Download lntraclonal mating in Trypanosoma brucei is

Microbiology (1997), 143, 909-920
Printed in Great Britain
lntraclonal mating in Trypanosoma brucei is
associated with out-crossing
Wendy Gibson,’ Kathleen Winters,’ Ginny Mizen,’ Julia Kearns’
and Mick Bailey3
Author for correspondence : Wendy Gibson. Tel :
e-mail: [email protected]
1
Department of Genetics,
University of Leicester,
Leicester LE1 7RH, UK
2.3
Department of Pathology
and Microbiology,z and
Department of Clinical
Veterinary Science,3
University of Bristol
Veterinary School,
Langford, Bristol
BS18 7DU, UK
+44 116 252 2956. Fax : + 44 116 252 3378.
To examine whether mating can occur within as well as between clones of
Trypanosoma brucei, w e transformed three T. brucei subspecies stocks with
heterologous genes conferring resistance to either hygromycin or Geneticin
and carried out a series of inter- and intraclone matings in all possible double
drug combinations. Double drug-resistant hybrids were recovered from three
of the six out-crosses, but not from any of the three intraclone matings.
However, further analysis of cloned progeny trypanosomes from one of the
out-crosses using RFLP markers, molecular karyotyping and RAPD (random
amplification of polymorphic DNA) produced unequivocal evidence that intraas well as interclone mating had occurred. The progeny of interclone mating
were double drug-resistant and heterozygous a t 9 of 13 loci examined. In
contrast, the progeny of intraclone mating had no demonstrable input of
genetic material from the hygromycin-resistant parent and were similar to the
Geneticin-resistant parent for most markers, except for five loci which were
heterozygous in the Geneticin-resistant parent but homozygous in these clones
(aldolase, THTl glucose transporter, procyclin, tubulin and cDNA 23). In
addition, PFGE showed considerable karyotypic rearrangements in these clones
and loss of genetic material was evident from RAPD and VSG (variant surface
glycoprotein) gene fingerprint analysis. We conclude that intraclone mating
can occur in trypanosomes, but only during out-crossing, suggesting that
meiosis and/or fusion are triggered by a diffusible factor.
Keywords : Trypanosoma brucei, genetic exchange, transformation, trisomy, selfing
INTRODUCTION
Trypanosoma brucei is a protozoan parasite which
causes human and animal trypanosomiasis in tropical
Africa and is transmitted by bloodsucking tsetse flies
(genus Glossina). Genetic exchange was first demonstrated by Jenni et al. (1986) after cotransmission of two
different trypanosome clones through tsetse flies. In this
first successful laboratory cross and several subsequent
ones (Gibson, 1989; Turner et al., 1990; Gibson &
Abbreviations: hph, hygromycin phosphotransferase gene; neo, neomycin phosphotransferase gene; H, hygromycin-B-resistant; N, Geneticin
(G418)-resistant; ALD, aldolase; GAPDH, glyceraldehyde phosphate dehydrogenase; GPI, glucose phosphate isomerase; PARP,, procyclic acidic
repetitive protein; PGK, phosphoglycerate kinase; PLC, phospholipase C;
PYK, pyruvate kinase; RAPD, random amplification of polymorphic DNA;
RNAP, RNA polymerase; SSC, standard saline citrate; TIM, triose phosphate
isomerase; VSG, variant surface glycoprotein.
0002-1012 0 1997 SGM
Garside, 1991; Schweizer et al., 1994; Degen et al.,
1995), hybrid trypanosomes were detected by laboriously searching through cloned trypanosomes for those
with a non-parental phenotype or genotype. The development of methods for the stable transformation of
trypanosomes with heterologous genes (Ten Asbroek et
al., 1990; Lee & Van der Ploeg, 1991; Eid & SollnerWebb, 1991) led to a new approach: incorporation of
selectable markers for drug resistance into the parental
trypanosome clones and selection of hybrid trypanosomes by double drug resistance (Gibson &
Whittington, 1993). This methodology not only
facilitates the recovery of large numbers of hybrid
genotypes, but also enables rare genetic exchange events
to be detected. Using this approach we previously
achieved one successful backcross in a series of experimental back and F1 crosses (Gibson et al., 1995) in
an attempt to define the limits of genetic exchange in
trypanosomes.
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909
W. G I B S O N a n d OTHERS
Extensive comparisons of parental and hybrid clones by
a number of genetic techniques have elucidated many
aspects of the mechanism of genetic exchange in
trypanosomes. The process is non-obligatory, with both
parentals and hybrid clones represented among the
infective salivary gland forms (metacyclics) produced by
an individual fly (Schweizer et al., 1988). T . 6rucei is
diploid throughout its life cycle and no haploid gamete
stage has been detected (Shapiro et al., 1984; Tait et al.,
1989; Kooy et al., 1989), although the fleeting or rare
occurrence of a haploid life cycle stage cannot be ruled
out. Analysis of isoenzymes and RFLPs indicates that
a meiotic division probably occurs during genetic
exchange : segregation and reassortment of genetic
markers is observed (Jenni et al., 1986; Paindavoine et
al., 1986; Wells et al., 1987; Gibson, 1989; Sternberg et
al., 1989; Turner et al., 1990; Gibson & Garside, 1991;
Gibson et al., 1992,1995; Gibson & Whittington, 1993;
Schweizer et al., 1994; Gibson & Bailey, 1994; Degen et
al., 1995) and also a high frequency of chromosomal
recombination in hybrids (Gibson et al., 1992; Gibson
& Bailey, 1994). However, hybrids with a 3n DNA
content have been found in four of five crosses for which
DNA contents were measured (Paindavoine et al., 1986;
Wells et al., 1987; Gibson et al., 1992, 1995; Gibson &
Bailey, 1994), including two early crosses which did not
employ selectable markers. These 3n hybrids have been
interpreted as triploid, since DNA contents of independent clones clustered at 3n, rather than a range of
intermediate values, and in addition, two clones
analysed in depth showed trisomy for several chromosomes (Gibson et al., 1992; Gibson & Bailey, 1994).
That triploidy is a frequent error of trypanosome genetic
exchange indicates a mechanism involving meiosis
followed by fusion of multiple haploid nuclei or haploid
and diploid nuclei. Analysis of the inheritance of
kinetoplast DNA (kDNA), which is the mitochondrial
DNA of trypanosomes, has shown that one DNA
component (maxicircles) is inherited uniparentally,
while the other (minicircles) is inherited from both
parents (Gibson & Garside, 1990). The generation of
hybrid kDNA networks implies that both cellular and
mitochondrial fusion occur during genetic exchange. All
the above experimental observations have been consolidated into a model in which trypanosomes undergo
cell fusion and meiosis (not necessarily in that order),
followed by fusion of (normally) two haploid nuclei, the
rest disintegrating (Gibson, 1995).
N o sex or mating type barriers to genetic exchange in
trypanosomes have yet been demonstrated. A three-way
cross ruled out the possibility of a simple two sex mating
system (Turner et al., 1990) and all subspecies and
subgroups within T . brucei, except Type I T . 6 .
gambiense, have been successfully mated (Jenni et al.,
1986; Gibson, 1989; Turner et al., 1990; Gibson &
Garside, 1991; Schweizer et al., 1994; Degen et al.,
1995). Other lower eukaryotes have multiple mating
types, determined by loci which encode or control
production of specific pheromones ; such systems ensure
that mating occurs only between organisms of different
910
mating types and hence favour out-crossing. The question remains whether trypanosomes have mating types
and can thus recognize 'strangers'. If incapable of
distinguishing self from non-self, then intraclone mating
should occur. Experiments in which clones heterozygous
for isoenzyme markers were singly transmitted through
tsetse provided no evidence of intraclone mating, i.e.
conversion of heterozygous markers to homozygosity
(Tait et al., 1989). However, putative products of self
fertilization were found among hybrid trypanosomes
from an experimental cross: five clones, which were
identical to parent A for homozygous markers, showed
homozygosity at one or more loci where parent A was
heterozygous (Tait et al., 1993, 1996). These results
tentatively suggested that intraclone mating could occur
among out-crossing trypanosomes.
Here we have searched for intraclone mating events by
incorporating drug resistance markers into otherwise
identical clones of the same trypanosome stock and
selecting for double drug-resistant hybrids following
cotransmission through the tsetse fly.
METHODS
Trypanosomes. The cloned transformants used for crossing
are listed in Table 1. Hygromycin resistance (H) was obtained
by transformation with plasmid construct pBnsp-H-T (Lee &
Van der Ploeg, 1991) carrying the hph gene and Geneticin (Life
Technologies) resistance (N) by transformation with plasmid
construct pUCTbneo3 (Ten Asbroek et al., 1990) carrying the
neo gene using the procedures previously described (Gibson &
Whittington, 1993). Each antibiotic resistance gene was
integrated into one of the two allelic tubulin gene clusters by
homologous recombination.
Crosses. All pairwise H x N combinations of the six cloned
transformants were cotransmitted through tsetse flies as
described previously (Gibson, 1989) (Table 2). In addition, the
six transformants were singly transmitted during the same
experimental series ; one batch of cryopreserved bloodstream
form stabilates was used for all infective feeds of each clone.
Each cage of infected flies (35-45 male Glossina mousitans)
was maintained on membrane-fed sterile horse blood, except
for a weekly feed on two clean, anaesthetized (Hypnorm,
1.2 ml kg-' i.p.) mice, commencing 16-17 d after the infective
feed. These mice were monitored for parasitaemia ; trypanosomes were harvested when numbers were > 10' (ml blood)-'
and transformed to procyclics in Cunningham's medium
(Cunningham, 1977) with 20 mM HEPES (CM) and 20 pg
gentamycin ml-' ;these procyclic populations were then tested
for drug resistance as below. Flies were finally dissected 8
weeks after infection. Infected salivary glands were inoculated
into immunosuppressed mice (200 mg cyclophosphamide
kg-') and the corresponding infected midguts were cultured in
CM with 100 pg gentamycin ml-'. Bloodstream forms from
infected mice were harvested when parasitaemia exceeded 10'
(ml blood)-' and were transformed to procyclics in CM.
Drug tests. Midgut procyclics were tested for resistance to
Geneticin and/or hygromycin at 50 pg ml-' in CM in a
microtitre plate format. Bloodstream forms from mice were
transformed to procyclics and tested for drug resistance in 24well plates in the same way. Plates were generally examined
every 1 or 2 d for 2 weeks and individual wells passaged as
necessary.
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Intraclonal mating in Trypanosoma brucei
Table 1. Transformants
Stock
Cloned
transformants
Day of first
appearance of
infective forms
058H
058N
TH2H
TH2N
KP2H
KP2N
17
29
16
16
23
16
T . b. rhodesiense
MHOM/ZM/74/58 [CLONE B]
T . b. gambiense
MHOM/CI/78/TH2-78E (020)
T. b. brucei
GPAP/CI/82/KP2-1 [CLONE 231
Reference
Gibson (1989)
Mehlitz et al. (1982)
Letch (1984)
Table 2. Results of experimental crosses
Day of first appearance of
infective forms
No. of infections in fly salivary glands at
dissection (56-60 d)
H
N
Hybrid
HxN
H
Interclone
A 058H x KP2N
B 058H x TH2N
C TH2H x KP2N
D TH2H x 058N
E KP2H x 058N
F KP2H x TH2N
23
17
15
23
29
23
23
16
15
30
36
16
37
30
29
Intraclone
G 058H x 058N
H TH2H x TH2N
I KP2H x KP2N
23
16
36
23
16
22
Cross
N
Mix
H+N
Hybrid
HxN
Total
10
-
-
-
10
1
1
1
11
12
3
1
4
9
-
2
-
-
-
-
-
Flow cytometric analysis. Samples of procyclics were fixed,
stained and analysed using an EPICS-CS flow cytometer
(Coulter Electronics) as described previously (Gibson et al.,
1992) using a quartz tip to stabilize the particle stream. The
DNA contents of 12 clones from cross B were measured
relative to parental clones 058H and TH2N prepared at the
same time. Each sample was analysed three times, giving a
cumulative total of 90000 trypanosomes of each clone.
Analysis of trypanosome samples. Samples for PFGE were
made by lysing and deproteinizing trypanosomes in situ in
agarose blocks as described by Van der Ploeg et al. (1984).
Samples of DNA for restriction analysis were prepared by
standard methods (Van der Ploeg et al., 1982). Genomic
DNAs were restricted and size-fractionated using 0.7 or 1.5%
(w/v) agarose gels. Size separation of chromosomal DNAs
was carried out using an LKB Pulsaphor system with a
hexagonal electrode array and five-phase program (Gibson et
al., 1992) or a Biorad CHEF DR I11 system using a series of
ramped pulses from 60 to 1800 s.
Gels were blotted in the standard way (Southern, 1975) and
nitrocellulose filters were hybridized as previously described
(Gibson et al., 1988) with the following oligo-labelled DNA
probes : (1)neo gene amplified from plasmid pg3neo by PCR ;
(2) hph gene HindIII-ScaI fragment containing the gene from
plasmid pBabe; (3)p-tubulin, insert from cDNA clone pTbpTC2 (Thomashow et al., 1983); (4) variant surface glycoprotein
1
-
-
1
4
8
-
-
-
-
1
-
1
1
-
-
-
4
6
3
(VSG) 117, 1-5kb PstI fragment from cDNA clone TcV-117.5
(Bernardset al., 1981) ; ( 5 )purified 12 kb EcoRI fragment from
kinetoplast maxi-circle DNA of T . b. rhodesiense ;(6)phosphoglycerate kinase (PGK), 1.3 kb PCR-amplified insert from
cDNA pTcPGK8 (Osinga et al., 1985) ; (7) glucose phosphate
isomerase (GPI), 1-9kb HindIII-BamHI fragment from
genomic clone (Marchand et al., 1989) ; (8) pyruvate kinase
(PYK), 1.2 kb PstI-EcoRI fragment from genomic DNA clone
pTgPK5 (Allert et al., 1991) ; (9) cytosolic glyceraldehyde
phosphate dehydrogenase (cGAPDH), 1.7 kb BamHI-PstI
fragment from genomic DNA clone pTgGAPc6 (Michels et
al., 1991); (10) aldolase (ALD), 1.3 kb PCR-amplified insert
from cDNA pTcALD7 (Marchand et al., 1988); (11) triose
phosphate isomerase (TIM),460 bp amplified fragment spanning polymorphic KpnI site within gene from genomic clone
pTgTIM12 (Swinkels et al., 1986); (12) 650 bp insert from
cDNA 23 (De Lange et al., 1984); (13) 760 bp insert from
cDNA 8 (ibid.); (14) 180 bp PCR-amplified fragment from
clone puc-THT1-Tag spanning 5’ region of T H T l glucose
transporter gene (Bringaud & Baltz, 1994); (15) 300 bp PCRamplified insert from cDNA clone 116 (gift from R. A. S.
Abdelhalim & K. M. Hudson, Brunel University) ; (16) 2 kb
insert from procyclin (or procyclic acidic repetitive protein,
PARP) cDNA clone pPR02001 (Konig et al., 1989); (17) 2 kb
PvuII fragment Pap5 cloned from and unique to downstream
region of proA procyclin locus (subsequently designated PARP
B) (ibid.) ; (18) 1.4 kb insert from phospholipase C (PLC) gene
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91 1
W. G I B S O N a n d O T H E R S
-
from clone pDH4 (Mensa-Wilmot et al., 1990); (19) 8 kb
insert from clone Trp6 containing the RNA polymerase
(RNAP) I11 gene from T . brucei (Kock et al., 1988). All posthybridizational washes were to 0.1 x SSC (1x SSC = 0.15 M
NaCI, 0.015 M sodium citrate) at 65 "C, except for probe 4
where washes were to 3 x SSC only.
Random amplified polymorphic DNA analysis. Genomic
DNAs were used as substrate for random amplification of
polymorphic DNA (Welsh & McClelland, 1990; Williams
et al., 1990) using eight different arbitrary primers, which
produce multi-banded patterns with T . brucei subspecies
(Tibayrenc et al., 1993). Primers were as follows 5'-3': 1,
CAGGCCCTTC; 2, TGCCGAGCTG; 4, AATCGGGCTG;
5 , AGGGGTCTTG; 7, GAAACGGGTG; 8, GTGACGTAGC; 9, GGTAACGCC; 10, GTGATCGCAG. Amplification was carried out using approximately 25 ng DNA and
primers at a final concentration of 0.2 pM using a programme
of 94 "C/ 180 s followed by 35 cycles of 94 "C/60 s, 36 OC/60 s,
72 "C/120 s.
RESULTS
Experimenta I crosses
Six inter- and three intraclone mating experiments were
carried out (Table 2). Three of the six out-crosses (A, B,
C) produced double drug-resistant (i.e. hybrid) progeny.
Trypanosomes with single drug resistance only
(assumed to be parentals) were found in the other six
crosses attempted, including the three intraclone
matings. In the three successful out-crosses, hybrids
were found among trypanosomes derived from the
salivary glands but not midguts of individual flies. This
brings the total number of crosses in which hybrids have
been found only in the salivary glands to five and
strengthens the view that the salivary glands rather than
the midgut are the site of genetic exchange (Gibson &
Whittington, 1993; Gibson et al., 1995).
Flies were dissected a t 8 weeks to allow maximum time
for hybrid formation (Schweizer et al., 1988), although
survival of older flies was poor, with typically 50%
mortality by the end of the experiment. T o monitor the
development of infectivity, flies were fed on clean
anaesthetized mice weekly from approximately 2 weeks
post-infection. The resultant trypanosome populations
from cotransmission experiments were drug tested to
ascertain the time of first appearance of double drugresistant trypanosomes. In the single transmissions, four
of the transformants produced infective forms by day
16/17 (Table 1); two transformants were much later
however, maturing by day 23 (KP2H) or day 29 (058N).
These transformants were also later maturing in the
mixed transmissions (KP2H, day 23-36; 058N, day
23-30; other transformants, day 15-23) and neither was
successfully crossed, possibly for this reason. Double
drug-resistant hybrids were first detected on day 29/30
for crosses B and C, and on day 37 for cross A (Table 2).
From flies dissected at 8 weeks, the majority of midguts
had mixed infections of both parental trypanosomes as
judged by drug resistance tests (not shown). However,
the majority of the corresponding salivary glands
produced uniparental infections when drug tested
91 2
Fig. 1. Ethidium-stained PFGE karyotypes of parents (Pl, P2)
and progeny clones from cross B. Approximate sizes were
estimated from yeast and bacteriophage ;L markers.
(Table 2). In particular, TH2N was very dominant,
coming through by itself in 23 of 27 flies with mature
infections in crosses B, F and H. There were no
noticeable differences between the parental clones, i n
terms of infectivity and virulence to mice or ability to
transform to procyclics and grow in vitro, which would
explain the predominance of one or other parent in the
drug tested procyclic populations. The results can
therefore be taken to reflect the true composition of the
salivary gland populations.
Analysis of progeny
For crosses A-C, procyclics were cloned from cryopreserved aliquots of the original unselected and double
drug-selected wells from the drug test plates; these
trypanosome populations had been cultivated in mice
and subsequently in procyclic culture for a combined
period of 18-20 d following fly dissection. Each clone
was drug tested and a PFGE sample made to compare
the molecular karyotype with those of the appropriate
parental clones by PFGE. The double drug-resistant
clones had PFGE karyotypes which were composed of
chromosome bands from both parents, thus verifying
their hybrid status. In Fig. 1, clones 7-12 also have 3
non-parental band of approximately 1 Mb.
Progeny from cross B were examined further, since it
involved a new combination of parental strains; parental
transformants TH2N and 058H will be referred to as P1
and P2, respectively, in the following. Twelve clones
were isolated in all, six of which from the unselected
population were resistant to Geneticin only, the remainder from the selected population being double
drug-resistant as expected. Of the six Geneticin-resistant
(N) clones, five were found to have non-parental
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Intraclonal mating in Trypanosonia bnicei
.4
Progeny
clones
I
I
I
100
72
,
THZN
058H
I
200
m i
.8
.lo
.6
85
Parents
0
compared to hybrid progeny clones in previous crosses
(Gibson & Garside, 1991; Gibson et al., 1992, 1995;
Gibson & Bailey, 1994), clones 2-6 would be classified
unequivocally as hybrid on the basis of karyotype alone.
The 11 clones with non-parental karyotypes can be
divided into three karyotype groups: clones 2 and 3,
clones 4-6 and clones 7-12. Clones 4-6 and clones 7-12.
have an extra non-parental band in region 111 (Fig. 1 ) .
m11
t12
.3
I
400
300
I
DNA content
500
Measurement of DNA content by flow cytometry
showed that the six N clones had similar DNA contents
to the parents, i.e. 2n, while the six H x N clones were 311
with respect to the parents (Fig. 2). Despite small
differences between the DNA contents of the parents
and between those of the clones, the clustering of values
at either 2n or 3n clearly points to inheritance of DNA in
packets of n, i.e. exchange of haploid nuclei. The lack of
intermediate values indicates that high DNA contents
are not inherently unstable during cloning and subsequent vegetative growth.
Relative DNA content
. ..,.,,,,,,..,.,,,,.,..,,,.,..,.,.,..,.,.,....,....,....,......................................................................................
, ,
Fig. 2. Comparison of DNA contents of parent (THZN, 058H)
and progeny clones from cross B. Relative DNA contents (mean
G1 peak positions) in arbitrary units are plotted on a linear
scale.
karyotypes, only clone 1 being identical to parent P1
(Fig. 1). The six double drug-resistant (H x N ) hybrid
clones (7-12) also had non-parental karyotypes as
expected. However, by comparison, clones 2-6 showed
far more alterations in the size and/or number of bands
throughout the resolved size range relative to P1 and P2
(Fig. 1). Bearing in mind the low frequency and minor
nature of karyotypic rearrangements seen in parental
RFLP analysis
The 12 clones were compared with the parents by
analysis of polymorphic restriction sites in genes for six
Table 3. Analysis of progeny from cross B
FACS: D N A content, D = 2n (diploid), T = 3n (triploid). DrugR: drug resistance phenotype. R genes: presence of genes conferring
resistance to hygromycin (hph)or Geneticin (neo).PFGE: molecular karyotype parental P1 or P2, N P = non-parental. k D N A : type of
maxi-circles present.
~
~~
Progeny*
Parents
__
O58H
= P2
TH2N
= P1
1
2
3
5
4
6
7
8
T
HxN
hPh
neo
NP
P1
AABt
AABt
AB
AB
AB
AB
ABC
CD
AA
AACt
AA
ABD
AABt
T
HxN
hPh
neo
NP
P1
AABt
AABt
AB
AB
AB
AB
ABC
CD
AA
AACt
AA
ABD
AABt
9
10
11
12
-~
FACS
Drug"
R genes
PFGE
kDNA
GPI
PY K
cGAPDH
PLC
RNAP 111
PGK
116
ALD
TIM
THTl
cDNA 8
c D N A 23
PARP B
D
H
hPh
D
N
neo
D
N
neo
D
N
neo
I> D
N
N
neo neo
D
D
N
N
neo neo
P2
P1
P1
BB
BB
BB
BB
BB
BB
P1
P1
BB
BB
BB
BB
BB
BB
NP
P1
BB
BB
BB
BB
BB
BB
NP
P1
BB
BB
BB
BB
BB
BB
NP
P1
BB
BB
BB
BB
BB
BB
P2
AA
AA
AA
AA
AA
AB
AB
cc
AA
AA
AA
AB
AA
cc
CD
AB
BC
AB
CD
BC
NP
P1
BB
BB
BB
BB
BB
BB
NP
P1
BB
-
-
BB
cc cc cc cc cc cc cc
C D DD DD cc
AB AB AB AB
BC BB BB cc
AB AB AB AB
C D cc cc C D
BC BC BC BB
AB
AB
AB
CD
BB
-
cc cc
-
T
HxN
I? p I?
neo
NP
P1
-
AB
-
CD
AA
AABt
-
'' Bold letters indicate homozygous alleles derived from P1.
t Presence of three alleles deduced from band stoichiometry.
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W. G I B S O N a n d O T H E R S
............................................................................................... ..........................................................................................................
..........................................................................................
Fig. 3. RFLP analysis of two homozygous markers. (a) Hindlll digests hybridized with a probe for the GPI gene; (b) Kpnl
digests hybridized with a probe for the PYK gene. Sketch maps indicate positions of polymorphic restriction sites
(asterisk) in alleles A and B. H, Hindlll; K, Kpnl. Approximate sizes were estimated from Hindlll-digested bacteriophage
DNA.
glycolytic enzymes (PGK, ALD, TIM, PYK, cGAPDH,
GPI), the glucose transporter gene array (THTl), two
cDNAs encoding unknown single copy genes (cDNA 8
and 23), the phospholipase C (PLC) gene, the procyclin
genes, the RNAP I11 gene and a repetitive gene of
unknown function conserved in T . cruzi (116). Complete
results were obtained for clones 1-5, 7 and 8 only (Figs
3-6, Table 3). For five loci (GPI, PYK, cGAPDH, PLC,
RNAP 111) where both parents were homozygous for a
given gene, hybrid clones 7 and 8 were heterozygous as
expected, while clones 1-6 were identical to P1 with no
detectable input of genetic material from P2 (e.g. Fig. 3a,
b). Likewise for the PGK and 116 loci, where P1 was
homozygous and P2 was heterozygous, hybrid clones 7
and 8 were also heterozygous, while clones 1-6 were
indistinguishable from parent P1 (Table 3). The parental
origin of alleles in the progeny clones is uncertain for the
PGK genes, since both parents have allele B, but is
914
unequivocal for the 116 genes, where clones 1-6 have C
alleles from PI, and the triploid hybrid clones have a C
allele from P1 and both P2 alleles (Fig. 4a).
For the remaining six loci, P1 was heterozygous. Again
clones 1-6 were indistinguishable from P1 for TIM and
cDNA 8, but remarkably clones 2-6 showed hoinozygosity at the other four loci. For the T H T l locus, the
B and C alleles of P1 have segregated to homozygous BB
in clones 2 and 3, and C C in clones 4-6 (Fig. 4b). The B
and C alleles are unique to P1, while P2 is homozygous
for a slightly larger fragment, A. The H x N clones
inherited A alleles, together with either allele B (clones
9-12) o r C (clones 7, 8) from P1 (Fig. 4b). This latter
result was the only evidence of heterogeneity among the
H x N clones.
For ALD, where P1 was heterozygous with alleles CD,
clones 2 and 3 had only the D allele, while clones 4 and
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Intraclonal mating in Trypanosoma hriicei
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Fig, 4. RFLP analysis of heterozygous markers. (a) Kpnl digests hybridized with a probe for the 116 gene; (b) EcoRl
digests hybridized with a probe for the THTl gene. Approximate sizes were estimated from Hindlll-digested
bacteriophage 2 DNA.
Fig. 5. RFLP analysis of heterozygous markers. (a) Pstl digests hybridized with a probe for the ALD gene; (b) f s t l digests
hybridized with a probe for the procyclin pro2001 gene; (c) f s t l digests hybridized with a probe for cDNA 23. Sketch
maps indicate positions of polymorphic restriction sites (asterisk) in alleles C and D. P, f s t l . Approximate sizes were
estimated from Hind1II-digested bacteriophage A DNA.
5 had only the C allele (Fig. 5a). The origin of the D
allele from P1 is unequivocal, but the C allele could also
derive from P2; however, this is unlikely to be the case
for clones 4 and 5, since n o other input from P2 was
demonstrated in this study. For procyclin or PARP,
restriction fragments derived from the PARP A and B
loci could be distinguished using the downstream Pap5
DNA probe (Konig et al., 1989), revealing homozygosity
for clones 4 and 5 at the PARP B locus ;it is clear that the
8 kb fragment for PARP B/allele B derives from P1 (Fig.
5b). Clones 1-3 had 8 and 12 kb fragments for PARP B
(alleles B and C) like P1, while the H x N clones had
alleles A and B, in what looks like a 2 : 1 ratio. For cDNA
23, the paired fragments of P1 and P2 are taken to
represent four different alleles; clones 2 and 3 are
therefore homozygous CC, while clones 4 and 5 are CD-
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91 5
W. G I B S O N a n d O T H E R S
like P1. As for probe 116, the H x N clones clearly have
three alleles for cDNA 23 : A and B from P2 plus D from
P1 (Fig. 5c).
In summary, four heterozygous loci of P1 show homozygosity in progeny clones 2-6. Numerous independent
mutations would have been required to produce this
result for four separate loci in these two sets of clones.
Given the lack of demonstrable input of genetic material
from P2 for the five homozygous markers (GPI, PYK,
cGAPDH, PLC, RNAP 111),the most simple explanation
for these results is the segregation and reassortment of
P1 alleles alone, i.e. intraclonal mating. Meanwhile,
clone 1 was identical to P1 in all respects and there is no
reason to believe that this represents anything other than
asexual transmission of P1, since sexual reproduction is
not an obligatory part of the trypanosome life cycle. In
previous crosses, clones of parental genotype have also
been transmitted with high fidelity and karyotypic
rearrangements are associated far more frequently with
meiotic rather than mitotic division (Gibson & Garside,
1991; Gibson et al., 1992,1995; Gibson & Bailey, 1994).
Chromosomal linkage of markers
-
The ALD and PARP B loci were previously mapped to
the same
5 M b chromosome (Gottesdiener et al.,
1990) and our results map the THTl locus to the same
chromosomal location (Fig. 6a). Therefore, with the
proviso that resolving power is limited in this size range,
all three loci are probably on the same chromosome.
Looking at Table 3, the results for ALD and THTl both
show homozygosity for clones 2-6, with linkage of
alleles ALD D / T H T 1 B and ALD C / T H T l C. However,
for PARP B, clones 2 and 3 are heterozygous like parent
P1, while clones 4 and 5 are homozygous. T o explain
this anomaly, either the PARP B, ALD and THTl loci
are on different, comigrating chromosomes, or a crossing over event took place between the B and C alleles of
PARP B during the meiotic division from which clones 2
and 3 were derived.
cDNA 23 maps to much smaller chromosomes of about
1.5-1.8 Mb, which are well resolved by PFGE analysis
(Fig. 6b). Assuming that allele C resides on the smaller
chromosome (1.5M b ) of P1, the apparent loss of the
larger chromosome (1.8 Mb) in clones 2 and 3 agrees
with the observed homozygosity of these clones. Clones
4-6 had alleles CD and we interpret the 1.6 M b band as
a deletion product of the 1.8 Mb chromosome of P1
carrying allele D. The 1.5 M b chromosome of the H x N
clones (7-9) could derive from either parent, but the
faint 1.8 M b band is from P2 (underloaded on this gel)
and there is a non-parental band of approximately
1.6 Mb.
Hybridization of a PFGE blot with a probe for the ptubulin gene is shown in Fig. 6(c). P1 and P2 each have
two homologues carrying the tubulin array, which differ
slightly in size ( N l / N 2 and H l / H 2 , respectively).
Judging by size, clones 2 and 3 appear to have inherited
N2 only, while clones 1 and 4-6 are identical to P1 with
916
both N 1 and N2; triploid H x N clones 7-9 have H1 and
probably H 2 and N 2 comigrating. The hph gene was
previously found to be located on H1 of P2 (Gibson &
Whittington, 1993), while the neo gene is on N2 of P1
(not shown). The presence of H1 or N2 with their
assigned hph and neo genes concurred with the drug
resistance phenotypes of the clones.
Fingerprint anaIysis
Low stringency hybridization with VSG gene 117 and
PCR using eight arbitrary primers (RAPD) were used to
fingerprint the parental and progeny clones. These
analyses not only confirmed the hybrid status of
individual clones if the fingerprints were different to
those of both parents, but also established which clones
were identical. Hybridization with VSG gene 117 (Fig. 7)
divided progeny clones 1-8 into the same groups as
PFGE (Fig. 1). It can be seen that while clone 1 is
identical to P1, clones 2 and 3 lack two fragments of
approximately 3.5 kb relative to P1 and clones 4-6 lack
fragments of approximately 2.5 and 3.5 kb (Fig. 7 ) ;
neither of the prominent bands of P2 at 1.3 or 2 kb has
been inherited by clones 1-6. Loss of genetic material
was also clearly seen with two of the RAPD primers for
clones 2 and 3 (not shown). These results are consistent
with the hypothesis that clones 2-6 are products of
intraclonal mating of P1, which have effectively lost
allelic variation.
kDNA
The kDNA maxi-circle type of the 12 clones was
assessed by presence (P2 type) or absence (PI type) of an
extra EcoRI site. All clones had P1 type maxi-circles
without the extra EcoRI site. Preliminary RFLP analysis
of kDNA minicircles of clones 1-3 and 10 did not show
major differences between the minicircles of P1 and
clones 1-3, although the hybrid clone 10 was shown to
have some P2-derived minicircles by hybridization (M.
Crow & W. Gibson, unpublished results).
DISCUSSION
These experiments were designed to test the hypothesis
that intraclonal mating occurs in T . brucei subspecies.
Segregants explicable as products of intraclone mating
were produced in an out-cross, together with authentic
double drug-resistant hybrids. However, we failed to
detect double drug-resistant hybrids in three attempted
matings of single clones transformed with two different
drug resistance markers.
The evidence that these segregants were the products of
intraclone mating rests on (1)lack of detectable input of
genetic material from one parent, P2, in particular for
five marker genes for which both parents P1 and P2 had
unique homozygous alleles ; (2) homozygosity at four
loci (ALD, THT1, PARP B, cDNA 23) where P1 was
heterozygous ; (3) extensive reorganization of molecular
karyotype ; (4) homozygosity of the chromosomes
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Int r a clon a 1 mating in T r y p an oso mn b YU cei
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Fig. 7. Comparison o f VSG 117 gene family in parent and
progeny clones (Hincll digests). Approximate sizes were
estimated from Hindlll-digested bacteriophage A DNA.
and RAPD analysis using two primers. The generation
of homozygosity at a total of five loci is hard to explain
by mutation, since many independent mutations would
have been required. The karyotype alterations seen in
these clones were far more extensive than seen previously for progeny clones of parental genotype ; in four
different crosses only a handful of such clones have
shown small differences of mobility in at most one
chromosome band and the more usual finding is a high
degree of karyotype stability, except when meiosis is
involved (Gibson & Garside, 1991; Gibson et al., 1992,
1995; Gibson & Bailey, 1994). The DNA contents of
these clones were normal (i.e. 2n), ruling out the
possibility of wholesale chromosome loss.
Figrn6. PFGE size-fractionated chromosomal DNAs hybridized
with probes for (a) the THTl gene, (b) cDNA 23 and (c) the ptubulin gene. Approximate sizes were estimated from yeast and
bacteriophage imarkers.
Thus the simplest conclusion is that clones 2-6 were the
products of mating of parent P1 alone. The hybridsecreting fly from out-cross B therefore contained a
mixture of double drug-resistant hybrids (clones 7-12),
products of P1 alone (clones 2-6) and parental (clone 1)
trypanosomes. Results along similar lines were reported
by Tait and colleagues for another cross, where evidence
rested on the isoenzyme product for tyrosine kinase, for
which the genetic basis of variability is unknown, RFLP
analysis of an unidentified locus and differences for three
chromosomal markers in PFGE analysis (Tait et al.,
1993,1996). Taken together, the results strongly suggest
that intraclone mating can take place among a mixture
of trypanosomes undergoing interclone mating, but not
in a single clone, as our three failed attempts to find
double drug-resistant trypanosomes in crosses of the
same clone bearing different drug resistance markers
demonstrate. Tait and colleagues also failed to find
evidence of ~ o m o z y g o s ~in
t y heterozygous clones transmitted
via the tsetse fly (Tait et al., 1989, 1993,
1996).
carrying the P-tubulin and cDNA 23 loci, which were of
different sizes in P1; and (5) loss of genetic material
observed by RFLP analysis of the VSG 117 gene family
The observation that intraclone mating takes place
only during sexual reproduction among heterologous
trypanosomes has intriguing implications: (1) that
trypanosomes distinguish self and non-self, i.e. there is a
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91 7
W. GIBSON a n d O T H E R S
system of mating types; and (2) that recognition of nonself triggers mating. The possible occurrence and genetic
basis of mating types in 7'.brucei subspecies has not yet
been examined in detail. Crosses between all three
subspecies except Type I T . 6. gambiense, which is
difficult to transmit experimentally, have succeeded
(Jenni et al., 1986; Gibson, 1989; Turner et al., 1990).
The three-way cross of Turner et al. (1990) ruled out a
two sex mating system. Control of mating types in a
diploid organism presents particular problems
(Hodgkin, 1992; Judelson et al., 1995) and it will be
interesting to see how this ancient eukaryote has solved
them. The mating types of other protists control the
secretion of pheromones for self/non-self recognition
(Sleigh, 1989). Our results suggest that similar diffusible
factors are at work in trypanosomes, as mediators of
mating between trypanosomes of different mating types
and as triggers of meiosis/fusion in trypanosomes of the
same mating type. The observed segregation of alleles is
unequivocal evidence of meiosis, while the fact that not
all heterozygous markers of P1 became homozygous
points to the fusion of haploid gametic nuclei derived
from different meioses, i.e. probably fusion of two P1
individuals.
not incorporate drug resistance markers) of six in which
DNA contents have been measured (Paindavoine et al.,
1986; Wells et al., 1987; Gibson et al., 1992, 1995;
Gibson & Bailey, 1994). Trisomy of several chromosomes was demonstrated for two previously isolated
clones (Gibson et al., 1992) and three alleles were
demonstrated at several loci in the 3n hybrids from this
experiment. Although there are slight but consistent
differences in DNA content between clones, perhaps
reflecting variation in the numbers of small chromosomes, all clones analysed thus far conform unequivocally to 2n or 3n; intermediate DNA contents,
suggestive of random chromosome loss from a tetraploid
precursor, have not been found. Therefore, it is reasonable to conclude that 3n hybrids are triploid and most
likely derive from fusion of haploid and diploid nuclei,
with the important corollary that meiosis must be
involved. Triploidy is a commonly encountered but
usually lethal problem of eukaryote zygote formation
even in man, where nearly 1 % of all conceptuses are
triploid. Triploid trypanosome hybrids are only unusual
in terms of their vigour.
Products of intraclonal mating were not 'selectable in
this experimental set-up, since they had the same drug
resistance phenotype as one parent. The homozygosity
of deleterious mutations might also be expected to
reduce fitness relative to heterozygous clones. It is
therefore surprising that two different genotypes of such
clones were isolated in this experiment, suggesting either
high levels of virulence and/or large numbers in the
salivary glands. It is possible that diploid clones might
out-compete triploid hybrids during vegetative growth,
but doubtful whether this would also apply to the
diploid parents. If the pheromone hypothesis outlined
above is correct, then all responsive trypanosomes may
be triggered into meiosis by diffusible factors, with the
result that the majority of salivary gland ,trypanosomes
mate. The intraclonal mating products seen in this
experiment might thus simply reflect an imbalance in
parental types. Indeed, it is not unreasonable to suggest
that haploids might predominate in the salivary glands
under these particular circumstances, although epimastigotes and metacyclics are normally diplaid (Shapiro et
al., 1984; Tait et al., 1989; Kooy et al., 1989). Inclusion
of a third, non-self parent in an intraclonal cross might
thus trigger the H and N clones to mate; however, a
preliminary experiment in which TH2H and T H 2 N
were cotransmitted with 058 wild-type failed to produce
any double drug-resistant hybrids, although 058 wildtype predominated in the salivary glands in this experiment.
ACKNOWLEDGEMENTS
Concerning the general mechanism of sexual reproduction in trypanosomes, two additional points arise
from this study. (1)The salivary glands rather than the
midgut have now been shown to be the site of genetic
exchange in five independent crosses (Gibson &
Whittington, 1993; Gibson et al., 1995). (2 )3n hybrids
have now been found in five crosses (two of which did
918
This work was supported by the Medical Research Council;
M.B. is supported by T h e Wellconie Trust. W e thank Mrs
Jenny Berry and Vanessa Ferris for expert assistance with the
tsetse flies and Dr Hayley Whittington for trypanosome
transformants. We are extremely grateful to the following
colleagues for generously providing D N A probes and constructs : Nina Agabian, Anneloor Ten Asbroek, The0 Baltz,
Piet Borst, Frederic Bringaud, Albert Cornelissen, Raymond
Evers, Ken Hudson, John Kelly, Mary Gwo-Shu Lee, Kojo
Mensa-Wilmot, Paul Michels, Isabel Roditi and Lex Van der
Ploeg.
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Received 4 June 1996; revised 18 September 1996; accepted
27 September 1996.
920
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