The nucleolus organizers of diploid wheats revealed by in situ hybridization

Summary Labelled RNA, transcribed in vitro from wheat ribosomal DNA cloned in a bacterial plasmid, has been hybridised to metaphase chromosomes of five diploid wheats. Autoradiography of the chromosomes has provided unequivocal evidence that these genotypes possess two pairs of nucleolus organizer chromosomes. The diploid wheat accessions used possess widely differing numbers of ribosomal RNA genes.     

Detection of Y-bearing spermatozoa by DNA-DNA in situ hybridisation

In situ hybridisation of a Y chromosome-specific DNA probe to preparations of decondensed spermatozoa revealed approximately 46.7% labelled spermatozoa among 3,900 scored. This is not significantly different from the 50% expected if only the Y chromosome-bearing spermatozoa are hybridised. Control hybridizations of Escherichia coli DNA and salmon testis DNA to decondensed sperm produced no significant labelling, whereas more than 99% of the spermatozoa were heavily labelled after hybridisation to total human DNA. These controls indicate that the methodology described in this paper renders the chromatin accessible for hybridisation and that the 50% hybridisation observed with the Y chromosome DNA probe was specific. In situ hybridisation with the Y probe therefore identifies the Y-bearing spermatozoa, and the protocol described should prove useful in evaluating methods of separating Y-bearing and X-bearing spermatozoa.     

二倍体、四倍体和六倍体小麦产量及水分利用效率

试验选用了6个不同染色体倍性的小麦进化材料（3个二倍体、2个四倍体和1个六倍体）,分别在不同水肥条件下研究其根系、地上生物量、产量、蒸腾耗水量和水分利用效率等指标,旨在阐明小麦进化材料产量及水分利用效率的差异及水肥条件对这些特性的影响。试验表明：不同倍性小麦进化材料的生物量、产量和水分利用存在显著的差异,而且水肥条件对其有显著影响。在染色体倍性由2n→4n→6n的进化过程中,小麦根系及地上生物量均先增加后降低,而产量却显著增加,这与收获指数的增加有关。小麦产量的大小顺序为：T.aestivum〉T.dicoccum〉T.dicoccoides〉Ae.squarrosa〉Ae.speltoides〉T.boeoticum。水分亏缺显著降低小麦的生物量、产量和收获指数;在不同水分条件下,增加施肥量有利于这些指标的增加。但是水分亏缺下,增加施肥却降低各小麦材料的根系生物量。随小麦的进化,蒸腾耗水量显著降低,这与其生育期缩短有关;而生物量水分利用效率和产量水分利用效率却显著升高,且后者的差异要大于前者。各小麦产量水分利用效率的大小排序与产量的完全一致。水分亏缺处理显著减少各小麦进化材料的蒸腾耗水量47％～52％,而显著增加生物量水分利用效率3％～40％;但水分亏缺对产量水分利用效率的促进作用却随染色体倍性的增加而降低,甚至降低六倍体小麦T.aestivum的产量水分利用效率19％。不同水分条件下,高肥处理均有利于蒸腾耗水量、生物量水分利用效率和产量水分利用效率的增加。     

Nucleolar morphology and rDNA in situ hybridisation in monocytes

Summary The aim of this study was to correlate morphological changes of nucleoli of non-proliferating monocytes to their functional activity, since nucleolar morphology is currently considered as a diagnostic marker for cell proliferation. Monocytes from healthy donors were fractionated by current counterflow centrifugation and kept in culture for 6 days. Cells were stimulated by the addition of 200 units/ml interferon (IFN). Under this stimulus the monocytes show no proliferation but a strongly augmented expression of type I Fc IgG receptor, human leucocyte antigen DR, human leucocyte antigen DP and human leucocyte antigen DQ. Morphological changes after stimulation included the appearance of multinucleated cells, typical signs of the activation of rRNA synthesis indicated by an increase in nucleolar size, and changes in nucleolar structure such as the appearance of reticulate and compact nucleoli. The number of nucleolus organiser regions (NORs) visualised by in situ hybridisation was compared with the position and number of nucleoli visualised by silverstaining in interphase cells. In comparison with control cultures, activated monocytes show a distinct increase in the number of those NORs that take part in the formation of nucleoli. Our results show that, in non-proliferating activated monocytes, the morphology of nucleoli and the increase of NOR activity are similar to those in proliferating cells. NOR activation is therefore an indicator for cellular activity, but is not necessarily correlated with proliferation.     

Relevance to prenatal diagnosis of the identification of a human Y/autosome translocation by Y-chromosome-specific in situ hybridisation

Routine cytogenetic analysis of an amniotic fluid sample revealed a large brightly fluorescent region in the short arm of chromosome 14 in an otherwise normal male karyotype (46,XY,14p+ + +). This site was also present in the paternal karyotype. In situ hybridisation to a Y-chromosome-specific DNA probe confirmed that the father had a Y/14 translocation. The incidence of two hybridisation bodies (large hybridisation sites), detecting both the translocated Y chromatin and the normal Y chromosome, was lower in interphase nuclei (44.3%) than in metaphase spreads (95.2%). The relevance of these observations to the potential use of in situ hybridisation to interphase nuclei for prenatal diagnosis is discussed.     

Nitrate reductases in hexaploid and tetraploid wheats and Aegilops

Summary Nitrate reductase activity (NR activity), protein content (NR protein) and polypeptides were compared in shoots of Triticum aestivum ssp. vulgare (L.) cv Fidel (bread wheat, AABBDD genome), Triticum dicoccum cv Vernal (AABB genome), Aegilops squarrosa var. strangulata (DD genome) and the amphiploid 365 (AABBDD genome), produced by crossing T. dicoccum cv Vernal and Ae. squarrosa var. strangulata. Constitutive NR protein and activity were found in shoots of all seedlings grown without nitrate, with the highest activity in the bread wheat. The inducible NR protein and activity developed upon the addition of nitrate. A 116-K polypeptide was identified as the main component of the NR from the bread wheat, while a faint, sometimes discernable 94-K band appeared on Western blots. Only one NR polypeptide could be identified in Ae. squarrosa —the 94 K. An intermediary situation was observed with the tetraploid T. dicoccum and the amphiploid: The 94-K polypeptide was the only one separated from NR of seedlings grown in the absence of nitrate. The 116-K polypeptide appeared after the addition of nitrate. The intensity of its band on the gel increased with the duration of the nitrate treatment. When comparing Ae. squarrosa and T. dicoccum, the constitutive isozyme (94-K polypeptide) was found in the D as well as in the AB genomes, while the inducible NR (116-K polypeptide) was absent from the D genome. Addition of the D genome into the AB genome slightly reinforced the expression of the inducible form (AB genome expression) in the amphiploid wheat. We postulate that the inducible form of NR in the bread wheat resulted from an evolutionary selection pressure favoured by cultivation.     

In situ hybridisation in filamentous fungi using peptide nucleic acid probes

Fluorescent DNA and peptide nucleic acid (PNA) probes were used for in situ hybridisations in colonies of Schizophyllum commune and Aspergillus niger. DNA probes for 18S rRNA did not diffuse through the cell wall after mild chemical fixation. After permeabilising the cell wall with lysing enzymes or slow freezing and embedding, hybridisation was still poor and not reproducible. In contrast, PNA probes did diffuse through the cell wall after mild chemical fixation and reproducible fluorescent signals were obtained. The rRNA signal was most intense in the apical compartment of hyphae of S. commune. Within this compartment, the signal was lower at the extreme apex. Apparently, ribosomes are unevenly distributed in hyphae. In S. commune, the mRNA of the SC3 gene was also detected with a PNA probe. The ratio between 18S rRNA and SC3 mRNA signals were variable between hyphae and their compartments. This is the first report of using PNA probes for in situ hybridisation of mRNA in fungi. The method provides a powerful tool to study gene expression.     

Fluorescence in situ hybridisation (FISH)--the next generation

Fluorescence in situ hybridisation (FISH) has become one of the major techniques in environmental microbiology. The original version of this technique often suffered from limited sensitivity due to low target copy number or target inaccessibility. In recent years there have been several developments to amend this problem by increasing signal intensity. This review summarises various approaches for signal amplification, focussing especially on two widely recognised varieties, tyramide signal amplification and multiply labelled polynucleotide probes. Furthermore, new applications for FISH are discussed, which arise from the increased sensitivity of the method.     

Group-specific 16S rRNA targeted probes for the detection of type I and type II methanotrophs by fluorescence in situ hybridisation

The study of methane-oxidising bacteria (methanotrophs) is of special interest, because of their role in the natural reduction of methane emissions from many different sources. Therefore new probes were developed to detect specifically either type I (Methylococcaceae) or type II methanotrophs (Methylocystaceae). The probes have shown high specificity in fluorescence in situ hybridisations (FISH), as demonstrated by parallel hybridisation of target and reference strains as well as sequence data analysis. With these probes, methanotrophs were detected in soil and root samples from rice microcosms, demonstrating their applicability even in a complex environmental matrix.     

Distribution of hydroxyindole-O-methyltransferase mRNA in the rat brain: an in situ hybridisation study

Hydroxyindole-O-methyltransferase (HIOMT) is the enzyme involved in the last step of the melatonin synthesis pathway. Recently, a cDNA encoding HIOMT has been isolated from a rat pineal gland library. Using this cDNA, we developed a highly sensitive in situ hybridisation protocol to investigate the distribution of HIOMT mRNA in both the rat brain and dissociated pinealocytes maintained in primary cell culture. In the rat brain, HIOMT mRNA was only detected in the three parts of the pineal complex: the superficial pineal, the stalk and the deep pineal. No extra-pineal hybridisation labelling was observed. These results strongly suggest that melatonin synthesis also occurs in the deep part and the stalk of the pineal gland. HIOMT mRNA was markedly expressed in cultured pinealocytes. No particular subcellular area was observed to express HIOMT mRNA specifically, as the labelling was homogeneously distributed in the cytosol and in the axon-like processes. In conclusion, the use of in situ and in vitro hybridisation with a pineal riboprobe has detected notable HIOMT expression restricted to pinealocytes. Received: 26 June 1997 / Accepted: 15 September 1997     

Comparison of the chromosomes of Triticum timopheevi with related wheats using the techniques of C-banding and in situ hybridization

Summary The chromosomes of the tetraploid wheats Triticum timopheevi (Genome AAGG) and T. araraticum (Genome AAGG) were C-banded at mitosis. The identity of the banded and unbanded chromosomes was then established by firstly making comparisons with the hexaploid species T. zhukovskyi which has the genome formula AAAAGG. Secondly, the meiotic pairing in F₁ hybrids between T. timopheevi and diploid wheats was examined by means of C-banding. The results showed that the banded chromosomes belonged to the G genome, while the unbanded chromosomes belonged to the A genome. Only one of the two pairs of satellited chromosomes had strong heterochromatic bands. The relationship between the genomes of T. timopheevi and T. dicoccum (Genome AABB) was then assessed at meiosis in hybrids between these species, using the techniques of C-banding and in situ hybridisation of a cloned ribosomal RNA gene probe. It was concluded that there were differences both in the amount and distribution of heterochromatin and also translocation differences between the species.     

Confirmation of primitive chromosome structure in the hexaploid wheats

Summary Hybrids between Triticum aestivum cv Chinese Spring (CS) and T. dicoccoides of chromosome type E_1a showed only a few or no trivalents at meiosis, but both trivalents and quadrivalents were shown by hybrids with six other types. Since there is strong evidence that the E_1a type has the primitive chromosome structure of T. dicoccoides, Chinese Spring can be said to have the primitive chromosome structure of the hexaploid wheats in regard to reciprocal translocation.Contribution no. 51 from the Plant Germ-plasm Institute, Faculty of Agriculture, Kyoto University, Kyoto, Japan     

Introgression of chromosomes of Festuca arundinacea var. glaucescens into Lolium multiflorum revealed by genomic in situ hybridisation (GISH)

An F₁ hybrid (n=4x=28) between the tetraploid species Festuca arundinacea var. glaucescens (GGG′G′) and a synthetic tetraploid Lolium multiflorum (LmLmLmLm) was backcrossed to diploid L. multiflorum to produce triploid (2n=3x=21) BC₁ hybrids (LmLmG). At metaphase I of meiosis the triploids had a preponderance of ring bivalents and univalents with some linear and frying-pan trivalents. Genomic in situ hybridisation (GISH) differentiated the Festuca chromosomes from Lolium and revealed that the bivalents were exclusively between Lolium homologues, while the univalents were Festuca. Despite the limited amount of homoeologous chiasmata pairing in the triploids, some recombinant chromosomes were recovered in the second backcross when the hybrids were further crossed to diploid L. multiflorum. The progeny from the second backcross was predominantly diploid. Genotypes with recombinant chromosomes and chromosome additions involving an extra Festuca chromosome were identified using GISH. Changes in plant phenotype were related to the presence of Festuca chromatin. Received: 20 September 2000 / Accepted: 05 January 2001     

Expression of high zinc efficiency of Aegilops tauschii and Triticum monococcum in synthetic hexaploid wheats

The effect of varied zinc (Zn) supply on shoot and root dry matter production, severity of Zn deficiency symptoms and Zn tissue concentrations was studied in two Triticum turgidum (BBAA) genotypes and three synthetic hexaploid wheat genotypes by growing plants in a Zn-deficient calcareous soil under greenhouse conditions with (+Zn=5 mg kg^-1 soil) and without (−Zn) Zn supply. Two synthetic wheats (BBAADD) were derived from two different Aegilops tauschii (DD) accessions using same Triticum turgidum (BBAA), while one synthetic wheat (BBAAAA) was derived from Triticum turgidum (BBAA) and Triticum monococcum (AA). Visible symptoms of Zn deficiency, such as occurrence of necrotic patches on leaves and reduction in shoot elongation developed more rapidly and severely in tetraploid wheats than in synthetic hexaploid wheats. Correspondingly, decreases in shoot and root dry matter production due to Zn deficiency were higher in tetraploid wheats than in synthetic hexaploid wheats. Transfer of the DD genome from Aegilops tauschii or the AA genome from Triticum monococcum to tetraploid wheat greatly improved root and particularly shoot growth under Zn-deficient, but not under Zn-sufficient conditions. Better growth and lesser Zn deficiency symptoms in synthetic hexaploid wheats than in tetraploid wheats were not accompanied by increases in Zn concentration per unit dry weight, but related more to the total amount of Zn per shoot, especially in the case of synthetic wheats derived from Aegilops tauschii. This result indicates higher Zn uptake capacity of synthetic wheats. The results demonstrated that the genes for high Zn efficiency from Aegilops tauschii (DD) and Triticum monococcum (AA) are expressed in the synthetic hexaploid wheats. These wheat relatives can be used as valuable sources of genes for improvement of Zn efficiency in wheat. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of the rice D-genome chromosomes by genomic in situ hybridisation

 The 24 rice D-genome chromosomes were identified among the 48 chromosomes of O. latifolia, which comprise the C- and D-genomes, using genomic in situ hybridisation (GISH). The B-genome chromosomes were also discriminated from the C-genome chromosomes in O. minuta (BBCC) by GISH. A comparison of the differences in the fluorescence intensity between the C and D genomes within O. latifolia (CCDD), and between the B and C genomes within O. minuta, indicated that the overall nucleotide-sequence homology between the B and C genomes is less than that between the C and D genomes. The origin of the D genome and the phylogenetic relationship of the D genome among the rice genomes are discussed, based on the results obtained. Received: 5 June 1997 / Accepted: 19 June 1997     

The use of DNA probes in preimplantation and prenatal diagnosis

DNA probes are now widely used for prenatal diagnosis, but the prospect of preimplantation diagnosis of genetic disorders requires the development of sensitive genetic tests that can be performed on small numbers of cells removed from a preimplantation-stage pre-embryo. The sensitivity of molecular tests can now be increased by specifically amplifying the target DNA with the polymerase chain reaction. In situ hybridisation with chromosome-specific DNA probes to repeated sequences also permits the detection of particular numerical chromosome aberrations or the distinction of male and female pre-embryos when only a few interphase nuclei are available. We have used in situ hybridisation to a Y chromosome-specific DNA probe to sex preimplantation-stage pre-embryos and to sex fetuses from samples of chorionic villus cells, amniotic fluid cells, and fetal blood. These two approaches (amplification of target DNA and in situ hybridisation) provide suitable tests for improving prenatal diagnosis particularly when few cells are available and they offer the possibility of tests suitable for preimplantation diagnosis.     

Chromosome markers in the tetraploid wheat Aegilops ventricosa analysed by in situ hybridization

 Three lines of the tetraploid wheat Aegilops ventricosa Tausch (2n=4x=28), which contains good resistance to eyespot, were analysed using fluorescent in situ hybridization. Probes used included rDNA, cloned repeated sequences from wheat and rye, simple-sequence repeats (SSRs) and total genomic DNA. The banding patterns produced could be used to distinguish most chromosome arms and will aid in the identification of Ae. ventricosa chromosomes or chromosome segments in breeding programmes. All lines had a single major 18S-25S rDNA site, the nucleolar organizing region (NOR) in chromosome 5N and several minor sites of 18S-25S rDNA and 5S rDNA. A 1NL.3DL, 1NS.3DS translocation was identified, and other minor differences were found between the lines. Received: 11 August 1998 / Accepted: 28 November 1998     

Proteome analysis of diploid,tetraploid and hexaploid wheat: towards understanding genome interaction in protein expression

Hexaploid wheat (Triticum aestivum L.) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. The proteome patterns of diploid, tetraploid and hexaploid wheat were analyzed to explore the genome interaction in protein expression. At least two species from each of the diploid and tetraploid were used to compare their proteome maps with a hexaploid wheat cv. Chinese Spring. The ancestral cultivars were selected based on their history of closeness with the cultivated wheat. Proteins were extracted from seed flour and separated by two-dimensional electrophoresis (2-DE) with isoelectric focusing of pH range from 4-10. 2-DE maps of cultivated and ancestral species were analyzed by computer assisted image analyzer. The region of high molecular weight glutenin subunits of hexaploid wheat showed similarity with those of the diploid donors, BB and DD genomes. The omega gliadin, which is controlled by B genome in common wheat, was assumed to have evolved as a result of interaction between AA and BB genomes. The low molecular weight glutenins and alpha and beta gliadin regions were contributed by the three genomes. This result suggests that the function of donor genomes particularly in the expression of proteins in hexaploid wheat is not totally independent; rather it is the product of interactions among the diploid genomes in the hexaploid nuclear constitutions. The expression of nonstorage proteins was affected substantially due to the removal of the D genome from hexaploid constitution. Location of the structural gene controlling one of the alpha amylase inhibitor proteins in the nonstorage protein region was identified in the short arm of chromosome 3D.