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MOTIVATION: A formulation of a new problem of the restriction map construction based on a simplified digestion experiment and a development of an algorithm for solving both ideal and noisy data cases of the introduced problem. RESULTS: A simplified partial digest problem and a branch and cut algorithm for finding the solution of the problem.
A simple method for the ordering of fragments and the determination of relative positions of restriction sites when constructing pair restriction maps of linear and circular DNAs is described. The major advantages of the suggested approach as compared to the routine strategy of exhaustive search are as follows: (1) One does not need to begin the construction of the map by searching for the restrictases that produce the least number of fragments; (2) the method allows one to construct quite complicated maps, the main limiting parameter being the number of fragments during double digestion (no more than 10-12); and (3) construction of the map using this approach is less labor- and time-consuming than the routine strategy.
Several Staphylococcus aureus strains were lysogenized by the phages of serological group B (phages phi 53, phi 85) as well as by some of serological group F (phages phi 77, phi 84) and macrorestriction fragment patterns of genomic DNA were estimated in the lysogenized, non-lysogenic and delysogenized (cured of prophages) strains. It was shown that the integration of phage DNA into chromosome of S. aureus leads to specific changes in restriction fragment pattern in all the lysogenized strains. These changes correlate well with the SmaI restriction map of S. aureus NCTC 8325 since they concern the restriction fragments defined in this map. Phages phi 53 and phi 85 integrate into SmaI fragment B. On the other hand, phages phi 77 and phi 84 integrate into SmaI fragment E of the S. aureus restriction map. The prophages of strain NCTC 8511 have their integration sites, as follows: the phage designated by us phi M integrates in fragment A, whereas the integration site for phage phi J lies in fragment E. Phage phi M was estimated to be genetically related to phages of serological group A and phage phi J to those of serological group F. Evidence was given that lysogenization of S. aureus strains by at least four prophages does not cast any doubt upon the estimation of their genetic relatedness based on their similarity in restriction pattern.
In the present study, molecular cloning, sequencing and restriction mapping of the genomic sequence encoding human proacrosin is described. The full-length cDNA encoding human proacrosin was utilized to recover a 17-kb human genomic clone which was sequenced without further subcloning. The nucleotide sequences of the exons agree with the sequence of the cDNA reported previously. More than 500 bases of the promoter region were sequenced and found to be highly GC rich but devoid of an identifiable TATA box. These findings are generally consistent with a recently published report [Keime, S., Adham, I. M. & Engel, W. (1990) Eur. J. Biochem. 190, 195-200]. However, further sequence analysis revealed discrepancies between our clone and that previously reported. Sequencing of the first intron showed similarity with the published data for 54 bases of the 5' region, beginning with the donor splice site, and for 114 bases at the 3' end. However, 500 bases sequenced distal to the initial 54 bases at the 5' end of intron 1 showed no similarity with the published sequence. In addition, the boundaries of intron 3 differed such that a cytosine residue previously reported to be in exon 3 was found to be the first base of exon 4. Detailed studies were undertaken to confirm that our clone constitutes the authentic sequence of human proacrosin. Cloning and characterization of the human proacrosin gene may allow for informative studies of its regulation, and for a more detailed examination of its role in fertilization.
A series of plaque-forming lambda h80 transducing phages carrying various portions of the malA region were isolated. A 5,800-base pair HindIII-EcoRI DNA fragment from one of these phages was cloned into pBR322 and shown to contain malT, which is the positive regulator gene of the maltose regulon, and most of malP, the structural gene for maltodextrin phosphorylase. A restriction map of the HindIII-EcoRI fragment was established, and it was correlated with the genetic map of the malA region (i) by mapping deletions which had been generated in vitro on the plasmid and (ii) by locating on the restriction map a DNA insertion of known genetic position. A 600-base pair HincII-HaeII segment was shown to contain all or part of the promoters for malT and malP, which are known to be transcribed in opposite directions. Strains carrying gene malT on a plasmid synthesized a 94,000-dalton polypeptide which was not produced by identical strains carrying similar plasmids in which malT was partially deleted. Estimates of the size of the malT gene support the conclusion that the 94,000-dalton polypeptide is the malT product.
Restriction site mapping of the genomes of eight different isolates of adenovirus serotype 7 (Ad7) has been performed with six different restriction endonucleases. In this analysis, 37 different restriction sites were localized. Three distinctly different cleavage patterns of the genomes of the Ad7 strains were observed. These strains could not be distinguished by serological techniques. The following three subtypes were defined on the basis of their restriction site patterns: the Ad7 prototype, represented by strain Gomen originally isolated from a case of pharyngitis; subtype Ad7a, represented by the Ad7 vaccine strain and strains isolated from undifferentiated respiratory disease and from a healthy carrier; and a third subtype of Ad7, represented by three strains which were isolated from Swedish patients, all having pronounced clinical symptoms indicating severe systemic infection. A comparison of the restriction site maps of the protype of Ad3 and the three subtypes of Ad7 indicated greater differences in the position of restriction sites between strains of Ad7 than between strains of the two serotypes. This technique is consequently recommended to obtain a more precise definition of distinct entities of viruses.
A restriction map of the 61 kb virulence plasmid of Salmonella enteritidis was constructed and compared with the Salmonella typhimurium virulence plasmid map. Polymerase chain reaction was used to detect a region homologous to rck gene in the S. enteritidis plasmid which was localized in the map by hybridization. Regions homologous to traT and vagD/vagC loci were not detected in this plasmid by this method.
Restriction maps of cervid herpesviruses 1 and 2 which are antigenetically related to bovine herpesvirus 1, were deduced from Southern blot hybridization with HindIII restriction fragments of BHV-1 DNA as probes.
An HPL program is described which constructs and draws circular restriction maps given a set of cleavage sites, together with deletion of insertion data if required. This program is of great use in the routine interpretation of restriction patterns for the analysis of recombinant DNA molecules.
Five cosmids containing inserts that comprise the complete genome of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata were mapped with four restriction enzymes (Bg/II, ClaI, SstI, XhoI). From these cosmid maps, composite maps of the complete genome were constructed for each restriction enzyme. A region containing repeats of the sequence GGC downstream of the polyhedrin gene was used to probe the genome. It cross-hybridized with a region which, upon sequence analysis, was found to be a highly repetitive GC-rich region of nearly 500 nucleotides. The two GC-rich regions appeared to be evolutionarily unrelated.
A restriction map of the genomic DNA of Lactobacillus casei phage PL-1 was constructed using the restriction endonucleases BamHI, EcoRI, HindIII, KpnI, NruI and XhoI. The PL-1 genome was 42.2 kb in size and had complementary cohesive ends forming a ring-like monomer. The cohesive ends, analysed with exonuclease III and S1 nuclease, were 3'-terminated single strands protruding from both ends. The nucleotide sequence of the cohesive ends, determined by the dideoxynucleotide method, comprised four A + T and 10 G + C pairs: 5' GAGGCCGACCGTTC 3'/3' CTCCGGCTGGCAAG 5'. Thus, the cohesive ends of PL-1 DNA were higher in G + C content than those of other known bacteriophage DNAs.
In the present study, 60 avian Chlamydia psittaci isolates were characterized using restriction fragment length polymorphism as well as serovar-specific monoclonal antibodies, enabling a comparison between the two characterization methods. Sixty avian C. psittaci isolates were characterized by Alul restriction mapping of the major outer membrane protein gene omp1 obtained after amplification by the polymerase chain reaction. The 60 avian C. psittaci strains were also characterized using serovar-specific monoclonal antibodies in a microimmunofluorescence test. Digestion of 60 avian C. psittaci omp1 amplicons by Alul generated 5 of the 6 known distinct restriction patterns (A, B, D, E and F). Restriction pattern C was not observed. Serotyping revealed 4 avian C. psittaci serovars (A, B, C and D). None of the 60 isolates was typed as serovar E. AluI restriction patterns A, B, D and E corresponded in 98% of the cases to serovars A, B, C and D, respectively. One isolate, classified as serovar A, generated restriction pattern F instead of A. Genotyping enabled a more precise differentiation of avian C. psittaci serovar A strains. Serovar A strains were divided into two groups according to their Alul restriction pattern (A or F). For epidemiological studies, genotyping can thus be a highly valuable alternative to serotyping, especially when applied directly to the clinical samples.
The Xp11.2 region o the human X chromosome contains genes involved in a number of inherited diseased, with at least one locus that escapes X chromosome inactivation, as well as abnormal methylation polymorphism. We isolated a series of yeast artificial chromosome (YAC) clones by hybridization screening with DNA probes localized within this region and assembled them into a 4.3 Mb contig spanning from Xp11.21 to Xp11.23 by a combination of Alu-PCR fingerprinting, STS-PCR and DNA probe cross hybridization. On the basis of these overlapping YAC clones we have constructed the long-range restriction map of this interval and placed exactly some DNA markers. Four CpG-dense regions between ARAF1 and OATL2 were identified based on the long-range restriction mapping, which indicated the distribution of genes within this interval. It should assist in the future nucleic acid sequence analysis and novel gene identification in this region.
A restriction endonuclease cleavage map of bacteriophage T3 DNA has been constructed. The enzymes used and, within parentheses, the number of their cleavage sites on T3 DNA are: HindIII (1), XbaI (1), BglII (1), KpnI (2), MboI (9), and HpaI (17). The size and the relative location of each fragment have been established, defining an accurate physical map of T3 DNA.
Chromosomal DNA restriction fragments carrying the nitrogen fixation (nif) and his genes of Klebsiella pneumoniae were identified in hybridization experiments using a plasmid derived from pRD1 as a radioactive probe. Restriction mapping of 26 genetically characterized chromosomal nif deletions provided a map showing the physical location of nif genes along the chromosome.
Molecular genetic research on the Mediterranean fruit fly, Ceratitis capitata, will provide tools to permit determination of source populations for new pest infestations. Restriction fragment length polymorphism (RFLP) of mitochondrial DNA provides some interpopulation discrimination. A restriction map, including the informative variable EcoRV and XbaI restriction sites, is constructed for the Mediterranean fruit fly, and several restriction sites are associated with specific gene regions based on polymerase chain reaction-RFLP and sequence analyses. A partial sequence of the mitochondrial 16S ribosomal RNA gene is reported.
A restriction map has been constructed for Anastrepha suspensa mitochondrial DNA. One HaeIII site was found to be polymorphic among individuals in highly inbred colonies and a feral population. Based on mapping information, the polymorphic site was determined to be in the ATPase 6 gene. Primers TK-J-3804 and C3-N-5460 amplified this region. The amplicon was cut by HaeIII in flies of one haplotype and not cut in flies of the other haplotype. From 30 to 43% of the individual flies studied had this additional HaeIlI site. After cloning of the approximately 5200 bp XbaI fragment, the two mitotypes were identified. A 988 base fragment, coding for the entire tRNA-Lys(AAG), tRNA-Asp(GAC), and ATPase 8genes, and a partial ATPase 6gene was sequenced Four silent mutations, including the one at the informative site were located. The HaeIII polymorphism and other sequence differences may prove useful as a diagnostic for identification of the origin of introduced fruit flies.
The capsule plasmid pTE702 of Bacillus anthracis has been physically mapped with the restriction endonucleases HindIII, PstI, BamHI, SalI, and XhoI. A HindIII fragment map of pTE702 (96.5 kb) was obtained by analysis of the recombinant plasmids and cosmids containing overlapping fragments partially digested with HindIII. The physical map for PstI, BamHI, SalI, and XhoI was obtained by double digestion mapping of these sites in relation to the HindIII sites. The replication region of pTE702 was determined by in vitro genetic replicon labeling in B. subtilis.