Chromosomal duplication in Giardia duodenalis

CHRO~OSO~AL

A. UPCROFT,

JACQUELINE The Bancroft

Centre,

Queensland

DUPLICATION IN GRANDPA DU~D~~A~r~

Institute

of Medical

ANDREW Research,

HEALEY 300 Herston

and

PETER

Road,

UPCROFT

Brisbane,

Queensland

4029, Australia

(Received 16 October 1992; accepted 9 December 1992) J. A., HEALEYA. and UPCROFTP. 19%. Chromosomal duplication in Gimdiu duodenalis. ~~ziernotio~o~Journalfor Parasitology 23: 609-616. Strains of Giardia duodenalis from diverse parts of the world have three or four chromosomal bands in the range of 65@8@0kb as defined by field inversion gel electrophoresis. The extra chromosome band in this range defined a group of strains which are geographically distinct from other strains missing this band. The cloned line WB-IB has three chromosome bands in this size range and chromosome band 3 was used to construct a library of chromosome-specific probes. In some strains examined, including BRIS/83/HEPU/106, a subset of these WB-1B probes hybridized to chromosome band 3 and to the extra chromosome band 4, indicating a partial duplication of chromosome 3 in BRlS/83/HEPU/106. This duplication was estimated to be at least 500 kb when the sizes of Not1 chromosome segments which hybridized with chromosome band 3-specific probes were added. A second subset of WB-I B chromosome 3-specific probes hybridized to a fifth chromosome of strain BRIS/83/ HEPU/l06, in the size range 650-800 kb, which was not visible by ethidium bromide staining. The two subsets of WB- 1B probes hybridized to a variety of chromosome combinations in this size range in other Giardia strains and included apparent reversal of chromosome 3 and 4 mobility as well as identification of other minor chromosomes. These data indicate that chromosome band 3 of the line WB-l B is a cluster of at least two different chromosomes that cannot be electrophoretically separated but genetic rearrangements in other strains allow separation of linkage groups carried by chromosome band 3 of WB-IS. The apparent uniformity of the electrophoretic karyotype in Giardia d~~de~ff~j~ as judged by chromosome size, or electrophoretic migration, masks an underlying genetic plasticity which is becoming evident as this organism is more thoroughly studied. AbStIX-UPCROFT

INDEX KEY WORDS: duplication; chromosomal

Giardia duodenalis; chromosomal DNA library; DNA hybridization.

chromosome

mapping;

genetic

somes (Upcroft et al., 1989a). We identified strains, predominantly derived from our locally sampled collection, with four major chromosome bands in the size range 650-800 kb. Most of the stocks established in North America appeared to have only three chromosome bands in this size range. Using a specific library of probes from chromosome band 3 of the cloned line WB-IB, we show here that the extra chromosome band of the strain BRIS/83/HEPU/I06 (designated chromosome band 4) contains a chromosome which is in part a duplication of a chromosome in band 3. However, other strains with an extra chromosome band do not generally carry such a duplication.

INTRODUCTION CHROMOSOMES

separation;

of the intestinal

protozoan parasite Giardin duodmalis have been visualized in several electrophoretic pulsed field gel systems and some controversy has been generated as to the number of chromosomes in the genome (Adam, Nash 2%Wellems, 1988; de Jonckheere, Majewska & Kasprzak, 1990; Campbell, van Keulen, Erlandsen, Senturia & Jarroll, 1990; Upcroft, Boreham & Upcroft, 1989a). Estimates of four to eight chromosome bands in the genome have been made with general agreement that some appear to contain more than one identical or different chromosomes (Adam et al., 1988; de Jonckheere et al., 1990). It has been suggested that as many as 30-50 chromosomal DNA molecules may be present in each trophozoite (Adam et ai., 1988). The karyotypes of G. duodenulis strains worldwide appeared to fall into two groups depending on an extra chromosome band visualized in field inversion gel electrophoretic (FIGE) separations of their chromo-

MATERIALS

AND METHODS

Stocks and cuhres. The terms stock, strain and line are used according to the nomenclature suggested by Baker, Brown, Godfrey, Herbert, Ketteridge, Lapage, Lumsden, Mattern, van Meirvenne, Njogu, de Raadt, Somerville, Steinert & Vickerman (1978) for the trypanosomes. Stocks 609

610

J. A. UPCKOFT, A. HEALEY and P. UPCROFT

BRlS/S3/HEPU/99~ BRIS/83~HEPU~lO6. BRIS/83/‘HEPUj 136. BRIS/87~HEPU/724, BRIS/S~~HEPU/782, BRlS/89,/ HEPU/t065, Ad1 13, OASI and BAC2 and the cloned line WB-IB were used in this study. The origins of ail stocks except BRIS/89/HEPU/li)65 have been previously documented (IJpcroft et ul., 1989a; Capon. Upcroft, Boreham. Cottis & Bundesen, 1989; Upcroft, Upcroft & Boreham. 1990). BRIS/89/HEPU/1065, derived from a duodenal biopsy, was established in culture after passage through neonatal mice. The biopsy sample was from a male child from Northgate, Queensland. Stocks were maintained in modified TYI-S-33 medium containing I mg ml ’ of bile as previously described (Boreham, Phillips & Shepherd, 1986). Cllronm?ume separations. Field inversion gel eiectrophoresis (FIGE) of Giardia chromosomes was carried out as previously described (Upcroft rr al., 1989a). Contour-cramped homogeneous gel electrophoresis (CHEF) was performed on a CHEF-DRT”ll Bio-Rad apparatus aLvording to the manufacturer’s instructions. Sacchar0~~~ce.s cfwvisiae strain YNN295 DNA size markers were from BioRad.

Southern

trunsfer

and hybridization

ofchrorno.vomul

DNA.

Gels with multiple lanes of separated chromosomes from Giurdia strains were transferred (Southern. 1975) on to nylon membrane (Amersham, Hybond-N) and individual lanes were hybridized with labelled probes as described (Upcroft. Dunn, Dommett, Healey, Upcroft & Boreham, 1989). Membranes were stripped between hybridizations in 0.4 M-NaOH for 30 min at 45°C. neutralized in 0.2 M-Tris (tris(hydroxymethyl)amino methane)~HCl, pH 7.5, 0.1 x SSC (standard saline citrate, 0.5 M-NaCI. 0.015 M-sodium citrate), 0.1% SDS for 30min at 45°C. rinsed several times in 2 x SSC at RT and air dried. Not1 &UVU~P (?f intact chromosomes. Whole blocklets containing intact Giurdiu chromosomes (Upcroft et cd., 1989a) were washed extensively with TE (IO mM-Tris/IICi, I mM-EDTA, pH 7.5) over several hours, several times with Not1 restriction enzyme buffer (New England Biolabs) and then cleaved overnight with Not1 (Boehringer Mannheim) in a total volume, including the blocklet, of 30 ~1. Other blocklets were similarly cleaved with Kpnl (New England Biolabs buffer conditions). Three sets of cleaved and uncleaved blocklets with KpnI cieaved bacteriophage T4 DNA (Amersham), lambda DNA (New England Bioiabs) and TS DNA (Sigma) molecuiar weight markers were separated by CHEF. The separated chromosome segments were Southern transferred and hybridized with probes as above. Membranes were stripped between hybridizations. as above. Chromosome specffic library und probes. Chromosomes to be extracted from agarose for recombinant DNA library construction were separated by FIGE in 0.7% SeaPlaque agarose (Marine Colloids). The gel and running buffer concentrations were increased from 0.5 x TBE (5 x TBE is 330 mM-Tris, 100 mM-boric acid, 20 mM-EDTA) to 0.75 x TBE and the rest phase between forward and backward pulses was doubled to improve separation in this agarose (Upcroft et al., 1989a: Upcroft, Boreham & Upcroft, 1989b). Bands of chromosome 3 from multiple lanes of an agarose gel, i.e. from approximately IO8 trophozoites of Ginrdia Iine WB-IB. were excised from an ethidium bromide stained gel transilluminated with u.v. light (302 run). Excised agarose

blocks were washed extensively in TE, in 0.5 x TE and finally in double distilled H,O. The blocks were centrifuged. excess liquid removed and melted at 70°C for 10 min. One-tenth of the final volume of IO x Hind111 buffer (New England Biolabs buffer conditions) was added and the molten blocks were incubated with Hind111 (200 U, New England Biolabs) at 37°C overnight. One-third of the volume ofTE was added and DNA extracted three times with equal volumes of buffered phenol followed by phenol/chloroform (I: I) and chloroformiisoamyl alcohol (24: I) (Upcroft & Healey. 1987). Several cycles of extraction with equal volumes of 2-butanol were used to reduce the aqueous phase to one-fifth the original volume. Ten per cent (v/v) of 2 M-ammonium acetate, pH 5.3, was added and the DNA precipitated with two volumes of ethanol at - 20°C. overnight. Precipitated DNA was centrifuged at 4°C for 5 min, dried and rcsuspended in IOplofTE,A DNAconcentrationof IOng~l ’ was estimated after I pl was electrophoresed and stained. The DNA remaining from the above extraction (9 ~11)was ligated with 80 ng of HindIII-cleaved, calf intestinal alkaline phosphatase (Boehringer-Mannheim)-treated Bluescript plasmid vector (Stratagene) in a total of 12 ~1 containing 3.3”;~ polyethyleneglycolhO00 (Sigma) and 20 U ofT4 DNA ligasc (New England Biolabs) in ligase buffer (according to the manufacturer’s instructions). One-tenth of the ligation mix was transformed into Eschrrichi~~ w/i DHSa (Bethesda Research Labordtorics) (Hanahan, 1983). The efficiency of transformation of the control supercoiled Bluescript vector was 3.8 x IO”cfu pg ’ of vector. Colonies were preserved as described by Han&an & Meselson (1980). The untransformed ligation mix was stored at - 20°C. Twenty colonies were picked at random and DNA prepared (Birnboim di Daly, 1979). Nine of the 20 had inserts larger then 500 hp. These nine were labelled with “P-dC’TP (I 10 TBq mm01 I_ Amersham) by random priming (Feinberg & Vogelstein. 1984) for hybridization to Southern transferred G~LIT&/ chromosomes. Other DNA probes used in this study were probe G6;i (Upcroft et cd..I989a; Upcroft. Capon. DharmkrongAt, Healey, Boreham & Ilpcroft, 1987; Upcroft. Hcalcy, Murray. Boreham & Upcroft. 1992) and probe Gl5:4 (Upcroft E/ ul., 1987, 1992). RESULTS

Chromosome band 3 of the G&d& line WB- I B was separated by FIGE and a library of recombinant clones from this band was constructed (see Materials and Methods). From IO’ trophozoites we recovered 100 ng of chromosome band 3 DNA and obtained a total number of lo4 transformed colonies from 90% of this DNA (10% was used to estimate the DNA content). Of nine randomly selected probes with inserts > 500 bp, seven specifically hybridized with chromosome band 3 of WB-I B (Fig. 1F). The eighth probe hybridized with chromosome band 2 of WB-I B and strain BR~S~83~~~PU~lO6 and the ninth hybridized to multiple chromosome bands of several strains including WB-1 B (data not shown).

Chromosomal

duplic :ation in G. duodenalis

611

E Ch

2l-

1

23

123

123

123

1

23

1 23

FIG. I. Hybridization of chromosome band 3-specific probes with chromosomes of different G. duodenalis strains. Chromosomes of different strains were separated by FIGE, stained with ethidium bromide, Southern transferred and hybridized. Each panel represents the hybridization pattern of chromosomes of different strains compared with the ethidium bromide stained chromosome bands of the same strain. Chromosome bands (Ch) are numbered 1,2,3 and 4 and are in the size range 65(f800 kb compared with yeast chromosomes separated in the same system (Upcroft et al., 1989a). Panels A. BRIS/87/ HEPU/106; B, BRIS/83/HEPU/136; C, BRIS/83/HEPU/99; D, BACZ; E, BRIS/89/HEPU/l065; F, WB-IB. Lane 1 in all cases is the ethidium bromide stained chromosomes of each strain; lanes 2 and 3 in all panels represent the hybridization patterns ofgroup I and group 2 probes, respectively. The hybridization patterns ofgroup 1 probes, C3/11, C3/19, C3/28, G6/1 and G15/4, were identical in each strain so only one representative is shown. Similarly, group 2 probes, C3i20, C3/25, C3/29 and C3/31, were identical in each strain. Hybridization probes

of WB- 1B chromosome

with chromosomes

of Giardia

band 3-specific strain BRIS/83/

HEPUI106

We have Giardia DNA

previously

shown,

by hybridization

of a

probe G6/1 to chromosomes of strain BRIS/83/HEPU/l06, that this sequence is duplicated in chromosome bands 3 and 4 (Upcroft, Upcroft & Boreham, 1989; Upcroft et al., 1992). In order to determine-the extent of this duplication we hybridized the WB-1B chromosome band 3-specific probes to the electrophoretically separated chromosomes of the Giardia strain BRIS/83/HEPU/l06 (Fig. lA, lane 1). The probes hybridized to a number of chromosome sites in this strain. Probes C3/11, C3/28 and C3/19 hybridized with chromosome bands 3 and 4, similarly to probe G6/1. Probe Gl5/4 generated from a genomic library but which encodes a Giardia protein (data not shown) also hybridized to chromosome bands 3 and 4 (Upcroft et al., 1992). The hybridization patterns of the above probes were the same and they are referred

to as group

1 probes (Fig. lB, lane 2). Probes C3/20, C3/25, C3/29 and C3/31 hybridized to DNA which migrated more slowly than chromosome band 4 (Fig. lB, lane 3). Again the hybridization pattern of these latter probes (referred to as group 2 probes) were identical, i.e. the probes hybridized to the same chromosome bands but to chromosome bands that were different from the group 1 probes. Comparison

of Giardia strains Previously, we characterized a number of isolates as similar to BRIS/83/HEPU/l06 in having an extra chromosome band between 650 and 800 kb (Upcroft et a/., 1989a) and in this study have used some of these strains to determine whether the duplication in band 3 in BRIS/83/HEPU/l06 is similarly duplicated in these other strains. The chromosomes in strain BRIS/87/ HEPU/724 hybridize identically to BRIS/83/IIEPU/ 106 (Fig. 1A) with both group 1 and group 2 probes. These two strains were from the Brisbane area but

.I. A, UPCROFT, A. HEALEY and P. UP~ROFT

612

A

B

kb

200-

100502314-

123456

7

1 2 3 4 5 6

7

8 9 IO

FIG. 2. Cleaved whole chromosomes of strain BRIS/~3~H~PU~iO6 hybridized with chromosome band 3-specific probes. Intact chromosomes were cleaved with Kpnf and Not1 and together with appropriate markers were separated by CHEF and ethidium bromide stained (Panel A). The gel was Southern transferred and whole chromosome cleavages with Not1 were hybridized with chromosome 3-specific DNA probes (Panel B). Panel A. lane 1, T4 DNA (166 kb); lane 2, TS DNA (105 kb); lane 3, Lambda chromosomes (48.5,97 kb. etc.): lane 4, Kpnl cleaved T4 DNA (14,23,36/38 doublet and 46 kb): lane 5. Not1 cleaved Girzrdiuchromosomes; lane 6, KpnI cleaved Giardia chromosomes; lane 7, uncleaved chromosomes. Panel B, lane I. hybridization pattern observed with all probes in uncleaved chromosome control. Lane 2. Not1 cleaved chromosomes hybridized with probe Gh; I; lane 3. probe C3:‘l I; lane 4, C3/ 19; lane 5. C3/28: lane 6, G I S,‘4; lane 7. C3,‘20: lane 8, C3.‘2S: lane 9, C3:29; lane 10, C/3 I

were separated by 4 years between establishment in culture. The strain BRIS~88/HEPU~l36, previously characterized as having an extra chromosome band, band 4 (U~roft PI al.. 1989a), does not carry the duplication seen in BRISi83/HEFU~IO6 when hybridized with the group 1 probes (Fig. IB, lane 2). The strain BRISj83/HEPU/99, which we characterized as having a faint chromosome band 4 (Fig. IC). and BAC2 (Fig. ID) clearly have chromosome bands 3 and 4 by electrophoretic karyotyping but these two do not contain duplications of the region defined by the group I probes. The group 1 and 2 probes hybridize to different chromosome bands in the strains BRlS/88/ HEPUjl36, BRIS/83/HEPU/99, BACZand BRISj89j HEPUi1065 (Fig. I). In BACZ(Fig. ID, lanes2and 3) these probes identify chromosomes with reversed mobility to the other strains. A more recently established strain, BRIS~89~H~PU~lO65, which has three visible chromosome bands in the range 650MOO

kb in FIGE separations, appears to have a fourth chromosome which migrates slightly slower in FIGE (Fig. IE). This chromosome is not visible in stained gels but hybridization with the group 2 probes indicates its presence (Fig. 1E, lane 3). Other strains with only three visibly stained chromosome bands in the range 650-800 kb, such as Ad1 13. OASI and BRLS! 87/HEPU/782, share similar hybridization patterns with the group I and 2 probes to that of WB-1 B (Fig. IF, lanes 2 and 3).

Whole chromosomes of the strain BRIS/83/HEPU: 106 were cleaved with Not1 and separated by CHEF. DNA markers were used for size estimates of the Not1 segments (Fig. 2A). Controls included KpnI-cleaved whole chromosomes and uncleaved chromosomes. Probes specific for chromosome band 3 of the cloned

Chromosomal TABLE I-SIZES

duplication

OF Not1 AND KpnI

in G.

DNA

106 CHROMOSOMESTO WHICH WB-1B

613

duodenalis

SEGMENTSOF BRIS/83/HEPU/

CHROMOSOME

BAND 3-SPECIFIC

DNA

PROBESHYBRIDIZED Group

1

Probe

Group Size (kb)

2 Size (kb)

Not1

KpnI

G6/1

160

23

C3/20

40

c3/11

120

12

C3/25

140

c3/19

150

20

C3/29

140

25

C3/28

10

c3/31

140

14

Gl5/4

4.3

300, 140,70

line WB-1B were hybridized to Not1 segments of whole BRIS/83/HEPU/106 chromosomes (Fig. 2B) and size estimates of the segments to which they hybridized are listed in Table 1. Sizes were estimated from the original autoradiograph by measuring the band distance from the origin and comparing these with the original photograph of the ethidium bromide stained gel. The sizes of the KpnI segments are also given in Table 1. These sizes were estimated from KpnI-cleaved Giurdia DNA separated in a uniform field gel (data not shown) as well as the same CHEF gel shown in Fig. 2A (hybridization data not shown). Sequences recognized by probes in group 1 (Table 1) appear on both chromosome bands 3 and 4 of the strain BRIS/83/HEPU/l06 (Fig. 1A) and the sizes of Not1 segments to which the probes hybridized account for at least 500 kb. The first four probes in the group are located at unique sites in the genome according to the Not1 cleavages and also the KpnI cleavages (Table 1). Probe G15/4 (Fig. 2B, lane 6) hybridized predominantly with one 70 kb Not1 segment and to two KpnI segments (Table 1). Since G15/4 hybridized to chromosome bands 3 and 4 equally we assumed that this 70 kb segment is represented in both chromosome bands and estimate that the minimum length of the duplicated chromosome in bands 3 and 4 is 500 kb. Probes which did not hybridize to chromosome bands 3 and 4 of BRIS/83/HEPU/106 but which did hybridize to chromosome band 3 of WB-I B (group 2 probes) elucidate a chromosome not always visible in ethidium bromide stained gels of chromosomes of BRIS/83/HEPU/106 (Fig. lA, lane 3). These probes localized to a 140 kb Not1 segment (Fig. 2B, lanes 8,9 and 10) and a 40 kb Not1 segment (Fig. 2B, lane 7). The group 2 probes identify a chromosome not normally visualized in BRIS/83/HEPU/106 but resolved by hybridization studies (Fig. lA, lane 3).

Comparison of’ CHEF chromosomes We have separated

Probe

and FIGE separated by CHEF

a total

Giardia of eight

Not1

KpnI 9 9.5

9, 7.5

different chromosome bands of the strain BRIS/83/ HEPU/ 106. Chromosome bands 6, 7 and 8 are larger than 2.2 Mb (megabase pairs) and chromosome band 2 is ofthe order of 1 Mb (Fig. 3A and C). Chromosome band 1, not shown in Fig. 3A and C, is identified under slightly different gel running conditions. We have been unable to consistently separate chromosome band 3 from chromosome band 4 of strain BRIS/83/HEPU/106 by CHEF as we have done by FIGE. However, chromosome band 3 specific probes of WB- 1B routinely hybridize to the chromosome 3 band in CHEF separated chromosomes of WB-1B (Fig. 3B, lane 5). In Fig. 3A two chromosome bands of BRIS/83/HEPU/106 equivalent in size to chromosome band 3 of WB- 1B can be distinguished. Probes of group 1 (of which C3/19 is a representative) hybridize to chromosome bands 3 and 4 of BRIS/83/ HEPU/106 (Fig. 3B, lanes 3 and 4) and to chromosome band 3 of WB-IB (Fig. 3B, lane 5). C3/20 which is a group 2 representative hybridizes to only chromosome band 3 of BRIS/83/HEPU/lO6 (Fig. 3D, lanes 2 and 3) and to chromosome band 3 of WB-1 B (Fig. 3D, lane 4). There is no evidence of hybridization of C3/20 to other chromosomes larger than chromosome bands 3 and 4 as there is in FIGE separated chromosomes of BRIS/83/HEPU/106 (Fig. IA, lane 3). It appears that some chromosomes in the 650-800 kb range, which separate well in FIGE gels, are tightly clustered in the CHEF system and are reversed in apparent size since the chromosomes recognized by the group 2 probes in FIGE separations migrate more slowly than chromosome band 4 of BRIS/83/HEPU/106 in CHEF gels (Fig. 3D, lanes 2 and 3). We have previously observed migration changes dependent on boundary conditions (Upcroft et al., 1989b). DISCUSSION

We have previously differentiated strains on the basis of possessing an extra chromosome band in the range 650-800 kb of their electrophoretic karyotype (Upcroft et al., 1989a). This observation was related to

614

Mb

J. A. UPCROFT, A. HEALEV and P. UPCROFT

Ch

Ch

-8 -7

-6 2.2-

1.6-

“6

-5

1; -2

l.O-

-5 1.6

-4 -3 -2

FIG. 3. Hybridization of CHEF separated G&r&a chrolnosom~s with WB-IB chromosome band 3-specific probes. Chromosomes of the line WB- 1B and the strain BRIS/83~HEPU~ 106 (Panels A and C) were hybridized with the probes C3: 19. a group 1 probe (Panel B), and C3/20, a group 2 probe (Panel D). Yeast chromosomes were used as markers and sizes are indicated in Mb at the left of the panels. Chromosome band designation (Ch) is indicated at the right of the panels. Panels A and C represent ethidium bromide stained chromosomes. Panel A: lanes I and 2, yeast; lanes 3 and 4, BRIS/83/HEPU/lOh: lane 5, WB-IB. Panel B: lanes 3,4 and 5 are as in Panel A. Panel C: lane 1, yeast; lanes 2 and 3. BRIS/83/HEPU!l06: lane 4. WB-IB. Panel D: lanes 2. 3 and 4 are as Panel C.

the geographical origin of the strain. Further examination of the strain BRIS/83/HEPU/106 showed that the extra chromosome band contained a large duplication of a band 3 chromosome. In strains such as Ad 113 and the cloned line WB-IS chromosome band 3 is a cluster of at least two different chromosomes. A grade in the separation of chromosomes exists between these latter strains and those represented by BRISj83j HEPU/106 with three distinct chromosome bands of similar size but all distinct by hydridization with WBI B chromosome band 3-specific probes. In FIGE and CHEF separations of chromosomes of WB- 1B, Ad I 13, OASl and BRIS/83/HEPU/728, from diverse parts of the world, the chromosome band 3-specific probes derived from WB- 1B (group 1 and 2 probes) recognize a single chromosome band, band 3. The gradation then occurs as follows; BRIS/89/H~PU~ 1065 has only one visible ethidium bromide stained chromosome band equivalent to chromosome band 3 of WB- 1B and Ad 113, but by hybridization with probes of group 2 a second chromosome was identified. The strains BRIS/

83/HEPU/99, BAC2 and BRIS/83/HEPU/ I36 have two distinct ethidium bromide stained chromosome bands of a similar size to chromosome band 3 of WB1B but the probes of group I and 2 distinguish these as con~dining different chromosomes with no detectable duplicated segments. The strains BRIS~83/~EPU/lO6 and BRIS~87~HEPU/724 also have two distinct ethidium bromide stained chromosome bands of similar size to chromosome band 3 of WB-IB but hybridization with group 1 probes indicated these two contain duplicated segments and hybridization with group 2 probes revealed a third chromosome band not normally distinguishable in ethidium bromide stained FIGE gels. The probes of group 1 are clearly linked, as they segregate together in the duplications in chromosomes of BRIS~83/HEPU~lO6 and BRIS~87/HEPU~724. Additionally, three of the group 1 probes are unique site sequences in the genome of BRIS~83~HEPU~lO6 by both Kpnl and Not1 cleavage analyses indicating identical flanking regions of the probes up to 160 kb in

Chromosomal duplication in G. duodenalis length. The duplications occur in the same chromosome band in the two strains and cover at least 0.5 Mb of chromosomes in bands 3 and 4. The group 2 probes cover a smaller region but they clearly segregate independently from group 1 probes. Thus we suggest the simplest interpretation is that in the strains with three chromosome bands in the range 650-800 kb band 3 is a cluster of at least two different chromosomes, one recognized by group 2 probes and a second chromosome recognized by group 1 probes as described here. This latter chromosome may well be duplicated but remains unchanged by further rearrangements. In the case of strains such as BRIS/S3/ HEPU/l06 rearrangements in the chromosomes with duplicated segments allow electrophoretic separation of this cluster. The duplication observed in BRIS/S3/HEPU/106 may be present but remains unaltered in some strains such as BRIS/S3/HEPU/99 which has a faint ethidium bromide stained chromosome band 4 and a more intensely stained chromosome band 3. That some chromosome gel bands comprised more than one different chromosome has been previously suggested by Adam et al. (1988) and de Jonckheere et al. (1990) where densitometer analysis of DNA content of chromosome bands suggested unequal numbers of chromosomes in the bands. Adam et al. (1988) reported faintly staining chromosome bands which they estimated contained two copies of a chromosome. Other bands contained many chromosomes. We have also described faintly staining bands in some strains (Upcroft et al., 1989a). In FIGE gels the size of chromosome band 3 when compared with yeast chromosomes is 0.85 Mb (Upcroft et al., 1989a). Thus the duplication in BRIS/S3/ HEPU/106 appears to constitute the majority of a chromosome in bands 3 and 4. However we have shown that chromosome band 3 of WB- 1B is 1.5 Mb when compared with yeast chromosomes in CHEF separated gels. It is apparent that FIGE may underestimate the size of some Giardia chromosomes when compared with yeast and Giardiu chromosomes separated in CHEF. Nevertheless it has proved very useful in separating Giurdia chromosomes, specifically the duplication in chromosome bands 3 and 4 of BRIS/S3/HEPU/I06, and initiating these preliminary mapping studies. In 1989 we (Upcroft et al., 1989) showed that although Giardia chromosome separations deteriorated when subjected to electrophoretic runs longer than 48 h, chromosome band 4 of the strain BRIS/S3/ HEPU/106 could be seen to separate into two bands; chromosome band 4, identified by group 1 probes in this report and another identified here with group 2 probes. Thus we have separated at least seven different

615

chromosome bands in this strain by FIGE and have separated eight using CHEF. From the simple patterns of DNA fingerprint bands that we observe in strains used in this study, we conclude that they are effectively clonal (Upcroft, 1991). Furthermore, continual culturing of some strains does not lead to an alteration in the DNA fingerprinting pattern or numbers of characteristic bands when compared with the cloned line WB-1B (Upcroft, Upcroft & Boreham, 1990; Upcroft, Mitchell & Boreham, 1990; Boreham, Upcroft & Upcroft, 1990). Thus the results presented here are concluded to have arisen from one predominant genotype in each stock or strain and are not a result of a heterogeneous population in the culture. Our conclusion is that chromosome band 3 in strains such as WB-IB, Ad113, OASI and BRIS/S7/ HEPU/782 is a cluster of at least two different chromosomes and, possibly, a third chromosome which is wholly or in part a duplication of one of these two chromosomes. Other strains, such as BRIS/S3/ HEPU/106 carry three different chromosomes belonging to the band 3 chromosome cluster, one a partial duplication of another. In strains such as these, genetic rearrangements have occurred which allow the electrophoretic separation of this cluster. It is unknown whether the duplication of the chromosome described here is advantageous to the parasite. An increase in copy number of a sequence involved in drug resistance might confer an advantage on the parasite. Indeed the probe :G6/ I is a marker for a rearrangement which is associated with the induction of drug resistance in the strain BRIS/S3/ HEPU/106 and is absent in some strains which grow poorly and fail to complete division (Upcroft et al., 1992). Thus it appears that chromosome rearrangements do occur in response to stress in Giardia. What appears as a uniform Giardia duodenalis karyotype from pulsed field gel studies can mask a variety of chromosomal rearrangements and genome plasticity involving a number of chromosomes. Additionally, chromosome localization and copy number of the rDNA genes vary from strain to strain (Upcroft, Healey, Mitchell, Boreham & Upcroft, 1990; Boothroyd, Wang, Campbell & Wang, 1987) and among subclones of WB (Le Blancq, Korman & van der Ploeg, 1991). Thus there appears to be a great deal of coding sequence mobility in the Giardia genome. In other organisms such activity usually underlies adaptive and evasive responses. Perhaps in the Giardia genome plasticity provides adaptation to its environmental niche in many different host species. Acknowledgements-This from the National

Health

work was supported by a grant and Medical Research Council of

616

J. A. UPCROFT. A. HEALEY and P. UPCROFT

Australia. We would like to thank Peter Boreham and our colleagues in the Giardia field for Giardia stocks. REFERENCES

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