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A strategy for genome-wide gene analysis: integrated procedure for gene identification.

ABSTRACT: We have developed a technique called the Integrated Procedure for Gene Identification that modifies and integrates parts from several existing techniques to increase the efficiency for genome-wide gene identification. The procedure has the following features: (i) Only the 3' portion of the expressed templates is used to ensure a match to 3' expressed sequence tag (EST) sequences; (ii) the 3' portion of the cDNA is poly dA/poly dT minus, which maintains complete representation of the expressed copies, particularly the rare copies, which otherwise would be lost heavily because of random poly dA/poly dT hybridization in the subtraction reaction; (iii) redundancy is decreased substantially by the subtraction reaction to reduce the effort for sequencing analysis; (iv) the nonsubtracted templates that largely contain the rare copies are amplified selectively with suppression PCR and are sequenced directly or through serial analysis of gene expression (SAGE); and (v) the identified sequences are matched to databases to determine whether they are cloned genes, ESTs, or novel sequences. Using this procedure in a model system, we showed that the redundant copies were largely removed, and the rates of EST matches and the novel sequence identification were significantly increased. Most of the plasmids containing the matched EST are readily available from the IMAGE consortium. This technique can be used to index genome-wide expressed genes and to identify differentially expressed genes in different cells. Compared with the existing techniques, this procedure is relatively efficient, simple, less expensive, and labor intensive. It is especially useful for standard molecular laboratories to perform genome-wide studies.

SUBMITTER: Wang SM

PROVIDER: S-EPMC21739 | biostudies-literature | 1998 Sep

REPOSITORIES: biostudies-literature

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A strategy for genome-wide gene analysis: integrated procedure for gene identification.

Wang S M SM Rowley J D JD

Proceedings of the National Academy of Sciences of the United States of America 19980901 20

We have developed a technique called the Integrated Procedure for Gene Identification that modifies and integrates parts from several existing techniques to increase the efficiency for genome-wide gene identification. The procedure has the following features: (i) Only the 3' portion of the expressed templates is used to ensure a match to 3' expressed sequence tag (EST) sequences; (ii) the 3' portion of the cDNA is poly dA/poly dT minus, which maintains complete representation of the expressed co ...[more]

PMID: 9751764

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Project description:BACKGROUND: Aspartic proteases (APs) are a large family of proteolytic enzymes found in almost all organisms. In plants, they are involved in many biological processes, such as senescence, stress responses, programmed cell death, and reproduction. Prior to the present study, no grape AP gene(s) had been reported, and their research on woody species was very limited. RESULTS: In this study, a total of 50 AP genes (VvAP) were identified in the grape genome, among which 30 contained the complete ASP domain. Synteny analysis within grape indicated that segmental and tandem duplication events contributed to the expansion of the grape AP family. Additional analysis between grape and Arabidopsis demonstrated that several grape AP genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of grape and Arabidopsis. Phylogenetic relationships of the 30 VvAPs with the complete ASP domain and their Arabidopsis orthologs, as well as their gene and protein features were analyzed and their cellular localization was predicted. Moreover, expression profiles of VvAP genes in six different tissues were determined, and their transcript abundance under various stresses and hormone treatments were measured. Twenty-seven VvAP genes were expressed in at least one of the six tissues examined; nineteen VvAPs responded to at least one abiotic stress, 12 VvAPs responded to powdery mildew infection, and most of the VvAPs responded to SA and ABA treatments. Furthermore, integrated synteny and phylogenetic analysis identified orthologous AP genes between grape and Arabidopsis, providing a unique starting point for investigating the function of grape AP genes. CONCLUSIONS: The genome-wide identification, evolutionary and expression analyses of grape AP genes provide a framework for future analysis of AP genes in defining their roles during stress response. Integrated synteny and phylogenetic analyses provide novel insight into the functions of less well-studied genes using information from their better understood orthologs.

Project description:The completion of the grape genome sequencing project has paved the way for novel gene discovery and functional analysis. Aldehyde dehydrogenases (ALDHs) comprise a gene superfamily encoding NAD(P)(+)-dependent enzymes that catalyze the irreversible oxidation of a wide range of endogenous and exogenous aromatic and aliphatic aldehydes. Although ALDHs have been systematically investigated in several plant species including Arabidopsis and rice, our knowledge concerning the ALDH genes, their evolutionary relationship and expression patterns in grape has been limited.A total of 23 ALDH genes were identified in the grape genome and grouped into ten families according to the unified nomenclature system developed by the ALDH Gene Nomenclature Committee (AGNC). Members within the same grape ALDH families possess nearly identical exon-intron structures. Evolutionary analysis indicates that both segmental and tandem duplication events have contributed significantly to the expansion of grape ALDH genes. Phylogenetic analysis of ALDH protein sequences from seven plant species indicates that grape ALDHs are more closely related to those of Arabidopsis. In addition, synteny analysis between grape and Arabidopsis shows that homologs of a number of grape ALDHs are found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the speciation of the grape and Arabidopsis. Microarray gene expression analysis revealed large number of grape ALDH genes responsive to drought or salt stress. Furthermore, we found a number of ALDH genes showed significantly changed expressions in responses to infection with different pathogens and during grape berry development, suggesting novel roles of ALDH genes in plant-pathogen interactions and berry development.The genome-wide identification, evolutionary and expression analysis of grape ALDH genes should facilitate research in this gene family and provide new insights regarding their evolution history and functional roles in plant stress tolerance.

Dataset Information

A strategy for genome-wide gene analysis: integrated procedure for gene identification.

Publications

A strategy for genome-wide gene analysis: integrated procedure for gene identification.

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