reference strains and 35 randomly selected clinical MAI isolates gave highly diverse LRF patterns when their DNAs were digested with XbaI or AsnI. The LRF patterns of random isolates identified to be the same species by DNA probe analysis were not similar. The LRF patterns of random isolates of the same serotype were also different. In contrast, all isolates recovered from the same patient gave identical patterns. This included 28 isolates from nine patients. One isolate from sputum, one isolate from bone marrow, and two isolates from blood recovered over a 27-month period from a patient with AIDS were identical. Seven isolates recovered from the sputum of a second patient over 37 months also had identical patterns. The LRF patterns of unrelated MAI strains are highly polymorphic, appear to be strain specific, are relatively stable, and offer exciting promise as epidemiologic markers for the study of MAI infections.

Genomic restriction map of the extremely thermophilic bacterium Thermus thermophilus HB8

A physical map of the chromosome of the extremely thermophilic eubacterium Thermus thermophilus HB8 has been constructed by using pulsed-field gel electrophoresis techniques. A total of 26 cleavage sites for the rarely cutting restriction endonucleases HpaI, MunI, and NdeI were located on the genome. On the basis of the sizes of the restriction fragments generated, the genome size was estimated to be 1.74 Mbp, which is significantly smaller than the chromosomes of Escherichia coli and other mesophiles. Partial digestion experiments revealed the order of the six HpaI bands on the chromosome. Hybridization of isolated restriction fragments to pulsed-field gel-separated restriction digestions confirmed the deduced order of the HpaI fragments and allowed ordering and alignment of the NdeI and MunI fragments. In addition, 16 genes or gene clusters cloned from several different Thermus strains were located on the T. thermophilus HB8 chromosomal map by hybridization of gene probes to pulsed-field gel-resolved restriction digestions.

X-linked retintis pigmentosa: Isolation and characterization of the gene at Xp21 (RP3)

Microsatellites are widely recognised as providing a rich source of polymorphic markers for genetic mapping.1–3 Consequently, highly polymorphic CA repeats tightly linked to a disease locus are invaluable tools in linkage studies. We have developed an efficient technique for cloning microsatellite repeats from a region of interest contained within a yeast artificial chromosome (YAC). The YAC material is digested with a frequent cutting restriction endonuclease and ligated to polymerase chain reaction (PCR) amplifiable catch-linkers. A 5′ biotinylated (CA)11 oligonucleotide is then used to select fragments containing a complementary repeat by binding to streptavidin-coated magnetic beads. The catch-linkers enable these fragments to be PCR amplified, cloned and sequenced. Primers are then designed to amplify the repeat locus and to confirm its genomic localization and heterozygosity. We have successfully used this technique to clone a new (CA)18 microsatellite from a 360-kb YAC. The YAC contains the CYBB locus in Xp21·1 and is thought to contain at least part of the RP3 gene responsible for X-linked retinitis pigmentosa. This new CA repeat is highly polymorphic with nine alleles identified so far and a heterozygosity of 0·75.4

Amplification of unknown DNA sequences by sequence-independent nested polymerase chain reaction using a standardized adaptor without specific primers

A new procedure for sequence-independent PCR amplification of DNA fragments is described. DNA from pUC18 plasmid was used as a test DNA. It was digested with a frequently cutting restriction enzyme ( Sau3A), generating sticky ends. The DNA was ligated to a synthetic, non-phosphorylated adaptor and subsequently amplified in a nested PCR using two oligonucleotides with sequences derived from the adaptor. As little as 1 fg of pUC18 DNA could be detected by this procedure. The product was analyzed on a gel and hybridized with a pUC18-specific probe. The sequence-independent nested PCR was repeated with different amounts of pUC18 DNA in the presence of an excess of non-specific DNA. In these experiments, pUC18 DNA fragments were amplified in a concentration-dependent manner. After hybridization with a digoxigenine dUTP-labelled pUC18 DNA probe, 1 fg of pUC18 DNA could still be detected. This method allows rapid screening of blood for low titred and mutated viruses in which primer binding sites are not conserved.

Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene.

Restriction fragment length polymorphisms, Western blot (immunoblot) analysis, and fluorescence-labelled signature probes were used for the characterization of methanotrophic bacteria as well as for the identification of methanotrophs which contained the soluble methane monooxygenase (MMO) gene and were able to degrade trichloroethylene (TCE). The gene encoding a soluble MMO component B protein from Methylosinus trichosporium OB3b was cloned. It contained a 2.2-kb EcoRI fragment. With this cloned component B gene as probe, methanotroph types I, II, and X and environmental and bioreactor samples were screened for the presence of the gene encoding soluble MMO. Fragments produced by digestion of DNA with rare cutting restriction endonucleases were separated by pulsed-field gel electrophoresis and transferred to Zeta-Probe membrane (Bio-Rad) for Southern blot analysis. Samples were also analyzed for the presence of soluble MMO by Western blot analysis and the ability to degrade TCE. The physiological groups of methanotrophs in each sample were determined by hybridizing cells with fluorescence-labelled signature probes. Among twelve pure or mixed cultures, DNA fragments of seven methanotrophs hybridized with the soluble MMO B gene probe. When grown in media with limited copper, all of these bacteria degraded TCE. All of them are type II methanotrophs. The soluble MMO component B gene of the type X methanotroph, Methylococcus capsulatus Bath, did not hybridize to the M. trichosporium OB3b soluble MMO component B gene probe, although M. capsulatus Bath also produces a soluble MMO.

Patent Evaluation: The combination ofin situhybridization and the use of rare restriction enzymes facilitates gene isolation

SummaryNovelty: Methods for mapping discrete DNA sequences complementary to regions of a eukaryotic chromosome and for isolating a gene of interest are disclosed.Biology: Methods for ordering a set of genomic clones complementary to a region of a eukaryotic chromosome are provided. The linear order of clones from a chromosome-specific library is determined by in situ hybridization, the clones are then analysed for the presence of rare cutter restriction enzyme sites and for restriction fragment length polymorphism. This method substantially reduces the number of clones which must be analysed to define markers of appropriate spacing. Specific genes of interest may be isolated by identifying candidate genomic clones which contain a rare cutter site and have a map position near the locus identified previously by genetic mapping.

Clonal differences within M-types of the group A streptococcus revealed by pulsed field gel electrophoresis

Digestion of chromosomal DNA with the rare cutting restriction enzyme SfiI in association with pulsed field gel electrophoresis was used to observe restriction fragment length polymorphisms (RFLP) among isolates of group A Streptococcus. Streptococci examined included isolates belonging to the same M-type (epidemiologically related and unrelated), and isolates from other M-types. RFLP patterns were quite distinct between all serotypes tested. More importantly, isolates from within a serotype could be differentiated by this technique.

Methylation and sequence analysis around EagI sites: identification of 28 new CpG islands in XQ24-XQ28.

Thirty-two probes for CpG islands of the distal long arm of the human X chromosome have been identified. From a genomic library of DNA of the hamster-human cell hybrid X3000.1 digested with the rare cutter restriction enzyme EagI, 53 different human clones have been isolated and characterized by methylation and sequence analysis. The characteristic pattern of DNA methylation of CpG islands at the 5' end of genes of the X chromosome has been used to distinguish between EagI sites in CpG islands versus isolated EagI sites. The sequence analysis has confirmed and completed the characterization showing that sequences at the 5' end of known genes were among the clones defined CpG islands and that the non-CpG islands clones were mostly repetitive sequences with a non-methylated or variably methylated EagI site. Thus, since clones corresponding to repetitive sequences can be easily identified by sequencing, such libraries are a very good source of CpG islands. The methylation analysis of 28 different new probes allows to state that demethylation of CpG islands of the active X and methylation of those on the inactive X chromosome are the general rule. Moreover, the finding, in all instances, of methylation differences between male and female DNA is in very strong support of the notion that most genes of the distal long arm of the X chromosome are subject to X inactivation.