to introduce a certain amount of randomness into minicircle segregation. In other words, sister minicircles may not necessarily end up in different daughter cells. A pulse–chase experiment performed with C. fasciculata cells at the light microscope level previously showed that newly replicated minicircles are spread throughout the network after one cell cycle is completed (Simpson et al., 1974).
In the case of T. brucei, the network apparently does not rotate and two dumbbell-shaped masses of nicked replicated minicircles accumulate at either end of the nucleoid body, which then divides in half into the daughter cells (Ferguson et al., 1994; Robinson and Gull, 1994). In this case there does not appear to be a mechanism for randomization throughout the network, other than the possible random selection and migration to the antipodal replisomes.
The number of minicircles per network in L. tarentolae was assayed by counting 4′,6-diamidino-2-phenylindole (DAPI)-stained networks in a cell counting chamber using a fluorescent microscope and measuring the DNA concentration spectrophotometrically. Quantitative dot blot hybridization using an oligonucleotide probe complementary to the conserved CSB-3 12-mer sequence yielded values of 12,600 ± 300 and 12,700 ± 800 for the UC and LEM125 strains, respectively. Similar dot blot hybridization analysis showed that the copy number of maxicircle DNA molecules was very similar in the UC and LEM125 strains (32 ± 2 and 25 ± 2 copies per network, respectively).
Quantitation of the copy numbers of 17 specific minicircle sequence classes in the UC strain was previously performed by Southern blot analysis using specific oligonucleotide probes for specific gRNAs. We have repeated these analyses with both UC strain kDNA and LEM125 strain kDNA, by dot blot hybridization of MspI-digested kDNA (all minicircles have at least one MspI site), and a known amount of specific cloned minicircles using primers specific to each gRNA. A primer to the conserved 12-mer sequence was used as a loading control. The results in Table 1 show that homologous minicircle sequence class frequencies are extremely variable, both between strains and between different kDNA isolates from the same strain taken after several years of culture. In general, the LEM125 strain kDNA exhibited lower copy numbers for the sequence classes in common between the strains, which is consistent with LEM125 possessing a more complex minicircle repertoire.
In the UC strain kDNA as mentioned above, two gRNAs, pLtl9 (= G4-III) and pB4 (= gND3-IX), are nonfunctional, in that all of the other