cDNA expression and human two-dimensional gel protein databases: towards integrating DNA and protein information.
The rapid progress in characterizing genes and mRNAs (expressed sequence tags, ESTs) as a result of the Human Genome Project makes it imperative to develop strategies to interface DNA mapping and sequencing data with protein information, as the latter orchestrate most cellular functions. Presently, the only technique able to resolve and record the thousands of proteins present in cells and tissues is two-dimensional (2-D) gel electrophoresis in combination with computer-aided technology to scan the gels, make synthetic images, assign numbers to individual spots as well as to enter qualitative and quantitative information. To date, comprehensive 2-D gel databases containing information about various properties of proteins (cellular localization, identification, regulatory properties, partial amino acid sequences, etc.) have been established (available on the internet: http:@biobase.dk/cgi-bin/celis). What remains is to provide a link between these data and the forthcoming information from the Human Genome Project. We are pursuing two approaches to achieve this goal: (i) microsequencing and mass spectrometry analysis of proteins resolved from 2-D gels and (ii) expression of cDNAs in the vaccinia virus expression system. Using the latter approach we have expressed about 60 cDNAs in human cells under conditions that faithfully reproduce post-translational trimmings and modifications of the proteins. The method, in combination with 2-D gel electrophoresis, allows precise matching of almost any cDNA to its protein product, irrespective of the protein abundance.
Leffers
H
H
Institute of Medical Biochemistry, Aarhus University, Denmark. lef@biobase.dk
Dejgaard
K
K
Honoré
B
B
Madsen
P
P
Nielsen
M S
MS
Celis
J E
JE
D000818:Animals; D019556:COS Cells; D003195:Computer Communication Networks; D004247:DNA / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D016208:Databases, Factual; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D016045:Human Genome Project; D006801:Humans; D011506:Proteins / Q000235:genetics; D012015:Reference Standards; D017421:Sequence Analysis; D014616:Vaccinia virus / Q000235:genetics
D018076:DNA, Complementary; D011506:Proteins; D004247:DNA