Histone fractions in chicken bursa of fabricius and thymus

Camp, Biochm. PkysioI,, 1973,

Vol.

44A, pp. 897to 901.PergamonPress. P&ted in Great Britain

HISTONE FRACTIONS IN CHICKEN BURSA OF FABRICIUS AND THYMUS N. SOTIROVl

and E. W. JOHN@

~Institute of Comparative Pathology of Animals, Bulgarian Academy of Sciences, Sofia-13, Bulgaria; and *Institute of Cancer Research, Royal Cancer Hospital, Chester Beatty Research Institute, Fulham Road, London SW3 BJB (Recei%ed 12 June 1972)

Abs$rsx%-1. Whole histone and the five main histone fractions were isolated from chicken bursa of Fabricius and thymus. 2. The proteins were all characterized by polyacrylamide gel electrophoresis and amino acid analyses and compared with the corresponding fractions from calf thyrnus. 3. Species specificity was noted with histone fractions Fl and F3. 4. The chicken erythrocyte specific fraction F2C was not present in chicken bursa of Fabricius or thymus.

INTRODUCTION THE SUGGESTIONadvanced

by Stedman & Stedman (1950) that the histones were cell specific has stimulated an intensive search for species, organ and cell specificity of these nuclear proteins. However, it is now generally accepted that there are only five main histones designated Fl and FZB (lysine-rich), FZAl and F3 (arginine-rich) and FZA2 ( an intermediate class) in the somatic cells of most species examined (Johns, 1971). Using sensitive chromatographic techniques, however, species specific heterogeneity has been demonstrated within the very lysine-rich histones Fl (Bustin & Cole, 1968; Kinkade, 1969). Recently, some changes during the evolution of the vertebrates has been observed in the electrophoretic mobility of histones F2B and F2A2 (Panyim et al., 1971). Cell specificity has also been established in nucleated erythrocytes in which an unusuaf histone, designated F2C, has been found (Neehn & Butler, 1961; Hnilica, 1964). In this respect, it is worth studying the histones of any organ which is unique in some species or class of animals. Bursa of Fabricius is such a unique avian organ localized not far from the caudat end of the alimentary canal of the birds. Its structure and function has been shown to be quite similar to that of the thymus. The bursa is involved in the development of the antibody producing system, and the thymus controls the cell-mediated immunity (Click et al., 1956; Aspinal & Meyer, 1964; Warner, 1967). Some recent studies have revealed that bursa of Fabricius exerts an effect on one of the histone fractions of the chicken spleen (Woods, 1966). 897

N. SOTIROVAND E. W. JOHNS

898

This paper describes the histones of chicken bursa of Fabricius compared with those of chicken and calf thymus. MATERIALS

AND METHODS

White Leghorn chickens (G&&s domes&us), 10 weeks old, were bled by heart puncture. Immediately afterwards the bursa of Fabricius and the thymus were excised and frozen by pressing between blocks of solid COs.

Preparation of nucleoprotein The excised glands were washed six times by blending in physiological saline as described previously (Johns, 1964). The sediment obtained after the last washing was used for the isolation of the histones.

Preparation of whole histune and histone fractions Whole histone was extracted from the nucleoprotein with 0.25 N HCl for 2 hr with shaking followed by two more similar extractions. The combined extract was clarified by filtration through a sintered glass funnel, grade 4, and the histone was precipitated from the clear supernatant with 6 vol. of acetone. The precipitate was washed three times in acetone and dried under vacuum. The various histone fractions were isolated according to Johns (1964) Method 2. Fl was also isolated from whole histone solutions by adding perchloric acid to a final concentration of 5% (0.75 N). All histone fractions except Fl were precipitated and Fl was isolated from the clarified supematant by the addition of trichloroacetic acid to a final concentration of 18% (l-1 M).

Polya~yl~ide

gel etectyoph~e~s

Electrophoresis in polyacrylamide gel was performed according to the method of Johns (1967) with the modification described by Dick & Johns (1969). For quantitative determinations of the various histones, the stained bands were cut out from the gels, broken up and extracted with dimethyl sulphoxide overnight. The colour yield of the histone fractions were converted into relative amounts of protein using standard curves constructed by running exact amounts of each histone fraction under the same experimental conditions. For comparison and identification of different histone fraction two samples were applied to the same gel according to the method of Johns (1969).

Amino acid analysis Total amino acids were measured by the method of Moore et at. (1958) using a Technicon auto-analyser apparatus. The samples were hydrolysed for 24 hr in 6 N HC1 at 11O’C. No corrections were made for hydrolytic losses. RESULTS

AND DISCUSSION

In polyacrylamide gel electrophoresis, histone fraction Fl extracted from chicken sources was separated into three subfractions (Fig. l-l, 24). Three subfractions of histone Fl were also obtained when it was isolated in a pure form (Fig. 4-2). Histone Fl isolated from various animal species has been reported (Bustin & Cole, 1968; Kinkade, 1969) to form three to five subfractions, which can be separated by very refined chromatographic techniques. In our studies separation of Fl subfractions from chicken tissues was obtained with a conventional technique for running histones on polyacrylamide gel. Under the same conditions, however, calf thymus histone Fl forms only one band. The resolving of histone

F2C

I FIG. 1. Polyacrylamide bursa

of Fabricius

FIG. 2. bursa

(l),

2

3

4

gel electrophoresis patterns of whole histone from chicken chicken thymus (2), calf thymus (3) and chicken erythrocytes (4).

Polyacrylamide gel electrophoresis patterns of Fabricius (1) together with histone fractions run in the same gel.

of whole histone of chicken F2C (2), F3 (3) and F2B (4)

2

I

Polyacrylamide gel electrophoresis patterns of 5?,-, perchloric acid extract of bursat whole histone (I) and the histones remaining after extraction (2).

FIG. 3.

I FK.

2

3

4

5

,6

4. Polya crylamide gel electrophoresis patterns of preparatio Ins of the five histones from chicken bursa of Fabricius: 1, whole histone; 2. Fl ; 3, F3;

4, F2B;

5, F2A2; 6, F2Al.

899

HISTONEFRACTIONS IN CHICKENBURSAOF FABRICIUS ANDTHYMUS

Fl into three subfractions using this system could be considered species characteristic as it was demonstrated in all chicken materials examined-erythrocytes, thymus and bursa of Fabricius. A distinct band was also observed between histone fractions Fl and F2B (Fig. l-l, 2) of chicken bursa of Fabricius and chicken thymus. It could be either histone F3 which, unlike calf thymus histones (Fig. l-3) was better resolved in the samples of the chicken organs studied, or histone F2C, as the latter showed a similar electrophoretic mobility in the chicken erythrocyte whole histone patterns (Fig. l-4). When pure preparations of histones F2B, F2C and F3 were run with bursal whole histone in the same gel, however, it can be seen that this band completely coincides with histone F3 (Fig. 2-3). There was no band corresponding to histone F2C (Fig. 2-2). It is well known that histone Fl and F2C are extracted with 5% perchloric acid (Johns, 1971). This extraction, however, only removed the three subfractions of histone Fl from the whole histone of chicken bursa of Fabricius and chicken thymus (Fig. 3-l). All the other histones, including the histone forming the above-mentioned band, were not dissolved in perchloric acid (Fig. 3-2). This demonstrates that it is histone F3 which gives a well-separated band in polyacrylamide gel under these conditions and not histone F2C. The separation of histone F3 from F2B seems to be complete as the amounts of these fractions estimated in this study (Table 1) were quite close to the corresponding values obtained by preparative methods in other materials (Johns, 1967). The quantitative determination of the various histone fractions has revealed a slightly increased value of histone Fl both in chicken thymus and in bursa of Fabricius as compared with calf thymus (Table 1). This difference, however, TABLE ~-RELATIVE YIELDS OF THE VARIOUSHISTONEFRACTIONSOF CHICKEN THYMUSAND BURSAOF FABRICIUS

_ Histone fractions Material Bursa of Fabricius Chicken thymus Calf thymus *

Fl

F3

F2B

F2A2

F2Al

22.6 21.6 18.4

18.2 18.1

25.4 25.8

16.5 16.7 17.6

17.3 17.8 17.6

46.4

*Johns (1967). The corresponding values of calf thymus histones are given for comparison. All values are expressed as percentages of total yields.

could be due to differences in the uptake of stain by histone Fl from different animal sources. The above-mentioned separation of chicken histone Fl into three subfractions does indicate differences in their composition. The five main histone fractions were isolated from chicken thymus and bursa of Fabricius in a relatively pure form (Fig. 4). Their amino acid composition did

4.5 6.3 4.2 11.1 4.6 6-4 14.2 5-l 0.4 0.8 5-l 9.4 1.9 2.5 10.1 1.6 11.8

3.0 4.9 6.8 5.3 8.4 6.4 22.8 4.8 0 0.2 1.9 5.1 1-l 1-1 24.4 0.5 3.3

5-7 5.7 5.5 8.6 4.8 8.6 13.5 6.1 Trace 0.2 4.6 7.9 2.2 l-9 14.5 1.8 8.4

ASP Thr Ser Glu Pro GUY Ala Val GYS Met Ilu Leu Tyr Phe LYS His Arg

5-5 6.4 8.6 9.4 5.0 6.3 10.7 6.2 0 0.9 5.3 5.7 3.2 l-9 16-l 2.3 6.5

F2B 6.4 4.2 3.6 8.9 3.2 10-o 13.0 6.7 0 0.2 4.8 11-l 2.2 1.3 11-l 2.3 10.4

5-4 6.3 2.3 7.3 l-4 16.9 8.1 7.7 0 0.5 5-6 8.1 3.1 2.0 10.1 l-9 12.8

F2Al 5-l 5.4 5-4 8.6 4.6 8.4 13.9 6-O Trace O-6 4.6 7.7 2.3 1.7 15.1 1.9 8.7 2.8 4.6 6-6 4-9 8.0 6.3 24.2 5.0 0 O-2 1.7 5-O O-8 O-8 25.3 o-5 3.3

Fl

OF CHICKEN

Whole histone

HISTONE FRACTIONS THYMUS

The amino acids are expressed as moles/100 moles of all amino acids found.

F3

Fl

F2A2

OF THE VARIOUS

Bursa of Fabricius

ACID COMPOSITION

Whole histone

%-AMINO

Amino acid

TABLE

F3

5.5 6-5 8.9 9.4 4-5 6-3 10.8 6-l 0 I.0 5.2 5.7 3.0 1.7 16.8 2.2 6.5

F2B

Thymus

OF FABRICIUS

4.5 6.6 4.2 10.5 4.8 6.4 14.6 4.7 0.3 O-6 4.8 8.9 1.8 2.5 11.5 1.7 11-6

BURSA

6.3 4.8 3.8 9-o 3-3 10.7 11-8 5.3 0 O-7 2.6 11.9 2.9 2.0 10.8 3.2 10.9

a ‘;s’

0 O-6 5.8 8.4 3.2 2-l 10.7 2.1 12-9

3 .m

14.7 8.2 8.0

5

?: 2 :! 4

5.4 6.4 2.4 7.1 l-6

F2Al

CHICKEN

F2A2

AND

HISTONE FRACTIONS IN CHICKENBURSAOF FABRICIUS ANDTHYMUS

901

not show any differences between the histone fractions of the chicken organs (Table 2) and those isolated from calf thymus (Johns, 1971). The conclusions are that the histones of chicken bursa of Fabricius are identical to those of chicken thymus, but that a species specificity does occur. Acknowledgements-This work has been supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council and the Cancer Research Campaign. One of us (N. S.) acknowledges a fellowship (SC/202/BUL/7110) from the International Atomic Energy Agency. REFERENCES R. L. & MEYER R. K. (1964) Effect of steroidal and surgical bursectomy and surgical thymectomy on the skin homograft reaction in chickens. In The Thymus in Immu~o~o~ogy (Edited by GOOD R. A. & GABRIEL~ENA. E.), pp. 376-394. Harper & Row, New York. BUSTINM. & COLE R. D. (1968) Species and organ specificity in very lysine-rich histones. J. biol. Chem. 243, 45004505. DICK C. & JOHNS E. W. (1969) The biosynthesis of the five main histone fractions of rat thymus. Biochim. biophys. Actu 174, 380-386. GL~CKB., CHANGT. S. & JAAP R. G. (1956) The bursa of Fabricius and antibody production. Poult. Sci. 35, 224-225. HNILICA L. S. (1964) The specificity of histones in chicken erythrocytes. Experientia 20, 13-14. JOHNSE. W. (1964) Studies on histones: preparative methods for histone fractions from calf thymus. Biochem.3. 92, 55-59. JOHNSE. W. (1967) The electrophoresis of histones in polyacrylamide gel and their quantitative determinations. Biochem. J. 104, 78-82. JOHNSE. W. (1969) A simple method for the application of two samples to polyacrylamide gel for comparative electrophoresis. J. Chromatogr. 42, 152-153. JOHNS E. W. (1971) The preparation and characterisation of histones. In Histones and Nucleohistones (Edited by PHILLIPS D. M. P.), pp. l-45. Plenum Press, London. KINKADEJ. M. (1969) Qualitative species differences and quantitative tissue differences in the distribution of lysine-rich histones. J. biol. Chem. 244, 3375-3386. MOORE S., SPACKMAND. H. & STEIN W. H. (1958) Chromatography of amino acids on sulphonated polystyrene resins. Anat. Biochem. 30, 1185-l 190. NEELYN J. M. & BUTLERG. C. (1961) A comparison of histones from chicken tissue by zone electrophoresis in starch get. Can. J. B&hem. Physiof. 39, 485491. PANYIM:S., BILEK D. & CHALKLEYR. (1971) An electrophoretic comparison of vertebrate histones. J. bioE. Chem. 246, 42064215. STEDMANE. & STEDMANE. (1950) Cell specificity of histones. Nature, Land. 166, 780-781. WARNERN. L. (1967) The immunological role of the avian thymus and bursa of Fabricius. Folia Biol. 13, l-17. WOODSR. (1966) The bursa of Fabricius and the immune response. Actu Path microbial. stand. 67, 220-228. ASPINAL

Ke$

Word Index-Histones;

histones-chicken;

histones-thymus;

histones-bursa.