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Articles Online (Volume 4, Issue 3)

Article

Functional Characterization of Gibberellin-Regulated Genes in Rice Using Microarray System

Asad Jan, Setsuko Komatsu

Gibberellin (GA) is collectively referred to a group of diterpenoid acids, some of which act as plant hormones and are essential for normal plant growth and development. DNA microarray technology has become the standard tool for the parallel quantification of large numbers of messenger RNA transcripts. The power of this approach has been demonstrated in dissecting plant physiology and development, and in unraveling the underlying cellular signaling pathways. To understand the molecular mechanism by which GA regulates the growth and development of plants, with reference to the monocot model plant—rice, it is essential to identify and analyze more genes and their products at the transcription and translation levels that are regulated by GA. With the availability of draft sequences of two major rice types, indica and japonica rice, it has become possible to analyze global expression profiles of genes on a genome scale. In this review, the progress made in finding new genes in rice leaf sheath using microarray system and their characterization is discussed. It is believed that the findings made in this regard have important implications for understanding the mechanism by which GA regulates the growth and development of rice.

Page 137–144

Article

Glia Maturation Factor Gamma (GMFG): A Cytokine-Responsive Protein During Hematopoietic Lineage Development and Its Functional Genomics Analysis

Ying Shi, , , Ling Chen, Lance A. Liotta, Hong-Hui Wan, Griffin P. Rodgers

Human hematopoiesis was evaluated using the techniques of controlled stem cell differentiation, two-dimensional gel electrophoresis-based proteomics, and functional genomics. We provide the first report that glia maturation factor gamma (GMFG) is a cytokine-responsive protein in erythropoietin-induced and granulocyte-colony stimulating factor-induced hematopoietic lineage development. Results from global functional genomics analysis indicate that GMFG possesses several other features: hematopoietic tissue-specific gene expression, a promoter concentrated with high-score hematopoiesis-specific transcription factors, and possible molecular coevolution with a rudimentary blood/immune system. On the basis of our findings, we hypothesize that GMFG is a hematopoietic-specific protein that may mediate the pluripotentiality and lineage commitment of human hematopoietic stem cells.

Page 145–155

Article

A Combined Computational and Experimental Study on the Structure-Regulation Relationships of Putative Mammalian DNA Replication Initiator GINS

Reiko Hayashi, Takako Arauchi, Moe Tategu, Yuya Goto, Kenichi Yoshida

GINS, a heterotetramer of SLD5, PSF1, PSF2, and PSF3 proteins, is an emerging chromatin factor recognized to be involved in the initiation and elongation step of DNA replication. Although the yeast and Xenopus GINS genes are well documented, their orthologous genes in higher eukaryotes are not fully characterized. In this study, we report the genomic structure and transcriptional regulation of mammalian GINS genes. Serum stimulation increased the GINS mRNA levels in human cells. Reporter gene assay using putative GINS promoter sequences revealed that the expression of mammalian GINS is regulated by 17β-Estradiol-stimulated estrogen receptor α, and human PSF3 acts as a gene responsive to transcription factor E2F1. The goal of this study is to present the current data so as to encourage further work in the field of GINS gene regulation and functions in mammalian cells.

Page 156–164

Article

Comparative Proteome Analysis of Breast Cancer and Adjacent Normal Breast Tissues in Human

Shi-Shan Deng, Tian-Yong Xing, Hong-Ying Zhou, Ruo-Hong Xiong, You-Guang Lu, Bin Wen, Shang-Qing Liu, Hui-Jun Yang

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS), incorporated with online database searching, were performed to investigate differential proteins of breast cancer and adjacent normal breast tissues. Considering that serum albumin is abundantly presented in normal control samples, 15 differential spots detected in 11 out of 12 (91.7%) breast cancer samples were identified by online SIENA-2DPAGE database searching and MALDI-TOF/TOF-MS analysis. The results indicate that pathological changes of breast cancer are concerned with augmentation of substance metabolism, promotion of proteolytic activity, decline of activity of some inhibitors of enzymes, and so on. Some important proteins involved in the pathological process of breast cancer with changed expression may be useful biomarkers, such as alpha-1-antitrypsin, EF-1-beta, cathepsin D, TCTP, SMT3A, RPS12, and PSMA1, among which SMT3A, RPS12, and PSMA1 were first reported for breast cancer in this study.

Page 165–172

Article

Evaluation of Six Methods for Estimating Synonymous and Nonsynonymous Substitution Rates

Zhang Zhang, Jun Yu

Methods for estimating synonymous and nonsynonymous substitution rates among protein-coding sequences adopt different mutation (substitution) models with subtle yet significant differences, which lead to different estimates of evolutionary information. Little attention has been devoted to the comparison of methods for obtaining reliable estimates since the amount of sequence variations within targeted datasets is always unpredictable. To our knowledge, there is little information available in literature about evaluation of these different methods. In this study, we compared six widely used methods and provided with evaluation results using simulated sequences. The results indicate that incorporating sequence features (such as transition/transversion bias and nucleotide/codon frequency bias) into methods could yield better performance. We recommend that conclusions related to or derived from Ka and Ks analyses should not be readily drawn only according to results from one method.

Page 173–181

Article

Phylogenetic Analysis of RhoGAP Domain-Containing Proteins

Marcelo M. Brandão, Karina L. Silva-Brandão, Fernando F. Costa, Sara T.O. Saad

Proteins containing an Rho GTPase-activating protein (RhoGAP) domain work as molecular switches involved in the regulation of diverse cellular functions. The ability of these GTPases to regulate a wide number of cellular processes conies from their interactions with multiple effectors and inhibitors, including the RhoGAP family, which stimulates their intrinsic GTPase activity. Here, a phylogenetic approach was applied to study the evolutionary relationship among 59 RhoGAP domain-containing proteins. The sequences were aligned by their RhoGAP domains and the phylogenetic hypotheses were generated using Maximum Parsimony and Bayesian analyses. The character tracing of two traits, GTPase activity and presence of other domains, indicated a significant phylogenetic signal for both of them.

Page 182–188

Article

Comparative Analysis of Transcription Start Sites Using Mutual Information

D. Ashok Reddy, Chanchal K. Mitra

The transcription start site (TSS) region shows greater variability compared with other promoter elements. We are interested to search for its variability by using information content as a measure. We note in this study that the variability is significant in the block of 5 nucleotides (nt) surrounding the TSS region compared with the block of 15 nt. This suggests that the actual region that may be involved is in the range of 5–10 nt in size. For Escherichia coli, we note that the information content from dinucleotide substitution matrices clearly shows a better discrimination, suggesting the presence of some correlations. However, for human this effect is much less, and for mouse it is practically absent. We can conclude that the presence of short-range correlations within the TSS region is species-dependent and is not universal. We further observe that there are other variable regions in the mitochondrial control element apart from TSS. It is also noted that effective comparisons can only be made on blocks, while single nucleotide comparisons do not give us any detectable signals.

Page 189–195

Article

Optimal Allocation of Replicates for Measurement Evaluation Studies

Stanislav O. Zakharkin, Kyoungmi Kim, Alfred A. Bartolucci, Grier P. Page, David B. Allison

Optimal experimental design is important for the efficient use of modern high-throughput technologies such as microarrays and proteomics. Multiple factors including the reliability of measurement system, which itself must be estimated from prior experimental work, could influence design decisions. In this study, we describe how the optimal number of replicate measures (technical replicates) for each biological sample (biological replicate) can be determined. Different allocations of biological and technical replicates were evaluated by minimizing the variance of the ratio of technical variance (measurement error) to the total variance (sum of sampling error and measurement error). We demonstrate that if the number of biological replicates and the number of technical replicates per biological sample are variable, while the total number of available measures is fixed, then the optimal allocation of replicates for measurement evaluation experiments requires two technical replicates for each biological replicate. Therefore, it is recommended to use two technical replicates for each biological replicate if the goal is to evaluate the reproducibility of measurements.

Page 196–202