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PubMed1998

Arabidopsis YAC restriction mapping.

The approach of partial restriction mapping and vector hybridisation has been used to restriction map and align six yeast artificial chromosomes (YACs) corresponding to the top arm (approximately 27.9 centiMorgans, cM) of Arabidopsis chromosome 5 and confirm the chimeric nature of a further four clones which map to this region. The restriction endonucleases Sma1 and Sfi1 which recognise rare-medium frequency sites in the Arabidopsis genome were used. This work has restriction mapped a 315 kb region that includes a number of genes implicated in floral development, namely PISTILLATA and TOUSLED, and a number of uncharacterized genes involved in male gametogenesis (e.g., Ms1 and Ms37). The information generated can be used to transcriptionally map genes to this contig and will provide data for the isolation of several uncharacterized floral development genes which lie in this region. This approach has demonstrated how large tracts of YAC DNA can be mapped and aligned to show the presence/absence of chimeric YAC clones and provide detailed restriction knowledge for a large genomic region to help facilitate the positional cloning of genes.

Arabidopsis↗

PubMed1990

Characterization of preearly genes in the terminal repetition of bacteriophage BF23 DNA by nucleotide sequencing and restriction mapping.

A finer restriction map of the terminal repetition of bacteriophage BF23 DNA was determined and used to localize a 3.4-kbp deletion in the terminal repetition and to determine the physical location of preearly gene A2-A3. The nucleotide sequence of gene A2-A3 was determined and shown to code for a protein of 125 amino acids with no indication of a membrane transport sequence. The beginning of an adjacent gene, probably gene A1, was also sequenced.

Amino Acid Sequence↗

PubMed1981

Alignment of a restriction map with the genetic map of bacteriophage T4.

A restriction map of the bacteriophage T4 genome was aligned with the T4 genetic map. Included were the cleavage sites for BamHI, BglII, KpnI, PvuI, SalI, and XbaI. The alignment utilized the fact that the T4 genetic map had been oriented previously with respect to a T2/T4 heteroduplex map. DNA fragments from a BglII digestion of cytosine-containing DNA from a T4 dCTPase- denA denB(rIIH23B) alc mutant were hybridized with full-length chromosomal strands of bacteriophage T2, and the heteroduplexes were examined by electron microscopy. From their lengths and patterns of substitution and deletion loops, the heteroduplexes formed with 6 of the 13 BglII fragments could be unambiguously identified and positioned on the T2/T4 heteroduplex map. The ends of the T4 DNA strands in the heteroduplexes directly identified the location of 10 BglII cleavage sites. The remaining three BglII cleavage sites could be assigned to the T2/T4 heteroduplex map based on their relative locations on the restriction map. It was also possible to identify the source of the DNA strands (i.e., T2 or T4) in four previously unassigned deletion loops on the T2/T4 heteroduplex. Among the BglII fragments identified in heteroduplexes was the fragment containing the rIIH23B deletion; this deletion was used as the primary point of reference for alignment of the T4 restriction map with the T2/T4 heteroduplex map and, hence, with the T4 genetic map.

Chromosome Mapping↗

PubMed1983

The four ribosomal DNA units of the malaria parasite Plasmodium berghei. Identification, restriction map, and copy number analysis.

The four ribosomal RNA genes (rDNA units) of the rodent malaria parasite, Plasmodium berghei, were identified and mapped by restriction enzyme analysis and Southern blot hybridization of genomic DNA. Although the four genes share common characteristics, they appear to be internally different from each other in expanse and sequence. One HindIII site near the 3' end of the coding region for the large rRNA is the only restriction site which we have detected that is conserved in all of the genes. The distance between the conserved HindIII site and the coding region for the small rRNA is at least 1-2 kilobases longer in two of the rDNA units than in a third unit. None of the four rDNA units were linked by restriction mapping where about 150 kilobases of the genome were accounted for. The copy number of two of the rDNA units was determined to be approximately 1 per haploid genome by quantitative analysis of cloned (plasmid) DNA versus genomic DNA digests on Southern blots. The other two genes also appear to be present in 1 copy. Restriction analysis confirms both the low copy number and that these genes are not in an easily recognizable tandem array. The low number of rDNA units requires that new copies of the genome produced during intraerythrocytic growth be active in RNA synthesis soon after their replication.

Animals↗

PubMed1990

Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome.

This paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from the Escherichia coli chromosome on the restriction map established by Kohara et al. (1987). The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Kohara et al. (1987). Therefore, the sequenced genes available in the EMBL library may be localized on the E. coli chromosome restriction map. This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions. Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between the E. coli K12 strains used by the various laboratories involved in DNA sequencing. The program is versatile enough to be used with other genomes.

Chromosome Inversion↗

PubMed1995

Restriction map of a 35-kb HLA fragment constructed by nested deletion 'drop-out' mapping.

An efficient method for generating detailed restriction maps of large cloned DNA segments is demonstrated. The mapping strategy entails comparing restriction fragments from a parent clone and from nested deletion derivatives of that clone. In a set of deletion plasmids of decreasing size, an individual fragment will be lost, or 'drop-out', according to its position in the cloned fragment. In this demonstration, nested deletions were generated in both directions in a 35-kb DNA segment from the human leukocyte antigen (HLA) region by intramolecular transposition of an engineered gamma delta (Tn1000) element present in a special 'deletion factory' cloning vector [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 7874-7878]. Fifteen plasmids with deletions extending in one direction and eleven plasmids with deletions extending in the opposite direction were digested singly by each of four restriction enzymes. A total of 36 cleavage sites were mapped in the 35-kb HLA fragment. This drop-out approach using nested deletions provides a simple and efficient means of mapping restriction sites, genes and other features of interest in cosmid-sized cloned DNA segments or DNAs.

Animals↗

PubMed2006

An algorithm for assembly of ordered restriction maps from single DNA molecules.

The restriction mapping of a massive number of individual DNA molecules by optical mapping enables assembly of physical maps spanning mammalian and plant genomes; however, not through computational means permitting completely de novo assembly. Existing algorithms are not practical for genomes larger than lower eukaryotes due to their high time and space complexity. In many ways, sequence assembly parallels map assembly, so that the overlap-layout-consensus strategy, recently shown effective in assembling very large genomes in feasible time, sheds new light on solving map construction issues associated with single molecule substrates. Accordingly, we report an adaptation of this approach as the formal basis for de novo optical map assembly and demonstrate its computational feasibility for assembly of very large genomes. As such, we discuss assembly results for a series of genomes: human, plant, lower eukaryote and bacterial. Unlike sequence assembly, the optical map assembly problem is actually more complex because restriction maps from single molecules are constructed, manifesting errors stemming from: missing cuts, false cuts, and high variance of estimated fragment sizes; chimeric maps resulting from artifactually merged molecules; and true overlap scores that are "in the noise" or "slightly above the noise." We address these problems, fundamental to many single molecule measurements, by an effective error correction method using global overlap information to eliminate spurious overlaps and chimeric maps that are otherwise difficult to identify.

Algorithms↗

PubMed1994

A restriction mapping engine using constraint logic programming.

Restriction mapping generally requires the application of information from various digestions by restriction enzymes to find solution sets. We use both the predicate calculus and constraint solving capabilities of CLP(R) to develop an engine for restriction mapping. Many of the techniques employed by biologists to manually find solutions are supported by the engine in a consistent manner. We provide generalized pipeline and cross-multiply operators for combining sub-maps. Our approach encourages the building of maps iteratively. We show how other techniques can be readily incorporated.

Animals↗

PubMed1994

Computer-assisted restriction mapping: an integrated approach to handling experimental uncertainty.

Building a map of restriction sites from double-digest gel data can be a complex and frustrating task, especially when many DNA fragments are detected or when the gel results are ambiguous. 'Double Digester' is an interactive, graphical computer program which helps researchers understand and resolve such data. It explicitly represents the experimental data, the associated uncertainties, the researcher's hypotheses and possible map interpretations. Alternative solutions are frequently possible, and the differences between them may help determine which additional experiments might resolve ambiguities. Initial use has confirmed the benefits of this approach, and has suggested ways in which it can be refined and extended. Double Digester meets the need for a practical tool to help build restriction maps, and also illustrates how a computer-based tool can confront experimental uncertainty in an integrated fashion.

Algorithms↗

PubMed1992

Mitochondrial DNA of Schizophyllum commune: restriction map, genetic map, and mode of inheritance.

Mitochondrial DNA (mtDNA) found in the basidiomycete Schizophyllum commune (strain 4-40) is a circular molecule 49.75 kbp in length. A physical map containing 61 restriction sites revealed no repeat structures. Cloned genes from Neurospora crassa, Aspergillus nidulans, and Saccharomyces cerevisiae were used in Southern hybridizations to locate nine mitochondrial genes, including a possible pseudogene of ATPase 9, on the restriction map. A probe from a functional ATPase 9 gene identified homologous fragments only in the nuclear genome of S. commune. Restriction fragment length polymorphisms (RFLPs) between mtDNA isolated from different strains of S. commune were used to show that mitochondria do not migrate with nuclei during dikaryosis.

Adenosine Triphosphatases↗

PubMed1997

Restriction mapping of genes by capillary electrophoresis with laser-induced fluorescence detection.

Restriction mapping is one of the essential steps in gene analysis and molecular biology studies. Slab gel electrophoresis is the traditional way to separate DNA fragments for restriction mapping. However, slab gel electrophoresis does not provide sufficient resolution as required in many mapping applications, and the use of radioisotopes in traditional mapping methods creates health hazards. In the present study, capillary electrophoresis coupled with laser-induced fluorescence detection and a modified partial digestion mapping procedure was developed to map DNA fragments. By using capillary electrophoresis, a restriction map of genomic lambda phage clone of human interleukin 5 receptor alpha chain (IL5R alpha) gene was constructed. The IL5R alpha gene was analyzed to have five XbaI enzyme cutting sites at locations 1370, 2290, 2950, 5430, and 9330. The system was further characterized by using pBluescript SK(+) phagemid DNA as a model. Using a sequence-derived map as a reference, the pBluescript SK(+) restriction map constructed by capillary electrophoresis had an accuracy greater than 90%.

DNA↗

PubMed1991

An SfiI restriction map of the Bacillus subtilis 168 genome.

A restriction map of 24 SfiI (GGCCN4/NGGCC) restriction fragments has been constructed for the Bacillus subtilis genome. The combined sizes of the fragments indicate a genome size of approx. 4.2 Mb. The SfiI fragments range in size from 7-730 kb. Genetic markers have been located on 19 of the SfiI fragments, and 69 genetic markers have been assigned to the SfiI restriction map.

Bacillus subtilis↗

PubMed1990

Restriction map of Thiobacillus versutus plasmid pTAV1.

A restriction map of Thiobacillus versutus plasmid pTAV1 was constructed using EcoRI, BamHI and SalI restriction enzymes. Knowledge of the restriction map is an obligatory starting point for genetic and molecular studies of this, so far cryptic, plasmid.

Deoxyribonuclease BamHI↗

PubMed1998

An estimate of phylogenetic relationships among culicine mosquitoes using a restriction map of the rDNA cistron.

Restriction maps of the rDNA cistron of twelve species of mosquitoes in six genera of the subfamily Culicinae were constructed using eight 6 bp recognition restriction enzymes. Anopheles albimanus was used as an outgroup. The size of the rDNA cistron ranged from 8.5 kb in Aedes katherinensis to 12.9 kb in Ae. polynesiensis. A total of twenty-six sites were scored; eighteen were polymorphic among ingroup taxa. The proportion of polymorphic nucleotide sites (Pnuc) was 0.059 and the heterozygosity per nucleotide site (Hnuc) was 0.028. Wagner and Fitch Parsimony, Dollo Parsimony and Nei-Li distance/neighbour-joining methods were used to construct phylogenetic trees. The rDNA RFLP dataset did not provide a well-supported phylogeny among culicine taxa. The RFLP phylogenies are incongruent with the morphology character based and molecular phylogenies and derived relationships did not correspond with current taxonomic classifications. The lack of resolution was due to homoplasy arising from frequent independent loss or gain of restriction sites among unrelated taxa.

Animals↗

PubMed1994

Non-isotopic restriction mapping of cosmid DNA.

A protocol for the non-isotopic restriction mapping of cosmid DNA was developed. After digestion with lambda terminase and partial digestion with restriction enzymes, DNA fragments containing right or left cohesive cos termini were selectively captured by hybridization with biotinylated oligonucleotides, bound to magnetic beads coated with streptavidin and recovered by heating. Recovered DNA fragments containing cos ends were resolved by agarose gel electrophoresis, and fluorescence from the DNA fragments in the ethidium bromide-stained gel was detected with the fluorescent image analyzer, FMBIO. Restriction maps were directly determined from the ladder of the partial digestion products. Two micrograms of cosmid DNA for each partial digestion were sufficient for mapping the restriction enzyme sites. This procedure provides a prototype for other cos sequence-mediated or other specific sequence-mediated DNA isolation technologies and a convenient method for non-isotopic DNA analysis. The rapid physical analysis of cosmid DNA that we present here will contribute to large DNA structural analyses like the human genome project.

Base Sequence↗

PubMed1991

Alignment of Sfi I sites with the Not I restriction map of Schizosaccharomyces pombe genome.

A Sfi I restriction map of the fission yeast Schizosaccharomyces pombe genome was aligned with the Not I restriction map. There are 16 Sfi I sites in the S. pombe genome. Three Sfi I sites are on chromosome III which is devoid of Not I sites. The sizes of the entire genome and individual chromosomes, calculated from the Sfi I fragment sizes, are consistent with that calculated from the Not I fragment sizes. The Sfi I map provides greater physical characterization of the S. pombe genome and further validates the use of S. pombe chromosomal DNA as size standard. These maps have allowed detection of polymorphism on all three chromosomes.

Base Sequence↗

PubMed1997

A complete NotI restriction map covering the entire long arm of human chromosome 11.

BACKGROUND: Human chromosome 11 is one of the autosomes on which many disease genes have been mapped. Many different types of map, including a radiation hybrid map, a genetic map, and an STS-content YAC map, have been constructed for the chromosome. However, a physical map providing accurate physical distances has not yet been established. A chromosome-wide NotI restriction map was constructed to understand the overall feature of the genome organization and to facilitate the positional cloning of disease genes. RESULTS: A complete NotI restriction map of the entire long arm of human chromosome 11 was constructed using linking-clone mapping. This physical map covers 77.6Mb, from a pericentromeric NotI site to the terminus, and provides the most accurate ordering and distance estimation to date. We also mapped 138 sequence markers in the q13 region that have been poorly mapped previously. CONCLUSIONS: The restriction map of the entire long arm of human chromosome 11 is the longest restriction map of the human genome. This mapping has disclosed unique features regarding the organization of the chromosome, indicating that restriction sites of NotI, a CpG-recognition enzyme, are primarily distributed in R (or T) bands and that genetic distance is considerably longer in R (or T) bands than in G bands. The mapping, as well as the dense concentration of mapped markers within the q13 region, should help with positional cloning of the genes associated with various diseases.

Bacteriophage P1↗

PubMed1987

Correlation between restriction map, genetic map and catalytic functions in the gene complex URA2.

We replaced the URA2 gene by six different deleted alleles constructed in vitro by Bg/II digestion in order to correlate the genetic map with the restriction map and to define the regions coding for the different functions of the carbamylphosphate synthetase--aspartate transcarbamylase complex (CPSase-ATCase). We also enlarged the collection of ura2 point mutations by using a positive selection method based on resistance to the toxic accumulation of ureidosuccinic acid (USA). Of the new independent mutations nine mapped in the intermediary zone, a previously defined mutationless region localized between regions coding for CPSase and ATCase. This shows that the former definition resulted from analysis of a limited number of mutants (40). The study of an allele deleted in the intermediary zone shows that this sequence codes for a protein region necessary for the feedback inhibition of the CPSase-ATcase enzyme complex. The CPSase- ATCase- phenotype of 26 mutants resistant to USA accumulation shows the importance of the in vivo channelling of carbamylphosphate in the CPSase-ATCase complex for USA and subsequent pyrimidine biosynthesis. Finally, our results confirm that the CPSase and ATCase activities are separate functions.

Aspartate Carbamoyltransferase↗