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Self-fertilisation in Trypanosoma brucei
MOLECULAR
EmcAL PARAsIToLoGy Molecular and Biochemical Parasitology 76 (1996) 31-42
Self-fertilisation Andrew
Tait”, Norma
in Trypanosoma brucei
Buchanana,
Geof Hide”, C. Michael
R. Turnerb.*
aWellcome Unit oj‘ Molecular Parasitology, The Anderson College, Universityof’ Glasgoas,56 Dumbarron Road, Glasgow GII 6NU. UK
bI.B.L.S.-D.I.I.. Joseph Bluck Building. Unitlersitysf Glasgobts, GlasgovvGl2 8QQ. UK Received 27 July 1995; revised 2 November 1995; accepted 3 November 1995
Abstract We have investigated whether Trypanosoma brucei can undergo self-fertilisation. A group of 27 metacyclic clones derived from the tsetse transmission of a mixture of two genetically marked stocks was analysed and 22 clones were observed to be of non-hybrid phenotype. A group of 10 clones from this non-hybrid subset were then analysed for one isoenzyme, one restriction fragment length polymorphism and three karyotype markers potentially informative for the detection of self-fertilisation. Five of the 10 clones were found to be recombinant for at least one marker and we interpret these recombination events as indicating the clones to be products of self-fertilisation. We have also analysed a limited number of metacyclic clones from stocks of T. brucei each singly transmitted through tsetse flies but, so far, no evidence of recombination has been detected. We conclude that T. brucei is able to self-fertilise but there may be a requirement for the presence of dissimilar stocks to initiate such an event. Keywords: Trypanosoma brucei; Genetics;
Self-fertilisation;
Karyotype
1. Introduction
Genetic exchange in Trypanosoma brucei has been directly demonstrated by laboratory crosses
_~ Abbreuiutions: STIB, Swiss Tropical Institute Basel; EATRO, East African Trypanosomiasis Research Organisation; RFLP, restriction fragment length polymorphism; PFGE, pulsed field gel electrophoresis; AP, alkaline phosphatase; ICD, isocitrate dehydrogenase; ME,, malic enzyme B; Tyr3. tyrosyl tyrosyl tyrosine peptidase; p/c, phospholipase C gene: pgk. phosphoglycerate kinase gene. * Corresponding author. Tel.: + 44 141 330 6629; Fax: + 44 141 307 8016; E-mail: [email protected]. 0166~6851/96/$15.00
0 1996 Elsevier
SSDI 0166-6851(95)02528-L
Science
Ireland
Ltd. All rights
[I -71 but its contribution to the epidemiology of African trypanosomiasis in the field remains a matter of debate [&13]. By infecting tsetse flies with two genetically marked cloned stocks of trypanosomes, it has been shown that mating takes place during development in the fly and that a proportion of such mixed-infected flies produce metacyclic trypanosomes which are hybrid for the range of markers examined [l-7]. A proportion of the trypanosomes emerging from such flies are, however, parental in genotype and phenotype implying that the mating events are non-obligatory during the life-cycle of the trypanosome [271.
In
reserved
order
to
investigate
the
rules
and
32
A. Tait et al. I Molecular and Biochemical Parasitology 76 (1996) 31-42
mechanisms governing this system of genetic exchange crosses between three different stocks in all pairwise combinations have been undertaken and it was shown that Fl progeny clones were produced from each cross [4]. These observations indicate either that there are no mating barriers between stocks or that there is a mating type system but at least one of the stocks must be heterozygous for alleles at a postulated mating type locus. On either hypothesis, these results imply that T. brucei can self-fertilise, but to date no evidence has been presented which establishes whether this occurs. We have directly examined the question of selffertilisation using a series of trypanosome clones derived from metacyclic stage parasites produced from the infection of tsetse flies with either a single cloned trypanosome line or a mixture of two genetically marked cloned trypanosome lines. If self-fertilisation occurs, then half the progeny of such an event will be homozygous at a locus for which the parental cloned line was heterozygous, but will remain identical to the parental line for any marker which is homozygous. These predictions provide a simple screen for the occurrence of self-fertilisation by analysis of loci in the clones derived from single or mixed-stock infected tsetse flies. In this paper we report the marker analysis of these clones and provide strong evidence for the occurrence of self-fertilisation.
2. Materials and methods 2.1. Parasites
.
A genetic cross was conducted between cloned stocks, STIB (Swiss Tropical Institute Basel) 247 and STIB 386, by co-transmitting populations of these stocks through tsetse flies. The methods for growing trypanosome populations, cyclically transmitting them through tsetse flies and deriving metacyclic clones from a fly producing hybrid trypanosomes have been described previously [4,14]. A series of 27 metacyclic clones were derived from a single fly (F492) on day 50 after infection with both parents and each clone was assigned a unique number.
Populations of the following cloned trypanosome lines were each individually transmitted through tsetse flies and clones derived from metacyclic forms: STIB 247, STIB 386, EATRO (East African Trypanosomiasis Research Organisation) 605, EATRO 1125, EATRO 2340, EATRO 795, F9/45 mcll0. This last line is a hybrid progeny clone from a cross between stocks STIB 247 and STIB 386 [2]. 2.2. Enzyme electrophoresis The preparation of trypanosome lysates, the conditions for starch gel electrophoresis and the enzyme-specific staining procedures were as previously described [151. 2.3. Restriction fragment analyses
length polymorphism
The methods for preparing DNA probes from libraries and using them to detect restriction fragment length polymorphisms (RFLPs) have been described previously [l]. T. brucei genomic
2.4. Karyo type analyses Pulsed field gel electrophoresis (PFGE) was performed in a contour-clamped homogeneous electric field apparatus. Gels were poured at 1% agarose in 0.089 M Tris-borate, pH 7.6, 1 mM EDTA buffer and run in the same buffer at 3.0 V cm-’ and a temperature of 13°C using buffer recirculation. Two sets of pulse times were used (duration of each pulse time in hours given in brackets): A, 900 s (24) 750 s (24) 600 s (24), 500 s (24); B, 1200 s (24), 1100 s (30), 900 s (32), 800 s (24). After completion of the gel run, chromosome bands were stained in 1 pg ml - ’ of ethidium bromide, destained in running buffer and visualised on a UV-transilluminator. Ethidium bromide stained gels were photographed and the negatives scanned in a Joece-Loebl scanner at wavelength 530 nm. Chromosome blocks were prepared by embedding whole purified trypanosomes at a concentration of 0.5 x lo9 ml --I in 1% low melting agarose at 37°C in phosphatebuffered saline, pH 7.4, and casting in a mould at
A. Tait et al.
1Molecular and Biochemical Parasitology 76 (1996) 31-42
4°C. The blocks were deproteinised by resuspending in 0.5 M EDTA, 0.5 mg ml -’ proteinase K, 1% sodium sarkosyl, pH 9.0, at room temperature for 20 min followed by incubation at 56°C for 48 h. Blocks were washed 3 x in 50 mM EDTA, pH 8.0, and stored at 4°C in the same solution until use. Gels were blotted onto Hybond-N membranes and hybridised with a series of probes, labelled by random primer extension. Hybridisation was at 65°C and the filters were washed in 1 x SSC (0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0) at 65°C before exposing to X-ray film. Blots were reprobed under the same conditions, after stripping the blot by boiling in 1% SDS for 10 min. Probes for the T. brucei phospholipase C gene, plc [16], the phosphoglycerate kinase gene, pgk [171, and the tbcrk2 protein kinase gene [18] were prepared by gel purification of the insert from restriction digests of purified recombinant plasmids. Twenty-five pg of the insert was excised from the gel and labelled with 32P by random priming (Stratagene).
3. Results 3.1. Phenotypic analysis of the progeny of a mixed transmission
An analysis of a series of progeny clones derived from the co-transmission of two stocks (STIB 247 and STIB 386) through tsetse flies was undertaken. Twenty-seven clones were screened by analysis of isoenzyme markers to define their phenotype and the results are shown in Table 1. The clones can be divided into three classes: Class 1 is identical to the 247 parent (Pl), Class 2 is the equivalent of the Fl progeny obtained from the cross between 247 and 386 while Class 3 are recombinant at the single heterozygous locus for the tyrosyl-tyrosyl-tyrosine peptidase (Tyr3) but identical to the 247 parent for the remaining three homozygous markers (alkaline phosphatase, AP; isocitrate dehydrogenase, ICD; malic enzyme B, ME,). Fig. 1 illustrates the results for the enzymes ICD and Tyr3. These results suggest that, in addi-
33
tion to the expected Fl progeny (class 2) recombinants related to STIB 247 had been generated in this experiment. No clones of the parental 386 phenotype were obtained but we attach no particular significance to the disparity in numbers of clones of the two parents as such disparities have been regularly observed in previous co-transmission experiments [4]. To investigate further the occurrence of recombinant STIB 247 genotypes, a group of clones from the mixed transmission experiment showing either the 247 recombinant phenotype (clones 57 and 58) or the 247 parental phenotype (clones 38, 40, 41, 53, 54, 55, 60) were analysed for a further heterozygous marker and for their molecular karytope. 3.2. Restriction fragment length polymorphism anal_wes Restriction digests of DNA from clones 38, 40, 41, 53, 54, 55, 57, 58 and 60 were Southern blotted and the blots probed with the single copy gene probe, pBE9, for which the parent 247 is heterozygous for the presence/absence of a PstI site [l]. These results showed that clone 53 (Class 1 parental by isoenzymes) was homozygous for this RFLP while the remaining clones were heterozygous and identical to the parental stock 247 (Fig. 2, Table 2). 3.3. Karyotype anaIyses Previous studies have shown that some homologous chromosomes differ sufficiently in size to be separated by PFGE [19,20] and these size differences can be used as markers for chromosome segregation and recombination [5,6]. Chromosome separations, for the size range 700 kb to 2.5 Mb, of STIB 247 and nine progeny clones are illustrated in Fig. 3a. STIB 247 shows nine bands of DNA; the most rapidly migrating band constitutes the mini-chromosomes (m), followed by the intermediate chromosomes (int z 700 kb) and a further six bands: band 1 ( > 1.3 Mb), bands 2, 3 and 4 (2.0-2.5 Mb) and bands 5 and 6 (> 2.5 Mb). The molecular karyotypes of clones 53, 54 and 58 are different from that of the parent STIB
A. Tait et al. / Molecular and Biochemical Parasitology 76 (1996) 31-42
34 Table 1 Analysis of the enzyme and STIB 386) Clone
electrophoretic
Enzyme ICD
Parental (Class 1) 34 36 38 39 40 41 42 44 45 46 47 52 53 54 55 59 60 61 62 Hybrids (Class 2) 43 48 51 56 68 Recombinant (Class 35 57 58 Parents 247 (P 1) 386 (P 2)
phenotypes
of progeny
AP
1 I
I 1 1
I I 1 1 1 1 1
1 1 1 1 1
from a tsetse fly infected
with two stocks
(STIB 247
phenotype
1 1 1
clones derived
1 nd 1 1 1 1 1 1
Tyr3
ME,
2-4 2-4 2-4 224 2-4 224 2-4 2-4 224 2-4 2-4 224 2-4 224 224 2-4 224 224 224
3 3 3 3 3 3 3 3 nd 3 3 3 3 3 3 3 3 3 3 223 nd nd 223 2-3
1-2 l-2 l-2 l-2 l-2
l-2 nd nd l-2 1-2
2 4 4 2-4 2-4
1
1 1 1
4 2 4
1 2
2-4 2-4
3)
1 1
I 2
peptidase; ME,, ICD, isocitrate dehydrogenase; AP, alkaline phosphatase; Ty?, tyrosyl-tyrosyl-tyrosine determined. The numbers refer to allelic electrophoretic variants: homozygous phenotypes are indicated heterozygous phenotypes by two numbers linked by a dash.
247 (Fig. 3a) and show the loss of either band 2 (clones 54 and 58) or band 4 (clone 53) with an increase in the intensity of staining of band 1 (clones 54 and 58) or band 3 (clone 53) as confirmed by densitometer scans of photographic negatives of these gels (Fig. 3b). Alterations are also observed in chromosomes of larger size by using different electrophoretic conditions: in Fig. 3c, bands 7 and 8 ( z 2.8 Mb) are clearly resolved in
3 2-3 malic enzyme,; nd, not by a single number and
STIB 247 which are identical to those observed in eight of the nine clones examined. Clone 57, however, showed a loss of band 8 with band 7 showing an increased intensity of fluorescence (Fig. 3d). To determine the relationship between the chromosome bands separated by PFGE, Southern blots of the separations were hybridised with probes for two single copy genes (plc and tbcrk2)
A. Tait et al. / Molecular and Biochemical Parasitology 76 (1996) 31-42
O-
-0 1
Pl
l-2 HYB
35
o-
-0
2
1
2-4
2-4
P2
REC
Pl
P2
4
2 REC
Fig. 1. Isoenzyme phenotypes of parental and progeny clones from the mixed transmission of stocks STIB 247 through tsetse flies. The left panel shows results for isocitrate dehydrogenase (ICD) and the right panel shows results detected using tyrosyl-tyrosly-tyrosine as substrate (Tyr3): Pl, STIB 247 parent: P2, STIB 386 parent; HYB, hybrid 43; REC, non-hybrid recombinant clone 58 (Tyr3 - 4) or clone 57 (Ty?-2). The inferred allelic combinations to homozygous (1, 2 or 4) or heterozygous (l-2 or 2-4) patterns are also shown
and a closely linked multiple copy gene (‘pgk): the results are illustrated in Fig. 4. The plc gene probe hybridises with bands 1 and 2, the pgk gene probe hybridises with bands 3 and 4, while the tbcrk2 gene probe hybridises with bands 7 and 8 in separations of STIB 247 chromosomes. These results suggest that the pairs of bands 1 and 2, 3 and 4, and 7 and 8 each represent the two homologues of three different chromosomes in which the two homologues differ in size. In each progeny clone where a chromosome band has been lost, only the remaining homologue hybridises to the relevant probe, i.e., no change in chromosomal size has occurred (Fig. 4). Densitometric scans of
and STIB 386 for a peptidase progeny clone generate these
such blots show that the intensity of hybridisation to the single chromosome bands from clones which have lost the corresponding homologue is equal to the sum of the intensities of hybridisation to the two homologues in the parental stock 247 (data not shown). The data on the molecular karyotype of the nine progeny clones, show that five (clones 38, 40, 41, 55 and 60) are identical to the parental STIB 247 stock in respect to three pairs of resolvable homologous chromosomes while four (53, 54, 57 and 58) show the loss of one homologue of a pair but the apparent gain of the other homologue. A similar interpretation can be used to explain the
A. Tait et al. 1 Molecular and Biochemical Parasitology 76 (1996) 31-42
36
pBE9 kb
8.1
homozygous enzyme phenotype of clones 35, 57 and 58 and the homozygous genotype detected by probe pBE9 (which hybridises to the unresolved region of the PFGE separations) in clone 53. The data for all ten clones examined are summarised in Table 2; five clones are identical by all criteria to the original parental STIB 247 stock while the other five (35, 53, 54, 57, 58) are recombinant for one or more markers. 3.4. Analysis of stocks transmitted singly through P ies
I.9
247
53
247
P 53
1 )
P 10.0 I
Fig. 2. Genotype of a parental and a recombinant clone determined by Southern blotting. PstI digests of STIB 247, probed with pBE9, show three fragments of 10, 8.1 and 1.9 kb which are interpreted as being due to the presence of a polymorphic PstI site within the IO-kb fragment for which 247 is heterozygous ( + / - ) while recombinant clone 53 shows a homozygous pattern ( - / - ). The sizes of the restriction fragments were determined using DNA size markers. The genomic organisation of the fragments detected by this probe (denoted by bar) are indicated diagrammatically below the blot.
The finding of novel recombinant karyotypes derived from a single parental stock in a mixed transmission experiment raised the question as to whether stocks could also undergo this process when singly transmitted through flies or after prolonged growth as bloodstream or procyclic stage trypanosomes. The karyotype of a series of derivatives of STIB 247 was therefore determined and the results are illustrated in Fig. 5. Clearly, the karyotype is stable to prolonged growth in either bloodstream or procyclic stages or after transfection and selection of drug resistance followed by re-cloning. Thus the karyotype changes observed in the metacyclic clones derived from the mixed infection cannot be due to alterations occurring during their vegetative expansion. Two metacyclic clones of STIB 247 from a tsetse fly infected only with that stock also show no alteration in karyotype. Equivalent data for STIB 386 also show no changes in karyotype (results not shown). As only three metacyclic clones of singly transmitted STIB 247 have been analysed for recombinant karyotypes, it was necessary to analyse further clones from singly transmitted stocks for evidence of recombination. We examined a series of metacyclic clones derived from stocks singly transmitted through tlies and each clone was screened for a series of markers which were heterozygous in the original clone used to infect the flies. Some of the data in this analysis have been reported previously [21] and are included here for completeness together with the new data and these are summarised in Table 3. None of the clones analysed showed any difference between the parental stock and the meta-
A. Tait et al. 1 Molecular and Biochemical Parasitology 76 (1996) 31-42
37
247
I
C
247 60
58
57
55 54
53
41
,
40 38
Fig. 3. Molecular karyotype of a parental and progeny clones determined by pulse field gel electrophoresis. Ethidium bromide stained gels showing the molecular karyotype of STIB 247 and the progeny clones 38, 40, 41, 53, 54, 55, 57, 58 and 60 under two different separation conditions are shown in (A) and (C). The sizes given are estimates (Mb) derived from comparison to standard yeast chromosomal markers (SC). The bands referred to in the text (Bl-8) are indicated as are the intermediate size chromosomes (int) and mini-chromosomes (m). Densitometric scans of negatives from photographs of tracks of the gels are shown in B and D. and the band numbers correspond to those shown in A and C.
38 a
A. Tait et al. / Molecular and Biochemical Parasitology 76 (1996) 31-42 247
58
57
54
53
b
d
247
60
58
57
247
58
57
54
53
247
60
84 B3 B2
55
e
58
57
55
B8 87
B5,6 Bit-4
Fig. 4. Identification of homologous chromosomes in STIB 247 and progeny clones 53, 54, 55, 57, 5s and 60. Southern blots of the pulse field gels (Fig. 3) were probed with either single copy genes (plc and tbcrk2) or a gene probe for the closely linked multicopy gene @gk). The panels show the Ethidium bromide stained gels (a) and (d) resulting from different separation conditions. Panels (b) and (c) are blots of gel (a) probed with plc and pgk gene probes respectively. Panel (e) is a blot of gel (d) probed for tbcrk2. The chromosome bands are labelled Bl -8 as described in the text.
A. Tait et al. / Molecular and Biochemical Parasitology 76 (1996) 31-42
39
Table 2 Phenotype, genotype and karyotype of ten progeny clones derived from the mixed infection of a tsetse fly with two different stocks of T. brucei Clone
Marker
Chromosomes
Tyr’ Parental 2-4 38 2-4 40 2-4 41 2-4 55 224 60 Recombinant 4 35 2-4 53 2-4 54 2 57 4 58 Original parent 241 2-4 The enzyme indicated as respectively. and absence
.
pBE9
1
2
3
4
I
8
+,‘+/+/+,:-
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+i-
+
+
+
+
+
+
nd
-l+i+/+I-
nd + ++ + ++
nd + _
nd _
+ _
nd ++ + + +
+ + +
nd + + ++ +
nd + + _
+I-
+
+
+
+
+
+
+
phenotype is described using the nomenclature shown in Table 1; the genotype determined by the probe pBE9 is described in Fig. 2. The pairs of homologous chromosomes which differ in size are indicated as: 1, 2; 3, 4; and 7, 8, The presence of a single copy of each is indicated as + while two copies of a single homologue are indicated as + + of a homologue by -.
cyclic clones derived after tsetse transmission, i.e., all the heterozygous loci remained heterozygous. The difference between the observation of recombinant parental types in the mixed transmission (Table 2) but the lack of such types in stocks transmitted singly (Table 3) raises the question of whether parental recombination only occurs in mixed transmissions. Table 3 appears to indicate that recombination is absent when stocks are transmitted singly but it can be argued that a number of these stocks (EATROs 605, 1125, 2340, 795 and F9/45mcl/lO) may be ‘sterile’ as they have never been used in genetic experiments. Using only the data from the known fertile stocks (STIB 247 and 386) provides too small a data set to enable meaningful statistical comparisons with the data from the mixed-transmission experiments to be made.
4. Discussion Analysis of a series of metacyclic clones from the infection of tsetse flies with two genetically
marked stocks, STIB 247 and 386, has shown that they can be divided into three classes on the basis of marker analysis. One class is typical of the products of mating between the two parental stocks as has been described previously [l-7]. The second class is identical to one of the parental clones (STIB 247) and can be interpreted as being the products of transmission without mating. Such clones have been observed in most crosses reported and form the basis for the conclusion that mating is non-obligatory [2-41. The third class is novel in terms of genotype and phenotype in that these clones are similar to the parental clone STIB 247 except that they are homozygous for one or more of the loci for which 247 is heterozygous. Karyotype analysis of these clones shows that some have apparently ‘lost’ one homologue of a chromosome but ‘gained’ an extra copy of the other homologue. From the marker and karyotype analysis it is clear that they have not arisen by any exchange of genetic information with the other parent, STIB 386. These observations raise the question of the mechanism by which these non-hybrid recombinants have arisen.
A. Tait et al. I Molecular and Biochemical Parasitology 76 (1996) 31-42
40
Two mechanisms can be considered. Firstly, the deletion of one homologue of a chromosome with a concomitant amplification of the remaining homologue. This mechanism would have to be meiosis-specific as the karyotype is stable at mitosis (Fig. 5). Secondly, the occurrence of self-fertilisation between individuals of STIB 247. This process, analogous to cross-fertilisation, would involve meiosis either before or after fusion of nuclei of the parental stock STIB 247 and lead to the segregation of alleles of a locus and homologues of each chromosome. As both mechanisms 247
b
PD
ml
m2
SC
sr
ml
pr
Mb
2.2
1.3
int
Table 3 Absence of evidence for self-fertilisation in lines of trypanosomes singly transmitted through tsetse flies Trypanosome stock/line
No. of metacyclic clones
No. of heterozygous loci screened
No. of potentially informative events
STIB 241 STIB 241” STIB 386 STIB 386 F9/45 mcl 10 EATRO 605 EATRO 605” EATRO 1125” EATRO 2340” EATRO 795”
3 4 4 4 10 4 9 1 1 5
3 2 1 2 2 I 3 1 6 6
9 8 4 8 20 28 27 1 6 30
Results from screening metacyclic clones from seven lines for markers for which they were heterozygous before cyclical transmission or in which homologous chromosomes of different size could be identified. No markers became homozygous in any metacyclic clone thus providing no evidence for self-fertilization.The number of informative events is the product of the numbers of clones and loci screened. “Indicates data given in Ref. [21]. Three STIB 247 clones were screened for chromosome homologue bands l/2, 3/4 and 7/8. Eight STIB 386 clones were screened for chromosome homologue bands 4/5. The clones resulting from cyclical transmission of the hybrid progeny clone F9/45mcllO [2] were screened for isoenzymes AP and ICD and nine clones of EATRO 605 were screened for Tyr3, AP and ICD.
m
Fig. 5. Stability of the molecular karyotype of STIB 247 after extensive passaging, transmission through tsetse flies or selection for drug resistance. Ethidium bromide stained pulse field gel separations of 247 (low passage): bp, bloodstream population after 50 passages in mice; pp. procyclic population after > 100 passages in in vitro culture; ml and m2, metacyclic clones after transmission of STIB 247 through tsetse flies; sr, bloodstream stage after selection for suramin and re-cloning of the drug resistant population; pr, cloned procyclic stage trypanosomes after transfection with the ble gene and selection on phleomycin; SC, yeast chromosomal markers, sizes of two chromosomes are indicated in megabases (Mb). The bands referred to in the text (Bl-8) are indicated together with the intermediate size chromosomes (int) and the minichromosomes (m). Different separation conditions were used in the left and right panels.
lead to an identical outcome, it is formally impossible to distinguish between the two. However, given that our previous data on the ability of three stocks to undergo genetic exchange in all three combinations, predict the occurrence of selffertilisation, we would favour self-fertilisation as the mechanism of generating the non-hybrid recombinants. The failure to detect self-fertilisation in previous crosses [2-71 probably results from a lack of analysis with informative markers. In this study, ten of the fifteen clones analysed in detail were recombinant (five each as a result of cross- and self-fertilisation), and five were identical to the parent STIB 247. It is instructive to consider these data in light of a Mendelian model of meiosis followed by fusion. (This model has
A. Tait et al. 1 Molecular and Biochemical Parasitology 76 (1996) 31-N
not yet been proven to apply to T. brucei but the data in this and our previous publications [2,4] are consistent with such a model). In such a model, the probability of the diploid meiotic products of a single nucleus fusing with each other is equal to that of them fusing to the meiotic products of the nucleus from another cell. Thus at any single locus 50% of the progeny of self-fertilisation will be identical to the original parental stock while 50% will be recombinant. Similar arguments apply in a model whereby fusion precedes meiosis, with which our data are also consistent. On the assumption of equal probability of fusion, our finding that half of the ‘parental’ progeny clones analysed in detail are recombinant (Table 2) suggests that meiosis occurs at high frequency. However, the occurence of clones with identical phenotypes, karyotypes and genotypes to the parental stock STIB 247 still support the conclusion that mating is non-obligatory. Our observation that recombinants are not found when progeny clones are examined after a single stock of trypanosomes has been used to infect tsetse flies suggests that the sexual process is only induced when two stocks interact with each other and can induce both self and cross-fertilisation. Establishing that T. brucei can undergo self- as well as cross-fertilisation when two stocks are present in a fly has implications for interpretation of field data. Selfing would be expected to lead to increases in homozygosity within a population leading to departures from Hardy-Weinberg equilibria and linkage disequilibria. The main evidence for the proposition of a clonal population structure in T. brucei has come from detection of such departures in population genetic data sets [9,10]. Conflicting conclusions have arisen, however, from analyses comparing field data sets to models in which all variation has been generated by mutation only [8,11- 131. An alternative view to these propositions of panmictic outbreeding or strict clonality is that T. brucei has an epidemic population structure, SenSu Ref. [12], whereby genetic exchange may be frequent but a particular clone may expand rapidly to dominate a population within a restricted ecological niche (defined either geographically, or by host or tsetse biology) [8,11- 131. Thus, we would envisage that in a
41
population where a particular genotype is favoured and predominates (an epidemic) most tsetse flies will be infected with a single stock and asexual reproduction will predominate. Where multiple genotypes occur in the population, however, tsetse flies will be infected with mixtures of stocks so that cross- and self-fertilisation occur allowing the generation of different combinations of alleles in both the homozygous and heterozygous state. Self-fertilisation provides a mechanism whereby the apparent conflict between reasonably frequent genetic recombination and failure to detect the predicted outcomes of such recombination (assuming random outbreeding) [9,10,13] can be resolved.
Acknowledgements
We would like to thank The Wellcome Trust and the UNDPjWORLD BANK/WHO Special Programme for Research and Training in Tropical Diseases for financial support. CMRT is a Royal Society University Research Fellow.
References
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42
A. Tait et al. 1 Molecular and Biochemical Parasitology 76 (1996) 31-42
[7] Gibson, W. and Whittington, H. (1993) Genetic exchange in Trypanosoma brucei: Selection of hybrid trypanosomes
by introduction of genes conferring drug resistance. Mol. Biochem. Parasitol. 60, 19-26. [8] Cibulskis, R.E. (1992) Genetic variation in Trypanosoma brucei and the epidemiology of sleeping sickness in the Lambwe Valley, Kenya. Parasitology 104, 99- 109. [9] Tibayrenc, M., Kjellberg, F. and Ayala, F.J. (1990) A clonal theory of parasitic protozoa: The population structures of Entamoeba, Giardia, Leishmania, Naegleria, Plasmodium, Trichomonas, and Trypanosoma and their medical and taxonomical consequences. Proc. Natl. Acad. Sci. USA 87, 2414-2418. [IO] Tibayrenc, M. and Ayala, F.J. (1991) Towards a population genetics of microorganisms: the clonal theory of parasite protozoa. Parasitol. Today 7, 228-232. [I 11 Cibulskis, R.E. (1988) Origins and organization of genetic diversity in natural populations of Trypanosoma brucei. Parasitology 96, 303-322. [12] Maynard-Smith, J.. Smith. N.H., O’Rourke, M. and Spratt, B.G. (1993) How clonal are bacteria? Proc. Natl. Acad. Sci. USA, 90, 4384-4388. [13] Hide, G., Welbum, SC., Tait, A. and Maudlin, I. (1994) Epidemiological relationships of Trypanosoma brucei stocks from South East Uganda: evidence for different population structures in human infective and non-human infective isolates. Parasitology 109, 95 - 111. [14] Hajduk, S.L., Cameron, C. R., Barry, J. D. and Vickerman, K. (1981) Antigenic variation in cyclically transmitTrypanosoma brucei. Variable antigen type ted
[15]
[16]
[17]
[18] [19]
[20]
[21]
composition of metacyclic trypanosome populations from the salivary glands of Glossina morsitans. Parasitology 83, 595-607. Tait, A.. Eldirdiri, A. and Le Ray. D. (1984) Enzyme variation in Trypanosoma brucei spp. Parasitology 89. 311-326. Carrington, M., Bulow, R., Remke, H. and Overath, P. (1989) Sequence and expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei. Mol. Biochem. Parasitol. 33, 289-296. Osinga. K.A., Swinkels, B.W., Gibson, W.C., Borst, P., Veeneman, G.H., Van Boom, J.H., Michels, P.A.M. and Opperdoes, F.R. (1985) Topogenesis of microbody enzymes: A sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei. EMBO J. 4, 381 l-3817. Mottram, J.C. and Smith, G (1995) A family of cdc2-related protein kinases. Gene 162, 147-152. Van der Ploeg, L.H.T., Smith, C.L., Polvere, R.I. and Gottesdiener, K.M. (1989) Improved separation of chromosome-sized DNA from Trypanosoma brucei, stock 42760. Nucleic Acids Res. 17, 3217-3227 Gottesdiener, K., Garcia-Auoveros. J., Lee, M.G. and Van der Ploeg, L.H.T. (1990) Chromosome organisation in the protozoan Tr.ypanosoma brucei. Mol. Cell Biol. 10, 6079-6083. Tait, A., Turner, C.M.R., Le Page, R.W.F. and Wells, J.M. (1989) Genetic evidence that metacyclic forms of Trypanosoma brucei are diploid. Mol. Biochem. Parasitol. 37, 2477256.
EmcAL PARAsIToLoGy Molecular and Biochemical Parasitology 76 (1996) 31-42
Self-fertilisation Andrew
Tait”, Norma
in Trypanosoma brucei
Buchanana,
Geof Hide”, C. Michael
R. Turnerb.*
aWellcome Unit oj‘ Molecular Parasitology, The Anderson College, Universityof’ Glasgoas,56 Dumbarron Road, Glasgow GII 6NU. UK
bI.B.L.S.-D.I.I.. Joseph Bluck Building. Unitlersitysf Glasgobts, GlasgovvGl2 8QQ. UK Received 27 July 1995; revised 2 November 1995; accepted 3 November 1995
Abstract We have investigated whether Trypanosoma brucei can undergo self-fertilisation. A group of 27 metacyclic clones derived from the tsetse transmission of a mixture of two genetically marked stocks was analysed and 22 clones were observed to be of non-hybrid phenotype. A group of 10 clones from this non-hybrid subset were then analysed for one isoenzyme, one restriction fragment length polymorphism and three karyotype markers potentially informative for the detection of self-fertilisation. Five of the 10 clones were found to be recombinant for at least one marker and we interpret these recombination events as indicating the clones to be products of self-fertilisation. We have also analysed a limited number of metacyclic clones from stocks of T. brucei each singly transmitted through tsetse flies but, so far, no evidence of recombination has been detected. We conclude that T. brucei is able to self-fertilise but there may be a requirement for the presence of dissimilar stocks to initiate such an event. Keywords: Trypanosoma brucei; Genetics;
Self-fertilisation;
Karyotype
1. Introduction
Genetic exchange in Trypanosoma brucei has been directly demonstrated by laboratory crosses
_~ Abbreuiutions: STIB, Swiss Tropical Institute Basel; EATRO, East African Trypanosomiasis Research Organisation; RFLP, restriction fragment length polymorphism; PFGE, pulsed field gel electrophoresis; AP, alkaline phosphatase; ICD, isocitrate dehydrogenase; ME,, malic enzyme B; Tyr3. tyrosyl tyrosyl tyrosine peptidase; p/c, phospholipase C gene: pgk. phosphoglycerate kinase gene. * Corresponding author. Tel.: + 44 141 330 6629; Fax: + 44 141 307 8016; E-mail: [email protected]. 0166~6851/96/$15.00
0 1996 Elsevier
SSDI 0166-6851(95)02528-L
Science
Ireland
Ltd. All rights
[I -71 but its contribution to the epidemiology of African trypanosomiasis in the field remains a matter of debate [&13]. By infecting tsetse flies with two genetically marked cloned stocks of trypanosomes, it has been shown that mating takes place during development in the fly and that a proportion of such mixed-infected flies produce metacyclic trypanosomes which are hybrid for the range of markers examined [l-7]. A proportion of the trypanosomes emerging from such flies are, however, parental in genotype and phenotype implying that the mating events are non-obligatory during the life-cycle of the trypanosome [271.
In
reserved
order
to
investigate
the
rules
and
32
A. Tait et al. I Molecular and Biochemical Parasitology 76 (1996) 31-42
mechanisms governing this system of genetic exchange crosses between three different stocks in all pairwise combinations have been undertaken and it was shown that Fl progeny clones were produced from each cross [4]. These observations indicate either that there are no mating barriers between stocks or that there is a mating type system but at least one of the stocks must be heterozygous for alleles at a postulated mating type locus. On either hypothesis, these results imply that T. brucei can self-fertilise, but to date no evidence has been presented which establishes whether this occurs. We have directly examined the question of selffertilisation using a series of trypanosome clones derived from metacyclic stage parasites produced from the infection of tsetse flies with either a single cloned trypanosome line or a mixture of two genetically marked cloned trypanosome lines. If self-fertilisation occurs, then half the progeny of such an event will be homozygous at a locus for which the parental cloned line was heterozygous, but will remain identical to the parental line for any marker which is homozygous. These predictions provide a simple screen for the occurrence of self-fertilisation by analysis of loci in the clones derived from single or mixed-stock infected tsetse flies. In this paper we report the marker analysis of these clones and provide strong evidence for the occurrence of self-fertilisation.
2. Materials and methods 2.1. Parasites
.
A genetic cross was conducted between cloned stocks, STIB (Swiss Tropical Institute Basel) 247 and STIB 386, by co-transmitting populations of these stocks through tsetse flies. The methods for growing trypanosome populations, cyclically transmitting them through tsetse flies and deriving metacyclic clones from a fly producing hybrid trypanosomes have been described previously [4,14]. A series of 27 metacyclic clones were derived from a single fly (F492) on day 50 after infection with both parents and each clone was assigned a unique number.
Populations of the following cloned trypanosome lines were each individually transmitted through tsetse flies and clones derived from metacyclic forms: STIB 247, STIB 386, EATRO (East African Trypanosomiasis Research Organisation) 605, EATRO 1125, EATRO 2340, EATRO 795, F9/45 mcll0. This last line is a hybrid progeny clone from a cross between stocks STIB 247 and STIB 386 [2]. 2.2. Enzyme electrophoresis The preparation of trypanosome lysates, the conditions for starch gel electrophoresis and the enzyme-specific staining procedures were as previously described [151. 2.3. Restriction fragment analyses
length polymorphism
The methods for preparing DNA probes from libraries and using them to detect restriction fragment length polymorphisms (RFLPs) have been described previously [l]. T. brucei genomic
2.4. Karyo type analyses Pulsed field gel electrophoresis (PFGE) was performed in a contour-clamped homogeneous electric field apparatus. Gels were poured at 1% agarose in 0.089 M Tris-borate, pH 7.6, 1 mM EDTA buffer and run in the same buffer at 3.0 V cm-’ and a temperature of 13°C using buffer recirculation. Two sets of pulse times were used (duration of each pulse time in hours given in brackets): A, 900 s (24) 750 s (24) 600 s (24), 500 s (24); B, 1200 s (24), 1100 s (30), 900 s (32), 800 s (24). After completion of the gel run, chromosome bands were stained in 1 pg ml - ’ of ethidium bromide, destained in running buffer and visualised on a UV-transilluminator. Ethidium bromide stained gels were photographed and the negatives scanned in a Joece-Loebl scanner at wavelength 530 nm. Chromosome blocks were prepared by embedding whole purified trypanosomes at a concentration of 0.5 x lo9 ml --I in 1% low melting agarose at 37°C in phosphatebuffered saline, pH 7.4, and casting in a mould at
A. Tait et al.
1Molecular and Biochemical Parasitology 76 (1996) 31-42
4°C. The blocks were deproteinised by resuspending in 0.5 M EDTA, 0.5 mg ml -’ proteinase K, 1% sodium sarkosyl, pH 9.0, at room temperature for 20 min followed by incubation at 56°C for 48 h. Blocks were washed 3 x in 50 mM EDTA, pH 8.0, and stored at 4°C in the same solution until use. Gels were blotted onto Hybond-N membranes and hybridised with a series of probes, labelled by random primer extension. Hybridisation was at 65°C and the filters were washed in 1 x SSC (0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0) at 65°C before exposing to X-ray film. Blots were reprobed under the same conditions, after stripping the blot by boiling in 1% SDS for 10 min. Probes for the T. brucei phospholipase C gene, plc [16], the phosphoglycerate kinase gene, pgk [171, and the tbcrk2 protein kinase gene [18] were prepared by gel purification of the insert from restriction digests of purified recombinant plasmids. Twenty-five pg of the insert was excised from the gel and labelled with 32P by random priming (Stratagene).
3. Results 3.1. Phenotypic analysis of the progeny of a mixed transmission
An analysis of a series of progeny clones derived from the co-transmission of two stocks (STIB 247 and STIB 386) through tsetse flies was undertaken. Twenty-seven clones were screened by analysis of isoenzyme markers to define their phenotype and the results are shown in Table 1. The clones can be divided into three classes: Class 1 is identical to the 247 parent (Pl), Class 2 is the equivalent of the Fl progeny obtained from the cross between 247 and 386 while Class 3 are recombinant at the single heterozygous locus for the tyrosyl-tyrosyl-tyrosine peptidase (Tyr3) but identical to the 247 parent for the remaining three homozygous markers (alkaline phosphatase, AP; isocitrate dehydrogenase, ICD; malic enzyme B, ME,). Fig. 1 illustrates the results for the enzymes ICD and Tyr3. These results suggest that, in addi-
33
tion to the expected Fl progeny (class 2) recombinants related to STIB 247 had been generated in this experiment. No clones of the parental 386 phenotype were obtained but we attach no particular significance to the disparity in numbers of clones of the two parents as such disparities have been regularly observed in previous co-transmission experiments [4]. To investigate further the occurrence of recombinant STIB 247 genotypes, a group of clones from the mixed transmission experiment showing either the 247 recombinant phenotype (clones 57 and 58) or the 247 parental phenotype (clones 38, 40, 41, 53, 54, 55, 60) were analysed for a further heterozygous marker and for their molecular karytope. 3.2. Restriction fragment length polymorphism anal_wes Restriction digests of DNA from clones 38, 40, 41, 53, 54, 55, 57, 58 and 60 were Southern blotted and the blots probed with the single copy gene probe, pBE9, for which the parent 247 is heterozygous for the presence/absence of a PstI site [l]. These results showed that clone 53 (Class 1 parental by isoenzymes) was homozygous for this RFLP while the remaining clones were heterozygous and identical to the parental stock 247 (Fig. 2, Table 2). 3.3. Karyotype anaIyses Previous studies have shown that some homologous chromosomes differ sufficiently in size to be separated by PFGE [19,20] and these size differences can be used as markers for chromosome segregation and recombination [5,6]. Chromosome separations, for the size range 700 kb to 2.5 Mb, of STIB 247 and nine progeny clones are illustrated in Fig. 3a. STIB 247 shows nine bands of DNA; the most rapidly migrating band constitutes the mini-chromosomes (m), followed by the intermediate chromosomes (int z 700 kb) and a further six bands: band 1 ( > 1.3 Mb), bands 2, 3 and 4 (2.0-2.5 Mb) and bands 5 and 6 (> 2.5 Mb). The molecular karyotypes of clones 53, 54 and 58 are different from that of the parent STIB
A. Tait et al. / Molecular and Biochemical Parasitology 76 (1996) 31-42
34 Table 1 Analysis of the enzyme and STIB 386) Clone
electrophoretic
Enzyme ICD
Parental (Class 1) 34 36 38 39 40 41 42 44 45 46 47 52 53 54 55 59 60 61 62 Hybrids (Class 2) 43 48 51 56 68 Recombinant (Class 35 57 58 Parents 247 (P 1) 386 (P 2)
phenotypes
of progeny
AP
1 I
I 1 1
I I 1 1 1 1 1
1 1 1 1 1
from a tsetse fly infected
with two stocks
(STIB 247
phenotype
1 1 1
clones derived
1 nd 1 1 1 1 1 1
Tyr3
ME,
2-4 2-4 2-4 224 2-4 224 2-4 2-4 224 2-4 2-4 224 2-4 224 224 2-4 224 224 224
3 3 3 3 3 3 3 3 nd 3 3 3 3 3 3 3 3 3 3 223 nd nd 223 2-3
1-2 l-2 l-2 l-2 l-2
l-2 nd nd l-2 1-2
2 4 4 2-4 2-4
1
1 1 1
4 2 4
1 2
2-4 2-4
3)
1 1
I 2
peptidase; ME,, ICD, isocitrate dehydrogenase; AP, alkaline phosphatase; Ty?, tyrosyl-tyrosyl-tyrosine determined. The numbers refer to allelic electrophoretic variants: homozygous phenotypes are indicated heterozygous phenotypes by two numbers linked by a dash.
247 (Fig. 3a) and show the loss of either band 2 (clones 54 and 58) or band 4 (clone 53) with an increase in the intensity of staining of band 1 (clones 54 and 58) or band 3 (clone 53) as confirmed by densitometer scans of photographic negatives of these gels (Fig. 3b). Alterations are also observed in chromosomes of larger size by using different electrophoretic conditions: in Fig. 3c, bands 7 and 8 ( z 2.8 Mb) are clearly resolved in
3 2-3 malic enzyme,; nd, not by a single number and
STIB 247 which are identical to those observed in eight of the nine clones examined. Clone 57, however, showed a loss of band 8 with band 7 showing an increased intensity of fluorescence (Fig. 3d). To determine the relationship between the chromosome bands separated by PFGE, Southern blots of the separations were hybridised with probes for two single copy genes (plc and tbcrk2)
A. Tait et al. / Molecular and Biochemical Parasitology 76 (1996) 31-42
O-
-0 1
Pl
l-2 HYB
35
o-
-0
2
1
2-4
2-4
P2
REC
Pl
P2
4
2 REC
Fig. 1. Isoenzyme phenotypes of parental and progeny clones from the mixed transmission of stocks STIB 247 through tsetse flies. The left panel shows results for isocitrate dehydrogenase (ICD) and the right panel shows results detected using tyrosyl-tyrosly-tyrosine as substrate (Tyr3): Pl, STIB 247 parent: P2, STIB 386 parent; HYB, hybrid 43; REC, non-hybrid recombinant clone 58 (Tyr3 - 4) or clone 57 (Ty?-2). The inferred allelic combinations to homozygous (1, 2 or 4) or heterozygous (l-2 or 2-4) patterns are also shown
and a closely linked multiple copy gene (‘pgk): the results are illustrated in Fig. 4. The plc gene probe hybridises with bands 1 and 2, the pgk gene probe hybridises with bands 3 and 4, while the tbcrk2 gene probe hybridises with bands 7 and 8 in separations of STIB 247 chromosomes. These results suggest that the pairs of bands 1 and 2, 3 and 4, and 7 and 8 each represent the two homologues of three different chromosomes in which the two homologues differ in size. In each progeny clone where a chromosome band has been lost, only the remaining homologue hybridises to the relevant probe, i.e., no change in chromosomal size has occurred (Fig. 4). Densitometric scans of
and STIB 386 for a peptidase progeny clone generate these
such blots show that the intensity of hybridisation to the single chromosome bands from clones which have lost the corresponding homologue is equal to the sum of the intensities of hybridisation to the two homologues in the parental stock 247 (data not shown). The data on the molecular karyotype of the nine progeny clones, show that five (clones 38, 40, 41, 55 and 60) are identical to the parental STIB 247 stock in respect to three pairs of resolvable homologous chromosomes while four (53, 54, 57 and 58) show the loss of one homologue of a pair but the apparent gain of the other homologue. A similar interpretation can be used to explain the
A. Tait et al. 1 Molecular and Biochemical Parasitology 76 (1996) 31-42
36
pBE9 kb
8.1
homozygous enzyme phenotype of clones 35, 57 and 58 and the homozygous genotype detected by probe pBE9 (which hybridises to the unresolved region of the PFGE separations) in clone 53. The data for all ten clones examined are summarised in Table 2; five clones are identical by all criteria to the original parental STIB 247 stock while the other five (35, 53, 54, 57, 58) are recombinant for one or more markers. 3.4. Analysis of stocks transmitted singly through P ies
I.9
247
53
247
P 53
1 )
P 10.0 I
Fig. 2. Genotype of a parental and a recombinant clone determined by Southern blotting. PstI digests of STIB 247, probed with pBE9, show three fragments of 10, 8.1 and 1.9 kb which are interpreted as being due to the presence of a polymorphic PstI site within the IO-kb fragment for which 247 is heterozygous ( + / - ) while recombinant clone 53 shows a homozygous pattern ( - / - ). The sizes of the restriction fragments were determined using DNA size markers. The genomic organisation of the fragments detected by this probe (denoted by bar) are indicated diagrammatically below the blot.
The finding of novel recombinant karyotypes derived from a single parental stock in a mixed transmission experiment raised the question as to whether stocks could also undergo this process when singly transmitted through flies or after prolonged growth as bloodstream or procyclic stage trypanosomes. The karyotype of a series of derivatives of STIB 247 was therefore determined and the results are illustrated in Fig. 5. Clearly, the karyotype is stable to prolonged growth in either bloodstream or procyclic stages or after transfection and selection of drug resistance followed by re-cloning. Thus the karyotype changes observed in the metacyclic clones derived from the mixed infection cannot be due to alterations occurring during their vegetative expansion. Two metacyclic clones of STIB 247 from a tsetse fly infected only with that stock also show no alteration in karyotype. Equivalent data for STIB 386 also show no changes in karyotype (results not shown). As only three metacyclic clones of singly transmitted STIB 247 have been analysed for recombinant karyotypes, it was necessary to analyse further clones from singly transmitted stocks for evidence of recombination. We examined a series of metacyclic clones derived from stocks singly transmitted through tlies and each clone was screened for a series of markers which were heterozygous in the original clone used to infect the flies. Some of the data in this analysis have been reported previously [21] and are included here for completeness together with the new data and these are summarised in Table 3. None of the clones analysed showed any difference between the parental stock and the meta-
A. Tait et al. 1 Molecular and Biochemical Parasitology 76 (1996) 31-42
37
247
I
C
247 60
58
57
55 54
53
41
,
40 38
Fig. 3. Molecular karyotype of a parental and progeny clones determined by pulse field gel electrophoresis. Ethidium bromide stained gels showing the molecular karyotype of STIB 247 and the progeny clones 38, 40, 41, 53, 54, 55, 57, 58 and 60 under two different separation conditions are shown in (A) and (C). The sizes given are estimates (Mb) derived from comparison to standard yeast chromosomal markers (SC). The bands referred to in the text (Bl-8) are indicated as are the intermediate size chromosomes (int) and mini-chromosomes (m). Densitometric scans of negatives from photographs of tracks of the gels are shown in B and D. and the band numbers correspond to those shown in A and C.
38 a
A. Tait et al. / Molecular and Biochemical Parasitology 76 (1996) 31-42 247
58
57
54
53
b
d
247
60
58
57
247
58
57
54
53
247
60
84 B3 B2
55
e
58
57
55
B8 87
B5,6 Bit-4
Fig. 4. Identification of homologous chromosomes in STIB 247 and progeny clones 53, 54, 55, 57, 5s and 60. Southern blots of the pulse field gels (Fig. 3) were probed with either single copy genes (plc and tbcrk2) or a gene probe for the closely linked multicopy gene @gk). The panels show the Ethidium bromide stained gels (a) and (d) resulting from different separation conditions. Panels (b) and (c) are blots of gel (a) probed with plc and pgk gene probes respectively. Panel (e) is a blot of gel (d) probed for tbcrk2. The chromosome bands are labelled Bl -8 as described in the text.
A. Tait et al. / Molecular and Biochemical Parasitology 76 (1996) 31-42
39
Table 2 Phenotype, genotype and karyotype of ten progeny clones derived from the mixed infection of a tsetse fly with two different stocks of T. brucei Clone
Marker
Chromosomes
Tyr’ Parental 2-4 38 2-4 40 2-4 41 2-4 55 224 60 Recombinant 4 35 2-4 53 2-4 54 2 57 4 58 Original parent 241 2-4 The enzyme indicated as respectively. and absence
.
pBE9
1
2
3
4
I
8
+,‘+/+/+,:-
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+i-
+
+
+
+
+
+
nd
-l+i+/+I-
nd + ++ + ++
nd + _
nd _
+ _
nd ++ + + +
+ + +
nd + + ++ +
nd + + _
+I-
+
+
+
+
+
+
+
phenotype is described using the nomenclature shown in Table 1; the genotype determined by the probe pBE9 is described in Fig. 2. The pairs of homologous chromosomes which differ in size are indicated as: 1, 2; 3, 4; and 7, 8, The presence of a single copy of each is indicated as + while two copies of a single homologue are indicated as + + of a homologue by -.
cyclic clones derived after tsetse transmission, i.e., all the heterozygous loci remained heterozygous. The difference between the observation of recombinant parental types in the mixed transmission (Table 2) but the lack of such types in stocks transmitted singly (Table 3) raises the question of whether parental recombination only occurs in mixed transmissions. Table 3 appears to indicate that recombination is absent when stocks are transmitted singly but it can be argued that a number of these stocks (EATROs 605, 1125, 2340, 795 and F9/45mcl/lO) may be ‘sterile’ as they have never been used in genetic experiments. Using only the data from the known fertile stocks (STIB 247 and 386) provides too small a data set to enable meaningful statistical comparisons with the data from the mixed-transmission experiments to be made.
4. Discussion Analysis of a series of metacyclic clones from the infection of tsetse flies with two genetically
marked stocks, STIB 247 and 386, has shown that they can be divided into three classes on the basis of marker analysis. One class is typical of the products of mating between the two parental stocks as has been described previously [l-7]. The second class is identical to one of the parental clones (STIB 247) and can be interpreted as being the products of transmission without mating. Such clones have been observed in most crosses reported and form the basis for the conclusion that mating is non-obligatory [2-41. The third class is novel in terms of genotype and phenotype in that these clones are similar to the parental clone STIB 247 except that they are homozygous for one or more of the loci for which 247 is heterozygous. Karyotype analysis of these clones shows that some have apparently ‘lost’ one homologue of a chromosome but ‘gained’ an extra copy of the other homologue. From the marker and karyotype analysis it is clear that they have not arisen by any exchange of genetic information with the other parent, STIB 386. These observations raise the question of the mechanism by which these non-hybrid recombinants have arisen.
A. Tait et al. I Molecular and Biochemical Parasitology 76 (1996) 31-42
40
Two mechanisms can be considered. Firstly, the deletion of one homologue of a chromosome with a concomitant amplification of the remaining homologue. This mechanism would have to be meiosis-specific as the karyotype is stable at mitosis (Fig. 5). Secondly, the occurrence of self-fertilisation between individuals of STIB 247. This process, analogous to cross-fertilisation, would involve meiosis either before or after fusion of nuclei of the parental stock STIB 247 and lead to the segregation of alleles of a locus and homologues of each chromosome. As both mechanisms 247
b
PD
ml
m2
SC
sr
ml
pr
Mb
2.2
1.3
int
Table 3 Absence of evidence for self-fertilisation in lines of trypanosomes singly transmitted through tsetse flies Trypanosome stock/line
No. of metacyclic clones
No. of heterozygous loci screened
No. of potentially informative events
STIB 241 STIB 241” STIB 386 STIB 386 F9/45 mcl 10 EATRO 605 EATRO 605” EATRO 1125” EATRO 2340” EATRO 795”
3 4 4 4 10 4 9 1 1 5
3 2 1 2 2 I 3 1 6 6
9 8 4 8 20 28 27 1 6 30
Results from screening metacyclic clones from seven lines for markers for which they were heterozygous before cyclical transmission or in which homologous chromosomes of different size could be identified. No markers became homozygous in any metacyclic clone thus providing no evidence for self-fertilization.The number of informative events is the product of the numbers of clones and loci screened. “Indicates data given in Ref. [21]. Three STIB 247 clones were screened for chromosome homologue bands l/2, 3/4 and 7/8. Eight STIB 386 clones were screened for chromosome homologue bands 4/5. The clones resulting from cyclical transmission of the hybrid progeny clone F9/45mcllO [2] were screened for isoenzymes AP and ICD and nine clones of EATRO 605 were screened for Tyr3, AP and ICD.
m
Fig. 5. Stability of the molecular karyotype of STIB 247 after extensive passaging, transmission through tsetse flies or selection for drug resistance. Ethidium bromide stained pulse field gel separations of 247 (low passage): bp, bloodstream population after 50 passages in mice; pp. procyclic population after > 100 passages in in vitro culture; ml and m2, metacyclic clones after transmission of STIB 247 through tsetse flies; sr, bloodstream stage after selection for suramin and re-cloning of the drug resistant population; pr, cloned procyclic stage trypanosomes after transfection with the ble gene and selection on phleomycin; SC, yeast chromosomal markers, sizes of two chromosomes are indicated in megabases (Mb). The bands referred to in the text (Bl-8) are indicated together with the intermediate size chromosomes (int) and the minichromosomes (m). Different separation conditions were used in the left and right panels.
lead to an identical outcome, it is formally impossible to distinguish between the two. However, given that our previous data on the ability of three stocks to undergo genetic exchange in all three combinations, predict the occurrence of selffertilisation, we would favour self-fertilisation as the mechanism of generating the non-hybrid recombinants. The failure to detect self-fertilisation in previous crosses [2-71 probably results from a lack of analysis with informative markers. In this study, ten of the fifteen clones analysed in detail were recombinant (five each as a result of cross- and self-fertilisation), and five were identical to the parent STIB 247. It is instructive to consider these data in light of a Mendelian model of meiosis followed by fusion. (This model has
A. Tait et al. 1 Molecular and Biochemical Parasitology 76 (1996) 31-N
not yet been proven to apply to T. brucei but the data in this and our previous publications [2,4] are consistent with such a model). In such a model, the probability of the diploid meiotic products of a single nucleus fusing with each other is equal to that of them fusing to the meiotic products of the nucleus from another cell. Thus at any single locus 50% of the progeny of self-fertilisation will be identical to the original parental stock while 50% will be recombinant. Similar arguments apply in a model whereby fusion precedes meiosis, with which our data are also consistent. On the assumption of equal probability of fusion, our finding that half of the ‘parental’ progeny clones analysed in detail are recombinant (Table 2) suggests that meiosis occurs at high frequency. However, the occurence of clones with identical phenotypes, karyotypes and genotypes to the parental stock STIB 247 still support the conclusion that mating is non-obligatory. Our observation that recombinants are not found when progeny clones are examined after a single stock of trypanosomes has been used to infect tsetse flies suggests that the sexual process is only induced when two stocks interact with each other and can induce both self and cross-fertilisation. Establishing that T. brucei can undergo self- as well as cross-fertilisation when two stocks are present in a fly has implications for interpretation of field data. Selfing would be expected to lead to increases in homozygosity within a population leading to departures from Hardy-Weinberg equilibria and linkage disequilibria. The main evidence for the proposition of a clonal population structure in T. brucei has come from detection of such departures in population genetic data sets [9,10]. Conflicting conclusions have arisen, however, from analyses comparing field data sets to models in which all variation has been generated by mutation only [8,11- 131. An alternative view to these propositions of panmictic outbreeding or strict clonality is that T. brucei has an epidemic population structure, SenSu Ref. [12], whereby genetic exchange may be frequent but a particular clone may expand rapidly to dominate a population within a restricted ecological niche (defined either geographically, or by host or tsetse biology) [8,11- 131. Thus, we would envisage that in a
41
population where a particular genotype is favoured and predominates (an epidemic) most tsetse flies will be infected with a single stock and asexual reproduction will predominate. Where multiple genotypes occur in the population, however, tsetse flies will be infected with mixtures of stocks so that cross- and self-fertilisation occur allowing the generation of different combinations of alleles in both the homozygous and heterozygous state. Self-fertilisation provides a mechanism whereby the apparent conflict between reasonably frequent genetic recombination and failure to detect the predicted outcomes of such recombination (assuming random outbreeding) [9,10,13] can be resolved.
Acknowledgements
We would like to thank The Wellcome Trust and the UNDPjWORLD BANK/WHO Special Programme for Research and Training in Tropical Diseases for financial support. CMRT is a Royal Society University Research Fellow.
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