REPLICATION INITIATES AT MULTIPLE DISPERSED SITES IN THE RIBOSOMAL DNA PLASMID OF THE PROTOZOAN PARASITE ENTAMOEBA-HISTOLYTICA

In the protozoan parasite Entamoeba histolytica (which causes amoebias is in humans), the rRNA genes (rDNA) in the nucleus are carried on an extrachromosomal circular plasmid, For strain HM-1:IMSS, the size of t he rDNA plasmid is 24.5 kb, and 200 copies per genome are present, Eac h circle contains two rRNA transcription units as inverted repeats sep arated by upstream and downstream spacers, We have studied the replica tion of this molecule by neutral/neutral two-dimensional gel electroph oresis and by electron microscopy, All restriction fragments analyzed by two-dimensional gel electrophoresis gave signals corresponding to s imple Y's and bubbles, This showed that replication initiated in this plasmid at multiple, dispersed locations spread throughout the plasmid , On the basis of the intensity of the bubble arcs, initiations from t he rRNA transcription units seemed to occur more frequently than those from intergenic spacers. Multiple, dispersed initiation sites were al so seen in the rDNA plasmid of strain HK-9 when it was analyzed by two -dimensional gel electrophoresis. Electron microscopic visualization o f replicating plasmid molecules in strain HM-I:IMSS showed multiple re plication bubbles in the same molecule. The location of bubbles on the rDNA circle was mapped by digesting with PvuI or BsaHI, which lineari ze the molecule, and with SacII, which cuts the circle twice. The dist ance of the bubbles from one end of the molecule was measured by elect ron microscopy. The data corroborated those from two-dimensional gels and showed that replication bubbles were distributed throughout the mo lecule and that they appeared more frequently in rRNA transcription un its. The same interpretation was drawn from electron microscopic analy sis of the HK-9 plasmid, Direct demonstration of more than one bubble in the same molecule is clear evidence that replication of this plasmi d initiates at multiple sites, Potential replication origins are distr ibuted throughout the plasmid. Such a mechanism is not known to operat e in any naturally occurring prokaryotic or eukaryotic plasmid.