|Functional Genomics Section
DOE Human Genome Program Contractor-Grantee Workshop
146. One Gene - How Many Proteins?
Raymond F. Gesteland, Chad Nelson,
Mike Giddings, Norma Wills, Jiadong Zhou, Barry Moore, Mike Howard, and
This is a new pilot project to use mass spectrometry methods to determine the multiplicity and character of proteins coming from individual mRNAs. Many processes contribute to the complexity of gene products that come from one gene. In addition to alternate splicing and RNA editing that increase mRNA complexity, protein modification and alternative translation can all expand the population of proteins that come from one mRNA species. Although we know a good deal about protein modifications on a protein by protein basis, we know little in a genome-wide sense. We know even less about the frequency of occurrence of unusual translation events. Alternative translations, or recoding, include programmed frameshifts, bypassing of mRNA regions, and redefinition of stop codons to encode one of the twenty amino acids or selenocysteine the 21st amino acid.
We are developing technology to ask how many different protein products come from each mRNA species. We are using Electrospray Liquid Chromatography Mass Spectrometry. With a genome of known sequence, such as yeast, we can fractionate proteins and by accurately determining their masses, see if they can be accounted for by predicted molecular weights from the known open reading frames. Identification with genes can be verified by mass analysis of tryptic digests. If initial molecular weights do not conform to any known ORF, alternate origins must be considered. Protein modifications will add predictable masses up to a few hundred Daltons, and again confirmation can be obtained by analysis of tryptic peptides. Recoding events such as frameshifting or bypassing will often result in more drastic changes in mass. Tryptic peptide analysis will identify the genome origin from which a limited number of possible masses due to recoding events can be predicted. Again, analysis of tryptic peptides should allow identification of the specific recoding event.
We are initially analyzing mitochondria of the yeast Saccharomyces cerevisiae since this will limit complexity to a fraction of the whole yeast genome - perhaps 500 genes out of 6,500. We are also pursuing tagging methods that are suited for examining one gene at a time and that will be more suited for analysis of the complexity of proteins coming from human genes. From this approach we hope to define the real complexity of the genome products.
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