Structural organization and expression of amplified chromosomal sequences, which include the rudimentary gene, in cultured Drosophila cells resistant to N-(phosphonacetyl)-l-aspartate

Abstract

We have used 160 kilobases of cloned Drosophila genomic DNA from the rudimentary (r) region to examine the organization of amplified DNA in Drosophila cells resistant to 10 mM N-(phosphonacetyl)-L-aspartate (PALAr cells) obtained by stepwise selection. Evidence for the direct tandem linkage of the amplified sequences is presented. The pattern and intensity of amplified bands as well as the presence of novel junctions in the DNA sequence of PALAr cells indicate that there are two types of units of 150 and 120 kilobases long. The sequence of the smaller unit is entirely included within the larger one. The longer of the two units is present twice while the shorter one is amplified eightfold as compared to the level of the relevant DNA sequences in the wild-type. These data are consistent with a model in which successive crossing-over events over several cell cycles lead to amplification of the selected r gene and flanking sequences. However, these data can also be accounted for by a totally different mechanism in which multiple copies of DNA are generated by rolling circle replication. Transcription units other than the r gene are present within the 150 kilobase region of amplified DNA. These are found to be overexpressed in PALAr cells, though some transcripts are underrepresented relative to the copy number of the corresponding coding sequences.

Sequence of the novel joints present in the amplified DNA of N‐phosphonacetyl‐L‐aspartate resistant Drosophila cells: Implication on the mechanisms of amplification in these cells

“…We have previously shown by Southern analysis that the amplified DNA from PALA r cells contained two additional EcoRl restriction fragments as compared to wildtype DNA [32]. They correspond to novel joints that result from the arrangement in head-to-tail fashion of the 120 kb unit (represented by letters c-f in fig lB) and the 150 kb unit (letters b-f in fig IB).…”

“…Southern blot analyses were performed as previously described using EcoRl-digested DNA from PALA r and wild-type cells [32]. The blots were probed with recombinant phages encompassing the rudimentao, region, obtained from the Canton S library [36] by a chromosomal walk performed in this region [32]. Probes were 32p-labeled by nick-translation.…”

“…Because of the polymorphism between Oregon R and Canton S flies [32], the sequence of the wild-type counterparts of the junctions was not determined on clones from the Canton S library [36], but on clones from wild-type cell DNA libraries that we…”

“…The analysis of inverted joints [23,33,35,41,44] leads to the proposal of models based on chromosomal [23] or extrachromosomal [41,44,63] double rolling circle to explain the formation and amplification of these joints. The analysis of joints between two unrelated regions, in some cases, allows the construction of linear maps of the amplified DNA [32,35]. In other cases, analysis of this type of joint did not give a lot of information as some joints appeared as rearrangements between amplified units or because only one of the wild-type counterparts is known and localized on the wild-type genome [4,15,22,33].…”

“…Two novel joints (J 1 and J2) have been characterized as junctions linking the repeated units. The presence of only two novel joints in the amplified DNA allows us to postulate that the amplification arose at the resident gene [32]. In this system, the investigation of the amplification process was facilitated by the low complexity of the amplified DNA and by the lack of any evidence of superimposed rearrangement events.…”