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.

p21^(WAF1/CIP1) Gene DNA Sequence Change and Their Relationship with the Phenotype of Human Osteosarcoma

Objective: To investigate the p21WAF1 /CIP1gene DNA sequence change and their relationship with the phenotype of human osteosarcoma. Methods: p21WAF1 /CIP1gene DNA of 36 osteosarcoma spec- imens was examined by using polymerase chain reaction-single strand conformation polymorphism (PCR- SSCP) method. The PCR products were sequenced directly. Results: In p21WAF1 /CIP1 gene exon3 of 36 cases of human osteosarcoma, the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 17 cases with the incidence being 44.4%. In 10 normal blood samples, DNA sequence analysis showed the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 8 cases with the incidence being 80%. Conclusion: The novel location of p21WAF1 /CIP1gene polymorphism of osteosarcoma, but not mutation was de?ned, and this location might provide the meaningful reference for the further research of p21WAF1/CIP1 gene.p2lWAF1/CIP1基因DNA序列分析及其与骨肉瘤表型的关系

Sequence Analysis of Mitochondrial DNA D-loop Region in Xinjiang Goose

[Objective] The sequences of mitochondrial DNA D-loop region of Xinjiang Goose with three different colors of plumage were analyzed in order to study the genetic diversity of Xinjiang Goose, as well as the phylogeny and evolution. [Method] Ten geese were selected randomly from the core populations of grey-, mosaic- and white-plumaged Xinjiang Goose respectively with a total number of thirty as experi- mental materials, of which the blood samples were collected from the largest vein under the wing （brachial vein） for DNA extraction. Sequences of mitochondrial DNA D-loop regions were determined using DNA sequencing technology to analyze the polymorphism. In addition, the genetic distances among different populations were estimated through the comparison with the reference sequences. [Resull] The con- tents of A, G, C and T nucleotides in the D-loop region of Xinjiang Goose were 28.85%, 17.05%, 25.38% and 28.72%, respectively. The average haplotype diversity and nucleotide diversity of Xinjiang Goose were 0.583 and 0.056. Xinjiang Goose and Greylag Goose were clustered into the same group. [Conclusion] The results showed that Xinjiang Geese with three different colors of plumage all descend from Greylag Goose （Anser anser）.

Characterization of Two Groups of Low_copy and Specific DNA Sequences Isolated from Chromosome 7B of Common Wheat

Recent work revealed that, in the genomes of polyploid wheat, there exists a class of low_copy and chromosome_specific sequences that are labile upon polyploid formation. This class of sequences was proposed to play a critical role in the stabilization and establishment of nascent plant polyploids as new species. To further study this issue, five wheat chromosome 7B_specific sequences, isolated from common wheat (Triticum aestivum L.) by chromosome microdissection, were characterized. The sequences were studied by genomic Southern hybridizations on a collection of polyploid wheats and their diploid progenitors. Four sequences hybridized to all polyploid species, but at the diploid level to only species closely related to the B_genome of polyploid wheat. This indicates that these sequences originated with the divergence of the diploid species, and was then vertically transmitted to polyploids. One sequence hybridized to all species at both the diploid and polyploid levels, suggesting its elimination after the polyploid wheat formation. The hybridization of this sequence to two synthetic polyploid wheats indicated that sequence elimination is a rapid event and probably related to methylation status of the sequence. Based on the above results, we suggest that selective changes of low_copy sequences occur rapidly after polyploid formation, which may contribute to the differentiation of chromosomes in newly formed allopolyploid wheats.

Preliminary Studies on Identification of Lycium Linn.Germplasm Resources by nrDNA ITS Sequencing

[Objective] The study aimed to identify woltberry （Lycium Linn.） germplasm resources at molecular level by analyzing the nrDNA ITS sequence. [Method] Genomic DNA from woltberry leaves extracted by modified CTAB method were regarded as templates for PCR amplification by specific primer, clone and sequencing. [Result] The nrDNA ITS sequences were obtained and then differentiated among three tested materials. [Conclusion] PCR amplification and sequencing on nrDNA ITS is a feasible approach to identify different woltberry germplasm resources.

Molecular phylogeny and evolution of Scomber(Teleostei:Scombridae)based on mitochondrial and nuclear DNA sequences

A molecular phylogenetic analysis of the genus Scomber was conducted based on mitochondrial(COI,Cyt b and control region) and nuclear(5S rDNA) DNA sequence data in multigene perspective.A variety of phylogenetic analytic methods were used to clarify the current taxonomic classification and to assess phylogenetic relationships and the evolutionary history of this genus.The present study produced a well-resolved phylogeny that strongly supported the monophyly of Scomber.We confirmed that S.japonicus and S.colias were genetically distinct.Although morphologically and ecologically similar to S.colias,the molecular data showed that S.japonicus has a greater molecular affinity with S.australasicus,which conflicts with the traditional taxonomy.This phylogenetic pattern was corroborated by the mtDNA data,but incompletely by the nuclear DNA data.Phylogenetic concordance between the mitochondrial and nuclear DNA regions for the basal nodes supports an Atlantic origin for Scomber.The present-day geographic ranges of the species were compared with the resultant molecular phylogeny derived from partition Bayesian analyses of the combined data sets to evaluate possible dispersal routes of the genus.The present-day geographic distribution of Scomber species might be best ascribed to multiple dispersal events.In addition,our results suggest that phylogenies derived from multiple genes and long sequences exhibited improved phylogenetic resolution,from which we conclude that the phylogenetic reconstruction is a reliable representation of the evolutionary history of Scomber.

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.

Identification of p63 expression in human lung cancer: analysis by complementary DNA and tissue microarray

Objective: To evaluate p63 expression at mRNA transcripts and protein levels in lung squamous cell cancer (SCC), adenocarcinoma, large cell lung cancer (LCLC) and small cell lung cancer (SCLC) and their matched metastatic tumors. The association between p63 expression and p63 locus at chromosomal 3q27 q29 was also investigated. Methods: p63 mRNA expression levels in a large series of lung cancers including SCC, adenocarcinoma, LCLC, SCLC and their matched metastatic tumors were analyzed by cDNA microarray technology. A tissue microarray from 150 primary lung cancer specimens was constructed and used for immunohistochemical detection of p63 protein expression. Chromosomal imbalances at the p63 locus in 70 primary lung cancers samples were studied by comparative genomic hybridization (CGH) technology. Results: mRNA levels were 10 fold in SCC compared to LCLC, SCLC, and adenocarcinoma. Interestingly, the mRNA expression of p63 in metastatic carcinomas was significantly higher than that in their matched primary tumors ( P <0 001). Immunohistochemistry demonstrated that p63 expression was 94.64% in SCC but only 1 79% in lung adenocarcinoma and 2 of 4 LCLC were positive staining. All the results in of SCLC were negative. There was a statistically significant difference for p63 positivity between pT1 tumors and those of higher stage ( P =0 035). The CGH results indicated that p63 locus at chromosomal 3q27 q29 was overrepresented in SCC. p63 immunopositivity correlated significantly with pronounced gains of the p63 locus at chromosomal 3q27 q29 (P=0.0001), indicating that strong expression of p63 in lung SCC correlated with increased gene amplification. Conclusion: p63 might play an important role not only in squamous differentiation of lung cancer but also in tumor development and progression.

DNA barcoding discriminates Pampus minor(Liu et al.,1998) from Pampus species

Although Pampus minor has been classified as a new species, it still remains controversial. Was used a DNA barcoding technique based on homologous sequence analysis of the16S and CO1 genes to clarify the confusion over the identification of this species. Among 12 individuals whose genetic distance was 0.002, two haplotypes were found. According to the 16S sequences, the genetic distances ranged from 0.121 to 0.133 between P. minor and other Pampus species. Although the same the genetic distance between the two P minor haplotypes was generated using CO1 sequences, the haplotype of Pm22-23, Pm28, and Pm32-33 was the same as that of Pci EF607462 and EF607466, while the haplotype of Pm24-27 and Pm29-31 was the same as that of Pci EF607461 and EF607463-65. In addition, the genetic distance ranged only from 0.002 to 0.005 between P minor and Pa EF607460 and EF607458. Apart from this, the interspecies genetic distances varied from 0.135 to 0.143 between P minor and other t＇ampus species according to the C01 sequences. Phylogenetic trees, using combined 16S and CO1 data, strongly support the viewpoint that all the P. minor individuals form one clade that is in a sister position to Pampus sp. individuals （EU357803, FJ434342-FJ434343, and FJ652423-FJ652427）.

Relationship between Mutations in DNA Sequences Loci Coding Pre-miRNAs and Genes Related to Biogenesis of SncRNAs with MiRNA Expression in Endometrial Carcinoma Tissues

Endometrial cancer （EC） is the most common and lethal gynaecological cancer type in Europe and in North America. Frequently EC arises more in the corpus proper and manifests as round, polypoid expansile masses, but it may also originate in the lower uterine segment or spread in endometrium with necrosis and hemorrhage. The analysis was performed using a custom panel containing all DNA sequences loci coding pre-miRNAs and genes related to biogenesis and regulation of sncRNAs in normal and tumor tissues extracted from 6 unrelated patients with endometrial carcinoma. The identified variations were correlated with mature miRNAs differentially expressed in the same normal and tumor endometrial tissues. The comparison analysis confirmed the high degree of cellular and genetic intratumoral heterogeneity with a temporal and spatial miRNA expression distribution in association with genomic variants identified. The classification of specific DNA mutations, onto the loci identified, should be suitable to characterize possible instability genome regions and help classification of tumors to ameliorate the clinical management of patients affected by endometrial carcinoma.

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.

An Efficient Procedure for DNA Isolation and Profiling of the Hyper Variable MtDNA Sequences

Present study describes the results of an efficient protocol for the isolation of good quality DNA from human saliva. The protocol includes collection of saliva in sterile specimen tubes, followed by the cell lysis. After formation of cell lysate, proteins wereextracted by phenol chloroform treatment for purification of DNA. The purified DNA was precipitated by adding equal volume of isopropanol to the treated supernatant. After isolation DNA pellet was washed with 70% ethanol, air-dried and was suspended in 30 pL of double distilled water. Best quality of DNA was extracted from the saliva samples and the PCR product was amplified for hyper-variable regions （HVI＆ HV2） of the mitochondrial DNA. The genes were cleaned with GeneAll gel elution kit （Gel SV） （Cat. No. 102-10） and sequenced accordingly. The DNA isolation protocol presented here is recommended for the isolation, best quality and yield of DNA from the human saliva.

裂殖酵母核心启动子结构的初步研究

位于转录起始位点附近的核心启动子是调控真核生物基因转录的关键区域。近年来发现的高等真核生物核心启动子具有多样的形状、结构和调控机制,使得对核心启动子多样化结构的研究成为了该领域新的研究热点。为研究简单真核生物启动子的结构及其参与调控的机制,我们利用CAGE技术在全基因组范围内捕获裂殖酵母(Schizosaccharomyces pombe)的转录起始位点,从而鉴定其核心启动子序列,并对其形状和序列进行分析。我们的研究揭示了低等真核生物酵母的核心启动子结构具有类似于高等真核生物的多样性,预示着其转录调控可能具有前所未知的复杂性。

Study on Genomic Changes in Partial Amphiploids of Common Wheat_Wheatgrass

According to conventional theory, little genomic changes should occur in homozygous and stable amphiploids of the grass family, particularly those involving polyploid wheat as a parent. In the present study, however, extensive genomic changes were detected in two octoploid partial amphiploids of common wheat (Triticum aestivum L.)_wheatgrass (Agropyron intermedium (Host) P.B.=Elytrigia intermedia (Host) Nevski=Thinopyrum intermedium (Host) Barkworth and Dewey), namely Zhong 3 and Zhong 5, by RFLP analysis using 10 low_copy, wheat chromosome_specific sequences and 33 representative homoeologous group_specific sequences as probes. Genomic changes involved loss of wheat hybridization fragment(s) and/or acquisition of new fragment(s). Uniformity of the RFLP patterns among 5 individual plants taken respectively from Zhong 3 and Zhong 5 in two successive generations, suggested that genomic changes probably had occurred in the early few generations after octoploid amphiploid formation, and remained essentially static thereafter. The highly similar RFLP patterns between Zhong 3 and Zhong 5, which had identical genomic constitution but differed from each other due to involvement of different wheat varieties as parents imply that genomic changes were probably not at random. Possible causes for the extensive and rapid genomic changes in the newly formed plant amphiploids, as well as their implications for polyploid genome evolution and breeding application are discussed.

Identification of Stellaria media by PCR

Aim To provide a rapid and reliable method for identifying the fork medicine Stellaria media （Linn. ） Cyr. （Herba Stellariae mediae） （Caryophyllaceae） from its adulterant Myosoton aquaticure （L.） Fries （Herba Myosoti aquatici） （Caryophyllaceae） by polymerase chain reaction （PCR） technology. Methods A molecular genetic approach has been developed to identify S. media for the first time. 5S-rRNA spacer domain was amplified by PCR from the isolated genomic DNA, and the PCR products were then sequenced. Results The nucleotide sequences of S. media and M. aquaticum were measured to determine their identity. Furthermore, the nucleotide sequences of three Stellaria species, S. vestita, S. longifolia and S. radians, were also measured for the sake of providing the evidence of the biological phylogeny of SteUaria. Diversity between DNA sequence and restriction enzyme mapping among a variety of the species was found in their 5S-rRNA spacer domains. Conclusion The 5S-rRNA spacer domains can be used as a molecular marker for differentiating S. media from M. aquaticum and in phylogenetie studies of Stellaria.

Epigenetic regulation in alcoholic liver disease

Alcoholic liver disease （ALD） is characterized by steatosis or fat deposition in the liver and inflammation, which leads to cirrhosis and hepatocellular carcinoma. Induction of target genes without involving changes in DNA sequence seems to contribute greatly to liver injury. Chromatin modifications including alterations in histones and DNA, as well as post-transcriptional changes collectively referred to as epigenetic effects are altered by alcohol. Recent studies have pointed to a significant role for epigenetic mechanisms at the nucleosomal level influencing gene expression and disease outcome in ALD. Specifically, epigenetic alterations by alcohol include histone modifications such as changes in acetylation and phosphorylation, hypomethylation of DNA, and alterations in miRNAs. These modifications can be induced by alcoholnduced oxidative stress that results in altered recruitment of transcriptional machinery and abnormal gene expression. Delineating these mechanisms in initiation and progression of ALD is becoming a major area of interest. This review summarizes key epigenetic mechanisms that are dysregulated by alcohol in the liver. Alterations by alcohol in histone and DNA modifications, enzymes related to histone acetylation such as histone acetyltransferases, his-tone deacetylases and sirtuins, and methylation enzymes such as DNA methyltransferases are discussed. Chromatin modifications and miRNA alterations that result in immune cell dysfunction contributing to inflammatory cytokine production in ALD is reviewed. Finally, the role of alcohol-mediated oxidative stress in epigenetic regulation in ALD is described. A better understanding of these mechanisms is crucial for designing novel epigenetic based therapies to ameliorate ALD.

A new mutation (1062 del 16) of iduronate-2-sulfatase gene from a Chinese patient with Hunter syndrome

Objective: To identify the mutations of iduronate-2-sulfatase (IDS) gene, to reveal its mutation features, and to establish a basis for genetic counseling and prenatal gene diagnosis of Hunter syndrome. Methods: Urine glycosaminoglycans (GAGs) assay, PCR and DNA sequencing were performed to detect mutation of IDS gene of the patient and his parents. Results: The result showed that the patient was: DS(++), HS(++), KS(-), CS(-), and that both of his parents were negative. A frame-shift deletion mutation (1062 del 16) was identified in exon 7 of the patient's IDS gene. His parents' genotypes were normal. Conclusion:The patient's mutation was not inherited by his parents but a novel one. The mutation probably altered the primary structure and tertiary structure of IDS enzyme protein remarkably and lowered the activity of IDS enzyme greatly. Therefore it is supposed to be the direct cause of the disorder.

Genomic Signal Enhancement by Clustering

Weight matrix models for signal sequence motif are simple. A main limitation of the models is the assumption of independence between positions. Signal enhancement is achieved by taking the total likelihood as the objective function for maximization to cluster sequences into groups with different patterns. As an example, the initial and terminal signals for translation in rice genome are examined.

Molecular Characterization of the Duck Enteritis Virus UL4 Gene

Duck enteritis virus(DEV) is a herpesvirus that causes an acute,contagious and fatal disease. In the present article,the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction(PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame(ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups,and the duck enteritis virus branched separately,closely related to the Mardiviruses group comprising Gallid herpesvirus 2(GaHV-2) ,Gallid herpesvirus 3(GaHV-3) and Meleagrid herpesvirus 1(MeHV-1) . The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.