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Immunolocalization of chromosomal proteins on polytene chromosomes of Drosophila
0047-7206/82/030301-02%03.00/0 Pergamon Press Ltd.
M~cron Vol.13, No.3, pp.30]-302, 1982. Printed in Great Britain
IMMUNOLOCALIZATION OF CHROMOSOMAL PROTEINS ON POLYTENE CHROMOSOMES OF DROSOPHILA
MARGARET R. MOTT, R.J. HILL and E.J. BURNETT Unit of Molecular and Cellular Biology, CSlRO, P.O. Box 184, North Ryde, N.S.W. 2113
The chromosomal proteins, both histones and non-histones are located at the ultrastructural level on the microdissected polytene chromosomes of Drosophila with the use of ferrltin labelled antibodies. The proteins of chromosomes are of particular interest in relation to their role in the regulation of gene expression, and the individual proteins both histones and non-histones, can be isolated and purified biochemically, and antlsera to these proteins raised in mice. Previous work I on sectioned material in the electron microscope has shown that these chromosomes, isolated by micromanipulation and avoiding the deleterious effect of the classical acid squash technique, demonstrate proteins in the native state, with excellent preservation of ultrastructure and ease of cytological mapping. These chromosomes are therefore excellent targets for immunolocalization of chromosomal proteins and can be treated readily with antibody to a particular protein, as has been2demonstrated previously at the light microscope level using fluorescent labelled antibody. For localization at the ultrastructural level with ferritin labelled antibody, structures on chromosomes are located from preliminary phase contrast light micrographs, and after treating with the labelled antibody, embedding and sectioning, these structures c a n b e identified readily in the electron microscope and related to the classical chromosome map of Bridges. Chromosome 3L (Fig. I) can be seen at a known site 65A-D to split into two chromatids, on each of which can be seen the puff 68C, the site of transcription of an active gene. This chromosome preparation has been exposed to ferritin labelled antibody against one of the major non-hlstone chromosomal proteins A2, and at higher magnification ferritin is seen to be present in the puffs and interbands and associated with the ribonucleoprotein (RNP) particles (Fig. 2). Chromosome preparations exposed to labelled antibody to another major non-histone A4 (Fig. 3) show ferritin again associated with the RNP in the puffs and interbands and not with the condensed bands, the outer edges of which can be seen to be free of ferrltin. In preparations exposed to labelled antibody to hlstone HI ferritin appears to be present along the chromatin threads. To visualize the arrangement in depth of the chromatln and ferritin labelling in the active 68C puff, stereo-palrs of electron mierographs were taken at 100kV at a tilt O angle of ± 5 using 0.1 um sections. The resulting three dimensional fused image demonstrates in depth the chromatin threads and RNP with ferritln labelling (Fig. 4). Stereo microscopy of thick serial sections, and the use of monoclonal antibodies to both hlstones and non-hlstones at present being prepared, will give more specific information on transcription at known active genetic locl. I.
Mott, M.R., Burnett, E.J. and Hill, R.J. (1980). somes of Drosophila isolated by microdissection.
Ultrastructure of polytene chromoJ. Cell Scl. 45: 15-30.
2.
Hill, R.J. and Watt, F. (1977). Chromosomal proteins of Drosophila melano~aster and an approach for their localization on polytene chromosomes. Chromosoma 63: 57-78. 30!
302
M.R.
Mott, R. J. Hill and E. J. Burnett
10 68C m ~pm.
Fig. i.
Chromosome
Fig. 2. Puff 68C with ferritin associated with RNP. Anti A2. Heavy counterstaining.
Fig. 4. Stereo-pair Tilt angle ± 5 ° .
of puff 68C.
3L
3L split to 2 chromatids
each with puff 68C.
Fig. 3. Ferritin associated with RNP of puff and interbands, absent from bands. Anti A4. Light counterstaining.
Ferritin labelling
to a major non-histone
protein A2.
M~cron Vol.13, No.3, pp.30]-302, 1982. Printed in Great Britain
IMMUNOLOCALIZATION OF CHROMOSOMAL PROTEINS ON POLYTENE CHROMOSOMES OF DROSOPHILA
MARGARET R. MOTT, R.J. HILL and E.J. BURNETT Unit of Molecular and Cellular Biology, CSlRO, P.O. Box 184, North Ryde, N.S.W. 2113
The chromosomal proteins, both histones and non-histones are located at the ultrastructural level on the microdissected polytene chromosomes of Drosophila with the use of ferrltin labelled antibodies. The proteins of chromosomes are of particular interest in relation to their role in the regulation of gene expression, and the individual proteins both histones and non-histones, can be isolated and purified biochemically, and antlsera to these proteins raised in mice. Previous work I on sectioned material in the electron microscope has shown that these chromosomes, isolated by micromanipulation and avoiding the deleterious effect of the classical acid squash technique, demonstrate proteins in the native state, with excellent preservation of ultrastructure and ease of cytological mapping. These chromosomes are therefore excellent targets for immunolocalization of chromosomal proteins and can be treated readily with antibody to a particular protein, as has been2demonstrated previously at the light microscope level using fluorescent labelled antibody. For localization at the ultrastructural level with ferritin labelled antibody, structures on chromosomes are located from preliminary phase contrast light micrographs, and after treating with the labelled antibody, embedding and sectioning, these structures c a n b e identified readily in the electron microscope and related to the classical chromosome map of Bridges. Chromosome 3L (Fig. I) can be seen at a known site 65A-D to split into two chromatids, on each of which can be seen the puff 68C, the site of transcription of an active gene. This chromosome preparation has been exposed to ferritin labelled antibody against one of the major non-hlstone chromosomal proteins A2, and at higher magnification ferritin is seen to be present in the puffs and interbands and associated with the ribonucleoprotein (RNP) particles (Fig. 2). Chromosome preparations exposed to labelled antibody to another major non-histone A4 (Fig. 3) show ferritin again associated with the RNP in the puffs and interbands and not with the condensed bands, the outer edges of which can be seen to be free of ferrltin. In preparations exposed to labelled antibody to hlstone HI ferritin appears to be present along the chromatin threads. To visualize the arrangement in depth of the chromatln and ferritin labelling in the active 68C puff, stereo-palrs of electron mierographs were taken at 100kV at a tilt O angle of ± 5 using 0.1 um sections. The resulting three dimensional fused image demonstrates in depth the chromatin threads and RNP with ferritln labelling (Fig. 4). Stereo microscopy of thick serial sections, and the use of monoclonal antibodies to both hlstones and non-hlstones at present being prepared, will give more specific information on transcription at known active genetic locl. I.
Mott, M.R., Burnett, E.J. and Hill, R.J. (1980). somes of Drosophila isolated by microdissection.
Ultrastructure of polytene chromoJ. Cell Scl. 45: 15-30.
2.
Hill, R.J. and Watt, F. (1977). Chromosomal proteins of Drosophila melano~aster and an approach for their localization on polytene chromosomes. Chromosoma 63: 57-78. 30!
302
M.R.
Mott, R. J. Hill and E. J. Burnett
10 68C m ~pm.
Fig. i.
Chromosome
Fig. 2. Puff 68C with ferritin associated with RNP. Anti A2. Heavy counterstaining.
Fig. 4. Stereo-pair Tilt angle ± 5 ° .
of puff 68C.
3L
3L split to 2 chromatids
each with puff 68C.
Fig. 3. Ferritin associated with RNP of puff and interbands, absent from bands. Anti A4. Light counterstaining.
Ferritin labelling
to a major non-histone
protein A2.