Chromosome identification and comparative karyotypic analyses of four Pinus species.

Chromosomal landmarks in four Pinus species: P. densiflora, P. thunbergii, P. sylvestris, and P. nigra were identified by fluorescence in situ hybridization (FISH) using hapten- or fluorochrome-labeled probes for the plant telomere repeat, centromeric repeat ( PCSR), and rDNA. FISH landmarks were located at the interstitial and proximal regions of chromosomes and allowed us to identify nearly all of the homologous chromosomes in each species. A comparative analysis of the FISH karyotypes among the four species showed that the interstitial FISH signals obtained by hybridization with the telomere and rDNA sequences were stable and could be used to identify homologous chromosomes among species. The identification of homologous chromosomes among species facilitated a detailed comparative karyotype analysis. The results suggest that the degree of chromosomal differentiation among the four Pinus species is very low and that the proximal regions vary in their DNA sequences. The similarities and differences among FISH karyotypes are discussed in relation to phylogeny.

Cloning of DNA sequences localized on proximal fluorescent chromosome bands by microdissection in Pinus densiflora Sieb. & Zucc.

Japanese red pine, Pinus densiflora, has 2n=24 chromosomes, of which most carry chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) bands at their centromere-proximal regions. It was proposed that these regions contain highly repetitive DNA. The DNA localized in the proximal fluorescent bands was isolated and characterized. In P. densiflora, centromeric and neighboring segments of the somatic chromosomes were dissected with a manual micromanipulator. The centromeric DNA was amplified from the DNA contained in dissected centromeric segments by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) and a cloned DNA library was constructed. Thirty-one clones carrying highly repetitive DNA were selected by colony hybridization using Cot-1 DNA from this species as a probe, and their chromosomal localization was determined by fluorescent in situ hybridization (FISH). Clone PDCD501 was localized to the proximal CMA band of 20 chromosomes. This clone contained tandem repeats, comprising a 27 bp repeat unit, which was sufficient to provide the proximal FISH signal, with a 52.3% GC content. The repetitive sequence was named PCSR (proximal CMA band-specific repeat). Clone PDCD159 was 1700 bp in length, with a 61.7% AT content, and produced FISH signals at the proximal DAPI band of the remaining four chromosomes. Four clones hybridized strongly to the secondary constriction and gave weak signals at the centromeric region of several chromosomes. Clone PDCD537, one of the four clones, was homologous to the 26S rRNA gene. A PCR experiment using microdissected centromeric regions suggested that the centromeric region contains 18S and 26S rDNA. Another 24 clones hybridized to whole chromosome arms, with varying intensities and might represent dispersed repetitive DNA.

Differentiation and the polymorphic nature of the Y chromosomes revealed by repetitive sequences in the dioecious plant, Rumex acetosa.

The dioecious plant Rumex acetosa has a multiple sex chromosome system: females are 2n = XX + 12, males are 2n = XY1Y2 + 12, and the two Y chromosomes are heterochromatic. A DNA sequence abounded in the mare genome was isolated and analyzed. The sequence (RAE180) was a 180-bp-long tandemly arranged repetitive sequence, distributed in chromosomes Y1 and Y2, and two pairs of autosomes. Both Y chromosomes contained large amounts of RAE180 and the sequence formed many DAPI bands, while, on the two pairs of autosomes, RAE180 did not form DAPI bands. The internal structure and morphological changes of the Y chromosomes were analyzed by FISH, using RAE180 and the Y-chromosome-specific sequence RAYSI as probes. The pattern of the FISH signals caused by the accumulation of RAE180 and RAYSI suggested the structural change in the Y chromosomes during the process of sex chromosome evolution, and the morphological change in the Y chromosomes was explained by reciprocal translocation and inversion.

Molecular cytogenetic analysis of supernumerary heterochromatic segments in Rumex acetosa.

The dioecious plant Rumex acetosa shows intraspecific karyotype variation, caused by supernumerary heterochromatic segments or DAPI (4',6-diamidino-2 phenylindole)-bands at the ends of the short arms of three pairs of autosomes. A DNA sequence (RAE730) specific to the supernumerary heterochromatic segments was cloned and sequenced. RAE730 was about 730 bp and AT-rich (71% AT-content). Using fluorescence in situ hybridization (FISH), RAE730 was localized in the supernumerary DAPI-positive heterochromatic segments on several mitotic chromosomes and chromocenters in interphase nuclei, but not in the DAPI-bands of Y or B chromosomes. RAE730 was tandemly arranged in the genome, and the copy number varied between plants from 40000 to 304000 copies per 2C, corresponding to the relative amount of supernumerary heterochromatic segments per genome. These results indicate that the karyotype variation caused by the supernumerary heterochromatic segment was generated by amplification or reduction of the tandem repeats of RAE730.

Chromosome painting of Y chromosomes and isolation of a Y chromosome-specific repetitive sequence in the dioecious plant Rumex acetosa.

The dioecious plant Rumex acetosa has a multiple sex chromosome system: XX in female and XY(1)Y(2) in male. Both types of Y chromosome were isolated from chromosome spreads of males by manual microdissection, and their chromosomal DNA was amplified using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). When the biotin-labeled DOP-PCR product was hybridized with competitor DNA in situ, the fluorescent signal painted the Y chromosomes. A library of Y chromosome DNA was constructed from the DOP-PCR product and screened for DNA sequences specific to the Y chromosome. One Y chromosome-specific DNA sequence was identified and designated RAYSI (R. acetosa Y chromosome-specific sequence I). RAYSI is a tandemly arranged repetitive DNA sequence that maps to the 4',6-diamidino-2-phenylindole bands of both Y chromosomes.

[New mental development screening test for use in health examinations of infants].

OBJECTIVE: This study is designed to produce a new mental development screening test with predictive validity for future developmental disorders. METHOD: 1. The items of mental development screening test were selected provisionally, and the results of the items were stored in the 7 months, 10 months, 1 year, 1 year and 6 months of health examinations in Suzaka city, Nagano Prefecture. 2. All the tested infants were prospectively followed until 5 years of age (the middle year of day nurseries or kindergartens), when their conditions were diagnosed for developmental disorders (mental retardation, pervasive developmental disorder, attention deficit hyperactivity disorder). 3. The relation between 1. and 2. was analyzed. RESULTS: 1. Acceptable levels for items of the mental development screening test were seen in 90% of the infants, except for anxiety with unfamiliar faces at 7 months, finger pointing at 1 year, correct picture recognition response at 1 year and 6 months, etc. 2. The items with high significance levels for chi 2 test and high sensitivity were: 4 items at 7 months of age such as anxiety with unfamiliar faces, sitting ability, parachute reaction, and others; 5 items at 10 months such as anxiety with unfamiliar faces, creeping ability, turning around and looking when called; 8 items at 1 year such as use of words with meaning, auditory imitation, finger pointing; 7 items at 1 year and 6 months such as correct picture recognition response, turning around and looking when called, auditory imitation. 3. The items effective for a screening test were selected based on clinical experience and on the following criteria: public health nurses can conveniently use them at the site of primary screening, and that the items can determine the possibility of future developmental disorders. 4. The cut off points were set for high specificity and sensitivity on scale points to which all the selected items were synthesized, and by which follow up rates were within appropriate ranges. CONCLUSIONS: 1. Items of mental development screening test with predictive validity for future developmental disorders were selected. 2. This study showed that items related to interpersonal relationships are highly important. 3. Possibility of future developmental disorder can be better determined by using cut off points on a scale which utilizes all the selected items, rather than by using individual items separately.

Allodiploid nature of Allium wakegi Araki revealed by genomic in situ hybridization and localization of 5S and 18S rDNAs.

Allium wakegi Araki could be originated from cross hybridization between close relatives of a form of A. cepa and A. fistulosum. Chromosomes of each parental haploid set were identified in the chromosome complement of A. wakegi by genomic in situ hybridization using probes of the total, parental genomic DNAs of A. cepa and A. fistulosum, respectively. The results of GISH suggested significant differentiation of genomic DNAs between these closely related species. Allium cepa had two 5S rDNA loci at the interstitial regions of the short arms of small chromosome pair (the 7th pair) and A. fistulosum had one locus at the interstitial region of the homologous short arms. Allium wakegi had two chromosomes carrying one and two 5S rDNA loci which appeared to correspond to those of A. fistulosum and A. cepa, respectively. Chromosomes carrying 18S rDNA loci originated from those of both A. cepa and A. fistulosum were also observed in the chromosome complement of A. wakegi.

Cytological characterization of NOR in the bivalent of Saccharomyces cerevisiae.

We cytologically characterized the nucleolar organizer region (NOR) on the bivalent in the yeast Saccharomyces cerevisiae. We used staining with 4'-6-diamidino-2-phenylindole (DAPI), chromomycin A3, and silver nitrate and in situ hybridization technique and utilized a video-intensified microscope system with an ultra-high-sensitive video camera. The results showed that of 16 bivalents of S. cerevisiae, the longest was a recognizable nucleolar chromosome which has an annular and synaptonemal complexless NOR in its submedian portion. The NOR was comprised of 2.65 X 10(9) D DNA which corresponded to 118 copies per haploid of rDNA repeating units. This evidence is discussed in terms of the possible participation of the annular NOR in suppressing the meiotic recombination of the rDNA gene clusters.

A highly reproducible method of nucleolus organizing regions staining in plants.

A method for the specific detection of the nucleolus organizing regions (NORs) of plant chromosomes has been developed employing enzymatic maceration and successive flame-drying for chromosome spreading and incubation with aqueous 50% AgNO3 at 55-60 C. When this method was applied to metaphase chromosomes the NORs were specifically discriminated as heavily stained segments in all the plant species examined. In the satisfactory results obtained by monitoring the reaction under a microscope during the course of the silver treatment, the chromosome arms were stained yellow to light brown while the NORs were dark brown to black. The present method has the advantage of yielding highly reproducible results for the specific detection of the NORs in plant materials.

Studies on mitochondrial structure and function in Physarum polycephalum. V. Behaviour of mitochondrial nucleoids throughout mitochondrial division cycle.

The fine structure of mitochondria and mitochondrial nucleoids in exponentially growing Physarum polycephalum was studied at various periods throughout the mitochondrial division cycle by light and electron microscopy. The mitochondrial nucleoid elongates lingitudinally while the mitochondrion increases in size. When the nucleoid reaches a length of approximately 1.5 mum the mitochondrial membrane invaginates at the center of the mitochondrion and separates the mitochondrial contents. However, the nucleoid does not divide even when the mitochondrial sections are connected by a very narrow bridge. Just before division of the mitochondrion, the nucleoid divides by constriction of the limiting membrane of the dividing mitochondrion. After division, one end of the nucleoid appears to be associated with the inner mitochondrial membrane. The nucleoid then again becomes situated in the center of the mitochondrion before repeating these same processes.