Oligonucleotide microarray data mining: search for age-dependent gene expression

Information on gene expression in colon tumors versus normal human colon was recently generated by an oligonucleotide microarray study. We used the associated database to search for genes that display age-dependent variations in expression. Statistically significant evidence was obtained that such genes are present in both the tumor and normal tissue databases. Besides the analysis of all genes included in the database, three subsets of genes were analyzed separately: genes controlled by p53, and genes coding for ribosomal proteins and for nuclear-encoded mitochondrial proteins. Among the genes controlled by p53 some show an age-dependent change in expression in tumor tissues, in the sense compatible with an activation of p53 at higher age. A decreased expression of some ribosomal genes at advanced age was detected both in tumor and normal tissues. No significant age-dependent expression could be detected for genes encoding mitochondrial proteins.     

微生物基因数据库在氮循环功能基因注释中的应用

氮循环是微生物和化学过程介导的生物地球化学循环。利用基因测序技术研究环境中参与氮循环的微生物群落、微生物及功能基因,是环境基因组学和微生物生态学的重要研究热点。近年来,各种类型的数据库被开发并应用到功能分析中。本文结合时下最新研究成果,聚焦由微生物引起的同化硝酸盐还原作用、异化硝酸盐还原作用、反硝化作用、固氮作用、硝化作用(包括完全氨氧化作用)和厌氧氨氧化作用等6种无机氮循环途径的功能基因,对比了National Center for Biotechnology Information (NCBI)、Integrated Microbial Genomes (IMG)、Universal Protein (UniProt)、Kyoto Encyclopedia of Genes and Genomes (KEGG)、Protein Families (Pfam)、Functional Gene (FunGene)、Clusters of Orthologous Groups (COG)和NCycDB等数据库的设计理念和功能特点,并结合环境介质、表征基因、分析方法和比对方法等影响因素,分析了以上数据库在氮循环功能基因注释中的选择及应用方式,展望了未来氮循环基因数据库的发展方向,以期为研究人员了解氮循环基因家族和选择合适的数据分析平台提供参考。     

Oligonucleotide microarray analysis of age-related gene expression profiles in miniature pigs

Miniature pigs are useful model animals for humans because they have similar anatomy and digestive physiology to humans and are easy to breed and handle. In this study, whole blood microarray analyses were conducted to evaluate variations of correlation among individuals and ages using specific pathogen-free (SPF) Clawn miniature pigs. Whole blood RNA is easy to handle compared to isolated white blood cell RNA and can be used for health and disease monitoring and animal control. In addition, whole blood is a heterogeneous mixture of subpopulation cells. Once a great change occurs in composition and expressing condition of subpopulations, their associated change will be reflected on whole blood RNA. From 12 to 30 weeks of age, fractions of lymphocytes, monocytes, neutrophils, eosinophils, and basophils in white blood cells showed insignificant differences with age as a result of ANOVA analysis. This study attempted to identify characteristics of age-related gene expression by taking into account the change in the number of expressed genes by age and similarities of gene expression intensity between individuals. As a result, the number of expressed genes was less in fetal stage and infancy period but increased with age, reaching a steady state of gene expression after 20 weeks of age. Variation in gene expression intensity within the same age was great in fetal stage and infancy period, but converged with age. The variation between 20 and 30 weeks of age was comparable to that among 30 weeks individuals. These results indicate that uniformity of laboratory animals is expected for miniature pigs after 20 weeks of age. Furthermore, a possibility was shown that whole blood RNA analysis is applicable to evaluation of physiological state.     

Oligonucleotide microarray for identification of Enterococcus species

For detection of most members of the Enterococcaceae, the specificity of a novel oligonucleotide microarray (ECC-PhyloChip) consisting of 41 hierarchically nested 16S or 23S rRNA gene-targeted probes was evaluated with 23 pure cultures (including 19 Enterococcus species). Target nucleic acids were prepared by PCR amplification of a 4.5-kb DNA fragment containing large parts of the 16S and 23S rRNA genes and were subsequently labeled fluorescently by random priming. Each tested member of the Enterococcaceae was correctly identified on the basis of its unique microarray hybridization pattern. The evaluated ECC-PhyloChip was successfully applied for identification of Enterococcus faecium and Enterococcus faecalis in artificially contaminated milk samples demonstrating the utility of the ECC-PhyloChip for parallel identification and differentiation of Enterococcus species in food samples.     

Oligonucleotide microarray for the identification of potential mycotoxigenic fungi

Background  

Mycotoxins are secondary metabolites which are produced by numerous fungi and pose a continuous challenge to the safety and quality of food commodities in South Africa. These toxins have toxicologically relevant effects on humans and animals that eat contaminated foods. In this study, a diagnostic DNA microarray was developed for the identification of the most common food-borne fungi, as well as the genes leading to toxin production.     

禽流感病毒分型基因芯片的研制

[目的]禽流感病毒是一种全球重要的人和动物呼吸道病病原,快速确定其不同亚型对于全球流感监测具有重要的意义.本研究意在研制一种可同时鉴定禽流感病毒所有亚型的方法.[方法]根据GenBank上已发表的禽流感病毒不同亚型(16个HA亚型和9个NA亚型)的基因序列,设计合成了25对特异性引物和1对通用引物,然后以各亚型病毒的参考株RNA作为模板,建立扩增不同亚型的多重RT-PCR方法.参考各亚型病毒靶cDNAs区域的保守序列设计了52条亚型特异的探针,进而利用扩增的各亚型病毒的靶cDNAs对其特异性进行评价.在此基础上,将设计好的探针点制到处理好的玻片上,制备了禽流感病毒分型鉴定基因芯片,结合所建立的扩增不同亚型的多重RT-PCR方法,开发了禽流感病毒亚型鉴定基因芯片试剂.利用收集自49个地区的2653份标本对其特异性和敏感性进行了初步评价.[结果]用于评价的各亚型参考毒株均出现良好的特异性杂交信号,检测的敏感度可达2.47 PFU/mL或2.5 ng靶DNA片段,而且与禽类常见的IBV、NDV等6种病毒均无交叉反应.[结论]证明该病毒分型基因芯片具有良好的特异性、敏感性.     

Oligonucleotide microarray for 16S rRNA gene-based detection of all recognized lineages of sulfate-reducing prokaryotes in the environment

For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).     

Development of a whole community genome amplification-assisted DNA microarray method to detect functional genes involved in the nitrogen cycle

A novel DNA microarray analysis targeting key functional genes involved in most nitrogen cycling reactions was developed to comprehensively analyze microbial populations associated with the nitrogen cycle. The developed microarray contained 876 oligonucleotide probes based on the nucleotide sequences of the nif, amo, hao/hzo, nap, nar, nirK, nirS, nrf, cnor, qnor and nos genes. An analytical method combining detection by the designed microarray with whole community genome amplification was then applied to monitor the nitrogen cycling microorganisms in river water and wastewater treatment sludge samples. The developed method revealed that nitrogen cycling microorganisms in river water appeared to become less diverse in response to input of effluent from municipal wastewater treatment plants. Additionally, the nitrogen cycling community associated with anaerobic ammonium oxidation and partial nitrification reactors could be reasonably analyzed by the developed method. However, the results obtained for two activated sludge samples from municipal wastewater treatment plants with almost equivalent wastewater treatment performance differed greatly from each other. These results suggested that the developed method is useful for comprehensive analysis of nitrogen cycling microorganisms, although its applicability to complex samples with abundant untargeted populations should be further examined.     

Oligonucleotide microarray for the subtyping of influenza virus a neuraminidase

A microarray for the subtyping of influenza A neuraminidase is presented. The selection of oligoprobes proceeded in two steps. The first step included the selection of peptides specific for each subtype of neuraminidase. At the second step, the oligoprobes were calculated using the found peptide structures with the subsequent additional selection of the most specific and representative probes. From 19 to 24 probes were used for the determination of each neuraminidase subtype. The microarray testing for 19 samples with the most widespread types (N1 and N2) specifies in an unequivocal definition 18 of them and only 1 isolate has not been identified.     

Oligonucleotide microarray for subtyping of influenza virus A neuraminidase

Microarray for influenza A neuraminidase subtyping was presented. Selection of oligoprobes proceeded in two steps. First step included selection of peptides specific for each subtype of neuraminidase. At the second step oligoprobes were calculated using found peptides structures with the subsequent additional selection of the most specific and representative probes. From 19 to 24 probes were used for determination of each subtype of neuraminidase. Microchip testing for 19 samples with the most widespread types (N1 and N2) specifies in unequivocal definition 18 of them and only one isolate has not been identified.     

温度和水分变化对冻土区泥炭地土壤氮循环功能基因丰度的影响

以大兴安岭多年冻土区泥炭地为研究对象,通过室内模拟增温实验,研究温度升高对不同深度（0-150 cm）土壤氮循环功能基因丰度的影响。同时针对0-20 cm和20-40 cm土壤设置两个水分处理,分别为土壤原始含水量和淹水状态,研究水分变化对表层土壤氮循环功能基因丰度的影响。结果表明温度升高显著提高了活动层（0-60 cm）、过渡层（60-80 cm）、永冻层（80-100 cm）中nifH、nirK基因丰度,温度升高显著提高了活动层（0-40 cm）和过渡层（60-80 cm）中nirS基因丰度。温度升高显著提高了过渡层（60-80 cm）NH₄⁺-N和较深永冻层（140-150 cm）NO₃^--N的含量,但降低了过渡层（60-80 cm）NO₃^--N和较深永冻层（120-150 cm）NH₄⁺-N的含量,相关性分析表明,NH₄⁺-N含量与nifH和nirS基因丰度呈显著正相关,NO₃^--N含量与nirK基因丰度呈显著正相关,说明温度升高能够通过改变微生物丰度促进过渡层固氮作用和反硝化作用。在增温条件下,淹水处理使表层土壤nirS和nirK基因丰度及NH₄⁺-N含量降低,但提高了NO₃^--N含量,说明淹水造成了过度还原的条件使反硝化底物浓度降低,降低反硝化微生物活性进而抑制了土壤反硝化作用。该结果对于明确未来气候变化影响下冻土区泥炭地土壤氮循环过程具有重要意义。     

海岸带沉积物中氮循环功能微生物多样性

海岸带生境类型多样,环境梯度明显,是研究微生物多样性、群落结构与功能关系及调控机制的天然实验场.沉积物是海岸带环境中营养盐再生与转化发生的重要场所,其中多种微生物类群在氮素循环过程中扮演重要角色.本文重点介绍海岸带沉积物中固氮菌、氨氧化菌、厌氧氨氧化菌、反硝化与硝酸盐铵化微生物的基于16SrRNA基因的物种多样性和基于关键酶基因nifH、amoA、narG、nirS、nirK、nosZ、nrfA、hzo、hzs等的功能多样性;总结了在海岸带特有生境(如河口、潮间带、海草藻床、红树林、盐沼、珊瑚礁、浅海等)及污染胁迫、生物扰动等条件下各功能类群的群落组成特征及时空变化规律,并提出今后需要重点关注新的培养技术和方法的开发,以进一步提高微生物的可培养性,将单细胞基因组测序与分析技术、DNA和RNA结合起来研究,以全面了解氮循环微生物多样性、参与介导硝酸盐铵化过程的微生物多样性等方面.     

A microtiter plate procedure for evaluating fungal functional diversity on nitrogen substrates

Ascertaining the effects of anthropogenic disturbance on belowground diversity is of paramount importance because pollution from agricultural practices and industrialization are increasing worldwide. Although we have methods for evaluating soil microbial function with respect to carbon use our ability to evaluate use of other compounds is limited. Because N cycling is of paramount importance in ecosystem stability, evaluation of the ability of saprophytic soil fungi to use a variety of N sources would provide important information on possible alterations in ecosystem stability with disturbance. Herein is described a procedure (soil Nitrolog) for evaluating fungal functional diversity on a suite of 95 different N substrates. The soil Nitrolog procedure was evaluated by testing fungal functional diversity at two sites in Big Bend National Park (Chihuahuan Desert), differing in elevation and plant community composition. The soil Nitrolog procedure distinguished between the two sites based on overall use of the 95 N substrates. In addition the procedure detected differences in individual substrate use based on site specific plant compounds in response to changes in the amount of N entering these ecosystems from anthropogenic inputs.     

Oligonucleotide microarray analysis of genomic imbalance in children with mental retardation

The cause of mental retardation in one-third to one-half of all affected individuals is unknown. Microscopically detectable chromosomal abnormalities are the most frequently recognized cause, but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause. Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants. We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis, by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays. We found de novo deletions as small as 178 kb in eight cases, de novo duplications as small as 1.1 Mb in two cases, and unsuspected mosaic trisomy 9 in another case. This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation.     

Development of a lab-made microarray for analyzing the genetic diversity of nitrogen fixing symbionts Sinorhizobium meliloti and Sinorhizobium medicae

Some bacterial species, like nitrogen-fixing Sinorhizobium that interact with Medicago plants, are prone to frequent horizontal gene transfers. Investigation of their genetic structure requires to study polymorphism patterns at many loci. Although DNA microarrays represent a method of choice for high throughput analysis of polymorphisms, this technology yet remains an expensive and heavy approach, thus depriving most of research groups from this powerful tool. In an attempt to overcome this limitation, we have developed a simple genotyping procedure by DNA microarrays, and have evaluated its ability to characterize a Sinorhizobium population. Thirty 18- to 24-mer oligonucleotide probes were designed to target the most frequent mutations in three polymorphic loci of Sinorhizobium meliloti and S. medicae. Probe hybridization efficiency was compared on two spotting surfaces: nylon membranes and epoxy-coated glass slides. Epoxy-coated glass slides revealed more sensitive than nylon membranes and allowed discrimination of single mismatches. Using this procedure, an uncharacterized population consisting of 33 S. meliloti/S. medicae isolates was successfully genotyped.     

Oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips

A possibility of using oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips was studied. The oligonucleotide conjugates contain a hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues with an aminocaproic acid residue as a linker and bound to the oligonucleotide duplex AT tract in a site-specific manner. We used as (5'-3') probes GACAAGAp, GACAAAAp, GACAAGA-MGB, and GACAAAA-MGB. The oligonucleotides labeled with Cy3 cyanine dye, Cy3-ACTAATTTTGTC and Cy3-ACTAATCTTGTC, were used as targets. The maximal MGB effect on the fluorescence level of microarray chip spots, which caused its fourfold increase as compared with the initial unmodified duplex, was observed for the duplex containing only AT pairs in the ligand binding site. The presence of A-C and G-T mutations in the binding site (imperfect duplexes) or a C-G pair (perfect duplex) affects the change in fluorescence level to a considerably lesser degree.     

Oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips

A possibility of using oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips was studied. The oligonucleotide conjugates contain a hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues with an aminocaproic acid residue as a linker and bound to the oligonucleotide duplex AT tract in a site-specific manner. We used as (5′-3′)-probes: GACAAGAp, GACAAAAp, GACAAGA-MGB, and GACAAAA-MGB. The oligonucleotides labeled with the Cy3 cyanine dye, Cy3-ACTAATTTTGTC and Cy3-ACTAATCTTGTC, were used as targets. The maximal MGB effect on the fluorescence level of microarray chip spots, which caused its fourfold increase as compared with the initial unmodified duplex, was observed for the duplex containing only AT pairs in the ligand binding site. The presence of AC and GT mutations in the binding site (imperfect duplexes) or a CG pair (perfect duplex) affect the change in fluorescence level to a considerably lesser degree.