Preparation and characterization of DNA from rodent malarias

LABORATORY MEETING

3

possible in the absence of oxygen; the nature of the final electron acceptor in these conditions is unknown.

? Substrate - - D

Rotenone

Antimycin A

V

V

. electron

transport

Cyanide

chain-

(Main branch) V ? acceptor V electron

V transport

chain

D 02

& (Minor branch) Substrate REFERENCES

BOWMAN, I. B. R., GRANT, P. T., KERMACK, W. O. & OGSTON, D. (1961). Biochem. J., 78, 472. BRYANT, C., VOLLER, A. & SMITH, M. J. H. (1964). Am. J. trop. Med. Hyg., 13, 515. FULTON, J. D. & SPOONER, D. F. (1956). ExpL Parasit., 5, 59. HOWELLS, R. E., PETERS, W. & FULLARD, J. (1969). Milit. Med., 134, 893. SCHEIBEL, L. W. & MILLER, J. (1969). Ibid., 1074. WARmmST, D. C. & BAGGALEY,V. C. (1972). Trans. R. Soc. trop. Med. Hyg., 66. We thank Professor W. Peters for encouragement. T h e study was supported by the Medical Research Council and in part by a grant from the U.S. Army Medical Research and Development Command, Department of the Army, under Contract DAJA37-72-C0489.

Preparation and characterization of D N A f r o m rodent malarias M. L. CHANCE,-~ D. C. W A R H U R S T , * V. C. BAGGALEYT AND W. P E T E R S t

*MRC Research Group on the Chemotherapy of Protozoal Diseases and Drug Resistance tDepartment of Parasitology, Liverpool School of Tropical Medicine, Liverpool L3 5QA D N A was extracted from white cell free preparations (BAGGALEY and ATKINSON, 1972) of erythrocytes from mice infected with various rodent malaria strains and species by the technique previously described (WARHURST et al., 1971). In addition to preparing D N A from the main extract, the compact surface layer of denatured protein ("skin") discarded in the original technique was homogenised in 1% sodium dodecyl sulphate and incubated with predigested pronase (1 mg./ml.) at p H 7 for 15 minutes at 37°C. This extract was adjusted to a density of 1.7 g./ml, with caesium chloride (CsC1), centrifuged at 30,000 g. for 15 minutes, and the skin discarded. D N A was recovered by diluting the CsC1 and sedimenting at 90,000 g. overnight. In some cases the preparations were further purified by CsC1 preparative centrifugation or hydroxyapatite column chromatography. T h e inclusion of the pronase digestion step resulted, in some cases, in the appearance of satellite species of D N A presumably released by the breakdown of some sequestering structure. T h e satellites of P. berghei N67 are D N A ase sensitive and ~ amylase resistant. T h e occurrence of these species is indicated in the Table. Further information on the presence and nature of satellite D N A in P. b. yoelii 17 × was obtained by hydroxyapatite column chromatography of denatured and renatured

4

LABORATORYMEETING

material. Presumptive renatured satellite, buoyant density 1.680 g./ml, was found in the 0.3M fraction.

Main D N A Parasite

P. b. berghei

Density g./ml.

Base composition % GC

SateUite D N A Density g./ml.

I

Base composition % GC

11 "2, 17"3

N (K173) NS NSL3 NK65 RC

• 683 '683 • 683 • 683 • 683

(2) (1) (2) (1) (2)

23" 5 23.5 23" 5 23-5 23"5

P. berghei

N67

• 685 (4)

25.5

1"671,1"677

P. b. yoelii

17X

• 683 (5)

23.5

?

P. vinckei

LPL5

• 683 (2)

23' 5

P. chabaudi

LPLS9

• 686 (2)

26-5

,, ,, ,, ,,

1.677 r

17 "3

The figures in parentheses represent the n u m b e r of determinations. Extraction of the satellite is not achieved on every occasion and it is difficult to make generalizations about its occurrence or distribution. GUTTERIDGEet al. (1971) have, however, noted the occurrence of a satellite D N A of similar buoyant density in P. knowlesi and P. falciparum. The occurrence of the satellite in these species and in P. chabaudi suggests that it is not directly related to chloroquine resistance. REFERENCES

BAGGALEY,V. C. & ATKINSON, E. M• (1972). Trans. R. Soc. trop. Med. Hyg. 66, 4. GUTTERIDGE,W. E., TRIGG, P. I. & WILLIAMSON,D. S . (1971). Parasitology, 62, 209. WARHURST, D. C. BAGGALEY, V. C• & ROBINSON, B. L. (1971)• Trans. R. Soc. trop. Med. Hyg., 65, 9. This work was supported in part by the Medical Research Council and in part by the U.S. Army Medical Research and Development Command, Department of the Army, under Contract DAJA37-72-C-0489.

U s e o f C F 12 c o l u m n s for p r e p a r a t i o n s o f D N A f r o m r o d e n t m a l a r i a s V. C. B A G G A L E Y AND E. M . A T K I N S O N (INTRODUCED BY ~r. PETERS)

Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool L3 5QA Leucocytes can be satisfactorily removed from monkey blood infected with Plasmodium knowlesi using sucrose gradients (WILLIAMSON and COVER, 1966), but the method is not as successful with malaria infected mouse Mood. The cellulose column method (FULTON and GRANT, 1956) used by COOK et al. (1969) for P. knowlesi preparations has been used for P. berghei (RIcHARDS and WILLIAMS, 1971)• A modification of this technique is useful in preparing pure D N A from rodent malarias. A plastic column (made from a 20 ml. syringe plunger) with a piece of fine nylon gauze fastened over the base, was packed to a depth of 6 cm. with 6.3 g. Whatman C F 12 (Cat. N. 11121). 20 ml. of heparinized infected blood (day 3 to day 7 after inoculation) was collected at 4°C, mixed with an equal volume of saline and applied to the dry C F 12 column, under slight pressure from a 30 ml. syringe, the needle of which was inserted through a tightly fitting rubber bung at the top of the column. By diluting the blood before putting it on the column less material was lost in the retention volume (20%) and the