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Journal of Clinical Microbiology, May 1999, p. 1656-1657, Vol. 37, No. 5
0095-1137/99/$04.00+0
LETTERS TO THE EDITOR
Structure of Leishmania Minicircle Kinetoplast DNA
Classes
 |
LETTER |
In a recent paper (5), Noyes et al. discussed a
nested-PCR-based approach for identification and analysis of
Leishmania kinetoplast DNA (kDNA) minicircle classes using
the analysis of minicircle variable-region (VR) restriction profiles.
The kDNA of trypanosomatids consists of catenated minicircle
(approximately 5 × 103 to 5 × 104
per associate) and maxicircle (20 to 50 per associate) molecules (8). It was shown that the minicircles encode guide RNA
(gRNA) molecules involved in the RNA editing of maxicircle cryptogenes (for a review, see reference 9). To date, only this
one important cellular function has been demonstrated for minicircles
but the enigma of their redundancy remains topical. The prognosticated number of minicircle classes in Leishmania is more than 60 (4). This number comes from the hypothetical number of
maxicircle cryptogenes requiring editing of pre-mRNA transcripts.
Recently the nonrandom distribution of minicircle classes has been
demonstrated for the following set of Leishmania tropica
isolates: MHOM/SU/74/SAF/K-27, MHOM/TU/85/TAT3,
MHOM/KE/84/NLB299, MRAT/IQ/72/MRCB-JBF, and MHOM/NA/76/ROSSI-II (3). Moreover, it has been proposed that some minicircle
classes might be preferentially amplified for expression of required
gRNA. The results obtained by Noyes et al. lead to the same conclusion. We think that analysis of the sequence (5) (GenBank
accession no. AF032997) (which is in fact a minicircle VR) for the
presence of specific gRNA genes (2a) or regulatory sequences
(2) may shed more light on the functional role of these molecules.
We have analyzed the presented sequence (5) (GenBank
accession no. AF032997) using the Parasite Genome Blast Server (5a) computational tool. The results of our search have
revealed a rather high level of homology of the L. infantum
MHOM/TN/80/IPT1 minicircle (5) with L. donovani
MHOM/IQ/88/RTC6 (clone 11; GenBank accession no. AJ010074), L. donovani MHOM/SD/85/FORSTER (clone 7; GenBank accession no.
AJ010080), and L. infantum (clone 10543; GenBank accession
no. Z32846) minicircle DNAs (up to 90, 68, and 68%, respectively). We
speculate that these minicircles might present one interspecies class
for expression of the one certain gRNA type.
All Trypanosomatidae kDNA minicircles are organized by a uniform scheme
and contain one or several highly conserved regions (CR). There are
three highly conserved blocks (CSB) within CRs: CSB1 (GGGCGT),
CSB2 (CCCCGTTC), and CSB3 (GGGGTTGGTGTA)
(with interspecies homology of ca. 90 to 100%). CSB1 and CSB3
are almost identical in all species investigated to date, whereas CSB2
is less universal (6, 7). Such an organization model
provides us with at least two different strategies for PCR analysis of minicircles with the outward-oriented PCR primers specific to CSB1 and
CSB3 regions (for amplification of VRs adjacent to the neighboring CRs)
(1) or to the CSB3 region (for amplification of CRs and VRs
simultaneously) (3).
Noyes et al. (5) used another two-step-PCR approach. The
first step was amplification of the minicircle template using the
nested CSB2 primers. The second round was performed by using CSB1-CSB3
primers. We think it would be of interest to discuss the possibility of
nonrandom amplification of a single minicircle class during the nested
(CSB2) PCR. Our experience shows that using outward-oriented CSB1 and
CSB3 primers leads to the detectable amplification of all minicircular templates.
| |
REFERENCES |
| 1.
|
de Bruijn, M. H., and D. C. Barker.
1992.
Diagnosis of New World leishmaniasis: specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA.
Acta Trop.
52:45-58[Medline].
|
| 2.
|
Fu, G., and A. A. Kolesnikov.
1994.
Leishmania gymnodactyli and Leishmania infantum minicircles contain the same guide RNA genes as do minicircles of Leishmania tarentolae.
Mol. Biochem. Parasitol.
67:171-174[Medline].
|
| 2a.
| Guide RNA Database. 19 October 1998, revision date.
[Online.] http://www.biochem.mpg.de/~goeringe/. [6 March 1999, last date accessed.]
|
| 3.
|
Kolesnikov, A. A.,
V. Y. Yurchenko,
L. P. Martinkina, and G. Schoenian.
1997.
A new class of mitochondrial minicircular DNA in Leishmania tropica.
Mol. Biol. (Moscow)
31:458-462.
|
| 4.
|
Maslov, D. A.,
N. R. Sturm,
B. M. Niner,
E. S. Gruszynski,
M. Peris, and L. Simpson.
1992.
An intergenic G-rich region in Leishmania tarentolae kinetoplast maxicircle DNA is a pan-edited cryptogene encoding ribosomal protein S12.
Mol. Cell. Biol.
12:56-57[Abstract/Free Full Text].
|
| 5.
|
Noyes, H. A.,
H. Reyburn,
J. W. Bailey, and D. Smith.
1998.
A nested-PCR-based schizodeme method for identifying leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan.
J. Clin. Microbiol.
36:2877-2881[Abstract/Free Full Text].
|
| 5a.
| Parasite Genome Blast Server. 3 February 1999, revision date. [Online.] Parasite Genome Blast Server computational
tool.
http://www.ebi.ac.uk/parasites/parasite_blast_server.html.
[4 March 1999, last date accessed.]
|
| 6.
|
Ray, D. S.
1989.
Conserved sequence blocks in kinetoplast minicircles from diverse species of trypanosomes.
Mol. Cell. Biol.
9:1365-1367[Abstract/Free Full Text].
|
| 7.
|
Sheline, C., and D. S. Ray.
1989.
Specific discontinuities in Leishmania tarentolae minicircles map within universally conserved sequence blocks.
Mol. Biochem. Parasitol.
37:151-157[Medline].
|
| 8.
|
Simpson, L.
1986.
Kinetoplast DNA in trypanosomid flagellates.
Int. Rev. Cytol.
99:119-179[Medline].
|
| 9.
|
Simpson, L.
1997.
The genomic organization of guide RNA genes in kinetoplastid protozoa: several conundrums and their solutions.
Mol. Biochem. Parasitol.
86:133-141[Medline].
|
| | | | |
V. Y. Yurchenko
E. M. Merzlyak
A. A. Kolesnikov
Department of Molecular Biology
Moscow State University
Vorob'evi Hills
119899 Moscow, Russia
|
| | | | |
L. P. Martinkina
Y. Y. Vengerov
Engelhardt Institute of Molecular Biology
Russian Academy of Science
117984 Moscow, Russia
|
| |
AUTHOR'S REPLY |
My colleagues and I thank Yurchenko et al. for their constructive and
useful letter and in particular for showing that the minicircle
sequence that we discovered is similar to a number of other minicircle
sequences from L. donovani and L. infantum. This
confirms that the sequence that we amplified was indeed of minicircle
origin and not an artefact.
Yurchenko et al. ask whether the ability to amplify a single minicircle
class is due to the nonrandom distribution of minicircles into the
different classes or alternatively to nonrandom amplification of
minicircle classes. In principle it seems likely that both these
factors are involved; however, we did not have time to investigate these points since our primary objective was to demonstrate the sensitivity of the nested PCR and not to investigate minicircle organization.
Nonrandom distribution of minicircles into the different classes has
been demonstrated, as Yurchenko et al. point out. Nonrandom amplification is also likely for two reasons. First, although the
amount of variability in the conserved region is small, it is possible
that there are sufficient differences between minicircle classes to
affect amplification efficiency of some of them. The greater effective
primer length in the nested PCR will increase the risk of not
amplifying some minicircle classes. Secondly, it seems likely that some
minicircle classes will be thermodynamically or kinetically favored in
the PCR, particularly given the extensive secondary structure of
minicircle DNA. These minicircles will be more efficiently amplified
and will be the major products of any PCR; they may be the only product
of the nested PCR given the very small quantities of template.
We attempted to minimize the risk of the first problem by designing the
primers from an alignment of a wide range of Leishmania species. Our alignment (Fig. 1) shows
that we used regions that are conserved across the main
Leishmania species. This strategy was based on the
assumption that regions that were conserved across many species would
also be conserved between classes in the same species. The inclusion of
multiple sequences from L. infantum suggests that this is
often the case. The complex fingerprints produced by the digestion of
the PCR products from amounts of template greater than 1 fg showed that
the nested PCR did indeed amplify multiple minicircle classes.
Furthermore, it has been shown that the six 3' bases of a primer are
the most important for specificity and that some mismatches in the 5'
region are tolerated by the Taq enzyme (1). Our
primers were anchored in regions in which the six 3' bases were
invariable. The alignment also shows that we designed the first set of
primers against both CSB2 and CSB3 and not against CSB2 alone, as
Yurchenko et al. seem to suggest.
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FIG. 1.
Alignment of the conserved region of
Leishmania kDNA minicircles showing the highly conserved
sequence blocks (bold). The primers used in the nested PCR are shown.
The reverse primers have been reversed and complemented. The alignment
shows that the regions selected for the primers are highly conserved
across the wide range of species used. It is therefore assumed that
they will also be conserved across most minicircle classes in a given
strain. The Genbank accession numbers of the strains are as follows:
L. braziliensis, M87315; L. guyanensis, M87316;
L. infantum 1, Z32847; L. infantum 2, Z32846;
L. infantum 3, Z35501; L. peruviana, M87317;
L. tropica, Z32843; L. infantum 4, Z35269;
L. infantum 5, Z35270; L. infantum 6, Z35272;
L. infantum 7, Z35273; L. infantum 8, Z35274;
L. infantum 9, Z35292; L. infantum 10, Z35501-2;
L. infantum 11, Z35501.
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|
The different efficiencies of amplification of different minicircle
classes were largely beyond our control; however, it may be interesting
to investigate the effect of different solvent systems, particularly
dimethyl sulfoxide, on the amplification.
Consequently, we think it is likely that our primers will not amplify
all minicircle classes and will not amplify any minicircle class in
proportion to its original abundance. This is a point that merits
further attention for the light it might shed on minicircle organization. Nevertheless, we would like to emphasize that our experiments showed that the nested PCR is the most sensitive method available for detecting and identifying kDNA without the use of isotopically labelled probes. This has recently been confirmed by
trials in Guatemala, where both L. braziliensis and L. mexicana are endemic. Furthermore, our data showed that the nested
PCR reproducibly amplified the same minicircle classes from different members of a presumed clonal population. Consequently, although some
selection may be involved it is reproducible and can be managed by
using suitable controls.
| |
REFERENCE |
| 1.
|
Caetano-Anollés, G.
1993.
Amplifying DNA with arbitrary oligonucleotide primers.
PCR Methods Appl.
3:85-94[Medline].
|
| | | | |
Harry Noyes
Liverpool School of Tropical Medicine
Pembroke Place
Liverpool L3 5QA, United Kingdom
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Journal of Clinical Microbiology, May 1999, p. 1656-1657, Vol. 37, No. 5
0095-1137/99/$04.00+0
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