A simple, rapid method for colony hybridization has been developed. The DNA probes were labeled by digo xigenin. The signal of hybridization was detected by streptavidin and poly (AP) system. The results showed that this method is sensitive, specific and repoducible, it can be used for colony hybridization instead of isotopic.

cholesterol 7α-hydroxylase gene ( CYP7A 1 ) plays a key role in the catabolism of cholesterol into bile acids. To investigate whether the A-204C polymorphism in CYP7A1 gene affects the gene expression,using luciferase as the reporter gene, four recombinants were constructed by inserting forward or reverse sequence with A or C allele at the polymorphism site into the promoter-less vector pGL3-basic. The constructs were then transfected into four cell lines and the luciferase activity of each expression vector was examined by dual luciferase reporter gene assay system. The results showed that activities of the forward sequence of both genotypes were higher than that of reverse sequence. Promoter activity of the recombinants with A allele was about one third lower than that with C allele. According to the analysis with TRANSFAC database, there may exist a Zic3 binding site when there is the C allele at -204. Our study indicates that the A-204 C polymorphism in CYP7A1 promoter region decreases its promoter activity and thus represses the gene expression, possibly due to the lack of a potential Zic3 binding site.

Objective. To study the associations of IDDM3, IDDM4, IDDM5 and IDDM8 with insulin-dependent diabetes mellitus (IDDM). Methods. The polymorphisms of short tandem repeat (STR) loci D15S657, D11S1369, D6S2420 and D6S503, linked to IDDM3, IDDM4, IDDM5 and IDDM8 respectively, were studied by polymerase chain reaction and polyacrylamide gel electrophoresis (PCR-PAGE) followed by direct sequencing of PCR products in 105 normal Chinese Han nationality subjects and 48 patients with IDDM. Results. The allele frequencies of allele A5 at D15S657 locus, allele A5 at D11S1369 locus and allele A4 at D6S2420 locus were increased significantly in patients with IDDM compared to those in the control group. No difference in the allele frequencies at D6S503 locus was observed between IDDM and control group. Conclusion. IDDM3, IDDM4 , IDDM5 but not IDDM8 may be associated with IDDM in Chinese Han nationality population.

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss by modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. This is a review aimed at analyzing the function of fertilization promoting peptide during this process. The possible molecular basis is also discussed.
Acrosome ; drug effects ; Adenylyl Cyclases ; metabolism ; Animals ; Cyclic AMP ; metabolism ; Humans ; Male ; Pyrrolidonecarboxylic Acid ; analogs & derivatives ; Signal Transduction ; drug effects ; Spermatozoa ; drug effects ; physiology ; Thyrotropin-Releasing Hormone ; analogs & derivatives ; pharmacology

OBJECTIVETo investigate polymorphisms in the gene for lipoprotein lipase (LPL) in Chinese populations with coronary heart disease (CHD) and to inquire into the relationship between these polymorphisms in LPL gene and CHD.

METHODSGenomic DNA was extracted from patients with CHD and normal control subjects using a salting out method. The entire coding region and flanking sequences of all coding exons of the LPL gene were amplified by PCR technique and PCR products were detected by denaturing high-performance liquid chromatography (DHPLC) and sequenced with a dideoxy terminal termination method.

RESULTSA novel polymorphic site, G830A, that is within the fifth exon of the LPL gene was found. The 192 codon CGA was changed into CAA and resulted in the substitution of glutamine for arginine. Between the control and CHD groups, chi-square test showed no significant difference in the frequencies of the A/A genotype and A allele (P > 0.05). However, the frequencies of A/A genotype and A allele (0.653 and 0.786) in CHD patients with high plasma triglyceride/lowed plasma high density lipoprotein cholesterol were higher than those (0.415 and 0.642) in CHD patients without hyperlipidemia (P < 0.05).

CONCLUSIONNo direct association was found between the LPL Arg192-->Gln substitution polymorphism and CHD, but there is a significant positive correlation between the A/A genotype of the LPL gene and CHD associated with high triglyceride/lowed high density lipoprotein cholesterol. This study may provide new data for exploring the molecular mechanism of CHD.

Alleles ; Apolipoproteins ; blood ; Cholesterol, HDL ; blood ; Chromatography, High Pressure Liquid ; methods ; Coronary Disease ; blood ; enzymology ; genetics ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Gene Frequency ; Humans ; Hypertriglyceridemia ; blood ; genetics ; Lipoprotein Lipase ; genetics ; Lipoproteins ; blood ; Polymorphism, Genetic

Objective To understand with greater clearness the genetic polymorphism of (GCT)n repeat of MHC class I chain-related gene A (MICA)in some Chinese populations and provide preliminary genetic evidence for the independent origin of Chinese Baima Tibetan (BMT). Methods ACD-blood or saliva specimens of 411 unrelated individuals from four Chinese populations were collected. A primer pair spanning exon 5 of MICA gene was used to amplify the GCT region. Alleles were detected by PCR and denaturing PAGE. Comparison of the allelic distributions among the four populations was carried out. Results Five previously reported alleles have been observed in all the four populations, but the allelic distributions are different from one another. The most frequent allele is the A5 in all the four populations (0.325 in BMT, 0.345 in Tibetans, 0.390 in Chengdu Hans and 0.319 in Qiangs). A5.1 allele is the second most frequent allele in Chengdu Hans (0.230) and in Qiangs (0.293), while the second most frequent alleles for BMT and Tibetans are A4 (0.254) and A9 (0.272) respectively. The distribution of alleles in BMT is significantly different from that in the other three populations. Conclusion Alleles of MICA gene exon 5 are conservative in all populations studied so far. The results suggest that genetically BMT might be an independent ethnic population.

OBJECTIVETo develop a simple and reliable method for intensifying the hybridization signals of gene chips.

METHODSThe authors added EDTA and another FAM-labeled probe to the normal PCR products, denatured the mixture by heat, and then let the mixture hybridize with the fastened probes on the chip.

RESULTSWith the use of EDTA and another FAM-labeled probe, the hybridization signals increased by 6 times or greater.

CONCLUSIONAdding EDTA and another probe to the normal PCR products is a simple and efficient method to intensify the hybridization signal of chips.

Base Sequence ; DNA Probes ; chemistry ; genetics ; Edetic Acid ; chemistry ; Fluorescent Dyes ; chemistry ; Microscopy, Fluorescence ; Molecular Sequence Data ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; methods ; Reproducibility of Results

OBJECTIVETo investigate the polymorphisms of four microsatellites, D4S1534, D4S1563, D4S423 and D4S414, which are tightly linked to polycystic kidney disease 2 (PKD2) gene, and hence to provide a basis for studying the heterogeneity of adult polycystic kidney disease (APKD).

METHODSAn analysis on the DNA of some unrelated Chinese people was performed using polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and silver staining.

RESULTSIn Chinese Hans, there are 11 alleles of D4S1534, and their sizes are 142-162 bp 14 alleles of D4S1563, 205-235 bp 17 alleles of D4S423, 103-135 bp; and 15 alleles of D4S414, 236-264 bp. The polymorphism information contents of the four microsatellites are 0.872, 0.844, 0.921 and 0.871 respectively.

CONCLUSIONIn the Chinese Han people studied above, the four microsatellite markers that have many alleles are highly polymorphic genetic markers and may serve as the data of population genetics, suggesting that all four microsatellites could be used in studies on heterogeneity of APKD, linkage analysis of APKD and forensic personal identification.

Alleles ; Asian Continental Ancestry Group ; genetics ; Genetic Linkage ; Genetic Predisposition to Disease ; Humans ; Membrane Proteins ; genetics ; Microsatellite Repeats ; genetics ; Polycystic Kidney Diseases ; genetics ; Polymorphism, Genetic ; TRPP Cation Channels

OBJECTIVETo standardize the experimental procedure of the gene test for autosomal dominant cerebellar ataxias (ADCA), and provide the basis for quantitative criteria of the dynamic mutation of spinocerebellar ataxia (SCA) genes in Chinese population.

METHODSGenotyping of the dynamic mutation loci of the SCA1, SCA2, SCA3, SCA6 and SCA7 genes was performed, using florescence PCR-capillary electrophoresis followed by DNA sequencing, to investigate the variation range of copy number of CAG tandem repeat of the genes in 263 probands of ADCA pedigrees and 261 non-related normal controls. Based on the sequencing result, the bias of the CAG copy number estimation using capillary electrophoresis with different DNA controls was compared to analyze the technical detailes of the electrophresis method in testing the dynamic mutation sites.

RESULTSPCR products containing dynamic mutation loci of the SCA genes showed significantly higher mobility than that of molecular weigh marker with relatively balanced GC content. This was particularly obvious in the SCA2, SCA 6 and SCA7 genes whereas the deviation of copy number could be corrected to +/-1 when known CAG copy number fragments were used as controls. The mobility of PCR products was primarily related to the copy number of CAG repeat when the fragments contained normal CAG repeat. In the 263 ADCA pedigrees, 6 (2.28%) carried SCA1 gene mutation, 8 (3.04%) had SCA2 mutation and 81 (30.80%) harbored SCA3 mutation. The gene mutation of SCA6 and SCA7 was not found. The normal variation range of the CAG repeat was 17-36 copies in SCA1 gene, 13-30 copies in SCA2, 14-39 copies in SCA3, 6-16 copies in SCA6 and 6-13 copies in SCA7. The heterozygosity was 76.1%, 17.7%, 74.4%, 72.1% and 41.3%, respectively. The mutation range of the CAG repeat was 49-56 copies in SCA1 gene, 36-41 copies in SCA2, 59-81 copies in SCA3. Neither homozygous mutation of an SCA gene nor double heterozygous mutation of the SCA genes was observed in the study.

CONCLUSIONThe copy number of the CAG repeat in SCA genes could be calculated accurately based on the result of florescence PCR-capillary electrophoresis when limited amount of known repeat copy number controls were used. Our result supported that the notion that SCA3 gene mutation was the most common cause for ADCA, and the obtained data would be helpful for establishing quantitative criteria of the dynamic mutation of the SCA genes in Chinese.

Adolescent ; Adult ; Aged ; Ataxin-7 ; Ataxins ; Base Sequence ; Calcium Channels ; genetics ; Cerebellar Ataxia ; genetics ; Gene Dosage ; Genes, Dominant ; Genetic Variation ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Nerve Tissue Proteins ; genetics ; Trinucleotide Repeats ; Young Adult

OBJECTIVETo analyze the pattern and prevalence of partial copy deletion of deleted-in-azoospermia (DAZ) gene in the azoospermia factor C(AZFc) region of patients with idiopathic azoospermia or severe oligozoospermia.

METHODSsY581 and sY587 in DAZ gene region were analyzed by polymerase chain reaction-restriction length polymorphism(PCR-RFLP) for its deletion in 197 patients with azoospermia, 166 patients with severe oligozoospermia, and 210 fertile men as controls.

RESULTSDeletion of both DAZ1 and DAZ2 was detected in 18 patients with azoospermia and 10 with severe oligozoospermia, and the prevalence was 9.1% and 6.0% respectively. There was significant difference in deletion rate between the cases and controls.

CONCLUSIONThe frequency of partial copy deletion of DAZ gene in Chinese idiopathic azoospermia or severe oligozoospermia patients is much higher than that of fertile controls, suggesting that the deletion of DAZ1/DAZ2 may be one of the important genetic etiological factors of spermatogenesis damage. The pattern and prevalence of DAZ partial copy deletion are similar to those of Caucasians populations, and detection of DAZ gene partial copy deletion by PCR-RFLP may be adopted as an additional clinical gene diagnostic measure after AZF microdeletion detection.

Azoospermia ; complications ; genetics ; Chromosomes, Human, Y ; genetics ; Deleted in Azoospermia 1 Protein ; Gene Deletion ; Humans ; Infertility, Male ; etiology ; genetics ; Male ; Models, Genetic ; Polymerase Chain Reaction ; RNA-Binding Proteins ; genetics