Abstracts
The binding of toluidine blue molecules to nucleic acid phosphates under conditions of competition with Mg2+ ions was studied in Trichosia pubescens polytene chromosome regions differing in composition and organization (RNA-rich areas and puffs, DNA puffs, heterochromatin). The aim was to find variability in the Mg2+ concentration at which metachromasy was completely prevented (= critical electrolyte concentration; CEC) and that could reflect differences at the level of nucleoprotein complexes in particular chromosome regions and developmental phases. Although high CEC values have been found in tightly packed chromatin such as that of heterochromatin zones, the CEC values for the other regions have proven to be affected not only by availability and proximity of DNA phosphates but also by RNA richness and other factors. Among these, changes in DNA geometry and packing state have been assumed for explaining increased values after RNA removal in DNA puffs and RNA-rich zones. Based on CEC values it has been suggested that alterations at the level of nucleoprotein complexes may occur in puffs before they are morphologically detectable. CEC results for polytene chromosomes were thus revealed to vary considerably with slight variations in nucleoprotein composition and organization. However, since a complex and apparently contradictory rationale has to be used for explaining part of the results, CEC is not recommended as a useful tool for extensive and comparative studies of this particular model.
A ligação de moléculas de azul de toluidina a grupamentos fosfatos de ácido nucléico em condições de competição com íons Mg2+ foi estudada em regiões de cromossomos politênicos de Trichosia pubescens que diferem em composição e organização (áreas ricas em RNA, pufes de RNA e DNA, heterocromatina). O objetivo foi encontrar variabilidade na concentração de Mg2+ na qual a metacromasia fosse completamente abolida (= concentração crítica de eletrólitos; CEC) e que refletisse diferenças ao nível dos complexos nucleoprotéicos em regiões cromossômicas e fases do desenvolvimento particulares. Embora valores altos de CEC tenham sido encontrados na cromatina altamente empacotada, como nas zonas heterocromáticas, os valores de CEC para outras regiões mostraram-se afetados não apenas pela disponibilidade e proximidade de fosfatos do DNA mas também pela riqueza em RNA e outros fatores. Entre estes, mudanças na geometria e no estado de empacotamento do DNA foram consideradas para explicar valores aumentados após remoção de RNA em pufes de DNA e zonas ricas em RNA. Baseando-se em valores de CEC foi sugerido que possam ocorrer alterações ao nível dos complexos nucleoprotéicos em pufes antes que estes sejam detectáveis morfologicamente como tal. Os valores de CEC nos cromossomos politênicos revelaram-se, pois, altamente variáveis com as variações em composição e organização nucleoprotéica do substrato. Contudo, uma vez que um raciocínio complexo e aparentemente contraditório tem que ser utilizado para a interpretação de parte dos resultados, a CEC não é recomendável como ferramenta para estudos extensivos e comparativos neste modelo em particular.
Critical electrolyte concentration of chromatin in polytene chromosomes of Trichosia pubescens (Diptera, Sciaridae)* * Part of a thesis presented by A.L.P.M. to the Instituto de Biologia, UNICAMP, Campinas, in partial fulfillment of the requirements for the Masters' degree.
Ana Lúcia P. Monteiro and Maria Luiza S. Mello
Departamento de Biologia Celular, Instituto de Biologia, UNICAMP, 13083-970 Campinas, SP, Brasil. Fax: 0055-19-2893124. Send correspondence to M.L.S.M.
ABSTRACT
The binding of toluidine blue molecules to nucleic acid phosphates under conditions of competition with Mg2+ ions was studied in Trichosia pubescens polytene chromosome regions differing in composition and organization (RNA-rich areas and puffs, DNA puffs, heterochromatin). The aim was to find variability in the Mg2+ concentration at which metachromasy was completely prevented (= critical electrolyte concentration; CEC) and that could reflect differences at the level of nucleoprotein complexes in particular chromosome regions and developmental phases. Although high CEC values have been found in tightly packed chromatin such as that of heterochromatin zones, the CEC values for the other regions have proven to be affected not only by availability and proximity of DNA phosphates but also by RNA richness and other factors. Among these, changes in DNA geometry and packing state have been assumed for explaining increased values after RNA removal in DNA puffs and RNA-rich zones. Based on CEC values it has been suggested that alterations at the level of nucleoprotein complexes may occur in puffs before they are morphologically detectable. CEC results for polytene chromosomes were thus revealed to vary considerably with slight variations in nucleoprotein composition and organization. However, since a complex and apparently contradictory rationale has to be used for explaining part of the results, CEC is not recommended as a useful tool for extensive and comparative studies of this particular model.
INTRODUCTION
The binding of toluidine blue (TB) molecules to DNA or RNA and DNA- or RNA-protein complexes in vitro and in situ under conditions of competition with inorganic cations has been proposed for studies of the critical electrolyte concentration (CEC) of nucleic acids. The inorganic cation concentration at which metachromasy is completely prevented has been considered the CEC value for the nucleic acid in nucleoprotein complexes of chromatin treated with TB solutions (Mello and Vidal, 1989; Vidal and Mello, 1989). Since RNA presents a CEC point higher than that for DNA, RNA and DNA can be distinguished from each other by the fact that RNA remains stained metachromatically at the DNA CEC point (Mello et al., 1993). Therefore, although TB molecules bind directly to nucleic acid phosphates, it is nucleoprotein which determines CEC of a specific chromatin region, with RNA being an important component as well (Mello, 1997).
Under in vitro conditions, the CEC of a DNA-Hl histone complex has been demonstrated to be lower than that of pure DNA, while higher than that of a DNA-protamine complex (Vidal and Mello, 1989). Additionally, the CEC of DNA-protein complexes in situ has been shown to be also affected by the chromatin packing state related to nuclear/cellular physiological changes (Amaral and Mello, 1989; Mello and Vidal, 1989; Mello and Falco, 1996).
Polytene chromosomes are special forms of chromatin known to exhibit changes in composition and supra-organization (puffs) associated with gene expression during development and differentiation and in response to hormones and stress agents (Korge, 1987). In sciarid flies certain types of puffs not only carry out RNA transcription but are also involved in gene amplification. These puffs, originally described by Breuer and Pavan (1955), were later demonstrated to code for the mRNA transcription responsible for the synthesis of the salivary gland secretion proteins, and were also found to exhibit additional amounts of histone and nonhistone proteins (Vidal, 1977; Winter et al., 1977b, 1980; Toledo and Lara, 1978; Glover et al., 1982).
In the present investigation using competitive TB/Mg2+ staining conditions, a comparative evaluation of CEC was carried out in bands, inter-bands, and puffs of salivary gland polytene chromosomes of a sciarid fly through different developmental phases in which DNA puffs are particularly evident (Amabis, 1983a).
MATERIAL AND METHODS
Specimens of Trichosia pubescens (Diptera, Sciaridae) at phases L5, L7, and P1 of the 4th larval instars were supplied by Dr. José Mariano Amabis (Laboratório de Sciarídeos, Departamento de Biologia, Universidade de São Paulo). These developmental phases have been characterized by Amabis (1983a) in terms of larval developmental characteristics (L5, beginning of the cocoon construction; L7, eyespot region at its maximum size and larva assuming a straight position inside the cocoon; P1, beginning of eyespots migration) associated with expansion and regression of large but slowly developing DNA puffs. At least three specimens of each phase were used under each experimental condition. The larvae were reared at 18oC in plastic cages containing a glass plate bottom covered with a layer of sterilized ground moistened with distilled water. The larvae were fed a meal consisting of a mixture of sweet potato branches or fermented crushed alfalfa, swine ration, and corn meal, moistened with an aqueous honey solution containing yeast.
Polytene chromosomes were obtained from the larval salivary glands. These glands are divided into different regions named S1, S2a, S2b, and S3, which exhibit polytene cells of various sizes (Amabis, 1983b). Only cells of the S1 region were studied due to their larger size.
The salivary glands were dissected in the insect's own hemolymph. The S1 region was fixed in an absolute ethanol-acetic acid mixture (3:1, v/v) for 2 min and then squashed in a drop of 50% acetic acid. The preparations were immediately frozen in liquid nitrogen and the coverslips removed, after which the material was immersed in 70% ethanol for 5 min, and air-dried.
For the establishment of the CEC point, staining solutions supplemented with increasing concentrations (0.05 M through 0.50 M) of MgCl2 were used (Vidal and Mello, 1989). Staining was carried out with a 0.025% toluidine blue (Merck) solution in McIlvaine buffer at pH 4.0 (Lison, 1960) for 15 min at room temperature. The material was then rinsed in distilled water, air-dried, cleared in xylene and mounted in Eukitte according to Vidal's method (Mello and Vidal, 1980). Some preparations were treated with a 0.l% RNase III (Sigma) aqueous solution at 37oC for 1 h prior to staining.
Cytological maps of polytene chromosomes as described by Amabis (1983b) for lacto-acetic orcein- stained preparations were used for the identification of chromosomes (A, B, C, X), chromosome regions (A, 28 sections; B, 25 sections; C and X, 23 sections) and puffs.
Observations of the color response in the stained chromosomes were made visually using a Zeiss standard binocular microscope. Since visual observations have usually proven efficient in discriminating the green color corresponding to the microspectrophotometrically established CEC point (Mello and Vidal, 1989), detection of the wavelength of the absorption peaks was not deemed necessary in this study.
RESULTS
All the chromosome regions treated with the TB solution in the absence of MgCl2 stained metachromatically, differing only in violet color intensity (Figure 1b-d). A banding pattern similar to that described for lacto-acetic orcein-stained chromosomes was detected after TB staining, although TB is known to bind to DNA and RNA phosphates (Mello, 1997), whereas orcein is hypothesized to stain proteins (Kiernan, 1990). Some slight differences verified in banding pattern and related to subdivisions of the polytene chromosome regions or more conspicuous staining with one of the dyes were not sufficient to prevent the identification of the various regions previously described by Amabis (1983a,b).
- Acetic ethanol-fixed salivary gland polytene chromosome views in 4th instar larvae (L7 stage) of Trichosia pubescens. The metachromasy which appeared all along the chromosomes and at the cytoplasm in preparations stained in the absence of Mg2+ (b-d) was abolished in many chromosome regions but remained detected in other regions (arrows) when staining was carried out in the presence of 0.10 M MgCl2 (a). The chromosome regions are indicated on the basis of Amabis' (1983a,b) data. Bars, 10 mm.
When subjected to treatment with the Mg2+-containing dye solution, most of the chromosome regions exhibited a CEC point (green color, Figure 1a) equal to 0.10 M. However, some chromosome regions remained stained metachromatically under this staining condition and exhibited higher CEC values varying with the chromosome region and/or developmental phase (Tables I-IV). Nearly one half of these sites were coincident with those reported to develop RNA puffs or represent RNA-rich sites according to descriptions by Amabis (1983a,b). Others corresponded to heterochromatin areas (J.M. Amabis, personal communication) or were coincident with areas which undergo gene amplification (DNA puffs) (Amabis, 1983a).
- CEC values (M) of MgCl2 for regions of the A chromosome which stained metachromatically while the other regions had attained their CEC point (0.1 M). Data for different developmental phases (L5, L7, P1).
Table II - CEC values (M) of MgCl2 for regions of the B chromosomes which stained metachromatically while the other regions had attained their CEC point (0.1 M). Data for different developmental phases (L5, L7, P1).
Table III - CEC values (M) of MgCl2 for regions of the C chromosome which stained metachromatically while the other regions had attained their CEC point (0.1 M). Data for different developmental phases (L5, L7, P1).
Table IV - CEC values (M) of MgCl2 for regions of the X chromosome which stained metachromatically while the other regions had attained their CEC point (0.1 M). Data for different developmental phases (L5, L7, P1).
RNase treatment prior to staining was found to induce a general increase in CEC values all along the chromosomes, with a usual value equal to or even higher than 0.20 M. Values > 0.20 M were also detectable in some RNA-rich zones (Table V). DNA puffs, with the exception of some present in the C chromosome, were found to maintain CEC values higher than those of most chromosome zones (underlined characters, Table V). Among heterochromatin sites, about one half were characterized by CEC values higher than 0.20 M (boldface, Table V).
- Polytene chromosome regions which stained metachromatically with a TB solution supplemented with 0.20 M MgCl2 after RNase treatment.
*RNA-rich sites and/or RNA puffs; boldface, heterochromatin; underlined characters, DNA puffs.
Finally, it should be mentioned that nearly 30% of the polytene chromosome regions exhibiting CEC > 0.10 M (Tables I-IV) could not be directly associated with a special composition and/or physiological event (except for proximity to regions containing DNA or RNA puffs or heterochromatin). One half of these regions continued to stain metachromatically at 0.20 M of MgCl2 after RNase treatment (Table V).
Summarizing results presented in Tables I-V:
l. specific polytene chromosome regions exhibited higher CEC than did other regions;
2. these regions in some cases exhibited a higher CEC only during some of the developmental stages of the fourth larval instar assayed;
3. a significant proportion of the higher CEC regions corresponded to sites of one of the following - RNA puff, heterochromatin, and DNA puff;
4. RNase treatment of fixed chromosomes prior to staining was associated with an overall increase in CEC for chromosomal regions, including increases in CEC for some RNA-rich regions.
DISCUSSION
The finding of a relatively lower CEC value in most polytene chromosome regions of T. pubescens in comparison with heterochromatin, puffs and RNA-rich zones could be explained by the loosely packed chromatin organization being present in the former, presumably RNA-poor zones. The data obtained here and in other studies indicate that heterochromatin usually exhibits high CEC values, probably due to its tightly packed state (Amaral and Mello, 1989; Mello and Vidal, 1989).
However, despite being known to consist of loosely packed chromatin, puffs and RNA-rich zones were also found to exhibit high CEC values, which could be attributed to RNA richness, since the CEC for RNA is known to be much higher than that for DNA (Scott, 1973; Mello et al., 1993). In addition, since DNA puffs are not only characterized by DNA amplification but also by expression of the amplified genes (Winter et al., 1977a,b; Toledo and Lara, 1978), RNA is also present in these structures, affecting CEC values.
The general increase in CEC values in the various polytene chromosome regions after RNase treatment is in agreement with published findings for other materials (Amaral and Mello, 1989) (Table VI). One tentatively explanatory hypothesis is that while RNA was removed by the enzyme, the DNA structure may have changed, also with an increase in its compactness state within the chromatin filament and a consequent increase in the amount of available DNA phosphate per unit area, thus requiring a higher concentration of Mg2+ cations to prevent metachromasy. In favor of this hypothesis is the report for chromosomal DNA of polytene chromosomes of Rhynchosciara americana and Bradysia hygida showing an increase in anomalous dispersion of birefringence and linear dichroism after RNase treatment (Almeida et al., 1974). These optical anisotropy findings have been attributed to recovery of the DNA double-helix structure and removal of the steric hindrances introduced by RNA presence, making many DNA phosphates more suitable for TB binding after enzymic RNA removal (Almeida et al., 1974). Note also that the participation of an RNA class in the architectural organization of chromatin has been proposed (Baskin, 1995). Thus, an increase in CEC values in the polytene chromosome regions, especially puffs and RNA-rich zones of T. pubescens after RNase treatment, could be induced by changes in DNA/chromatin geometry promoted by RNA removal.
- CEC values in M MgCl2 for the chromatin of polytene chromosomes in Trichosia pubescens and in some other materials similarly fixed and subjected to RNase treatment.
1Mello and Vidal, 1989; 2Mello and Falco, 1996; 3Amaral and Mello, 1989; 4Mello, M.L.S., unpublished data.
A decrease in CEC after RNase treatment in a few heterochromatin zones specifically corresponding to chromosome break-points (Amabis, 1983b) could not be explained. Also unexplainable is the decrease in CEC values after this treatment in the NOR-containing region 10 of the X chromosome, involved in RNA transcription and exhibiting small micronucleolar bodies (Amabis, 1983b).
It is not to be overlooked that variation in CEC values with chromosome regions and insect development as shown here was detected in preparations treated with acetic acid-containing fixative and squashing solutions. These are assumed to induce extraction of some proteins from the chromosomes (Dick and Johns, 1968; Brody, 1974; Retief and Rüchel, 1977) and introduce some change in DNA and RNA phosphate availability. In fact, unpublished data reveal that when the chromosomes studied here had been fixed in a 1.75% saline paraformaldehyde solution for l min, a CEC value equal to 0.2 M was found all along the chromosomes and RNA removal by RNase was prevented. However, since CEC variability was revealed in acetic acid-fixed preparations, it is inferred that this variation reflects underlying differences in nucleoprotein complexes even after partial protein removal, which may be advantageous when one intends to detect them.
Alterations at the level of nucleoprotein complexes in puff regions before they are morphologically detectable were assumed from changes in CEC values (examples, regions 13B and 28BC in A chromosome, and regions 2AB and 13E in C chromosome).
In conclusion, the interpretation of CEC values for polytene chromosomes seems to be a rather complex matter. On the one hand, a high availability of nucleic acid phosphates as well as the tight package of available nucleic acid phosphates are in fact crucial agents in determining higher CEC values. On the other hand, despite the fact that the presence of RNA is also responsible for high CEC values in chromatin, RNA removal could be found to induce an increase or maintenance of high CEC values, by affecting the DNA geometry and increasing its packing state in chromatin. Indeed, no particular type of chromatin organization in the polytene chromosomes could be characterized by a unique CEC value differing from what happens in in vitro models and in sperm cell nuclei of different species containing different types of basic proteins making a complex with the DNA (Vidal and Mello, 1989; Mello and Falco, 1996; Taboga et al., 1996; Falco and Mello, 1997). Probably, CEC is too sensitive even to a slight variation in nucleoprotein complex composition and organization, especially when RNA is involved. CEC is thus not recommended as a useful tool for extensive and comparative studies involving polytene chromosomes.
ACKNOWLEDGMENTS
We thank Dr. José Mariano Amabis (USP, São Paulo) for providing the insects, for laboratory facilities and for helpful criticism. Thanks are also due to Dr. Benedicto de Campos Vidal (UNICAMP, Campinas) for critically reading the manuscript. Financial support by the Brazilian agencies CAPES and CNPq is also acknowledged. Publication supported by FAPESP.
RESUMO
A ligação de moléculas de azul de toluidina a grupamentos fosfatos de ácido nucléico em condições de competição com íons Mg2+ foi estudada em regiões de cromossomos politênicos de Trichosia pubescens que diferem em composição e organização (áreas ricas em RNA, pufes de RNA e DNA, heterocromatina). O objetivo foi encontrar variabilidade na concentração de Mg2+ na qual a metacromasia fosse completamente abolida (= concentração crítica de eletrólitos; CEC) e que refletisse diferenças ao nível dos complexos nucleoprotéicos em regiões cromossômicas e fases do desenvolvimento particulares. Embora valores altos de CEC tenham sido encontrados na cromatina altamente empacotada, como nas zonas heterocromáticas, os valores de CEC para outras regiões mostraram-se afetados não apenas pela disponibilidade e proximidade de fosfatos do DNA mas também pela riqueza em RNA e outros fatores. Entre estes, mudanças na geometria e no estado de empacotamento do DNA foram consideradas para explicar valores aumentados após remoção de RNA em pufes de DNA e zonas ricas em RNA. Baseando-se em valores de CEC foi sugerido que possam ocorrer alterações ao nível dos complexos nucleoprotéicos em pufes antes que estes sejam detectáveis morfologicamente como tal. Os valores de CEC nos cromossomos politênicos revelaram-se, pois, altamente variáveis com as variações em composição e organização nucleoprotéica do substrato. Contudo, uma vez que um raciocínio complexo e aparentemente contraditório tem que ser utilizado para a interpretação de parte dos resultados, a CEC não é recomendável como ferramenta para estudos extensivos e comparativos neste modelo em particular.
(Received November 8, 1996)
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Publication Dates
-
Publication in this collection
06 Jan 1999 -
Date of issue
June 1998
History
-
Received
08 Nov 1996
