Rare-cutting Enzymes Research Articles

Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing

Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.

IDENTIFICATION OF CONSERVED FEATURES OF LAGLIDADG HOMING ENDONUCLEASES

LAGLIDADG family of homing endonucleases are rare-cutting enzymes which recognize long target sequences and are of great interest in genome engineering. Despite advances in homing endonuclease engineering, effective methods of broadening the range of cleaved sequences are still lacking. Here, we present a study of conserved structural features of LAGLIDADG homing endonucleases that might aid further development of such methods. The protein-DNA interface of LAGLIDADG homing endonucleases differs considerably with the particular nuclease, and the analysis of conserved protein-DNA interactions could not identify any residues crucial for DNA binding and common to most nucleases of the family. For the homing endonuclease PI-SceI, a comparison of structural and experimental data derived from literature helped to identify 23 residues that are likely to be important for DNA binding. Analysis of the LAGLIDADG domain dimerization interface allowed the choosing of six positions that contribute to dimerization specificity most, while comparison of 446 sequences of LAGLIDADG endonucleases revealed groups of residues in these positions that appear to be most favorable for dimerization.

New transposons to generate GFP protein fusions in Candida albicans

Transposon elements are important tools for gene function analysis, for example they can be used to easily create genome-wide collections of insertion mutants. Transposons may also carry sequences coding for an epitope or fluorescent marker useful for protein expression and localization analysis. We have developed three new Tn5-based transposons that incorporate a GFP (green fluorescent protein) coding sequence to generate fusion proteins in the important fungal pathogen Candida albicans. Each transposon also contains the URA3 and Kan R genes for yeast and bacterial selection, respectively. After in vitro transposition, the insertional allele is transferred to the chromosomal locus by homologous recombination. Transposons Tn5-CaGFP and Tn5-CaGFP-URA3::FLIP can generate C-terminal truncated GFP fusions. A URA3 flipper recycling cassette was incorporated into the transposon Tn5-CaGFP-URA3::FLIP. After the induction of Flip recombinase to excise the marker, the heterozygous strain is transformed again in order to obtain a GFP-tagged homozygous strains. In the Tn5-CaGFP-FL transposon the markers are flanked by a rare-cutting enzyme. After in vitro transposition into a plasmid-borne target gene, the markers are eliminated by restriction digestion and religation, resulting in a construct coding for full-length GFP-fusion proteins. This transposon can generate plasmid libraries of GFP insertions in proteins where N- or C-terminal tagging may alter localization. We tested our transposon system by mutagenizing the essential septin CDC3 gene. The results indicate that the Cdc3 C-terminal extension is important for correct septin filament assembly. The transposons described here provide a new system to obtain global gene expression and protein localization data in C. albicans.

ENGINEERING HOMING ENDONUCLEASES TO MODIFY COMPLEX GENOMES

Gene targeting to selected chromosomal loci is greatly stimulated when free DNA ends are created that initiate double-strand break repair. Gene therapy reagents can be developed by engineering DNA endonucleases that cleave genomes at desired target sequences. Homing endonucleases are naturally occurring rare-cutting enzymes that have well understood DNA binding and DNA cleavage properties. Rational design methods as well as directed evolution strategies that involve genetic selections and screens using combinatorial libraries generate homing endonucleases with altered sequence specificities. Molecular switches are being introduced into these enzymes to regulate their activity. This article reviews the progress that has been made in constructing homing endonucleases for gene therapy and genome engineering, and discusses the challenges that remain.

Genetic diversity of Xanthomonas axonopodis pv. citri based on plasmid profile and pulsed field gel electrophoresis

Xanthomonas axonopodis pv. citri strains that cause disease in citrus were investigated by pulsed field and plasmid profile analysis. For the first method, genomic DNA was digested by the rare-cutting enzymes Xba I and Vsp I. The strains evaluated were collected in seven different States of Brazil and in Argentina, Bolivia, Paraguay and Uruguay. Genetic variability was found among strains of X. axonopodis pv. citri from different geographical areas Argentina, Bolivia and Uruguay, with similarities varying from 0.62 to 0.83. However, the strains collected in Brazil, despite being from different States, have shown a genetic similarity ranging from 0.83 to 1.00. Cluster analysis showed a relationship between genomic similarity and geographical origin of the strains. Plasmids were observed in all strains, with a total of five different plasmids, with sizes between 57.7 and 83.0 kilobases. The 72.6 kb plasmid was the most frequent, present in 15 out of 22 strains, while the 68.1 kb plasmid was observed in two strains only. Although the plasmid diversity detected in the present study was not very great, the X. axonopodis pv. citri strains evaluated showed a considerable degree of diversity with regard to this extrachromosomal genetic element.

Polyphasic study of the genetic diversity of lactobacilli associated with 'Almagro' eggplants spontaneous fermentation, based on combined numerical analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoresis patterns

The goal of this study was to assess the genetic diversity of lactic acid bacteria (LAB) from the complex natural ecosystem present in the spontaneous fermentation of 'Almagro' eggplants by a polyphasic approach based on molecular techniques. Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) were applied to 149 Lactobacillus isolates obtained from that fermentation process. Two random primers, OPL-05 and ArgDei-For, and two rare-cutting enzymes, SfiI and SmaI, chosen after preliminary testing on the basis of band intensity and distribution, were used. RAPD and PFGE generated electrophoretic patterns suitable for strain discrimination, but further discrimination was achieved when combined numerical analysis of the results from both methods and the results previously obtained by SDS-PAGE whole cell protein analysis, was carried out. The findings indicated a considerable degree of genomic diversity in the LAB microbiota studied and especially in the Lactobacillus plantarum isolates. In terms of species assignment, the polyphasic study allowed a definite and well-founded identification of 98.7% of the isolates. The combined numerical analysis of RAPD and PFGE patterns represented a useful tool to discriminate the diversity of the Lactobacillus strains responsible for the spontaneous fermentation of this pickle. The species identification and strain typing results from the polyphasic study were regarded as the most exact compromise yielding the fewest contradictions based on the available data. Combined numerical analysis of RAPD-PCR and PFGE patterns has not yet been employed to study the genetic diversity of LAB from an ecosystem like that found in fermenting vegetables.

Recombining overlapping BACs into a single larger BAC

BackgroundBAC clones containing entire mammalian genes including all the transcribed region and long range controlling elements are very useful for functional analysis. Sequenced BACs are available for most of the human and mouse genomes and in many cases these contain intact genes. However, large genes often span more than one BAC, and single BACs covering the entire region of interest are not available. Here we describe a system for linking two or more overlapping BACs into a single clone by homologous recombination.ResultsThe method was used to link a 61-kb insert carrying the final 5 exons of the human CFTR gene onto a 160-kb BAC carrying the first 22 exons. Two rounds of homologous recombination were carried out in the EL350 strain of bacteria which can be induced for the Red genes. In the first round, the inserts of the two overlapping BACs were subcloned into modified BAC vectors using homologous recombination. In the second round, the BAC to be added was linearised with the very rare-cutting enzyme I-PpoI and electroporated into recombination efficient EL350 bacteria carrying the other BAC. Recombined BACs were identified by antibiotic selection and PCR screening and 10% of clones contained the correctly recombined 220-kb BAC.ConclusionThe system can be used to link the inserts from any overlapping BAC or PAC clones. The original orientation of the inserts is not important and desired regions of the inserts can be selected. The size limit for the fragments recombined may be larger than the 61 kb used here and multiple BACs in a contig could be combined by alternating use of the two pBACLink vectors. This system should be of use to many investigators wishing to carry out functional analysis on large mammalian genes which are not available in single BAC clones.

Prevalence of Listeria monocytogenes in Broilers at the Abattoir, Processing Plant, and Retail Level

The environment and products from two broiler abattoirs and processing plants and raw broiler pieces at the retail level were sampled for Listeria monocytogenes in order to evaluate the contamination level of the broiler carcasses and products. Sampling started in the slaughtering process and finished with raw broiler meat or ready-to-eat cooked product. Sampling sites positive for L. monocytogenes at the broiler abattoir were the air chiller, the skin-removing machine, and the conveyor belt leading to the packaging area. The L. monocytogenes contamination rate varied from 1 to 19% between the two plants studied. Furthermore, 62% (38 of 61) of the raw broiler pieces, bought from retail stores, were positive for L. monocytogenes. Altogether, 136 L. monocytogenes isolates were obtained for serotyping and pulsed-field gel electrophoresis(PFGE) characterization performed with two rare-cutting enzymes (ApaI and AscI). Altogether three serotypes (1/2a, 1/2c, and 4b) and 14 different PFGE types were obtained using information provided from both ApaI and AscI patterns for discrimination basis. The two broiler abattoirs studied did not share the same PFGE types. However, the same PFGE types found in the raw broiler pieces at the retail level were also found in the broiler abattoirs where the broilers had been slaughtered.

A gene-enriched BAC library for cloning large allele-specific fragments from maize: isolation of a 240-kb contig of the bronze region.

A generic bacterial artificial chromosome (BAC) library from a complex plant genome like maize may not be suitable for some types of genomic analysis, for example, for establishing correlations between the genetic and the physical organization of a given chromosome region. Previously, we carried out extensive genetic analysis of the bronze (Bz) region in Zea mays using a W22 inbred line carrying the Bz-McC allele; however, BAC libraries of that line are neither available nor under construction. Here, we report the isolation of large, adjacent BAC clones of this region from a partial BAC library of W22. We developed a BAC vector suitable for cloning NotI fragments and used it to clone size-fractionated genomic DNA that had been cut to completion with the methylation-sensitive, rare-cutting enzyme NotI. This strategy resulted in a very significant enrichment of large genic DNA. From a library of about 20,000 BACs, containing just two-thirds of a maize genome, we isolated 16 BAC clones of the 110-kb distal Bz fragment and 10 BAC clones of the 130-kb proximal Bz fragment. This recovery means that our strategy resulted in a 15- to 24-fold enrichment of specific sequences. The order of the BAC clones in the 240-kb contig, predetermined from an internal NotI site in the Bz-McC allele was confirmed by hybridization with sequences from sites previously mapped proximal and distal to Bz and by sequencing. To show the general utility of our approach and the value of our partial BAC library, we also isolated BAC clones of other sequences, such as tub4 and the complex R-r allele, contained in the same size fraction of DNA. This is the first report of the use of a BAC vector to clone allele-specific large DNA fragments from a plant with a large genome, circumventing the need to construct a complete BAC library.

Campomelic Dysplasia Translocation Breakpoints Are Scattered over 1 Mb Proximal to SOX9: Evidence for an Extended Control Region

Campomelic dysplasia (CD), a skeletal malformation syndrome with or without XY sex reversal, is usually caused by mutations within the SOX9 gene on distal 17q. Several CD translocation and inversion cases have been described with breakpoints outside the coding region, mapping to locations >130 kb proximal to SOX9. Such cases are generally less severely affected than cases with SOX9 coding-region mutations, as is borne out by three new translocation cases that we present. We have cloned the region extending 1.2 Mb upstream of the SOX9 gene in overlapping bacterial-artificial-chromosome and P1-artificial-chromosome clones and have established a restriction map with rare-cutter enzymes. With sequence-tagged-site-content mapping in somatic-cell hybrids, as well as with FISH, we have precisely mapped the breakpoints of the three new and of three previously described CD cases. The six CD breakpoints map to an interval that is 140-950 kb proximal to the SOX9 gene. With exon trapping, we could isolate five potential exons from the YAC 946E12 that spans the region, four of which could be placed in the contig in the vicinity of the breakpoints. They show the same transcriptional orientation, but only two have an open reading frame (ORF). We failed to detect expression of these fragments in several human and mouse cDNA libraries, as well as on northern blots. Genomic sequence totaling 1,063 kb from the SOX9 5'-flanking region was determined and was analyzed by the gene-prediction program GENSCAN and by a search of dbEST and other databases. No genes or transcripts could be identified. Together, these data suggest that the chromosomal rearrangements most likely remove one or more cis-regulatory elements from an extended SOX9 control region.

Genetic and Physical Analyses of the Centromeric and Pericentromeric Regions of Human Chromosome 5: Recombination across 5cen

Human centromeres are poorly understood at both the genetic and the physical level. In this paper, we have been able to distinguish the alphoid centromeric sequences of chromosome 5 from those of chromosome 19. This result was obtained by pulsed-field gel electrophoresis after cutting genomic DNA with restriction endonucleasesNcoI (chromosome 5) andBamHI (chromosome 19). We could thus define a highly polymorphic marker, representing length variations of the D5Z1 domain located at the q arm boundary of the chromosome 5 centromere. The centromeric region of chromosome 5 was then analyzed in full detail. We established an approximately 4.6-Mb physical map of the whole region with five rare-cutting enzymes by using nonchimeric YACs, two of which were shown to contain the very ends of 5cen on both sides. The p-arm side of 5cen was shown to contain an alphoid subset (D5Z12) different from those described thus far. Two genes and several putative cDNAs could be precisely located close to the centromere. Several L1 elements were shown to be present within alpha satellites at the boundary between alphoid and nonalphoid sequences on both sides of 5cen. They were used to define STSs that could serve as physical anchor points at the junction of 5cen with the p and q arms. Some STSs were placed on a radiation hybrid map. One was polymorphic and could therefore be used as a second centromeric genetic marker at the p arm boundary of 5cen. We could thus estimate recombination rates within and around the centromeric region of chromosome 5. Recombination is highly reduced within 5cen, with zero recombinants in 58 meioses being detected between the two markers located at the two extremities of the centromere. In its immediate vicinity, 5cen indeed exerts a direct negative effect on meiotic recombination within the proximal chromosomal DNA. This effect is, however, less important than expected and is polarized, as different rates are observed on both arms if one compares the 0 cM/Mb of the p proximal first 5.5 Mb and the 0.64 cM/Mb of the q proximal first 5 Mb to the sex-average 1.02 cM/Mb found throughout the entire chromosome 5. Rates then become close to the average when one goes further within the arms. Finally, most recombinants (21/22), irrespective of the arm, are of female origin, thus showing that recombination around 5cen is essentially occurring in the female lineage.

Characterization of Listeria monocytogenes from an ice cream plant by serotyping and pulsed-field gel electrophoresis

One dominating strain of serotype 1/2b was found when serotyping and pulsed-field gel electrophoresis (PFGE) patterns were used for the characterization of 41 Listeria monocytogenes isolates originating from an ice cream plant. Samples were taken from the production environment, equipment and ice cream during the years 1990–1997. Serotyping divided the isolates into two serovars, 1/2b and 4b. Three rare-cutting enzymes ( ApaI, AscI and SmaI) were used in the creation of PFGE patterns. AscI resulted in the best restriction enzyme digestion patterns (REDPs) for visual comparison. Eight different AscI REDPs were obtained, whereas ApaI produced six and SmaI seven banding patterns. When one-band differences are taken into account, 12 different PFGE types were distinguished based on information obtained with all three enzymes. The dominant PFGE type was found to have persisted in the ice cream plant for seven years. Improved and precisely targeted cleaning and disinfection practices combined with structural changes making for easier cleaning of the packaging machine, resulted in eradication of L. monocytogenes from this plant.

A 6p22 reference map of leukocyte DNA: exclusion of rearrangement in four cases of atypical haemochromatosis.

We describe a 4 Mb reference map of the haemochromatosis gene region in leukocyte DNA from seven controls and four atypical haemochromatosis patients. Three patients had normal coding sequence for HFE, the candidate gene for genetic haemochromatosis (GH). The fourth patient had classical GH but was heterozygous for Cys282Tyr with otherwise normal coding sequence. The genomic DNA was mapped by pulsed-field gel electrophoresis (PFGE) using five rare-cutting enzymes. Seventeen probes including HFE were positioned on the map. Despite proximity to the highly polymorphic major histocompatibility complex (MHC), no polymorphism was observed in the control group with these telomeric probes. Furthermore, major rearrangement of the HFE region was excluded as a mutation contributing to iron overload in these atypical patients. Maps of cloned DNA are linked through genes and other probes to this reference map of the HFE region in uncloned genomic DNA.

Establishing the Cryptosporidium parvum karyotype by NotI and SfiI restriction analysis and Southern hybridization.

The molecular karyotype of the coccidian parasite Cryptosporidium parvum has proven difficult to study because chromosomes of similar sizes migrate together when submitted to pulsed-field gel electrophoresis (PFGE). In the present work, the karyotype was studied by restriction of chromosome-sized DNA with the rare-cutting enzymes NotI and SfiI, followed by PFGE separation of the restriction fragments and Southern hybridization. These experiments showed that the C. parvum karyotype is formed by eight chromosomes, ranging in size from approximately 0.95 to 1.45 million base pairs (Mbp), accounting for a genome size of 9.6Mbp. As a first step towards the construction of a physical map of the C. parvum genome, a total of 20 probes, including 16 genes and the ribosomal DNA (rDNA) sequence, was mapped to intact chromosomes and to their restriction fragments. In this way, all chromosomes, but one, were identified by specific markers. A comparison of mapping data of homologous genes from different species belonging to the phylum Apicomplexa showed differences in the distribution of rDNA sequences and in the chromosomal localization of alpha- and beta-tubulin genes. The variation in genome size among these parasites is also discussed.

Homing endonucleases: keeping the house in order.

Homing endonucleases are rare-cutting enzymes encoded by introns and inteins. They have striking structural and functional properties that distinguish them from restriction enzymes. Nomenclature conventions analogous to those for restriction enzymes have been developed for the homing endonucleases. Recent progress in understanding the structure and function of the four families of homing enzymes is reviewed. Of particular interest are the first reported structures of homing endonucleases of the LAGLIDADG family. The exploitation of the homing enzymes in genome analysis and recombination research is also summarized. Finally, the evolution of homing endonucleases is considered, both at the structure-function level and in terms of their persistence in widely divergent biological systems.

High frequency transposition of the Tn5 derivative Tn 5493 in Streptomyces lividans

The mini-Tn5 derivative transposon Tn5493 transposes at a frequency as high as 3% in the Gram-positive bacterium Streptomyces lividans. The use of a thiostrepton-resistance gene and a temperature-sensitive delivery system carrying the transposase gene allows an easy selection for stable transposition events. Insertions into the S. lividans genome seem to be fairly random, as shown by Southern blot and pulsed-field gel electrophoresis (PFGE). Transpositions were easily mapped by PFGE using the restriction sites of rare-cutting enzymes present in the transposon. Because of its characteristics, Tn5493 is a promising tool for genetic and molecular analysis and manipulation of the antibiotic-producing streptomycetes.

Targeted integration of foreign genes into repetitive sequences of theBrevibacterium lactofermentumchromosome

Directed integration of heterologous DNA into the insertion sequence IS13869 of Brevibacterium lactofermentum has been achieved by using integrative vectors that contain a 600 bp fragment from IS13869 and a kanamycin resistance cartridge with unique PacI, SwaI and PmeI sites. The incorporation of these restriction sites for rare-cutting enzymes, which recognize octanucleotide sequences, allowed rapid mapping of the location of plasmid insertion into the chromosome. Mapping by pulsed-field gel electrophoresis and hybridization with an IS13869 probe showed four types of transformants. In three of these types, integration of the vector occurred at each of three different sites in the chromosome. These vectors have the advantage of increasing the number of integration events due to the presence of multiple targets in the genome.

Targeted integration of foreign genes into repetitive sequences of the Brevibacterium lactofermentum chromosome

Directed integration of heterologous DNA into the insertion sequence IS13869 of Brevibacterium lactofermentum has been achieved by using integrative vectors that contain a 600 bp fragment from IS13869 and a kanamycin resistance cartridge with unique PacI, SwaI and PmeI sites. The incorporation of these restriction sites for rare-cutting enzymes, which recognize octanucleotide sequences, allowed rapid mapping of the location of plasmid insertion into the chromosome. Mapping by pulsed-field gel electrophoresis and hybridization with an IS13869 probe showed four types of transformants. In three of these types, integration of the vector occurred at each of three different sites in the chromosome. These vectors have the advantage of increasing the number of integration events due to the presence of multiple targets in the genome.

Characterization of Lactobacillus sake strains associating with production of ropy slime by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) patterns

Randomly amplified polymorphic DNA (RAPD) patterns and pulsed-field gel electrophoresis (PFGE) patterns were used for the characterization of ropy slime-producing Lactobacillus sake strains. The two most revealing commercially available primers (OPJ 12 and OPJ 16, Operon Inc. Alameda, USA) and two rare-cutting enzymes ( AvrII and SmaI) were chosen from a pretested lot for the typing of 69 ropy slime-producing strains, 7 non-ropy isolates and 4 non-ropy reference strains. Both RAPD and PFGE patterns confirmed the group division established in previous studies and provided new information concerning ropy slime-producing strains. PFGE patterns were found to have the greatest discriminatory power, revealing the genetic variation of the main group of ropy slime-producing L. sake strains and distinguishing all non-ropy strains from slime-producers.

1.8-megabases fine physical map encompassing IFNAR and AML1 loci on human chromosome 21q22.1.

A long-range restriction map of the 1.8-megabases (mb) region encompassing the area between the interferon-alpha receptor and the acute myelogenous leukemia loci on human chromosome 21q22.1 was constructed after analysis of both the contiguous yeast artificial chromosome (YAC) clones and genomic DNA. Analysis of pulsed-field gel electrophoresis of lymphoblastoid DNA digested with three rare-cutting enzymes, Not I, Mlu I, and Nru I, revealed the positions of 17 markers on each restriction map. The 1.8-mb YAC contig that covers this region was obtained through YAC walking mediated by sequence-tagged sites (STSs), with 29 STSs including 12 newly generated YAC end-specific STSs. The consensus restriction map from 15 overlapping YACs and the positioning of the STS markers on each clone allowed 24 markers including 4 Not I-linking STSs to be ordered and mapped physically. Comparison of the maps revealed that the proximal region contains more unmethylated CpG islands than the distal region, which suggests that many expressed genes are in the proximal region. This fine consensus physical map will be informative and useful for construction of contigs of cosmid, P1, or BAC clones for further large-scale sequencing in this gene-rich region.