Specific Deavage of Idnetoplast Minidrde Dna from Leishmania Tarentolae by Mung Bean Nudease and Identification of Several Additional Minidrde Sequence Classes

Multiple sequence classes of kinetoplast miniclrcle DNA from Le^iihmar^ia tarentolae were cleaved by mung bean nuclease In the presence of formamide, yielding unit length linear molecules which retained the anomalous electrophoretic mobility in acrylamide characteristic of minicircle DNA. No specific cleavage site sequence common to all minicircle sequence classes was apparent, although the main region of nuclease cleavage was localized approximately 350 bp from the unique Smal restriction site of the conserved region found in all minicircle sequence classes. Covalent closure of the minicircle substrate was not a requirement for cleavage, as linearized network-derived or cloned minicircles were also cleaved by mung bean nuclease at similar locations. The partial sequences of several new minicircle sequence classes released from the network by mung bean nuclease are also reported. INTRODUCTION The structurally unique mltochondrial genome of the hemoflagellate protozoa, termed kinetoplast DNA (kDNA), consists of a catenated network of 5-20 X 10 minicircles and 20-50 maxicircles (1-3). The maxicircle represents the informational mitochondrial DNA of these cells. It consists of a conserved transcribed region encoding sequences homologous to known mitochondrial genes (4-10) and a nontranscribed region which diverges extensively in size and sequence among the various trypanosomatid genera (1114). The function of minicircle DNA is not known. Minicircles range in size from 465 to 2300 bp, depending on the species (3); however, within any one species minicircle size is fairly uniform. A single network typically consists of multiple sequence classes of minicircles, each containing a small, 150-270 bp, region of species spec ific, conserved nucleotide sequence (15, 16). Rapid rates of minicircle sequence evolution have been C IRL Press Limited, Oxford, England. 5531 Nucleic Acids Research documented in different strains of Tr^25S2£°S5 ^£H£*i (17. 18) and Tj:yj>ano«joma ££H£i (19, 20), and in different species of Leishmania (21-23) and Cri.thidia (24, 25). Minicircle DNA is not transcribed in detectable amounts and sequences of cloned minicircles from T^ brucei (26), T^ lewi.8j. (27) and L̂_ tarento^ lae (16) reveal only short open reading frames. There is, however, a preliminary report of a possible protein product of Crithldla minicircle DBA (28), which, if confirmed and extended, would imply a genetic role for at least the large minicircles of this species. Mung bean nuclease has been used as an endonucleolytic probe of altered DNA helix conformation for both prokaryotic and eukaryotic DNAs. The sites of mung bean nuclease single strand nicking of supercoiled PM2 phage and pBR322 DNAs have been mapped to the non-coding regulatory regions of these molecules (29-32); cleavage by mung bean nuclease occurred predominantly within A+Trich regions and at inverted repeat sequences capable of forming hairpin structures (31, 32). Mung bean nuclease is capable of double strand cleavage of jLla£nodi.urn nuclear DNA, and possibly nuclear DNA from other lower eukaryotes, at sites before and after genes (33). Under partially denaturing conditions cleavage occurred on linear Plasmodium DNA at A+T-rich sites, which, however, were no more A+T-rich than surrounding sequences which were not cleaved by the nuclease (33). We report here the specific double strand cleavage of multiple L_̂ t'rentoUs minicircle sequence classes by mung bean nuclease. The sites of nung bean nuclease cleavage on the previously sequenced KSR1/Ltl9 minicircle (16) were mapped. Cleavage site specificity was shown to be independent of the nucleotide sequence of the cleavage site and the topology of the minicircle, but vas related, in those cases that could be determined, to the distance from the conserved region with the adjacent conformatlonal 'bend*. Ve also present the partial sequences of five additional classes of minicircles released from the kinetoplast network by mung bean nuclease. MATERIALS AND METHODS Cell culture and DHA iaolation Cultures of L^ tarentolae were grown to stationary phase in