EXPRESSION OF HLA-CLASS Ⅱ GENESOF IDDM PATIENTS ON THE SURFACE OF THE LTK- CELLS

To investigate the function of HLA-class Ⅱ genes in the autoimmune response of insulin dependent diabetes mellitus(DDM),the HLA-class Ⅱ gene of IDDM patients was introd uced into Ltk^-cells with pSV2-neo plas-mid,using the calcium phosphate precipitation technique.We obtained a stable cell line expressing the HLA-class Ⅱ gene from lymphocytes of IDDM patients.Expression was identified by direct ox erythocyte-CrCl3-HLA DR monoclonal antibody rosetting.

Genetic Differentiation Analyses Based on mtDNA COⅡ Gene Sequences Among Different Geographic Populations of Aphis glycines(Hemiptera: Aphididae) in Northeast China

Aphis glycines （Hemiptera： Aphididae） is considered as a cosmopolitan pest of cultivated soybean, major difficulties in its control measures may be due to its higher genetic diversity; however, the knowledge about population genetic diversity of this species is limited. This study aimed to represent the genetic differentiation among different geographic populations of soybean aphid in Northeast China. In order to investigate and assess the genetic diversity, genetic differentiation, molecular variance, population structure, ecological importance and evolutionary history of A. glycines, we sequenced a fragment of one protein-coding gene, the cytochrome c oxidase I/of mitochondrial DNA gene. The results showed that four haplotypes were defined among CO 11 gene of 180 sequences of soybean aphid in Northeast China including H1 shared by all the populations. Lower haplotype diversity （Hd=0.3590± 0.0420） and nucleotide diversity （Pi=0.0012±0.0002） were observed and high gene flow was detected in every two populations, while most of the variation （80.81%） arose from variability within A. glycines from individuals. Low genetic differentiation and high gene flow （Nm=2.106） indicated a high migration rate between the populations, which might reveal that gene flow in different geographic populations did not affect by geographical distance. The phylogenetic tree and the haplotype network ofA. glycines were obtained based on sequences of CO Ⅱ gene, there were no significant genealogical branches or clusters recognized in NJ tree, and no clear distribution, delineation of haplotypes were demonstrated in the haplotype network according to geographical location. This study rejected the vicariance hypothesis： geographic isolation could be a barrier and it restricted A. glycines gene flow among 10 populations.

The relation between HLA-DQA1 genes and genetic susceptibility to duodenal ulcer in Wuhan Hans

AIM To study the genetic susceptibility of HLA-DQA1 alleles to duodenal ulcer in Wuhan Hans.METHODS Seventy patients with duodenalulcer and fifty healthy controls were examinedfor HLA-DQA1 genotypes.HLA-DQA1 typing wascarried out by digesting the locus specificpolymerase chain reaction amplified productswith alleles specific restriction enzymes(PCR-RFLP),i.e.,Apal Ⅰ,Bsaj Ⅰ,Hph Ⅰ,Fok Ⅰ,Mbo Ⅱ and Mnl Ⅰ.RESULTS The allele frequencies of DQA1 * 0301and DQA1 * 0102 in patients with duodenal ulcerwere significantly higher and lower respectivelythan those in healthy controls(0.40 vs 0.20,P = 0.003,mcorret = 0.024)and(0.05 vs 0.14,P = 0.012,but Pcorret】0.05),respectively.CONCLUSION DQA1 * 0301 is a susceptiblegene for duodenal ulcer in Wuhan Hans,andthere are immunogenetic differences in HLA-DQA1 locus between duodenal ulcer patients andhealthy controls.

Cloning of Human Uroplakin Ⅱ Gene from Chinese Transitional Cell Carcinoma of Bladder and Construction of Its Eukaryotic Expression Vector

Summary: To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T 3N 0M 0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between XbaⅠ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UPⅡ for Uroplakin Ⅱ was successfully constructed. After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

Expression of IGF-Ⅱ,p53,p21 and HBxAg in precancerous events of hepatocarcinogenesis induced by AFBI and/or HBV in tree shrews

INTRODUCTIONIn order to study the relationship between oncogeneexpression and HCC generation,we observed theprecancerous hepatic GGT loci,IGF-Ⅱ,p53 andp21 expression during hepatocarcinogenesis of treeshrew induced by hepatitis B virus (HBV) and/oraflatoxin B1 (AFB1).

Cloning and expression of HLA-B7 gene

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Association of Polymorphisms in Angiotensin-converting Enzyme and Type 1 Angiotensin Ⅱ Receptor Genes with Coronary Heart Disease and the Severity of Coronary Artery Stenosis

To explore the relation of angiotensin-converting enzyme （ACE） and angiotensin Ⅱ type 1 receptor （AT1R） gene polymorphism with coronary heart disease （CHD） and the severity of coronary artery stenosis, 130 CHD patients who underwent coronary angiography were examined for the number of affected coronary vessels （≥75% stenosis） and coronary Jeopardy score. The insertion/deletion of ACE gene polymorphism and AT1R gene polymorphism （an A→C transversion at nucleotide position 1166） were detected by using polymerase chain reaction （PCR） and restriction fragment length polymorphism （RFLP） in CHD patients and 90 healthy serving as controls. The resuits showed that DD genotype and of ACE were more frequent in CHD patients than that in control group （38.5% vs 14.4%, P〈0.001）. The frequency of the ATIR A/C genotypes did not differ between the patients and the controls （10% vs 13.1%, P〉0.05）. The relative risk associated with the ACE-DD was increased by AT1R-AC genotype. Neither the number of affected coronary vessels nor the coronary score differed among the ACE I/D genotypes （P〉0.05）. But the number of affected coronary vessels and the coronary score were significantly greater in the patients with the AT1R-AC genotype than in those with the AA genotype （P〈0.05）. In conclusion, DD genotype may be risk factor for CHD and MI in Chinese people, and is not responsible for the development of the coronary artery stenosis. The AT1R-C allele may increase the relative risk associated with the ACE-DD genotype, and may be involved in the development of the stenosis of coronary artery.

Genetics of coronary heart disease with reference to ApoAICⅡI-AIV gene region

Cardiovascular diseases are affected by multiple factors like genetic as well as environmental hence they reveal factorial nature. The evidences that genetic factors are susceptible for developing cardiovascular diseases come from twin studies and familial aggregation. Different ethnic populations reveal differences in the prevalence coronary artery disease(CAD) pointing towards the genetic susceptibility. With progression in molecular techniques different developments have been made to comprehend the disease physiology. Molecular markers have also assisted to recognize genes that may provide evidences to evaluate the role of genetic factors in causation of susceptibility towards CAD. Numerous studies suggest the contribution of specific "candidate genes", which correlate with various roles/pathways that are involved in the coronary heart disease. Different studies have revealed that there are large numbers of genes which are involved towards the predisposition of CAD. However, these reports are not consistent. One of the reasons could be weak contribution of genetic susceptibility of these genes. Genome wide associations show different chromosomal locations which dock, earlier unknown, genes which may attribute to CAD. In the present review different ApoAI-CⅡI-AIV gene clusters have been discussed.

RFLP OF HLA CLASS I GENES IN CHINESE POPULATION AND ITS CORRELATION WITH SEROLOGICALLY DEFINED ANTIGEN SPECIFICITIES

The restriction fragnent length polymorphisms （RFLPs） of HLA class I genes in 104 unrelated healthy Chinese individuals were analyzed with Southern blot assay. The DNAs from peripheral blood leucocytes were digested with EcoRI, EcoRV and XbaI respectively, and hybridized with 32P-labeled HLA class I probe （ 1.4Kb, B7 cDNA ）. The results showed that 3 EcoRI fragments （13.7, 8.1 and 5.2 Kb ）, 3 EcoR V fragments （11.3, 7.8 and 4.1 Kb） and 7 Xbal fragmnents （21.9, 19.2, 16.3, 6.0, 3.8, 1.8 and 1.5 Kb） were polymorphic. Ten of the fragments were found to be correlated significantly with the serologically de fined antigen specificities. The significance of this kind of correlation is discussed.

Allelic Polymorphism,Gene Duplication and Balancing Selection of MHC Class ⅡB Genes in the Omei Treefrog(Rhacophorus omeimontis)

The worldwide declines in amphibian populations have largely been caused by infectious fungi and bacteria. Given that vertebrate immunity against these extracellular pathogens is primarily functioned by the major histocompatibility complex（MHC） class Ⅱ molecules, the characterization and the evolution of amphibian MHC class Ⅱ genes have attracted increasing attention. The polymorphism of MHC class Ⅱ genes was found to be correlated with susceptibility to fungal pathogens in many amphibian species, suggesting the importance of studies on MHC class Ⅱ genes for amphibians. However, such studies on MHC class Ⅱ gene evolution have rarely been conducted on amphibians in China. In this study, we chose Omei treefrog（Rhacophorus omeimontis）, which lived moist environments easy for breeding bacteria, to study the polymorphism of its MHC class Ⅱ genes and the underlying evolutionary mechanisms. We amplified the entire MHC class ⅡB exon 2 sequence in the R. omeimontis using newly designed primers. We detected 102 putative alleles in 146 individuals. The number of alleles per individual ranged from one to seven, indicating that there are at least four loci containing MHC class ⅡB genes in R. omeimontis. The allelic polymorphism estimated from the 102 alleles in R. omeimontis was not high compared to that estimated in other anuran species. No significant gene recombination was detected in the 102 MHC class ⅡB exon 2 sequences. In contrast, both gene duplication and balancing selection greatly contributed to the variability in MHC class ⅡB exon 2 sequences of R. omeimontis. This study lays the groundwork for the future researches to comprehensively analyze the evolution of amphibian MHC genes and to assess the role of MHC gene polymorphisms in resistance against extracellular pathogens for amphibians in China.

Autosomal dominant osteopetrosis typeⅡresulting from a de novo mutation in the CLCN7 gene:A case report

BACKGROUND Osteopetrosis is a family of extremely rare diseases caused by failure of osteoclasts and impaired bone resorption. Among them, autosomal dominant osteopetrosis type Ⅱ(ADO Ⅱ), related to the chloride channel 7(CLCN7) gene, is the most frequent form of osteopetrosis. In this study, we report a de novo mutation of CLCN7 in a patient without the family history of ADO Ⅱ.CASE SUMMARY A 5-year-old Chinese boy with ADO Ⅱ was found to have a de novo mutation in the CLCN7 gene [c.746 C>T(p.P249 L)]. Typical clinical manifestations, including thickening of the cortex of spinal bones and long bones, non-traumatic fracture of the femoral neck, and femoral head necrosis, were found in this patient. The patient is the first reported case of ADO Ⅱ with the missense mutation c.746 C>T(p.P249 L) of the CLCN7 gene reported in China. We also review the available literature on ADO Ⅱ-related CLCN7 mutations, including baseline patient clinical features, special clinical significance, and common mutations.CONCLUSION Our report will enrich the understanding of mutations in ADO Ⅱ patients. The possibility of a de novo mutation should be considered in individuals who have no family history of osteopetrosis.

Plant regeneration of transgenic China Rose (Rosa chinensis Jacq.) from organogenic callus

Different types of explants of China Rose （Rosa chinensis Jacq.） were placed on a Schenk and Hildebrandt （SH） medium containing L-proline and 2,4-dichlorophenoxyacetic acid （2,4-D）. Organogenesis was observed on callus induced from both whole leaf and petiole and the high frequency of organogenesis was observed on the whole leaf. Shoot regeneration was obtained via or- ganogenesis. The effects of pH and concentrations of antibiotics on maintenance of organogenesis capacity were investigated in sub- sequent subcultures. The pH value was found to play a critical role in retaining organogenesis capacity. The binary vector pBI121, carrying the gus gene coding for fl-glucuronidase （GUS） and the npt II gene mediated by Agrobacterium tumefaciens, was used for transformation of organogenic callus using 50 mg·L^-1 geneticin for selection. Six regenerated lines showed GUS activity, of which five were verified for the presence ofnpt Ⅱ gene by PCR.

Deleterious Nonsynonymous SNP Found within <i>HLA-DRB1</i>Gene Involved in Allograft Rejection in Sudanese Family: Using DNA Sequencing and Bioinformatics Methods

Renal transplantation provides the best long-term treatment for chronic renal failure. Single-nucleotide polymorphisms (SNPs) play a major role in the understanding of the genetic basis of many complex human diseases. Also, the genetics of human phenotype variation could be understood by knowing the functions of these SNPs. It is still a major challenge to identify the functional SNPs in a disease-related gene. This work explored how SNPs mutations in HLA-DRB1 gene could affect renal transplantation rejection. This study was carried out in Ahmed Gasim Hospital, Renal Dialysis Center during the period, from September 2012 to November 2013. Blood samples from five Sudanese patients (different families) with known renal transplantation rejection were collected before hemodialysis, furthermore one blood sample for control. DNA sequences results and detected SNPs were analyzed using bioinformatics tools (BLAST, SIFT, nsSNP Analyzer, PolyPhen, I-mutant, BioEdit, CPH, Chimera, Box shade and Project Hope). In addition, international databases were used for datasets [NCBI, Uniprot]. Results showed that, three SNPs were detected;two of three SNPs were predicted as tolerant or benign (rs1059575, novel) and one was deleterious (rs17885437). This study concluded that the identification of pathological SNPs could be an answer to unknown causes for a lot of organ transplantation rejection cases.

Relationship between polymorphism of class Ⅱ transactivator gene promoters and chronic hepatitis B

AIM: To investigate the relationship between the polymorphism of class Ⅱ transactivator (CⅡTA) gene promoters and chronic hepatitis B (CHB).METHODS: Genomic DNA was prepared from peripheral blood leukocytes. Promoters Ⅰ, Ⅲ and Ⅳ of gene were analyzed respectively with polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) in 65 patients with CHB, 26 patients with acute hepatitis B (AHB) and 85 normal controls.RESULTS: No abnormal migration was found in PCR-SSCP analysis of the three promoters in the three groups. Also,no sequential difference was observed at the three promoters among the CHB patients, AHB patients and normal controls.CONCLUSION: No polymorphism in promoters Ⅰ, Ⅲ and Ⅳ of CⅡTA gene exists in CHB patients, ABH patients and normal controls, suggesting that the promoter of CⅡTA gene might be a conserved domain.

Expression of CⅡTA Gene in Five Human Malignant Hematological Cell Lines and Its Significance

The role of MHC class Ⅱ transactivator (CⅡTA) in constitutive or IFN-γ inducible expression of HLA molecules in human malignant hematological cell lines was investigated. The expression of HLA molecules and CⅡTA protein was detected by Western blot, immunohistochemistry and flow cytometry. The expression of CⅡTA gene was determined by RT-PCR. The capability of peripheral blood T cell reaction stimulated by tumor cells was monitored by mixed lymphocyte reaction. It was found that the HLAⅡ-positive tumor cells expressed the CⅡ TA quite well, and the expression of HLAⅠ+Ⅱ was increased in the tumor cells with constitutive or inducible expression of CⅡ TA after induced by IFN-γ. The tumor cells which did not express CⅡ TA after induced by IFN- γ were not response to the expression of HLAⅡ promoted by IFN- γ. It suggests a correlation between the inability of some malignant hematological cell lines in response to IFN-γ for HLA expression and the deficiency in the inducible expression of CⅡTA, indicating CⅡ TA might take part in the regulation of HLA Ⅰ+Ⅱ expression in the tumor cells, which might play an important role in tumor immunologic escape.

Mutations in Exons of the CYP17-Ⅱ Gene Affect Sex Steroid Concentration in Male Japanese Flounder(Paralichthys olivaceus)

As a specific gene of fish,cytochrome P450c17-Ⅱ(CYP17-Ⅱ) gene plays a key role in the growth,development and reproduction level of fish.In this study,the single-stranded conformational polymorphism(SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-Ⅱ gene in a population of 75 male Japanese flounder(Paralichthys olivaceus).Three single nucleotide polymorphisms(SNPs) were identified in CYP17-Ⅱ gene of Japanese flounder.They were c.G594A(p.G188R),c.G939A and c.G1502A(p.G490D).SNP1(c.G594A),located in exon 4 of CYP17-Ⅱ gene,was significantly associated with gonadosomatic index(GSI).Individuals with genotype GG of SNP1 had significantly lower GSI(P < 0.05) than those with geno-type AA or AG.SNP2(c.G939A) located at the CpG island of CYP17-Ⅱ gene.The mutation changed the methylation of exon 6.Indi-viduals with genotype AA of SNP2 had significantly lower serum testosterone(T) level and hepatosomatic index(HSI) compared to those with genotype GG.The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder.How-ever,the SNP3(c.G1502A) located in exon 9 did not affect the four measured reproductive traits.This study showed that CYP17-Ⅱ gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.

Structural analysis of DMD gene and its clinical application in Chinese.Ⅰ. Bgl Ⅱ exon-containing fragment,RFLP and carrier detection

This article is one of the serial studies oll the characteristics of the molecular structure for dystrophin gene in Chinese. By using the entire dystrophin cDNA (14 kb) as a probe- the number and RFLPs of Bgl Ⅱ exon-containing fragments of the dystrophin gene were analysed. Four new Bgl Ⅱ fragments were found, two of them (3.7 and 6.2 kb) detected by comparing the hybridization patterns with cDNA1-2a. 1a and 2a, one (9.3 kb) from the hybridization pattern with cDNA 9 by lengthening migrating distance of DNA fragments in electrophoresis. and another one (4.0 kb) by comparing the patterns with cDNA 11-14,11a- 11b’ 11c-12a and 14. The results indicated that the number of Bgl Ⅱ exon-containing fragments should be 59 rather than 55 reported previously, which laid the foundation of the Bgl Ⅱ partial restriction map for dystrophin gene. Three of the four RFLPs found in Caucacian appear in the hybridization patterns of three subclones, i.e.cDNA 2b-3. cDNA 4-5, and cDNA 5b-7.’ The values of expected heterozygote frequency (EHF) were 0.33, 0.33and 0.40 and the observed heterozygote frequency (OHF)were 0.40. 0.40 and 0.48 respectively. Meanwhile, two new rare allelic fragments (15 kb) were found in RFLPs from Bgl Ⅱ/2b-3 and Bgl Ⅱ/4-5a patterns respectively. These Bgl Ⅱ RFLPs and four XbaI RFLPs documented in our laboratory, have been used to detect the carrier in 7 DMDfamilies and 1 BMD family. Of the 69 individuals from the 8 families- 11 females were diagnosed as the carriers with DMD mutation, 4 females as the doubtful carriers, 12 females were defined as normal genotype and 2 females as probably normal. The results suggest that the carrier testing method based on dosage intensity analysis and genotype analysis by using dystrophin cDNA as a probe will be more sensitive and accurate.

Carcinogenesis of nasopharyngeal carcinoma:an alternate hypothetical mechanism

Current proposed mechanisms implicate both early and latent Epstein-Barr virus(EBV)infection in the carcinogenic cascade,whereas epidemiological studies have always associated nasopharyngeal carcinoma(NPC)with early childhood EBV infection and with chronic ear,nose,and sinus conditions.Moreover,most patients with NPC present with IgA antibody titers to EBV capsid antigen(VCA-lgA),which can precede actual tumor presentation by several years.If early childhood EBV infection indeed constitutes a key event in NPC carcinogenesis,one would have to explain the inability to detect the virus in normal nasopharyngeal epithelium of patients at a high risk for EBV infection.It is perhaps possible that EBV resides within the salivary glands,instead of the epithelium,during latency.This claim is indirectly supported by observations that the East Asian phenotype shares the characteristics of an increased susceptibility to NPC and immature salivary gland morphogenesis,the latter of which is influenced by the association of salivary gland morphogenesis with an evolutionary variant of the human ectodysplasin receptor gene(EDAR),EDARV370A.Whether the immature salivary gland represents a more favorable nidus for EBV is uncertain,but in patients with infectious mononucleosis,EBV has been isolated in this anatomical organ.The presence of EBV-induced lymphoepitheliomas in the salivary glands and lungs further addresses the possibility of submucosal spread of the virus.Adding to the fact that the fossa of Rosen Muller contains a transformative zone active only in the first decade of life,one might be tempted to speculate the possibility of an alternative carcinogenic cascade for NPC that is perhaps not dissimilar to the model of human papillomavirus and cervical cancer.

The Construction of the Probe for Amylase n Gene Cloning from Bacillus halodurans Strain 38C1-1

Primers and probes were established according to the sequences of the alpha-amylase genes of Bacillus. halodurans C-125, Therrnus sp. IM6501, B. stearothermophilus ET-1, and B, acidopullulytics. Primers were designed and a 0.2 kb DNA fragment was amplified, the fragment was successfully used for the detection of the amylase Ⅱ gene in a 2 842 bp region from Bacillus halodurans strain 38C1-1.