A multicopy, extrachromosomal DNA in Leishmania infantum contains two inverted repeats of the 27.5-kilobase LD1 sequence and encodes numerous transcripts

Molecular and Biochemical Parasitology, 55 (1992) 39-50 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00

39

MOLBIO 01804

A multicopy, extrachromosomal DNA in Leishmania infantum contains two inverted repeats of the 27.5-kilobase LD1 sequence and encodes numerous transcripts C y n t h i a A. Tripp*, W e n d y A. Wisdom, Peter J. Myler and K e n n e t h D. Stuart Seattle Biomedical Research Institute, Seattle, WA, USA (Received 16 January 1992; accepted 26 May 1992)

Leishmania D N A 1 (LD1) is a 27.5-kb sequence that occurs as an inverted repeat in a 55-kb multicopy, circular D N A in Leishmania infantum ITMAP263. The sequence is also found with a different genomic organization, possibly a tandem array, within a 1.5-Mb chromosome in all Leishmania isolates. About 26 stable transcripts of LDI sequence, ranging from 0.6 to 15 kb, are found in ITMAP263. Transcripts were detected from both strands of the entire LD 1 sequence, but the inverted repeat nature of the circular molecule prevented determination of whether transcription proceeded in one or both directions. Nine abundant transcripts (0.6-8.4 kb) from adjacent regions on the same strand of the repeat unit may represent mature mRNAs. One of these transcripts was shown to contain the 39-nucleotide spliced leader sequence characteristic of the 5' termini of trypanosomatid mRNAs. Several transcripts from the other strand of the repeat unit are also abundant and contain sequence complementary to some of the putative mRNAs. Less abundant, larger transcripts that span sequences encoding abundant mRNAs are also present, suggesting that transcription of LDI is polycistronic. Key words: Leishmania; Genomic organisation; Circular DNA; Multicopy DNA

Introduction

Leishman& species cause a wide range of human diseases of varying severity and clinical manifestations. The Leishmania genome appears to be diploid [1,2] containing numerous chromosomes [3], ranging in size from 200 kb to several megabase pairs. While different Leishmania species generally have distinctive molecular karyotypes [3,4], DNA rearrangements resulting in the generation of chromosome-sized linear DNAs [5] and amplification Correspondence address." K.D. Stuart, Seattle Biomedical Research Institute, 4 Nickerson St., Seattle, WA, 98109-1651, USA. Tel.: (206) 284 8846; Fax: (206) 284 0313, *Present address: Department of Pathology, Colorado State University, Fort Collins, CO 80523, USA. Abbreviations: EtBr, ethidium bromide; CsC1, caesium chloride.

of distinct regions of Leishmania DNA as extrachromosomal molecules [6] can result in intra-species variation in molecular karyotype. In the latter instance, some sequences are amplified in drug-resistant mutants or in wild-type stocks. Not unexpectedly, in drug-resistant Leishmania, the amplified sequences have been found to contain the genes encoding enzymes targeted by the drugs. For example, the dihydrofolate reductase/thymidylate synthase gene is amplified as R DNA in methotrexate resistant Leishmania [7,8]. Similarly, Leishmania resistant to tunicamycin overproduce N-acetylglucosaminyltransferase and contain an amplified extrachromosomal element (G DNA) that probably encodes the gene for this enzyme [9]. Amplification of H DNA has been re-ported in Leishmania resistant to methotrexate [7,10,11], arsenite [12], primaquine and terbinafine [13], as well as in unselected stocks

40

of Leishmania [10,11,14]. H DNA contains a gene related to the P-glycoprotein genes [15] involved in multiple drug resistance in mammals [16], although the mechanism of methotrexate resistance in Leishmania does not appear to be linked with this gene on the H circle [13,15]. Other multicopy circular DNAs have been detected in different stocks of Leishmania not intentionally subjected to drug selection [6]. These include the D D N A in Leishmania tropica [11], T D N A in Leishmania tarentolae [17], LD1 (or CD1) in Leishmania donovani, L. infantum and Leishmania amazonensis [18,19] and a related D N A in Leishmania mexicana [20]. The function(s) of these circular DNAs have not yet been identified. Almost all the extrachromosomal D N A s described in Leishmania have a homologous megabase-sized source chromosome [6], suggesting that they are generated by gene amplification. The structural organization of some of these amplified DNAs have been determined. The R region D N A that is amplified in methotrexateresistant L. major occurs as a direct repeat [7], while the H D N A in several species of Leishmania [7,10,11,14] and the D D N A in L. tropica [11] occur as inverted repeats with up to 5 kb of unique flanking sequence separating the repeats. Small (250-550 kb) linear amplified chromosomes have been described in several different isolates of Leishmania [3-5] and subsequently shown to contain LD1 sequence [5,18,21]. In addition to its presence in amplified circular and small linear molecules in some isolates, LD1 sequence, as well as other sequence from the small amplified linear chromosomes, is integrated in a 1.5-Mb chromosome(s) in all Leishmania stocks [5,18]. Thus, LD1 sequences can occur in three different genomic organizations [18]. We have cloned and mapped the entire 27.5-kb LD1 sequence and characterized the structural and transcriptional organization of circular LD1 D N A in L. infantum ITMAP263. We find that the multicopy, circular molecule consists of 2 copies of the 27.5-kb LD1 sequence arranged as an inverted repeat. We determined the transcription map and show that both strands

of the entire sequence are transcribed. A set of transcripts that are probably mature mRNAs, map to adjacent sites on the same strand of one repeat unit, suggesting that LD1 encodes at least 9 genes. Abundant transcripts from the opposite strand are also detected, the implications of which are discussed.

Materials and Methods

Leishmania parasites and in vitro cultivation. The stocks used in this study include L. infantum ITMAP263 clone 10, L. infantum LRC-L336, L. donovani 1S, L. donovani 1S clone 2D, L. donovani FBD5, and L. braziliensis M2903. Leishmania promastigotes were cultivated at 24°C [18], in either modified HOM E M media supplemented with 10% Serumax (Sigma) or in Schneider's medium supplemented with 100 units ml-1 penicillin, 100/~g ml-1 streptomycin, and 15% heat inactivated fetal calf serum (Gibco/BRL). Nucleic acid preparation. Total Leishmania D N A was prepared by embedding promastigotes in low melting ajgarose blocks at a concentration of 1 × 10"parasites per block (approx. 1.5 /~g of nuclear DNA) and lysing the cells with SDS and proteinase K [3]. Genomic D N A was also prepared as previously described [22]. To obtain large amounts of purified circular LD1, total D N A from L. infantum ITMAP263 promastigotes was fractionated on isopycnic CsCI gradients. Cells (approx. 6 × 10 l°) were washed in PBSG (10 m M Na2HPO4/10 m M NaH2PO4/145 m M NaCI/2% glucose), lysed by incubation with 50 #g m l - 1 proteinase K and 0.5% SDS for 4 h at 37°C, and dialyzed against 2 changes of 50 mM Tris, pH 8.0/0.1 M NaC1/10 m M EDTA for 24 h at room temperature. CsC1 was added to 1.55 ~ m l - ethidium bromide (EtBr) to 600 ~g m l - ' , and the sample centrifuged in a VTi 50 rotor at 45000 rev./min for 22 h at 20°C. The 3 bands present in the gradients were shown by slot-blot analysis to contain chromosomal DNA, LD1 circular DNA, and kinetoplast DNA, respectively [23]. Fractions

41

containing the circular LD1 (middle band) and chromosomal DNA (uppermost band) were pooled separately, recentrifuged, and refractionated. Samples were then extracted with 1butanol and dialyzed against several changes of TE (10 mM Tris, pH 8.0, 1 mM EDTA). DNA was precipitated by addition of sodium acetate to 0.3 M and 2 volumes of ethanol, washed with 70% ethanol, and subsequently resuspended in TE. Circular LD1 DNA was also isolated from L. infantum 1TMAP263 using an alkaline lysis purification procedure described previously [18]. LD1 DNA in this preparation migrating with an apparent size of 27.5 kb was gel-purified using an Elutrap apparatus according to the manufacturer's specifications (Schleicher and Schuell). Total RNA was purified from 2 x 10 9 L. infantum ITMAP263 promastigotes using an acid guan-

idinium phenol chloroform extraction [24].

Construction of clones. DNA purified from L. infantum ITMAP263 by alkaline lysis treatment was digested with EcoRI, BamHI, SalI, HindlII or KpnI, ligated into similarly restricted pBS or pBluescript S K - plasmid vectors (Stratagene) and transformed into Escherichia coli XL1-Blue (Stratagene) according to the supplier's recommendations. LD1 clones were identified by screening the transformants with 32p-labeled, gel-purified 27.5 kb LD1 DNA and subcloned using standard techniques. Two clones, H35 and $32, were prepared by gel purification of 3.5-kb HindlII and 3.2-kb SalI restriction fragments from alkaline lysis DNA, respectively; ligation into pBluescriptlI S K - , and transformation into E. coli SURE cells (Stratagene). Restriction maps and locations of

H CE , / c ,s ~'~

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/.~

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-N

40 kD 20 kb

x

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/

B

K

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~

"

-

S f3

B

Fig. 1. Restriction map of 55-kb circular LD1 molecule in L. infantum ITMAP263. The location of cloned LD1 sequences is shown inside the circle. The junctions of the 27.5 kb inverted repeats are demarcated by dotted lines. For simplicity, all clones are shown derived from one of the repeats. Restriction enzyme abbreviations: B, BamHI; C, ClaI; E, EcoRI; H, HindllI; K, KpnI; N, NotI; S, Sail; and Sf, SfiI.

42

the clones relative to the circular LD1 molecule are shown in Fig. 1. Electrophoresis and hybridization. Total genomic DNA, D N A fractionated on CsC1/ EtBr equilibrium gradients, and LD1 DNA prepared by the alkaline lysis method were digested with restriction enzymes according to the manufacturer's specification (BRL). Digested DNA was separated by agarose gel electrophoresis under non-denaturing conditions in TBE (90 mM Tris-borate/2 mM EDTA), or under denaturing conditions in alkali buffer (50 mM NaOH/1 mM EDTA) [25]. For enhanced separation of large circular DNAs, samples were electrophoresed at high field strength (5 V cm-1) in agarose/TBE gels. A 66-kb plasmid, pSM409, was included as a circular DNA marker in high field intensity gels. Following electrophoresis, DNA was transferred to Nytran (Schleicher and Schuell) according to the manufacturer's specifications, and hybridized as previously described [3]. Radiolabeled (3Zp) probes were prepared from gel-purified 27.5 kb LDI DNA or recombinant plasmid clones by random priming with mixed hexanucleotides [26]. Total RNA (8 #g) from L. infantum ITMAP263 was fractionated on 1.0% agarose gels containing 20 mM 4-morpholine propanesulfonic acid (Mops) and 0.66 M formaldehyde, transferred to Nytran and hybridized as described by Fourney et al. [27]. Radiolabeled, strandspecific probes were prepared from LD1 clones using T3 or T7 RNA polymerase according to the manufacturer's protocol (Stratagene).

Results

L. infantum I T M A P 2 6 3 contains LD1 sequences on a circular molecule. Previous analyses using pulsed field gradient electrophoresis [18,19,21] suggested that LD1 sequences in L. infantum ITMAP263 occur in multicopy, circular DNA molecules, as well as integrated into a megabase pair-sized chromosome(s) [18]. The presence of LD1 sequences within

circular molecules was confirmed by agarose gel electrophoresis under high field intensity. Supercoiled DNA molecules may be resolved from linear DNA molecules under these conditions. High field intensity gel electrophoresis of total DNA from L. infantum ITMAP263 (Fig. 2A, lanes 5 and 6), reveals an EtBr-staining band (small arrow) with properties (i.e. migration above the chromosomal DNA, large arrow) of a supercoiled circular DNA molecule. A similar molecule is seen in L. donovani 1S (lane 4), but not L. infantum LRC-L336, L. braziliensis M2903 or L. donovani 1S clone 2D (lanes 1-3, respectively). Hybridization with a LD1 clone probe ($32) (Fig. 2B) indicates that the circular molecule in L. infantum ITMAP263 (lanes 5 and 6) (and L. donovani 1S, lane 4) contains LD1 sequence. In these stocks, the intense hybridization signal in the well (Fig. 2B, asterisk) represents nicked circular LD1 molecules which do not migrate out of the agarose blocks under these conditions. Hybridization to LD1 sequences integrated into chromosomal DNA (Fig. 2B, large arrow) is seen in all stocks, but is most intense in L. braziliensis M2903 (lane 2) and L. donovani 1S clone 2D (lane 3), which contain multicopy LD1 sequences in a small chromosome (250 and 200 kb, respectively) [3,18]. The circular LD1 molecule contains two inverted repeats o f a 27.5-kb sequence. Circular molecules containing LD1 sequences from L. infantum ITMAP263 can be enriched by alkaline lysis treatment or by CsC1/EtBr gradient centrifugation. Digestion of circular LD1 molecules from isopycnic CsC1/EtBr gradients with various restriction enzymes results in fragments that add up to different apparent total sizes. For example, EcoRI digestion (Fig. 3, lane 2) produces fragments of 18.5, 13.0, 2.4, and 1.3 kb (a total of 35.2 kb). The results are explained by the organization of LDI sequences as inverted 27.5 kb repeats within the circular molecule. The restriction fragments spanning the junctions between inverted repeats are responsible for the apparent variation in the sum of restriction

43

B

A 1

2

3

4

5

6

1

2

3

4

5

6

-'k kb 66-

48.5-

9.46.6"

Fig. 2. Analysis of the multicopy circular LD1 molecule by electrophoresis at high field intensity. (A) EtBr stained gel after electrophoresis (0.6% agarose, 5.0 V c m - l, 5 h) of agarose blocks containing Leishmania DNA (approx 1.5 #g per block). Lane (1) L. infantum LRC-L336 (LD1 sequences integrated into a 1.5-Mb chromosome only); lanes (2) and (3) L. braziliensis M2903 and L. donovani 1S clone 2D, respectively (LD1 sequences in a 1.5 Mb chromosome and integrated into 250 and 200 kb chromosomes, respectively); lane (4) L. donovani 1S (LD1 sequences in both a circular DNA and the large chromosome); lanes (5) and (6) L. infantum ITMAP263 (LD1 sequences in both a circular DNA and the large chromosome). (B) The gel in panel A was blotted and probed with LD1 clone $32. The small arrows indicate the circular LD1 molecules, the large arrows indicate chromosomal DNA, and the asterisk denotes DNA remaining in the wells. Size markers include undigested 2DNA (48.5 kb), HindlII restricted 2DNA (9.4 and 6.6 kb), and pSM409, a supercoiled, 66-kb plasmid DNA.

fragments, since these are represented only once per molecule while other fragments occur twice. The 13.0- and 2.4-kb EcoRI fragments (Fig. 3, large arrows) which are seen only in strains containing circular LD1 molecules (compare lanes 1-4 with lanes 5 and 6) represent such junction fragments (see Fig. 4A). Their presence in chromosomal ,DNA (lane 3) from the CsC1/EtBr gradients reflects contamination with nicked circular LD 1 DNA. The migration of the circular LD 1 molecule on high field intensity gels (slightly ahead of the 66-kb plasmid marker, Fig. 2) are consistent with a size (55 kb) resulting from two inverted copies of the 27.5-kb LD1 sequence. Denaturation of nicked circular 55-kb LD1 molecules during preparation by alkali lysis resulted in folding back of the inverted repeats of each individual strand to form molecules which migrate as double-stranded, 27.5-kb linear DNA. EcoRI digestion of alkaline lysis

DNA (Fig. 3, lane 1), resulted in the same restriction pattern seen with digestion of circular LD1 molecules from CsC1/EtBr gradients, plus 6.5- and 1.2-kb fragments (Fig. 3, asterisks). These fragments are homologous to (but are half the apparent size of) the l 3.0- and 2.4-kb junction fragments as previously noted [18], and thus are the result of their folding back upon themselves to form double stranded molecules (see Fig. 4B). The 1.2-kb and 2.4-kb EcoRI fragments were gel purified and electrophoresed under denaturing and non-denaturing conditions (Fig. 5). The 2.4- and 1.2-kb fragments migrated as expected (relative to double stranded DNA markers) in nondenaturing gels (Fig. 5A, lanes 1 and 2, respectively), but both fragments migrated with an apparent size of 2.4 kb (relative to single stranded DNA) in denaturing gels (Fig. 5B). Similar analysis of the adjacent 1.3-kb EcoRI fragment (see Fig. 4B) showed that it

44

1.3 kb

2,4 kb ' 1.3 kb

A

1

2

34

56 +i ++!++i ++!i++++

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18.5 kb

-19.3 kb 13.0 kb

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E

!

-5.7

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H35

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C 1.3 kb ! E~E

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-0.7 Fig. 3. Genomic organization of LD1 sequences. Southern blot of EcoRI-digested DNA probed with LDI clones H35 and $85. Lane (1) LD1 DNA prepared by alkaline lysis treatment of L. infantum ITMAP263 (LD1 sequences in circular DNA); lanes (2) and (3) L. infantum ITMAP263 DNA from the middle (circular DNA) and uppermost bands (chromosomal and nicked circles) of the CsC1/EtBr equilibrium gradient, respectively; lane (4) L. infanturn ITMAP263 genomic DNA (LD1 sequences in multicopy circular molecule and large chromosome); lane (5) genomic DNA from L. donovani IS clone 2D (LD1 integrated in both a 1.5-Mb and a small multicopy chromosome); lane (6) genomic DNA from L. donovani FBD5 (LDI integrated in a large chromosome only). The large arrows indicate 13.0-kb and 2.4-kb EeoRI fragments containing the junctions between inverted repeats in the 55-kb circular molecule. The small arrow indicates a 7.7-kb fragment containing the junction between these sequences in integrated LDI sequence. The asterisks indicate the 6.5- and 1.2-kb fragments seen only in the 27.5-kb alkaline lysis LD1 DNA. Molecular weight markers were derived from a BstElI digest of 2DNA.

18.5 kb

E

7,7kb

E E

-'II

I.

J . .' .' .

H35

$8,5

H35

"

Fig. 4. The organization of LDl sequences in the circular and integrated forms. Restriction maps of LDI sequences in the 55-kb circular molecule (A), 27.5-kb doublestranded, fold back molecule formed during alkaline treatment of nicked circular LDI molecules (B), and chromosomal integrated copy (C) are shown indicating the size of EcoRI (E) fragments. The locations of the probes used in Fig. 3 are indicated by the thin barred lines. The thin lines in panel B indicate that only a single 55-kb strand is shown, folded back on itself. The dotted lines in panel C indicate the boundaries of the of 27.5 kb LDI tandem repeat unit. m i g r a t e d w i t h t h e s a m e a p p a r e n t size (1.3 k b ) o n b o t h n o n - d e n a t u r i n g a n d d e n a t u r i n g gels ( d a t a n o t s h o w n ) . T h u s , b o t h t h e 1.2- a n d 2.4kb bands represent two inverted copies of a 1.2-kb repeat, but the 2.4-kb band contains b o t h s t r a n d s in a d u p l e x , w h i l e t h e 1 . 2 - k b b a n d c o n t a i n s a single s t r a n d o f t h e 2 . 4 - k b f r a g m e n t in a f o l d e d b a c k d u p l e x ( F i g . 4B). S i m i l a r results were obtained with fragments which mapped to the other end of the 27.5-kb LD1 sequence (data not shown), confirming that the 1.2- a n d 6 . 5 - k b f r a g m e n t s s e e n o n l y in a l k a l i n e lysis D N A a r e a c o n s e q u e n c e o f p r e p a r a t i o n b y a l k a l i n e lysis. L D 1 sequences in large c h r o m o s o m e s are not

45

B

A 1

chromosome(s) are organized in a tandem linear array of the 27.5-kb sequence (Fig. 5C). The 7.7-kb EcoRI band was also seen in digests of total DNA from a Leishmania stock which contains LD1 sequences in a small (<550 kb), multicopy chromosome (Fig. 4, lane 5), indicating that the amplified LD1 sequences in this stock have a similar organization.

2

1

2

2.4 kb

Fig. 5. The multicopy circular LD1 molecule contains inverted repeats. Gel-purified 2.4 kb (lane 1) and 1.2 kb (lane 2) EcoRl fragments were separated on non-denaturing (A), or denaturing (B) agarose gels, blotted and hybridized with LDI clone H35.

organized as inverted repeats. LD1 sequences in L. infantum ITMAP263 are present in a multicopy circular DNA and in megabase pairsized chromosome(s) [18]. Hybridization of cloned LD1 probes to EcoRI-digested total Leishmania genomic DNA (Fig. 3, lanes 4-6) or chromosomal DNA from CsCI/EtBr gradients (lane 3) revealed a restriction fragment (7.7 kb, small arrow) not present in digests of circular LD 1 DNA from either CsCI gradients (lane 2) or alkaline lysis preparation (lane 1). This indicates that the organization of LD1 sequences integrated into chromosomes differs from that within circular molecules. The 7.7-kb EcoRI fragment is detected by both H35 and $85 probes which hybridize with the 6.5 kb and 1.2 kb inverted repeat sequences, respectively (see Fig. 4B). Similar results are obtained with other restriction enzymes. Thus, these sequences (which are at opposite ends of the inverted repeats in the circular molecule) are adjacent to each other in the chromosomal DNA. One possible explanation for these results is that LD1 sequences integrated into the large

LD1 sequences encode numerous transcripts in L. infantum ITMAP263. In order to assess the gene coding capacity of LD 1 sequences in L. infantum ITMAP263, we probed Northern blots with strand-specific probes from a series of LD1 clones encompassing all of the 27.5-kb LD1 sequence. Representative results using 6 probes (clones El3, H30, BH5, B30, S10 and $32) covering most of the 27.5-kb sequence are shown in Fig. 6. While riboprobes from both strands of each clone were used separately, analysis of the results was complicated by the inverted repeat nature of the circular LD1 molecule. Sequences detected by each riboprobe would be present on both strands of the circular LD1 molecule. Each riboprobe hybridized with several transcripts, some of which were detected by adjacent probes (e.g., a 3.0-kb transcript was detected by strand I probes from BH5 and B30, and a 6.5-kb transcript hybridized with strand II probes from these clones). Some transcripts were detected by probes from both strands of the same clone (8.4- and 6.3-kb transcripts hybridized with both strands from El3 and H30, and 10- and 7.0-kb transcripts were detected by both strands of S10 and $32). When RNA from stocks which contain LD1 sequences integrated into large chromosome(s) alone was probed, the transcripts were not detected, or were present at much reduced levels [23], indicating that the transcripts are derived from the circular LD1 molecule. Similar results obtained with riboprobes from numerous LD1 clones (data not shown) enabled us to construct an initial transcription map for the multicopy 55-kb circular LD1 DNA (Fig. 7). The map shows the size, relative abundance, location, and strand to which the

46

E13 II

I

9 . 5 :,,.-

7.5"-

H30 I

BH5 II

I

$10

B30 II

I

II

I

S32 II

I

II

I0.0

9.5 ~7.5~-

8.4 6.3

ao zo 5.0

44:""

4.4

24~-

1.4~,-

1.4 ~"

i!~ii !~i~i i ~ ~

~ ~

~ ~ ~i ~

i i ii i!ii~ii I

Fig. 6. Circular LD1 molecules encode numerous transcripts. Northern blots of total R N A from L. infantum ITMAP263 were hybridized with strand-specific probes (labeled I or II) from the LDI clones indicated. The location of the clones and strands I or II are indicated in Fig. 7. Sizes of the high-molecular-weight R N A markers (BRL) are indicated in kb, and the sizes of the major transcripts are indicated.

RNAs map. There appear to be about 26 transcripts, varying in size (0.6-15 kb) and abundance, encoded by LD1, covering both inverted repeat units of the 55-kb LD1 molecule. While we have shown only one strand of the molecule being transcribed, the inverted repeat nature of the molecule makes it impossible to determine whether the other strand of the molecule is also transcribed (in the opposite direction), since both sets of transcripts would appear identical. Nine adjacent transcripts, ranging in size from 0.68.4 kb, mapping to strand I of the 27.5 kb inverted repeat (Fig. 7, thickest lines) are more abundant, and thus, some are probably mature mRNAs. Northern blot analysis using poly(A) + RNA showed that these transcripts are polyadenylated and PCR amplification of the 0.6-kb transcript (data not shown) indi= cated the presence of the 39 nucleotide spliced

leader (SL) sequence [28] at the 5' end, which is characteristic of all trypanosomatid messenger RNAs. A n u m b e r . o f less abundant, overlapping transcripts, generally larger in size, mapped throughout the 55-kb molecule and may represent processing precursors and intermediates. These results suggest that sequences from both strands of the inverted repeat are transcribed. Indeed, five transcripts (8.4, 6.3, 5.0, and two measuring 3.5 kb) from strand II of the inverted repeat sequence are present in relatively high abundance (Figs. 6 and 7), and are not detected in strains lacking the circular LDI molecule.

Discussion

We have determined the genetic organization of the amplified LD1 sequence in L.

47

J Fig. 7. Transcription map of the 55-kb circular LDI molecule in L. infantum ITMAP263. The dashed lines demarcate the inverted repeat units. The location, size and relative abundance (indicated by the thickness of the lines) of transcripts detected by Northern blot analysis are shown. Transcripts on the right half of the molecule were detected by strand I probes, those on the left were detected by strand II probes. The location of clones used as probes are indicated by thin barred lines within the circle.

infantum ITMAP263 and report here that it occurs as inverted repeats of a 27.5-kb sequence within a 55-kb multicopy, circular molecule. The circularity of the multicopy DNA was confirmed by a variety of methods: anomalous migration on PFGE, presence in DNA prepared by alkaline lysis of L. infantum ITMAP263, banding in CsC1/EtBr equilibrium gradients, and separation in high field intensity agarose gels. Restriction fragments corresponding to the junctions between the 27.5 kb inverted repeat units within the circular molecule (3.5-kb HindlII and 3.2-kb SalI fragments) have been cloned (clone H35 and clone $32, respectively) and shown to contain inverted repeats by restriction mapping and preliminary DNA sequencing (unpublished

results). Restriction mapping of cellular DNA and LD 1 clones as well as sequence analysis of the latter suggest that the inverted repeats within the circular DNA molecule are joined directly to each other. This contrasts to the organization seen in other multicopy circular DNAs in Leishmania: H DNA in methotrexate-resistant L. major [7,11], L. tarentolae [10,14] and L. tropica [11]; and D DNA in unselected L. tropica [11], in which there is a region of unique sequence that separates the inverted repeats. The unusual inverted repeat organization of the circular molecule resulted in the production of an apparently linear, 27.5kb LD1 molecule following alkaline lysis treatment [18]. Circular 55-kb LD1 molecules that were nicked or cleaved denatured during

48

alkali treatment, and the two inverted repeats within the single D N A strands reannealed to form double stranded molecules which migrated as 27.5-kb linear D N A (see Fig. 4A and B). The resistance of these molecules to S 1, ExoIII, and BAL 31 nucleases substantiates their unusual, non-linear structure and confirms the absence of unique intervening sequence, which would be sensitive to S1 nuclease. LD1 sequences are found in a 1.5 Mb chromosome in L. infantum ITMAP263 and in all other Leishmania stocks examined [5,18], suggesting the circular LDI molecules are generated by amplification of endogenous sequences. Southern blot analyses of the integrated LD1 sequence indicates that it has a different genetic organization from the circular molecule. Sequences at opposite ends of the sequence t h a t i s inverted in the circular molecule are adjacent to each other in the chromosomal DNA. Thus, the LD1 sequences integrated into the large chromosome(s) may be organized in a tandem linear array (see Fig. 4C). The LD1 sequences that are present in a 1.5 Mb chromosome in all Leishmania are amplified in different molecules in several different stocks [5,18,20]. Two different circular molecules have been seen: the 55-kb inverted repeat described here and elsewhere [18,19] in L. infantum, L. donovani and L. amazonensis, and a 26-kb molecule which has been described in L. mexicana M379 [20,29]. While these molecules share some sequence, they both appear to contain sequence not present in the other [29]. Multicopy small (180-550 kb) chromosomes containing LD1 sequences have been described in L. major [5], L. mexicana [20], L. donovani, Leishmania chagasi, Leishmania braziliensis, and Leishmania amazonensis [18]. In addition, the chromosomes amplified in L. major have been shown to contain sequences which are adjacent (within 40 kb) to the LD1 sequences in the 1.5-Mb chromosomes, but are not amplified in the 55-kb circular molecule [5] Thus, it appears that the LD1 and adjacent sequences within the 1.5 Mb chromosomes act as a 'reservoir' which can be amplified in a number of different ways. Although both

circular [20] and linear [5] molecules have been shown to arise spontaneously in the laboratory, a role for the amplified LD1 sequences in drug resistance cannot be ruled out, since most of the Leishmania stocks examined were isolated from humans who were likely to have been treated with drugs. Further studies will be required to characterize the integration site of LD1 within the large chromosome(s) and elucidate the mechanism of amplification. Northern blot analysis of LD1 sequences in L. infantum ITMAP263 indicates there are nine abundant, polyadenylated transcripts from one strand of the 27.5-kb LD1 sequence derived from the circular inverted repeats. At least one of these transcripts (0.6 kb) contains the 39 nucleotide SL sequence characteristic of the 5' termini of trypanosomatid mRNAs. Analysis of cloned LD1 sequences (unpublished data) shows open reading frames (ORFs) corresponding to several of these transcripts, suggesting that most are mature m R N A s that encode proteins. Several transcripts that are generally larger and less abundant than the putative mRNAs, map throughout the circular LD1 molecule. Some of these RNAs may represent processing precursors and/or intermediates. The transcripts from the complementary strand of the 27.5-kb sequence may represent read-through transcription from the putative m R N A coding strand since this half of the molecule is complementary to the putative m R N A coding strand. Several transcripts (see Fig. 7) span the junction between the inverted repeats that would be used by such read-through. Thus, these initial analyses suggest that circular LD1, or each strand thereof, may be a single polycistronic transcription unit. Of particular interest are the abundant 6.3and 8.4-kb transcripts from the strand that is complementary to the putative m R N A coding strand. These RNAs are the same size and map to similar positions as two transcripts from the putative coding strand, but are transcribed from the opposite strand and are thus 'antisense' RNAs. A similar situation has been reported for R region transcription [30].

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Other RNAs (e.g., two 3.5 and one 5.0 kb RNA) are also abundant 'antisense' RNAs but their size and/or coding location does not match that of the nine putative mRNAs. Although the 6.3- and 8.4-kb transcripts are abundant, sequence analysis indicates they contain no sizeable ORFs [23], indicating a role other than protein coding function.

Acknowledgements We thank Dr. Arnold Bendich for the 66-kb pSM409 plasmid DNA. This work is supported by NRSA AI08116 to C.T. and NIH grant AI24771 to K.S. who is a Burroughs Wellcome scholar in Molecular Parasitology.

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