Journal of Cell Science 107, 3515-3520 (1994) 3515 Printed in Great Britain © The Company of Biologists Limited 1994 Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle Sally G. Pasion, Grant W. Brown, Lisa M. Brown and Dan S. Ray* Molecular Biology Institute and Department of Biology, University of California, Los Angeles, California 90024, USA *Author for correspondence SUMMARY In trypanosomatids, DNA replication in the nucleus and in drial topoisomerase, was disrupted by the insertion of a the single mitochondrion (or kinetoplast) initiates nearly NEO drug-resistance cassette was found to express both a simultaneously, suggesting that the DNA synthesis (S) truncated TOP2 mRNA and a truncated topoisomerase phases of the nucleus and the mitochondrion are coordi- polypeptide. The truncated mRNA was also expressed peri- nately regulated. To investigate the basis for the temporal odically coordinate with the expression of the endogenous link between nuclear and mitochondrial DNA synthesis TOP2 mRNA indicating that cis elements necessary for phases the expression of the genes encoding DNA ligase I, periodic expression are contained within cloned sequences. the 51 and 28 kDa subunits of replication protein A, dihy- The expression of both TOP2 and nuclear DNA replication drofolate reductase and the mitochondrial type II topoiso- genes at the G1/S boundary suggests that regulated merase were analyzed during the cell cycle progression of expression of these genes may play a role in coordinating synchronous cultures of Crithidia fasciculata. These DNA nuclear and mitochondrial S phases in trypanosomatids. replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone Key words: cell cycle, replication, topoisomerase, kinetoplast, (pdN-1) in which TOP2, the gene encoding the mitochon- mitochondrion INTRODUCTION 1993). Minicircle replication intermediates also have been shown by in situ hybridization methods to be present in two Trypanosomes contain a novel form of mitochondrial DNA or antipodal sites on the periphery of the kinetoplast disc in vivo kinetoplast DNA (kDNA), which consists of thousands of (Ferguson et al., 1992). The antipodal location of the newly copies of minicircles and a small number of maxicircles topo- synthesized minicircles coincides with that of two kinetoplast- logically interlocked to form a cup-shaped sheet of DNA associated replication proteins, a type II DNA topoisomerase termed a DNA network (Simpson, 1987). Maxicircles encode (Melendy and Ray, 1989; Melendy et al., 1988) and a DNA rRNAs and proteins essential for mitochondrial function. polymerase (Ferguson et al., 1992), and suggests that these Expression of maxicircle genes involves extensive editing of sites may represent multiprotein replication complexes. primary transcripts in which U residues are added and/or The temporal link between nuclear and kinetoplast DNA deleted to create functional mRNAs (Stuart, 1991). The speci- replication raises the possibility that nuclear and kinetoplast S ficity of the editing process is determined by small ‘guide phases are coordinately regulated in trypanosomes. This RNAs’ encoded in both maxicircles and minicircles and which feature of kDNA replication differs from that observed for serve as templates for the editing process (Sturm and Simpson, other mitochondrial DNAs in which replication occurs 1990). throughout the cell cycle (Guttes et al., 1967). The coordina- Replication of kinetoplast DNA occurs in approximate tion of nuclear DNA synthesis with the cell cycle in eukary- synchrony with nuclear S phase (Cosgrove and Skeen, 1970) otic organisms appears to be under the control of a p34cdc2 and involves a mechanism in which individual minicircles are protein kinase that directly or indirectly modulates the released from the kDNA network prior to their duplication expression and/or activity of numerous proteins required for (Englund, 1979). Upon replication the daughter minicircles are DNA replication (Norbury and Nurse, 1992). In the budding rejoined to the periphery of the network while still containing yeast, one level of control involves the regulation of genes nicks and gaps in the newly synthesized strands (Kitchin et al., encoding DNA replication proteins. At least eighteen Saccha- 1984). Newly synthesized minicircles have been observed to romyces cerevisiae genes required for DNA synthesis are be concentrated in two sites on opposite sides of the network expressed under cell cycle control at, or near to, the G1/S (Simpson and Simpson, 1976; Perez-Morga and Englund, boundary (Johnston and Lowndes, 1992). All of these genes 3516 S. G. Pasion and others are coordinately regulated through a common cis-acting supernatant was decanted and the cells were resuspended in the sequence recognized by a specific transcription factor residual media and plated onto 1% agar plates containing BHI plus (Johnston et al., 1991). Since all kinetoplast DNA replication 10 µg hemin/ml, 100 µg streptomycin sulfate/ml and 80 µg G418/ml. proteins are likely to be nuclear encoded, their expression may The plates were wrapped in parafilm and incubated at 28°C. G418- be under the same controls as those of genes encoding nuclear resistant colonies appeared within 4 to 5 days. replication proteins. To investigate this possibility, we have Construction of the pdN-1 plasmid examined the cell cycle expression of genes encoding both The vector pX.2 was derived from pX (LeBowitz et al., 1990), which nuclear and kinetoplast replication proteins in the trypanoso- contains a mutant neomycin phosphotransferase (NPT) gene by matid Crithidia fasciculata. replacing the 350 bp BalI-NcoI fragment contained within the NPT gene with the corresponding 350 bp BalI-NcoI fragment from the wild-type NPT gene from pSV2neo (Yenofsky et al., 1990). ′ MATERIALS AND METHODS Two oligonucleotides (Kpn 1: 5 -CGCGGATCCTCTAGAG- GTAC-3′ and Kpn 2: 5′-CTCTAGAGGATCCGCGGTAC-3′) were synthesized, annealed, and subcloned into the unique KpnI site of Cell synchronization pX.2 introducing BamHI, SacII and XbaI sites flanked by KpnI sites C. fasciculata was synchronized using a modification of the method and generating the vector pX.2-KO. This construct allows the described for synchronizing Leishmania tarentolae (Simpson and movement of the 3.3 kb NEO cassette (the wild-type NPT gene with Braly, 1970). Cells were grown to late log phase (~108 cells/ml) at flanking Leishmania major DHFR-TS sequences) as a BamHI, SacII 28°C in Difco brain heart infusion medium (BHI) supplemented with or XbaI fragment. 10 µg hemin/ml and 100 µg streptomycin sulfate/ml prior to the To create a disruption of the plasmid-encoded CfaTOP2 gene, the addition of 200 µg hydroxyurea/ml for 6 hours. Growth was continued NEO cassette was subcloned as a SacII fragment from pX.2-KO into at 28°C in fresh medium lacking hydroxyurea, and samples were the pt2-1 plasmid (Pasion et al., 1992), which contains the CfaTOP2 removed every 30 minutes. coding region and 883 bp 5′ flanking and 1.5 kb 3′ flanking sequences. The pt2-1 plasmid was constructed by inserting a 6.1 kb genomic Measurement of DNA synthesis fragment into the bacterial cloning vector pGEM-7Zf(+), which Total DNA synthesis was measured by labeling 65 µl of culture with contains no other trypanosomatid sequences. The inserted NEO 10 µl of [3H]thymidine (1 mCi/ml; 79 Ci/mmol) at 28°C for 10 cassette replaces a 1.4 kb SacII fragment within the CfaTOP2 coding minutes. Cells were lysed and incorporation of [3H]thymidine into sequence to generate pdN-1 in which the CfaTOP2 gene and the NPT trichloroacetic acid-precipitable form was measured. Mitochondrial gene are in the same orientation. DNA synthesis was measured by scintillation counting of [3H]thymidine label in minicircle DNA bands on Southern blots Western blot analysis following digestion with XhoI and agarose gel electrophoresis. Cell lysates prepared from C. fasciculata strains were analyzed on 7% acrylamide-SDS gels as described previously (Pasion et al., 1992). Northern blots Each lane contained lysate from 2.5×106 cells. Blots were probed with RNA extracted from 109 cells by guanidinium isothiocyanate lysis polyclonal antisera (1:5000) raised against purified topoIImt was used for northern blotting and hybridization as described (Pasion (Melendy and Ray, 1989) and detected with alkaline phosphatase con- et al., 1992). Either 50 µg (for CfRPA1) or 25 µg (all others) of RNA jugated to goat anti-rabbit IgG. was fractionated. Probes for analysis of C. fasciculata RNA were a 980 bp EcoRI-MluI fragment of the CfaRPA1 gene (EMBL accession number Z23163), a 606 bp HaeII fragment of the CfaRPA2 gene RESULTS (EMBL accession number Z23164), a 630 bp EcoRI-AvaI fragment of the CfaLIG1 gene (EMBL accession number Z23078), a 2000 bp XhoI fragment of the CfaTOP2 gene encompassing amino acid Expression of DNA replication genes in residues 311 to 1007 (Pasion et al., 1992) and a 260 bp fragment of synchronized cells a C. fasciculata gene designated CaBP due to its sequence similarity C. fasciculata cultures undergoing synchronous progress to trypanosome calcium-binding proteins (Lee et al., 1990). The probe through the cell cycle were prepared by a hydroxyurea arrest for the analysis of Cf (pdN-1) RNA was a 2.1 kb HindIII-SacII method (Simpson and Braly, 1970). Cells were released from ′ fragment of pdN-1 designated 5 TOP2 US and CfaTOP2 (1-1186) in the hydroxyurea block and sampled every 30 minutes to Fig. 2. Northern blots were quantitated using a Molecular Dynamics determine the cell number, the percentage of dividing cells and PhosphorImager. the incorporation of pulse label into DNA. Cultures released Transformation of Crithidia fasciculata from hydroxyurea treatment entered S phase immediately, as C. fasciculata cells were cultured in BHI media supplemented with indicated by the high level of DNA synthesis at 0 and 30 10 µg hemin/ml and 100 µg streptomycin sulfate/ml to a density of minutes (Fig.