J . Protozool., 29(3), 1982, pp. 438441 @ 1982 by the Society of Protozoologists

Ribosomal DNA of Leishmania brasiliensis: Number of Ribosomal Copies and Gene Isolation1

ELENA VILLALBA’ and JOSE LUIS RAMIREZ3 Departamento de Biologia Celular, Escuela de Biologia de la Universidad Central de Venezuela,

Apartado 47525, Los Chaguaramos 1041A, Caracas, Venezuela

ABSTRACT. The DNA content of the hemoflagellate Leishmania brasiliensis, strain Y , has been determined by colorimetric reactions and found to be nearly 0.226 pg/cell. When this DNA is bound to filters and hybridized with labeled rRNA from the same organism, saturation is reached at 0.47% of the DNA, corresponding to an estimated 160 ribosomal gene copies. When the DNA is sheared and centrifuged to equilibrium in CsCl gradients, two major satellites of the main band (p = 1.712 g/cm3) are observed: a heavy one (1.720 g/cm3), which hybridizes with labeled rRNA, and a light one (1.699 g/cm3) with the electron microscopic characteristics of the kinetoplast DNA network.

NDERSTANDING of host-parasite interactions in kine- U toplastids is seriously limited by our lack of knowledge about chromosomal morphology, genetic markers, and sexual exchange. Such a situation may be overcome by molecular studies of the genetic material of these organisms (1 , 17). In this context, the present work reports some basic characteris- tics of the genome of the hemoflagellate Leishmania brasilien- sis, strain Y, such as DNA content per cell, the number of gene copies for rRNA per cell, and, in addition, the possibility of the physical isolation of these genes.

MATERIALS AND METHODS Culture of cells. Our experimental organism, Leishmania

brasiliensis, strain Y, was first isolated in Venezuela by Dr. Felix Pifano of the Instituto de Medicina Tropical, Universidad Central de Venezuela. It was grown in our laboratory in bi- phasic Davies medium (25) at 28°C. Cells were usually collected at the end of the log phase, and cell counts were made with a Coulter counter, Model ZBI.

Isolation of total DNA. Cells were collected by centrifugation at 10,000 g for 10 min, and washed twice with an ice-cold 0.85% NaCl solution. The cells were lysed by resuspending in SDS buffer (10 mM Tris-HC1, 1 mM EDTA, 0.1 M NaCI, 0.2 M sodium acetate, 0.5% SDS, pH 7.4) at 4°C. The lysate was further treated as described by Dawid (7).

Isolation of rRNA. Cells were collected and lysed as men- tioned above. The lysate was centrifuged at 10,000 g for 10 min, and the supernatant solution was then centrifuged at 105,000 g for 2 h. The ribosomal pellet was resuspended in a 0.5 M NaCl solution and deproteinized by adding an equal volume of cold phenol-chloroform (1: I), 0.02% hydroxyquinoline in ANE buffer (0.1 M NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 8). This treatment was repeated several times. The aqueous phase was finally recovered and mixed with 2.5 volumes of cold ethanol and left overnight at -20°C; the rRNA was recovered by centrifugation at 20,000 g for 10 min, resuspended in 0.5 M NaCl, and kept at -70°C. All solutions were autoclaved except detergents, which were incubated at 65°C for 1 h.

Sucrose gradients for rRNA purijication. The rRNA used in hybridization reactions was routinely purified by centrifugation in a linear 15-30% sucrose gradient in 0.5% SDS, 0.1 M NaCl, 10 mM Tris-HCI, 1 mM EDTA, pH 7.4. The gradient was cen- trifuged at 80,000 g for 18 h at 20°C. Fractions corresponding to the major rRNA species were pooled and precipitated by

Supported by a research grant from the C.D.C.H. de la Universidad

Present address: Facultad de Ciencias de la Universidad Centro-

To whom all correspondence should be addressed.

Central de Venezuela No C-O1.1/78.

Occidental, Coro. Edo. Falcon.

adding cold ethanol as before. This rRNA was essentially free of 4 and 5 s RNA.

Gel electrophoresis of rRNA. Flat composite polyacrylam- ide-agarose gels (2.7%-0.5%) were prepared in acetate buffer (40 mM Tris-HC1, 0.2 M sodium acetate, 2 mM EDTA, pH 8) and fractionated at 20 mA for 12 h with the same buffer, plus 0.5% SDS. The gels were stained with an ethidium bromide solution (5 pg/ml) and observed in a U.V. transilluminator (Ul- tra-Violet Products Inc., California). Photographs were taken with a Kodak 25A red filter.

Filter hybridization. DNA was fixed to S & S nitrocellulose filters as described by Ivanov & Markov (13). Hybridization conditions were those reported by Dawid (8). Samples were either counted in a gamma counter or in a scintillation counter, both from Packard Instruments Co.

CsCl equilibrium gradients. DNA samples were mixed with a CsCl stock solution (1.790 g/cm3 in 20 mM Tris-HC1, 1 mM EDTA, pH 8) and adjusted to a density of 1.700 g/cm3 in a final volume of 5 ml. The centrifugation was performed either in a Beckman SW-65 or Ti-50 rotor at 33,000 g for 60 h at 20°C. The gradient was fractionated after piercing the tube at the bottom. Aliquots were taken for both refractive index deter- minations and radioactive counting, while the remainder of the fractions was used for filter hybridization.

DNA colorimetric estimation. The determination of total DNA was carried out as described by De Torres & Pogo (9).

Electron microscopy. The fractions of the light satellite were dialyzed against TE-buffer (10 mM Tris-HC1, 1 mM EDTA, pH 8), and prepared for electron microscopy following the proce- dure of Davis et al. (6).

Radioactive labeling of rRNA and DNA. The labeling in vivo of DNA was done by adding 20 pCi/ml of [6-3H]-thymidine to a culture in early log phase, and then left for three days before collecting the cells. For RNA labeling, 20 pCilml of [32Pl-or- thophosphate were added to the cell culture and left for two days before processing.

rRNA was also labeled in vitro by iodination with [1251]-io- dine, according to Commerford (3) as modified by Prensky et al. (18).

TABLE I. DNA content per cell estimated by colorimetric reactions.

Total DNAkell Experiment cell number a DNA (Pd

I 1.36 x 109 3 54 0.260 2 8.6 x 108 188 0.218 3 4 x 108 128 0.320

438

r R N A

Ecoli

VILLALBA AND RAM~REZRIBOSOMAL DNA OF LEISHMANIA

Mda 1.2

-0.83 -0.63 -0.54

Ilt

Fig. 1. Flat composite 2.7% acrylamide-0.5% agarose gel electro- phoresis of partially purified Leishmania brasiliensis (Y) rRNA. The sample was not heated. The Escherichia coli rRNA used as a standard was fractionated in the same gel. Details in Materials and Methods.

E C

0 W N a

I

I0 20 30 40 40

BOTTOM FRACTION NUMBER TOP Fig. 2. Sedimentation profile of partially purified rRNA from L.

brusiliensis (Y). 100 p g of rRNA were layered on top of the gradient. Labeled rRNA of E. cofi (broken line) was included as an internal standard. Details in Materials and Methods.

43 9

1.8

1.7

1.6 1.8

- m

1.7 5 \ 0, v

4 1.6 1.8

I .75

I .7

1.6 5 I 0 I 5 20 25 30

BOTTOM FRACTION NUMBER TOP

Equilibrium CsCl gradients of [6-3H]-thymidine labeled DNA for buoyant density determination. From top to bottom are shown: a) Micrococcus lysodeikticus DNA, b) L. brusiliensis (Y) total DNA, and c) M . lysodeikticus plus L . brusiliensis (Y) DNA. The density line represents the linear regression analysis of the values. The cen- trifugation was performed in a Beckman SW-65 rotor. Details in Ma- terials and Methods.

Fig. 3.

RESULTS Ribosomal RNA. The rRNA from L. brasiliensis (Y) shows

three major bands after electrophoresis in 2.7% polyacryl- amide-0.5% agarose composite gels (Fig. 1). Their molecular weights, using Escherichia coli rRNA as a standard, are 0.83, 0.63, and 0.54 Mdaltons. A fourth band of higher molecular weight is usually observed when samples are not heated prior to electrophoresis. This component corresponds to the nondis- sociated rRNA from the larger ribosomal subunit (3) since it disappears when heated, thus enriching the other rRNA species. Furthermore, when rRNA samples are heated and centrifuged in a 15-30% density sucrose gradient, a broad peak with a sedi- mentation coefficient of about 18s is observed (Fig. 2). This peak contains only the three species mentioned above.

Cell DNA content. The total DNA per cell was estimated in three experiments, the average content being 0.266 pg/cell _t

0.051 (mean * SD) (Table I).

440 J. PROTOZOOL., VOL. 29, NO. 3, AUGUST 1982

I 5 10 15 20 25

BOTTOM FRACTION NUMBER TOP

Fig. 4. Equilibrium CsCl gradients of [6-3H]-thymidine labeled DNA from L. brusiliensis. The different fractions were filter hybridized with [32P]-rRNA. The DNA profile was obtained by scintillation counting, whereas the radioactivity after hybridization was measured in a gamma counter (see Materials and Methods). Arrows indicate: 1. heavy sat- ellite, 2. main band, 3 . light satellite.

Isolation of rRNA in CsCl gradients. The buoyant density of the main DNA band of L . brasiliensis was calculated in CsCl equilibrium gradients with Micrococcus lysodeikticus DNA (1.731 g/cm3) as a density marker (21). Figure 3 shows three gradients made in parallel, from which the buoyant density of the main band was estimated in 1.712 g/cm3. When a similar experiment is performed and the different fractions hybridized with labeled rRNA, most of the radioactivity is localized at the heavy side of the main band (Fig. 4). The buoyant density for this radioactivity peak is about 1.720 g/cm3, which corresponds to a G+C content of 60% (20). The shoulder at the light side of the main band (buoyant density 1.699 g/cm3) was observed by electron microscopy to contain the typical kinetoplast DNA array. A better resolution of the different DNA bands is achieved when the centrifugation is performed using a fixed angle rotor like the Ti-50 (data not shown; see 10).

Number of rDNA copies per cell. The number of rDNA cop- ies was calculated on the basis of the fraction of DNA hybrid- ized when saturation with rRNA is achieved (Fig. 5). The sat- uration point was calculated from a reciprocal plot of the values shown in Fig. 5 (insert). The average value from three experi- ments was 0.47%, which is equivalent to 160 copies of rDNA, using the sum of the rRNA molecular weights (6.06 kilobases- 2 Mdaltons) as the unit, and correcting for the kinetoplast DNA content (15).

DISCUSSION The transcription products of the ribosomal genes of Leish-

mania brasiliensis, when analyzed by gel electrophoresis, ap- pear as three major bands with molecular weights of 0.83,0.63, and 0.54 Mdaltons. In Leishmania donovani the presence of a 5.8 S rRNA has also been shown (15). The above rRNA pattern seems to be common to all kinetoplastids studied so far (3, 5 , 1 1 , 15).

/ ? j 0.02 0.06 0.1 0.14 0.18

I / N g rRNA 0 I I I

I 2 3 4

rRNA DNA -

Fig. 5. Hybridization of purified rRNA from L . brusiliensis (Y) to total DNA. Nitrocellulose filters with 30 pg of DNA were incubated with increasing amounts of r2P]-rRNA. The specific activity of the rRNA was 20,000 cpdpg . The radioactivity has been corrected for background. Insert: reciprocal plot of the values corresponding to three independent experiments. Saturation was achieved when 0.47% of the DNA was hybridized. The number of nuclear ribosomal copies, cal- culated after subtraction of the kinetoplast DNA, was 160.

In L . brasiliensis (Y ) , an average of 160 copieskell code for these RNAs. This value is within the range reported for other protozoa (3 , 16) and is similar to the 160 copies/haploidgenome found in L . donovani (15). In the present study, we have pur- posely avoided referring our values to ploidy, since there is only indirect evidence of diploid y in other hemoflagellates such as Trypanosoma brucei (24). Although the number of ribosomal copies is similar to that of L . donovani, two important differ- ences between the two species are observed: the DNA content, 0.266 pgkell (24.2 x lo7 base pairs) vs. 0.166 pgkell(lO.5 x lo7 bp) in L. donovani; and the percent of DNA that hybridizes with labeled rRNA, 0.47% in L. brusiliensis vs. 0.8% in L. donovani. The total DNA/cell reported in the present study agrees with the values reported for other flagellated protozoa: Trypanosoma cruzi, 18 x lo7 bp (4); and Euglena gracilis, 24.6 x lo7 bp (12). We do not know if these differences could be explained by large interspecific differences in DNA content, as has been found to occur in amphibia (2).

The buoyant densities of the main and light satellite bands are in the range reported for other kinetoplastids (14, 22), while the heavy ribosomal satellite is comparable to the ribosomal satellite of Xenopus laevis, whose G+C content is 63% (23). No other rDNA satellite has been reported in kinetoplastids. Nonetheless, an uncharacterized heavy satellite has been re- ported in Trypanosoma lewisi (19).

The DNA recovered from the CsCl gradients is highly frag- mented (average size = 4 Mdaltons), being unsuitable for elec- tron microscopic analysis of the ribosomal repeating units (26). However, it could be used for other biochemical experiments, including partial cloning of ribosomal genes.

LITERATURE CITED 1. Arnheim, N . , Krystal, M. , Schmickel, T., Wilson, G . , Ryder, 0.

& Zimmer, E. 1980. Molecular evidence for genetic exchanges among

VILLALBA AND RAM~REZRIBOSOMAL DNA OF LEISHMANIA 44 1

ribosomal genes of nonhomologous chromosomes in man and apes. Proc. Natl. Acad. Sci. U.S .A. , 77: 7323-7327.

2. Britten, R. J. & Davidson, E. H. 1969. Gene regulation for higher cells: a theory. Science, 165: 349-357.

3. Castro, C., Hernandez, R. & Castaneda, M. 1981. Trypanosoma cruzi ribosomal RNA: internal break in the large-molecular-mass species and number of genes. Mol. Biochem. Parasitol., 2: 219-233.

1971. Iodination of nucleic acids in vitro. Biochemistry, 10: 1993-1999.

1980. Isolation and charac- terization of polysomes from Trypanosoma brucei. Parasitology, 81: 537-55 1.

6. Davis, R. W., Simon, M. & Davidson, N. 1971. Electron mi- croscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, in Grossman, L. & Moldave, K. eds., Meth- ods in Enzymology, Academic Press, New York, 21 (D): 413-428.

7. Dawid, I. B. Evidence for mitochondria1 origin of frog cytoplasmic DNA. Proc. Natl. Acad. Sci. U.S.A., 56: 269-276.

8. - 1977. DNA-DNA hybridization on membrane filters: a convenient method using formamide. Biochim. Biophys. Acta, 477: 1 9 1- I 94.

1965. Factors affecting the estimation of nucleic acids in Euglena gracilis. Anal. Biochem., 13: 281-293.

10. Flamm, W. G. & Burr, H. E. 1966. Density-gradient centrif- ugation of DNA in fixed angle rotor. A higher order of resolution. Biochim. Biophys. Acta, 129: 310-319.

11. Garvin, R. T., Hill, R. C. & Weber, M. M. 1978. The atypical RNA components of cytoplasmic ribosomes from Crithidia fasciculata. Arch. Biochem. Biophys., 191: 774-781.

12. Gruol, D. J. & Halselkorn, R. 1976. Counting the genes for stable RNA in the nucleus and chloroplast of Euglena. Biochim. Bio- phys. Acta, 477: 82-95.

13. Ivanov, I. G. & Markov, G. G. 1975. RNA-DNA hybridization on membrane filters with fragmented mammalian DNA. Biochim. Bio- phys. Acta, 383: 123-130.

14. Kleisen, C. M., Borst, P. & Weijers, P. J. 1975. The structure of kinetoplast DNA. Biochim. Biophys. Acta, 390: 155-167.

4. Commerford, S. L .

5 . Cordingley, J. S. & Turner, M. J.

1966.

9. De Torres, R. A. & Pogo, A. 0.

15. Leon, W., Fouts, D. L. & Manning, J. 1978. Sequence ar- rangement of the 16s and 26s rRNA genes in the pathogenic haemo- flagellate Leishmania donovani. Nucl. Acid. Res. , 5: 491-504.

16. Long, E. 0. & Dawid, I. B. 1980. Repeated genes in eukary- otes. Annu. Rev. Biochem., 49: 727-764.

17. Petes, T. 1980. Unequal meiotic recombination within tandem arrays of yeast ribosomal DNA genes. Cell, 19: 765-774.

18. Prensky, W., Steffensen, D. M. & Hughes, W. 1973. The use of iodinated RNA for gene localization. Proc. Natl. Acad. Sci. U.S .A. , 70: 1860-1864.

19. Renger, H. C. & Wolstenholme, D. R. 1970. Kinetoplast de- oxyribonucleic acid of the hemoflagellate Trypanosoma lewisi. J . Cell Biol., 47: 689-702.

20. Schildkraut, C. A., Marmur, J. & Doty, P. 1962. Determination of the base composition of DNA from its buoyant density in CsCl. J . Mol. Biol., 4: 430-443.

1974. Isolation of the kinetoplast DNA of Leishmania tarentolae in the form of a network. J . Protozool., 21: 382-393.

22. Simpson, L. & Da Silva, A. 1971. Isolation and characteriza- tion of kinetoplast DNA from Leishmania tarentolae. J . Mol. Biol., 56: 443-473.

1971. Retention of common nucleotide sequences in the ribosomal deoxyribonucleic acid of eu- karyotes and some of their physical characteristics. Biochemistry. 10: 2761-2769.

24. Tait, T. Evidence for diplody and mating in trypano- somes. Nature, 287: 536-538.

25. Ulrich, M., Trujillo, D. & Convit, J. 1968. The effect of fresh serum on the leptomonads of Leishmania. I . Preliminary report. Trans. R . Soc. Trop. Med. Hyg.. 62: 825-830.

26. Wellauer, P. K., Reeder, R. H., Dawid, I. B. & Brown, D. D. 1976. The arrangement of length heterogeneity in repeating units of amplified and chromosomal ribosomal DNA from Xenopus laevis. J . Mol. Biol., 10.5: 487-505.

Received I2 X 81; accepted 17 I l l 82

21. Simpson, L. & Berliner, J.

23. Sinclair, J. H. & Brown, D. D.

1980.

J . Protozool., 29(3). 1982, pp. 41-45 0 1982 by the Sociely of Protozoologhts

Surface Changes Induced by Immune Serum on Eimeria tenella Sporozoites and Merozoites

DALE R. WITLOCK and HARRY D. DANFORTH U S . Department of Agriculture, Agriculture Research Service, Northeastern Region,

Animal Parasitology Institute, Beltsville, Maryland 20705

ABSTRACT. The surface of merozoites and sporozoites of Eimeria tenella was affected by incubation with E. tenella-immune chicken serum (ICS). Normal chicken serum (NCS) and heat-inactivated ICS had no effect on the pellicular surface of either developmental stage. Sporozoites formed surface bulges or swellings after 10 min of incubation with ICS, and by I5 min postincubation, the morphology of the sporozoites was distorted by a surface coating of fibrinous material. Merozoites exposed to ICS were similarly coated, but surface swelling was not as severe. The coating formed rapidly and was seen as early as 5 min postincubation. Sporozoites incubated with heat-inactivated ICS supplemented with normal chicken serum were coated with a fibrinous material and in some cases lysed. These data indicated that complement must be present for the surface interaction to occur.

YTIC effects o f immune sera on motile forms of Eimeria gressive effects of exposure to antibody on the surface of spo- L tenella have been reported (3,7, 13). Sporozoites and mer- rozoites and merozoites of E. tenella. ozoites were also reported to be immobilized after 1-2 h of incubation in hyperimmune serum (7, 13). Agglutination of the MATERIALS AND METHODS parasites by immune serum suggested the presence of surface antigens (1 1). However , electron microscopic studies detailing these events are lacking. The purpose of the present investi- gation is to assess by scanning electron microscopy the pro-

Production of immune and normal sera. White Leghorn chickens (raised coccidia-free) were immunized against E. te- nella (Beltsville strain #lo) b y six oral doses of 15,000 oocysts each twice a week for three weeks. Blood was collected from