Influence of heat shock, drugs, and radiation on karyotype of <i>Leishmania major</i>

, Seo, M; , Chun, D K; , Hong, S T; , Lee, S H

doi:1993.31.3.277

Korean J Parasito > Volume 31(3):1993 > Article

Original Article


Korean J Parasitol. 1993 Sep;31(3):277-283. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1993.31.3.277
Copyright © 1993 by The Korean Society for Parasitology


Influence of heat shock, drugs, and radiation on karyotype of Leishmania major
M Seo,D K Chun,S T Hong,^* and S H Lee
Department of Parasitology and Institute of Endemic Diseases, Seoul National University College of Medicine, Seoul 110-799, Korea.

Received July 03, 1993; Accepted July 22, 1993.
Abstract
Leishmaniasis is one of the important tropical diseases in the world. Although it is not prevalent in Korea, imported cases have been recorded. The karyotype of Leishmania sp. has been observed to be variable by localities or by strains, but the karyotype of a strain is known to be stable. This study was performed to observe if the karyotype of a Leishmania sp. would be changed under some stressful conditions. The karyotype, analyzed by pulsed field gradient gel electrophoresis, was not grossly changed by heat shock, chemotherapeutics, UV illumination, and gamma irradiation. Radiation destroyed the chromosomes mechanically, but subcultured organisms after irradiation showed unaffected karyotype. The present findings suggest that the karyotype of a Leishmania strain is so stable that it is not altered by temporary stimulation with heat, drugs, and radiation.

Figures


	Fig. 1 Population growth of Leishmania jamor in N.N.N. media at 25℃

Fig. 2
Field inversion gel electrophoresis (FIGE) gel run under the parameters of 1% agarose, 100 seconds forward and 50 seconds backwards, 105 V, 72 hours, 1/2 × TBE, 14℃. 1) Saccharomyces cerevisiae AB972: 2-6) L. major, 2) primarily cultured; 3) subcultured after 72 hours heat shock at 37℃. 4) 100 hours heat shocked; 5) 72 hours heat shocked, every other day; 6) 56 hours heat shocked. The karyotype is constant under various heat shock cultivations.

Fig. 3
Contour clamped homogeneous electric field electrophoresis (CHEF) gel run in 1% agarose under 90 V for 115 hours. Initial A time 50 seconds and final A time 300 seconds, A/B ratio 1, 1/2 × TBE buffer at 15℃. 1) Saccharomyces cerevisiae AB972: 2-9) L. major, 2) 37℃.1 week & room temperature (RT) 1 week; 3) 37℃.one overnight & RT 2 weeks; 4) RT 2 weeks; 5) cultured in media with trimethoprim-sulfamethoxazole (Bactrm ®, B) 10 µm/ml for 1 week; 6) cultured in media containing amphotericin B (Fungizone ®, F) 0.5 mg/ml 1 week; 7) culture in F 1 mg/ml for 48 hours & normal media 1 week; 8) control cultured at RT; 9) 1 week heat shock by alternate day. All lanes display the same band pattern.

Fig. 4
CHEF gel run in 1% agarose under 90 V for 115 hours. Initial A time 50 seconds and final A time 300 seconds. A/B ratio 1, 1/2 × TBE buffer at 15℃. 1) Saccharomyces cerevisiae AB972 size marker; 2-9) L. major, 2) control; 3) room temperature (RT) 2 weeks; 4) one week heat shock alternately; 5) 37℃.one overnight & RT 2weeks; 6) cultured in media with trimethoprim-sulfamethoxazole (Bactrim ®, B) 10 mg/ml for 1 week; 7) amphotericin B (Fungizone ®) 1 mg/ml for 48 hours & normal media 1 week; 8) UV overnight in a clean bench; 9) control. All samples are in the same pattern of bands.

Fig. 5
CHEF gel run in 1% agarose under 115 V for 96 hours. Initial A time 50 seconds and final A time 400 seconds. A/B ratio 1, 1/2 × TBE buffer at 15℃. 1) Saccharomyces cerevisiae AB972 size marker; 2-7) L. major, 2) control; 3) 30 Gy irradiated; 4) 60 Gy Gy irradiated; 5) 100 Gy irradiated; 6) 200 Gy irradiated; 7) 300 Gy irradiated. The lane 6 shows decreased amount of DNA in large bands and bands over 800 kb are missing on the lane 7.


	Fig. 6 CHEF gel run in 1% agarose under 115 V for 96 hours. Initial A time 50 seconds and final A time 400 seconds. A/B ratio 1, 1/2 ×TBE buffer at 15℃. 1) Saccharomyces cerevisiae AB972 size marker; 2-5) L. major, 2) control; 3) subcultured after cryopreservation; 4) 500Gy irradiated; 5) 1000 Gy irradiated. All of the bands irregularly degraded to be thick smear on lanes 4 and 5.


	Fig. 7 FIGE gel run under parameters of 1% agarose, 100 seconds forward and 50 seconds backwards, 95 V for 64 hours in 1/2 × TBE at 14℃. 1) Saccharomyces cerevisiae AB972 size marker; 2-4) L. major, 2) control; 3) 500 Gy irradiated; 4) 1000 Gy irradiated. All of the irradiated specimens show no discrete chromosomal bands.

Fig. 8
FIGE gel run under parameters of 1% agarose, 100 seconds forward and 50 seconds backwards, 95 V for 64 hours in 1/2 × TBE at 14℃. All lanes vere loaded with L. major samples, 1) 50 Gy irradiated; 2) subcultured after 50 Gy irradiated; 3) 100 Gy irradiated; 4) subcultured after 100 Gy irradiation; 5) 300 Gy irradiated; 6) subcultured after 300 Gy irradiation. All of the irradiated specimens show same pattern of chromosomal bands. The plugs on lanes 4 and 6 include less DNA amount than others.

Tables


	Table 1 Heat shock schedule for L. major in N.N.N. media


	Table 2 The effects of chemotherapeutics on L. major


	Table 3 Gamma irradiation on L. major in N,N.N. media

References


1.	Babiker HA, Creasey AM, Bayoumi RA, Walliker D, Arnot DE. Genetic diversity of Plasmodium falciparum in a village in eastern Sudan. 2. Drug resistance, molecular karyotypes and the mdr1 genotype of recent isolates. Trans R Soc Trop Med Hyg 1991;85(5):578–583.

2.	Giannini SH, Curry SS, Tesh RB, Van der. Size-conserved chromosomes and stability of molecular karyotype in cloned stocks of Leishmania major. Mol Biochem Parasitol 1990;39(1):9–21.

3.	Janse CJ, Ramesar J, van den Berg FM, Mons B. Plasmodium berghei: in vivo generation and selection of karyotype mutants and non-gametocyte producer mutants. Exp Parasitol 1992;74(1):1–10.

4.	Kidane GZ, Samaras N, Spithill TW. Cloning of developmentally regulated genes from Leishmania major and expression following heat induction. J Biol Chem 1989;264(7):4244–4250.

5.	Maresca B, Carratu L. The biology of the heat shock response in parasites. Parasitol Today 1992;8(8):260–266.

6.	Oliver JH Jr, Stanley MA. Effects of gamma radiation on spermatogenesis and fertility of male Amblyomma americanum (Acari: Ixodidae). J Parasitol 1987;73(2):309–313.

7.	Bishop RP, Miles MA. Chromosome size polymorphisms of Leishmania donovani. Mol Biochem Parasitol 1987;24(3):263–272.

8.	Samaras N, Spithill TW. Molecular karyotype of five species of Leishmania and analysis of gene locations and chromosomal rearrangements. Mol Biochem Parasitol 1987;25(3):279–291.