Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

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 anl 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.In a number of systems, drug resistance is mediated by gene amplification, and in all cases studied, a sequence much larger than the selected gene is amplified. The first estimates of the average sizes of amplified units-defined as extra DNA sequences containing one copy of the selected geneindicate that they range from about 200 to 3,000 kilobases (kb) in highly resistant cells which have experienced several independent amplification events (for reviews, see references 18, 35, and 39). More recently, a study of the structures formed in the first step of amplification of the CAD gene in Syrian hamster cells has led to the hypothesis that, in this case, the average size of the amplified unit may be 10,000 kb or more (15).Several different techniques have been used to study the structure of the coamplified DNA: cloning of amplified sequences by differential screening (2,4,15) or from purified double minutes (5,14) or homogeneously staining regions (19,43), chromosome walking from the cloned selected genes (11,22), direct visualization of ethidium bromidestained amplified restriction fragments in agarose gels (24,40) and in gel renaturation (12, 31). In most cases, the results point to remarkable differences between the sequences coamplified in independent highly amplified mutants: the amplified units include a region comprising the selected gene and sequences immediately surrounding it in the wild-type line, but also include sequences which are amplified uniquely in each cell line (2,5,12, 34,36,43). Thus, during the amplification process, pieces of DNA from different parts of the genome are joined by recombination to form novel structures and novel join...

Evolution and stability of chromosomal DNA coamplified with the CAD gene.

“…In the recent work of Debatisse et al (5), the AMP deaminase gene was cloned in this way. It is also interesting that two striking examples of preferential reamplification were observed by Debatisse et al (5). Two novel joints, one on either side of the AMP deaminase gene, generated independently in different first-step mutants, were amplified in every secondand third-step mutant studied by these authors.…”

Two extended arrays of a satellite DNA sequence at the centromere and at the short-arm telomere of Chinese hamster chromosome 5

“…Cos9-1 is a random cosmid clone isolated from a Chinese hamster library of genomic DNA partially digested with MboI (Debatisse et al, 1988). The insert of cos9-1 comprises about 30 kb and does not contain any recognition site for the restriction enzymes EcoRI, HindIII, BamHI, and SacI, suggesting that it contains a large block of tandemly repeated DNA.…”

The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification.

“…It is highly reamplified (up to 150fold) in the third step mutant HC50474 which concomitantly A: AMPD gene; W,X,Yl,Y2: co-amplified genes. The W-A-Y, linkage has been confirmed by a cosmid walk (Debatisse et al, 1988). Middle line: restriction map of the wild-type (GMA32) W region (Hyrien et al, 1987) suffered a mass deletion of pre-existing amplified units (Debatisse et al, 1986).…”