Objective] To analyze the sequence difference of the SSU rDNA variable regions of Leishmania isolates from hilly foci and plain foci of China. [Methods] Specific SSU rDNA fragments from nuclear DNA of five Leishmania species and isolates were amplified by PCR. The amplified DNA fragments were cloned into pGEM R\|T Easy vector. The specific fragments were sequenced by the automated DNA sequencer. [Results] Sequence analysis showed that the amplified DNA fragments of five Leishmania species and isolates were all 392 bp in length, point mutations were located in the two unique sequence (UQ\|Ⅰ and UQ\|Ⅱ); L.d.SC10 and L.d.GS7 had two same point mutations in UQ\|Ⅱ, only L.d.GS7 had one in UQ\|Ⅰ; no insertion/deletion. [Conclusion] Sequence difference of the SSU rDNA variable region existed between Leishmania isolates from hilly foci and plain foci; The sequences of the SSU rDNA variable regions of L.d. SD2 isolate and L.infantum were identical.

The LACK gene from Leishmania, an analogue of the receptor of activated protein kinase C, was discovered recently. In this study, the LACK gene of Leishmania donovani was obtained from the recombinant plasmid T-LACK by PCR. The gene was cloned into eukaryotic expressed plasmid pcDNA3.1(+) to construct recombinant plasmid. This recombinant plasmid then was transfected into the eukaryotic cell COS-7, and the expression of LACK gene in eukaryotic cell was detected by RT-PCR and immunofluorescent staining. Both RT-PCR and immunofluorescent staining of recombinant plasmid transfected COS-7 showed positive reaction, thus indicating that the recombinant plasmid pcDNA3-LACK can express LACK protein in euka ryotic cell COS-7.
Animals ; Antigens, Protozoan ; biosynthesis ; genetics ; immunology ; COS Cells ; Cloning, Molecular ; DNA, Recombinant ; biosynthesis ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Leishmania donovani ; Plasmids ; genetics ; Protozoan Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vaccines, DNA

Leishmaniasis (Kala-azar) from different endemic regions of China expresses different clinic and epidemiological features, and traditionally is classified as hilly, plain and desert types/foci. We concentrated our review on whether the pathogens from those foci were different at molecular level, if so, whether there are were molecular markers readily identifiable by molecular technologies. This was a review of a 20-year search for such markers by using kinetoplastic DNA (kDNA), nDNA hybridization, PCR-SSCP, RAPD and sequence analysis of SSU rDNA variable regions and LACK gene. The results showed that heterogeneities at molecular level exist in Leishmania isolated from different foci of China, which could be used as markers for different types of Leishmaniasis in China.
China ; DNA Fingerprinting ; DNA, Protozoan ; analysis ; genetics ; Genotype ; Humans ; Leishmania donovani ; classification ; genetics ; isolation & purification ; Leishmaniasis, Visceral ; classification ; parasitology ; Mutation

OBJECTIVETo confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L.d.) isolates from different epidemic foci in China.

METHODSSpecific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM(R)-T Easy Vectors. After that, the specific fragments were sequenced by an automated DNA sequencer.

RESULTSSequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length. All 5 point mutations were located in two unique sequence blocks (UQ-I and UQ-II), and no insertions or deletions were found. The identities of comparison of Leishmania in GeneBank were more than 98%.

CONCLUSIONFive point mutations exist in the SSU rDNA variable region of 5 L.d. isolates from different epidemic foci of visceral leishmaniasis (VL) in China. Sequence differences of the SSU rDNA variable region exist among L.d. isolates from different foci.

Animals ; DNA, Protozoan ; chemistry ; DNA, Ribosomal ; chemistry ; Humans ; Leishmania donovani ; genetics ; Leishmaniasis, Visceral ; parasitology ; Point Mutation ; Polymerase Chain Reaction