126 E. Lincoln Avenue, Rahway, NJ 07065, USA. Received October 20, 1995; ... Trypanosoma brucei is one of the causative agents of African trypanosomiasis.
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Nucleic Acids Research, 1996, Vol. 24, No. 4
Artificial linear mini-chromosomes for Trypanosoma brucei Pradeep K. Patnaik*, Nancy Axelrod1,+, Lex H. T. Van der Ploeg2 and George A. M. Cross Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA, 1Department of Genetics and Development, Columbia University, 630 N. 168th Street, New York, NY 10032, USA and 2Department of Genetics and Molecular Biology, Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway, NJ 07065, USA Received October 20, 1995; Revised and Accepted December 22, 1995
ABSTRACT We have constructed artificial linear mini-chromosomes for the parasitic protozoan Trypanosoma brucei. These chromosomes exist at ∼2 copies per cell, are indefinitely stable under selection but are lost from 50% of the transformed population in ∼7 generations when grown in the absence of selective pressure. Consistent with results obtained earlier with natural chromosomes in T.brucei, the telomeres on these artificial chromosomes grow, adding ∼1–1.5 telomeric repeats per generation. The activity of a procyclic acidic repetitive protein (parp) gene promoter on these elements is unaffected by its proximity to a telomere, implying the lack of a telomere-proximal position effect (TPE) in procyclic trypanosomes. Among other things, these autonomously replicating dispensable genetic elements will provide a defined system for the study of nuclear DNA replication, karyotypic plasticity and other aspects of chromosomal behavior in this ancient eukaryotic lineage. INTRODUCTION The long range objective of this study was to lay the groundwork for an investigation of chromosomal behavior in Trypanosoma brucei. Parasitic protozoa, such as T.brucei, Plasmodium falciparum, Giardia lamblia, Trypanosoma cruzi and Leishmania demonstrate an unusually plastic karyotype (1–15; for recent reviews see 16,17). In a study of T.brucei and certain sub-species, Gibson and Borst observed a ‘remarkable fluidity of chromosome organization’ and saw ‘no stocks with an identical karyotype’(2). An inherited alteration in the karyotype is by definition a mutation and the range of these changes and the tolerance shown by the organism for these alterations seems truly remarkable. An understanding of such a process in trypanosomatids, for which a fairly sophisticated repertoire of molecular techniques are already
available, could have general significance for other parasitic protozoa. Trypanosoma brucei is one of the causative agents of African trypanosomiasis. Phylogenetic analyses demonstrate that these organisms are among the most ancient eukaryotic lineages known (18–21). The parasite has a digenetic life-cycle, alternating between the midgut and salivary glands of its insect vector (Glossina: tsetse) and the mammalian bloodstream. Nuclear DNA replication is an unexplored aspect of trypanosome biology. In an earlier attempt to develop models that would aid in the delineation of critical replication control elements, we had constructed a panel of autonomously replicating episomes for T.brucei (22). These were made by inserting random pieces of its genomic DNA onto a molecule that could not otherwise exist as a stable replicon in this organism. We call the inserted pieces of DNA the plasmid maintenance sequence or PMS. One of these plasmids (pT13-11), has been extensively characterized (22–24). It exists as a single-copy episome in procyclic (insect mid-gut form) T.brucei, but nonetheless demonstrates substantial mitotic stability in the absence of selection (50% loss in ∼12 generations). In addition, we have shown that autonomous plasmid replication was dependent on interactions between the PMS and a 108 bp region encompassing the procyclic acidic repetitive protein (Parp or procyclin) gene promoter that serves to drive the transcription of a selectable marker on this episome (24). Parp is the major surface protein in procyclics and is transcribed from two unlinked loci, parpA and B (25). Parp transcription is resistant to α-amanitin and is thought to be mediated by RNA polymerase I (pol I), which is capable of supporting mRNA transcription in T.brucei (26,27; also see review by 28). A 39 nt spliced leader is trans-spliced onto each mRNA in trypanosomatids and provides the 5′ cap that is a necessary feature of all eukaryotic mRNA. Although α-amanitin sensitive transcription units are known, no RNA polymerase II (pol II) promoters have been yet identified in T.brucei. An important additional motivation for the construction of these linear mini-chromosomes lay in our continuing interest to develop ‘promoter-traps’ for the isolation
*To whom correspondence should be addressed at present address: Division of Parasitology, National Institute of Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK +Present
address: Nature Medicine, 1234 National Press Building; 529, 14th Street NW Washington, DC 20045-2200, USA
669 Nucleic Acids Acids Research, Research,1994, 1996,Vol. Vol.22, 24,No. No.14 Nucleic and characterization of pol II promoters of T.brucei. A previous attempt to obtain such a trap, by deleting the parp promoter from pT13-11 and seeking to complement it with another promoter from the T.brucei genome, was complicated by the essential role played by the parp promoter region in plasmid replication (24). An artificial chromosome would provide us with a means to overcome this complication, by allowing us to direct the parp promoter mediated transcription on the linear element away from a promoter-less selection unit. This would avoid potential problems of read-through transcription that would occur on a circular plasmid as a result of polycistronic transcription, which is the norm in trypanosomatids. MATERIALS AND METHODS Trypanosomes Culture adapted procyclic forms of T.brucei strain 427 were grown at 27C in SDM-79 medium (29) supplemented with 10% heat-inactivated fetal bovine serum (Sigma) as previously described (22). Constructs For pTacA, a 1530 bp fragment consisting of an Escherichia coli chloramphenicol acetyl transferase (cat) gene cartridge (787 bp HindIII fragment from plasmid pCM7; Pharmacia), flanked on its 5′ end by the parpB promoter and splice acceptor signals (SAS) and on its 3′ end by a section of the β-α tubulin (tub) intergenic region, was inserted between the HindIII and SpeI restriction sites of Bluescript SK+. The promoter and SAS correspond to sequences between –312 and +93 with respect to the initiation site of parp-B transcription (30). The intergenic region comprises the last 108 bp of the β-tub coding sequence and 227 bp of the 3′ untranslated region (31). The PMS and the neomycin phosphotransferase (Neo) expression unit were derived from a modified version of pT13-11 where the BglII restriction site within the PMS had been destroyed, and a KpnI site had been introduced at the unique EcoNI restriction site of pT13-11, as part of a polylinker. These alterations do not affect the ability of the plasmid to replicate autonomously (Patnaik, unpublished observations). The PMS (∼4.8 kb) consists of the entire insert from the SalI restriction site to the left most EcoRI restriction site of pT13-11 and the Neo expression unit is the ∼2.1 kb ApaI–KpnI fragment (22). These were inserted at the XhoI restriction site, and between the ApaI and KpnI restriction site of the Bluescript vector respectively. The PMS is in the same orientation with respect to the Neo expression unit as it is in pT13-11. A ∼4 kb SacI fragment consisting of two head to head ‘telomeres’ (1.6 kb each) separated by a 650 bp spacer with BglII linkers at either end, was inserted into the SacI restriction site of the Bluescript vector. The 1.6 kb ‘telomeric’ DNA corresponds to the EcoRI–PstI fragment from pT4 (32,33) and consists of 55 copies of the 6 bp T.brucei telomeric repeat, in addition to other related sequences, and ∼1 kb of ‘sub-telomeric’ DNA (33). The spacer was expected to inhibit recombination between the inverted telomeric repeats (34). The construct pTacB is identical to pTacA, except that three tandem copies of a 1.5 kb fragment, consisting of a hygromycin phosphotransferase (hyg) gene flanked by the ParpB SAS (+6 to
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+93 with respect to the start site of ParpB transcription) and the same β-α tub intergenic region that abuts the 3′ end of the cat gene, were inserted into the XbaI restriction site of Bluescript. Construct pTacC is a SalI deletion derivative of pTacB. This deletion removes the ParpB promoter and SAS flanking the cat coding sequence. Construct pTacD was obtained by inserting a synthesized polylinker comprising of sites for restriction enzymes SalI, AflIII, BclI, PmeI, SfiI, AscI and SacI into a SalI partial–SacI digest of pTacB. The sequence constituting these sites is 5′-GTCGACTTAAGTGATCAGTTTAAACGGCCTAACTGGCCGGCGCGCCGAGCTC-3′. The SalI, PmeI, SfiI and AscI sites are unique on pTacD. Construct pTacAm is identical to pTacA except for a 286 bp sequence (derived from pTacB and described in the text) inserted into the NotI site of the Bluescript polylinker. This site is located just upstream of the SacI site flanking the right sub-telomeric repeats. As there is a NotI site in the PMS (22), insertion was achieved through a partial-NotI digest of pTacA. Construction of pTacE involved several steps. The HindIII– BamHI fragment comprising the luciferase coding sequences in pHD16 (35) was replaced with an 411 bp HpaI–StuI fragment from the plasmid pUT56 (kindly provided by D. Drocourt, CAYLA Laboratories, France) comprising the Streptoalloteichus hindustanus phleomycin resistance (ble) gene (36). The plasmid obtained is called pHD16-ble. A ble-expression cassette was generated by polymerase chain reaction (PCR) mediated amplification of the appropriate fragment using primers complementary to the T.brucei aldolase (ald) SAS and PAS respectively that flanked the ble gene in pHD16-ble. These primers were designed with additional XhoI, PmeI (SAS primer) and KpnI (PAS primer) sites. The mobilized cassette was then inserted into XhoI–KpnI digested pEV-luc described earlier (24) giving rise to the plasmid pBLN (Ble.Luc.Neo). Note, pEV-luc had been previously obtained in several versions with one or the other AscI site flanking the actin SAS and PAS respectively having been lost as a result of aberrant ligation. In the clone used for the construction of pBLN, only the downstream AcsI site flanking the actin PAS was present. A PmeI–AscI digest of pBLN was used to mobilize the ∼3.7 kb combined ble-luc expression unit with their corresponding SAS and PASs. This unit was inserted into a similarly digested pTacD to give pTacE. Construct pTacE-R was derived from pTacE by inserting an ∼550 bp BamHI–HindIII fragment corresponding to the rRNA promoter on the plasmid polINeo (37) into the unique PmeI site of pTacE. The only BglII sites in any of these constructs are the ones flanking the spacer that separates the telomeric repeats.
Transformation Procyclic trypanosomes were transfected by electroporation using 10 µg of BglII-linearized DNA. Electroporator settings and other conditions were as previously described (38). Transformed procyclic clones were obtained following plating of cells on media containing agarose and 50 µg/ml of G418 (GIBCO-BRL), as previously described (39). Where appropriate, 50 µg/ml of hygromycin B (Sigma) or phleomycin (CAYLA,) was substituted for G418. Transfection efficiencies were similar to those previously determined for episomes (22).
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Figure 1. Schematic representation of artificial chromosomes. Both circular and linear (BglII-digested) forms of pTacA are shown. The linear forms of pTacB, pTacAm, and pTacE are abbreviated to only show their difference from pTacA. The arrows indicate telomeric and sub-telomeric DNA. PMS, plasmid maintenance sequence derived from pT13-11; Ble, Streptoalloteichus hindustanus phleomycin resistance gene; Bls, Bluescript SK+; Cat, chloramphenicol acetyltransferase; Hyg, hygromycin phosphotransferase; Luc, firefly luciferase; Neo,neomycin phosphotransferase. A,AscI; B, BamHI; Bg, BglII; N, NotI; P, PmeI; S, SalI; Sc, SacI. The linear maps are not to scale.
DNA and RNA isolation Except in the preparation of samples for FIGE gels (see later), DNA was isolated using the commercially available reagent DNA STAT60 and the manufacturer’s protocol (TEL TEST ‘B’ Inc., Friendswood, TX). RNA STAT60 from the same manufacturer was used to isolate total RNA. DNA analysis Restriction digests, gel electrophoresis, Southern and Northern hybridizations were performed using standard protocols (40). Final washes following hybridizations were done under stringent conditions (0.1× SSC, 1% SDS, 65C). Separation of large DNA fragments was performed using a field inversion gel electrophoretic (FIGE) system (Minipulse programmable polarity switching system obtained from IBI New Haven, CT). Agarose blocks containing ∼1 × 107 cells were prepared as previously described (41). These were electrophoresed through a 1.2% agarose gel in 44 mM Tris-base, 2 mM EDTA, 40 mM boric acid at an electrical gradient of 5 V/cm using the INV output mode of the minipulse generator and a forward (fwd) and reverse (rev) pulse interval ranging from 0.025 to 0.505 (fwd)/s and 0.01 to 0.202 (rev)/s. This constituted program #1 of the manufacturer’s protocol. Bal31 exonuclease digestion To digest DNA isolated from 2 ×108 cells, 5 U of Bal31 exonuclease (NEB) was used. Reactions were done in duplicate at 30C. Aliquots were removed at 0, 5, 10, 20 and 30 min into tubes containing EDTA (20 mM final concentration). Each of these tubes was kept on ice until all the aliquots had been obtained after which they were placed at 68C for 10 min to destroy the
exonuclease. Following ethanol precipitation, the DNA was dissolved in TE (10 mM Tris pH 8.0, 1 mM EDTA). This DNA was then digested with BamHI and electrophoresed through agarose, Southern transferred and probed as shown. Stability and copy number of artificial chromosomes Trypanosomes bearing pTacA or pTacB were subcultured (1:150 dilution at each passage, equivalent to ∼7 generations between passages) in media without G418. Aliquots were withdrawn at each passage. Genomic DNA was isolated, digested with BamHI, electrophoresed through agarose, Southern transferred and probed with a 110 bp PflMI fragment from pEV (24) corresponding to the ParpA promoter. The intensities of the signals corresponding to the genomic ParpA locus (arrow G) and the artificial chromosome (arrow AC) were measured using a phosphorimager. RESULTS Construction and characterization of artificial chromosomes Constructs pTacA and pTacB (Fig. 1) were made by adding telomeric and subtelomeric sequences derived from a T.brucei chromosome (32,33) to derivatives of pT13-11 (22). In addition to the PMS, this plasmid bears a neo gene whose transcription is driven by a parpA promoter. The splice-acceptor and polyadenylation signals (SAS and PAS, respectively) flanking neo in pT13-11 are also from the parpA locus (22,38,42). In addition, both constructs contain the bacterial chloramphenicol acetyl transferase (cat) gene, which is present either adjacent to the telomeric DNA (pTacA) or separated from it by three copies of
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Figure 2. Field inversion gel electrophoretic (FIGE) analysis of trypanosome clones bearing pTacA or pTacB. Lanes 1 and 2, uncut (supercoiled) plasmids pTacA and pTacB, respectively; lanes 3 and 4, BglII linearized input plasmids pTacA and pTacB, respectively; lanes under the heading pTacA represent total genomic DNA from 7 clones bearing pTacA; lane labeled WT represents total genomic DNA from wild type trypanosomes; lanes under the heading pTacB represents total genomic DNA from 6 clones bearing pTacB. Following FIGE and Southern transfer, the filter was hybridized with a probe corresponding to the neo coding sequence (A). (B) and (C) are 1 day and 1 week exposures, respectively, of the same filter, re-probed with telomeric (tel) DNA after the initial label had been stripped off. The position of T.brucei mini-chromosomes (∼50–150 kb) and chromosomes 2, 3, 4 and 5 (∼150–500 kb) are provided for reference. Chromosome numbers and sizes are as previously assigned (56).
a hygromycin phosphotransferase (hyg) gene and flanking sequences (pTacB). Cat and hyg transcription are mediated by a parp promoter derived from the B locus (30). The SAS and PAS flanking cat and hyg are identical, the former being derived from the parpB locus and the latter from the β-α tub intergenic region. Following transfection of procyclic trypanosomes with BglIIlinearized pTacA or pTacB DNA, cells were plated on media containing agarose and G418 (39). Several clones from each transfection were picked and grown in liquid culture under selection. Agarose blocks containing genomic DNA from 7 clones of pTacA-transformed trypanosomes and 6 clones of pTacB-transformed trypanosomes were prepared. Following field inversion gel electrophoresis (FIGE) and Southern transfer, the filter was hybridized to a probe corresponding to the neo coding sequence (Fig. 2A). Figure 2B and C represent a short (overnight) and a long (1 week) exposure of the same filter, re-probed with T.brucei telomeric DNA.
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Results (Fig. 2A) indicated the presence of a neo-hybridizing element in each of the transformed clones (arrows a and b) that was not present in DNA from wild-type (WT) trypanosomes. These elements migrated ahead of the smallest T.brucei minichromosomes (note position of smallest hybridizing band in the WT lane in panel C), at a position closely corresponding to the linear transfecting input molecules (lanes 3 and 4 corresponding to linear pTacA and pTacB, respectively). The hybridizing elements in the transformed clones are not re-circularized plasmid molecules as these would have migrated to a position corresponding to the supercoiled input DNA (lanes 1 and 2). The smearing seen in Figure 2A is an artifact of the FIGE, and the very long exposure. We concluded that there were putative linear elements (artificial chromosomes) in each of the transformed trypanosome clones. In addition, a few clones may also bear an integrated or rearranged plasmid (arrow x). These were clearly a minority and have not been examined further. To determine if these artificial chromosomes were identical to their respective input DNAs, we isolated total DNA from WT and transformed trypanosome clones and digested these with BamHI. A BamHI digest of the artificial chromosomes was expected to yield 3 fragments, each of which could be individually visualized with a probe specific for the left, central and right arms of the chromosomes. Following electrophoresis, Southern transfer and hybridization with the three probes (Fig. 3A), it was clear that the central region of these artificial chromosomes was unchanged with respect to the input DNA (central panel, neo probe), but the right and left arms (probed with cat and Bluescript, respectively) were each 500–800 bp longer, as a consequence of telomeric growth (see later). This was most evident in this experiment with the right arm of pTacA (Fig. 3A bottom panel), which is the smallest of the fragments generated and, consequently, is in a region of the gel showing very good separation. We have also documented growth of the left arm of these elements by an essentially similar experiment where we generated smaller terminal fragments by using an enzyme (SspI) that digests within Bluescript sequence. Results (not shown) corroborate the conclusions drawn above. Telomere growth Clones bearing pTacA or pTacB were kept in continuous culture for 3 months under G418 selection. DNA analysis (Fig. 3B top panel) indicated that the right arm of pTacA grew by 1.2–1.6 kb over a 3 month period (∼200 generations) of continuous culture (compare lanes 1 and 4), corresponding to a growth rate of ∼6–8 bp per generation. Also, the measured growth between any two time points suggests that this growth rate was consistent over the entire period. In the experiment shown in the bottom panel of Figure 3B, genomic DNA isolated at different times from a continuous culture of clones harboring either pTacA (lanes 1 and 2) or pTacB (lanes 3 and 4) was digested with BamHI and SacI before analysis. SacI digestion of these chromosomes was expected to remove the telomeric DNA from the fragment seen by the cat probe (see Fig. 1). Following this digest, the artificial chromosome band visualized by the cat probe was identical in size to the corresponding fragment derived from the input plasmid (compare lanes 1 and 2 with lane A and lanes 3 and 4 with lane B). Thus, these results indicated that the growth of artificial chromosomes was confined to their telomeric/sub-telomeric region. Although
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Figure 3. Characterization of artificial chromosomes. (A) Total genomic DNA from wild-type trypanosomes (lane 3) and two clones bearing pTacA (lanes 4 and 5) or pTacB (lanes 6 and 7), were digested with BamHI. Lanes 1 and 2 have DNA corresponding to the input plasmids pTacA and pTacB, respectively, which were linearized with BglII and then digested with BamHI. Following electrophoresis and Southern transfer the filters were probed as shown. (B) Top panel: a clone bearing pTacA was kept in continuous culture for 3 months. Samples were withdrawn soon after cloning (lane 1) and then∼3, 6 and 12 weeks later (lanes 2–4). Genomic DNA corresponding to each sample was digested with BamHI, electrophoresed through agarose, Southern transferred, and then probed with cat. Lanes A and a are control lanes with two different dilutions of input pTacA DNA that has been linearized with BglII and then digested with BamHI. Bottom panel: genomic DNA was isolated from a clone bearing pTacA (lanes 1 and 2) or pTacB (lanes 3 and 4) after ∼3 and 12 weeks of continuous culture subsequent to cloning. This DNA was digested with BamHI and SacI, electrophoresed through agarose, Southern transferred, and probed with cat. Lanes A and B correspond to the input plasmids pTacA and pTacB, respectively, which have been linearized with BglII and then digested with BamHI and SacI.
we have not shown that this growth is confined precisely to the ends of telomeres, that is the most likely scenario. The estimated growth rate would indicate that ∼1–1.5 telomeric repeats are added at every generation. Artificial chromosomes have exonuclease-accessible ends The electrophoretic mobility of pTacA and pTacB isolated from transformed procyclic clones, and the growth of their telomeric DNA, strongly suggests that these are linear extrachromosomal elements. We have confirmed this by Bal31 sensitivity experiments (Fig. 4), which demonstrated the presence of exonuclease accessible ends on this artificial chromosome. As a control for non-specific exonuclease activity, the filter was stripped and reprobed with a fragment corresponding to the parpA promoter. As shown, the band corresponding to the genomic parpA locus, and the central BamHI fragment from the artificial chromosome seen by the same probe, were both unaffected by this period of Bal31 exposure, indicating their chromosome-internal position. Stability of artificial chromosomes Clones bearing pTacA or pTacB have been kept in continuous culture under G418 selection for >5 months without loss of the artificial chromosomes or any discernible effect on trypanosome growth. To determine the stability of these elements in the absence of selective pressure, cultures of trypanosomes bearing pTacA or pTacB were subcultured in media without G418. Aliquots were withdrawn after each subculture. Measurements (Fig. 5) indicated that pTacA and pTacB were present at an initial copy number of ∼2 per cell and, in the absence of selection, 50% of these molecules are lost from a transformed population in ∼7
Figure 4. Artificial chromosomes have exonuclease-accessible ends. DNA isolated from a pTacA transformed procyclic clone was exposed to Bal31 for 0, 5, 10, 20 and 30 min. Following inactivation of the exonuclease and digestion with BamHI, the DNA was electrophoresed, Southern transferred and probed with a fragment corresponding to the cat coding sequence (A). Following autoradiography, this label was stripped off and the filter reprobed with a 110 bp fragment corresponding to the parpA promoter (B). Arrows indicate the genomic (G) and pTacA-derived fragment (AC).
generations. Consequently, these linear elements are less stable than the parent episome from which they were derived. An artificial chromosome as a potential promoter-trap? An important motivation for the construction of these elements was for their anticipated use as promoter-traps. pTacB-transformed procyclics are able to survive selection with either G418 or hygromycin. A SalI deletion removed the parpB promoter on
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Figure 5. Stability of artificial chromosomes. Procyclic clones bearing pTacA or pTacB were passaged (1:150 dilution at each passage, equivalent to ∼7 generations between passages) in medium without G418. Aliquots were withdrawn at each passage. Genomic DNA was isolated, digested with BamHI, electrophoresed through agarose, Southern transferred and probed with a fragment corresponding to the parpA promoter. Lane 0 corresponds to samples from cultures grown in media with G418. The ratio of the signal strength (AC/G) for each lane is a measure of the number of copies of the artificial chromosome (AC) relative to the genomic parpA locus (G). Note: the ParpA promoter probe used [110 bp PflMI–PflMI fragment from pEV (24)], did not hybridize with the ParpB promoter bearing fragment from either the genome or these artificial chromosome under the conditions used.
pTacB giving the plasmid pTacC. Procyclic trypanosomes transformed with BglII-linearized pTacC maintain the construct as an artificial chromosome in the presence of G418 but could not survive hygromycin selection, indicating the absence of fortuitous transcription into the hyg coding region, and the potential use of constructs such as pTacC as promoter traps. We further refined this vector to improve its convenience for such a use. A SalI–partial SacI digest removes the entire cat-hyg expression unit, which we replaced with a polylinker (pTacD). Construct pTacE (Fig. 1) has a promoter-less cassette inserted at this polylinker. The cassette consists of firefly luciferase (luc) and phleomycin resistance (ble) genes with flanking SAS and PAS derived from T.brucei actin (act) and aldolase (ald) loci, respectively. Very low levels of ble transcription can be selected for, as indicated by our success in being able to introduce and select for this marker at ‘silent Vsg’ expression sites (Navarro and Cross unpublished data). Consequently, pTacE should enable us to ‘trap’ fairly weak promoters. The luc unit facilitates analysis of the strength of a trapped promoter in a transient assay. Procyclic trypanosomes transformed with BglII-linearized pTacE could not withstand ble selection, but could if transformed with linear pTacE-R with an rRNA promoter in the polylinker of pTacE.
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Figure 6. The activity of a parpB promoter is unaffected by its distance from the telomeres. Total RNA obtained from pTacAm or pTacB transformed clones was electrophoresed and transferred onto nitrocellulose. The same filter was successively probed with cat, neo and β-tub coding sequences (in that order). The label was stripped off the filter each time before hybridization with the next probe. Exposure was for 36 (cat), 12 (neo) and 3 h (tub), and band intensities were measured using a phosphorimager. Signal strengths of cat, neo and tub mRNA from pTacB transformed procyclics were ∼2.7-, 2.3- and 1.6-fold higher than the corresponding signal from the pTacAm transformed cell line.
the demonstrated need of a downstream SAS in 3′ end formation of mRNA in trypanosomatids (46–48). Southern analysis of genomic DNA derived from pTacAmtransformed clones indicated that this construct, like its parent pTacA, existed as an autonomous linear element. No integrated, or otherwise rearranged molecules were detected (data not shown). RNA analysis indicated that the cat mRNA derived from pTacAm and pTacB-tansformed clones were of the same size (Fig. 6 top panel), indicating that the transcripts from both clones were similarly processed as expected. Phosphorimager analysis of band intensities showed that, when corrected for loading differences, the amounts of cat mRNA in the two populations were almost identical. Similar results were obtained when we measured transcriptional initiation from the parp promoter driving cat expression, by doing nuclear run-on assays with lysolecithin permeabilized cells (data not shown). We conclude that transcription from the parpB promoter in pTacAm is not significantly different from its level in pTacB despite the fact that it is ∼4.5 kb closer to the telomere in the former construct. DISCUSSION
The activity of a parpB promoter on these chromosomes is unaffected by its relative distance from the telomeres We were concerned that the proximity of a ‘trapped’ promoter to a telomere on these constructs might result in the repression of transcription, as has been demonstrated in the yeast Saccharomyces cerevisiae (43–45). We addressed this concern by comparing cat transcription in pTacB and pTacAm-transformed procyclic trypanosomes. Construct pTacAm (Fig. 1) is a derivative of pTacA and was obtained by the insertion of a 286 bp fragment, consisting of the parp-SAS and the first 169 bp of the hyg coding sequence, downstream of the tub intergenic region. The 286 bp insert was derived from pTacB, and was used to fulfill
We report the development and characterization of a set of linear artificial mini-chromosomes for T.brucei. A similar construct has been described for Leishmania (S. Beverley, personal communication). In addition, linear or supercoiled DNA injected into the macronucleus of the ciliated protozoan Paramecium tetraurelia are maintained as linear molecules. The injected DNA is cleaved at random points and sequences characteristic of Paramecium telomeres are added. However, neither replication nor telomere addition shows any defined sequence requirement and the linear elements are maintained at a copy number of ∼10 000–50 000 molecules/cell (49). In contrast, and as in yeast where they were first constructed (50,51), the artificial chromosomes described
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here exist at a copy number characteristic of natural chromosomes, and the parental episome from which these were derived displays very specific sequence requirements for autonomous replication (23,24). Although these elements are indefinitely stable under selection and do not integrate into the genome, they are lost fairly quickly on the removal of selective pressure. We do not know the basis for this loss in stability compared to that of their parent pT13-11. However, a similar phenomenon has been documented in the yeast Saccharomyces cerevisiae, where circular CEN plasmids (i.e. plasmids with centromeric DNA) are substantially more stable than the linear chromosomes derived from them (50). Finally, the estimated growth rate of the telomeric repeats on these artificial chromosomes agrees with previous data on the growth of natural chromosomes in T.brucei (32,52). It is interesting to note that the band corresponding to the growing telomeric fragment in pTacA does not show substantial smearing on prolonged culture, indicating a fairly uniform growth of telomeres over the entire population. As small dispensable genetic elements that bear all the cis-acting signals necessary for autonomous replication, these defined linear mini-chromosomes should be useful models for the study of trypanosome nuclear DNA replication and chromosomal behavior. We expect such studies in this ancient lineage to provide a unique perspective on the evolution of the DNA replication and segregation apparatus in eukaryotes. A potential concern in the use of these small linear elements as promoter-traps was the possibility of telomere mediated repression (silencing) of transcription. A telomeric position effect (TPE) whereby several pol II promoters are silenced in the vicinity of telomeres has been extensively studied in the yeast S.cerevisiae (43–45). TPE is mitotically heritable but reversible, and a telomere-proximal promoter shows a variegated pattern of activity, such that some cells initiate transcription from this promoter while others do not. TPE is thought to be mediated by a change in chromatin structure, which establishes a gradient of transcriptional repression originating at the telomeres and spreading inwards (for ∼5 kb in S.cerevisiae) along the chromosome. The extent of this silenced domain is affected by many factors, including the strength of the promoter (45). Our observations indicate that initiation of transcription from the parpB promoter is unaffected by its distance from the telomere in procyclic T.brucei. At least three earlier studies have reported data consistent with these observations (53–55). In each instance, a parp or rRNA promoter showed high activity at a telomere-proximal (∼4–5 kb from sub-telomeric repeats) position in procyclic T.brucei. The absence of a gradient of transcriptional repression between positions that are ∼1.7 (pTacAm) to ∼6.2 kb (pTacB) away from the subtelomeric repeats on these artificial chromosomes is inconsistent with the phenomena of TPE as described in yeast implying a putative difference in the make-up of telomeric chromatin between yeast and procyclic-form trypanosomes. However, we cannot discount the possibility that weaker promoters than the one studied here might be subject to telomere-mediated repression. Delineation of Pol II promoters will play a key role in the understanding of gene expression in trypanosomes. ACKNOWLEDGEMENTS NA thanks Mary Gwo-Shu Lee for her hospitality in making laboratory space available to continue her research at Columbia
University. This work was supported by NIH grants AI 21729 (to GAMC), AI 21784 (to LHTVdP) and by a grant from the John D. and Catherine T. MacArthur Foundation to LHTVdP.
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