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Preferential amplification of rearranged sequences near amplified adenylate deaminase genes
M Debatisse1, I Saito, G Buttin
1Laboratory of Molecular Biology, Imperial Cancer Research Fund, London, United Kingdom.
Abstract:
In a previous study of three independent families of mutants selected for overproduction of adenylate deaminase (AMPD), we were not able to isolate a cDNA probe for the gene and so could not demonstrate its amplification directly. In addition to overproduction of AMPD, four proteins of unknown function, designated W, X, Y1, and Y2, accumulated, and by using the corresponding cDNA probes, we demonstrated amplification of all four genes. In independent mutant clones, sometimes all and sometimes only a subset of these genes were amplified. Assuming that all five genes are linked, the pattern of their coamplification suggested a genetic map in which AMPD lies between W and Y1. We show here that a two-step chromosome walk joins the W and Y1 genes, that the AMPD gene is the only expressed sequence between them, and that its amplification is indeed responsible for overproduction of the AMPD protein. In the course of this work, we cloned and studied two novel joints which mark rearrangements on either side of the AMPD gene. Each joint was generated independently in a single first-step mutant at single or low copy number. Remarkably, each joint was amplified preferentially in every second- and third-step mutant derived from the first-step line in which it was originally present, suggesting that the two independent rearrangements each generated amplification-prone structures.
Insights
Gene amplification causes adenylate deaminase (AMPD) overproduction. Researchers confirmed AMPD gene amplification is responsible for increased protein levels, revealing its location between other amplified genes.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Previous studies identified mutants overproducing adenylate deaminase (AMPD) but couldn't directly prove gene amplification.
- Four uncharacterized proteins (W, X, Y1, Y2) also accumulated, with their genes shown to be amplified.
- Coamplification patterns suggested AMPD gene linkage with W, X, Y1, and Y2, with AMPD located between W and Y1.
Purpose of the Study:
- To directly demonstrate gene amplification of adenylate deaminase (AMPD) in overproducing mutants.
- To elucidate the genetic organization and physical linkage of the AMPD gene with other amplified genes.
- To identify the molecular basis for gene amplification and protein overproduction.
Main Methods:
- Performed a two-step chromosome walk to physically link the W and Y1 genes.
- Utilized cDNA probes to identify expressed sequences within the amplified region.
- Cloned and analyzed novel DNA joints marking genomic rearrangements flanking the AMPD gene.
Main Results:
- Confirmed that the adenylate deaminase (AMPD) gene is the sole expressed sequence located between the W and Y1 genes.
- Demonstrated that amplification of the AMPD gene is directly responsible for the observed overproduction of AMPD protein.
- Identified two independent, novel joints associated with rearrangements flanking the AMPD gene, which were preferentially amplified in subsequent mutant steps.
Conclusions:
- The amplification of the adenylate deaminase (AMPD) gene is the cause of its overproduction.
- The AMPD gene is physically located between the W and Y1 genes, confirming the proposed genetic map.
- Specific genomic rearrangements (joints) may predispose the AMPD gene locus to amplification.