Genes Frequently Coexpressed with Hoxc8 Pro-vide Insight into the Discovery of Target Genes

Myoung Hee Kim,Ruthala Kalyani,Ji-Yeon Lee,민혜현,Heejei Yoon 한국분자세포생물학회 2016 Molecules and cells Vol.39 No.5

Identifying Hoxc8 target genes is at the crux of under-standing the Hoxc8-mediated regulatory networks underlying its roles during development. However, identification of these genes remains difficult due to intrinsic factors of Hoxc8, such as low DNA binding specificity, context-dependent regulation, and unknown cofactors. Therefore, as an alternative, the present study attempted to test whether the roles of Hoxc8 could be inferred by simply analyzing genes frequently coexpressed with Hoxc8, and whether these genes include putative target genes. Using archived gene expression datasets in which Hoxc8 was differentially expressed, we identified a total of 567 genes that were positively coexpressed with Hoxc8 in at least four out of eight datasets. Among these, 23 genes were coexpressed in six datasets. Gene sets associated with extracellular matrix and cell adhesion were most significantly enriched, followed by gene sets for skeletal system development, morphogenesis, cell motility, and transcriptional regulation. In particular, transcriptional regulators, including paralogs of Hoxc8, known Hox co-factors, and transcriptional remodeling factors were enriched. We randomly selected Adam19, Ptpn13, Prkd1, Tgfbi, and Aldh1a3, and validated their coexpression in mouse embryonic tissues and cell lines following TGF-2 treatment or ectopic Hoxc8 expression. Except for Aldh1a3, all genes showed concordant expression with that of Hoxc8, suggesting that the coexpressed genes might include direct or indirect target genes. Collectively, we suggest that the coexpressed genes provide a resource for constructing Hoxc8-mediated regulatory networks.

Transcriptome-based identification of the optimal reference genes as internal controls for quantitative RT-PCR in razor clam (Sinonovacula constricta)

Xuelin Zhao,Jianping Fu,Liting Jiang,Weiwei Zhang,Yina Shao,Chunhua Jin,Jinbo Xiong,Chenghua Li 한국유전학회 2018 Genes & Genomics Vol.40 No.6

Quantitative real-time PCR (qRT-PCR) is a standard method to measure gene expression in function exploring. Accurate and reproducible data of qRT-PCR requires appropriate reference genes, which are stably expressed under different experimental conditions. However, no housekeeping genes were validated as internal controls for qRT-PCR in Sinonovacula constricta. In this study, we classified the transcriptome data of two tissues for Vibrio infection and Cd2+ stress into ten clusters based on the gene expression patterns. Among them, cluster 5 had the most stable gene expression patterns regardless of tissues and treatments as the database for candidate reference genes. A total of 55 orthologs of classical housekeeping genes in the clam transcriptome were annotated. Combined the expression profiles and housekeeping genes in S. constricta, we chose eight candidate reference genes and validated their expression in Vibrio-infected samples and different tissues by qRT-PCR. Their expression stability was analyzed by three different algorithms geNorm, NormFinder and BestKeeper. Although the rank of the eight candidate reference genes is different in different treatments using different software, RS9 could be the best reference genes for normalization of qRT-PCR expression data in S. constricta under various treatments considering the above analysis. Meanwhile, the ranking of genes based on the CV values of transcriptomic data was similar to the validation results. This study provides for the first time a list of suitable reference genes for S. constricta and a valuable resource for further studies of clam immune defense systems.

Isolation of cold-responsive genes from garlic, Allium sativum

손재한,박경철,이성일,김행훈,김종화,김선형,김남수 한국유전학회 2012 Genes & Genomics Vol.34 No.1

Cold stress discourages development of the full genetic potential of plants and results in serious adverse effects on plant growth and limits agricultural productivity. Garlic (Allium sativum) requires low temperatures for the induction of flowering and bulb development. However, low or freezing temperatures can often cause physiological damage. Cold-responsive genes were isolated from a Korean garlic variety through systematic analyses using differential display (DD)-PCR, reverse transcription (RT)-PCR, and quantitative (Q)-PCR. Of the 2470 transcripts observed, 76 transcripts were up-regulated and 20 transcripts were down-regulated in response to cold temperatures (4°C)as determined by DD-PCR analysis. The differentially-expressed genes were further narrowed to 15 up-regulated and four down-regulated genes through subsequent RT-PCR and Q-PCR analyses. Of the 15 up-regulated genes, 11 genes were homologous to previously-reported abiotic-stress regulated genes in other species, and four genes did not match any known genes. Of the four down-regulated genes, three genes matched function-annotated genes and one gene did not match any genein the BLAST analysis. While most of the up-regulated poly-peptides were hydrophilic, the down-regulated polypeptides were neutral or hydrophobic. Of the 19 differentially-regulated genes, four genes showed different expression profiles in different tissues upon cold stress. Genetic manipulation of the CR genes obtained may provide molecular tool for overcoming frost damage of the garlic plants during hibernation.

Genome-wide identification and analysis of rice genes to elucidate morphological agronomic traits

Anil Kumar Nalini Chandran,Nikita Bhatnagar,김범기,정기홍 한국식물학회 2016 Journal of Plant Biology Vol.59 No.6

Molecular understanding of morphological agronomic traits is very important to improve grain yield and quality. According to the literature information summarized in Overview of Functionally Characterized Genes in Rice online database, 430 genes related to these traits have been functionally characterized in rice, while the functions of other genes remain to be elucidated. Gene indexed mutants are available for at least half of the genes identified in the rice genome, and are very useful resources to study gene function. To suggest candidate genes for functional studies associated with morphological agronomic traits, we identified genes with tissue/organ-preferred expression patterns through meta-analysis of microarray data, and identified 781 genes for roots, 1,084 for leaves, 1,029 for calluses, 927 for anthers, 241 for embryos, and 343 for endosperms. Additionally, 4,243 genes expressed in all tissue types were allocated to a ubiquitously-expressed gene group (‘housekeeping’ genes). The estimated tissue/organ-preferred and housekeeping genes accounted for 40% of the characterized genes associated with morphological agronomic traits, indicating that identification of tissue/organ-preferred genes is an effective way to provide putative gene function. In this study, we reported the information of gene-indexed mutants for 84% of the identified candidate genes. Our candidate genes and relating indexed mutant resources can potentially be used to improve morphological agronomic traits in rice.

Use of p-value plots to diagnose and remedy problems with statistical analysis of microarray data

이태원,Robert R. Delongchamp,김원국,Robert J. Shmookler Reis 한국유전학회 2016 Genes & Genomics Vol.38 No.1

Microarray and RNA-sequencing technologies measure thousands of genes per biological sample. Because of the large number of genes, empirical distributions over genes for statistics computed over samples resolve properties of the data that can be exploited to define the expressed genes, diagnose problems with hypothesis tests, and even remedy some of these problems. The empirical distribution of the average expressions for genes was first used to partition the interrogated genes into two subsets, ‘non-expressed’ genes and ‘expressed’ genes. The p-values from tests for treatment effects were computed for both subsets and their empirical distributions were examined next. A plot of empirical distributions of p-values (p-value plot) indicated that the ‘non-expressed’ genes do not follow the anticipated null distribution, which implies that the pvalues for expressed genes may also misrepresent their true significance. In simulations we were able to produce a similar departure from the null distribution with dye effects, suggesting that comparable confounding may account for the observed discrepancies. By using the empirical distribution of non-expressed genes as the null distribution, p-values for the expressed genes were adjusted with the goal of mitigating biases introduced by systematic distortions such as a dye effect. A plot of the empirical distribution for a statistic computed per endpoint provides a succinct visualization of the distributional properties, which can be compared to expected properties. Such comparisons are effective at identifying problems with analyses, and indicating adjustments that can be applied to generate more reliable lists of affected genes based on false-discovery criteria.

Selection and evaluation of reference genes for gene expression using quantitative real‐time PCR in Mythimna separata walker (Lepidoptera: Noctuidae)

Bai‐Zhong Zhang,Jun-Jie LIU,Xi-Ling CHEN,Guo-Hui YUAN 한국곤충학회 2018 Entomological Research Vol.48 No.5

In order to precisely assess gene expression levels, the suitable internal reference genes must be served to quantify real‐time reverse transcription polymerase chain reaction (RT‐qPCR) data. For armyworm, Mythimna separata, which reference genes are suitable for assessing the level of transcriptional expression of target genes have yet to be explored. In this study, eight common reference genes, including β‐actin (β‐ACT), 18 s ribosomal (18S), 28S ribosomal (28S), glyceraldehyde‐3‐phosphate (GAPDH), elongation fator‐alpha (EF1α), TATA box binding protein (TBP), ribosomal protein L7 (RPL7), and alpha‐tubulin (α‐TUB) that in different developmental stages, tissues and insecticide treatments of M. separata were evaluated. To further explore whether these genes were suitable to serve as endogenous controls, three software‐based approaches (geNorm, BestKeeper, and NormFinder), the delta Ct method, and one web‐based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized HSP70, and MsepCYP321A10 gene expression data. We found that the most suitable reference genes for the different experimental conditions. For developmental stages, 28S/RPL7 were the optimal reference genes, both RPL7/EF1α were suitable for experiments of different tissues, whereas for insecticide treatments, 28S/α‐TUB were suitable for normalizations of expression data. In addition, 28S/α‐TUB were the suitable reference genes because they have the most stable expression among different developmental stages, tissues and insecticide treatments. Our work is the first report on reference gene selection in M. separata, and might serve as a precedent for future gene expression studies.

Genes Frequently Coexpressed with Hoxc8 Provide Insight into the Discovery of Target Genes

Kalyani, Ruthala,Lee, Ji-Yeon,Min, Hyehyun,Yoon, Heejei,Kim, Myoung Hee Korean Society for Molecular and Cellular Biology 2016 Molecules and cells Vol.39 No.5

Identifying Hoxc8 target genes is at the crux of understanding the Hoxc8-mediated regulatory networks underlying its roles during development. However, identification of these genes remains difficult due to intrinsic factors of Hoxc8, such as low DNA binding specificity, context-dependent regulation, and unknown cofactors. Therefore, as an alternative, the present study attempted to test whether the roles of Hoxc8 could be inferred by simply analyzing genes frequently coexpressed with Hoxc8, and whether these genes include putative target genes. Using archived gene expression datasets in which Hoxc8 was differentially expressed, we identified a total of 567 genes that were positively coexpressed with Hoxc8 in at least four out of eight datasets. Among these, 23 genes were coexpressed in six datasets. Gene sets associated with extracellular matrix and cell adhesion were most significantly enriched, followed by gene sets for skeletal system development, morphogenesis, cell motility, and transcriptional regulation. In particular, transcriptional regulators, including paralogs of Hoxc8, known Hox co-factors, and transcriptional remodeling factors were enriched. We randomly selected Adam19, Ptpn13, Prkd1, Tgfbi, and Aldh1a3, and validated their coexpression in mouse embryonic tissues and cell lines following $TGF-{\beta}2$ treatment or ectopic Hoxc8 expression. Except for Aldh1a3, all genes showed concordant expression with that of Hoxc8, suggesting that the coexpressed genes might include direct or indirect target genes. Collectively, we suggest that the coexpressed genes provide a resource for constructing Hoxc8-mediated regulatory networks.

Prioritizing candidate genes by weighted network structure for the identification of disease marker genes

Miyoung Shin,Hyungmin Lee 한국바이오칩학회 2011 BioChip Journal Vol.5 No.1

The use of microarray gene expression profiles for gene ranking is one of the most popular approaches to find marker genes associated with specific diseases. In addition, recently, other types of biological resources, such as gene annotations, bio-litera-ture, and so forth, have been also explored along with the expression profiles. The GeneRank algorithm is one of such approaches that employs gene annotation data as well as expression scores to prioritize genes. Particularly, the GeneRank algorithm constructs an unweighted network structure from gene annotation data. Based on such network, it calculates ranking scores for individual genes according to their associated links and expression scores. In this work, our interest is to investigate the effectiveness of the weighted network structure generated from gene annotations for gene prioritization. For this purpose, we propose two novel weighting schemes to define the link strength between genes, called the Shared Functions (SF) link-weighting scheme and the Weighted Shared Functions (WSF) link-weighting scheme. The evaluation of the proposed schemes was done by applying them to prioritize candidate genes associated with prostate cancer. That is, from microarray expression profiles and gene annotation data, we produced ranking scores of individual genes based on the weighted network structure built by our proposed link-weighting schemes. As results, the top n-ranked genes were taken as our selection of marker genes associated with prostate cancer. For biological validation of the identified marker genes, we searched for a priori known list of genes related to prostate cancer disease from bio-literature and used them as the gold standard. Then, from the top n-ranked genes, we counted how many genes in the gold standard were identified by using the proposed schemes. According to our experiments, the proposed link-weighting schemes improved the perfor-mance of the detection of disease marker genes, compared to original GeneRank algorithm. Consequently, it is observed that the use of the weighted network structure for gene ranking can be very effective to identify marker genes involved in specific diseases.

Isolation of Defense-Related Genes from Nicotiana glutinosa Infected by Tobacco Mosaic Virus Using a Modified Differential Screening

Park, Kyung-Soon,Suh, Mi-Chung,Cheong, Jong-Joo,Park, Doil The Korean Society of Plant Pathology 1999 Plant Pathology Journal Vol.15 No.5

Many of plant defense responses are consequence of transcriptional activation of related genes. We have developed a modified differential screening procedure to isolate tobacco genes that are involved in the defense responses against TMV infection. A cDNA library was constructed from Nicotiana glutinosa leaves infected by TMV under temperature shift conditions. Each of plasmid DNA in the library was hybridized on a set of slot blots to a pool of cDNA probes prepared from either TMV-infected or mock-treated tobacco leaves. Among 900 plasmid DNAs, 81 clones exhibiting significantly enhanced or reduced level of hybridization to either probe were selected for nucleotide sequencing. The clones were listed into 61 genes considering redundancy between the sequences. The genes were identified to be defense-related genes including PR-genes and genes involved in primary or secondary metabolisms. This results supports the implication that plant defense process entails a major shift in total cellular metabolisms rather than activation of a limited number of defense-related genes. Expression patterns of a number of defense-related genes. Expression patterns of a number of selected genes were examined in northern blot analyses. It is notable that the clone 630 of unknown function exhibits expression pattern similar to those of previously known PR-genes. Experiments to elucidate the roles in defense mechanism of a couple of genes newly identified in this study are in progress.

RFLP Analysis of cry1 and cry2 Genes of Bacillus thuringiensis Isolates from India

( Patel Ketan D. ),( Sanjay S. Ingle ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.6

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.