New experimental and computational approaches to the analysis of gene expression.
Public and private EST (Expressed Sequence Tag) programs provide access to a large number of ESTs from a number of plant species, including Arabidopsis, corn, soybean, rice, wheat. In addition to the homology of each EST to genes in GenBank, information about homology to all other ESTs in the data base can be obtained. To estimate expression levels of genes represented in the DuPont EST data base we count the number of times each gene has been seen in different cDNA libraries, from different tissues, developmental stages or induction conditions. This quantitation of message levels is quite accurate for highly expressed messages and, unlike conventional Northern blots, allows comparison of expression levels between different genes. Lists of most highly expresses genes in different libraries can be compiled. Also, if EST data is available for cDNA libraries derived from different developmental stages, gene expression profiles across development can be assembled. We present an example of such a profile for soybean seed development. Gene expression data obtained from Electronic Northern analysis can be confirmed and extended beyond the realm of highly expressed genes by using high density DNA arrays. The ESTs identified as interesting can be arrayed on nylon or glass and probed with total labeled cDNA first strand from the tissue of interest. Two-color fluorescent labeling allows accurate mRNA ratio measurements. We are currently using the DNA array technology to study chemical induction of gene expression and the biosynthesis of oil, carbohydrate and protein in developing seeds.
DuPont Agricultural Biotechnology - Genomics. Delaware Technology Park, Newark 19714-6104, USA. j-antoni.rafalski@usa.dupont.com
J A
JA
Rafalski
M
M
Hanafey
G H
GH
Miao
A
A
Ching
J M
JM
Lee
M
M
Dolan
S
S
Tingey
D015152:Blotting, Northern / Q000379:methods; D003198:Computer Simulation; D018076:DNA, Complementary / Q000032:analysis; D016208:Databases, Factual; D020224:Expressed Sequence Tags; D015870:Gene Expression; D017343:Genes, Plant; D008967:Molecular Biology / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000378:metabolism
D018076:DNA, Complementary; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors