Identification of Novel Genes Differentially Expressed in PMA-induced HL-60 Cells Using cDNA Microarrays

Identification of normal growth and differentiation-inducing proteins and their interaction in normal development have made it possible to elucidate the molecular basis of normal development and the mechanisms uncoupling growth and differentiation during tumor development. The development of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. cDNA microarrays provide a powerful tool for studying these phenomena. In the present study, a high-density microarray of human cDNA elements was used to search for differences in gene expression associated with differentiation of human promyelic leukemia HL-60 cells. Microarrays containing 3,063 human cDNAs were printed on glass slides with high-speed robotics. These DNA chips were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. The identification of known and novel phorbol ester-regulated genes in hematopoietic progenitor cells demonstrates the sensitivity of the assay.     

Identification of Novel Genes Differentially Expressed in PMA-induced HL-60 Cells Using cDNA Microarrays

Identification of normal growth and differentiation-inducing proteins and their interaction in normal development have made it possible to elucidate the molecular basis of normal development and the mechanisms uncoupling growth and differentiation during tumor development. The development of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. cDNA microarrays provide a powerful tool for studying these phenomena. In the present study, a high-density microarray of human cDNA elements was used to search for differences in gene expression associated with differentiation of human promyelic leukemia HL-60 cells. Microarrays containing 3,063 human cDNAs were printed on glass slides with high-speed robotics. These DNA 'chips' were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. The identification of known and novel phorbol ester-regulated genes in hematopoietic progenitor cells demonstrates the sensitivity of the assay.     

A Model-Based Joint Identification of Differentially Expressed Genes and Phenotype-Associated Genes

Over the last decade, many analytical methods and tools have been developed for microarray data. The detection of differentially expressed genes (DEGs) among different treatment groups is often a primary purpose of microarray data analysis. In addition, association studies investigating the relationship between genes and a phenotype of interest such as survival time are also popular in microarray data analysis. Phenotype association analysis provides a list of phenotype-associated genes (PAGs). However, it is sometimes necessary to identify genes that are both DEGs and PAGs. We consider the joint identification of DEGs and PAGs in microarray data analyses. The first approach we used was a naïve approach that detects DEGs and PAGs separately and then identifies the genes in an intersection of the list of PAGs and DEGs. The second approach we considered was a hierarchical approach that detects DEGs first and then chooses PAGs from among the DEGs or vice versa. In this study, we propose a new model-based approach for the joint identification of DEGs and PAGs. Unlike the previous two-step approaches, the proposed method identifies genes simultaneously that are DEGs and PAGs. This method uses standard regression models but adopts different null hypothesis from ordinary regression models, which allows us to perform joint identification in one-step. The proposed model-based methods were evaluated using experimental data and simulation studies. The proposed methods were used to analyze a microarray experiment in which the main interest lies in detecting genes that are both DEGs and PAGs, where DEGs are identified between two diet groups and PAGs are associated with four phenotypes reflecting the expression of leptin, adiponectin, insulin-like growth factor 1, and insulin. Model-based approaches provided a larger number of genes, which are both DEGs and PAGs, than other methods. Simulation studies showed that they have more power than other methods. Through analysis of data from experimental microarrays and simulation studies, the proposed model-based approach was shown to provide a more powerful result than the naïve approach and the hierarchical approach. Since our approach is model-based, it is very flexible and can easily handle different types of covariates.     

Generation and envelope protein analysis of internalization defective Brucella abortus mutants in professional phagocytes, RAW 264.7

Brucella abortus is a facultative intracellular bacteria that replicates within a macrophage without producing any classical virulence factors. It can become internalized to cells by zipper-like and/or swimming internalization mechanisms. However, the bacterial proteins involved in internalization remain unclear. To define these bacterial proteins, random insertion mutants of B. abortus were generated by the Tn5 transposome complexes. In all, 132 mutants were screened, cellular internalization-defective mutants were selected, and these genomic and envelope proteomic features were identified. The transposon insertion sites were ccmC,ppk and BruAb2_0168 for the mutant C10, C29 and D7, respectively. Mutant C10 showed a deficiency in internalization without any changes in expression of the cell envelope proteins; however, mutant C29 showed a reduced expression of OMP25, and a mutant D7 also showed reduced expression of OMP25, OMP28 and Porin2b. These results suggest OMP25 is not an essential factor, but might be involved in host cellular internalization. We identified the ppk gene and BruAb2_0168 locus which are associated to expression of OMP25, OMP28 and Porin2b as well as pleiotropic effects of ccmC gene.     

汉坦病毒感染细胞整合素受体信号转导相关差异表达基因筛选

通过基因芯片技术观察汉坦病毒感染细胞后整合素受体信号转导相关基因的差异表达,以探讨汉坦病毒进入细胞的分子机制.用汉滩病毒76-118株感染原代人胚肺成纤维细胞,同时设立正常对照细胞,于感染后第6h、24h和96h同时抽提感染组与正常对照组细胞总RNA,利用荧光标记dUTP逆转录制备cDNA探针,与人类受体及信号转导类表达谱芯片杂交,并对Cy3、Cy5荧光信号做扫描分析.结果显示有37项基因出现差异表达,其中31项基因在感染后6h出现差异表达,13项下调、18项上调;但在感染后24h和96h分别只有5项和4项基因出现表达差异;而且整合素信号转导通路中的重要基因如一些蛋白激酶相关基因在感染6h表达增强,丝化增强子1在感染6h和96h表达下调,微管相关蛋白1A在感染6h表达上调、感染24h和96h表达下调.这些进一步说明汉坦病毒感染细胞与整合素有关.     

汉坦病毒感染细胞整合素受体信号转导相关差异表达基因筛选

通过基因芯片技术观察汉坦病毒感染细胞后整合素受体信号转导相关基因的差异表达,以探讨汉坦病毒进入细胞的分子机制。用汉滩病毒76—118株感染原代人胚肺成纤维细胞,同时设立正常对照细胞,于感染后第6h、24h和96h同时抽提感染组与正常对照组细胞总RNA,利用荧光标记dUTP逆转录制备cDNA探针,与人类受体及信号转导类表达谱芯片杂交,并对Cy3、Cy5荧光信号做扫描分析。结果显示有37项基因出现差异表达,其中31项基因在感染后6h出现差异表达,13项下调、18项上调;但在感染后24h和96h分别只有5项和4项基因出现表达差异;而且整合素信号转导通路中的重要基因如一些蛋白激酶相关基因在感染6h表达增强,丝化增强子1在感染6h和96h表达下调,微管相关蛋白1A在感染6h表达上调、感染24h和96h表达下调。这些进一步说明汉坦病毒感染细胞与整合素有关。     

Microarray-Based Approach Identifies Differentially Expressed MicroRNAs in Porcine Sexually Immature and Mature Testes

Background

MicroRNAs (miRNAs) are short non-coding RNA molecules which are proved to be involved in mammalian spermatogenesis. Their expression and function in the porcine germ cells are not fully understood.

Methodology

We employed a miRNA microarray containing 1260 unique miRNA probes to evaluate the miRNA expression patterns between sexually immature (60-day) and mature (180-day) pig testes. One hundred and twenty nine miRNAs representing 164 reporter miRNAs were expressed differently (p<0.1). Fifty one miRNAs were significantly up-regulated and 78 miRNAs were down-regulated in mature testes. Nine of these differentially expressed miRNAs were validated using quantitative RT-PCR assay. Totally 15919 putative miRNA-target sites were detected by using RNA22 method to align 445 NCBI pig cDNA sequences with these 129 differentially expressed miRNAs, and seven putative target genes involved in spermatogenesis including DAZL, RNF4 gene were simply confirmed by quantitative RT-PCR.

Conclusions

Overall, the results of this study indicated specific miRNAs expression in porcine testes and suggested that miRNAs had a role in regulating spermatogenesis.     

Identification of Differentially Expressed Genes in Distinct Skeletal Muscles in Cattle using cDNA Microarray

The 788-gene microarray was manufactured using selected elements from three different cDNA libraries in order to identify molecular processes that determine phenotypic characteristics between loin (M. longissimus thoracis) and round (M. semimembranosus) muscles. Microarray analyses identified 24 differentially expressed genes between the two muscles investigated. Five of the genes were verified by quantitative RT-PCR and three of them were mapped on bovine chromosomes using 5,000 rad bovine radiation hybrid (RH) panel. The map locations indicated that they were mapped in the same chromosomal regions where IMF and growth QTLs were located, suggesting that they are most possible positional candidate genes for the traits.     

Powers of Multiple-Testing Procedures for Identification of Genes Significantly Differentially Expressed in Microarray Experiments

Because of the high operation costs involved in microarray experiments, the determination of the number of replicates required to detect a gene significantly differentially expressed in a given multiple-testing procedure is of considerable significance. Calculation of power/replicate numbers required in multiple-testing procedures provides design guidance for microarray experiments. Based on this model and by choice of a multiple-testing procedure, expression noises based on permutation resampling can be considerably minimized. The method for mixture distribution model is suitable to various microarray data types obtained from single noise sources, or from multiple noise sources. By using the biological replicate number required in microarray experiments for a given power or by determining the power required to detect a gene significantly differentially expressed, given the sample size, or the best multiple-testing method can be chosen. As an example, a single-distribution model of t-statistic was fitted to an observed microarray dataset of 3 000 genes responsive to stroke in rat, and then used to calculate powers of four popular multiple-testing procedures to detect a gene of an expression change D. The results show that the B-procedure had the lowest power to detect a gene of small change among the multiple-testing procedures, whereas the BH-procedure had the highest power. However, all multiple-testing procedures had the same power to identify a gene having the largest change. Similar to a single test, the power of the BH-procedure to detect a small change does not vary as the number of genes increases, but powers of the other three multiple-testing procedures decline as the number of genes increases.     

Identification of Genes Differentially Expressed Between Ochratoxin-Producing and Non-Producing Strains of Aspergillus westerdijkiae

Approximately 70 % of Aspergillus westerdijkiae strains are able to produce ochratoxin A (OTA), a nephrotoxic and carcinogenic mycotoxin which have been found in cereal and food commodities. Despite of its importance there is, up to now, no information available about which genes are differentially expressed between A. westerdijkiae ochratoxin-producing and non-producing strains. Using cDNA RDA approach we successfully sequenced 231 raw ESTs expected to be enriched in the ochratoxin-producing strain. BLASTX searches against the public databases showed that of these, 205 ESTs (79 %) exhibited significant similarities with proteins of known functions, 28 ESTs (11 %) had matches to hypothetical proteins, and the remaining 27 ESTs (10 %) had no significant hits. EST alignment resulted in a total of 14 non-redundant consensus sequences. Three putative genes encoding oxidoreductases were validated as up-expressed in the OTA producer strain using RT-qPCR approach. The expression of the putative genes encoding a cytochrome P450 family protein, 3-hydroxyphenylacetate-6-hydroxylase, and endoplasmic reticulum oxidoreductin were higher (32-, 2.8- and 20-fold respectively) in the OTA producer strain compared to the non-producer strain.     

多重检验法在基因芯片研究中鉴定差异表达基因的统计功效

鉴于基因芯片实验的造价,在基因芯片实验设计中,首要考虑的因素是需要多少重复才能检测出一个具有显著差异表达的基因。计算多重检验法要求的重复数（样本大小）或功效可为基因芯片实验设计提供重要的参考。为此,本文基于置换重抽样法构建了一种基因表达噪声混合分布模型。该方法适用各类基因表达数据,即无论是基因表达单噪声源或是多噪声源都可行。应用混合模型和多重检验法并给定统计功效。研究者能在基因芯片实验中获得所需要的最少生物学重复数：或者根据样本大小来确定测定一个显著差异表达的基因所具有的检验功效;或者根据样本大小和统计检验功效,选择最好的统计测验方法。本文以一组在老鼠中与中风有关的3000个基因的基因芯片实验所获得的数据为例,应用该方法拟和后组建了一个单分布模型（即表达单噪声源的分布模型）。根据该模型,我们计算了4种多重检验法在鉴定一个具有表达差异（D）值的基因中所需要的统计功效。结果表明。检测一个小的差异D值,4种多重检验法中B方法的统计功效最低,而BH方法最高。但是,对于鉴定一个具有最大表达差异的基因时,4种方法有相同的鉴定功效。与传统的单个检验法一样,BH方法检测一个小的变化所需要的效率不会随基因数目增加而改变,其他3种多重检验法的检测功效则随基因数目增加而降低。     

Identification of the Genes Specifically Expressed in Orally Tolerized T Cells

Oral tolerance is the systemic immunological unresponsiveness that occurs after feeding protein antigens. Its physiological role is thought to be the prevention of hypersensitivity to food antigens, and its therapeutic use to treat inflammatory diseases has been suggested. Although it has been shown that CD4⁺ T cells mediate oral tolerance, the precise molecular mechanisms remain unclear. In the present study, we employed suppression subtractive hybridization and identified 10 genes specifically expressed in orally tolerized T cells. These included genes that were interesting in terms of their putative functions in the negative regulation of T cell activation, e.g. Culin 1, LAX, and Zfhx1b, as well as four genes that encoded unknown proteins. We further investigated the expression of these genes in hyporesponsive T cells induced in vitro (in vitro anergized T cells). We found that six of the 10 genes were highly expressed in these cells, and kinetic studies suggested that one was associated with the induction of anergy, while the other five were associated with the maintenance of anergy. The remaining 4 genes that were not expressed in in vitro anergized T cells are also of interest as they may play a specific role in in vivo T cell tolerance. Functional analysis of these genes should help to understand the complex mechanisms underlying the induction and maintenance of oral tolerance, and moreover, in vivo immune tolerance in general.     

Intracellular Production of Brucella L Forms I. Recovery of L Forms from Tissue Culture Cells Infected with Brucella abortus

Hatten, Betty A. (The University of Texas Southwestern Medical School, Dallas), and S. Edward Sulkin. Intracellular production of Brucella L forms. I. Recovery of L forms from tissue culture cells infected with Brucella abortus. J. Bacteriol. 91:285-296. 1966.-Infectivity of virulent Brucella abortus strain 3183 was less for hamster macrophages after a 2-hr adsorption period than for an attenuated strain (S19) and its tissue culture variant (30). Both strains S19 and 30 were very toxic for the cells, but 3183 was not toxic. Two types of L forms were recovered from a large percentage of hamster kidney cell cultures when disintegration of infected cells was accelerated by tissue culture medium of high pH. One type grew in finely granular microcolonies, was isolated from cells infected for short periods of time, and often reverted to the bacterial form. The other type occurred in small irregularly shaped forms which later developed into round bodies. Both stained specifically with fluorescein-conjugated B. abortus antiserum. Semisolid media containing 0.7% agar provided optimal subsurface L-form growth. L forms also grew well in Thioglycollate Medium but grew poorly in other liquid media. Surface L-form growth was supported by several agar media, but CO(2) was required for optimal growth. Monolayers infected with strain 3183 and examined immediately after adsorption contained occasional small, round bodies. Bizarre forms increased in number with time and, after 24 to 72 hr, large pink-staining inclusions were often present which persisted for several days. Also appearing at about the same time were smaller, dark-staining forms which were first seen in clusters but later dispersed and finally occurred in chainlike configurations. Direct fluorescent-antibody stains of infected cells established that the intracellular forms were related to the infecting strain of B. abortus.     

Identification of Differentially Expressed Genes in Breast Muscle and Skin Fat of Postnatal Pekin Duck

Lean-type Pekin duck is a commercial breed that has been obtained through long-term selection. Investigation of the differentially expressed genes in breast muscle and skin fat at different developmental stages will contribute to a comprehensive understanding of the potential mechanisms underlying the lean-type Pekin duck phenotype. In the present study, RNA-seq was performed on breast muscle and skin fat at 2-, 4- and 6-weeks of age. More than 89% of the annotated duck genes were covered by our RNA-seq dataset. Thousands of differentially expressed genes, including many important genes involved in the regulation of muscle development and fat deposition, were detected through comparison of the expression levels in the muscle and skin fat of the same time point, or the same tissue at different time points. KEGG pathway analysis showed that the differentially expressed genes clustered significantly in many muscle development and fat deposition related pathways such as MAPK signaling pathway, PPAR signaling pathway, Calcium signaling pathway, Fat digestion and absorption, and TGF-beta signaling pathway. The results presented here could provide a basis for further investigation of the mechanisms involved in muscle development and fat deposition in Pekin duck.