ANALYSIS OF JUNCTION SEQUENCES RESULTING FROM INTEGRATION AT NONHOMOLOGOUS LOCI IN NEUROSPORA-CRASSA

We have analyzed the junctions involved in two examples of ectopic int egration of plasmids containing the am+ (glutamate dehydrogenase) gene into a strain of Neurospora crassa bearing a complete deletion of the am locus. In one transformed strain a single copy of plasmid DNA had been integrated into linkage group (LG) III DNA without the loss of ch romosomal DNA. In contrast, 450 bp had been lost from plasmid sequence s at the site of integration. The transforming DNA used was circular, so we postulate that the plasmid was linearized and truncated prior to its integration by end joining into a break in LG III DNA. There was no significant homology between the incoming DNA and DNA at the site o f integration. The second transformed strain resulted from transformat ion with a linearized plasmid. It contained multiple integrated copies of plasmid DNA, one of which was recloned, together with adjacent chr omosomal DNA, by plasmid rescue in Escherichia coli. Prior to integrat ion into chromosomal DNA, the linear plasmid had been truncated by 64 bp on one end and 3.2 kbp on the other end. One end of the integrated DNA was adjacent to DNA from the right arm of LG I, while the other en d was integrated into a copy of a repetitive sequence. Restriction fra gment length polymerisim mapping showed that integration was in a copy of the repetitive sequence that is linked to the previously unassigne d telomere M11 and is distantly linked to the LG VI marker con-11. Gen etic analysis revealed that a long segment of LG I containing all mark ers from un-1 to the right tip has been translocated to the right end of LG VI. Tetrad analysis showed that the integrated DNA was closely l inked to the translocation. We conclude that the transforming DNA was truncated and joined to DNA from two different chromosomes by end join ing during the formation of a quasiterminal translocation, T(IR --> VI R) UK-T12. We also conclude that the previously unassigned telomere, M 11, is the right end of LG VI.