Microdissection of the Y chromosome and fluorescencein situ hybridization analysis of the sex chromosomes of lake trout,Salvelinus namaycush

Lake trout,Salvelinus namaycush, is one of the few salmonids with morphologically differentiated sex chromosomes. Genetic analysis suggested that the sex-determining region of this species lies on the short arm of the Y chromosome. The differential arm of the Y chromosome was microdissected and the resulting DNA amplified in a sequence-independent manner. Amplified DNA was biotin labeled as a probe for fluorescencein situ hybridization (FISH). Strong hybridization signals were seen covering defined regions of both the Y and X chromosomes. Homeologous chromosomes of the ancestrally tetraploid genome were not identified by FISH with the Y probe, indicating diploidization of this region of the genome.     

Detection of aneuploidy in human spermatozoa using fluorescencein situ hybridization (FISH)

Summary Fluorescencein situ hybridization (FISH) with chromosome specific alpha-satellite DNA probes was used to estimate the rates of aneuploidy of chromosomes 1, 17, 18, X and Y in human ejaculated sperm. Sperm samples were collected from six donors, and biotinylated DNA probes, D1Z5, D17Z1, D18Z1, DXZ1 and DYZ3 were hybridized to interphase sperm which had been pretreated with dithiothreitol to expand their nuclei. A minimum of 3,000 sperm per donor were analyzed. The hybridization efficiency was 99.68% for all the five probes. The frequencies of aneuploidy for chromosomes 1, 17 and 18 were 0.65%, 0.66% and 0.61% respectively. For XX-and YY-sperm the frequencies were 0.28% and 0.27% respectively. To estimate the diploidy and disomy rates, a mixture of D17Z1 and D18Z1 were used as probes, and the frequency of diploid sperm was calculated to be 0.27%. After subtraction of the diploidy rate, the disomy rates for chromosomes 1, 17, and 18 were estimated to be 0.38%, 0.39% and 0.33%, respectively. The proportion of X- and Y-bearing sperm were 49.9% and 49.66%, consistent with an expected 1: 1 ratio.     

Autosomal location of a new subtype of 1.688 satellite DNA ofDrosophila melanogaster

During the screening of aDrosophila melanogaster YAC library with DNA from the minichromosomeDp(1;f)1187 we isolated a clone, yw20D5, which contains a new subtype of 1.688 satellite DNA. Although the sequences of several monomers subcloned from the YAC show a considerable variation in length, the derived consensus sequence is 356-bp long. This new subtype and the one constituted by the 353-bp repeats are both located on the left arm heterochromatin of chromosome 3, arranged in separate arrays. Despite their autosomal location, phylogenetic relationships among 1.688 satellite sequences suggest that they may have originated from the 359-bp repeats of the X chromosome heterochromatin. We have used the new 356-bp repeats to investigate whether sequences related to the 1.688 satellite are dispersed along the euchromatic arms of the autosomes in a similar way to that in which they are found along the X chromosome euchromatin.accepted for publication by D. Ward     

Analysis of rye B-chromosome structure using fluorescencein situ hybridization (FISH)

Fluorescencein situ hybridization (FISH) has been used to analyse the structure of the rye B chromosome. Genomicin situ hybridization (GISH) demonstrates the high level of overall similarity between A and B chromosomes of rye, as well as the presence of a number of specific sequences. The B-specific repeat families D1100 and E3900 have been analysed in terms of their physical location and possible contiguity. Rye Bs contain members of the rye-specific dispersed repetitive family R173, as well as centromeric regions similar to those of the As. The B chromosomes analysed in our study lack detectable rDNA sequences. Anomalous results have been obtained with a number of subtelomeric repetitive probes from rye. Bs usually lack these sequences, but evidence is presented that in some cases A–B translocation events may relocate such sequences from the As to the Bs. These data are discussed in the context of current models for the origin of the B chromosome.     

Identification of iso(18p) marker chromosome by fluorescencein situ hybridization with single-copy DNA probe

Summary The patient displayed the clinical features consistent with tetrasomy (18p) syndrome, who had an extra small metacentric iso(18p) chromosome in otherwise normal karyotype. Identification of the marker chromosome used the chromosome 18 band-specific fluorescencein situ hybridization strategy.     

Sensitivity enhancement of fluorescencein situ hybridization on plant chromosomes

An improvedin situ hybridization procedure is presented, based on synchronization of root meristems of barley and wheat, enzymatic digestion, a protoplast drop technique, and the use of the fluorescent dye Cy3. The combination of these approaches resulted in a significant increase of well-spread metaphases suitable forin situ hybridization as compared to squash preparations, and to a significantly enhanced number and intensity of hybridization signals as demonstrated for a B-hordein-specific lowcopy probe of barley. In the case of Cy3 all metaphases displayed a signal, more than 60% of them on both chromatids of each gene-bearing chromosome.     

Reexamination of chromosomal loci of human muscle actin genes by fluorescencein situ hybridization

Summary Human genes for cardiac (ACTC), skeletal (ACTA), and vascular type (aortic type or -) smooth (ACTSA) muscle actins have been localized to chromosomes 15q14, 1q42.1, and 10q23.3, respectively, by fluorescencein situ hybridization.     

Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescencein situ hybridization (FISH)

Fluorescencein situ hybridization (FISH) is an increasingly powerful tool with a variety of applications in both basic and applied research. With excellent genetic, cytogenetic and molecular maps available, the tomato genome provides a good model to benefit from the full potential of FISH. Tomato chromosomes at mitotic metaphase are small and not particularly suitable for high-resolution FISH. In contrast, chromosomes at meiotic pachytene are about 15 times longer, and easier to identify by their differences in chromosome arm lengths and chromomere pattern. We have developed a technique for preparing chromosomal spreads of young pollen mother cells at midprophase I which is suitable for FISH. In a first series of experiments, the hybridization patterns of three classes of repetitive DNA sequences were studied in single and multicolour FISH.accepted for publication by J. S. (Pat) Heslop-Harrison     

Comparison of ribosomal DNA sites inLolium species by fluorescencein situ hybridization

The position of the 18S-5.8S-26S and 5S rRNA genes have been physically mapped on the chromosomes of sevenLolium taxa. 18S-5.8S-26S sites were seen on two pairs of chromosomes in the inbreeding taxa. In the outbreeding taxa six sites were found in theL. multiflorum, seven inL. perenne and nine inL. rigidum var.rigidum. Two 5S sites were found in each of the taxa. In the inbreeders, the 5S sites were found adjacent to the 18S-5.8S-26S sites on chromosome 2. InL. multiflorum andL. perenne the 5S sites were on the short arm of chromosome 3. However, inL. rigidum var.rigidum the 5S rDNA site was found in either of the two positions.accepted by J.S. (Pat) Heslop-Harrison     

Ribosomal RNA location in the Antarctic fishChampsocephalus gunnari (Notothenioidei,Channichthyidae) using banding and fluorescencein situ hybridization

A biotinylated 28S rDNA probe was prepared from the genomic DNA of the Antarctic ice-fishChampsocephalus gunnari and hybridized to metaphase chromosomes of the same species by fluorescencein situ hybridization (FISH). The hybridization signal appeared over the whole heterochromatic arm of the submetacentric chromosomes bearing the nucleolar organizer regions. The results of rDNA/FISH are compared with those coming from classical cytogenetic (C, Q, Ag-NOR, chromomycin A3) banding techniques. Thein situ detection of a specific DNA sequence offers a new more precise perspective for understanding the evolving process in chromosomes of Antarctic fish and will provide an interesting contribution to comparative cytogenetics of lower vertebrates.accepted for publication by M. Schmid     

Analysis using dual-colour fluorescencein situ hybridization of meiotic chromosome segregation in male mice heterozygous for a reciprocal translocation

Meiotic chromosome behaviour was investigated in male mice heterozygous for the translocation T(7;16)67H. At metaphase I, chain-of-four quadrivalents were present in approximately 80% of the spermatocytes; the bulk of remaining cells contained a ring quadrivalent, with only a few having either a trivalent plus univalent configuration or two bivalents. A low rate of non-disjunction, approximately 5%, was found through analysis of C-banded metaphase II spermatocytes. Using fluorescencein situ hybridization with differentially labelled whole chromosome paints, a wide array of segregation products were observed at metaphase II, depending on whether they arose from alternate, adjacent I, adjacent II orientation at metaphase I or were uninformative for alternative/adjacent I because of the presence of a chiasma in an interstitial pairing segment. Some 62% of the cells fell into this latter category, with only small proportions clearly arising through alternate (1.8%) or adjacent I (0.7%) orientations. Approximately 30% of the cells contained the products of adjacent II orientation. Consideration of the data suggested that most of these cells arose from metaphase I cells that contained a chain quadrivalent. Ring quadrivalents appeared predominantly to orientate in an alternate/adjacent I manner.for publication by J. S. (Pat) Heslop-Harrison     

Detection of chromosomal abnormalities of chromosome 12 in uterine leiomyoma using fluorescencein situ hybridization

Summary Fifty uterine leiomyomas were examined using conventional cytogenetic method and fluorescencein situ hybridization (FISH) for detection of chromosomal, abnormalities of chromosome 12. Of the 50 tumors, nine were examined using FISH on the non-cultured samples. Two (4.0%) of 50 tumor samples examined showed chromosomal abnormalities of chromosome 12 by the conventional cytogenetic analysis. For FISH, the whole-chromosome painting probe and D12Z3 probe specific for the centromeric region were used. Of the 50 cultured samples, 10 showed structural aberrations and four showed numerical aberrations of chromosome 12 by FISH analysis. Of the nine non-cultured samples, four showed structural abnormalities of chromosome 12, all of which also showed structural abnormalities of chromosome 12 on the cultured samples. These results indicate that chromosomal abnormalities of chromosome 12 are important in the biology of at least some types of uterine leiomyoma, and that FISH is a useful complement to the conventional cytogenetic analysis in the study of solid tumors.     

Interstitial deletion of the long arm of chromosome 11 determined by fluorescencein situ hybridization

Summary An interstitial deletion, del(11)(q14q22), found in a female infant was examined by fluorescencein situ hybridization with cosmid DNA markers mapped on the long arm of chromosome 11. Three cosmids mapped on 11q14.1-11q22.1 region were not hybridized to the del(11) chromosome, while all the other DNA markers mapped on 11cen-11q14.1 and 11q23.1-11 qter region gave hybridization signals on the del(11) chromosome. Cytogenetic analysis after R-banding confirmed an apparent deletion of 11q14-q22, but containing a small R-negative band, a part of 11q22.3 and/or 11q14.1, in the middle part of del(11) chromosome. The karyotype thus was determined to be 46, XX, del(11)(q14.1q22.3).     

Cytogenetic study of a severe case of Pallister-Killian syndrome using fluorescencein situ hybridization

Summary Usually, the supernumerary isochromosome 12p characterizing Pallister-Killian syndrome patients was detected in cultured skin fibroblasts but not in cultured blood lymphocytes. The proband of this study was a one-day-old female, who presented with major clinical characteristics of the Pallister-Killian syndrome, and had severe malformations in the form of anal atresia, cleft palate, and severe laryngomalacia. Chromosome preparations from cultured blood lymphocytes and skin fibroblasts, as well as buccal smears, from this patient were analyzed by fluorescencein situ hybridization (FISH) using a chromosome 12-specific alpha satellite probe. The proportions of cells showing positive signals for i(12p) in these samples were found to be 20, 62.5, and 70% respectively. Repeated FISH studies of buccal smears from this patient showed considerable decreases in the proportions of i(12p) containing cells to 40% at one year of age and to 32% at the age of one year and five months. The decline in the percentage of i(12p)-containing cells in buccal smears with aging supports the concept ofin vivo loss of the marker during repeated cell division.     

Comparative genomic in situ hybridization (cGISH) analysis on plant chromosomes revealed by labelled Arabidopsis DNA

A new approach for comparative cytogenetic banding analysis of plant chromosomes has been established. The comparative GISH (cGISH) technique is universally applicable to various complex genomes of Monocotyledonae (Triticumaestivum, Agropyronelongatum, Secalecereale, Hordeumvulgare, Alliumcepa, Muscariarmenaticum and Liliumlongiflorum) and Dicotyledonae (Viciafaba, Betavulgaris, Arabidopsisthaliana). Labelled total genomic DNA of A. thaliana generates signals at conserved chromosome regions. The nucleolus organizing regions (NORs) containing the majority of tandemly repeated rDNA sequences, N-band regions containing satellite DNA, conserved homologous sequences at telomeres and additional chromosome-characteristic markers were detected in heterologous FISH experiments. Multicolour FISH analysis with repetitive DNA probes simultaneously revealed the chromosome assignment of 56 cGISH signals in rye and 61 cGISH signals in barley. Further advantages of this technique are: (1) the fast and straightforward preparation of the probe; (2) the generation of signals with high intensity and reproducibility even without signal amplification; and (3) no requirement of species-specific sequences suitable for molecular karyotype analysis. Hybridization can be performed without competitive DNA. Signal detection without significant background is possible under low stringency conditions. The universal application of this fast and simple one-step fluorescence banding technique for plant cytogenetic and plant genome evolution is discussed.     

Assignment of the human cytochrome P-450 nifedipine oxidase gene (CYP3A4) to chromosome 7 at band q22.1 by fluorescencein situ hybridization

Summary We have used a full length cDNA clone (2.2 kb) for the human cytochrome P-450 nifedipine oxidase (CYP3A4) enzyme as a probe to determine its chromosome localization by fluorescencein situ hybridization. CYP3A4 was mapped on R-banded human prometaphase chromosomes, and the precise localization of CYP3A4 on chromosome 7 was further confirmed by a delineation of G-banded pattern on the same prometaphase chromosomes through a combination of UV-filter. We assigned CYP3A4 to chromosome 7 at q22.1.     

A 169-base pair tandem repeat DNA marker for subtelomeric heterochromatin and chromosomal rearrangements in aphids of the Myzus persicae group

Numerous copies of a 169-base pair DNA sequence (Myzus persicae group repeat; MpR) occur at subtelomeric locations on all chromosomes of three members of the Myzus persicae species group (Myzus persicae, M. antirrhinii, M. certus). MpR occurs in large tandem arrays at both ends of all autosomes of the standard 2n = 12 karyotype, and near one end of the X chromosome (the end opposite to the nucleolar organizer) and is estimated to make up about 5% of the genome (a total of about 200 000 copies). Locations of MpR were compared in various karyotypes to determine the likely nature of the rearrangements (fusions, dissociations, translocations) that are found in this species group which, like other Hemiptera, has holocentric chromosomes that are devoid of morphological markers. Aphid clones heterozygous for autosome dissociations do not have any detectable MpR at 'new' chromosome ends, indicating that this sequence is not involved in 'capping' of chromosomes. However, a clone with a de novo autosome fusion had an interstitial block of MpR marking the point of fusion, and clones heterozygous for an autosomal 1,3 translocation had MpR from autosome 1 translocated to a new site on autosome 3. The isolation from M. antirrhinii of the telomeric repeat TTAGG, which is found in several insect groups, is also reported.     

Mapping of the porcine urate oxidase and transforming growth factor beta 2 genes by fluorescencein situ hybridization

We have mapped two genes from human chromosome 1, urate oxidase (UOX) and transforming growth factor beta 2 (TGFB2), by fluorescencein situ hybridization (FISH) in the pig genome. Porcine-specific polymerase chain reaction (PCR) primers for both genes were designed from the porcine cDNA sequence. With the help of these primers yeast artificial chromosome (YAC) clones forUOX andTGFB2 were isolated from a pig YAC library. These DNA probes were used for FISH analysis.TGFB2 was localized to SSC 10p16. With the YAC probe forUOX two porcine chromosome regions 6q26 and 6q32, revealed specific signals. These results, help to refine the comparative mapping data between human and pig.accepted for publication by H. Schmidt