No abstract

Polytene chromosomes of Chironomus thurnmi were treated with antisera elicited by purified calf thymus histone fractions, and the location of each histone type was visualized by the indirect immunofluorescence technique. Each of the antisera produced specific and distinct patterns of fluorescence, suggesting that it is possible to use the indirect immunofluorescence technique to study the in situ organization of each histone in the various regions of the chromosomes. H1 and H2A antisera produced diffuse fluorescence patterns in acetic acid-fixed chromosomes which become more defined in formaldehyde-fixed preparations. Antisera to H2B, H3 and H4, when reacted with either formaldehyde-or acetic acid-fixed chromosomes, produce distinct banding patterns closely resembling the banding of acetoorcein-stained or phase-contrast-differentiated chromosomal preparations. These antisera produce corresponding patterns of fluorescence for each chromosome, suggesting that the overall organization of the histones is similar in the various bands. Because the dense band regions stain more brightly with antihistone sera than the less compacted interband areas, we believe that the number of antigenic sites of chromosome-bound histones is related to the amount of DNA present, and that the accessibility of histone determinants does not differ between the bands and interbands.KEY WORDS histone localization antihistone sera polytene chromosomes 9 Chironomus thummi immunofluorescence Polytene chromosomes provide an excellent system for studying the structural organization of chromosomal constituents because their diameter has been amplified by several consecutive duplications of the basic chromatid fiber without ensuing separation of the sister chromatids. These amplified organelles permit examination not only at the chromosomal level, but also at the level of the gene.Chironomus thummi is an ideal source of these chromosomes because it is easily grown (14) and its chromosomal banding patterns have been well characterized (12). An additional advantage in using Chironornus chromosomes is that they are not fused at the chromocenter as are the chromosomes of Drosophila. This permits easy visualization and analysis of individual chromosomes.The availability of well-characterized antibodies to isolated histones (10, 22) allows the study of the in situ organization of histories within polytene chromosomes. Antihistone sera bind to chromatin (3) and can be used to probe the arrangement of 910 J. CELL BIOLOGY. ~ The Rockefeller University Press 9

Treatment of Chironomus polytene chromosomes with the ultimate carcinogen benzo[a]pyrene diol epoxide I or in vivo administration of the parent hydrocarbon to larvae indicates that the carcinogen interacts with the genome in a nonrandom manner. Visualization of the carcinogen-DNA binding sites by immunofluorescence reveals that, in vivo, some sites are preferentially modified. The combined effects of DNA sequence, chromatin structure, and gene localization may lead to selective targeting of carcinogens to specific genomic regions. In polytene chromosomes the targeting effect is amplified, thereby making these chromosomes a uniquely suitable system for visualizing and studying site-specific interactions of carcinogens with the genome.

CHANGES in transcriptional activity at defined loci are often correlated with significant local structural changes in the genome(1), and in polytene chromosomes, such changes are thought to be associated with compositional or conformational changes in the protein complement at these particular bands(2,3). Thus, various studies on Balfoiani rings and specific 'puffs' in such chromosomes are useful for elucidating the role of defined chromosomal components in both chromosome structure and gene activity. Such studies require specific probes which will allow in situ localisation of a chromosomal component during the various stages of puffing. Antibodies specific to purified histone fractions(4-7), HMG proteins(8), RNA polymerase(9) and non-histone protein subfractions(10) have been used in studies on chromatin and chromosome structure. We reported previously that concanavalin A (Con A) specifically binds to three types of non-histone proteins present in chromatin purified from rat liver nuclei and suggested that derivatives of Con A might serve as specific probes to study the in situ organisation of these non-histone proteins(11). We have now reacted fluorescein-labelled Con A with polytene chromosomes isolated from different developmental stages of Chironomus thummi and visualised the location of the bound Con A by fluorescence microscopy. We observed that the fluorescent lectin, which has an affinity for glucose- and mannose-containing molecules, specifically bound to the transcriptionally active regions of chromosome IV. The extent of binding of Con A to the Balbiani rings present in regions b and c of chromosome IV is proportional to the size of the respective ring. Our results indicate that glucose- or mannose-containing molecules are present in these Balbiani rings and that the availability of these sugars to interact with Con A can be correlated with the developmental stage of a puff. We suggest that lectins can be useful cytological tools with which to study the in situ organisation of defined chromosomal components during various functional states of the genome.

The distribution of accessible antigenic sites in the chromosomal protein high mobility group one (HMG-1) in Chironomus thummi polytene chromosomes is visualized by immunofluorescence . The results indicate that (a) HMG-1 is distributed in a distinct banding pattern along the entire length of the chromosomes; (b) the banding pattern obtained with fluorescent antibody does not strictly correspond to that observed by phase-contrast microscopy ; and (c) the amount of HMG-1 increases, and the fluorescent banding pattern changes, during the development of the organism . Our findings suggest that the protein may be involved in the modulation of the structure of selected loci in the chromosome .The nonhistone chromosomal proteins are an integral part of the eucaryotic genome . Although the function of these proteins is not well understood, there is evidence that some proteins belonging to this group are important in maintaining the structure (1, 36) and regulating the function (9, 35, 41) of chromatin and chromosomes. The difficulties in purifying homogeneous molecular species from this group of proteins are a major obstacle in elucidating their function. Chromosomal protein high mobility group one (HMG-1) is one of the few nonhistone proteins that has been purified to homogeneity (11). This protein is ubiquitous in its distribution, as it is found in several eucaryotic kingdoms (15,28,37,38,40). Sequence studies revealed that it is unusually rich in charged amino acid residues, and that the negatively and positively charged residues are clustered on the polypeptide chain (39) . The proteins bind to histones and DNA and can induce changes in the DNA helical structure (17,19,44). Evidence has been presented that this protein is associated with isolated nucleosomes (13) .Antisera, elicited by HMG-1 protein purified from calf thymus, cross react immunologically with HMG-1 derived from several species (33) . The antibodies bind to chromatin (4), allowing these antisera to serve as useful cytological tools to study the cellular function of this protein.In the present study, we investigate the distribution of protein HMG-1 in polytene chromosomes of Chironomus thummi. Polytene chromosomes have the same fundamental chromatin fiber structure as that present in all eucaryotic systems (43) . Their large size offers the advantage of amplification in studying the location of a particular chromosomal protein and in following structural alterations associated with functional changes in the genome. Antisera to RNA polymerase (18), nonhistone proteins (36), histones (5), and fluorescent concanavalin A (22) have already been successfully used for such studies. The present study is the first investigation on the chromosomal localization of a structurally defined nonhistone protein that has been detected in several eucaryotic kingdoms . Affinity-purified antibodies are used to demonstrate that Chironomus contains a protein that is indistinguishable from its homologue purified from calf thymus . Its organization in the chromosome...