Rheumatoid arthritis: from Rubens to restriction maps.
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Mapping of restriction enzyme cuts by a new two-dimensional procedure.
A new procedure has been worked out to establish restriction maps. The method is fast, does not in general require labeled DNA and has been applied to map the linear palindromic rDNA of Physarum with the restriction enzyme Bst EII.
DNA↗
In vitro construction of gshB::kan in Escherichia coli and use of gshB::kan in mapping the gshB locus.
The Escherichia coli structural gene for glutathione synthetase, gshB, was cloned into pBR322. Plasmids containing gshB were able to complement the glutathione requirement of a trxA gshB double mutant, and cells containing the plasmids were found to have elevated levels of glutathione synthetase. A mutant gshB allele was constructed by inserting the kan gene from pUC4K into a unique HpaI site located within gshB. The resulting plasmid-encoded allele was used to replace a genomic gshB+ by homologous recombination. The resulting strain had no detectable glutathione synthetase activity. The gshB allele containing the kan insertion was used to map gshB on the E. coli chromosome by P1 transduction. The results indicated that gshB is located at 63.4 min, between metK and speC. The allele was further localized to a region of 3,100 to 3,120 kilobase pairs on the physical map (restriction map) of E. coli by DNA-DNA hybridization to a series of lambda bacteriophages (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987).
Chromosome Mapping↗
Use of yeast artificial chromosome clones for mapping and walking within human chromosome segment 18q21.3.
Well-characterized large genomic clones obtained from yeast artificial chromosome (YAC) libraries provide the framework to localize genes and approach genetic disease. We developed universally applicable approaches to establish authenticity, localize and orient internal genes, map restriction sites, and rescue the distal ends of large human genomic DNA inserts. We selected human chromosome segment 18q21.3 as a model system. Molecular cloning of this segment was initiated by characterizing three plasminogen activator inhibitor type 2 (PAI-2) clones [290, 180, and 60 kilobases (kb)] isolated from a YAC library. Comparison of YAC and bacteriophage lambda genomic DNA clones confirmed the fidelity of the PAI-2 locus. Detailed rare cutting restriction maps were generated by ramped contour-clamped homogeneous electric field electrophoresis. The PAI-2 locus was located and oriented within the YACs, which span a distance 70 kb 5' to 220 kb 3' of PAI-2. Moreover, both left and right ends of the YAC genomic DNA inserts were rescued by amplifying circularized cloning sites with an inverted form of the polymerase chain reaction. These unique terminal genomic DNA fragments were used to rescreen the YAC library and isolate overlapping clones that extend the map. These approaches will enable neighboring loci to be definitively linked and establish the feasibility of using YAC technology to clone and map chromosomal segments.
Base Sequence↗
A novel tandem repeat sequence located on human chromosome 4p: isolation and characterization.
In an effort to analyze the genomic region of the distal half of human chromosome 4p, to where Huntington disease and other diseases have been mapped, we have isolated the cosmid clone (CRS447) that was likely to contain a region with specific repeat sequences. Clone CRS447 was subjected to detailed analysis, including chromosome mapping, restriction mapping, and DNA sequencing. Chromosome mapping by both a human-CHO hybrid cell panel and FISH revealed that CRS447 was predominantly located in the 4p15.1-15.3 region. CRS447 was shown to consist of tandem repeats of 4.7-kb units present on chromosome 4p. A single EcoRI unit was subcloned (pRS447), and the complete sequence was determined as 4752 nucleotides. When pRS447 was used as a probe, the number of copies of this repeat per haploid genome was estimated to be 50-70. Sequence analysis revealed that it contained two internal CA repeats and one putative ORF. Database search established that this sequence was unreported. However, two homologous STS markers were found in the database. We concluded that CRS447/pRS447 is a novel tandem repeat sequence that is mainly specific to human chromosome 4p.
Animals↗
Chromosomal assignment and imprinting tests for the mouse delta subunit of the cytosolic chaperonin containing TCP-1 (Cct4) gene to proximal chromosome 11.
The CCT (chaperonin containing TCP-1) complex functions as a molecular chaperone in the eukaryotic cytosol. This complex consists of several species of related polypeptides. The chromosomal localization of the mouse Cct4 gene encoding the delta subunit of CCT was assigned in this study to proximal chromosome 11 by genetic mapping. Restriction mapping analysis using YAC and pulse-field gel electrophoresis showed that Cct4 is located within a region about 300 kb from the imprinted gene U2af1-rs1. Expression of Cct4 was biallelic, and therefore Cct4 is not imprinted in neonatal mice. The localization of the human homologue of Cct4 on chromosome 2 corresponds well with the fact that homologues of other genes in the proximal region of mouse chromosome 11 also map to the region of conserved synteny in human chromosome 2.
Alleles↗
ISWAC: proposed system for the integrated assembly of chromosomes.
The generation of a physical map as an integral part of sequence project management is a problem that present computer systems do not address. Primarily, the analysis performed is based solely on the information available from a single knowledge level. Management systems that are currently available do not adequately model the multi-layer top down strategy that is most often utilized to manage large scale sequencing projects. Single layered approaches reflect an algorithmic inadequacy since interacting data sets are required to provide a good solution. The analysis tool that is currently under development termed ISWAC, the Integrated System for Wholistic Assembly of Chromosomes, overcomes these limitations by integrating information available from five layers of knowledge. These knowledge layers utilize information from the linkage map, physical map, restriction map, clone strategy map and the DNA sequence itself. The approach we are implementing, reviews current project status and continually refines the experimental strategy necessary to efficiently complete the sequencing task. To facilitate project completion the system is designed to interactively recommend strategies based on partial information. The utility of this tool is enhanced by implementing knowledge representation techniques that allow reasoning with approximate concepts characteristic of these data-sets. In addition, the raw physical data is maintained within an integrated map database to ease data verification. This paper presents the first discussion of the design specifications for a computer system to assimilate the various forms of data that are being generated as part of the human genome project. It was specifically written to stimulate discussion regarding data standardization, translation, analysis and most important, an understandable user-interphase for the molecular biologist. We would hope that interested readers would respond by assisting in the definition of a set of universal data standards and adopting them in their laboratories.
Algorithms↗
Partial endonuclease digestion mapping of restriction sites using PCR-amplified DNA.
Although direct DNA sequencing is now readily available, restriction enzyme analyses are still widely used in population genetics and molecular systematics studies. These analyses provide cheaper and faster ways to assay patterns of nucleotide differentiation across a large number of individuals. In this paper, we introduce a new approach to restriction enzyme analyses in which high-resolution restriction site maps are obtained from partial digestions of PCR products. This procedure increases the level of resolution at least an order of magnitude over the double-digestion method for restriction enzyme mapping, can target specific DNA regions with the use of specific primers, and, because it uses chemiluminescent detection of DNA, can be easily implemented in laboratories that lack the necessary setups to handle radioactive substances.
Animals↗
Automatic construction of restriction site maps.
A computer program is described which constructs maps of restriction endonuclease cleavage sites in DNA molecules, given only the fragment lengths. The program utilizes fragment length data from single and double restriction enzyme digests to generate maps for linear or circular molecules. The search for a map can be limited to the unknown (insert) region of a recombinant phage or plasmid. Typical restriction maps with four or five enzymes which cut at three to five unknown sites can be calculated in a few minutes.
Autoanalysis↗
Restriction analysis of the prototype strain of enteric adenovirus type 41 using exonuclease III.
Enteric adenoviruses 40 and 41 (Ad40 and Ad41) are a prominent cause of gastroenteritis in young children. Diagnosis of these enteric types by conventional means is complicated by their fastidious growth characteristics. Enteric adenovirus growth was enhanced by cocultivation. Typing of enteric isolates currently entails analysis of the extracted viral DNA with restriction enzymes. Restriction endonuclease fragments of the Ad41 strain Tak genome were ordered by (i) double digestion, (ii) release of restriction fragments from plasmids containing 84% of the Ad41 genome in EcoRI fragments A, B and C, (iii) hybridization of Southern blotted Ad41 fragments with EcoRI fragment containing plasmids and various segments of the Ad2 genome, (iv) sequential reduction of the genome beginning with terminal restriction fragments with exonuclease III and S1 nuclease. The termini of adenovirus genomes are difficult to clone and the use of exonuclease III is a useful alternative to conventional restriction mapping. DNA restriction patterns, fragment sizes and restriction maps of the Ad4 1 strain Tak with enzymes BamHI, BglII, ClaI, EcoRI, HindIII, PstI, SalI, SmaI and XhoI are presented. Prototype strain restriction maps should enable better understanding of adenovirus type 41 and its epidemiology.
Adenoviruses, Human↗
Application of long-distance PCR to restriction site mapping of a cloned DNA fragment on the lambda EMBL3 phage vector.
Long-distance PCR was applied to rapidly map the restriction sites of long inserts cloned on lambda EMBL3 phage vector. The restriction sites of 9 of 15 enzymes were completely assigned in a model experiment within 14 h, including 8h for the PCR amplification. This method was found particularly useful for genomic DNA cloning when the partial sequence of the corresponding cDNA is known.
Bacteriophage lambda↗
Restriction endonuclease mapping of the HI2 incompatibility group plasmid R478.
A restriction map of the 272-kb IncHI2 plasmid R478 was constructed by using the enzymes ApaI, XbaI, SalI, and XhoI. The map was derived from cloned restriction fragments from R478 inserted into cosmid and plasmid vectors as well as from double-digestion analysis of R478 and R478 miniplasmids. All previously known resistance determinants were cloned from R478, and their positions were located on the restriction map. A region involved in incompatibility was cloned and mapped. The location of a previously unreported arsenite resistance gene was also determined. The genes encoding tellurite resistance, colicin B resistance, and phage inhibition were found to be associated with a 6.7-kb SalI fragment of R478.
Cosmids↗
Conserved organization of the murine immunoglobulin epsilon gene region: restriction endonuclease maps and switch-region nucleotide sequence.
Various inbred mouse strains showed remarkable conservation of organization in the epsilon germline region as determined by restriction mapping. Restriction fragment length polymorphisms (RFLP) were seen in the epsilon gene region for only two of eight restriction enzymes tested. Furthermore, the RFLP did not correlate with the IgE-response phenotype for the murine strains SJL, SJA, C57BL/6, BALB/c, A/ST, and A/J. The IgE class-switch region (S epsilon) DNA from an SJL genomic clone was sequenced and was compared with S epsilon sequences from BALB/c mice. These S epsilon sequences were at least 95% homologous. Most of the S epsilon sequence differences observed between the two strains were single base pair substitutions, deletions, or insertions. The largest difference between the S epsilon sequences resulted from an insertion of seven contiguous bases seen in the SJL S epsilon region.
Animals↗
Methods for comparing sequence data such as restriction endonuclease maps or nucleotide sequences of viral nucleic acid molecules.
Maps of restriction endonuclease sites in the DNA genomes of orthopoxviruses were compared by computer classification methods. Data from such maps were recorded as qualitative binary information, and gave closely similar dendrograms when different dissimilarity measures and sorting strategies were used for the computations. A method for calculating dendrograms from small data sets, by 'hand', is described in detail to assist those who do not have access to suitable computer programs. The same calculation strategy gave useful classifications of partial nucleotide and amino acid sequences of orthomyxovirus haemagglutinins.
Animals↗
Polymorphisms and fluctuations in copy number of amplified esterase genes in Culex pipiens mosquitoes.
In Culex pipiens mosquitoes, A2 esterase alleles are co-amplified with B2 esterase alleles in response to selection with organophosphate insecticides. In this study the amplified A2 and B2 sequences were compared between twelve strains from four continents by restriction mapping. The restriction maps were almost identical in each strain throughout 22 kb surrounding the genes, suggesting that this represents a constant core sequence. A polymorphism was found in two strains collected from Egypt and Kenya in the mid 1980s. This polymorphism was present in all copies of the amplicon, which suggests that a mechanism of sequence homogenization was operating, i.e. concerted evolution. These two strains were almost certainly descendants from the same population and migration probably occurred along the River Nile. Although the maps were almost identical in each strain, dot blotting demonstrated that amplification levels differed by up to 13-fold between strains. Thus the presence of the A2-B2 haplotype cannot be used to indicate the level of amplification or any particular degree of resistance.
Animals↗
Restriction endonuclease mapping of linear unintegrated proviral DNA of bovine leukemia virus.
A detailed restriction map was deduced for the genome of the exogenous bovine leukemia virus. The cleavage sites for nine restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with bovine leukemia virus was isolated. The linear viral DNA had a unique restriction map, indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-enriched probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. Restriction enzyme analysis of integrated bovine leukemia virus information present in animals with enzootic bovine leukosis provided evidence for the existence of genetic variants of the virus.
Chromosome Mapping↗
Genome organization and restriction site map of the mitochondrial DNA of the housefly (Musca domestica).
A restriction map of the housefly mitochondrial DNA was constructed. The putative arrangement of the major coding regions was determined by two methods. In the first, Drosophila clones of known gene content were hybridized to housefly restriction fragments on Southern blots. In the second, rare restriction sites and clusters of restriction sites were used to align the housefly restriction map with that of Drosophila yakuba. Both methods produced the same apparent gene arrangement.
Animals↗
Baboon endogenous virus genome. I. Restriction enzyme map of the unintegrated DNA genome of a primate retrovirus.
A detailed restriction map was deduced for the genome of an endogenous retrovirus of a higher primate, that of baboon. The cleavage sites for 12 restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with baboon endogenous virus was isolated. Hybridization with a strong-stop complementary DNA probe demonstrated presence of a terminal repetition in the linear viral DNA. The positions of restriction sites for two particular enzymes, SmaI and XhoI, near each end were consistent with this result and indicated that the length of the repetition is 0.55 +/- 0.01 kilobase. The linear viral DNA had a unique restriction map indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-specific probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. We observed a minor heterogeneity in a BamHI recognition site 1.95 kilobases from the right end of the linear map.
Animals↗