Immunogold electron microscope localization of proteins in Drosophila polytene chromosomes: applications and limitations of the method (original) (raw)
Related papers
Chromosoma, 1977
Chromosomal proteins have been prepared from embryos of Drosophila melanogaster and separated into histone and nonhistone fractions by a procedure which completely avoids exposure to extremes of pH. These fractions have been characterised by amino acid analysis and gel electrophoresis. Antisera have been prepared against whole chromatin and against the two chromosomal protein fractions.-A new method is described for the preparation of Drosophila salivary chromosomes. This method employs microdissection techniques and completely avoids the use of acid fixatives. Preservation of fine structure in these preparations is comparable to, if not better than, that in classical acid-fixed preparations. Antisera against embryo chrornatin and chromosomal protein fractions react with the salivary chromosome preparations. These reactions exhibit selectivity with different chromosomal structures. Evidence is presented suggesting a specific distribution of protein antigens along the chromosome.
Immunological Method for Mapping Genes on Drosophila Polytene Chromosomes
Proceedings of The National Academy of Sciences, 1982
A method is described for localizing DNA sequences hybridized in situ to Drosophila polytene chromosomes. This procedure utilizes a biotin-labeled analog of TTP that can be incorporated enzymatically into DNA probes by nick-translation. After hybridization in situ, the biotin molecules in the probe serve as antigens which bind affinity-purified rabbit antibiotin antibodies. The site of hybridization is then detected either fluorimetrically, by using fluorescein-labeled goat anti-rabbit IgG, or cytochemically, by using an anti-rabbit IgG antibody conjugated to horseradish peroxidase. When combined with Giemsa staining, the immunoperoxidase detection method provides a permanent record that is suitable for detailed cytogenetic analysis. This immunological approach offers four advantages over conventional autoradiographic procedures for detecting in situ hybrids: (i) the time required to determine the site of hybridization is decreased markedly; (ii) biotin-labeled probes are chemically stable and give reproducible results for many months; (iii) biotin-labeled probes appear to produce less background noise than do radiolabeled probes; and (iv) the resolving power is equal to and often greater than that achieved autoradiographically.
2010
Hybridomas secreting monoclonal antibodies have been produced by fusion of NS-1 mouse myeloma cells with the spleen cells of mice inoculated with a 60-65,000-mol wt fraction of proteins released from Drosophila embryo nuclei treated with DNase I. The antibodies secreted by the hybridomas were examined with polytene chromosomes of formaldehyde-fixed salivary gland squashes by an immunofluorescence assay. Most of the clonal antibodies obtained resulted in specific staining of the chromosomes relative to the cytoplasmic debris . In the case of clone 28, the antibodies showed a preferential association with sites of gene activity, both puffs and loci identified as puffing at some time during the third instar and prepupal period . In larvae that were heat shocked (exposed to 35°C for 15 min before removal and fixation of the glands), the antibodies of clone 28 stained preferentially the induced heatshock loci while continuing to stain most of the normal set of loci . The antigen for clone 28 was identified as a single protein of 62,000 mol wt by using the antibodies followed by 1211_ rabbit anti-mouse Ig to stain nitrocellulose replicas of SDS polyacrylamide gels of total chromosomal proteins . This study demonstrates that monoclonal antibodies can be used successfully in immunofluorescence staining of formaldehyde-fixed polytene chromosomes. The results verify the hypothesis that a specific nonhistone chromosomal protein is preferentially associated with the set of loci that includes both active sites and those scheduled to be active at some time in this developmental program. Such proteins may play a general role in the mechanisms of cell determination and gene activation .
2009
The giant polytene chromosomes from Drosophila third instar larval salivary glands provide an important model system for studying the architectural changes in chromatin morphology associated with the process of transcription initiation and elongation. Especially, analysis of the heat shock response has proved useful in correlating chromatin structure remodeling with transcriptional activity. An important tool for such studies is the labeling of polytene chromosome squash preparations with antibodies to the enzymes, transcription factors, or histone modifications of interest. However, in any immunohistochemical experiment there will be advantages and disadvantages to different methods of fixation and sample preparation, the relative merits of which must be balanced. Here we provide detailed protocols for polytene chromosome squash preparation and discuss their relative pros and cons in terms of suitability for reliable antibody labeling and preservation of high resolution chromatin structure.
Locations of chromosomal proteins in polytene chromosomes
Proceedings of the National Academy of Sciences, 1976
DI, a nonhistone chromosomal protein rich in both basic and acidic amino acids, has been localized at a limited number of specific loci in polytene chromosomes of Drosophila melanogaster. H2B, a nucleosomal histone, and Hi, a nonnucleosomal histone, are both found throughout most chromosomal regions. Study of the role of proteins in gene function in eukaryotes has been hampered by the complexity of chromatin. Thus, whereas many proteins have been detected in purified chromatin preparations, none of them has yet been shown to be associated with specific genetic loci. We have now been able to localize by immunofluorescence a nonhistone protein in the polytene chromosomes of Drosophila.
Native" Salivary Chromosomes of Drosophila melanogaster
Cold Spring Harbor Symposia on Quantitative Biology, 1978
A method has been developed which, for the first time, allows the preparation of mappable cytological spreads of salivary chromosomes from D. melanogaster without exposure to acid fixatives. These isolated native chromosomes show the best preservation of ultrastructure observed to date-ribonucleoprotein particles may be seen to be organized in linear arrays in transcriptionally active puffs and the repeating nucleosome module is present. Native salivary chromosomes are proving useful for the localization of nuclear proteins both at the light microscope and ultrastructural levels. They display only background-level binding of antibodies specific for the Z-DNA conformation. However, Z-DNA immunoreactivity is activated by exposure to acid fixative, first in interbands and then in bands. The Z-conformation in the chromosomes is held in place by elastic torsional strain which appears in the DNA following acid fixation. Native D. melanogaster salivary chromosomes offer promise for enabling the probing of the chromatin of known genetic loci for properties dependent on the preservation of macromolecular integrity.
BMC Genomics, 2011
Background: Despite many efforts, little is known about distribution and interactions of chromatin proteins which contribute to the specificity of chromomeric organization of interphase chromosomes. To address this issue, we used publicly available datasets from several recent Drosophila genome-wide mapping and annotation projects, in particular, those from modENCODE project, and compared molecular organization of 13 interband regions which were accurately mapped previously.
Russian Journal of Genetics, 2011
In eukaryotic genomes, genetic material is represented by two types of chromatin-euchromatin, which is decondensed in interphase nucleus, and heterochromatin, epigenetically inactivated chromosomal material that is always densely packed. Giant polytene chromosomes in Drosophila salivary glands contain pericentromeric heterochromatin (PH) regions, which are formed primarily by highly repeated DNA sequences, as well as approximately 250 intercalary heterochromatin (IH) regions. These regions are scattered along chromosome arms and share many properties with PH, such as chromatin compaction, delayed replication completion, and binding to the SUUR protein . Application of microchip technology revealed 52 regions in IH in which DNA is underreplicated. Each of these regions contains blocks numbering 5 to 40 unique genes whose replication finishes late both in the differentiated cells of salivary gland, which contain polytene chromosomes, and in the cells undergoing mitosis (Kc cultured cells). In half of these clusters genes are expressed in a similar way by forming transcriptional territories . However, the functional relationships in IH regions remain largely obscure, and there are data for only some of them. In particular, it was established that bands 56F and 39DE contain clusters of tandemly arranged repeated genes encoding 5S rRNA and histones (HIS-C), respectively [1]; bands 89E1-4 and 84AB, clusters of homeobox-containing genes Bithorax-Complex and Antennapedia-Complex , respectively [4]; the most underreplicated zone of band 75C1-2, four genes that are together involved in the regulation of apoptosis [2]; and band 83E, a cluster of 20 Osiris genes encoding related proteins containing a conserved transmembrane domain . Thus, available data suggest that some IH regions contain genes that are grouped together in accordance with certain principles that ensure coordinated regulation. In view of above, a question arises as to whether this conclusion applies to other IH regions. In this study, as a result of combination of data on bioinformatics and cytogenetic analysis of one IH region, band 10A1-2, we provide evidence for inhomogeneous distribution of genes in Drosophila genome. We showed that gene density is high on the boundaries of IH regions but within the IH region as such it corresponds to the average values characteristic of X chromosome. The level of gene expression in high-density regions is higher than in IH regions.
Chromosoma, 1981
Fragments from section 3 of the salivary gland X chromosome of D. melanogaster were dissected with a micromanipulator. The DNA was extracted, cut and ligated to a λ vector in a volume of a few nanoliters in an oil chamber monitored through a microscope. From about 10 pg of DNA we obtained 80 recombinant clones, a sample of which were analysed and shown to contain Drosophila DNA which hybridises in situ to the region of section 3 of the X chromosome. With this technique we can isolate clones from any desired region as small as 200 kb from the euchromatic arms of polytene chromosomes.