SSR - PCR反应体系建立与优化的研究概述

SSR标记是基于PCR基础上的一种分子标记,其扩增效果直接影响到SSR分析,近年来从不同方面对SSR - PCR反应体系的建立与优化进行了大量的研究.该文简要介绍SSR - PCR扩增反应体系建立的方法,综述SSR - PCR扩增反应的应用及其近展,并对存在的问题进行探讨,对今后的发展进行展望,以期为从事该方面的研究奠定基础.     

麂属动物陈旧皮张标本的DNA提取及PCR扩增

本实验用改进的方法从保存于标本馆的动物皮张标本中提取ＤＮＡ,所得ＤＮＡ片段的分子量从１００ｂｐ到１ｋｂ以上。利用线粒体ＤＮＡ细胞色素ｂ通用引物和ＰＣＲ技术,从小麂、印度麂、贡山麂、费氏麂、黑麂ＤＮＡ中扩增出３０７ｂｐ的细胞色素ｂ特异片段。用２８种限制性内切酶对从新鲜血样和从陈旧皮张标本中所得扩增片段进行酶切分析,发现只有４个酶在这个片段上有切点,其中ＨａｅⅢ和ＨａｐⅡ的识别位点在各种麂中有所不同。     

用PCR法检测献血员单个核细胞中的CMV—DNA

107份自愿献血新鲜血标本和22份库存血标本分别用PCR检测单个核细胞中巨细胞病毒DNA（CMV－DNA）和用ELISA检测其血浆中CMV－IgG。结果CMV－DNA阳性率达80．4％和77．3％,CMV－IgC阳性纺为65．9％。其中CMV－IgG阳性者,基本上都携带CMV－DNA;CMV－IgG阴性者,亦有部分携带CMV－DNA。因此认为献血员血液中CMV高带毒率应引起临床有关部门的高度重视;PCR是筛选无传播CMV危险性血液制品的最可靠方法。     

血沉标本中人巨细胞病毒DNA的检出

用聚合酶链反应（ＰＣＲ）和地高辛标记探针（ｄｉｇ－ｐｒｏｂｅ）检测临床血沉标本中人巨细胞病毒（ＨＣＭＶ）ＤＮＡ。结果表明,人群血标本中ＨＣＭＶ－ＤＮＡ携带率较高;ＰＣＲ技术较ｄｉｇ－ｐｒｏｂｅ更敏感、快速、简便,二者检出ＨＣＭＶ－ＤＮＡ阳性率分别为８３．３％和６０．３％;ＨＣＭＶ－ＤＮＡ检出率、ＨＣＭＶ－ＩｇＭ检出率与血沉值高低之间无相关性。     

纵纹腹小鸮DNA指纹谱探针的筛选和初步应用

以人工合成的微卫星序列 (GTG) 5,(GT) 8,(CAC) 5和人源小卫星 33 1 5作引物 ,扩增纵纹腹小的基因组DNA ,产生多态性DNA片段 ,回收了 8个表现个体特异性的片段。当用小的基因组总DNA探针与它们杂交时 ,其中 2个表现阳性 ,说明PCR方法扩增出的高变异产物含有重复序列。用含重复序列的个体特异性PCR产物作探针 ,与无关个体小基因组DNA的HaeⅢ酶切产物进行DNA印迹 ,获得了变异性较高的DNA指纹图谱。且通过对京白鸡家系分析表明 ,用小基因组DNA的PCR产物分离制备的探针所获得的DNA指纹图带能够稳定的遗传。因此 ,高变异的PCR产物可以有效地用作DNA指纹探针。     

Analysis of ribosomal DNA restriction patterns in the genusKluyveromyces

Riboprinting was used to determine the relationships among strains belonging to 15 species of the genusKluyveromyces. The small subunit ribosomal RNA gene (SSU rDNA) was amplified using the Polymerase Chain Reaction (PCR) and subjected to a battery of nine restriction enzymes. Similarity coefficients between strains were calculated based on shared and unique restriction fragments. Cluster analysis revealed three major groups that generally correlated with previously reported relationships based on other molecular data. Variations in SSU rDNA restriction fragments may be used for differentiation of theKluyveromyces strains included in this study.The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Relevance of the branch point adenosine, coordination loop, and 3' exon binding site for in vivo excision of the Sinorhizobium meliloti group II intron RmInt1

Excision of the bacterial group II intron RmInt1 has been demonstrated in vivo, resulting in the formation of both intron lariat and putative intron RNA circles. We show here that the bulged adenosine in domain VI of RmInt1 is required for splicing via the branching pathway, but branch site mutants produce small numbers of RNA molecules in which the first G residue of the intron is linked to the last C residue. Mutations in the coordination loop in domain I reduced splicing efficiency, but branched templates clearly predominated among splicing products. We also found that a single substitution at the EBS3 position (G329C), preventing EBS3-IBS3 pairing, resulted in the production of 50 to 100 times more RNA molecules in which the 5' and 3' extremities were joined. We provide evidence that these intron molecules may correspond to both, intron circles linked by a 2'-5' phosphodiester bond, and tandem, head-to-tail intron copies.     

PCR analysis to distinguish between alleles of a member of a multigene family correlated with wheat bread-making quality

Good or poor wheat bread-making quality is associated with two allelic pairs at theGlu-D1 complex locus, designated 1Dx5-1Dy10 and 1Dx2-1Dy12, respectively. The polymerase chain reaction (PCR) verified the presence of the HMW-glutenin 1Dx5 gene from genomic DNA extracted from part of the endosperm of a single dry seed, or a small amount of leaf or root tissue, of several bread-wheat cultivars. This easy, quick, and non-destructive PCR-based approach is proposed as a very efficient and safe alternative to standard procedures for selecting bread-wheat genotypes with good bread-making properties.     

Importin beta interacts with the endoplasmic reticulum-associated degradation machinery and promotes ubiquitination and degradation of mutant alpha1-antitrypsin

The mechanism by which misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for proteasomal degradation is still poorly understood. Here, we show that importin β, a well established nucleocytoplasmic transport protein, interacts with components of the retrotranslocation complex and promotes ER-associated degradation (ERAD). Knockdown of importin β specifically inhibited the degradation of misfolded ERAD substrates but did not affect turnover of non-ERAD proteasome substrates. Genetic studies and in vitro reconstitution assays demonstrate that importin β is critically required for ubiquitination of mutant α1-antitrypsin, a luminal ERAD substrate. Furthermore, we show that importin β cooperates with Ran GTPase to promote ubiquitination and proteasomal degradation of mutant α1-antitrypsin. These results establish an unanticipated role for importin β in ER protein quality control.     

Hierarchal gene trees and molecular phylogeny of the Rotifera: use of the Polymerase Chain Reaction (PCR) to dissect ecological and evolutionary patterns

The study of rotifer phylogenies and the analysis of population-level processes historically have been disjunct. This is despite a growing recognition that there are many ways in which rotifer population biologists and ecologists might profit from the availability of a comprehensive phylogeny of the group. New molecular methods which can be applied to a wide range of genetic systems and systematic grades will shortly eliminate the methodological (and perhaps conceptual) distinction between these fields. Of particular importance is the development of the Polymerase Chain Reaction (PCR), a technique of synthetic DNA amplification which produces concentrated preparations of selected genes from complex mixtures of nuclear and mitochondrial genomes. Analysis of PCR products can provide hierarchal genetic comparisons from the level of local rotifer populations through broad evolutionary (at least molecular) phylogenies.     

Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.     

T-LAK Cell-originated Protein Kinase (TOPK) Phosphorylation of Prx1 at Ser-32 Prevents UVB-induced Apoptosis in RPMI7951 Melanoma Cells through the Regulation of Prx1 Peroxidase Activity

Protein kinases are potential targets for the prevention and control of UV-induced skin cancer. T-cell-originated protein kinase (TOPK) is highly expressed in skin cancer cells, but its specific function is still unknown. We investigated the role of TOPK in UVB-induced apoptosis in RPMI7951 human melanoma cells. Liquid chromatography-tandem mass spectrometry analysis was used to identify proteins that bind with TOPK. Immunofluorescence, Western blot, and flow cytometry were used to assess the effect of UVB on TOPK, peroxiredoxin 1 (Prx1), and apoptosis in RPMI7951 cells. TOPK binds with Prx1 and its phosphorylation of Prx1 at Ser-32 is important for regulation of H₂O₂-mediated signal transduction. Analysis of the CD spectra of Prx1 and mutant Prx1 (S32A) proteins showed that the secondary structure of Prx1 was significantly altered by phosphorylation of Prx1 at Ser-32. UVB irradiation induced phosphorylation of TOPK in RPMI7951 human melanoma cells and phosphorylated TOPK co-localized with Prx1 in the nucleus. UVB induced the peroxidase activity of Prx1 in vitro and ex vivo. Following treatment with UVB, H₂O₂ levels and apoptosis were increased in RPMI7951 cells stably expressing TOPK siRNA or stably mutant Prx1 (S32A). Phosphorylation of Prx1 (Ser-32) by TOPK prevents UVB-induced apoptosis in RPMI7951 melanoma cells through regulation of Prx1 peroxidase activity and blockade of intracellular H₂O₂ accumulation.     

Differential expression of sodium channel β subunits in dorsal root ganglion sensory neurons

The small-diameter (<25 μm) and large-diameter (>30 μm) sensory neurons of the dorsal root ganglion (DRG) express distinct combinations of tetrodotoxin sensitive and tetrodotoxin-resistant Na(+) channels that underlie the unique electrical properties of these neurons. In vivo, these Na(+) channels are formed as complexes of pore-forming α and auxiliary β subunits. The goal of this study was to investigate the expression of β subunits in DRG sensory neurons. Quantitative single-cell RT-PCR revealed that β subunit mRNA is differentially expressed in small (β(2) and β(3)) and large (β(1) and β(2)) DRG neurons. This raises the possibility that β subunit availability and Na(+) channel composition and functional regulation may differ in these subpopulations of sensory neurons. To further explore these possibilities, we quantitatively compared the mRNA expression of the β subunit with that of Na(v)1.7, a TTX-sensitive Na(+) channel widely expressed in both small and large DRG neurons. Na(v)1.7 and β subunit mRNAs were significantly correlated in small (β(2) and β(3)) and large (β(1) and β(2)) DRG neurons, indicating that these subunits are coexpressed in the same populations. Co-immunoprecipitation and immunocytochemistry indicated that Na(v)1.7 formed stable complexes with the β(1)-β(3) subunits in vivo and that Na(v)1.7 and β(3) co-localized within the plasma membranes of small DRG neurons. Heterologous expression studies showed that β(3) induced a hyperpolarizing shift in Na(v)1.7 activation, whereas β(1) produced a depolarizing shift in inactivation and faster recovery. The data indicate that β(3) and β(1) subunits are preferentially expressed in small and large DRG neurons, respectively, and that these auxiliary subunits differentially regulate the gating properties of Na(v)1.7 channels.     

Prospecting metagenomic enzyme subfamily genes for DNA family shuffling by a novel PCR-based approach

DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64-99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering.     

Linkage Mapping of DNA Markers Generated with Specific and Non-Specific Gene Primers in Soybean

Polymerase chain reaction (PCR) has been used extensively in the construction of linkage maps for many cultivated crops including soybean, [Glycine max (L.) Merr]. In this study, four sets of oligonucleotide primer pairs of known genes (pearl millet Adh 1, nodule specific proline-rich protein, Drosophila homeobox, heat shock protein), several different combinations from kits A, D, E, and J of arbitrary primers and five primer pairs of soybean simple sequence repeats of varying length (Satt 9, Satt 20, Satt 42, Satt 64, and Satt 30) were utilized in PCR to identify molecular markers which were then used to construct a genetic linkage map. DNA for the PCR reactions was isolated from 65 recombinant inbred soybean lines resulting from crossing PI 290,136 and BARC-2 (Rj ₄ ), followed by self-pollination for seven generations without selection. Mapmaker 3.0, a computer package, was used for construction of the linkage map. A total of 43 polymorphic markers were identified; 30 markers were linked and distributed among 5 linkage groups while 13 markers were unlinked. Arbitrary primers revealed more polymorphisms than specific primers. A combination of arbitrary primers A5 and A18 revealed the maximum number of polymorphic bands. Five observed linkage groups can be expanded in future soybean research by using additional markers.     

Transport of phenylalanine into vacuoles isolated from barley mesophyll protoplasts

The energy-dependent transport of phenylalanine into isolated vacuoles of barley (Hordeum vulgare L.) mesophyll protoplasts has been studied by silicone-layer floatation filtering. The uptake of this aromatic amino acid into the vacuolar compartment is markedly increased by MgATP, showing saturation kinetics; the K _m values were 0.5 mM for MgATP and 1.2 mM for phenylalanine. V _max for phenylalanine transport was estimated to 140 nmol phenylalanine·(mg·Chl)^-1·h^-1. The transport shows a distinct pH optimum at 7.3 and is markedly inhibited by 40 mM nitrate. Azide (1 mM) and vanadate (400 M) had no or little effect on rates of transport while p-fluorophenylalanine seemed to be an effective inhibitor, indicating a possible competition at an amino-acid carrier. Ionophores such as valinomycin, nigericin or gramicidin were strong inhibitors of phenylalanine transport, indicating that this process is coupled to both the transmembrane pH gradient (pH) and the transmembrane potential ().Abbreviations and symbols BSA bovine serum albumin - Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - pH transmembrane pH gradient - transmembrane potential     

In Vitro Activities of the Multifunctional RNA Silencing Polymerase QDE-1 of Neurospora crassa

QDE-1 is an RNA- and DNA-dependent RNA polymerase that has functions in the RNA silencing and DNA repair pathways of the filamentous fungus Neurospora crassa. The crystal structure of the dimeric enzyme has been solved, and the fold of its catalytic core is related closely to that of eukaryotic DNA-dependent RNA polymerases. However, the specific activities of this multifunctional enzyme are still largely unknown. In this study, we characterized the in vitro activities of the N-terminally truncated QDE-1ΔN utilizing structure-based mutagenesis. Our results indicate that QDE-1 displays five distinct catalytic activities, which can be dissected by mutating critical amino acids or by altering reaction conditions. Our data also suggest that the RNA- and DNA-dependent activities have different modes for the initiation of RNA synthesis, which may reflect the mechanism that enables the polymerase to discriminate between template nucleic acids. Moreover, we show that QDE-1 is a highly potent terminal nucleotidyltransferase. Our results suggest that QDE-1 is able to regulate its activity mode depending on the template nucleic acid. This work extends our understanding of the biochemical properties of the QDE-1 enzyme and related RNA polymerases.