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Characterization of HMG2 complement changes in chicken tissues
EXPERIMENTAL
CELL RESEARCH
197,333-334 (1991)
SHORT NOTE Characterization of HMG2 Complement Changes in Chicken Tissues J. BOIX’ Department of Physiology, University of Barcelona School of Medicine, Barcelona, Spain
MATERIALS
Variations in the content of nonhistone proteins high mobility group 2a (IIMG2a) and IIMGBb have been determined in several cell types of chicken. HMG2a was found to accumulate during erythrocyte maturation. IIMGBb is the major IIMG2 subtype in testis and reaches the highest proportion, detected so far, in spermatid cells obtained by centrifugal elutriation. In hepatocytes HMG2b is barely detectable and HMG2a is the major subtype. In conclusion, the pattern of HMG2 composition is different in three quiescent and terminally differentiated cell types, no correlation between the state of cell proliferation and HMG2 composition Q isei AWIW~C pray, he. can be established.
AND
METHODS
Hubbard White Mountain roosters (25-50 weeks old) were used throughout this study. Erythrocytes were obtained from peripheral blood. Beticulocytes were obtained from peripheral blood after treatment with phenylhydracine for 1 week [8]. Blood was collected into a pH 7.4 solution containing 15 mM sodium citrate and 150 mM sodium chloride at 0-4’C. Blood cells were pelleted and washed once in this medium before being extracted. The liver was perfused with the same solution in order to eliminate contaminating erythrocytes. Testes were carefully decapsulated before being used. Testicular cell suspensions were prepared and populations of spermatids were obtained by means of centrifugal elutriation [9]. These different cells and tissues were extracted following the perchloric acid procedure [lo]. The resulting proteins were resolved in urea-acetic polyacrylamide gel electrophoresis (PAGE) [ll], with minor modifications [12]. After Coomassie brillant blue (CBB) staining [13], the gels were scanned by means of a Hoeffer densitometer.
INTRODUCTION RESULTS
The high mobility group proteins (HMG) are a class of abundant nonhistone chromosomal proteins. Although a structural role in chromatin has been proposed, their precise functions remain unknown [l]. HMGl and HMG2 belong to the high molecular weight subgroup of HMG proteins and show sequence homologies and a common conformation [2]. The HMG2 protein has been found to be composed of two variants or sequence subtypes: the HMG2a and the HMG2b [3]. The HMG2a was initially thought to be unique to avian erythrocytes and was named HMGE [4]. Later, however, it was found in other avian and mammalian cell lines [5]. High levels of HMG2 were initially correlated to cell proliferative activity [6]; further studies weakened this correlation [7], but HMG2 complement was never considered. The purpose of this work has been to find out if any correlation exists between cell proliferation and changes in the proportions of HMG2 subtypes.
i Present address: C/. Pujada Part de1 Castell, Barcelona, Spain.
18, Manresa
08240
AND
DISCUSSION
When a perchloric acid extract from chicken cells or tissues is resolved in high-resolution urea-acetic PAGE, HMGl and HMG2 are easily identified by their migration above Hl histone. In addition, both HMG2 components are separated and can be quantified. Following this procedure, the HMG2 complement has been characterized in reticulocytes, erythrocytes, testis, and liver (Fig. 1). In avian erythropoiesis, nuclei become progressively condensed as the cell stops proliferation and differentiates. An accumulation of H5 histone has been detected and related to this process [ 141. During the reticulocyte maturation to erythrocyte, I have found a 10% relative increase in HMG2a protein (Table 1). This is consistent with the high amount of HMG2a referred to before in erythrocyte chromatin [4]. In chicken liver HMG2a accounts for nearly all the HMG2 protein in hepatocyte chromatin. Hepatocytes are terminally differentiated cells with no proliferative activity. As a consequence of the aforementioned results, a correlation between high levels of HMG2a and cell quiescence could be suggested. In chicken thymus HMG2b is the major subtype and
333
Copyright 0 1991 by Academic Press, Inc. All righta of reproduction in any form reserved.
334
SHORT
cells are very active proliferating [3,5]. In the nonproliferating chicken hepatocytes there is a low amount of HMG2b. When rooster testes are studied, HMG2b is found to be the major component (Table 1). Testis contains cell populations with a high proliferative activity (spermatogonia), thus a correlation between high levels of HMG2b and cell proliferation could be proposed. After preparing a testicular cell suspension, the quiescent populations of spermatids are available by centrifugal elutriation. HMG2b is found to increase during early to late spermatid maturation. In late spermatids, I have found the highest proportion of HMG2b detected so far in any cell or tissue (over 60%). Therefore, the correlation between HMG2b and cell proliferation can no longer be sustained. Interestingly, a band with the characteristic mobility of HMG2b accounts for the high amount of HMG2 found in mammalian spermatids [15, 161. The results presented in this paper, together with previous results [7], lead to the conclusion that not only is
NOTE
TABLE
1
Relative Amounts (%) of the Three Molecular
Weight
HMG
HMGl Reticulocyte Erythrocyte Liver Testis Early spermatids Late spermatids
55 -c 5 50 f 3 60 f 6 50 2 3 20 + 2 8+4
High
Proteins HMG2a
HMG2b
25 + 35 f 38 k 20 * 21+ 30 It
20 f 15 * 2+1 30 f 53 * 62 iz
3 2 3 1 1 3
1 1 3 4 5
the amount of HMG2 unrelated to proliferation, but neither HMG2a nor HMG2b loss or accumulation can be correlated with the proliferative state of the cell. REFERENCES 1.
Goodwin, G. H., and Mathew, C. G. P. (1982) in The HMG Chromosomal Proteins (Johns, E. W., Ed.), pp. 193-221, Academic Press, London/New York.
2.
Reeck, G. R., Isackson, P. J., and Teller, D. C. (1982) Nature 300.76-18. Mathew, C. G. P., Goodwin, G. H., Gooderham, K., Walker, J. M., and Johns, E. W. (1979) B&hem. Biophys. Res. Commun. 87,1243-1251. Sterner, R., Boffa, L. C., and Vidali, G. (1978) J. Biol. Chem. 263,3830-3836. Gordon, J. S., Rosenfeld, B. I., Kaufman, R., and Williams, D. L. (1980) Biochemistry 19,4395-4402. Seyedin, S. M., and Kistler, W. S. (1979) J. Biol. Chem. 254, 11,264-11,271. Bucci, L. R., Brock, W. A., Goldknopf, I. L., and Meistrich, M. L. (1984) J. Bid. Chem. 259,8840-6846. Rapoport, S. M. (1986) in The Retyculocyte, pp. 91-102, CRC Press, Boca Raton, FL. Rota, J., and Mezquita, C. (1989) EMBO J. 8, 1855-1860. Goodwin, G. H., and Johns, E. W. (1977) in Methods in Cell Biology (Stein, G., Stein, J., and Kleinsmith, L. J., Eds.), Vol. 15, pp. 257-267, Academic Press, New York. Panyim, S., and Chalkley, R. (1969) Arch. Biochem. Biophys.
3.
4. 5. 6. 7. 8. 9. 10.
11.
130,337-346. 12. 13. 14. 15.
FIG.
1. Densitometric
tracings obtained from urea-acetic gels showing HMGl, HMG2a, and HMG2b. cytes, (E) erythrocytes, (T) testis, and (L) liver. Received June 18,199l
CBB-stained (R) Reticulo-
16.
Shannon, M. F., and Wells, J. R. E. (1987) J. Biol. &em. 262, 9664-9668. Lennox, R. W., Oshima, R. G., and Cohen, L. H. (1982) J. Biol. Chem. 257,5183-5189. Affolter, M., Cot&, J., Renaud, J., and Ruiz-Carrillo, A. (1987) Mol. Cell. Biol. 7, 3663-3672. Bucci, L. R., Brock, W. A., and Meistrich, M. L. (1985) B&hem. J. 229,233-240. Faire, R. J., and Cooper, D. W. (1987) Comp. Biochem. Physiol. B 87,423-433.
CELL RESEARCH
197,333-334 (1991)
SHORT NOTE Characterization of HMG2 Complement Changes in Chicken Tissues J. BOIX’ Department of Physiology, University of Barcelona School of Medicine, Barcelona, Spain
MATERIALS
Variations in the content of nonhistone proteins high mobility group 2a (IIMG2a) and IIMGBb have been determined in several cell types of chicken. HMG2a was found to accumulate during erythrocyte maturation. IIMGBb is the major IIMG2 subtype in testis and reaches the highest proportion, detected so far, in spermatid cells obtained by centrifugal elutriation. In hepatocytes HMG2b is barely detectable and HMG2a is the major subtype. In conclusion, the pattern of HMG2 composition is different in three quiescent and terminally differentiated cell types, no correlation between the state of cell proliferation and HMG2 composition Q isei AWIW~C pray, he. can be established.
AND
METHODS
Hubbard White Mountain roosters (25-50 weeks old) were used throughout this study. Erythrocytes were obtained from peripheral blood. Beticulocytes were obtained from peripheral blood after treatment with phenylhydracine for 1 week [8]. Blood was collected into a pH 7.4 solution containing 15 mM sodium citrate and 150 mM sodium chloride at 0-4’C. Blood cells were pelleted and washed once in this medium before being extracted. The liver was perfused with the same solution in order to eliminate contaminating erythrocytes. Testes were carefully decapsulated before being used. Testicular cell suspensions were prepared and populations of spermatids were obtained by means of centrifugal elutriation [9]. These different cells and tissues were extracted following the perchloric acid procedure [lo]. The resulting proteins were resolved in urea-acetic polyacrylamide gel electrophoresis (PAGE) [ll], with minor modifications [12]. After Coomassie brillant blue (CBB) staining [13], the gels were scanned by means of a Hoeffer densitometer.
INTRODUCTION RESULTS
The high mobility group proteins (HMG) are a class of abundant nonhistone chromosomal proteins. Although a structural role in chromatin has been proposed, their precise functions remain unknown [l]. HMGl and HMG2 belong to the high molecular weight subgroup of HMG proteins and show sequence homologies and a common conformation [2]. The HMG2 protein has been found to be composed of two variants or sequence subtypes: the HMG2a and the HMG2b [3]. The HMG2a was initially thought to be unique to avian erythrocytes and was named HMGE [4]. Later, however, it was found in other avian and mammalian cell lines [5]. High levels of HMG2 were initially correlated to cell proliferative activity [6]; further studies weakened this correlation [7], but HMG2 complement was never considered. The purpose of this work has been to find out if any correlation exists between cell proliferation and changes in the proportions of HMG2 subtypes.
i Present address: C/. Pujada Part de1 Castell, Barcelona, Spain.
18, Manresa
08240
AND
DISCUSSION
When a perchloric acid extract from chicken cells or tissues is resolved in high-resolution urea-acetic PAGE, HMGl and HMG2 are easily identified by their migration above Hl histone. In addition, both HMG2 components are separated and can be quantified. Following this procedure, the HMG2 complement has been characterized in reticulocytes, erythrocytes, testis, and liver (Fig. 1). In avian erythropoiesis, nuclei become progressively condensed as the cell stops proliferation and differentiates. An accumulation of H5 histone has been detected and related to this process [ 141. During the reticulocyte maturation to erythrocyte, I have found a 10% relative increase in HMG2a protein (Table 1). This is consistent with the high amount of HMG2a referred to before in erythrocyte chromatin [4]. In chicken liver HMG2a accounts for nearly all the HMG2 protein in hepatocyte chromatin. Hepatocytes are terminally differentiated cells with no proliferative activity. As a consequence of the aforementioned results, a correlation between high levels of HMG2a and cell quiescence could be suggested. In chicken thymus HMG2b is the major subtype and
333
Copyright 0 1991 by Academic Press, Inc. All righta of reproduction in any form reserved.
334
SHORT
cells are very active proliferating [3,5]. In the nonproliferating chicken hepatocytes there is a low amount of HMG2b. When rooster testes are studied, HMG2b is found to be the major component (Table 1). Testis contains cell populations with a high proliferative activity (spermatogonia), thus a correlation between high levels of HMG2b and cell proliferation could be proposed. After preparing a testicular cell suspension, the quiescent populations of spermatids are available by centrifugal elutriation. HMG2b is found to increase during early to late spermatid maturation. In late spermatids, I have found the highest proportion of HMG2b detected so far in any cell or tissue (over 60%). Therefore, the correlation between HMG2b and cell proliferation can no longer be sustained. Interestingly, a band with the characteristic mobility of HMG2b accounts for the high amount of HMG2 found in mammalian spermatids [15, 161. The results presented in this paper, together with previous results [7], lead to the conclusion that not only is
NOTE
TABLE
1
Relative Amounts (%) of the Three Molecular
Weight
HMG
HMGl Reticulocyte Erythrocyte Liver Testis Early spermatids Late spermatids
55 -c 5 50 f 3 60 f 6 50 2 3 20 + 2 8+4
High
Proteins HMG2a
HMG2b
25 + 35 f 38 k 20 * 21+ 30 It
20 f 15 * 2+1 30 f 53 * 62 iz
3 2 3 1 1 3
1 1 3 4 5
the amount of HMG2 unrelated to proliferation, but neither HMG2a nor HMG2b loss or accumulation can be correlated with the proliferative state of the cell. REFERENCES 1.
Goodwin, G. H., and Mathew, C. G. P. (1982) in The HMG Chromosomal Proteins (Johns, E. W., Ed.), pp. 193-221, Academic Press, London/New York.
2.
Reeck, G. R., Isackson, P. J., and Teller, D. C. (1982) Nature 300.76-18. Mathew, C. G. P., Goodwin, G. H., Gooderham, K., Walker, J. M., and Johns, E. W. (1979) B&hem. Biophys. Res. Commun. 87,1243-1251. Sterner, R., Boffa, L. C., and Vidali, G. (1978) J. Biol. Chem. 263,3830-3836. Gordon, J. S., Rosenfeld, B. I., Kaufman, R., and Williams, D. L. (1980) Biochemistry 19,4395-4402. Seyedin, S. M., and Kistler, W. S. (1979) J. Biol. Chem. 254, 11,264-11,271. Bucci, L. R., Brock, W. A., Goldknopf, I. L., and Meistrich, M. L. (1984) J. Bid. Chem. 259,8840-6846. Rapoport, S. M. (1986) in The Retyculocyte, pp. 91-102, CRC Press, Boca Raton, FL. Rota, J., and Mezquita, C. (1989) EMBO J. 8, 1855-1860. Goodwin, G. H., and Johns, E. W. (1977) in Methods in Cell Biology (Stein, G., Stein, J., and Kleinsmith, L. J., Eds.), Vol. 15, pp. 257-267, Academic Press, New York. Panyim, S., and Chalkley, R. (1969) Arch. Biochem. Biophys.
3.
4. 5. 6. 7. 8. 9. 10.
11.
130,337-346. 12. 13. 14. 15.
FIG.
1. Densitometric
tracings obtained from urea-acetic gels showing HMGl, HMG2a, and HMG2b. cytes, (E) erythrocytes, (T) testis, and (L) liver. Received June 18,199l
CBB-stained (R) Reticulo-
16.
Shannon, M. F., and Wells, J. R. E. (1987) J. Biol. &em. 262, 9664-9668. Lennox, R. W., Oshima, R. G., and Cohen, L. H. (1982) J. Biol. Chem. 257,5183-5189. Affolter, M., Cot&, J., Renaud, J., and Ruiz-Carrillo, A. (1987) Mol. Cell. Biol. 7, 3663-3672. Bucci, L. R., Brock, W. A., and Meistrich, M. L. (1985) B&hem. J. 229,233-240. Faire, R. J., and Cooper, D. W. (1987) Comp. Biochem. Physiol. B 87,423-433.