Enzymes NotI Research Articles

Isolation and regional localization of cosmid linking clones from human chromosome 12.

We have developed a new method for constructing cosmid linking libraries. The method is based on the insertion of a selection gene, beta-lactamase, in genomic cosmid clones containing recognition sites for rare-cutting enzymes. The selection gene is maintained as a gene cassette in a plasmid and may be excised by the enzymes NotI, SacII, SplI, MluI, BssHII, and NarI or combinations of these enzymes. Using this gene cassette and a genomic cosmid library made from a human-hamster cell line containing the human chromosome 12 as its only human component, a chromosome 12-specific NotI linking library was constructed. The NotI linking clones contained recognition sites for other rate-cutting enzymes, SacII and BssHII, at high frequency, indicating the presence of CpG islands. Thirty cosmid linking clones were regionally localized by FISH and were found to be clustered to chromosome bands 12p13, 12q13, and 12q24.

Physical and genetic map of the Serpulina hyodysenteriae B78T chromosome.

A combined physical and genetic map of the Serpulina hyodysenteriae B78T genome was constructed by using pulsed-field gel electrophoresis and DNA blot hybridizations. The S. hyodysenteriae genome is a single circular chromosome about 3.2 Mb in size. The physical map of the chromosome was constructed with the restriction enzymes BssHII, EclXI, NotI, SalI, and SmaI. The physical map was used to constructed a linkage map for genes encoding rRNA, flagellum subunit proteins, DNA gyrase, NADH oxidase, and three distinct hemolysins. Several flaB2-related loci, encoding core flagellum subunit proteins, were detected and are dispersed around the chromosome. The rRNA gene organization in S. hyodysenteriae is unusual. S. hyodysenteriae has one gene each for 5S (rrf), 16S (rrs), and 23S (rrl) rRNAs. The rrf and rrl genes are closely linked (within 5 kb), while the rrs gene is about 860 kb from the other two rRNA genes. Using a probe for the S. hyodysenteriae gyrA gene, we identified a possible location for the chromosomal replication origin. The size and genetic organization of the S. hyodysenteriae chromosome are different from those of previously characterized spirochetes.

Long-range restriction map of human chromosome 22q11-22q12 between the lambda immunoglobulin locus and the Ewing sarcoma breakpoint.

A long-range restriction map of the region between the immunoglobulin lambda locus and the Ewing sarcoma breakpoint has been constructed using the rare-cutting enzymes NotI, NruI, AscI, and BsiWI. The map spans approximately 11,000 kb and represents about one-fifth of the long arm of chromosome 22. Thirty-nine markers, including seven NotI junction clones as well as numerous genes and anonymous sequences, were mapped to the region with a somatic cell hybrid panel. These probes were then used to produce the map. The seven NotI junction clones each identified a possible CpG island. The breakpoints of the RAJ5 hybrid and the Ewing sarcoma t(11;22) were also localized in the resulting map. This physical map will be useful in studying chromosomal rearrangements in the region, as well as providing the details to examine the fidelity of the YAC and cosmid contigs currently under construction. Comparisons of this physical map to genetic and radiation hybrid maps are discussed.

Recognition of Leuconostoc oenos strains by the use of DNA restriction profiles.

The chromosome of 41 Leuconostoc oenos strains obtained from collections in different countries was analysed with the aim of differentiating the strains. Pulsed field electrophoresis (TAFE) was used to separate large DNA fragments created by the restriction enzymes NotI, SfiI and ApaI, which specifically recognize guanines or cytosines. The genomic DNA of 11 strains was analysed initially with NotI and only four different restriction profiles were observed. The genome size ranged from 1.8 to 2.1 megabase pairs (Mbp). Constant field electrophoresis applied to DNA treatment with 19 different restriction enzymes showed that the size of the fragments obtained increased proportionally to the percentage G + C present at the site of restriction. EcoRI and HindIII profiles revealed that the zone between 9 and 23 kbp allowed differentiation of the strains tested. Thus, the 41 strains fell into 30 restriction groups using only two enzymes. Hybridization with a non-radioactive DNA probe coding for 16S rRNA revealed that there were two 16S genes on the chromosome.

A complete physical map of a Bacillus thuringiensis chromosome

Bacillus thuringiensis is the source of the most widely used biological pesticide, through its production of insecticidal toxins. The toxin genes are often localized on plasmids. We have constructed a physical map of a Bacillus thuringiensis chromosome by aligning 16 fragments obtained by digestion with the restriction enzyme NotI. The fragments ranged from 15 to 1,350 kb. The size of the chromosome was 5.4 Mb. The NotI DNA fingerprint patterns of 12 different B. thuringiensis strains showed marked variation. The cryIA-type toxin gene was present on the chromosome in four strains, was extrachromosomal in four strains, and was both chromosomal and extrachromosomal in two strains. A Tn4430 transposon probe hybridized to 5 of the 10 cryIA-positive chromosomal fragments, while cryIA and the transposon often hybridized to different extrachromosomal bands. Ten of the strains were hemolytic when grown on agar plates containing human erythrocytes. Nine of the strains were positive when assayed for the presence of Bacillus cereus enterotoxin. We conclude that B. thuringiensis is very closely related to B. cereus and that the distinction between B. cereus and B. thuringiensis should be reconsidered.

Scattering of the rRNA genes on the physical map of the circular chromosome of Leptospira interrogans serovar icterohaemorrhagiae.

Leptospira interrogans is a pathogenic bacterium with a low G+C content (34 to 39%). The restriction enzymes NotI, AscI, and SrfI cut the chromosome of L. interrogans serovar icterohaemorrhagiae into 13, 3, and 5 fragments separable by one- and two-dimensional pulsed-field gel electrophoresis (PFGE). The genome is composed of a circular 4.6-Mbp chromosome and a 0.35-Mbp extrachromosomal element. A physical map of the chromosome was constructed for NotI, AscI, and SrfI by using single and double digests, or partial NotI digests obtained at random or by cross-protection of NotI sites by FnuDII methylase, and linking clones. rRNA genes were found to be widely scattered on the chromosome.

Generation and characterization of a human chromosome 9 cosmid library.

A cosmid library has been constructed from the hamster-human hybrid cell line PK-87-9, which contains chromosome 9 as its sole known human component. Ten thousand colonies were produced, of which approximately 200, or 2%, contain human material. Fifty of these 200 were regionally mapped by an Alu-primed PCR product hybridization procedure. These cosmids were localized to all regions of chromosome 9, but were especially concentrated in the distal portion of 9q. The map location derived by the Alu-primed PCR product hybridization procedure was compared to the map location derived by fluorescent in situ hybridization. Assignment of chromosomal location by the two methods was correspondent in all but a few cases. The presumptive presence of HTF islands was investigated for 130 cosmids by digestion with the restriction enzyme NotI. Twenty percent of cosmids contained at least one NotI site. A number of simple sequence repeat polymorphisms identified from the cosmid set were characterized and will provide a link between the genetic and physical maps for this chromosome.

Long-range mapping of the gene for the human α5(IV) collagen chain at Xq22–q23

The X-linked kidney disorder known as Alport syndrome (AS) has been shown to be due to mutations in the gene for an α5 chain of type IV collagen that maps to Xq22–23. Using overlapping cDNA clones that represent approximately 90% of this gene and pulsed-field gel electrophoresis, we have constructed a 2.4-Mb long-range restriction map around the locus. All of the cDNA clones lie within a 360-kb segment of DNA bounded by CpG islands that contain sites for the rare-cutting enzymes BssHII, MluI, NotI, NruI, SalI, and SfiI. High-resolution PFGE mapping with XhoI shows that the gene is at least 110 kb in size and is one of the largest collagen genes characterized to date. This map will prove useful in the characterization of mutations in individuals affected with AS and will also provide information as to the location of other genes in the region.

Methylation status of ribosomal RNA gene clusters in the flow-sorted human acrocentric chromosomes.

Southern blot analysis of the human acrocentric chromosomes that were flow-sorted from B-lymphoblastoid cell line GM130B revealed that the sensitivity of the ribosomal RNA (rDNA) gene clusters to the restriction enzyme NotI differs among these rDNA-containing chromosomes: the rDNA clusters of Chromosomes (Chr) 13, 14, and 15 are much more sensitive to NotI digestion than those of Chrs 21 and 22 in this particular cell line. Detailed analysis by use of methylation-sensitive enzymes HpaII and HhaI and methylation-insensitive enzyme MspI confirmed the significant variation in the methylation status of rDNA clusters among these chromosomes. Quantitative analysis by fluorescent in situ hybridization (FISH) indicated that copy number of rDNA varies among individual chromosomes, but the average copy number in the acrocentric Chrs 21 and 22 is significantly greater than that of the Chrs 13, 14, and 15 in GM130B cells. Similar analysis reveals that the methylation status of rDNA clusters in another B-lymphoblastoid cell line GM131 was different from that of GM130B. These data together indicate that the copy number and methylation patterns of rDNA clusters differ among individual acrocentric chromosomes in a given cell line, and they are different among cell lines.

Identification of multiple CpG islands and associated conserved sequences in a candidate region for the Huntington disease gene

The HD locus has been assigned to 4p16.3 distal to the DNA segment D4S10. However, the precise location of this gene is still unknown. At least three regions, together encompassing more than 3.5 Mb of DNA, can still be considered as candidate regions for the HD gene. Our efforts are directed toward the cloning and the complete characterization of one of these regions. Thus far we have cloned 460 kb of DNA in contiguously overlapping cosmids distal to D4S111 and have developed a detailed long-range restriction map orienting the contig within the terminal region of 4p16.3. We characterized 15 CpG-rich islands defined by tightly clustered rare cutter restriction sites for the enzymes NotI, BssHII, EagI, NruI, and SacII. In addition, we show that the sequences associated with the CpG-rich islands detect cross-species conservation. The detailed genetic analysis of the 460-kb contig provides a framework for the identification of genes, which can be assessed for the characteristics expected for the HD gene.

Large-scale physical mapping within the region 22q12.3–13.1 in meningioma

The lack of physical mapping data strongly restricts the analysis of the meningioma chromosomal region that was assigned to the bands 22q12.3-qter. Recently, we reported a new marker D22S16 for chromosome 22 that was assigned to the region 22q13-qter by in situ hybridization. Utilizing somatic cell hybrids we now sublocalized the marker D22S16 within the band region 22q12–13.1, thus placing it in the vicinity of the gene for the platelet derived growth factor (PDGFB). A physical map was established for the regions surrounding the PDGFB gene and the D22S16 marker. By means of pulsed-field gel electrophoresis (PFGE) D22S16 and PDGFB were found to be physically linked within 900 kb. We also identified two CpG clusters bordering the PDGFB gene. For the enzyme NotI, a variation of the PDGFB restriction pattern was found between different individuals. PFGE analysis of the two loci (PDFGB and D22S16) failed to identify major rearrangements in meningioma.

Long-range restriction mapping and linkage analysis of the Prader-Willi chromosome region ( PWCR)

In an attempt to elucidate the relationship between genetic alterations at chromosomal bands 15q11.2–12 and the Prader-Willi syndrome (PWS), we have constructed a long-range restriction map of this region using a combination of pulsed-field gel techniques and the infrequently cutting restriction enzymes NotI, MluI, SalI, SfiI, NruI, SacII, and BssHII. Four previously reported probes mapping to 15q11.2–12 and known to be deleted in PWS patients were used to construct the physical map of this region. The loci recognized by these four probes have been localized to a 2600-kb partial SalI restriction fragment and a 3200-kb partial EcoRI restriction fragment. Linkage studies were performed on nine families to estimate the recombination rates between these loci. The calculated lod scores did not indicate significant linkage between any of the four loci. The contrast between the physical distance and the observed recombination frequency suggests that these four loci are located in a recombinational “hot spot”.

パルスフィールドゲル電気泳動法（ＰＦＧＥ）によるＴｈｅｒｍｏｐｈｉｌｉｃ Ｃａｍｐｙｌｏｂａｃｔｅｒ３菌種のＤＮＡ解析

In order to determine the suitable conditions for gaining distribution of DNA fragments useful for the chromosomal DNA analysis of the thermophilic Campylobacter, C. coli, C. jejuni and C. laridis, we were interested in the poor G+C content of the chromosomal DNAs and the intact DNAs embedded in agarose blocks were digested with 15 restriction enzymes which can recognize sequences containing only G and C nucleotides (NotI, SfiI, ApaI, BglI, SacII, SmaI and HpaII), sequences rich with G and C (BamHI, KpnI, PstI, SalI and XhoI) and some other sequences (DraI, EcoRI and HindIII) respectively. The resulted restriction fragments were fractionated by pulsed-field gel electrophoresis.When the chromosomal DNAs prepared from the 3 Campylobacter strains were digested with two restriction enzymes (NotI and SfiI) available with 8-base recognition, electrophoretic profiles of the digested chromosomal DNAs were shown to be very similar to those of each undigested chromosomal DNAs, respectively.The DNAs digested with EcoRI migrated at the same level as each undigested chromosomal DNAs by unknown reason(s).Twelve other restriction enzymes were able to cut the DNAs from the 3 Campylobacter strains with the exception that KpnI did not cut the DNA from the C. jejuni strain. Among the enzymes, ApaI, SalI and SmaI cut the DNAs from the 3 Campylobacter strains into a relatively limited number of restriction fragments in the range of approximately 50-1, 500kb in length.Thus, three restriction enzymes, ApaI, SalI and SmaI, were found to produce distributions of DNA fragments useful for chromosomal DNA analysis of the 3 Campylobacter strains by pulsed-field gel electrophoresis.Moreover, the genome size of the 3 bacterial strains were preliminarily estimated to be approximately 2, 000kb for C. coli JCM 2529T, 1, 900kb for C. jejuni JCM 2013 and 1, 700kb for C. laridis JCM 2530T in length, respectively.

Physical map of chromosomal and plasmid DNA comprising the genome of Leptospira interrogans.

The size and physical structure of the Leptospira interrogans genome was characterized using contour-clamped homogenous electric field (CHEF) gel electrophoresis. The L. interrogans genome is approximately 4750 kb in size and is composed of two molecular species of DNA: a 4400 kb chromosome; and a 350 kb plasmid, pLIN1. A physical map of the chromosome was constructed with the restriction enzymes NotI and SfiI. A physical map of pLIN1 was constructed with ApaI, NotI, Sse83871, SgrAI, and SmaI. Both the L. interrogans chromosome and pLIN1 are circular.

Construction of a NotI restriction map of the Streptococcus mutans genome

Streptococcus mutans and Streptococcus sobrinus are the major causative organisms of human dental caries. Pulsed-field gel electrophoresis (PFG) showed that the restriction enzyme NotI produced ten and six DNA fragments from the genomes of S. mutans strain MT8148 and S. sobrinus strain 6715, respectively. The sizes of the chromosomes of S. mutans and S. sobrinus were each estimated to be about 2200 kb. The NotI restriction map of S. mutans MT8148 genome was constructed by Southern blot analysis with probes that overlapped two adjacent NotI fragments. Several virulence-associated genes of S. mutans were placed on the NotI restriction map. In addition, unique 'fingerprints' of S. mutans chromosomal DNA digested with NotI were produced by PFG, and these may be useful for epidemiological studies.

Use of pulsed-field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a SalI map of Campylobacter jejuni UA580

To determine the physical length of the chromosome of Campylobacter jejuni, the genome was subjected to digestion by a series of restriction endonucleases to produce a small number of large restriction fragments. These fragments were then separated by pulsed-field gel electrophoresis with the contour-clamped homogeneous electric field system. The DNA of C. jejuni, with its low G+C content, was found to have no restriction sites for enzymes NotI and SfiI, which cut a high-G+C regions. Most of the restriction enzymes that were used resulted in DNA fragments that were either too numerous or too small for genome size determination, with the exception of the enzymes SalI (5' ... G decreases TCGAG ... 3'), SmaI (5' .... CCC decreases GGG .... 3'), and KpnI (5' ... GGTAC decreases C .... 3'). With SalI, six restriction fragments with average values of 48.5, 80, 110, 220, 280, and 980 kilobases (kb) were obtained when calibrated with both a lambda DNA ladder and yeast Saccharomyces cerevisiae chromosome markers. The sum of these fragments yielded an average genome size of 1.718 megabases (Mb). With SmaI, nine restriction fragments with average values ranging from 39 to 371 kb, which yielded an average genome size of 1.726 Mb were obtained. With KpnI, 11 restriction fragments with sizes ranging from 35 to 387.5 kb, which yielded an average genome size of 1.717 Mb were obtained. A SalI restriction map was derived by partial digestion of the C. jejuni DNA. The genome sizes of C. laridis, C. coli, and C. fetus were also determined with the contour-clamped homogeneous electric field system by SalI, SmaI, and KpnI digestion. Average genome sizes were found to be 1.714 Mb for C. coli, 1.267 Mb for C. fetus subsp. fetus, and 1.451 Mb for C. laridis.

Size and physical map of the chromosome of Haemophilus influenzae

A variation of pulse-field electrophoresis, field-inversion gel electrophoresis, was used to determine the size and physical map of the chromosome of Haemophilus influenzae. The DNA of H. influenzae had a low G + C content (39%) and no restriction sites for the enzymes NotI or SfiI. However, a number of restriction enzymes (SmaI, ApaI, NaeI, and SacII) that recognized 6-base-pair sequences containing only G and C nucleotides were found to generate a reasonable number of DNA fragments that were separable in agarose gels by field-inversion gel electrophoresis. The sizes of the DNA fragments were calibrated with a lambda DNA ladder and lambda DNA restriction fragments. The sum of fragment sizes obtained with restriction digests yielded a value for the chromosome of 1,980 kilobase pairs. Hybridization of a labeled fragment with two or more fragments from a digest with a different restriction enzyme provided the information needed to construct a circular map of the H. influenzae chromosome.

Transposon-mediated restriction mapping of the Bacillus subtilis chromosome

Analysis of chromosomal DNA depends upon a knowledge of the locations of restriction sites over several thousand kilobases (kb). However determination of even a subset of these sites can be time-consuming, and it can be difficult to link genetic and physical maps. We describe here a significant improvement which can be used in concert with genetically mapped chromosomal insertions. The circular chromosome of Bacillus subtilis 168 was physically examined on contour-clamped homogeneous electric field (CHEF) gels using the restriction enzyme NotI. Restriction mapping of the 4.7-megabase (Mb) DNA was accomplished using a novel technique involving the transposon Tn917, which linked the genetic and physical maps and also significantly increased the rate at which this was performed. The DNA of 54 strains which contained Tn917 at genetically determined locations was cleaved with NotI and used to determine the approximate positions of 31 restriction fragments with sizes between 45 kb and 290 kb, totalling 3589 kb. This information should greatly assist in the construction of a more detailed map using standard methodology.

A physical map of the human pseudoautosomal region.

A physical map of the human pseudoautosomal region has been constructed using pulsed field gel electrophoresis and the infrequently cutting restriction enzymes BssHIII, EagI, SstII, NotI, MluI and NruI. This map extends 2.3 Mbp from the telomere to sex-chromosome-specific DNA, includes at least seven CpG islands and locates four genetically mapped loci. Five of the CpG islands are organized into two clusters. One cluster is adjacent to the telomere, the other extends into sex-chromosome-specific DNA. There is congruence between the genetic and physical maps which implies that the frequency of recombination is approximately uniform throughout the DNA.

Plasmodium falciparum: Mapping genes to nine parasite chromosomes

Using pulsed field gel electrophoresis with pulse time of 120 sec, eight chromosomal DNA molecules from clone 7G8 of the Plasmodium falciparum Brazilian isolate IMTM22 were resolved. A ninth chromosomal molecule which did not enter the gel was identified at the slot by hybridization to two DNA probes and by restriction enzyme analysis. Thirteen parasite DNA sequences were mapped to the nine chromosomes, with at least one sequence mapped to each chromosome. The restriction enzyme NotI appeared to produce only one cut in the entire IMTM22 genome.