Changes in DNA base sequences in the mutant of Arabidopsis thaliana induced by low-energy N+ implantation

To reveal the mutation effect of low-energy ion implantation on Arabidopsis thaliana in vivo, T80II, a stable dwarf mutant, derived from the seeds irradiated by 30 keV N⁺ with the dose of 80×10¹⁵ ions/cm² was used for Random Amplified Polymorphic DNA (RAPD) and base sequence analysis. The results indicated that among total 397 RAPD bands observed, 52 bands in T80II were different from those of wild type showing a variation frequency 13.1%. In comparison with the sequences of A. thalianain GenBank, the RAPD fragments in T80II were changed greatly in base sequences with an average rate of one base change per 16.8 bases. The types of base changes included base transition, transversion, deletion and insertion. Among the 275 base changes detected, single base substitutions (97.09%) occurred more frequently than base deletions and insertions (2.91%). And the frequency of base transitions (66.55%) was higher than that of base transversions (30.55%). Adenine, thymine, guanine or cytosine could be replaced by any of other three bases in cloned DNA fragments in T80II. It seems that thymine was more sensitive to the irradiation than other bases. The flanking sequences of the base changes in RAPD fragments in T80II were analyzed and the mutational “hotspot” induced by low-energy ion implantation was discussed.     

Isolation and Purification of Carp Mitochondrial DNA and Structural Analysis of Its tRNA~(Cys) Gene and the Light Strand Origin

A new method is presented with which we isolated milochondrial DNA from fresh carp liver usingdifferential centrifugation and DNase treatment that gave high yield of purified product with an easyand economical procedure. Highly distinct bands were displayed in agarose gel electrophoresls ofthe product digested with restrictlon enzymes, which were successfully used in constructingrestriction map and molecular clone of mitochondrial genes. With DNAs thus obtained, we havecloned cysteine tRNA gene (tRNA~(Cys) gene) of carp mitochondria, determined the nucleotide sequenceof it and the light strand origin, and depicted the cloverleaf secondary structure of tDNA~(Cya) and thelight strand origin. Analysis of nucleotide sequences of tRNA~(Cy) genes of 5 vertebrates has revealedunusual features of carp mitochondrial tRNA~(Cy) gene as compared with their cytoplasmic counter-parts, Altogether 36 bases were found in the light strand origin of carp mitochondriaf: 11 pairs in thestem; and 14 bases in the loop. As compared with those of other 11 vertebrate species, the sequenceof the stem is very conservative while both sequence and length of the loop are quite variable. Thestructure of the stem-loop may play an important role in light strand replication.     

Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein β-5

By homologous EST searching and nested PCR a new human gene GJB5 encoding gap junction protein β-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DNA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Charcot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two missense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR analysis in skin tissue. Mutation analysis in 199 unaffected individuals revealed that two of them were carriers with the same 18 bp deletion.     

Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein β-5

By homologous EST searching and nested PCR a new human gene GJB5 encoding gap junction protein p-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DMA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Char-cot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two mis-sense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR anal     

CHANGES OF GLYCEROL CONTENT IN DIAPAUSE LARVAEOF THE ORANGE WHEAT BLOSSOM MIDGE, SITODIPLOSIS MOSELLANA （GEHIN） IN VARIOUS SEASONS

The glycerol contents in diapause larvae of the orange wheat blossom midge, Sitodiplosis mosellana (Gehin), collected from various seasons, were measured. The results showed that there was less glycerol content in larvae during living on the wheat head. Content of glycerol began to increase significantly when the larvae left the wheat head and entered the soil. A change trend of upper- lower- upper- lower in larvae glycerol contents during diapause in soil was observed from June to April of next year. More glycerol could be examined in larvae collected in summer and winter than in spring and autumn. There was not more glycerol in cocooned larvae than that in non-cocooned larvae during various seasons from the point of statistics. Comparing the glycerol content of larvae being diapause in the first year with that of larvae in the second year, there was yet no obvious difference when larvae were collected in the same season belonged to different years. Therefore, it is shown that the content of glycerol in larvae of the wheat midge in diapause is affected mainly by the seasons or diapause intensity.     

Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell lines

This study aimed to investigate the effects of arsenic trioxide(As2O3) on the mitochondrial DNA(mtDNA) of acute promyelocytic leukemia(APL) cells.The NB4 cell line was treated with 2.0 μmol/L As2O3 in vitro,and the primary APL cells were treated with 2.0 μmol/L As2O3 in vitro and 0.16 mg kg-1 d-1 As2O3 in vivo.The mitochondrial DNA of all the cells above was amplified by PCR,directly sequenced and analyzed by Sequence Navigatore and Factura software.The apoptosis rates were assayed by flow cytometry.Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As2O3 use,but the mutation spots were remarkably increased after As2O3 treatment,which was positively correlated to the rates of cellular apoptosis,the correlation coefficient:rNB4-As2O3=0.973818,and rAPL-As2O3=0.934703.The mutation types include transition,transversion,codon insertion or deletion,and the mutation spots in all samples were not constant and regular.It is revealed that As2O3 aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo.Mitochondrial DNA might be one of the targets of As2O3 in APL treatment.     

一个氨基糖苷类抗生素致聋家系线粒体DNA突变研究

应用PCR、PCR－SSCP和DNA序列分析等分子生物学技术,对一个有明确氨基糖苷类抗生素应用史的母系遗传耳聋家系共8人(包括聋人和听力正常者) 的线粒体DNA进行研究,结果显示,家系中有4份样品存在线粒体DNA 12S rRNA 1 555位点A→G的突变。提示线粒体DNA点突变是导致该家系致聋的主要因素之一。 Abstract:Blood samples were obtained from a pedigree with aminoglycoside antibiotic induced deafness.DNA was extracted from the isolated leukocytes.The mitochondrial DNA fragments were detected by PCR－SSCP and DNA sequencing.It was found that four individuals from the pedigree carried 1 555 A→G mutation.From our results,mitochondrial DNA mutation may be one of major factors in aminoglycoside antibiotic induced deafness.     

Sequence analysis of lacZ~- mutations induced by ion beam irradiation in double-stranded M13mp18DNA

While M13mpl8 double-stranded DNA was irradiated with ion beam, and transfected into E. coli JM103, a decrease of transfecting activity was discovered. The lacZ-mutation frequency at 20% survival could reach (3.6-16.8) × 104, about 2.3-10 times that of unirradiated M13DNA. Altogether, 27 lacZ~ mutants were select-ed, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants contain more than one mutational base sites (some of them even have 5-6 mutational base sites in 250-bp region ex-amined) ; this dense distribution of base changes in polysites has seldom been seen in X-rays, γ-rays or UV induced DNA mutations. Our experiments also showed that the types of base changes include transitions( 50 % ), transversions (45% ) and deletion (5% ); no addition or duplication was observed. The transitions were mainly C→T and A→G; the transversion     

利用差减杂交分离具有进化保守性的睾丸表达的基因

The mechanisms of sexual development and regulation display extraordinary diversity between phyla. However, recent studies show that there must be underlying evolutionary conservatism between different mechanisms. In order to examine evolutionary conservatism in molecular mechanisms of sex determination, the natural sex reversal process of the rice field eel (Monopterus albus) was used to identify conserved functional genes involved in sex determination and differentiation. Firstly, a mini cDNA library was constructed from the cDNAs expressed specifically in the human testis. The cDNA library was screened with first strand cDNA probes from the intersex gonads of rice field eels. Secondly, likely positive clones were arrayed onto two nylon membranes, which were then hybridized with the first strand cDNAs probes from rice field eel intersex and female gonads respectively. Two positive clones were obtained, which showed obvious differential expression between intersex and female gonads. DNA sequencing analysis reveal that the insert fragments of these two clones were 895 bp and 596 bp long respectively. BLAST results indicate that the 895 bp fragment is the 3′ part of the No.AJ011779 gene, located on 6q21, encoding a homologue of yeast Sec63p protein. An interesting finding is that one member of the human Sec gene family, Sec61γ , is expressed in the testis and involved in the process of testis development. The 596 bp fragment was 97% identical with the No.BG723880 EST, located on clone RP11 12M5 of chromosome 1 This needs further study. We infer that these two genes should also play a role in sexual development.     

结核分枝杆菌rpoB基因突变的检测(简报)

结核病主要是由结核分枝杆菌(Mycobacterium tuberculosis)引起的一种慢性传染性疾病。利福平是结核病化疗方案中一个关键性的药物,它在结核病的短程化疗中起着重要的作用。但是,在我国结核菌对利福平的耐药发生率呈上升局势,而通过传统的依赖生物生长的药敏试验方法进行结核菌对利福平耐药性检测所需时间较长(4-8周),不能满足临床早期开展有效化疗的需要,所以迫切需要建     

Detection of point mutations in the chloroplast genome by single-stranded conformation polymorphism analysis

Single-stranded conformation polymorphism (SSCP) analysis was used to examine the mutations of the chloroplast 16S rRNA locus of streptomycin-resistant mutants in Nicotiana plumbaginifolia. DNA fragments of 121, 517, 968 and 1578 bp, each possessing a known point mutation, were generated by polymerase chain reaction (PCR). The resulting fragments were denatured and separated by nondenaturing polyacrylamide gel electrophoresis. Compared to the patterns of the wild-type DNA fragments, the bands of the single-stranded DNA fragments of 121 and 517 bp with base changes were shifted. However, no pattern variations were detected from the DNA fragments of 968 and 1578 bp generated from both wild-type and mutants.     

PCR-SSCP的效果分析

PCR-SSCP是一种以PCR为基础的单链构象多态性分析技术,是DNA已知突变的检测或未知变异分析中常用和实用的技术之一。影响SSCP试验效果的因素有很多,本研究主要对凝胶浓度和是否添加甘油两个因素进行分析与探讨。结果表明,凝胶浓度12%和添加甘油终浓度10%的条件下可以得到满意的结果。     

Detection of single-base mutations by reaction of DNA heteroduplexes with a water-soluble carbodiimide followed by primer extension: application to products from the polymerase chain reaction

A new method was developed for the detection of single-base mutations in DNA. The polymerase chain reaction was used to prepare DNA fragments of up to 1 kb. Fragments that differed by a single-base were combined, denatured and renatured to generate heteroduplexes. The heteroduplexes were reacted with a water-soluble carbodiimide under conditions in which the carbodiimide modified Gs and Ts that were not base paired. The DNA was then used as a template for primer extension with Taq DNA polymerase under conditions in which extension terminated at the site of the carbodiimide-modified base and generated a 32P-labeled fragment that was identified by polyacrylamide gel electrophoresis as a fragment smaller than the full length product. The procedure detected all four general classes of single-base mutations in several different sequence contexts. The site of the mutation was located to within about 15 bp. Extension with both a 5'- and a 3'-primer made it possible to confirm the site of the mutation in most DNA samples or detect a mutation in heteroduplexes even if a G or T in one strand was unreactive because of its sequence context. The procedure appears to have several advantages over previously published techniques.     

A simple method for non-radioactive PCR-SSCP using MDE gel solution and a midi gel format: application for the detection of variants in the GLUT1 and CTLA-4 genes

The need to identify disease-causing mutations and DNA polymorphisms has increased with the continuing identification of new candidate genes. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques most widely used to identify a mutant sequence or a polymorphism in a known gene. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis on sequencing gels for detection were labour intensive and time-consuming. Here we describe a simple SSCP protocol using MDE gel solution and a midi gel format to detect SSCP variations in the glucose transporter gene GLUT1, that we have previously analysed with the standard radioactive SSCP protocol, and we have also tested this method on the previously described point mutation (A/G transition in exon 1) of the CTLA-4 (cytotoxic T lymphocyte associated-4) gene. All known variants were detected. Based on the results, this technique appears to be simple, with no use of radioactive labels and with easy handling of the gel. Furthermore, it needs little optimisation, is relatively rapid and highly sensitive. We propose this method for the first screening for candidate gene variants.     

New mutant gene (transthyretin Arg 58) in cases with hereditary polyneuropathy detected by non-isotope method of single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) was analyzed to detect a mutation in the transthyretin (TTR) gene from the mother and son showing polyneuropathy with carpal tunnel syndrome. DNA segments containing TTR coding sequence were amplified by polymerase chain reaction, heat denatured and electrophoresed on a neutral polyacrylamide gel. The single-stranded DNA fragments in the gel were transferred to a nylon membrane and hybridized with biotinylated TTR cDNA probe, followed with chemiluminescent DNA detection. The mobility shift was found in the fragments of exon 3 from the patients' DNA. Sequencing analyses of the exon 3 confirmed a T----G base change, resulting in a Leu 58----Arg substitution. TTR Arg 58 is the first mutant TTR gene that has been detected by SSCP analysis. The rapid and sensitive detection of new mutations at various sites on the TTR gene is hereafter possible by the present method in the facilities for non-radioactive experiments.     

Purification and properties of nuclear and cytoplasmic DNA polymerases from JLS-V9 cells.

Four DNA polymerases, two enzymes from the nucleus and two from the cytoplasm, were purified 2000- to 7000-fold from continuous mouse cell-line (JLS-V9), by sequential column chromatography. Each of these polymerases require all the deoxynucleoside-5′-triphosphates in order to synthesize DNA, using activated DNA as a primer-template, and can copy the ribonucleotide strand of hybrid templates, but their rate of efficiency varies. The molecular weights of these DNA polymerases range from 35,000 to 160,000, as estimated by Sephadex column chromatography. Three out of the four DNA polymerases are probably a single polypeptide chain, since they have a single major band in polyacrylamide gel electrophoresis as well as one enzymatically active peak in guanidine hydrochloride gel filtration. The highly purified preparation of the high molecular weight cytoplasmic DNA polymerase contains two major bands in sodium dodecyl sulfate polyacrylamide gel electrophoresis and two enzymatically active peaks in guanidine hydrochloride gel filtration.     

Purification de l'alcool deshydrogénase de tomate par chromatographie d'affinité

Tomato alcohol dehydrogenase has been purified 99-fold by affinity chromatography on Blue Sepharose CL-6B with 37% yield. The enzyme so obtained is homogenous in polyacrylamide gel electrophoresis. By adding 20% glycerol to the extraction and purification buffers, an enzyme is obtained which is stable for several months at 4°. The molecular weight values determined by gel filtration (Sephadex G 200) and polyacrylamide gradient gel electrophoresis on one hand and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate on the other, show that the enzyme exists in dimeric form.     

Validation of fluorescent SSCP analysis for sensitive detection of p53 mutations

We have developed a fluorescence-based single strand conformation polymorphism (SSCP) method that offers fast and sensitive screening for mutations in exons 5-8 of the human p53 gene. The method uses an ABI 377 DNA sequencer for unique color detection of each strand, plus accurate alignment of lanes for better detection of mobility shifts. To validate the method, 21 cell lines with reported mutations in p53 exons 5-8 were analyzed by SSCP using various gel conditions. The sensitivity for mutation detection was 95% for all cell lines studied, and no false positives were seen in 10 normal DNA samples for all four exons. Experiments mixing known amounts of tumor and normal DNA showed that mutations were detected even when tumor DNA was mixed with 80% normal DNA. Fluorescent SSCP analysis using the ABI sequencer is a useful tool in cancer research, where screening large numbers of samples for p53 mutations is desired.     

SSCP和HMA方法在马MHC-Ⅰ类分子基因多态性研究中的应用

摘要: 文章使用SSCP和HMA两种基于聚丙烯酰胺凝胶电泳的方法对马MHC-I类分子基因多态性进行了分析。在应用SSCP法进行分析时, 尽管经过实验条件优化, 仍未得到对MHC-I基因理想的分离效果, 提示该方法对分离多态性较高的基因有一定局限性。在对HMA法用参考标准DNA对影响DNA分子构象的温度和变性剂浓度等实验条件进行优化后, 获得了对马MHC-I类分子基因较好的分离效果。6、7、8、9和10号马的样本在相对应泳道上分别出现了6、5、6、5和7个条带。从凝胶中进行DNA条带回收后克隆测序的结果表明, 这一方法可以有效地分离高度多态性的MHC-I类分子基因。