Abstract The maxicircle control region [also termed

divergent region (DR)] composed of various repeat

elements remains the most poorly studied part of the

kinetoplast genome. Only three extensive DR se-

quences demonstrating no significant similarity were

available for trypanosomatids (Leishmania tarentolae,

Crithidia oncopelti, Trypanosoma brucei). Recently,

extensive DR sequences have been obtained for

Leishmania major and Trypanosoma cruzi. In this

work we have sequenced DR fragments of Leishmania

turanica, Leishmania mexicana, Leishmania chagasi

and two monogenetic trypanosomatids Leptomonas

seymouri and Leptomonas collosoma. With the emer-

gence of the additional extensive sequences some

conserved features of DR structure become evident.

A conserved palindromic sequence has been revealed

in the DRs of the studied Leishmania species, L. sey-

mouri, and T. cruzi. The overall DR structure appears

to be similar in all the Leishmania species, their rela-

tive L. seymouri, and T. brucei: long relatively GC-rich

repeats are interspersed with clusters of short AT-rich

repeats. C. oncopelti, L. collosoma, and T. cruzi have a

completely different DR structure. Identification of

conserved sequences and invariable structural features

of the DR may further our understanding of the

functioning of this important genome fragment.

Keywords Leishmania Æ Leptomonas Æ Kinetoplast

maxicircle Æ Divergent region Æ Palindrome

AbbreviationsCSB-I, -II, -III Conserved sequence blocks-I, -II, -III

DR Divergent region

gRNA Guide RNA

Introduction

The divergent region (DR) was initially described as a

variable and presumably non-coding segment of the

kinetoplast maxicircle (Borst et al. 1980, 1982; Stuart

and Gelvin 1982; Muhich et al. 1983; Maslov et al.

1984). It proved to be resistant to cloning (Stuart and

Gelvin 1982; Simpson 1986) and sequencing. However,

complete or partial DR sequences for a few species

(Leishmania tarentolae, Crithidia oncopelti, Trypano-

soma brucei, Table 1) were obtained several years la-

ter. The sequences were composed of various repeat

arrays, and the DR structure seemed to be drastically

different in various species. Studies of the DR func-

Communicated by S. Hohmann

Nucleotide sequence data reported in this paper are available inthe GenBankTM, EMBL and DDBJ databases under the acces-sion numbers DQ107351, DQ107352, DQ107354-DQ107358,DQ239759-DQ239765, DQ492251-DQ492256.

Electronic Supplementary Material Supplementary materialis available to authorized users in the online version of thisarticle at http://dx.doi.org/10.1007/s00438-006-0145-5

P. N. Flegontov Æ Q. Guo Æ L. Ren Æ A. A. Kolesnikov (&)Department of Molecular Biology,Lomonosov Moscow State University,Vorobjevy Gory 1, build. 12, 119992 Moscow, Russiae-mail: sasha@protein.bio.msu.ru

M. V. StrelkovaDepartment of Medical Protozoology,Martsinovsky Institute of Medical Parasitologyand Tropical Medicine, Sechenov Moscow MedicalAcademy, M. Pirogovskaya 20, 119830 Moscow, Russia

Mol Gen Genomics (2006) 276:322–333

DOI 10.1007/s00438-006-0145-5

123

ORIGINAL PAPER

Conserved repeats in the kinetoplast maxicircle divergent regionof Leishmania sp. and Leptomonas seymouri

Pavel N. Flegontov Æ Qiang Guo Æ Lina Ren ÆMargarita V. Strelkova Æ Alexander A. Kolesnikov

Received: 5 May 2006 / Accepted: 22 June 2006 / Published online: 15 August 2006� Springer-Verlag 2006

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Mol Gen Genomics (2006) 276:322–333 323

123

tioning were also started. Promoters for the 12S rRNA

gene were roughly localized in the DR of T. brucei

(Michelotti et al. 1992) and Leptomonas seymouri

(Vasil’eva et al. 2004). Sequences essential for kine-

toplast DNA replication (conserved sequence blocks,

CSB I–III) were also found within the DR (Horvath

et al. 1990; Myler et al. 1993). But most research pro-

jects dealing with the DR were not completed, and the

DR remains the most poorly studied part of the kine-

toplast genome.

Partial but extensive DR sequences of Leishmania

major have been obtained by our group recently

(Flegontov et al. 2006); also maxicircle non-coding

regions of Trypanosoma cruzi strains CL Brener and

Esmeraldo have been assembled from whole-genome

shotgun sequences (Westenberger et al. 2006). In

addition, potential promoters have been located in the

DR of L. seymouri (Vasil’eva et al. 2004). Initially, the

5¢-terminal sequence of the 12S rRNA gene primary

transcript was determined by Primer Extension, Elu-

tion, Tailing, and Amplification (PEETA). By tran-

scription tests in isolated kinetoplasts (in organello) it

was demonstrated that a short fragment (24 bp)

immediately upstream of the transcription start site is

essential for transcription initiation. A fragment com-

prising the 5¢-end region of the primary transcript

(about 300 bp) and some part of the upstream se-

quence (from 200 to 24 bp) was cloned in a plasmid

vector. Then a linear construct containing this frag-

ment flanked by a vector sequence was obtained by

PCR and introduced into the isolated mitochondria of

L. seymouri. Transcription of this construct was as-

sessed by RT-PCR with transcript- and vector-specific

primers. It was demonstrated that an upstream se-

quence as short as 24 bp is still capable of promoting

effective transcription, shorter sequences being abso-

lutely ineffective (Vasil’eva et al. 2004).

Then a partial transcription map for the L. seymouri

maxicircle was constructed using hybridization, RT-

PCR, and RACE methods (Bessolitsyna et al. 2006 in

manuscript). According to this map some intergenic

spacers (before the ND7, ND1, COII, COI, ND4, and

RPS12 genes) must contain transcription initiation

sites. These spacers were also shown to promote tran-

scription in an in organello system (using the procedure

described in the previous paragraph) (Bessolitsyna

et al. 2006 in manuscript). The comparison of all such

transcription-promoting fragments revealed a con-

served motif containing the A5–6C sequence and sev-

eral TGW sequences upstream and downstream of the

A5–6C sequence. Moreover, the sequence A5CTTGT

has been shown to promote transcription of an artificial

construct (a tested sequence inserted into a plasmid

sequence) in the isolated mitochondria of L. seymouri

(E. Merzlyak et al. 2006, unpublished data).

In this work a nearly complete DR sequence of

L. seymouri and partial DR sequences of Leptomonas

collosoma, Leishmania turanica, Leishmania mexicana,

and Leishmania chagasi have been obtained (Table 1).

Now that the set of the available sequences has been

considerably expanded, a thorough comparison of the

DR sequences in different trypanosomatids becomes

possible. Preliminary results of such comparison have

already been reported for L. major, L. tarentolae, L.

(mexicana) amazonensis, and L. seymouri (Flegontov

et al. 2006). Here we provide a detailed analysis of all

the available DR sequences, which demonstrates that

the overall DR structure is quite conserved in the

species of the Leishmania–Leptomonas group (the

clade with slow-evolving 18S rRNA sequences

according to Merzlyak et al. 2001) (Fig. 1). T. brucei

belonging to a distant clade has a somewhat similar DR

structure. Furthermore, a conserved sequence (about

150–200 bp long) was found in the DRs of the inves-

tigated Leishmania species. PCR screening of various

Leishmana species revealed that this sequence may be

present in all members of this genus. A part of this

sequence is also shared by Leishmania sp. and L. sey-

mouri. A conserved sequence was also revealed in the

DR of two T. cruzi strains (Westenberger et al. 2006).

We believe that identification of conserved sequences

and invariable structural features of the DR will lead to

better understanding of the functioning of this poorly

studied but important genome fragment.

Materials and methods

Isolation of the Leptomonas seymouri and

Leptomonas collosoma DR fragments

Leptomonas seymouri ATCC 30220 and L. collosoma

ATCC 30261 promastigotes were cultured in the

STAR medium as previously described (Maslov et al.

1982). Kinetoplast DNA extraction and restriction

mapping of maxicircles were conducted according to

(Maslov et al. 1982). Restriction fragments were cloned

in the pBluescript II SK (KS)+ vector. The strategies of

DR fragments isolation (see Fig. S1 for L. seymouri;

Fig. S2 for L. collosoma) and PCR amplification

primers used for that purpose (Table S3) are described

in the Supplementary materials. A commercial kit

(Isogen, Moscow, Russia) designed for hot start

PCR was utilized for all amplification reactions. The

324 Mol Gen Genomics (2006) 276:322–333

123

reaction mixture contained Taq DNA polymerase

(1 U), primers (0.5 lM), total DNA (about 0.1 lg),

dNTPs (200 lM), MgCl2 (2.5 mM), and PCR-buffer

with betaine (1.5 M). Most reactions were carried out

according to the following program: 94�C—5 min;

(92�C—20–30 s; 42–55�C—30 s–1 min; 65�C—1–

5 min) · 30–35 cycles. The annealing temperature was

by 5–7�C lower than the primer melting temperature

according to the nearest-neighbor thermodynamic

formula. The amplification time was calculated in the

following way: 1 min/1 kb + 1 min. PCR-products

were purified using QIAquick PCR purification kit or

QIAquick gel extraction kit (QIAGEN, Hilden, Ger-

many) and sequenced on ABI Prism 310 and 3100

automated sequencers (Applied Biosystems, Warring-

ton, UK).

Divergent region sequences of the other species

The partial DR sequence of L. major and the com-

plete sequence of C. oncopelti were determined in

our laboratory and reported previously (Horvath

et al. 1990; Flegontov et al. 2006). Promastigotes and

amastigotes of several L. major strains were investi-

gated but only the longest sequence (obtained from

the strain MHOM/IL/2003/LRC-L952 at the pro-

mastigote stage) was used here for the interspecific

comparison. DR fragments (adjacent to the 12S

Fig. 1 The neighbor-joining majority consensus tree of trypan-osomatids (Kimura 2-parameter substitution model; gaps notexcluded; phylogeny tested by bootstrap method with 500replicates) inferred from partial 18S rRNA sequences (thealignment was constructed by ClustalW algorithm according toMerzlyak et al. 2001). Euglena was used as an outgroup(according to Hughes and Piontkivska 2003). Only bifurcationpoints with bootstrap values greater than 50% are shownproducing a majority consensus (condensed) tree. Dashed linesindicate species (notably Trypanosoma vivax, Leptomonascollosoma, Blastocrithidia triatoma) which position on the treevaries significantly depending on the tree construction method

applied (see also Merzlyak et al. 2001; Hughes and Piontkivska2003). Species with the available DR sequences are marked bytriangles; species with conserved palindromic sequences in theDR are marked by filled triangles. Stable clades are marked byletters: L(L) subgenus Leishmania (Leishmania), L(S) subgenusLeishmania (Sauroleishmania), L(V) subgenus Leishmania(Viannia), Lep the clade of typical insect trypanosomatidsincluding many Blastocrithidia (Bl.), Crithidia (C.), Leptomonas(Lep.), and Wallaceina (W.) species, H typical Herpetomonas(H.) species, P Phytomonas (P.), End the clade of endosymbiont-containing species, T Am the clade of American trypanosomes, TAf the clade of African trypanosomes

Mol Gen Genomics (2006) 276:322–333 325

123

rRNA gene) of L. turanica (reference strain MRHO/

UZ/83/KD051), L. mexicana (strain MHOM/??/94/

BEHA), and L. chagasi (reference strain MHOM/

BR/74/PP75) promastigotes were isolated following

the same procedures as employed for L. major

(Flegontov et al. 2006). The complete DR sequences

of T. brucei, T. cruzi and partial sequences of

L. tarentolae and L. (mexicana) amazonensis were

obtained from GenBank (Table 1). In the latter case

one short DR fragment (clone 29) was isolated from

arsenite- or tunicamycin-resistant clones; the other

(clone 94) from the wild-type strain LV78 (Lee et al.

1994).

Sequence analysis

Sequence alignment was carried out using the Clu-

stalW algorithm employed by the AlignX (Vector-

NTI 8 package) or MEGA 3.1 programs and the

Jotun Hein algorithm employed by the MegAlign

program (DNAStar 99 package). The following pro-

grams were employed for sequence analysis and

annotation: VectorNTI (annotation; detection of re-

peats, guide RNA genes, and conserved elements)

and BioAnnotator (detection of repeats, GC-profile

determination) of the VectorNTI 8 package; Gene-

Quest (identification of repeats and bent regions) of

the DNAStar 99 package. Intrinsically bent regions

were predicted according to the bending wedge

model (method parameters: arc length 100 bp,

threshold angle 72�). The method computes the

helical trajectory over the arc length. At the same

time, the angle formed by two vectors parallel to the

helical midpoint at the ends of the arc is measured.

All raw sequence files, alignments, and annotated

sequences are available upon request from Alexander

A. Kolesnikov. Appropriate accession numbers are

listed in Table 1.

PCR screening of Leishmania species

The investigated species and strains are listed in the

Supplementary materials (Table S4). The following

amplification primers were used: 12SR, DRF, DRR

(see Table S3). The latter two primers (DRF, DRR)

are complementary to each other and anneal within the

conserved DR fragment (Introduction, see also Fle-

gontov et al. 2006); the former (12SR) anneals within

the 5¢-end region of the 12S rRNA gene. Three

amplification reactions were performed with each

sample: DRF-12SR, DRR-12SR, and DRF-DRR.

For detailed reaction conditions see Isolation of the

L. seymouri and L. collosoma DR fragments.

Results

Structure of the divergent region in Leptomonas

seymouri and Leishmania sp.

The DR sequences of L. seymouri, L. tarentolae, L.

chagasi, L. mexicana, L. (mexicana) amazonensis, L.

major, and L. turanica (Table 1) were analyzed for the

presence of repeats, conserved elements (including

potential promoters, see Introduction), potential bent

regions and gRNA genes. Sequences involved in

transcription of several maxicircle genes of L. seymouri

(Bessolitsyna et al. 2006 in manuscript) including the

12S rRNA gene (Vasil’eva et al. 2004) have been

mapped recently, and a potential promoter consensus

(including the A5–6C sequence) has been described

(see Introduction). Multiple sequences (termed Le-

ptomonas-like, potential promoters or A5C-elements)

resembling this consensus have been found in the DRs

of the studied species. Moreover, their location ap-

pears to be non-random (see below).

Despite the initial discovery of intrinsic DNA

bending in the minicircles of L. tarentolae (Marini et al.

1983; Ntambi et al. 1984), maxicircles were never

analyzed for the presence of bent DNA. We performed

computer-assisted search for potential bent regions in

the maxicircle sequences of the studied species. It was

revealed that DNA bending is more pronounced in the

divergent than in the coding region (data not shown).

Long bent fragments in the DR were associated with

certain repeat types (see below).

The DRs of the studied Leishmania species and L.

seymouri are composed of alternate GC- and AT-rich

regions and share the same general arrangement of

repeats (Fig. 2a–c). Three repeat families occurring in

all seven species may be delineated (Table 2): (1) long

relatively GC-rich (25–50% GC) conserved repeats

containing three or four potential promoters and a long

predicted bent region (type I repeats); (2) shorter, also

relatively GC-rich (20–40% GC), less conserved re-

peats containing one or two possible promoters and

usually no bent regions (type II repeats); (3) very short

AT-rich (0–5% GC) repeats arranged in tandem clus-

ters of variable length and composition.

The repeats described above are arranged in a spe-

cific fashion, i.e., they form ‘‘superclusters,’’ which

have the following structure: a type I copy followed by

several type II copies separated by clusters of AT-rich

repeats (Fig. 2a–c). Such organization (long inter-

spersed repeats alternating with clusters of short re-

peats) is rarely observed in mitochondrial control

regions and appears to be unique for the maxicircle

control (divergent) region.

326 Mol Gen Genomics (2006) 276:322–333

123

Type I repeats

These repeats seem to be the most conserved part of

the DR. Type I copies are nearly identical within the

DR of a single strain and in different L. major (Fle-

gontov et al. 2006) and L. turanica strains. Further-

more, 5¢-end sequences of type I repeats are conserved

in the studied Leishmania species and the related

species L. seymouri. PCR-tests with DR-specific

primers have demonstrated that this conserved se-

quence is probably present in many species of the

Leishmania, Sauroleishmania, and Viannia subgenera

(see Supplementary materials, Table S4). A nearly

perfect palindrome (47 bp long for Leishmania sp.,

39 bp for L. seymouri) with two A5C-elements at its

center occurs at the 5¢-ends of at least some type I

copies (Fig. 3). Remarkably, this palindromic sequence

is the most conserved.

At some distance from the 5¢-end the sequence of

type I repeats becomes less conserved (Fig. S5). Se-

quence similarity is greater (about 200 conserved bp)

among L. mexicana, L. (mexicana) amazonensis, L.

major, L. turanica, and L. chagasi, which agrees with

the observed phylogenetic relationship (Schonian et al.

1996; Momen and Cupolillo 2000; Hughes and Pion-

tkivska 2003; see also Fig. 1). L. tarentolae belonging to

Fig. 2 The maps of the sequenced portions of the Leishmaniatarentolae (a), Leishmania major strain MHOM/IL/2003/LRC-L952p (b), Leptomonas seymouri (c), Trypanosoma ruzi strainsCL Brenner and Esmeraldo (d), Trypanosoma brucei strainEATRO 164 (e), Leptomonas collosoma (f), and Crithidiaoncopelti (g) DRs. All symbols are explained in the upper box(different symbols used on maps d–g are explained in the lowerbox). Predicted bent fragments are indicated by lines and

rectangles below the maps; A5C-elements are indicated byarrows above and below the maps. The squared A5C-elementoverlaps with the fragment essential for the 12S rRNA genetranscription initiation in Leptomonas seymouri (Vasil’eva et al.2004). Known and predicted (in the case of Leptomonasseymouri) gRNA genes and CSBs (designated I–III for CSB-I–III, respectively) are also mapped. A scale in kb is given in themiddle of the picture

Mol Gen Genomics (2006) 276:322–333 327

123

a different subgenus (Sauroleishmania) has a more

divergent sequence. Type I repeats in L. seymouri

share a shorter conserved sequence (about 80 bp) with

type I repeats in the studied Leishmania species, which

also agrees with the phylogenetic data (Maslov et al.

2001; Merzlyak et al. 2001; Hughes and Piontkivska

2003; see also Fig. 1).

Type I repeats contain at least three A5C-elements

in all the investigated species. Forward and reverse

A5C sequences are always located within the palin-

drome at the 5¢-end of the repeat sequence. One or two

forward or reverse A5C-elements are also located near

the 3¢-end (Fig. 2a–c, Table 2). However, these motifs

are absent in L. (mexicana) amazonensis. Type I copies

contain potential bent DNA stretches (about 280 bp in

L. tarentolae, 150–200 bp in L. seymouri, 50–60 bp in

L. major), which are usually absent from the other

repeats.

Type II repeats

Repeats of this class are always shorter than type I

repeats (Table 2). Their sequences are rather diver-

gent even within a single molecule and do not have

any significant similarity in different species. Despite

this sequence heterogeneity, type II repeats also

have some distinctive features. In L. tarentolae and

L. seymouri they usually contain only one forward

A5C sequence near the 3¢-end. In L. seymouri some

copies also include another forward A5C-element at

the 5¢-end. L. major type II sequences contain a

reverse A5C-element near the 3¢-end; a forward

A5C-element is frequently located at the 5¢-end or

within an upstream AT-rich cluster (Fig. 2b). In the

other species a reverse A5C-element is located in the

middle of the repeat sequence. Unlike type I

sequences type II repeats rarely contain bent DNA

fragments. Several type II copies usually follow a

Table 2 Repeat families peculiar to Leptomonas seymouri and Leishmania sp.

Species Type I repeats Type II repeats AT-rich clusters

Length(bp)

Number ofA5C-elements

BentDNA

Length(bp)

Number ofA5C-elements

BentDNA

Repeat unit Cluster length(bp)

Leptomonas seymouri 620–683 3F, 1R + 138–175 1-2F – A2T3–4 54–15142 bp 187–443

Leishmania tarentolae 434 2F, 1R + 239–294 1F – A3T2 36–95AATAATAT 268–297

Leishmania major 366–433 1F, 2R + 135–259 (1F), 1R – A4–6T, A3T2–3 62–158ATATT 121

Leishmania (mexicana)amazonensis WT

530 1F, 1R + ? ? ? A3T1–3 83? ?

Leishmania (mexicana)amazonensis R

295a ? + 144–177 (1F), 1R + A3–4T1–2, GATA 144–160? ?

Leishmania mexicana 370a 1F, 2R + 177 1F, 1R – A2–5T1–2 66–263? ?

Leishmania turanica 450a 1F, 2R + 149–204 1R – A5–7T1–2 119–274? ?

Leishmania chagasi 420a 1F, 2R – 193 1R – A1–5T1–5 90–124? ?

a The repeat sequences are incomplete at the 5¢-end, and probable repeat length has been calculated on the basis of the alignment withthe complete Leishmania tarentolae and Leishmania major type I repeat sequences. The number of A5C-elements (1F, one forwardelement; 1R, one reverse element, etc.) and the presence of long predicted bent DNA tracts (longer than 50 bp) within a repeat areindicated

Fig. 3 The most conserved sequence within type I repeats inLeishmania sp. (located at their 5¢-end). A possible hairpinconformation is shown. The positions marked by an asterisk arevariable. Leishmania tarentolae has a T–A pair; Leishmania(mexicana) amazonensis and Leishmania major have a G–C pairat the *K–M* position; most copies contain A, not C, at the M*position. The sequence AAAAAC is marked by an arrow

328 Mol Gen Genomics (2006) 276:322–333

123

type I copy within repeat superclusters. In L. seymo-

uri single inverted type II copies are located upstream

of type I repeats (Fig. 2c).

Other long repeats

Some repeated sequences resemble neither type I nor

type II repeats. Such unclassified repeats are not ob-

served in the partial DR sequences of Leishmania sp.

but have been described in the more extensive se-

quence of L. seymouri (Fig. 2a–c). L. seymouri

unclassified repeats fall into two groups. Rather short

relatively GC-rich type III repeats (about 70 bp long;

20–30% GC) separated by short AT-rich variable

spacers form a long cluster near the ND5 gene

(Fig. 2c). This cluster contains only one A5C sequence

but includes two long (213 and 76 bp) potential bent

fragments. Another peculiar tandem cluster is located

downstream of the type III cluster. It is composed of

three nearly identical copies (209–211 bp long) termed

CSBII-repeats (Fig. 2c). They contain the CSB-II

(C3GTTC) sequence typically occurring within the

minicircle origin of replication along with CSB-I and

CSB-III (Ray 1989). CSBII-repeats are not associated

with bent DNA, but each contains three forward A5C-

motifs.

Short AT-rich repeats

Short repeats take up about 30% of the DR sequence.

They are arranged in tandem clusters composed of

heterogeneous copies (Table 2). Most AT-rich clusters

(‘‘AnTn clusters’’) lie upstream of type II copies. In L.

major some AT-rich clusters contain A5C-elements.

Somewhat different AT-rich clusters (‘‘ATAT clus-

ters’’) are located upstream of type I repeats (Table 2,

Fig. 2a–c). They are generally longer and are com-

posed of AATAATAT, ATATATAA or ATATT re-

peat units (in L. tarentolae, L. (mexicana) amazonensis,

and L. major, respectively). In L. seymouri these

clusters are composed of longer divergent repeat units

(about 40 bp). Six long AT-rich regions have been

mapped to the DR of L. tarentolae (Simpson et al.

1982); two of them have been sequenced and proved to

be composed of AATAATAT repeats (Muhich et al.

1985; Simpson et al. 1987) (Fig. 2a).

Guide RNA genes

Genes for gRNAs are in some cases (e.g. in Leish-

mania sp.) located not only in minicircles, but also in

maxicircles. For example, gRNA genes have been

mapped to the ND5-proximal (the gG4-IV and

gRPS12-VI genes) and 12S-proximal (the gM150 and

gND3-I genes) parts of the L. tarentolae DR (Fig. 2a).

We have located a sequence homologous to the L.

tarentolae gG4-IV gRNA gene downstream of the ND5

gene of L. seymouri (Figs. 2c, 4). This gRNA gene has

similar position and sequence in both species. An A5C-

motif is located upstream of the potential gG4-IV gene

in L. seymouri and is incorporated into the gene in L.

tarentolae (Fig. 4).

Structure of the Leptomonas collosoma divergent

region

Leptomonas collosoma is rather distant from the

Leishmania–Leptomonas clade (Merzlyak et al. 2001)

and may even cluster with American trypanosomes,

although its position on the tree varies considerably

when different phylogenetic algorithms are applied

(Fig. 1). So it is not surprising that the L. collosoma

DR has a unique repeat arrangement (Fig. 2f).

Extensive AT-rich regions are lacking; long tandem

repeats take up the most part of the DR. A5C-elements

are rare: only three sequences occur upstream of the

12S rRNA gene. Multiple potential bent fragments (up

to 130 bp) were also detected. The 5¢-part of the DR

contains at least four peculiar sequences strongly

resembling the minicircle conserved region (see the

next section).

Conserved sequence blocks

The minicircle conserved region always contains three

almost invariable sequences (termed CSBs): CSB-I

(G3CGT), -II (C3GTTC), and -III (G4TTGGTGTA)

(Ntambi and Englund 1985; Ntambi et al. 1986; Ray

1989). CSB-III and CSB-III-like sequences were found

in the DRs of C. oncopelti (Horvath et al. 1990), T.

brucei (Myler et al. 1993), L. seymouri (Fig. 2c), and L.

collosoma (Fig. 2f). CSB-elements are clustered in a

repeat-free region near the center of the L. seymouri

Fig. 4 The alignment of the Leptomonas seymouri and Leishmania tarentolae gG4-IV gRNA genes and upstream sequences. Theputative promoter sequences A5C are marked by arrows. Nucleotide substitutions within the gene are typed in bold

Mol Gen Genomics (2006) 276:322–333 329

123

DR. They have the following arrangement: a CSB-III

sequence followed by four dispersed CSB-I copies and

two CSB-III copies on the complementary strand

(Fig. 2c). Three A5C-elements and several potential

bent fragments are also located within the repeat-free

segment. The DR of L. collosoma contains four se-

quences nearly identical to the minicircle conserved

region sequence and, therefore, including the complete

set of the CSB sequences (Fig. 2f). These minicircle-

like sequences overlap with potential bent regions.

Discussion

Overall structure of the divergent region

The DRs of only three Kinetoplastida species (L. tar-

entolae, C. oncopelti, T. brucei) were extensively se-

quenced until recently. No conserved motifs could be

traced in the available sequences. In this work con-

served repeat arrangement in the DR has been iden-

tified in several trypanosomatid species. These findings

pave the way for studies of the DR functioning, which

is virtually unknown today.

The investigated species of the Leishmania–Lepto-

monas group (Fig. 1) have a unique structure of the

maxicircle control region: long relatively GC-rich re-

peats separated by AT-rich spacers of variable length.

This arrangement is rarely observed in mitochondrial

control regions. However, the DR of T. brucei (Myler

et al. 1993) has a somewhat similar structure (Fig. 2e).

Its 5¢-end part (segment I according to Myler et al.

1993) contains long GC-rich repeats (type A according

to Myler et al. 1993) with less GC-rich spacers (type B

repeats according to Myler et al. 1993) between them.

These spacers have variable length (41–369 bp) and

are composed of various short repeats containing AnC

sequences. All type A copies have quite uniform length

(160–188 bp) and sequence. However, repeat supercl-

usters observed in the Leishmania–Leptomonas group

are lacking in T. brucei. The short repeats are also not

so AT-rich as in the case of Leishmania sp. and L.

seymouri.

Crithidia oncopelti (Horvath et al. 1990) of the

endosymbiont-containing clade (Fig. 1), L. collosoma,

and T. cruzi have a radically different DR structure

(Fig. 2d, f, g). Their DRs do not contain any extensive

AT-rich regions and are composed mainly of long

relatively GC-rich repeats arranged in tandem clusters.

Such a structure is quite common for mitochondrial

control regions, especially long ones. Thus, the DR

structure typical for the Leishmania–Leptomonas

group and T. brucei possibly appeared early in the

evolution of trypanosomatids. But then it apparently

disappeared in the L. collosoma, T. cruzi, and C. on-

copelti clades (Fig. 1). Investigation of additional try-

panosomatid species is needed to test this hypothesis.

Type I repeats

Type I copies have several distinguishing features: (1)

intra- and inter-specific sequence similarity: a quite

long (80–200 bp) conserved sequence containing a

palindrome (about 40 bp) is present; (2) distinctive

arrangement of A5C-elements (potential promoters):

two elements within the palindrome, the others near

the 3¢-end; (3) invariable association with relatively

long (at least 50 bp) predicted bent regions. All these

features are suggestive of some functional significance

of type I repeats, for example, they possibly take part

in transcription initiation. It is very interesting that the

most conserved element in the DRs of two T. cruzi

strains is also represented by a palindrome (39 bp)

(Westenberger et al. 2006) containing two A5C se-

quences and overlapping with potential bent fragments

(Fig. 2d). This conserved motif is a part of long repeats

in both CL Brenner and Esmeraldo strains (Fig. 2d).

Thus, the palindromic structure associated with A5C-

elements and bent fragments may be the most signifi-

cant element in the DR of many trypanosomatids.

The A5C-motif described here as a potential pro-

moter does not necessarily represent a promoter con-

sensus. On one hand, A5C-motifs occur in all DNA

fragments involved in maxicircle transcription initia-

tion (see Introduction) including the 24 bp fragment

upstream of the 12S rRNA gene (Vasil’eva et al. 2004)

and promote transcription in isolated kinetoplasts (E.

Merzlyak et al. 2006, unpublished data). On the other

hand, their promoter function has not been directly

demonstrated, therefore they may act as binding sites

for some transcription factors but not as actual pro-

moters. Additional studies are needed to clearly define

the maxicircle promoter consensus and to determine

the role of A5C sequences. Such studies will certainly

involve testing of various recombinant DR fragments

in an in organello transcription system. In any case,

A5C-elements are probably significant because they

are associated with conserved palindromes and non-

randomly located in the DR of some species. Long

primary transcripts extending into the coding region

may be transcribed from promoters within the DR.

These promoters may also function during DNA rep-

lication for the synthesis of short RNA primers. A set

of RNAs (from 0.5 to 2.3 kb) hybridizing with the DR

was revealed in C. oncopelti (Tarassoff et al. 1987). It

was also shown that transcription of the 12S rRNA

330 Mol Gen Genomics (2006) 276:322–333

123

gene starts within the DR in T. brucei (Michelotti et al.

1992) and L. seymouri (Vasil’eva et al. 2004). How-

ever, no DR transcripts were revealed by Northern

hybridization in L. tarentolae (Simpson et al. 1982;

Muhich et al. 1983). Undoubtedly, DR transcription

requires thorough investigation using more powerful

methods.

The long palindrome within type I repeats may as-

sume a hairpin conformation with an A5C-element

located at the end of the hairpin (Fig. 3). This

arrangement appears very interesting because pro-

moters are frequently associated with hairpins (Wad-

kins 2000), e.g., in the chicken (L’Abbe et al. 1991) and

the red alga Chondrus crispus (Richard et al. 1998)

mitochondrial DNA. In both cases promoters are lo-

cated in hairpin loop regions. Hairpin or cruciform

structures may also act as protein-binding sites (Wad-

kins 2000).

Transcription initiation sites are frequently associ-

ated with intrinsically bent DNA. Computer analyses

demonstrate that bent DNA sequences often occur

upstream of open reading frames (VanWye et al. 1991)

and strong Escherichia coli promoters (Plaskon and

Wartell 1987). Some studies have directly shown that

DNA bending is often associated with promoters

(Tanaka et al. 1991; Gaal et al. 1994). AT-rich up-

stream bent DNA may act as a transcriptional activator

(Bracco et al. 1989; Gartenberg and Crothers 1991).

However, bent DNA in the DR may have a totally

different function. It has been proposed that curved

DNA fragments determine the structural organization

of the kinetoplast DNA (Marini et al. 1983; Silver et al.

1986). Bent fragments of minicircles are usually ex-

posed at the surface of the kinetoplast DNA disc

contributing to packaging of these molecules (Silver

et al. 1986). Thus, bent fragments in the DR of maxi-

circles may play a certain structural role.

Type II and AT-rich repeats

These elements do not contain any sequences con-

served at the genus level. However, their general

arrangement is constant in the studied species (see

above). Thus, some general features like alternate GC-

and AT-rich segments rather than a defined sequence

may be essential for the DR functioning. Nearly all

type II copies contain at least one A5C-element (Ta-

ble 2, Fig. 2a–c). It has been shown that RNA

polymerases bind preferentially (and non-specifically)

with AT-rich tracts (Brack and Delain 1975; Grellet

et al. 1981; Gabrielsen and Oyen 1982; Beritashvili

et al. 1989; Harada et al. 1999; Tang et al. 2005). Thus,

such tracts may bind a pool of RNA polymerase

molecules waiting for ‘‘deployment’’ at nearby pro-

moters. The presence of long AT-rich tracts in the

vicinity of most A5C-elements, especially upstream of

the conserved palindromes in Leishmania sp. and

L. seymouri, agrees with this hypothesis.

Putative origin of replication

It has been demonstrated that the CSB-III sequence

acts as an origin of replication on the leading strand of

minicircles; CSB-I—on the lagging strand (Ryan et al.

1988). Despite the difference in replication mecha-

nisms between minicircles and maxicircles (Ryan et al.

1988; Shapiro and Englund 1995; Gull et al. 1997;

Lukes et al. 2005), minicircle CSBs are thought to play

a role also in the replication of maxicircles (Shapiro

and Englund 1995). Therefore, CSBs present in the DR

may act as replication origins. The DR of L. collosoma

contains four segments nearly identical to the mini-

circle conserved region, which includes CSB-I–III. The

DR of L. seymouri does not contain such long mini-

circle-like sequences. However, it contains a unique

region comprising several CSB-elements.

Acknowledgments We thank G. Schonian for providing someDNA samples, S.J. Westenberger for providing alternativeassemblies of the T. cruzi DR, T.A. Akopian and K. Shidlovskyfor assistance with DNA sequencing. The research was partiallysupported by grants from the International Association for thePromotion of Cooperation with Scientists from the New Inde-pendent States of the Former Soviet Union (INTAS No.2001-216) and from the Russian Foundation for Basic Research(RFBR No.05-04-48895a).

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