ABSTRACT
Objective To analyze the structures of atypical class 1 integrons in clinical Proteus isolates. Methods This study included 26 class 1 integron integrase gene-positive clinical Proteus isolates, from which the variable regions of class 1 integrons could not be amplified. Six isolates were chosen from them to amplify the flanking DNA segments of class 1 integron integrase gene using inverse PCR. The se-quences of PCR products were analyzed with BLAST to identify the target homologous sequences as well as their accession numbers in GenBank. Primers for overlap PCR were designed according to the flanking se-quences. Then the 26 clinical Proteus isolates were analyzed with overlap PCR and sequencing analysis. Re-sults The variable regions of class 1 integrons in 25 out of the 26 clinical Proteus isolates were completely or partly identified by using inverse PCR,overlap PCR and sequencing analysis. A gene cassette array of estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 was detected in 22 isolates. The variable regions of class 1 integrons in the other three isolates were identified to be estX-psp-aadA2-cmlA1,dfrA14 and IS26,respectively. All of the 25 isolates lacked the 3'conserved segements in class 1 integron. Conclusion Inverse PCR can be used to analyze the structures of atypical class 1 integrons. Gene cassette psp and gene cassette arrays of estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 and estX-psp-aadA2-cmlA1 in clinical Proteus isolates are reported for the first time.
ABSTRACT
Objective To precious localize DNase Ⅰ hypersensive sites exactly in the promoter region of CD133 of cell line SW480 by inverse-PCR.Methods The colonel cancer cell SW480 nuclei were suspended in digested buffer,treated with DNase Ⅰ at the concentration of 10 U/ml for 10 min.The inversePCR was performed as follows.DNA treated by DNase Ⅰ was purified,fragmented with restricted enzyme EcoRI and Xmal Ⅰ.Then the ends were blunted,ligated by T4 ligase.PCR was performed,and production was sequenced.The restricted enzymes cut sites were near DNase Ⅰ cleavage sites.Results 9 DNase Ⅰ cut sites were identified in CD133 promoter region.The DNaseI hypersensitive sites all distributed in a region -300 bp--700 bp up to transcription start site.Conclusion The DNase Ⅰ cleavage sites could identified preciously by application of inverse-PCR.These sites locate in a region of-300 bp--700 bp up to transcription start site.
ABSTRACT
Objective To map the deletion breakpoint of 9p21 in breast cell line MCF-7 use the inverse PCR .Methods After di-gestion ,ligation and PCR reaction ,the breakpoint was confirmed by sequencing .Results The deletion breakpoint started at chr9 :21819532 and ended at chr9 :21989622 with a small insertion of ACTGG ,which was consistent with the result confirmed by the long range PCR .Conclusion The inverse PCR is one good method for deletion breakpoint mapping and suitable for large sample size detection .
ABSTRACT
Based on a ?-tubulin gene EST sequence from Trichoderma harzianum mycelium cDNA library,a 1.74kb ?-tubulin gene coding region,together with a 1.5kb 5'-flanking region and a 1.0kb 3'-flanking region was cloned from T.harzianum by IPCR methods.The gene encodes a 446 amino acid polypeptide,which shows a high degree of homology with other fungal ?-tubulins.A three-dimensional model of the T.harzianum ?-tubulin was built based on the crystal structure of bovine tubulin using homology modeling method.Tertiary protein structure of the ?-tubulin should provide a basis for understanding a significant body of research on tubulin's properties and interactions with antimicrotubule agents.
ABSTRACT
DNA fragments of ? subunit and ? subunit were amplified by PCR from the genome of Acidianus tengchongensis with the degenerated primers designed from the consensus amino acid sequence of Sulfolobus chaperonins. The two DNA fragments about 500bp were used as probe for southern hybridization to determine the suitable restriction endonuclease. Restriction endonuclease digested genomic DNA was circularized by self-ligation, and complete sequences of ? subunit and ? subunit were obtained by inverse PCR. The primers oriented in the reversed direction of the usual orientation to amplify the DNA sequence that flank the known region. The complete genes of ? subunit and ? subunit were PCR amplified from genomic DNA using two pairs primers.