Showing papers on "Restriction map published in 1999"
TL;DR: A blunt-end linker, named SNX for its restriction sites, that allows the use of combinations of restriction enzymes to digest the majority of genomic DNA into the 200-1000-bp range, resulting in representative small-insert libraries with a very high proportion of positive clones.
Abstract: Microsatellite loci are highly informative genetic markers useful for population genetic studies, linkage mapping and parentage determination. Methods to identify novel microsatellite loci commonly use subtractive hybridization to enrich small-insert genomic libraries for repeat sequences. A critical step in enrichment is attachment of an oligonucleotide linker to genomic DNA fragments so that repeat-containing sequences can be recovered by PCR for cloning. Current linkers and ligation methods rely on single restriction enzymes to size-fraction genomic DNA and generate complementary ends. These restriction enzyme/linker combinations are often species-specific, give poor recovery of repeat-enriched DNA and yield library inserts that are not a broad sample of the genome. We have developed a blunt-end linker, named SNX for its restriction sites, that allows the use of combinations of restriction enzymes to digest the majority of genomic DNA into the 200-1000-bp range. SNX is attached to genomic DNA with a simultaneous ligation/restriction reaction that is highly efficient and improves recovery of sequences after subtractive hybridization. SNX can be used for microsatellite enrichment in any species, since ligation is independent of the restriction enzymes used to size-fraction genomic DNA. These methods improve current repeat-enrichment strategies, resulting in representative small-insert libraries with a very high proportion of positive clones.
812 citations
TL;DR: Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species.
Abstract: The Bacillus anthracis Sterne plasmid pXO1 was sequenced by random, "shotgun" cloning. A circular sequence of 181,654 bp was generated. One hundred forty-three open reading frames (ORFs) were predicted using GeneMark and GeneMark.hmm, comprising only 61% (110,817 bp) of the pXO1 DNA sequence. The overall guanine-plus-cytosine content of the plasmid is 32.5%. The most recognizable feature of the plasmid is a "pathogenicity island," defined by a 44.8-kb region that is bordered by inverted IS1627 elements at each end. This region contains the three toxin genes (cya, lef, and pagA), regulatory elements controlling the toxin genes, three germination response genes, and 19 additional ORFs. Nearly 70% of the ORFs on pXO1 do not have significant similarity to sequences available in open databases. Absent from the pXO1 sequence are homologs to genes that are typically required to drive theta replication and to maintain stability of large plasmids in Bacillus spp. Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species. Among the remaining ORFs, there are three sequences that may encode enzymes responsible for the synthesis of a polysaccharide capsule usually associated with serotype-specific virulent streptococci.
381 citations
TL;DR: The entire KIR/ILT region revealed a considerable degree of genetic polymorphism, and it is suggested the designation ``Leukocyte Receptor Cluster'' (LRC) for this chromosomal segment.
Abstract: A large number of cDNAs coding for killer cell inhibitory receptors (KIR) and immunoglobulin-like transcripts (ILT) have already been described, and some of the respective genes are known to map in 19q13.4. To understand the genetic relationships of these transcripts, some of which may be alleles from polymorphic loci, it is necessary to determine the genomic organization of the region. To do so, we performed long-range restriction enzyme mapping of the 19q13.4 region along with YAC and PAC contig construction. Eighteen genes could be assigned to a chromosomal segment of about 600 kb. Twelve KIR loci are contained within ∼200 kb, bordered by the locus for the Fc receptor for IgA (FCAR) at the telomeric side and by a 150-kb cluster containing ILT loci at the centromeric side. A further region with a maximal size of 135 kb containing at least one ILT gene was identified further centromeric, separated by ∼50 kb from the ILT region near the KIR cluster. The entire KIR/ILT region revealed a considerable degree of genetic polymorphism as shown, for example, by different restriction maps of two sets of PACs spanning the same region. We suggest the designation ``Leukocyte Receptor Cluster'' (LRC) for this chromosomal segment.
234 citations
TL;DR: Together, these data suggest that the chromosomal rearrangements most likely remove one or more cis-regulatory elements from an extended SOX9 control region.
Abstract: 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.
217 citations
TL;DR: It is concluded that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi.
Abstract: Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization of EcoRI-digested T. rubrum genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates of T. rubrum. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonuclease MvaI produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as T. rubrum-T. soudanense and T. quinkeanum-T. schoenlenii could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi.
210 citations
TL;DR: A whole-genome restriction map of Deinococcus radiodurans, a radiation-resistant bacterium able to survive up to 15,000 grays of ionizing radiation, was constructed without using DNA libraries, the polymerase chain reaction, or electrophoresis.
Abstract: A whole-genome restriction map of Deinococcus radiodurans, a radiation-resistant bacterium able to survive up to 15,000 grays of ionizing radiation, was constructed without using DNA libraries, the polymerase chain reaction, or electrophoresis. Very large, randomly sheared, genomic DNA fragments were used to construct maps from individual DNA molecules that were assembled into two circular overlapping maps (2.6 and 0.415 megabases), without gaps. A third smaller chromosome (176 kilobases) was identified and characterized. Aberrant nonlinear DNA structures that may define chromosome structure and organization, as well as intermediates in DNA repair, were directly visualized by optical mapping techniques after γ irradiation.
201 citations
TL;DR: In this article, short defined DNA segments are produced using specially designed primers to amplify a cDNA or genomic DNA template, which contain a recognition site for a restriction endonuclease.
Abstract: Genotyping can be accomplished by analysis of short, defined DNA segments using electrospray ionization mass spectrometry. The DNA segments are produced using specially designed primers to amplify a cDNA or genomic DNA template. The primers contain a recognition site for a restriction endonuclease. The amplification products are digested with the restriction endonuclease. Single nucleotide polymorphisms can be detected rapidly and reliably.
174 citations
TL;DR: Physical mapping has been rediscovered as an important component of large-scale sequencing projects and can be constructed from both large-insert clones and genomic DNA, and used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing.
158 citations
TL;DR: Results indicate that alleles with duplicated or multiduplicated genes have occurred through unequal crossover at a specific breakpoint in the 3'-flanking region of the CYP2D6*2B allele with a specific repetitive sequence.
153 citations
TL;DR: DNA blotting and restriction analysis suggest that the BACs include most members of the immediate tryptase family, and chromosome 16p13.3 harbors a cluster of known and previously undescribedMembers of the tryptases gene family.
151 citations
TL;DR: The chapter provides a detailed description of the method used to perform READS analysis on control and activated Jurkat T cells, which relies on specific amplification of only the 3'-end restriction fragments under stringent PCR conditions.
Abstract: Publisher Summary This chapter discusses a gel-based method for analysis of differential gene expression called READS 2 or restriction endonucleolytic analysis of differentially expressed sequences. This method uses stringent polymerase chain reaction (PCR) conditions to amplify only the extreme Y-end fragment of a restriction endonuclease-digested cDNA molecule without employing methods for physical separation of the fragments before or after PCR amplification. A comprehensive analysis of differentially expressed mRNAs between two physiological states can be done by comparing the gel patterns of their 3'-end cDNA restriction fragments. One of the important features of READS is the reproducibility of gel patterns in repeat experiments. Because the method relies on specific amplification of only the 3'-end restriction fragments under stringent PCR conditions, reproducibility of gel patterns can be expected because the set of cDNA molecules synthesized from a particular total RNA by a given anchored primer will always be the same and will, therefore, always generate the same pattern of 3'-end restriction fragments. The chapter provides a detailed description of the method used to perform READS analysis on control and activated Jurkat T cells.
TL;DR: Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of FokI (a type IIS restriction endonuclease) is fused to other DNA-binding motifs and one such chimeric nuclease, Zif-QQR-FN was shown to find and cleave its target in vivo.
Abstract: Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of Fokl (a type IIS restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely the helix-turn-helix motif, the zinc finger motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by microinjection of DNA substrates and the enzyme into frog oocytes (Carroll et al., 1999). The injected enzyme made site-specific double-strand breaks in the targets even after assembly of the DNA into chromatin. In addition, this cleavage activated the target molecules for efficient homologous recombination. Since the recognition specificity of zinc fingers can be manipulated experimentally, chimeric nucleases could be engineered so as to target a specific site within a genome. The availability of such engineered chimeric restriction enzymes should make it feasible to do genome engineering, also commonly referred to as gene therapy.
TL;DR: The Malaria Genome Consortium was formed to sequence and annotate the entire 24.6-Mb genome of the unicellular parasite Plasmodium falciparum.
Abstract: The unicellular parasite Plasmodium falciparum is the cause of human malaria, resulting in 1.7–2.5 million deaths each year1. To develop new means to treat or prevent malaria, the Malaria Genome Consortium was formed to sequence and annotate the entire 24.6-Mb genome2. The plan, already underway, is to sequence libraries created from chromosomal DNA separated by pulsed-field gel electrophoresis (PFGE). The AT-rich genome of P. falciparum presents problems in terms of reliable library construction and the relative paucity of dense physical markers or extensive genetic resources. To deal with these problems, we reasoned that a high-resolution, ordered restriction map covering the entire genome could serve as a scaffold for the alignment and verification of sequence contigs developed by members of the consortium. Thus optical mapping was advanced to use simply extracted, unfractionated genomic DNA as its principal substrate. Ordered restriction maps (BamHI and NheI) derived from single molecules were assembled into 14 deep contigs corresponding to the molecular karyotype determined by PFGE (ref. 3).
TL;DR: The presence of DprA homologs in bacteria known not to be naturally competent suggests a broad function in DNA processing, and while dprA is required for high-frequency transformation, transformation also may occur independently of DPRA.
Abstract: Helicobacter pylori, a microaerophilic, gram-negative bacterium that colonizes the human stomach, has been recognized as the major causative agent of chronic gastritis and as playing a role in peptic ulcer disease and gastric cancer (8). H. pylori strains have a high degree of diversity at the genetic level (2, 19, 34), including a high rate of point mutations in conserved genes such as ureB (28) and flaA (45), mosaicism within genes such as vacA (5), and the presence of nonconserved DNA fragments, in particular, the cag pathogenicity island (2, 10, 13, 50). Other factors that lead to further diversity among H. pylori strains are the presence of insertion sequences (10, 23) or plasmids (31), variation in gene order (25), and the complement of putative restriction endonucleases (2, 48).
The genetic diversity of H. pylori has clinical significance, since markers for strains with enhanced virulence have been identified (5, 13, 39, 50). Additionally, techniques such as restriction fragment length polymorphism and randomly amplified polymorphic DNA PCR have been used to exploit this heterogeneity for epidemiologic purpose (2, 3, 41, 51).
Horizontal DNA transfer within the reservoir for H. pylori, i.e., the stomach of primates, would contribute to the development of genetic diversity (33). There is substantial evidence that recombination among H. pylori strains has been an important feature of their evolution (5, 23, 46). Natural transformation in bacteria is a complex process involving DNA binding, uptake/translocation, and recombination. Many H. pylori strains are known to be naturally competent for transformation in vitro (32, 37, 43, 49, 55). However, the mechanisms for transformation of DNA have not been closely studied for H. pylori. Thus far, only recA (44, 47) and the comB locus (22) have been identified as having a role in H. pylori transformation. Recognition of the mechanisms involved in genetic exchange may help us to understand the adaptation of H. pylori to changing environments and could shed light on clinically important issues of virulence and development of antibiotic resistance (7, 21, 52). The complete genomic sequence of H. pylori 26695 (48) revealed a 810-bp open reading frame (ORF) (HP0333) encoding a deduced protein of 270 amino acids (30,470-Da molecular mass) with homology to DprA (encoded by dprA) of Haemophilus influenzae. In H. influenzae, dprA is required for transformation by chromosomal DNA (29). We therefore sought to determine whether HP0333 is required for natural transformation in H. pylori.
TL;DR: Positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzyme's ability to select cleavage sites randomly throughout the DNA molecule, thus, positive super coiling does not prevent DNA translocation.
Abstract: Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non-specific cleavage site. We have examined several potential blocks to DNA translocation, such as positive supercoiling or a Holliday junction, for their ability to trigger DNA cleavage by type I restriction enzymes. Introduction of positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzyme's ability to select cleavage sites randomly throughout the DNA molecule. Thus, positive supercoiling does not prevent DNA translocation. EcoR124II endonuclease cleaved DNA at Holliday junctions present on both linear and negatively supercoiled substrates. The latter substrate was cleaved by a single enzyme molecule at two sites, one on either side of the junction, consistent with a bi-directional translocation model. Linear DNA molecules with two recognition sites for endonucleases from different type I families were cut between the sites when both enzymes were added simultaneously but not when a single enzyme was added. We propose that type I restriction enzymes can track along a DNA substrate irrespective of its topology and cleave DNA at any barrier that is able to halt the translocation process.
TL;DR: In this article, the LTR-retrotransposon MAGGY was found sporadically in isolates of Pyricularia grisea (Magnaporthegrisea), and the MAGGY-related sequence was a degenerate form of MAGGY.
Abstract: The LTR-retrotransposon MAGGY is found sporadically in isolates of Pyricularia grisea (Magnaporthe grisea). Based on a dendrogram constructed by RFLP analysis of rDNA, isolates that carry MAGGY elements were classified into a single cluster that comprised four rDNA types. However, in a few members of this cluster, exemplified by isolates from common millet (Panicum miliaceum), the MAGGY element has distinct features. Southern analysis suggested that these isolates possessed a single copy of a MAGGY-related sequence whose restriction map differed from that of MAGGY itself. Sequence analysis revealed that the MAGGY-related sequence was a degenerate form of MAGGY, characterized by numerous C:G to T:A transitions, which have often been reported to result from RIP (Repeat-induced point mutation) or RIP-like processes. However, the favored target site for C:G to T:A transitions in this fungus, determined by examining a total of 501 sites, was (A/T)pCp(A/T), which differs from that for the RIP process originally reported in Neurospora (CpA), and from that reported in Aspergillus (CpG). The fact that certain members of the cluster of MAGGY carriers retain a single copy of a degenerate MAGGY element implies that the ancestor of these isolates successfully “captured” the invading MAGGY element.
TL;DR: A single 2.45 Mbp long continuum of genomic DNA cloned as 45 p1 artificial chromosomes, three bacterialificial chromosomes, and 34 cosmid clones is constructed to provide a bacterial clone resource that will enable further studies of genomic structure, transcriptional regulation, function and evolution of the epidermal differentiation complex.
TL;DR: It is shown that ClpXP-dependent regulation of the endonuclease activity enables bacteria that acquire unmodified chromosomal target sequences to survive and supports a model in which the HsdR component of a type I restriction endonUClease becomes a substrate for proteolysis after the end onuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage.
Abstract: ClpXP-dependent proteolysis has been implicated in the delayed detection of restriction activity after the acquisition of the genes (hsdR, hsdM, and hsdS) that specify EcoKI and EcoAI, representatives of two families of type I restriction and modification (R-M) systems. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an hsd+ bacterium as the result of replication errors or recombination-dependent repair. We show that ClpXP-dependent regulation of the endonuclease activity enables bacteria that acquire unmodified chromosomal target sequences to survive. In such bacteria, HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP. A mutation that blocks only the modification activity of EcoKI, leaving the cell with ≈600 unmodified targets, is not lethal provided that ClpXP is present. Our data support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage.
TL;DR: Endonucleases that require two copies of an 8-bp sequence may be widespread in nature, where, despite this seemingly inappropriate requirement, they may function in DNA restriction.
TL;DR: This chapter presents the theoretical analyses of the restriction-enzyme digestion experiments and the relationship among the kinetic analyses of restriction- enzyme cleavage and equilibrium and cooperative binding studies, and discusses important issues and controls for experimental studies using restriction enzymes as probes of nucleosome stability and dynamics.
Abstract: Publisher Summary Restriction enzymes have several attributes that make them particularly valuable reagents for studies of nucleosome stability and dynamics. Routine nucleosome mapping experiments often use restriction enzymes as probes, taking advantage of the inaccessibility of nucleosomal target sites. Sites that are digested readily are present in linker regions among nucleosomes or in larger nucleosome-free regions, whereas sites that cannot be cleaved are possibly present in nucleosomes. This chapter presents the theoretical analyses of the restriction-enzyme digestion experiments and the relationship among the kinetic analyses of restriction-enzyme cleavage and equilibrium and cooperative binding studies. It also discusses important issues and controls for experimental studies using restriction enzymes as probes of nucleosome stability and dynamics.
TL;DR: All the analyses indicate the existence of two different populations with a very limited gene flow across the Strait of Gibraltar, and no haplotype was shared between Mediterranean and Atlantic areas.
Abstract: We used mitochondrial DNA (mtDNA) restriction analysis to study genetic variation in 98 striped dolphins (Stenella coeruleoalba) stranded on coasts from different European countries and from animals caught by fisheries. A total of 63 different restriction sites was mapped after digestion of mtDNA with 15 restriction endonucleases that yielded a total of 27 haplotypes. No haplotype was shared between Mediterranean and Atlantic areas. All the analyses indicate the existence of two different populations with a very limited gene flow across the Strait of Gibraltar.
Proceedings Article•
06 Aug 1999TL;DR: In this article, a set of restriction maps are constructed from the images of individual genomic (unchmed) DNA molecules digested by restriction enzymes, and the resulting softwart, is a key component of our physical mapping automation tools and has been used to create complete maps of various microorganisms.
Abstract: In this paper, we describe our algorithmic approacl, to constructing an aligmnent of (contiging) a set of restriction maps created from the images of individual genomic (unchmed) DNA molecules digested by resl.riction enzymes. Generally, these DNA segments are sized in the range of 1 4Mb. The goal is to devise contiging algorithms capable of producing high-quality composite maps rapidly and in a scalable maturer. The resulting softwart, is a key component of our physical mapping automation tools and ha.s been used t.o create complete maps of various microorganisms (E. coll. P. falciparum and D. radiodurans). Experimental l’eSll]tS match known seqHent’e data.
TL;DR: The chromosomal arrangement of these genes appears to reflect the evolution of this multigene family and supports the theory that it arose by gene duplication.
Abstract: . A physical map of the CXC chemokine locus on chromosome 4 has been constructed by PCR analysis and PFGE mapping of YAC clones. The genes for IL8, GRO1, PPBP, PF4, SCYB5 (ENA-78)
TL;DR: Infection of Escherichia coli containing the type I restriction enzyme EcoKI by bacteriophage T7 0.3 mutants leads to restriction during the late stages of genome entry and during DNA replication, showing that the normal T7 DNA translocation process delays restriction.
Abstract: Infection of Escherichia coli containing the type I restriction enzyme EcoKI by bacteriophage T7 0.3 mutants leads to restriction during the late stages of genome entry and during DNA replication. Patterns of cleavage in vivo suggest that some cutting occurs near the midpoint of two recognition sites, consistent with the idea that EcoKI translocates DNA bidirectionally through itself and cuts when two enzyme molecules collide. Rapid ejection of a 0.3+ T7 genome from a bacteriophage λ particle results in degradation of the infecting DNA by EcoKI, showing that the normal T7 DNA translocation process delays restriction. A unique recognition site inserted at the genomic left end allows EcoKI to function as a molecular motor and to translocate the remaining 39 kilobases of T7 DNA into the cell.
TL;DR: The data show that development of amoxicillin resistance amongSalmonella, especially in serovar Typhimurium, results from both gene transfers and strain dissemination.
Abstract: A survey conducted between 1987 and 1994 at the University Hospital of Besancon, France, demonstrated a dramatic increase (from 0 to 42.5%) in the prevalence of amoxicillin resistance among Salmonella spp. Of the 96 resistant isolates collected during this period (including 77 Typhimurium), 54 were found to produce TEM-1 β-lactamase, 40 produced PSE-1 (equivalent to CARB-2), one produced PSE-1 plus TEM-2, and one produced OXA-1 in isoelectric focusing and DNA hybridization experiments. Plasmids coding for these β-lactamases were further characterized by (i) profile analysis, (ii) restriction fragmentation pattern analysis, (iii) hybridization with an spvCD-orfE virulence probe, and (iv) replicon typing. In addition, isolates of S. typhimurium were genotypically compared by pulsed-field gel electrophoresis of XbaI-macrorestricted chromosomal DNA. Altogether, these methods showed that 40 of the 41 PSE-1 producers were actually the progeny of a single epidemic S. typhimurium strain lysotype DT104. Isolates of that strain were found to harbor RepFIC virulence plasmids with somewhat different restriction profiles, but which all carried the blaPSE-1 gene. Of these virulence/resistance plasmids, 15 were transmissible to Escherichia coli. TEM-1-producing S. typhimurium displayed much greater genotypic and plasmidic diversities, suggesting the acquisition of the blaTEM-1 gene from multiple bacterial sources by individual strains. In agreement with this, 32 of the 35 S. typhimurium plasmids encoding TEM-1 were found to be conjugative. These data show that development of amoxicillin resistance among Salmonella, especially in serovar Typhimurium, results from both gene transfers and strain dissemination.
TL;DR: Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis strains and to a strain of B. alesti, and replication was found to be dependent on host-gene-encoded DNA polymerase I.
Abstract: A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.
TL;DR: It is proposed that the large A. vulgare mtDNA is produced by the tripling of a 14-kb monomer with a singular rearrangement: one monomer is linear and the other two form a circular dimer, which suggests that replication occurs during the monomer phase.
Abstract: The mitochondrial DNA (mtDNA) in animals is generally a circular molecule of approximately 15 kb, but there are many exceptions such as linear molecules and larger ones. RFLP studies indicated that the mtDNA in the terrestrial isopod Armadillidium vulgare varied from 20 to 42 kb. This variation depended on the restriction enzyme used, and on the restriction profile generated by a given enzyme. The DNA fragments had characteristic electrophoretic behaviors. Digestions with two endonucleases always generated fewer fragments than expected; denaturation of restriction profiles reduced the size of two bands by half; densitometry indicated that a number of small fragments were present in stoichiometry, which has approximately twice the expected concentration. Finally, hybridization to a 550-bp 16S rDNA probe often revealed two copies of this gene. These results cannot be due to the genetic rearrangements generally invoked to explain large mtDNA. We propose that the large A. vulgare mtDNA is produced by the tripling of a 14-kb monomer with a singular rearrangement: one monomer is linear and the other two form a circular dimer. Densitometry suggested that these two molecular structures were present in different proportions within a single individual. The absence of mutations within the dimers also suggests that replication occurs during the monomer phase.
TL;DR: A novel polymerase chain reaction/restriction digestion assay to screen for this polymorphism was developed and proved after comparison with a previously used method to be advantageous.
Abstract: Sporadic, iatrogenic and new variant forms of Creutzfeldt-Jakob disease are associated with a predisposition for disease depending on a homozygosity at amino acid residue 129 of the prion protein gene (PRNP). A novel polymerase chain reaction/restriction digestion assay to screen for this polymorphism was developed and proved after comparison with a previously used method to be advantageous. Furthermore, for prevention of incorrect results an internal control for the restriction digestion was constructed. The feasibility of this method was tested in a cohort of 300 healthy Caucasian subjects. Of this normal population, 48.7% were heterozygous at codon 129, 43% homozygous for methionine and 8.3% for valine.
TL;DR: The PCR-RFLP technique has provided us with a simple procedure which can be used to characterize DCV isolates and allowed easy assignment of isolates to one of the five viral genotypes identified in this study.
TL;DR: Cosedimentation assays performed with domains expressed as glutathione S-transferase fusion proteins demonstrate an actin-binding activity mediated specifically by the kelch repeat domain of IPP, a novel human gene that predicts a kel ch family protein homologous to the mouse Ipp gene.