School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK1
Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 7DU, UK2
Author for correspondence: Wendy C. Gibson. Tel: +44 117 928 8249. Fax: +44 117 925 7374. e-mail: w.gibson{at}bristol.ac.uk
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ABSTRACT |
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Keywords: Trypanosoma brucei, hybrids, meiosis, Green Fluorescent Protein, tsetse fly
Abbreviations: CM, Cunninghams medium; GFP, Green Fluorescent Protein; UTR, untranslated region
a Present address: School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
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INTRODUCTION |
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T. brucei undergoes a complex life cycle involving both mammalian and bloodsucking insect (tsetse fly) hosts. Trypanosomes in the blood of the mammal are taken up by the fly when it feeds; inside the fly, trypanosomes first differentiate and multiply within the midgut and then migrate to the salivary glands, where they again multiply and complete their development to the mammal-infective form. Jenni et al. (1986) showed that hybrids were produced after co-transmission of two parental trypanosome clones through the tsetse fly. However, genetic exchange is not an obligatory part of the trypanosome life cycle and, although it may occur quite frequently (3040%) in infected flies (Schweizer et al., 1988
; Turner et al., 1990
), experimental flies are refractory to infection. These factors, coupled with the relative inaccessibility of genetic exchange within the tsetse fly vector, have hampered direct observation of the process. After analysis of crosses using selectable drug resistance markers, hybrids were found only in trypanosome populations derived from the salivary glands, not midguts, suggesting that genetic exchange probably takes place in the salivary glands (Gibson & Bailey, 1994
; Gibson et al., 1997b
).
Analysis of the inheritance of genetic markers in hybrid progeny suggests that a meiotic division occurs at some stage (Paindavoine et al., 1986 ; Sternberg et al., 1988
, 1989
; Gibson, 1989
; Turner et al., 1990
; Gibson & Stevens, 1999
), but triploid hybrids also occur with some frequency (Paindavoine et al., 1986
; Wells et al., 1987
; Gibson et al., 1992
, 1997b
; Gibson & Bailey, 1994
; Hope et al., 1999
) and a haploid life cycle stage has not been found (Shapiro et al., 1984
; Tait et al., 1989
). The observation that kinetoplast (mitochondrial) DNA is inherited from both parents in hybrid progeny supports the surprising suggestion that somehow the complex parental kinetoplast DNA networks swap DNA (Gibson & Garside, 1990
; Turner et al., 1995
; Gibson et al., 1997a
). This in turn presupposes fusion of the parental mitochondria, and hence cells, during genetic exchange.
To get any further towards unravelling what happens during genetic exchange, we need to see the process within the fly. This means pinpointing the developmental stage and the region of the fly where genetic exchange takes place. To do this, we need to be able to visualize hybrids in situ and distinguish them from parental trypanosomes. The new approach described here is based on the tetracycline (Tet)-inducible expression system developed by Clayton and colleagues (Wirtz & Clayton, 1995 ; Biebinger et al., 1997
). The inducible expression system comprises a repressor construct targeted to the tandem array of tubulin genes in T. brucei, where it constitutively expresses the Tet-inducible bacterial repressor protein TetR at high level, and a reporter construct with the cognate operator sequence in its promoter. The reporter is Green Fluorescent Protein (GFP), which does not require any substrate for activity and can be visualized in live or fixed cells by fluorescence microscopy or flow cytometry. Beverley and colleagues first demonstrated the feasibility of using GFP as a marker in trypanosomatids, using both live and fixed cells of Leishmania (Ha et al., 1996
) and its use in T. brucei has also been reported (e.g. Vaidya et al., 1997
; Hill et al., 1999
; Marchetti et al., 2000
). In an experimental cross, segregation of the chromosomes carrying the GFP and repressor genes should uncouple repression, giving rise to a proportion of hybrid progeny expressing GFP (Fig. 1
). A proportion of haploid forms, should they occur, will also be fluorescent. This system should enable us to search for and detect very few hybrids against a background of non-fluorescent trypanosomes.
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In this paper our goal was to determine whether this challenging new approach would produce fluorescent hybrids as predicted.
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METHODS |
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T. brucei genomic DNA was obtained using a simple mini-prep procedure (Medina-Acosta & Cross, 1993 ). After digestion with appropriate restriction enzymes, DNA was fractionated by electrophoresis through 1% agarose gels. Samples for PFGE were prepared by lysing and deproteinizing trypanosomes in situ in agarose blocks (Van der Ploeg et al., 1984
). Chromosomes were separated using the Pulsaphor system (Pharmacia) with a four-phase programme (Phase I, 75 min pulses at 35 V for 150 h; Phase II, 20 min pulses at 60 V for 15 h; Phase III, 5 min pulses at 130 V for 15 h; Phase IV, 3 min pulses at 130 V for 15 h) or a five-phase programme at 130 V (Phase I, 15 min pulses for 15 h; Phase II, 5 min pulses for 15 h; Phase III, 3·5 min pulses for 10 h; Phase IV, 3 min pulses for 10 h; Phase V, 40 s pulses for 10 h). After acid depurination for 15 min in 0·25 M HCl, DNA was blotted from agarose gels onto nylon membrane (Zeta Probe; Bio-Rad) by alkaline transfer in 0·4 M NaOH.
DNA probes were labelled with [32P]dCTP and hybridization was carried out overnight in 0·25 M sodium phosphate buffer (pH 7·2), 7% (w/v) SDS at 60 °C; the membranes were then washed in 0·1xSSC, 0·5 % SDS at 60 °C.
Tsetse transmission and experimental cross.
Trypanosomes were transmitted through male tsetse flies (Glossina morsitans morsitans) as described previously (Gibson, 1989 ). Infected flies were maintained on membrane-fed sterile horse blood supplemented with 2·5% (w/v) bovine serum albumin (Sigma A4503) (Langley et al., 1978
). Flies were dissected between 7 and 9 weeks following the infective feed. Infected midguts were cultured in CM supplemented with 10% fetal calf serum and 100 µg gentamicin ml-1. Metacyclics from infected salivary glands were inoculated into mice; bloodstream forms were subsequently transformed back to procyclics if necessary, by incubation in standard CM at 27 °C.
For the experimental cross, flies were infected with a mixture of clones K11 (a GFP TetR transfectant derived from TH2) and T. b. brucei KP2N, a previously transfected derivative of GPAP/CI/82/KP2-I (CLONE 23) (Letch, 1984 ) carrying the NEO gene in the tubulin gene array (Gibson & Whittington, 1993
). Drug-resistant clones of these two parental stocks, TH2 and KP2, were successfully mated in a previous cross (Gibson et al., 1997b
).
Microscopy and flow cytometry.
Living trypanosomes were viewed as wet mounts in CM, blood or PBS, sometimes mounted in Vectashield (Vectalabs). Whole tsetse midguts or salivary glands were dissected into a drop of PBS and also viewed as wet mounts. Cells were generally fixed in 2% (w/v) paraformaldehyde at 4 °C for 30 min, if required. A DMRB microscope (Leica) equipped with either a Colour Coolview camera (Photonic Science) or Megaview II camera (Norfolk Analytical) was used for fluorescence and standard microscopy, with ImagePro Plus software (Photonic Science) or analySIS software (Norfolk Analytical), respectively.
Samples for flow cytometry were prepared by mixing a suspension of trypanosomes in PBS with an equal volume of 2% paraformaldehyde at 4 °C for 30 min; trypanosomes were recovered by centrifugation and resuspended in PBS at approximately 107 ml-1. Samples were analysed using an EPICS-CS or EPICS-XL flow cytometer with Elite software (Coulter Electronics). A total of 100000 cells were collected from each sample.
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RESULTS AND DISCUSSION |
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The correct integration of the GFP construct into the rRNA locus was confirmed by PCR across border regions and Southern analysis of restriction digests (Figs 3 and 4
). The chromosomal location was determined by Southern analysis of PFGE blots (Fig. 5
), since homologous chromosomes of trypanosomes often differ in size (Gibson & Borst, 1986
) and the rRNA loci are found on several pairs of chromosomes (Gottesdiener et al., 1990
). The majority of transfectants were found to have the reporter construct on chromosome I, the chromosome which also carries the tubulin array (data not shown). In clone E8, however, the construct had integrated into a larger chromosome (not identified) and this clone was therefore suitable for the second round of transfection, targeting the repressor construct to the tubulin array.
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Integration of the constructs was also analysed by Southern blotting of HindIII-digested genomic DNA from GFP transfectant clone E8 and the GFP-repressed transfectant clones K6 and K11 (Fig. 4). A GFP probe hybridized to fragments of approximately 11 kb in the E8 and K11 digests, consistent with integration of the pHD67E construct in the rRNA locus (Biebinger et al., 1996
, 1997
). In contrast, this probe hybridized to a smaller fragment (approx. 7 kb) in clone K6. This suggests that the construct pHD449ACT, which contains a HindIII restriction site, had integrated downstream of the GFP coding region (which also has a HindIII site at the 5' end) in this clone. A TetR coding region probe hybridized to different sized fragments in HindIII-digested genomic DNA from clones K1, K6 and K11 (Fig. 4
), suggesting integration of pHD449 at different loci in these clones. In the K11 digest the TetR probe hybridized to fragments of two different sizes; a band of approximately 9 kb presumably represents a border fragment, while the much stronger hybridization to a fragment of 6·5 kb (approximately the same size as linearized plasmid) suggests the presence of multiple copies of the repressor construct arranged in a tandem repeat.
Expression and repression of GFP through development in the tsetse fly
The next step was to ascertain that the GFP reporter would be adequately expressed throughout the trypanosome developmental cycle in the tsetse fly and that expression would be efficiently repressed in the parental clone. The GFP transfectant clones E8 and E21 were highly visible against a background of tsetse midgut or salivary gland tissues, despite some autofluorescence of the fly tissue, and stably expressed GFP throughout cyclical development in the tsetse fly. Procyclic forms were found to express functional GFP in the relatively anoxic environment of the tsetse midgut and could be clearly visualized against the background midgut autofluorescence. Similarly, GFP expression was observed in all trypanosomes in the salivary glands, i.e. both attached epimastigote and metacyclic forms (Fig. 7). In bloodstream-form trypanosomes, however, no expression of functional GFP could be detected by fluorescence microscopy. This finding was confirmed by flow cytometry: the peak green fluorescence of bloodstream forms was just slightly greater than that of wild-type trypanosomes (not shown). When these non-fluorescent bloodstream-form trypanosomes were transformed to procyclic forms in vitro, they regained bright fluorescence. The reporter construct pHD67E was derived from pHD676 (Biebinger et al., 1997
), which is designed for use in both procyclic and bloodstream-form trypanosomes; the GFP coding region is followed by 3' UTR sequences from the non-developmentally regulated actin gene. The extremely low levels of GFP expression we observed may be the result of the lower activity of the procyclin promoter in bloodstream-form trypanosomes (fivefold down-regulation compared with procyclic forms) (Biebinger et al., 1996
, 1997
) in combination with a reduced expression efficiency specific to GFP in this life cycle stage.
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Experimental cross
The GFP-repressed transfectant K11 was crossed with another trypanosome clone, KP2N, carrying the NEO gene, conferring geneticin resistance, integrated into the tubulin locus. A mixture of fluorescent and non-fluorescent trypanosomes were observed in the salivary glands of one fly (Fig. 8a,b
) and subsequently in the recovered procyclic forms (Fig. 8c
,d
). This population of procyclics was selected with hygromycin and geneticin separately and in combination, and 21 clones were obtained from either the drug-selected or starting populations. Of these clones, four were fluorescent and resistant to both hygromycin and geneticin, but not phleomycin. These clones were therefore unequivocal hybrids of the two parents. The phenotypes of all clones are given in Table 1
. Note that few (inferred) genotypes are represented and no phleomycin-resistant trypanosomes were recovered at all; the absence of the TetR gene in these clones was confirmed by PCR using primers C and D (not shown). Since the original salivary gland population was expanded both in vivo and in vitro, it experienced population bottlenecks on transfer from fly to mouse and then mouse to culture, and also certain genotypes may have been favoured by growth conditions. Therefore, these clones cannot be said to represent all genotypes present among metacyclics in the salivary glands. Importantly, the trypanosomes isolated from the midgut of the same fly were not fluorescent, indicating that genetic exchange occurred in (or possibly en route to) the salivary glands.
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ACKNOWLEDGEMENTS |
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Received 8 May 2001;
revised 20 July 2001;
accepted 9 August 2001.