中国部分地区结核分支杆菌耐利福平、异烟肼和链酶素耐药基因的检测

目的研究我国部分地区耐利福平(RFP)、异烟肼(INH)和链酶素(SM)结核分支杆菌临床分离株rpoB基因、KatGr、psL基因突变情况,评价其耐药分子机制。方法采用PCR和聚合酶链反应-单链构象多态性(PCR-SSCP)对373株结核分支杆菌临床分离株(其中敏感株148株,耐RFPI、NH、SM株共225株)rpoB基因、KatG和rpsL基因进行检测。并对其中19株SSCP阴性的耐RFP株用双脱氧末端终止法进行测序分析。结果安徽省、北京市、上海市、河北省、河南省、辽宁省、吉林省结核分支杆菌耐RFP临床分离株rpoB基因突变率分别为90%、91%、90%、91%、90%、93%、91%;KatG基因突变率分别为69%、63%、71%、68%、67%、68%、65%,rpsL基因突变率71%、69%、74%、68%、70%、61%、76%,分别为经统计学处理不同地区间无显著性差异(χ2=0.46,P>0.05)。同时rpoB基因敏感株均无突变,特异性为100%;KatG基因有3株发生突变,特异性为98%。19株SSCP阴性耐药株中经测序6株在531位密码子TCG→TTG,1株526位密码子CAC→TAC,7株发生在516位密码子GAC→GTC,5株未改变。结论我国不同地区耐利福平、异烟肼和链霉素的rpoB、KatGr、psL耐药基因突变率大致相同,rpoB基因、KatG和rpsL基因突变分别是耐RFP、INH和SM结核分支杆菌耐药性产生的主要分子机制,PCR-SSCP可快速检测结核分支杆菌RFPI、NH和SM耐药性。

Molecular Characterization of a Chinese Soybean Mosaic Virus Isolate by RT_PCR, cDNA Sequence Analysis and Direct Expression of PCR Products in Bacteria

Soybean mosaic virus (SMV) causes one of the most severe viral diseases in soybean ( Glycine max L.) and is known to contain many pathogenically and serologically related isolates. In the present study, the authors have obtained cDNAs to all cistrons of a Chinese SMV isolate, SMV_ZK, by RT_PCR. By analysing the nucleotide and amino acid sequence of the HC_PRO, NIb and CP cistrons, it was found that SMV_ZK was highly homologous to the G2 strain of SMV, thus confirming the existence of G2_like isolates in soybean crop in China. The amplified cDNAs were directly cloned into a bacterial expression vector. With the exception of the P3 cistron, expression of the cDNAs of all other cistrons in bacteria gave rise to polypeptides of expected molecular weight. The expressed viral proteins were subsequently purified by gel elution. The preparation of viral_specific cDNAs and gene products will be useful in future functional study of the SMV genome.

p21^(WAF1/CIP1) Gene DNA Sequence Change and Their Relationship with the Phenotype of Human Osteosarcoma

Objective: To investigate the p21WAF1 /CIP1gene DNA sequence change and their relationship with the phenotype of human osteosarcoma. Methods: p21WAF1 /CIP1gene DNA of 36 osteosarcoma spec- imens was examined by using polymerase chain reaction-single strand conformation polymorphism (PCR- SSCP) method. The PCR products were sequenced directly. Results: In p21WAF1 /CIP1 gene exon3 of 36 cases of human osteosarcoma, the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 17 cases with the incidence being 44.4%. In 10 normal blood samples, DNA sequence analysis showed the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 8 cases with the incidence being 80%. Conclusion: The novel location of p21WAF1 /CIP1gene polymorphism of osteosarcoma, but not mutation was de?ned, and this location might provide the meaningful reference for the further research of p21WAF1/CIP1 gene.p2lWAF1/CIP1基因DNA序列分析及其与骨肉瘤表型的关系

CLONING AND ANALYSIS OF THE GENOMIC DNA SEQUENCE OF AUGMENTER OF LIVERR EGENERATION FROM RAT

Objective.To search for genomic DNA sequence of the augmenter of liver regeneration(ALR)of rat.Methods.Polymerase chain reaction(PCR)with specific primers was used to amp lify the sequence from the rat genome.Results.A piece of genomic DNA sequence and a p iece of pseudogene of rat ALR were identified.The lengths of the gene and pseudogene are 1508bp and 442bp,respectively.The ALR gene of rat includes 3exons and 2introns.The 442bp DNA sequence may represent a pseudogene or a ALR-related peptide.Predicted amino acid sequence analysis showed that there were 14different amino acid residues between the gene and pseu do-gene.ALR-related peptide is 84amin o acid residues in length and relates closely to ALR protein.Conclusion.There might be a multigene family of A LR in rat.

内蒙古地区传统发酵乳中乳酸菌的分离与鉴定

对内蒙古地区传统发酵乳中乳酸菌进行分离并鉴定。以蒙古族传统发酵乳为材料,采用传统的细菌纯培养分离方法分离乳酸菌株,通过形态学特征及生理生化试验初步确定乳酸菌的基础上,进一步利用细菌16S r DNA基因序列分析和系统发育进化树构建,对其种属进行分析鉴定。分析鉴定结果显示,分离得到的29株乳酸菌共鉴定为2个属,6个种。2个属分别为乳酸杆菌属(Lactobacillus)和肠球菌属(Entercoccus),6个种分别为短乳杆菌(Levilaccillus brevis,4株)、植物乳杆菌(Lactiplantibacillus plantarum,8株)、粪肠球菌(Enterococcus faecalis,9株)、鸡肠球菌(Enterococcus gallinarum,3株)、蒙氏肠球菌(Enterococcus mundtii,1株)、耐久肠球菌(Enterococcus durans,4株)。该研究采集的发酵乳样品中乳酸菌资源丰富,代表着内蒙古蒙古族传统发酵乳制品中优势性乳酸菌菌种,为优良发酵剂的研发奠定了微生物资源基础。

A new mutation (1062 del 16) of iduronate-2-sulfatase gene from a Chinese patient with Hunter syndrome

Objective: To identify the mutations of iduronate-2-sulfatase (IDS) gene, to reveal its mutation features, and to establish a basis for genetic counseling and prenatal gene diagnosis of Hunter syndrome. Methods: Urine glycosaminoglycans (GAGs) assay, PCR and DNA sequencing were performed to detect mutation of IDS gene of the patient and his parents. Results: The result showed that the patient was: DS(++), HS(++), KS(-), CS(-), and that both of his parents were negative. A frame-shift deletion mutation (1062 del 16) was identified in exon 7 of the patient's IDS gene. His parents' genotypes were normal. Conclusion:The patient's mutation was not inherited by his parents but a novel one. The mutation probably altered the primary structure and tertiary structure of IDS enzyme protein remarkably and lowered the activity of IDS enzyme greatly. Therefore it is supposed to be the direct cause of the disorder.

Molecular Characterization of the Duck Enteritis Virus UL4 Gene

Duck enteritis virus(DEV) is a herpesvirus that causes an acute,contagious and fatal disease. In the present article,the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction(PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame(ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups,and the duck enteritis virus branched separately,closely related to the Mardiviruses group comprising Gallid herpesvirus 2(GaHV-2) ,Gallid herpesvirus 3(GaHV-3) and Meleagrid herpesvirus 1(MeHV-1) . The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.

Genomic structure analysis of SNC6, a progesterone-receptor associated protein gene, and cloning and characterization of its 5＇-flanking region

Objective: To analyze the genomic structure of SNC6, a progesterone\|receptor associated protein gene and its regulatory elements in its 5'\|flanking region. Methods: Genomic sequence from GenBank database (accession number: Z98048) covering the whole SNC6 gene was used to analyze the genomic structure of SNC6 and design primers for PCR amplification of its 5'\|flanking region. A 1894 bp fragment of the 5'\|flanking region \{(-1814\} to +75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments (1423 bp, 632 bp and 416 bp, which correspond to -1344 to +75, -552 to +75 and -337 to +75 respectively), were subcloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luciferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All transfected SW620 cells with the above 5\|flanking region\|containing constructs showed luciferase activities. The highest luciferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luciferase activities were measured in transfected cells with vectors containing 416 bp fragments. Luciferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in transfected cells with vectors containing 1423 bp fragments. Conclusion: The basic transcription\|promoting element (promoter) for SNC6 expression resides between 0 to -337, and two transcription\|enhancing elements (enhancer) resides between -337 to -552 and -1344 to -1814, whereas one transcription\|inhibiting element (silencer) exists between -552 to -1344.

Variation and Characterization Analysis of Partial Fragment of Fibroin Gene From Silkworm, Antheraea pernyi

A 1.4Kb DNA fragment containing 3’ flanking sequence of fibroin gene of silkworm, Antheraea pernyi, was obtained from the silk gland’s mRNA of 5 th larva. Analysis of this sequence with another A.pernyi fibroin protein (accession No. D83241) revealed that it consists of a completely open reading frame (ORF), which includes 14 polyalanine-containing units (motifs) and 100bp 3’-UTR. The sequence of the predicted amino acid reveals the highest level of overall identity (90%) with D83241. It was found that it loses a repeat region at the upstream of TAA codon and some mutations. A putative polyadenylation signal AATAAA tail was found in position 1300, which follows the termination codon.

Sequence analysis on drug-resistant rpoB gene of Mycobacterium tuberculosis L-form of isolated from pneumoconiosis workers

To study the characteristics of drug-resistant genetic mutation of rpoB on coal workers＇ pneumoconiosis complicated with L-form of Mycobacterium tuberculosis. Methods： A total of 42 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected, including 31 drug-resistant strains. Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator. Results： No mutation of rpoB gene was identified in 11 rifampicin-sensitive strains while conformation changes were found in 31 rifampicin-resistant strains. The mutation rate was 93.55% （29/31） in resistant strains, mainly concentrated in codon 531 （51.6%, 16/31） and 526 （32.26%, 10/31）. Base substitutions happened, including 27 unit point mutation and 2 two point mutation. The mutation of codon 516 that new found wasn＇t reported by internal and overseas scholars. Conclusion： The substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for rifampicin resistance in Mycobacterium tuberculosis L-forms. It also proves that rpoB gene is diversiform.

Single Nucleotide Polymorphism Genotyping of Calpastatin Gene Using the ARMS Compared with the RFLP

Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins, The association of Single Nucleotide Polymorphism （SNP） in the calpastatin gene with meat tenderness is an important topic in meat production. Therefore efficient procedure to investigate the SNP is necessary. The objectives of this study were to detect the SNP of calpastatin gene at domain L marker （G/C transversion） of the Kamphaengsaen beef breed （KPS cattle; n = 26） by the Amplification Refractory Mutation System （ARMS） compared with the Restriction Fragment Length Polymorphism （RFLP） methods and to determine the genotypes of the KPS cattle at that marker. Genomic DNA of calpastatin gene extracted from blood of the KPS cattle was detected with ARMS and RFLP methods. The ARMS system has utilized two primer pairs to amplify the two different alleles of a polymorphism in single PCR reaction to detected single base mutation. In this method, the alleles-specific primers had a mismatch at 3＇ terminal base and a second deliberate mismatch at position -2 from 3＇ terminus. While the RFLP method detected a polymorphism using PCR-base technique follow by RsaI restriction enzyme. Amplification of the ARMS method revealed that the results were not different from the conventional method of RFLP. Analysis of genotypes revealed that the KPS cattle inherited the CC, CG and GG genotypes at domain L marker. These were reliable when verified by nucleotide sequence analysis of PCR products. The animals were genotyped and determined for tenderness phenotype with this marker that predicted variation of an intronic polymorphism at domain L of the calpastatin gene. Therefore, the ARMS method was simple, efficient technique, and suitable for detecting SNP at domain L marker of the calpastatin gene.

Difference in DNA sequences in SSU rDNA variable regions among pathogens isola ted from different epidemic foci of visceral leishmaniasis in China

OBJECTIVE: To 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. METHODS: Specific 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. RESULTS: Sequence 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%. CONCLUSION: Five 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.