Determination of the genomic organization of human presenilin 1 by fiber-FISH analysis and restriction mapping of cloned DNA.
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Alzheimer Disease↗
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Direct cloning of the unknown flanking DNA fragments from a large insert without restriction mapping.
A simple technique for direct cloning of the target DNA fragments from a large insert according to its adjacent known sequence is described here. In this new subcloning method, a large DNA insert is digested and ligated with a linearized plasmid vector to construct a subclone library that is subjected to screening. The bacterial clones in this library are individually picked, grown in a 96-well plate, and then pooled across the rows or columns. Target clones are obtained from the ordered separate pools by PCR-screening with a set of primers, one specific for the adjacent known sequence and the other serving as "anchor primer" specific for the vector sequence. This direct subcloning procedure was efficiently demonstrated by cloning a specific DNA region from a large insert within 2 days without mapping the starting DNA or isolating the digested DNA fragment.
Cloning, Molecular↗
Rapid mini-scale plasmid isolation for DNA sequencing and restriction mapping.
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Bacteria↗
Detection of germline mutations in the von Hippel-Lindau disease gene by the primer specified restriction map modification method.
Von Hippel-Lindau disease (VHL) is an inherited disorder characterised by a predisposition to develop tumours in the eyes, central nervous system, kidneys, and adrenal glands. Recently the VHL gene was cloned and shown to be mutated in 75% of US and Canadian VHL families. To develop simple, rapid methods for the detection of mutations found in large numbers of affected people, we designed based on the primer specified restriction site modification method. These tests have proved useful in identifying asymptomatic mutated VHL gene carriers who have the nt 505 T to C mutation or the nt 686 T to C mutation. Together with an MspI digestion test which can detect a mutation hot spot in codon 238, polymerase chain reaction/restriction endonuclease based tests can now detect VHL mutations in more than 50% of VHL type 2 families.
Base Sequence↗
Restriction maps of the regions coding for methicillin and tobramycin resistances on chromosomal DNA in methicillin-resistant staphylococci.
Chromosomal BamHI DNA fragments containing both the mecA gene encoding the penicillin-binding protein responsible for methicillin resistance and the aadD gene encoding 4',4"-adenylyltransferase responsible for tobramycin resistance were cloned from three methicillin- and tobramycin-resistant strains of Staphylococcus aureus and one strain of Staphylococcus epidermidis. Physical maps of the fragments were similar, suggesting their unique origin.
Blotting, Southern↗
SARs, ARSs, and A,T-rich regions evidenced by restriction mapping on an 835-kb DNA fragment from Drosophila.
In Drosophila, sequences anchoring the DNA molecule to the scaffold (SARs) and sequences able to replicate autonomously (ARSs) had been shown to comap on an 835-kb DNA fragment (Brun et al. (1990) Mol. Cell. Biol. 10, 5455-5463). To investigate the question of whether this comapping results from the coincidental recruitment of SARs and ARSs in A,T-rich regions, A,T-rich regions of the 835-kb DNA fragment have been identified by restriction analysis with enzymes recognizing motifs made exclusively of A and T. Within the limits of sensitivity of this approach, the obtained data favor the idea of a noncoincidental recruitment: obviously a SAR and an ARS subpopulation are preferentially localized in the A,T-rich regions, but not every A,T-rich region displays a SAR activity, or an ARS activity, or both, nor are all SARs or ARSs localized in the A,T-rich regions. In addition, the data support the idea that a statistical assessment of base composition using restriction analysis might be developed into a general useful approach to genome organization.
Adenine↗
Molecular cloning and restriction mapping of human lymphotoxin gene.
In order to clone the human lymphotoxin (HuLT) gene, we practiced a concise and time-saving method: homologous recombination in vivo (1). By using the mouse lymphotoxin (MuLT) cDNA (1.3 kb) as a probe, we isolated the HuLT gene from a human genomic library which was constructed with cosmid pcos2EMBL as a vector. After linearization, the recombinant cosmid was partially digested with BamHI, EcoRI, PstI, and PvuII respectively, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence (2). The physical map of HuLT gene was made by this method.
Blotting, Southern↗
[Restriction mapping of a highly-repetitive DNA sequence in studying the genetic relationship of lower taxons of animal].
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Animals↗
New cosmid vectors for library construction, chromosome walking and restriction mapping in filamentous fungi.
New cosmid vectors were constructed for the ascomycete fungus, Magnaporthe grisea and the basidiomycete fungus, Ustilago maydis. These vectors are capable of transforming M. grisea at frequencies of up to 5 transformants/micrograms linear DNA and U. maydis at up to 25 transformants/microgram circular DNA for integrative transformation. In addition, 2800 transformants/microgram DNA are possible when using an autonomously replicating vector. Since the promoters used in these vectors function in other ascomycete and basidiomycete fungi, we anticipate that these vectors will be widely applicable.
Ascomycota↗
[Plasmid from Halobacterium halobium and its restriction map].
In this paper, ten strains of H. halobium were screened for the occurrence of plasmid by four methods. Six of the strains harbored plasmids of high molecular weight. Of all the detecting methods, the TENS method was the best for H. halobium. J7 strain contains only one type of plasmid named as pHH205 which showed stationary CCC form. The highest copy number of the plasmid was observed in late stationary growth phase, which was approximately 49 copies per cell. The molecular weight is 16.7kb. The sites of five endonucleases had been determined for pHH205 and three of them, namely BamHI, HindIII and XbaI, were single sites neighboring to each other.
Halobacterium salinarum↗
[Subcloning of K88ac antigen gene of enterotoigenic E. coli and the restriction map of recombinant plasmid].
We had reported a recombinant E. coli RR1(pNZ8801) which was obtained from a wild strain E. coli 79-1454. The recombinant plasmid was digested by EcoRI and generated three segments, medium segment (3.2Md) was removed, the largest and the smallest segment was ligased, then the mixture was transformed into E. coli RRI, screening Ap(r) Tc(s) clones, one of recombinants was named E. coli RR1(pNZ8802). The recombinant plasmid molecular weight is smaller, but expression of K88ac antigen is higher than first cloning. Subcloning can adhere to mucosae of piglet's intesting. Therefore, the recombinant can be use for oval living vaccine.
Antigens, Bacterial↗
Transformation and genomic restriction mapping of Rochalimaea spp.
Transformation procedures using electroporation were established for Rochalimaea quintana. Several cosmid/plasmids possessing the RK2 or RSF1010 origin of replication were successfully inserted. Plasmid retention and replication were verified by antibiotic resistance and Southern blot analysis. The highest level of transformation was obtained at a voltage field strength of 12.5 kV/cm with a pulse time of 10 miliseconds. Transformation efficiency was low (0.3%) with approximately 10(5) transformants/microgram of DNA. One construct, designated pAG10, reached sufficient levels in R. quintana to be isolated by density gradient centrifugation. Analysis of this plasmid after several cycles of growth in R. quintana revealed no obvious modifications. Physical maps of Rochalimaea spp. chromosomal DNA using pulse-field electrophoresis are being developed. Digestion of R. vinsonii chromosomal DNA with NotI or SfiI resulted in three and one fragments, respectively. When R. quintana was digested in a similar manner, both NotI and SfiI produced four fragments. Double digestion of R. quintana DNA with NotI and SfiI yield seven fragments ranging in size from 11 to 925 kb. Summing the fragments indicate an approximate genome size of 2.1 x 10(6) bp for R. vinsonii and 1.7 x 10(6) bp for R. quintana chromosomal DNA.
Electrophoresis, Gel, Pulsed-Field↗
Isolation and restriction map of the V-J interval of the human T cell receptor gamma chain locus.
The human T cell receptor gamma chain locus encodes the immunoglobulin-like gamma chain polypeptide and spans a distance of approximately 150 kb. Previous studies have not precisely characterized the interval separating variable regions from joining--constant regions which is excised during gamma gene rearrangement. We report a series of overlapping cosmids which includes the portion of the gamma chain locus beginning with V2 and extends to the second exon of C2. Sixteen kilobases separate the most 3' variable region gene, V4, from the most 5' joining segment, J1.1.
Cosmids↗
A continuous restriction map from HLA-E to HLA-F. Structural comparison between different HLA-A haplotypes.
The class I region of the human major histocompatibility complex contains genes encoding the classical transplantation antigens (HLA-A, B, and C), at least three new class I genes (HLA-E, F, and G) and many class I pseudogenes (including HLA-H). By pulse field gel electrophoresis and using five rare cutter enzymes, we have constructed a precise and continuous map of 1200 kilobases (kb) around HLA-A. The blots were hybridized with HLA-A, E, and F-specific probes and with new probes derived from yeast artificial chromosomes and cosmids of the class I region. We have compared the genomic organization of the same 1200 kb in three homozygous lymphoblastoid cell lines corresponding to three different HLA haplotypes (A3, A24, and A31). The differences in size observed may have been caused by insertions and deletions and may prove valuable in understanding the evolution of the HLA chromosomal region.
Genes, MHC Class I↗
[Molecular weight and restriction mapping of the DNA of cholera phages].
Molecular masses of cholera bacteriophages 493, 7226 and Eltor II were defined by electron microscopic technique. DNA of these bacteriophages was digested by the restriction endonucleases PstI, BglI, MluI and SalI. The number and molecular masses of the obtained restricts were identified. The physical map of bacteriophage 493 was constructed using three restriction endonucleases. The obtained data can be used for classification and molecular biology research of cholera bacteriophages.
Bacteriophages↗
Restriction map of large plasmid pFW1 in Bacillus sphaericus strain 10.
Some strains of Bacillus sphaericus have been found to be toxic to mosquito larvae (1). They differ from Bacillus thuringiensis var. israelensis (BTI) (2,3) in the following aspects: BTI is relatively ineffective in polluted water, and its residual activity in most habitats is limited to a few days after treatment, whereas BS isolates are effective in such habitats, particularly against species of Culex; BS also remains effective longer, as a result of either persistence or recycling. B. sphaericus strain 10 (BS10) was isolated by the Lixiahe Institute of Agricultural Sciences in Jiangsu Province. Its larvicidal activity is much higher than that of BS1593, which was recommended by the World Health Organization for widespread use as a standard strain (1). The mosquito-larvicide gene of BS1593 has been cloned and expressed in E. coli and B. subtilis (4, 5). In the present study, BS10 was investigated to establish whether it contains the plasmid and whether homology exists between the mosquito-larvicide gene in BS and that in BTI.
Animals↗
Analysis of respiratory syncytial virus in clinical samples by reverse transcriptase-polymerase chain reaction restriction mapping.
The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed.
Humans↗
Genomic organisation of 34 kb of the human immunoglobulin lambda locus (IGLV): restriction map and sequences of new V lambda III genes.
In order to improve our knowledge of the human immunoglobulin variable lambda locus (IGLV), we mapped one cosmid clone (designated as C40.2) isolated by screening a Colo320HSR genomic library. The 34 kb insert of the C40.2 clone was shown to contain six genes. One gene, IGLV2S1, belongs to the V lambda II subgroup. Four genes belong to the V lambda III subgroup. Two of them, IGLV3S1 and IGLV3S2, are potentially functional whereas the two others are pseudogenes. The size of the IGLV3S2 leader intron is four times longer than the classical intron size of 110 bp. The cosmid also contains a vestigial sequence lambda vg2. All these genes share the same orientation of transcription. Pulsed field gel electrophoresis analysis of the IGLV locus shows that most of the V lambda I subgroup genes are located at the 5' end of the locus.
Amino Acid Sequence↗