Current Genomics

ISSN: 1389-2029

Current Genomics
Volume 9, Number 4, June 2008

Contents


Application of TILLING and EcoTILLING as Reverse Genetic Approaches to Elucidate the Function of Genes in Plants and Animals Pp. 212-226
N.A. Barkley and M.L. Wang
[Abstract]


Plant Spliceosomal Introns: Not Only Cut and Paste Pp. 227-238
L. Morello and D. Breviario
[Abstract]


Nutrigenomic Analysis of Diet-Gene Interactions on Functional Supplements for Weight Management Pp. 239-251
F.C. Lau, M. Bagchi, C. Sen, S. Roy and D. Bagchi
[Abstract]


The Potential Role of Pharmacogenomic and Genomic in the Adjuvant Treatment of Early Stage Non Small Cell Lung Cancer Pp. 252-262
C. Schettino, M.A. Bareschino, P. Maione, A. Rossi, F. Ciardiello and C. Gridelli
[Abstract]


Mass Spectrometry-Based Approaches Toward Absolute Quantitative Proteomics Pp. 263-274
K. Kito and T. Ito
[Abstract]


NBS1 Heterozygosity and Cancer Risk Pp. 275-281
A. di Masi and A. Antoccia
[Abstract]


Episcopic 3D Imaging Methods: Tools for Researching Gene Function Pp. 282-289
W.J. Weninger and S.H. Geyer
[Abstract]




Abstracts


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Application of TILLING and EcoTILLING as Reverse Geneticv Approaches to Elucidate the Function of Genes in Plants and Animals
N.A. Barkley and M.L. Wang

With the fairly recent advent of inexpensive, rapid sequencing technologies that continue to improve sequencing efficiency and accuracy, many species of animals, plants, and microbes have annotated genomic information publicly available. The focus on genomics has thus been shifting from the collection of whole sequenced genomes to the study of functional genomics. Reverse genetic approaches have been used for many years to advance from sequence data to the resulting phenotype in an effort to deduce the function of a gene in the species of interest. Many of the currently used approaches (RNAi, gene knockout, site-directed mutagenesis, transposon tagging) rely on the creation of transgenic material, the development of which is not always feasible for many plant or animal species. TILLING is a non-transgenic reverse genetics approach that is applicable to all animal and plant species which can be mutagenized, regardless of its mating / pollinating system, ploidy level, or genome size. This approach requires prior DNA sequence information and takes advantage of a mismatch endonuclease to locate and detect induced mutations. Ultimately, it can provide an allelic series of silent, missense, nonsense, and splice site mutations to examine the effect of various mutations in a gene. TILLING has proven to be a practical, efficient, and an effective approach for functional genomic studies in numerous plant and animal species. EcoTILLING, which is a variant of TILLING, examines natural genetic variation in populations and has been successfully utilized in animals and plants to discover SNPs including rare ones. In this review, TILLING and EcoTILLING techniques, beneficial applications and limitations from plant and animal studies are discussed.


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Plant Spliceosomal Introns: Not Only Cut and Paste
L. Morello and D. Breviario

Spliceosomal introns in higher eukaryotes are present in a high percentage of protein coding genes and represent a high proportion of transcribed nuclear DNA. In the last fifteen years, a growing mass of data concerning functional roles carried out by such intervening sequences elevated them from a selfish burden carried over by the nucleus to important active regulatory elements. Introns mediate complex gene regulation via alternative splicing; they may act in cis as expression enhancers through IME (intron-mediated enhancement of gene expression) and in trans as negative regulators through the generation of intronic microRNA. Furthermore, some introns also contain promoter sequences for alternative transcripts. Nevertheless, such regulatory roles do not require long conserved sequences, so that introns are relatively free to evolve faster than exons: this feature makes them important tools for evolutionary studies and provides the basis for the development of DNA molecular markers for polymorphisms detection. A survey of introns functions in the plant kingdom is presented.


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Nutrigenomic Analysis of Diet-Gene Interactions on Functional Supplements for Weight Management
F.C. Lau, M. Bagchi, C. Sen, S. Roy and D. Bagchi

Recent advances in molecular biology combined with the wealth of information generated by the Human Genome Project have fostered the emergence of nutrigenomics, a new discipline in the field of nutritional research. Nutrigenomics may provide the strategies for the development of safe and effective dietary interventions against the obesity epidemic. According to the World Health Organization, more than 60% of the global disease burden will be attributed to chronic disorders associated with obesity by 2020. Meanwhile in the US, the prevalence of obesity has doubled in adults and tripled in children during the past three decades. In this regard, a number of natural dietary supplements and micronutrients have been studied for their potential in weight management. Among these supplements, (–)-hydroxycitric acid (HCA), a natural extract isolated from the dried fruit rind of Garcinia cambogia, and the micronutrient niacin-bound chromium(III) (NBC) have been shown to be safe and efficacious for weight loss. Utilizing cDNA microarrays, we demonstrated for the first time that HCA-supplementation altered the expression of genes involved in lipolytic and adipogenic pathways in adipocytes from obese women and up-regulated the expression of serotonin receptor gene in the abdominal fat of rats. Similarly, we showed that NBC-supplementation up-regulated the expression of myogenic genes while suppressed the expression of genes that are highly expressed in brown adipose tissue in diabetic obese mice. The potential biological mechanisms underlying the observed beneficial effects of these supplements as elucidated by the state-of-the-art nutrigenomic technologies will be systematically discussed in this review.


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The Potential Role of Pharmacogenomic and Genomic in the Adjuvant Treatment of Early Stage Non Small Cell Lung Cancer
C. Schettino, M.A. Bareschino, P. Maione, A. Rossi, F. Ciardiello and C. Gridelli

Although notable progress has been made in the treatment of non-small-cell lung cancer (NSCLC) in recent years, this disease is still associated with a poor prognosis. Despite early-stage NSCLC is considered a potentially curable disease following complete resection, the majority of patients relapse and eventually die after surgery. Adjuvant chemotherapy prolongs survival, altough the absolute improvement in 5-year overall survival is only approximately 5%.

Trying to understand the role of genes which could affect drug activity and response to treatment is a major challenge for establishing an individualised chemotherapy according to the specific genetic profile of each patient. Among genes involved in the DNA repair system, the excision repair cross-complementing 1 (ERCC1) is a useful markers of clinical resistance to platinum-based chemotherapy. In the International Lung Cancer Trial (IALT) adjuvant chemotherapy significantly prolonged survival among patients with ERCC1 negative tumors but not among ERCC1-positive patients. BRCA1 and ribonucleotide reductase M1 (RRM1), two other key enzymes in DNA synthesis and repair, appear to be modulators of drug sensitivity and may provide additional information for customizing adjuvant chemotherapy.

Several clinical trials suggest that overexpression of class III β-tubulin is an adverse prognostic factor in cancer since it could be responsible for resistance to anti-tubulin agents. A retrospective analysis of NCIC JBR.10 trial showed that high tubulin III expression is associated with a higher risk of relapse following surgery alone but also with a higher probability of benefit from adjuvant cisplatin plus vinorelbine chemotherapy.

Finally, the use of gene expression patterns such as the lung metagene model could provide a potential mechanism to refine the estimation of a patient’s risk of disease recurrence and could affect treatment decision in the management of early stage of NSCLC.

In this review we will discuss the potential role of pharmacogenomic approaches to guide the medical treatment of early stage NSCLC.


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Mass Spectrometry-Based Approaches Toward Absolute Quantitative Proteomics
K. Kito and T. Ito

Mass spectrometry has served as a major tool for the discipline of proteomics to catalogue proteins in an unprecedented scale. With chemical and metabolic techniques for stable isotope labeling developed over the past decade, it is now routinely used as a method for relative quantification to provide valuable information on alteration of protein abundance in a proteome-wide scale. More recently, absolute or stoichiometric quantification of proteome is becoming feasible, in particular, with the development of strategies with isotope-labeled standards composed of concatenated peptides. On the other hand, remarkable progress has been also made in label-free quantification methods based on the number of identified peptides. Here we review these mass spectrometry-based approaches for absolute quantification of proteome and discuss their implications.


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NBS1 Heterozygosity and Cancer Risk
A. di Masi and A. Antoccia

Biallelic mutations in the NBS1 gene are responsible for the Nijmegen breakage syndrome (NBS), a rare autosomal recessive disorder characterized by chromosome instability and hypersensitivity to ionising radiation (IR). Epidemiological data evidence that the NBS1 gene can be considered a susceptibility factor for cancer development, as demonstrated by the fact that almost 40% of NBS patients have developed a malignancy before the age of 21. Interestingly, also NBS1 heterozygotes, which are clinically asymptomatic, display an elevated risk to develop some types of malignant tumours, especially breast, prostate and colorectal cancers, lymphoblastic leukaemia, and non-Hodgkin’s lymphoma (NHL). So far, nine mutations in the NBS1 gene have been found, at the heterozygous state, in cancer patients. Among them, the 657del5, the I171V and the R215W mutations are the most frequently described. The pathogenicity of these mutations is presumably connected with their occurrence in the highly conserved BRCT tandem domains of the NBS1 protein, which are present in a large superfamily of proteins, and are recognized as major mediators of processes related to cell-cycle checkpoint and DNA repair.

This review will focus on the current state-of-knowledge regarding the correlation between carriers of NBS1 gene mutations and the proneness to the development of malignant tumours.


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Episcopic 3D Imaging Methods: Tools for Researching Gene Function
W.J. Weninger and S.H. Geyer

This work aims at describing episcopic 3D imaging methods and at discussing how these methods can contribute to researching the genetic mechanisms driving embryogenesis and tissue remodelling, and the genesis of pathologies. Several episcopic 3D imaging methods exist. The most advanced are capable of generating high-resolution volume data (voxel sizes from 0.5x0.5x1μm upwards) of small to large embryos of model organisms and tissue samples. Beside anatomy and tissue architecture, gene expression and gene product patterns can be three dimensionally analyzed in their precise anatomical and histological context with the aid of whole mount in situ hybridization or whole mount immunohistochemical staining techniques. Episcopic 3D imaging techniques were and are employed for analyzing the precise morphological phenotype of experimentally malformed, randomly produced, or genetically engineered embryos of biomedical model organisms. It has been shown that episcopic 3D imaging also fits for describing the spatial distribution of genes and gene products during embryogenesis, and that it can be used for analyzing tissue samples of adult model animals and humans. The latter offers the possibility to use episcopic 3D imaging techniques for researching the causality and treatment of pathologies or for staging cancer. Such applications, however, are not yet routine and currently only preliminary results are available. We conclude that, although episcopic 3D imaging is in its very beginnings, it represents an upcoming methodology, which in short terms will become an indispensable tool for researching the genetic regulation of embryo development as well as the genesis of malformations and diseases.