Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Restriction endonuclease (RE) in situ digestion (REISD) of human metaphase chromosomes and interphase nuclei may uncover cryptic polymorphisms. This technique can be applied to identify the individual origin of cells and thus analyze the hemopoietic chimerism that eventually results in leukemic patients after allogeneic bone marrow transplantation (BMT). In the current study, results of REISD with different REs are shown. In particular, the use of Sau 3A reveals a polymorphism for constitutive heterochromatin of chromosome 3 and may differentiate BMT donor (D) and recipient (R) cells. Once pre-BMT characterization shows a different Sau 3A digestion pattern of D and R cells, it is possible to monitor the development of hematopoietic cell populations in the R bone marrow after BMT. A panel of 24 patients who underwent BMT and their Ds were analyzed. The method presented here allowed cells from D and R to be distinguished, and therefore to quantify the post-BMT hemopoletic chimerism, in 6 (25%) of the cases. This quantitative and sex-independent genetic approach to the study of hemopoietic chimerism has already shown itself to be useful in patients with leukemia who require a BMT, but could also be extended to other transplant situations.
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PMID:Restriction endonuclease in situ digestion (REISD): a novel quantitative sex-independent method to analyze chimerism after bone marrow transplantation. 886 45

Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources. The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites. A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P. fluorescens LP6a. This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns. In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene. Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes. Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis. Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.
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PMID:Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens. 898 93

Mutations in the type VII collagen gene (COL7A1) have recently been established as the molecular basis of the inherited blistering skin disorder, dystrophic epidermolysis bullosa. We report a novel combination of COL7A1 mutations in a Japanese patient with an autosomal recessive form of dystrophic epidermolysis bullosa. Clinically, the patient had suffered from generalized trauma-induced blistering since the first week of life loss of most finger- and toenails, esophageal stenosis and partial fusion of the fingers and toes. Immunofluorescence microscopy of the dermal-epidermal junction in the patient's skin revealed reduced intensity of staining with an anti-type VII collagen antibody. Transmission electron microscopy showed only a few thin, poorly formed anchoring fibrils. The patient was a compound heterozygote for a nonsense mutation on one COL7A1 allele and a donor splice site mutation on the other allele. The mutations were identified by PCR amplification of genomic DNA, heteroduplex analysis, and nucleotide sequencing, and verified by restriction endonuclease digestion. Reverse transcriptase-PCR and sequencing of cDNA from the patient's cultured keratinocyte mRNA showed evidence of aberrant splicing resulting from the donor splice site mutation, due to activation of a cryptic intronic splice site that leads to a frameshift and a downstream premature termination codon. Knowledge of the genetic lesions in this patient is helpful in elucidating the molecular consequences of COL7A1 mutations in dystrophic epidermolysis bullosa and in providing information about the fundamental mechanisms involved in maintaining adhesion between the epidermis and the dermis.
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PMID:Compound heterozygosity for a nonsense mutation and a splice site mutation in the type VII collagen gene (COL7A1) in recessive dystrophic epidermolysis bullosa. 904 57

The incidence of Neisseria gonorrhoeae with reduced susceptibility to quinolones increased from 0.18% (63 of 3285) in 1992 to 0.56% (15 of 2663) in 1993 and 0.62% (46 of 2846) in 1994. In all, 65 of the 67 isolates of Neisseria gonorrhoeae with decreased susceptibility to quinolones were characterised by pulsed-field gel electrophoresis (PFGE), auxotyping, serotyping and plasmid content. The strains were distributed among 14 auxotype/serovar (A/S) classes. Thirty isolates (46.2%) which were penicillin-susceptible with ciprofloxacin MIC90 of 0.12 mg/L and norfloxacin MIC90 of 1.0 mg/L belonged to a single A/S class, OUHL/IA-2. All but two of the 30 isolates had identical PFGE restriction profiles with NheI restriction endonuclease. Fifteen isolates (23.1%) with MICs in the intermediate (or resistant) categories for penicillin and with ciprofloxacin and norfloxacin MIC90 of 0.25 and 4.0 mg/L and (0.5 and 4.0 mg/L) respectively, belonged to A/S class P/IB-1. The 15 isolates showed nine different patterns with NheI and eight patterns with SpeI restriction endonucleases. Two of three beta-lactamase-producing (PPNG) isolates belonged to A/S class P/IB-5 and had a dissimilar PFGE restriction profile with NheI endonuclease; the other isolate belonged to A/S class P/IB-8. The remaining 17 isolates were distributed among 11 A/S classes. Three isolates within the common A/S class NR/IB-1 were subdivided into two types by PFGE as were three isolates belonging to A/S class NR/IB-2. Overall the 65 isolates of N. gonorrhoeae were distributed into 30 NheI and 26 SpeI macrorestriction profiles. All but one isolate harboured the 2.6-MDa cryptic plasmid and 18 isolates carried the 24.5-MDa transferable plasmid. The three PPNG isolates carried the 4.5-MDa Asian beta-lactamase-producing plasmid and a 25.2-MDa conjugative plasmid was found in the two TRNG isolates.
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PMID:Analysis of Neisseria gonorrhoeae in Ontario, Canada, with decreased susceptibility to quinolones by pulsed-field gel electrophoresis, auxotyping, serotyping and plasmid content. 915 33

Thermus species YS45 harbors two small cryptic plasmids of 5.8 (pTsp45s) and approximately 12 kb (pTsp45I). Plasmid pTsp45s has been entirely sequenced, revealing three significant ORFs. In addition to a previously reported thermophilic plasmid-encoded replication protein (Rep), pTsp45s contains two genes for the Tsp45I methyltransferase (M.Tsp45I) and restriction endonuclease (Tsp45I). These two converging genes (tsp45IM and tsp45IR) overlap by 4 bp at their stop codons within an XbaI site. M.Tsp45I (413 aa, 47.0 kDa, recognizing 5'-GTSAC-3') is highly homologous to other m6A-methyltransferases, especially M.EcaI (recognizing 5'-GGTNACC-3'). Tsp45I (332 aa, 37.4 kDa, cleaving 5'-/GTSAC-3') is not homologous to M.Tsp45I, or to other restriction endonucleases. Recombinant Tsp45I is stably produced in E. coli, and cleaves DNA at 65 degrees C with the same specificity as the native enzyme. Therefore, the thermophilic Tsp45I restriction-modification system is plasmid-borne within its native host.
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PMID:The Tsp45I restriction-modification system is plasmid-borne within its thermophilic host. 942 49

NaeI is a remarkable type II restriction endonuclease. It must bind two recognition sequences to cleave DNA, forms a covalent protein-DNA intermediate, and is only 1 aa change away from topoisomerase and recombinase activity. The latter activities apparently derive from reactivation of a cryptic DNA ligase active site. Here, we demonstrate that NaeI has two protease-resistant domains, involving approximately the N-terminal and C-terminal halves of the protein, linked by a protease-accessible region of 30 aa. The domains were purified by cloning. The C-terminal domain was shown by gel mobility-shift assay to have approximately 8-fold lower DNA-binding ability than intact NaeI. Analytical ultracentrifugation showed this domain to be a monomer in solution. The N-terminal domain, which contains the catalytic region defined by random mutagenesis, did not bind DNA and was a mixture of different-sized complexes in solution implying that it mediates self-association. DNA greatly inhibited proteolysis of the linker region. The results identify the DNA-binding domain, imply that DNA cleavage and recognition are independent and separable, and lead us to speculate about a cleft-like structure for NaeI.
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PMID:The domain organization of NaeI endonuclease: separation of binding and catalysis. 952 Apr

Yersinia pestis, the etiologic agent of plague, carries three prototypic plasmids with sizes of 110 kb (pFra, pTox), 70 kb (pLcr, pVW, pCad), and 9.5 kb (pPla, pPst). Studies suggest that geographic isolates of Y. pestis may be differentiated by plasmid profiles. Yersinia pestis isolated from the western United States harbor an additional plasmid, estimated to be approximately 19 kb in size. This cryptic plasmid was characterized by restriction endonuclease digestion, amplification and sequencing of the plasminogen activator gene segment, Southern blotting, and visualized by electron microscopy. Results revealed that this cryptic plasmid is a supercoiled DNA plasmid, 18.85+/-0.59 (mean+/-SD) kb in length, and is a dimer of the 9.5-kb plasmid. The genetic reason for the appearance of this form of the 9.5-kb plasmid in Y. pestis from Arizona, California, Colorado, New Mexico, and Texas is under study.
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PMID:A cryptic 19-kilobase plasmid associated with U.S. isolates of Yersinia pestis: a dimer of the 9.5-kilobase plasmid. 984 May 81

Pseudomonas aeruginosa R-type pyocin particles have been described as bacteriocins that resemble bacteriophage tail-like structures. Because of their unusual structure, we reexamined whether they contained nucleic acids. Our data indicated that pyocin particles isolated from P. aeruginosa C (pyocin C) contain DNA. Probes generated from this DNA by the random-primer extension method hybridized to distinct bands in restriction endonuclease-digested P. aeruginosa C genomic DNA. These probes also hybridized to genomic DNA from 6 of 18 P. aeruginosa strains that produced R-type pyocins. Asymmetric PCR, complementary oligonucleotide hybridization, and electron microscopy indicated that pyocin C particles contained closed circular single-stranded DNA, approximately 4.0 kb in length. Examination of total intracellular DNA from mitomycin C-induced cultures revealed the presence of two extrachromosomal DNA molecules, a double-stranded molecule and a single-stranded molecule, which hybridized to pyocin DNA. Sequence analysis of 7,480 nucleotides of P. aeruginosa C chromosomal DNA containing the pyocin DNA indicated the presence of pyocin open reading frames with similarities to open reading frames from filamentous phages and cryptic phage elements. We did not observe any similarities to known phage structural proteins or previously characterized pseudomonal prt genes expressing R-type pyocin structural proteins. These studies demonstrate that pyocin particles from P. aeruginosa C are defective phages that contain a novel closed circular single-stranded DNA and that this DNA was derived from the chromosome of P. aeruginosa C.
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PMID:The R-type pyocin of Pseudomonas aeruginosa C is a bacteriophage tail-like particle that contains single-stranded DNA. 991 82

Directed open reading frame (ORF) disruption and a serial selection technique in Escherichia coli and the extremely thermophilic archaeon Sulfolobus solfataricus allowed the identification of otherwise cryptic crucial and noncrucial viral open reading frames in the genome of the archaeal virus SSV1. It showed that the 15. 5-kbp viral genome can incorporate a 2.96-kbp insertion without loss of viral function and package this DNA properly into infectious virus particles. The selection technique, based on the preferential binding of ethidium bromide to relaxed DNA and the resulting inhibition of endonuclease cleavage to generate a pool of mostly singly cut molecules, should be generally applicable. A fully functional viral shuttle vector for S. solfataricus and E. coli was made. This vector spreads efficiently through infected cultures of S. solfataricus, its replication is induced by UV irradiation, it forms infectious virus particles, and it is stable at high copy number in both S. solfataricus and E. coli. The classification of otherwise unidentifiable ORFs in SSV1 facilitates genetic analysis of this virus, and the shuttle vector should be useful for the development of genetic systems for Crenarchaeota.
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PMID:Genetic requirements for the function of the archaeal virus SSV1 in Sulfolobus solfataricus: construction and testing of viral shuttle vectors. 1043 May 70

Co-expression of multiple variants of the MLL/AF4 fusion transcript is a common phenomenon in patients with acute lymphoblastic leukemia (ALL) with t(4;11)(q21;q23). Different transcriptional and post-transcriptional mechanisms were found to contribute to the heterogeneity of the chimeric transcripts. Multiple splice variants are generated by utilizing alternative splice sites that result in the joining of different MLL-exons within the breakpoint cluster region to one of three exons in the AF4 fusion partner. To address the question of how splice site selection occurs during RNA processing, we investigated der(11) transcripts in 10 infants with t(4;11) positive ALL. Specific RT-PCR products were analyzed by Southern blot hybridization, SSCP, endonuclease digestion, cloning and sequencing. In patients co-expressing as many as six different chimeric mRNA species, activation of cryptic splice sites has been detected in MLL-exons 8 and 10. This led to the formation of four novel transcript variants, three of which maintained open reading frames (ORFs). Patients with cryptic donor site activation in MLL-exon 8 did not have any MLL-exon 8/AF4 transcripts using the authentic 5' splice site, although this site is 100% homologous to the consensus sequence. However, since MLL-exon 8 does not end in-phase, the use of the authentic splice site would result in loss of the ORF of the fusion message. The activated cryptic splicing sites are located in the vicinity of the polypurine stretches present in MLL-exons 8 and 10, which are known to function as splicing enhancers recognized by SR proteins. We postulate that both the nonsense-mediated decay eliminating correctly spliced MLL-exon 8/AF4 mRNAs and activation of suboptimal splicing sites contribute to the diversity of MLL/AF4 RNA species.
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PMID:Cryptic splice site activation during RNA processing of MLL/AF4 chimeric transcripts in infants with t(4;11) positive ALL. 1077 50


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