Detection of active genes in mouse metaphase chromosomes using DNAse I in situ

The active genes of rRNA were localized near the centromere region of metacentric translocation chromosome Rb(9, 19) 163H in early mouse embryos revealed by differential silver staining of NORs. Using nick-translation reaction in situ it was shown that rRNA genes in metaphase chromosomes were in a deoxyribonuclease I sensitive conformation. This method of nick-translation in situ can be used for visualization of various actively transcribed regions of genome at metaphase.     

Nucleolar organizer regions (NORs) distribution and behavior in spermatozoa and meiotic cells of the horse (Equus caballus)

Nucleolar organizing regions (NORs) containing rDNA gene clusters have been assigned to the equine autosomes ECA1, ECA28, and ECA31. Active NORs (Ag-NORs) are associated with argyrophilic proteins, which allow them to be readily identified using silver staining techniques. Fluorescence in situ hybridization (FISH) for rDNA can also be used to visualize all NOR clusters in the nucleus, regardless of whether they are active or inactive. The present study analyzed the distribution and behavior of equine Ag-NOR and NOR clusters in horse spermatozoa and during male meiosis by FISH and silver staining. The NOR foci were observed to be variable in number, size, and shape, but were usually located centrally and appeared as one or two nucleolus-like structures in the spermatozoa head. Three distinctive FISH signals identified the NOR-bearing chromosome pairs during the synaptic cell stage of meiosis I. At diakinesis/metaphase I, as well as different stages of meiosis II, FISH signals clearly depicted the NOR-bearing sister chromatids. The synaptonemal complexes of primary spermatocytes consistently showed three rDNA foci following FISH, but variably demonstrated two or three Ag-NOR bodies following silver staining. We propose rDNA loss and gain during unequal crossing-over events could be both a direct and indirect cause of variation in equine NOR foci. Additionally, our cytogenetic analysis did not confirm the presence of a fourth pair of NORs-bearing chromosomes in the horse, which is contrary to previously mitotic published data.     

Affinity isolation of active murine erythroleukemia cell chromatin: uniform distribution of ubiquitinated histone H2A between active and inactive fractions.

This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,3'-dithiopropionyl-3- aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragments which had bound to the affinity complex. Western blot assessment for ubiquitinate histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.     

Lack of correspondence between CMA3-, Ag-positive signals and 28S rDNA loci in two Iberian minnows (Teleostei, Cyprinidae) evidenced by sequential banding

Despite the growing outcome of results that put doubt upon the reliability of silver (Ag) staining and chromomycin A3 (CMA3) fluorescent banding in the detection of major ribosomal gene sites (NORs), these methods have been widely used, especially in fishes. In order to clarify the previous patterns obtained with those techniques, we performed fluorescence in situ hybridisation (FISH) with 28S rDNA probe followed by sequential CMA3 and Ag staining in diploid non-hybrid males of the Squalius alburnoides complex and in Squalius pyrenaicus. The results from all the studied specimens revealed a lack of correlation between classical and molecular techniques. Not just some other regions besides NORs were stained with CMA3 and Ag, but also the majority of the 28S rDNA sites were not detected. Care should then be taken in considering CMA3- and Ag-stained sites as NORs since their accuracy for that purpose may not always correspond to the expectations.     

Nuclease sensitivity of the mouse HPRT gene promoter region: differential sensitivity on the active and inactive X chromosomes.

We investigated the conformation of the X-linked mouse hypoxanthine-guanine phosphoribosyltransferase gene (HPRT) promoter region both in chromatin from the active and inactive X chromosomes with DNase I and in naked supercoiled DNA with S1 nuclease. A direct comparison of the chromatin structures of the active and inactive mouse HPRT promoter regions was performed by simultaneous DNase I treatment of the active and inactive X chromosomes in the nucleus of interspecies hybrid cells from Mus musculus and Mus caroli. Using a restriction fragment length polymorphism to distinguish between the active and inactive HPRT promoters, we found a small but very distinct difference in the DNase I sensitivity of active versus inactive chromatin. We also observed a single DNase I-hypersensitive site in the immediate area of the promoter which was present only on the active X chromosome. Analysis of the promoter region by S1 nuclease digestion of supercoiled plasmid DNA showed an S1-sensitive site which maps adjacent to or within the DNase I-hypersensitive site found in chromatin but upstream of the region minimally required for normal HPRT gene expression.     

DNase I-sensitive sites within the nuclear architecture visualized by immunoelectron microscopy

Nick-translation using mild digestion with DNase I allows preferential labeling of actively transcribing or potentially active genes, as compared with inactive genes. We have adapted this method to the level of electron microscopy to see the DNase I-sensitive regions in situ in Ehrlich tumor cells. In interphase cells treated with very low concentrations of DNase I, labeled sequences are found at the borders and in the close vicinity of condensed chromatin blocks. Labeling of condensed areas of chromatin requires higher DNase I concentrations and longer incubation in the nick-translation medium. In the nucleolus, the first sites to be nick-translated are the fibrillar centers and the interstices surrounding them, whereas the dense fibrillar component never contains labeled sequences. When cells are pretreated with actinomycin D, only a few perinucleolar clumps of condensed chromatin are labeled under the same conditions. This method provides a new tool for studying the functional organization of chromatin within a cell. The precise location of nick-translated sites in nucleolar components observed could change classical views concerning the functional organization of the nucleolus.     

DNAse I digestion of fixed chromosomes specifically reveals NORs in man

Chromosomal preparations were digested with different concentrations of DNAse I for various periods of time and then stained with Giemsa. It was found that this endonuclease rapidly modifies the structure of the chromosomes which then lose most of their stainability. However, small chromosomal segments corresponding to the nucleolar organizers (NORs) in man have been consistently observed. Although DNAse I treatment has permitted us to observe that some NORs are stained less intensely than others, comparisons with silver nitrate-stained NORs have shown a strict correspondence, in metaphase as well as in interphase. Chromosomal proteins seem responsible for this staining.