香蕉束顶病毒NS株系DNA组分6的克隆及序列分析

通过常规的基因克隆技术 ,对香蕉束顶病毒NS株系代表分离物的DNA组分 6进行了克隆和序列分析 ,并将该分离物这个组分的序列与先前报道的广州天河分离物 (属NSP株系 )的该组分序列进行比较 ,结果表明 :该组分全序列、ORF及其编码的氨基酸序列在 2个分离物间的变异率分别为 3 4%、1 7%和 2 6 % .表明该组分在 2个株系代表分离物间存在较显著差异 ,从而进一步支持了广东BBTV存在 2个株系的结论 .此外 ,NS株系代表分离物DNA组分 6的全序列、ORF及其编码的氨基酸序列与澳大利亚分离物的变异率分别为 14.4%、7.5 %和 7.1% .

香蕉束顶病毒DNA组分2、3的启动子区的组织特异性分析

香蕉束顶病毒（BBTV）基因组至少由6个大小约为1．0～1．1kb的单链环状DNA组分所组成，每一个DNA组分包含编码区与非编码区。在前人的研究基础上进一步了解BBTV DNA组分启动子的功能。首先根据BBTV海南分离物的全序列，通过常规PCR扩增出长为540bp的BBTV DNA3组分启动子序列BV3．1，同时通过重叠PCR扩增出646bp的DNA2与DNA3组分非编码区拼接的重组启动子序列BV23，分别替代pBll21 35S启动子序列与gus基因进行融合，构建植物表达载体pBIBV3．1、pBIBV23。农杆菌介导转化获得的pBIBV3．1转基因烟草经GUS化学组织染色后，在其叶片的叶脉处检测到微弱的GUS活性，证实了DNA3组分的韧皮部特异表达活性；而pBIBV23转基因烟草，其叶片经GUS组织化学染色后，在叶肉、叶缘及一些叶脉上检测到弱GUS活性，这表明由BV23驱动的gus基因在烟草中类似于组成型表达，则DNA2组分转录方式可能有异于DNA3组分。

The Distribution of Repetitive DNAs Along Chromosomes in Plants Revealed by Self-genomic in situ Hybridization

The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization （GISH） procedure, fluorescence in situ hybridization （FISH） with genomic DNA to their own chromosomes （called self-genomic in situ hybridization, self-GISH） was carried out in six selected plant species with different genome size and amount of repetitive DNA. Nonuniform distribution of the fluorescent labeled probe DNA was observed on the chromosomes of all the species that were tested. The signal patterns varied among species and were related to the genome size. The chromosomes of the small Arabidopsis genome were labeled almost only in the pericentromeric regions and the nucleolus organizer regions （NORs）. The signals in the relatively small genomes, rice, sorghum, and Brassica oleracea var. capitata L., were dispersed along the chromosome lengths, with a predominant distribution in the pericentromeric or proximal regions and some heterochromatic arms. All chromosomes of the large genomes, maize and barley, were densely labeled with strongly labeled regions and weakly labeled or unlabeled regions being arranged alternatively throughout the lengths. In addition, enhanced signal bands were shown in all pericentromeres and the NORs in B. oleracea var. capitata, and in all pericentromeric regions and certain intercalary sites in barley. The enhanced signal band pattern in barley was found consistent with the N-banding pattern of this species. The GISH with self-genomic DNA was compared with FISH with Cot-1 DNA in rice, and their signal patterns are found to be basically consistent. Our results showed that the self-GISH signals actually reflected the hybridization of genomic repetitive DNAs to the chromosomes, thus the self-GISH technique would be useful for revealing the distribution of the regions where repetitive DNAs concentrate along chromosomes and some chromatin differentiation associated with repetitive DNAs in plants.

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.

Interaction Study between DNA and Histone Proteins on Single-molecule Level using Atomic Force Microscopy

DNA and histone protein are important in the formation of nucleosomal arrays, which are the first packaging level of DNA into a more compact chromatin structure. To characterize the interactions of DNA and histone proteins, we reconstitute nucleosomes using lambda DNA and whole histone proteins by dialysis and perform direct atomic force microscopy （AFM） imaging. Compared with non-specific DNA and histone binding, nucleosomes are formed within the assembled “beads-on-a-string” nucleosomal array by dialysis. These observations facilitate the establishment of the molecular mechanisms of nucleosome and demonstrate the capability of AFM for protein-DNA interaction analysis.

指甲附着物DNA检测在凶杀案中的应用

报道了指甲附着物DNA的检验在刑事案件中的成功应用,并讲述了检验过程中的二组分混合DNA分析的一些问题和解决方法。

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.

Preparation of high molecular weight （HMW） genomic DNA from tea plant （Camellia sinensis） for BAC library construction

A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for constructing a BAC Library. In order to construct a BAC library for enhancing tea plant genomics research, a new method for the preparation of tea pant high molecular weight （HMW） genomic DNA must be developed due to young tea plant leaves and shoots are notably rich in both tea polyphenols and tea polysaccharides. In this paper, a modified method for preparing high quality tea plant HMW genomi~ DNA was optimized, and the quality of tea plant genomic DNA was evaluated. The results were as follows： Critical indicators of HMW DNA preparation were the appearance of the smooth nuclei in solution （as opposed to sticky-gummy） before agarose plug solidification, non-dark colored nuclei plugs after lysis with an SDS/proteinase K solution, and the quality and quantity of HMW DNA fragments after restriction enzyme digestion. Importantly, 1% dissolved PVP-40 and 1% un-dissolved PVP-40 during the nuclei extraction steps, in conjunction with the removal of PVP-40 from the plug washing and nuclei lysis steps, were critical for achieving HWM tea plant DNA suitable for BAC library construction. Additionally, a third PFGE fraction selection step to eliminate contaminating small DNA fragments. The modifications provided parameters that may have prevented deleterious interactions from tea polyphenols and tea polysaccharides. The HMW genomic DNA produced by this new modified method has been used to successfully construct a large-insert tea plant BAC library, and thus may be suitable for BAC library construction from other plant species that contain similarly interfering compounds.

Termination of DNA Replication and the Role of Enzymes in Recombination

DNA is the genetic material of all cells, containing coded information about cellular molecules and processes. DNA consists of two polynucleofide strands twisted around each other in a double helix. The first step in cellular division is to replicate DNA so that copies can be distributed to daughter cells. Additionally, DNA is involved in transcribing proteins that direct cell growth and activities. However, DNA is tightly packed into genes and chromosomes. In order for replication or transcription to take place, DNA must firstly unpack itself so that it can interact with enzymes. DNA packing can be visualized as two very long strands that have been intertwined millions of times, tied into knots, and subjected to successive coiling. However, replication and transcription are much easier to accomplish if the DNA is neatly arranged rather than tangled up in knots. Enzymes are essential to unpacking DNA. Enzymes act to slice through individual knots and reconnect strands in a more orderly way. Hypothesizing that Termination of DNA replication proteins gave rise to those of eukaryotes during evolution, we chose the DNA polymerase （which infects microalgae） as the basis of this analysis, as it represents a primitive recombination. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal DNA are near the root of the containing all recombination. DNA polymerase delta members but that this does not contain the polymerases of other DNA. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA. DNA can be visualized as a complicated knot that must be unknotted by enzymes in order for replication or transcription to occur. It is perhaps not surprising then that connections between mathematical knot theory and biology have been discovered. By thinking of DNA as a knot, we can use knot theory to estimate how hard DNA is to unknot. This can help us estimate properties of the enzymes that unknot DNA.

Isolation and Characterization of Microsatellites in Snap Bean

The objectives of this study were to isolate and characterize microsatellites from a heat tolerant variety of snap bean (Phaseolus vulgaris L.) in order to generate polymorphic genetic markers linked to quantitative trait loci for heat tolerance. A genomic library contained 400-800 bp inserts was constructed and screened for the presence of (GA/CT) n and (CA/GT) n repeats. The proportion of positive clones yielded estimated of 3.72×10 4 such dinucleotide repeats per genome, roughly comparable to the abundance reported in other eukaryotic genomes. Twenty_six positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) n motif was much more abundant than the (CA/GT) n motif in these clones. The (GA/CT) n repeats also showed longer average repeat length (mean n =10.4 versus 6.5), suggesting that they are better candidates for yielding polymorphic genetic markers in the snap bean genome.

Primary Identification of Alien Chromatin in T911289,a Maintainer of Wheat Male Sterile Line with Cytoplasm of Aegilops kotschyi

The genomic composition of 1911289, a wheat ( Tritium aestivum L.) maintainer of K-CMS, was examined by several methods, such as genomic in situ hybridization (GISH), biochemical marking, and DNA molecular marking. The results got by GISH and PCR amplification of dispersed rye-specific repetitive DNA sequence suggested that the alien chromatin in T911289 derived from rye. Specifically PCR amplification of the rye-specific microsatellite primers (SCM9) and seed storage protein analysis indicated that the alien chromatin in T911289 had developed from the short arm of 1R chromosome of rye (1RS). PCR amplification by using microsatellite primers locating on 1BS and seed storage protein analysis also revealed that 1911289 had lost the arm of 1BS or a small distal segment of it. We conclude that T911289 is a heterogeneous population which displays two distinct different types of translocation, i.e. the Robertsonian translocation and small segment translocation. The Robertsonian translocation type observed in our study is different from the 1BL/1RS translocation which is widely used in wheat production; it may be a novel and complex translocation form. Though the linkage between the desirable agronomic traits and the deleterious genes expressed as sticky dough has not got broken in T911289, the recovery of small segment translocation will still benefit the genetic study of wheat and rye.

Methods Comparison for Microsatellite Marker Development:Different Isolation Methods,Different Yield Efficiency

Microsatellite markers have become one kind of the most important molecular tools used in various researches. A large number of microsatellite markers are required for the whole genome survey in the fields of molecular ecology,quantitative genetics and genomics. Therefore,it is extremely necessary to select several versatile,low-cost,efficient and time-and labor-saving methods to develop a large panel of microsatellite markers. In this study,we used Zhikong scallop(Chlamys farreri) as the target species to compare the efficiency of the five methods derived from three strategies for microsatellite marker development. The results showed that the strategy of constructing small insert genomic DNA library resulted in poor efficiency,while the microsatellite-enriched strategy highly improved the isolation efficiency. Although the mining public database strategy is time-and cost-saving,it is difficult to obtain a large number of microsatellite markers,mainly due to the limited sequence data of non-model species deposited in public databases. Based on the results in this study,we recommend two methods,microsatellite-enriched library construction method and FIASCO-colony hybridization method,for large-scale microsatellite marker development. Both methods were derived from the microsatellite-enriched strategy. The experimental results obtained from Zhikong scallop also provide the reference for microsatellite marker development in other species with large genomes.

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.

Incidence of human papilloma virus in esophageal squamous cell carcinoma in patients from the Lublin region

AIM:To assess the prevalence of human papilloma virus(HPV) in esophageal squamous cell carcinoma(ESCC) in the south-eastern region of Poland.METHODS:The study population consisted of 56 ESCC patients and 35 controls.The controls were patients referred to our department due to other nonesophageal and non-oncological disorders with no gross or microscopic esophageal pathology as confirmed by endoscopy and histopathology.In the ESCC patients,samples were taken from normal mucosa(56 mucosa samples) and from the tumor(56 tumor samples).Tissue samples from the controls were taken from normal mucosa of the middle esophagus(35 control samples).Quantitative determination of DNA was carried out using a spectrophotometric method.Genomic DNA was isolated using the QIAamp DNA Midi Kit.HPV infection was identified following PCR amplification of the HPV gene sequence,using primers MY09 and MY11 complementary to the genome sequence of at least 33 types of HPV.The sequencing results were computationally analyzed using the basic local alignment search tool database.RESULTS:In tumor samples,HPV DNA was identified in 28 of 56 patients(50%).High risk HPV phenotypes(16 or/and 18) were found in 5 of 56 patients(8.9%),low risk in 19 of 56 patients(33.9%) and other types of HPV(37,81,97,CP6108) in 4 of 56 patients(7.1%).In mucosa samples,HPV DNA was isolated in 21 of 56 patients(37.5%).High risk HPV DNA was confirmed in 3 of 56 patients(5.3%),low risk HPV DNA in 12 of 56 patients(21.4%),and other types of HPV in 6 of 56 patients(10.7%).In control samples,HPV DNA was identified in 4 of 35 patients(11.4%) with no high risk HPV.The occurrence of HPV in ESCC patients was significantly higher than in the controls [28 of 56(50%) vs 4 of 35(11.4%),P < 0.001].In esophageal cancer patients,both in tumor and mucosa samples,the predominant HPV phenotypes were low risk HPV,isolated 4 times more frequently than high risk phenotypes [19 of 56(33.9%) vs 5 of 56(8.9%),P < 0.001].A higher prevalence of HPV was identified in female patients(71.4% vs 46.9%).Accordingly,the high risk phenotypes were isolated more frequently in female patients and this difference reached statistical significance [3 of 7(42.9%) vs 2 of 49(4.1%),P < 0.05].Of the pathological characteristics,only an infiltrative pattern of macroscopic tumor type significantly correlated with the presence of HPV DNA in ESCC samples [20 of 27(74.1%) vs 8 of 29(27.6%) for ulcerative or protruding macroscopic type,P < 0.05].The occurrence of total HPV DNA and both HPV high or low risk phenotypes did not significantly differ with regard to particular grades of cellular differentiation,phases in depth of tumor infiltration,grades of nodal involvement and stages of tumor progression.CONCLUSION:Low risk HPV phenotypes could be one of the co-activators or/and co-carcinogens in complex,progressive,multifactorial and multistep esophageal carcinogenesis.

A hybrid neural network system for prediction and recognition of promoter regions in human genome

This paper proposes a high specificity and sensitivity algorithm called PromPredictor for recognizing promoter regions in the human genome. PromPredictor extracts compositional features and CpG islands information from genomic sequence,feeding these features as input for a hybrid neural network system (HNN) and then applies the HNN for prediction. It combines a novel promoter recognition model, coding theory, feature selection and dimensionality reduction with machine learning algorithm.Evaluation on Human chromosome 22 was ～66% in sensitivity and ～48% in specificity. Comparison with two other systems revealed that our method had superior sensitivity and specificity in predicting promoter regions. PromPredictor is written in MATLAB and requires Matlab to run. PromPredictor is freely available at http://www.whtelecom.com/Prompredictor.htm.

Helicobacter species sequences in liver samples from patients with and without hepatocellular carcinoma

AIM:Only a minority of patients carrying a defined viral aetiologic agent develop cirrhosis and ultimately hepatocellular carcinoma(HCC),the mechanism underlying the worsening is still undefined.Experimental infection by Helicobacter hepaticus in mice causes chronic hepatitis and HCC and recently,more Helicobacterspecies(Helicobacter spp.)have been detected in the liver of patients suffering from cholestatic diseases and HCC arising from non-cirrhotic liver.We investigated whether Helicobacterspp.sequences could be detected in the liver of patients with cirrhosis and HCC compared to subjects with metastasis to liver from colon cancer. METHODS:Twenty-three liver samples from patients operated upon for HCC superimposed on hepatitis C virus (HCV)-related cirrhosis and 6 from patients with resected metastases from colorectal cancer,were tested by polymerase chain reaction for presence of genomic 16S rRNA of Helicobacter genus using specific primers.DNA sequencing and cag A gene analysis were also performed. RESULTS:Genornic sequences of Helicobacter spp.were found in 17 of 20(85%)liver samples from patients with HCC and in 2 of 6 samples from patients with liver metastasis. In three samples of the first group the result was uncertain. Hpyloriwas revealed in 16 out of 17 positive samples and Helicobacter pullorum in the other. CONCLUSION:Helicobacter spp.,carcinogenic in mice, were found at a higher frequency in the liver of patients with HCV-related cirrhosis and HCC than those in patients without primary liver disease.

Variations in Laboratory-Scale Actinomycete Communities Exposed to Cadmium as Assessed by Denaturing Gradient Gel Electrophoresis Profiles

The actinomycete populations and functions in cadmium （Cd） contaminated soil were investigated by the cultivation- independent molecular methods. The genomic DNA was extracted and purified from soil adulterated with various con- centrations of Cd in the laboratory. The partial 16S rDNA genes were amplified by polymerase chain reaction （PCR） using specific primers bound to evolutionarily conserved regions within these actinomycete genes. The diversity in PCR- amplified products, as measured by denaturing gradient gel electrophoresis （EGGE）, was used as a genetic fingerprint of the population. Principle component analysis and Shannon-Weaver diversity index （H） analyses were used to analyze the DGGE results. Results showed that the two principal components accounted for only a low level of the total variance. The value H in contaminated soil was lower than that in the control at later stages of cultivation, whereas at earlier stages it was higher. Among the six sampling time points, the first, fifth and sixth weeks had the highest values of H. Significantly negative correlations between bioavallable Cd concentration and H values existed in the samples from weeks 2 （R = 0.929, P 〈 0.05） and 4 （R = 0.909, P 〈 0.05）. These results may shed light on the effect of Cd on the soil environment and the chemical behavior and toxicity of Cd to actinomycetes.

The Biology of Chilo Iridescent Virus

Chilo iridescent virus (CIV) is the type species for genus Iridovirus, and belongs to the family Iridoviridae. Since the discovery of CIV in 1966, many attempts were made to elucidate the viral genome structure. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The genome of CIV has been entirely sequenced. The CIV virion consists of an unusual three-layer structure containing an outer proteinaceous capsid, an intermediate lipid membrane, and a core DNA-protein complex containing the genome. CIV has a broad host spectrum and has, in general, a limited mortality effect on its hosts. Up to now there have been several studies about CIV describing its structure, ecology, and molecular biology. In this review study we present all these studies together to describe the CIV.

Primary 4-3-oxosteroid 5β-reductase deficiency:Two cases in China

Aldo-keto reductase 1D1(AKR1D1) deficiency,a rare but life-threatening form of bile acid deficiency,has not been previously described in China.Here,we describe the first two primary 4-3-oxosteroid 5β-reductase deficiency patients in China's Mainland diagnosed by fast atom bombardment-mass spectroscopy of urinary bile acids and confirmed by genetic analysis.A high proportion of atypical 3-oxo-4-bile acids in the urine indicated a deficiency in 4-3-oxosteroid 5β-reductase.All of the coding exons and adjacent intronic sequence of the AKR1D1 gene were sequenced using peripheral lymphocyte genomic DNA of two patients and one of the patient's parents.One patient exhibited compound heterozygous mutations:c.396C>A and c.722A>T,while the other was heterozygous for the mutation c.797G>A.Based on these mutations,a diagnosis of primary 4-3-oxosteroid 5β-reductase deficiency could be confirmed.With ursodeoxycholic acid treatment and fat-soluble vitamin supplements,liver function tests normalized rapidly,and the degree of hepatomegaly was markedly reduced in both patients.

Analysis of Methylation-Sensitive Amplified Polymorphism and Prediction of Candidate Genes Infected by SCMV in Zea mays Genome

DNA methylation is an important component of the epigenetic network, and it plays important roles in gene expression regulation and epigenetic change response to various stresses. In this study, the authors assessed the methylation patterns stressed by SCMV （sugarcane mosaic virus） in maize by methylation-sensitive amplified polymorphism （MSAP）, and identified important candidate genes related to SCMV resistance through combining microarray analysis with CpG islands prediction. The results of MSAP indicated DNA methylation levels appeared dynamic changes inoculated for 0 d, 1 d, 4 d, 5 d and 10 d. 118 candidate genes were identified infected by SCMV, which may participate in DNA methylation modification. Among them, eight candidate genes were mapped on Scmvl and Scmv2 QTL regions, which are crucial for SCMV resistance. In conclusion, DNA methylation is closely related with maize resistance to SCMV and plays an important role in regulating gene expression responded to maize resistance.