Sequences of Two Kinetoplast DNA Minicircles of Trypanosoma Brucei (Recombinant DNA/Restriction Enzymes/Sequence Homology/DNA Sequencing) KENNETH K
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Proc. Natl. Acad. Sci. USA Vol. 77, No. 5, pp. 2445-2449, May 1980 Biochemistry Sequences of two kinetoplast DNA minicircles of Trypanosoma brucei (recombinant DNA/restriction enzymes/sequence homology/DNA sequencing) KENNETH K. CHEN AND JOHN E. DONELSON Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242 Communicated by William Trager, January 18, 1980 ABSTRACT Kinetoplast DNA of Trypanosoma brucei is kDNA network are about 23 kilobases in size and are homo- composed of a network of about 10,000 interlocked minicircle geneous in sequence (13). Several RNA species hybridize to DNA molecules (1.0 kilobase) that are catenated with about 50 maxicircles and it has maxicircle DNA molecules (23 kilobases). Several different (14-16), been proposed that maxicircle DNA-DNA hybridization techniques using individual minicircle DNA corresponds to normal mitochondrial DNA of other lower DNA sequences cloned in Escherichia coli have indicated that eukaryotes (17). The T. brucei minicircles are about 1 kilobase each minicircle molecule contains about one-fourth of its se- and heterogeneous in sequence, as determined by restriction quence in common with most other minicircles and the re- enzyme analysis (18) and renaturation kinetics (19). The early maining three-fourths in common with about 1 out of every 300 renaturation analyses (19) indicated that 100 or more different minicircles. We have determined the complete sequence of two minicircle sequences may occur among the cloned minicircle DNA molecules that were released from the 10,000 molecules total kinetoplast DNA network by different restriction enzymes; in a network. The biological function of minicircle kDNA is one minicircle is 1004 base pairs long, the other is 983 base pairs. completely unknown. Both are about 72% dA + dT. They share about 27% of their We have previously reported the isolation of individual T. sequences; the largest continuous region in common is 122 base brucei kDNA minicircle molecules in bacteria by recombinant pairs of near-perfect homology. Twelve other regions of perfect DNA techniques (20). Several of these cloned sequences were homology equal to or greater than 10 base pairs are also present. characterized renaturation Both sequences contain a large number of translation termi- by kinetics, filter hybridizations, nation codons in all potential translation reading frames. The and heteroduplex analyses in the electron microscope. These largest oligopeptide potentially specified by one minicircle studies indicated that each minicircle molecule has one-fourth sequence is 52 amino acids; the largest by the other minicircle of its sequence in common with most other minicircles and the sequence is 71 amino acids. One minicircle contains a decanu- remaining three-fourths in common with about 1 out of 300 cleotide sequence that is repeated in tandem five times. It is minicircle molecules. This suggested two possible models for proposed that massive recombination among the interlocked minicircles in the kinetoplast DNA network may account for the sequence organization of the minicircles; one in which small much of the homology observed in the two minicircle se- homologies are scattered throughout each minicircle and one quences. in which the homology is confined to a specific 25% of each molecule. Trypanosomes are unicellular parasites which include the We present here the complete sequence of two cloned kDNA species that causes sleeping sickness in Africa and Chagas dis- minicircles, one of 1004 base pairs and the other of 983 base ease in South America (1). Those species that have been inves- pairs. A comparison of the two sequences reveals that both tigated have a single large mitochondrion per cell that is models for sequence organization are partially correct; the two characterized by a specialized region called the kinetoplast (2, molecules share about 12% homology within a nearly contin- 3) located near the basal body of the cell's single flagellum. The uous stretch and another 15% that is scattered randomly kinetoplast contains one of the most unusual DNA structures throughout the rest of the two sequences for a total of 27% ho- found in nature. mology. Kinetoplast DNA (kDNA) consists of about 10,000 minicircle molecules and 50 maxicircle molecules, all of which are inter- MATERIALS AND METHODS locked together to form a massive disk-shaped network whose width is about the diameter of a minicircle (see refs. 4-6.for Recombinant plasmid DNAs were isolated from Escherichia recent reviews). Transmission electron micrographs of purified coli HB101 cells as described (21). Purified DNAs were digested kDNA show a massive tangle of catenated supercoiled mole- with various restriction enzymes according to protocols sug- cules (7, 8). Replication of the kDNA network occurs via gested by the commercial suppliers (New England BioLabs, mechanisms that are only partially understood but appear to Beverly, MA or Bethesda Research, Rockville, MD). The re- involve the release and reattachment of individual DNA circles striction fragments were resolved on 5% polyacrylamide gels (6, 9, 10) until a double-sized kDNA network is obtained which (22) and eluted from the gels. separates into two as daughter cells are formed. Some of the All of the restriction fragments whose sequences were de- common trypanocidal drugs, such as Berenil and ethidium termined were generated-by restriction enzymes that cleave compounds, strongly interact with the kDNA network (11, 12) DNA to produce staggered ends with recessed 3' termini. These and probably kill the trypanosome by interfering with the 3' termini were radiolabeled by incubation of the restriction kDNA replication or function. fragment with E. coli DNA polymerase I (Boehringer In Trypanosoma brucei the 50 maxicircle molecules in the Mannheim) and deoxynucleoside [a-32P]triphosphates (Am- ersham; specific activity 2000 mCi/,mol; 1 Ci = 3.7 X 1010 The publication costs of this article were defrayed in part by page becquerels) and the radioactive ends were separated as de- charge payment. This article must therefore be hereby marked "ad- scribed (23). The Maxam and Gilbert sequencing method (24) vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: kDNA, kinetoplast DNA. 2445 Downloaded by guest on September 23, 2021 2446 Biochemistry: Chen and Donelson Proc. Natl. Acad. Sci. USA 77 (1980) was used, and the chemical cleavage products were resolved by electrophoresis through 0.38-mm-thick gels of 6% (85 cm long) or 20% (40 cm long) acrylamide (25). Sequences were analyzed by the computer program of Kom et al. (26) with the IBM 370/168 computer available at the University of Iowa. Experiments involving recombinant plasmids containing kDNA inserts were conducted under P2 + EKI containment as specified by the National Institutes of Health Recombinant DNA Guidelines of December, 1978. RESULTS AND DISCUSSION DNA Sequencing Strategy. Preliminary restriction enzyme *. analyses were performed on six different minicircle kDNA I inserts cloned at the Pst I site of pBR322 (20) to identify two cloned minicircle kDNA sequences that possessed a distribution of restriction enzyme cleavage sites suitable for total sequence 11 V determination by the Maxam and Gilbert procedure (24). Taq b * I was the only enzyme tested that cleaved all six kDNA se- I It quences at least once, although several other enzymes cleaved I more than one kDNA. The kDNA inserts of pkT51 and pkT201 SS* were chosen for further characterization. Neither kDNA insert ... contains a Hpa II site. However, Hpa II cleaves the vector, pBR322, sequence about 50 base pairs on either side of the Pst I site (27, 28) at which the kDNAs were cloned. Therefore, the I.- -9. vii largest Hpa II fragment in both pkT51 and pkT201 contains ** the complete kDNA insert. About 400 ,ug of these two Hpa II S. * - 0 fragments was eluted from preparative 5% acrylamide gels to * *. a w serve as the starting material for the sequence determina- * 'S1 tions. Fig. 1 shows the strategy used to determine the two se- 3-eu quences. The minicircle kDNA sequence in pkT51 was initially 5 ,' released from the kDNA network of T. brucet clone 18E2 by digestion with a mixture of Hha I, Hpa II, and Alu 1 (20). It was - Cl cloned at the Pst I site of pBR322 via the poly(dG)-poly(dG) tailing method (20), which regenerates Pst I sites at the two * *r boundaries between the kDNA insert and the pBR322 se- FIG. 2. Example of an auto- quence. Sequences of 250-350 nucleotides were obtained from radiogram of a DNA sequencing the several different labeled termini, as shown in Figs. 1 and ...* gel. The two Hpa II/Taq I restric- 2, to provide the complete continuous sequence shown in Fig. tion fragments of kDNA 51 labeled 3B. This minicircle is 983 base pairs long and is 71.2% dA + dT. _* at the external Hpa II sites (Fig. 1) are shown. The four cleavage re- The base composition of the strand shown is 37.4% A, 33.8% T, B In 4 actions of each of the two frag- 20.1% G, and 8.6% C. recombinant plasmid pKT51 this ments were applied three different minicircle sequence is flanked by a 26-base-pair dG-dC times to the gel at 10-hr intervals. :*... * boundary on one side and 29 dG-dC base pairs on the other side, *a From left to right the order of the as indicated in Fig. 1 but not shown in Fig. 3B. Because of these a. ** four cleavage reactions are A, G, C, 9 and C + T. Approximately the first kDNA 21 75 nucleotides can be determined 5 .9 a from the last application, the next I -F] ... 125 from the second application, 4x1 Pa ftl HpoN and the next 125 from the first 8- 0 application. Prominent features of kDNA 51 these two sequences are the 26- 9 5.- r a nucleotide poly(dC) boundary of the left fragment (third column 2 olTinqI PitlHp from the left) and the 29-nucleo- tide poly(dC) boundary of the Number of Base Pairs right fragment (the extreme right FIG.