Molecular cytogenetic analysis of the highly repetitive DNA in the genome of Apodemus argenteus, with comments on the phylogenetic relationships in the genus Apodemus.

The DNA of Apodemus argenteus was digested with DraI, and the resultant DraI fragment of highly repetitive DNA was isolated and analyzed by DNA filter hybridization, cloning, sequencing, and fluorescence in situ hybridization (FISH). Southern blot hybridization and nucleotide sequencing revealed that most of the DraI fragment consisted of a 230-bp repeating unit and contained no sex-chromosome-specific nucleotide sequences. The DraI fragment included the CENP-B box-like sequence, with a strong homology to the human CENP-B box sequence. FISH revealed that the DraI fragment was specific to all pericentromeric C-band-positive regions, as well as to the C-block of the X chromosome. No hybridization signals were obtained from A. speciosus, A. peninsulae peninsulae, A.p. giliacus, A. agrarius, A. sylvaticus, A. semotus, or Mus musculus when the DraI fragment was used as probe. Peptide nucleic acid (PNA)-FISH using the CENP-B box-like sequence in the DraI fragments as probe suggested that this nucleotide sequence was also specific to all pericentromeric C-heterochromatic regions of A. argenteus chromosomes. Zoo-blot hybridization using DraI-digested genomic DNA from three species of Apodemus (namely, A. argenteus, A. speciosus, and A. peninsulae) and from Mus musculus strongly suggested that the consensus DraI fragment contained nucleotide sequences that were species-specific for A. argenteus. These results also suggest that A. argenteus is phylogenetically distant from other Apodemus species examined, as well as the possibility that the DraI fragment might be related directly to the delayed quinacrine mustard fluorescence of many pericentromeric C-heterochromatic regions of the chromosomes in A. argenteus.     

Modeling heterochromatin regions in transgenic mice

Transgenic mice carrying bovine satellite DNA IV were obtained. The size of the transgene integrated into the mouse genome was approximately 390 kb (about 100 transgene copies) as determined by a semiquantitative PCR. Restriction analysis with isoschizomeric restrictases HpaII and MspI, showed that the alien DNA was methylated. In the genome of a transgenic founder male, two integration sites for satellite DNA IV were revealed by in situ hybridization and in situ PCR. These sites are situated on two different chromosomes: in pericentromeric heterochromatin and within a chromosomal arm. In transgenic mice, de novo formation of heterochromatin regions (C-block and the CMA3 disk within the centromeric heterochromatin of another chromosome) was revealed by C-banding and staining with chromomycin A3. This formation is not characteristic of mice, because their chromosomes normally contain no interstitial C-blocks or sequences intensely stained by chromomycin A3.     

B chromosomes of Korean field mouse Apodemus peninsulae (Rodentia,Murinae) analysed by microdissection and FISH

Organization of B chromosomes in the Korean field mouse Apodemus peninsulae was analyzed. We painted its metaphase chromosomes with whole and partial chromosome paints generated by microdissection and DOP-PCR. The results of the painting indicated that all B chromosomes contained a large amount of repeated DNA sequences. The repeats could be classified in terms of their homology and predominant location. Pericentromeric repeats of B chromosomes were present in many copies in pericentromeric C-blocks of all autosomes and in non-centromeric C-blocks of the sex chromosomes. B arm specific type 1 repeats comprised the main body of the arms of almost all B chromosomes and were present in the arms of A chromosomes as interspersed sequences. B arm-specific type 2 repeats were found at the ends of some B chromosomes that did not undergo compaction at the interphase- metaphase transition and remained uncondensed. On the basis of comparative analysis of localization of B chromosome repeats in the chromosomes of two related species, A. peninsulae and A. agrarius, we suggest a hypothesis of B chromosome origin and evolution in the genus Apodemus.     

Methylation-sensitive restriction endonuclease digestion patterns revealed in Vicia faba L. chromosomes by in situ nick-translation

Restriction endonucleases sensitive to cytosine methylation (HpaII, MspI and HhaI) and 5-azacitidine were used to study the localization of target sequences in Vicia faba metaphase chromosomes by in situ digestion and radioactive or non-radioactive nick-translation. In control experiments, neither isolated DNA nor chromosomes in situ were digested by HpaII and MspI. Pretreatment with demethylating agent, 5-azacitidine resulted both in increased effectiveness of in situ digestion and nick-translation. In 5-azacitidine-treated material, negative bands in M chromosomes appeared. HhaI cleaved isolated DNA, digested it in situ and gave positive signals as a result of nick-translation procedure in metaphase chromosomes. In S chromosomes containing heterochromatin without target sequences for HpaII and MspI, negative bands were shown after nick-translation. Such heterochromatin contains FokI sequences and in situ nick-translation driven by that restriction enzyme resulted in positive bands.     

Eimerians from different karyotypes of the Japanese wood mouse (Apodemus spp.), with descriptions of two new species and a redescription of Eimeria montgomeryae Lewis and Ball, 1983

Examination of 131 wood mice (Apodemus spp.) representing 2 species and 6 subspecies collected from the Japanese islands of Hokkaido, Honshu, Kyushu, and Tsushima showed that 70 mice (53%) had coccidian oocysts in their feces. These included 21 of 42 (50%) Apodemus argenteus argenteus; 7 of 14 (50%) Apodemus argenteus hokkaidi; 2 of 3 (67%) Apodemus argenteus sagax; 3 of 9 (33%) Apodemus speciosus ainu; 36 of 61 (59%) Apodemus speciosus speciosus; and 1 of 2 (50%) Apodemus speciosus tusimaensis. Four distinct coccidians were identified: Eimeria argenteus n. sp. from A. a. argenteus, A. a. hokkaidi, A. a. sagax, and A. s. speciosus; Eimeria inuyamensis n. sp. from A. a. argenteus, A. s. speciosus, and A. s. tusimaensis; Eimeria montgomeryae Lewis and Ball, 1983, from A. a. argenteus, A. a. hokkaidi, A. a. sagax, A. s. ainu, and A. s. speciosus; and Eimeria uptoni Lewis and Ball, 1983, from A. a. argenteus, A. a. hokkaidi, and A. s. speciosus. Standard karyotypes were prepared from selected specimens of each host subspecies. All 3 subspecies of A. argenteus and A. s. tusimaensis have a 2n = 46; A. s. ainu, from Hokkaido, has a 2n = 48; and A. s. speciosus has at least 2 chromosomal races, 1 on northern (2n = 48) and 1 on southern (2n = 46) Honshu. Both chromosomal races of A. s. speciosus, as well as the other subspecies of Apodemus examined, shared their coccidian parasites freely.     

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.     

In situ hybridization with fluoresceinated DNA.

We have used fluorescein-11-dUTP in a nick-translation format to produce fluoresceinated human nucleic acid probes. After in situ hybridization of fluoresceinated DNAs to human metaphase chromosomes, the detection sensitivity was found to be 50-100 kb. The feasibility and the increase in detection sensitivity of microscopic imaging of in situ hybridized, fluoresceinated DNA with an integrating solid state camera for rapid cosmid mapping is illustrated. Combination of fluoresceinated DNA with biotinated and digoxigeninated DNAs allowed easy performance of triple fluorescence in situ hybridization. The potential of these techniques for DNA mapping, cytogenetics and biological dosimetry is briefly discussed.     

A highly repeated FCP centromeric sequence from chaffinch (Fringilla coelebs: Aves) genome is revealed within interchromosomal connectives during mitosis

A highly repeated FCP (Fringilla coelebs PstI element) sequence was localized by FISH in centromeric regions of all chromosomes of the chaffinch. Besides, FISH signal was found also in interchromosomal connectives linking centromeres of non-homologous chromosomes in mitotic cells. The presence of DNA in the connectives was confirmed by immunostaining with anti-dsDNA antibodies as well as in experiments on nick-translation and random primed labeling in situ. Non-denaturing FISH with FCP probe and random primed labeling of non-denatured chromosomes resulted in fluorescence signal on both centromeres and intercentromeric connectives, thus providing evidence for the availability of single-strand DNA tracts in FCP sequence. It is suggested that the highly repeated FCP centromeric sequence may be respondible for interconnection of mitotic chromosomes and may by involved in nuclear architecture maintenance in the chaffinch.     

Laboratory Hints from the Literature

The employment of certain DNA-specific fluorescent stains on unhanded and C-banded chromosomes of two species of grasshoppers shows remarkable differences among C-heterochromatic regions supposed to be similar in their base pair composition, according to their response to the standard fluorescence techniques. The possible interspersion of the opposite DNA base pairs in these regions as well as the role played by proteins in chromosome banding are discussed.     

New sites of methylcytosine-rich DNA detected on metaphase chromosomes

In situ immunofluorescence detection of antibodies against 5-methylcytosine on metaphase chromosomes prepared by a new procedure allows the display of new 5-methylcytosine-rich sites as compared to previously published methods. In short-term culture lymphocytes, the immunofluorescent signals give a recurrent pattern in which four types of binding sites can be distinguished. Type I sites are the secondary constrictions and a few juxtacentromeric regions, type II sites correspond to T-bands. Both types I and II sites emit a strong fluorescence. Type III sites form an R-band pattern and emit a weaker fluorescence. Type IV sites are the short arms of acrocentrics, they emit strong but polymorphic signals. The results obtained from control experiments suggest that the pattern observed is rather the expression of an uneven distribution of 5-methylcytosine-rich sites than a consequence of the various treatments used. In a lymphoblastoid cell line known to have a reduced 5-methylcytosine content, it was possible to demonstrate a heterogeneous hypomethylation among chromosome structures, principally involving type I sites. The method opens the possibility of studying in situ on chromosomes, regional variations of methylation in pathological conditions.     

First record of the larval parasitic nematode Rhabditis orbitalis from Japanese wood mice (Apodemus spp.)

Parasitic larvae of Rhabditis orbitalis (formerly confused with Pelodera strongyloides) were found in the conjunctival sacs of the eyes of Apodemus argenteus and A. speciosus from Japanese islands.     

The mammalian model for population studies of B chromosomes: the wood mouse (Apodemus)

The presence of B chromosomes was reported in six species of the genus Apodemus (A. peninsulae, A. agrarius, A. sylvaticus, A. flavicollis, A. mystacinus, A. argenteus). High frequencies of Bs were recorded particularly in A. peninsulae and A. flavicollis. The origin of Bs in Apodemus seems to be rather ancient, and it is possible that the supernumerary elements, and/or a tendency for their appearance, were inherited from the common ancestor of the extant species. We have not found any correlated changes between frequencies of Bs and the level of protein polymorphism and/or heterozygosity assessed in electrophoretic studies. No measurable effect of Bs on overall genetic variability was thus revealed in studied populations. The pattern of evolutionary dynamics of Bs can be distinctly different between geographical populations, and both the parasitic and the heterotic models can be applied to explain the maintenance of Bs in different populations. Further studies are desirable to improve our understanding of the complicated evolutionary dynamics of Bs in the Apodemus species. An essential condition for success in this respect is much more detailed information on inheritance and the molecular structure of Bs.     

C-Banding with Specific Fluorescent DNA-Ligands: a New Approach to Constitutive Heterochromatin Heterogeneity

The employment of certain DNA-specific fluorescent stains on unhanded and C-banded chromosomes of two species of grasshoppers shows remarkable differences among C-heterochromatic regions supposed to be similar in their base pair composition, according to their response to the standard fluorescence techniques. The possible interspersion of the opposite DNA base pairs in these regions as well as the role played by proteins in chromosome banding are discussed.     

Color Differential Staining of Nor-Associated Heterochromatic Segments Using Acridine Orange

A new staining technique has been evaluated for detecting heterochromatic segments accompanying nucleolus organizing regions (NORs). This technique essentially consists of C-banding followed by acridine orange staining. When the technique was applied to five species of plants, the NOR-associated heterochromatic segments (NOR H-segments) were differentiated from other segments of the chromosomes as regions emitting yellowish green fluorescence. An incubation of at least 30 min in hot 2 × SSC was required to make the NOR H-segments emit yellowish green fluorescence in Nothoicordum fragrans. Fluorescence on other heterocnromatic segments varied from reddish orange to bright yellow; euchromatic segments emitted orange or yellowish orange fluorescence. The technique permits identification of NOR H-segments throughout mitosis based on the characteristic fluorescent color.     

Color differential staining of NOR-associated heterochromatic segments using acridine orange

A new staining technique has been evaluated for detecting heterochromatic segments accompanying nucleolus organizing regions (NORs). This technique essentially consists of C-banding followed by acridine orange staining. When the technique was applied to five species of plants, the NOR-associated heterochromatic segments (NOR H-segments) were differentiated from other segments of the chromosomes as regions emitting yellowish green fluorescence. An incubation of at least 30 min in hot 2 x SSC was required to make the NOR H-segments emit yellowish green fluorescence in Nothoscordum fragrans. Fluorescence on other heterochromatic segments varied from reddish orange to bright yellow; euchromatic segments emitted orange or yellowish orange fluorescence. The technique permits identification of NOR H-segments throughout mitosis based on the characteristic fluorescent color.     

Restriction enzyme banding and in situ nick-translation on different types of hetero- and euchromatin.

We studied the role of chromatin accessibility and methylation in the banding patterns produced by means of in situ nick-translation (NT) and restriction enzyme (RE) banding techniques. For these studies we used the X chromosomes of Microtus cabrerae because of their large segment with four different types of constitutive heterochromatin and because in these chromosomes we can also compare active and inactive euchromatin. The results demonstrate that constitutive heterochromatin in the X chromosomes of M. cabrerae is methylated at specific sequences in both active and inactive Xs. They also show that NT-based techniques are suitable for detecting weak differences in chromatin accessibility, such as differences between active and inactive euchromatin, and are able to distinguish methylation only at the accessible sites. Thus, when methylation has to be mapped in situ, additional experiments have to be performed in order to distinguish findings due to differential accessibility. RE banding seems less sensitive to slight differences in chromatin accessibility, and might thus be more suitable than in situ NT-based techniques for methylation mapping. In harmony with these results, HpaII-based RE banding is able to distinguish between active and inactive euchromatin, possibly depending on its methylation status.     

Karyotypic variation between wood mouse species: banded chromosomes ofApodemus alpicola andA. microps

The banding pattern (G-, C-, AgNOR-staining) was described in karyotypes ofApodemus alpicola Heinrich, 1952 andA. microps Kratochvil et Rosicky, 1952 collected from the Alps and central Europe. Distinct differences between the two species were revealed in the distribution of C-heterochromatic regions in autosomes and the sex chromosomes, and the distribution of nucleolar organizer regions (NORs). Extensive variation in the distribution pattern of C-heterochromatin and NORs obviously exists among the wood mice of the subgenusSylvaemus, and individual species can be distinguished according to a specific variation pattern. However, it seems premature to designate individual karyotypic forms as separate species, because the extent of overall geographical interpopulation variation is still not sufficiently known.     

Sorting of chromosomes by magnetic separation

Summary Chromosomes were isolated from Chinese hamster x human hybrid cell lines containing four and nine human chromosomes. Human genomic DNA was biotinylated by nick translation and used to label the human chromosomes by in situ hybridization in suspension. Streptavidin was covalently coupled to the surface of magnetic beads and these were incubated with the hybridized chromosomes. The human chromosomes were bound to the magnetic beads through the strong biotin-streptavidin complex and then rapidly separated from nonlabeled Chinese hamster chromosomes by a simple permanent magnet. The hybridization was visualized by additional binding of avidin-FITC (fluorescein) to the unoccupied biotinylated human DNA bound to the human chromosomes. After magnetic separation, up to 98% of the individual chromosomes attached to magnetic beads were classified as human chromosomes by fluorescence microscopy.     

Uneven extraction of protein in Chinese hamster chromosomes during G-staining procedures

To elucidate the role of chromosomal protein in G-band production, changes of protein distribution in chromosomes were studied in situ at each step of G-staining procedures. As a highly specific stain for protein, dansyl Cl was used, which conjugated with amino groups in polypeptide to emit bright fluorescence under UV irradiation, so that the pattern of fluorescence of dansyl-stained chromosomes was expected to reflect the distribution of protein. Uniform fluorescence pattern observed in untreated, dansyl-stained chromosomes indicated even distribution of protein in the ordinary air-dried chromosomes. The pattern of fluorescence representing the distribution of chromosomal protein after pretreatments of G-staining showed brighter outlines of chromatids, reduced fluorescence of chromosome body, and a slight difference in intensity along chromosome arms which corresponded to G-bands. This correspondence was confirmed when Giemsa stain was removed from G-banded chromosomes and the chromosomes were stained with dansyl Cl. The resulting dim fluorescence pattern conformed to G-bands previously observed in the same chromosomes. Similar events were observed in HCl-extracted chromosome slides, although the fluorescence was considerably reduced in this case. Our results inferred that chromosomal protein was partially lost during pretreatments of G-staining, that acid-soluble protein assumed less significant role in G-staining mechanism, and that uneven deprivation of acid-insoluble protein may occur during G-staining procedures.     

Delineation of marker chromosomes by reverse chromosome painting using only a small number of DOP-PCR amplified microdissected chromosomes

A new procedure for determining the chromosomal origin of marker chromosomes has been carried out. The origin of marker chromosomes that were unidentifiable by standard banding techniques could be verified by reverse chromosome painting. This technique includes microdissection, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH). A number of marker chromosomes prepared from unbanded and from GTG-banded lymphocyte chromosomes were collected with microneedles and transferred to a collection drop. The chromosomal material was amplified by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The resulting PCR products were labelled by nick-translation with biotin-11-dUTP and used as probes for FISH. They were hybridized onto normal metaphase spreads in order to determine the precise regional chromosomal origin of the markers. Following this approach, we tested 2–14 marker chromosomes in order to determine how many are necessary for reverse chromosome painting. As few as two marker chromosomes provided sufficient material to paint the appropriate chromosome of origin, regardless of whether the marker contained heterochromatic or mainly euchromatic material. With this method, it was possible to identify two marker chromosomes of a healthy proband [karyotype: 48,XY, +mar₁,+mar₂] and an aberrant Y chromosome of a mentally retarded boy [karyotype: 46,X, der(Y)].