Interchromosomal effects for chromosome 21 in carriers of structural chromosome reorganizations determined by fluorescence in situ hybridization on sperm nuclei

We have used dual color fluorescence in situ hybridization (FISH) on decondensed sperm heads from four carriers of structural chromosome reorganizations, viz. t(3;15), t(Y;7), t(13;22) and inv(9), to assess the possible existence of an interchromosomal effect (ICE) on the segregation of chromosome 21. In the carriers of t(Y;7), t(13;22) and inv(9), all results were within the limits described in controls. A highly significant increase (P<0.0001) of disomy 21 (1.90% v 0.37%), which could be considered as a positive ICE, was observed in the t(3;15) carrier. Significantly higher percentages (P<0.0001) of diploid sperm (5.71% v. 0.27%) were also observed in this patient. Our results suggests that the occurrence of an ICE may depend on the reorganization and on the chromosome and chromosome regions involved, resulting in a particular meiotic behaviour (presence of unsynapsed regions, preferential meiotic configurations) that could lead to the observed increase in chromosome 21 disomies. Further studies with this technical approach in a wide range of structural reorganizations could help to elucidate the actual occurrence of ICEs.     

Incidence of mosaic cell lines in vivo and malsegregation of chromosome 21 in lymphocytes in vitro of trisomy 21 patients: detection by fluorescence in situ hybridization on binucleated lymphocytes

In order to detect aneuploidy in interphase human lymphocytes, both in vivo and in vitro, fluorescence in situ hybridization (FISH) was carried out on binucleated cells cytokinesis-blocked by cytochalasin B at the first mitosis after phytohemagglutinin stimulation. A pericentric chromosome-21-specific DNA probe prepared from yeast artificial chromosome clone 881D2 by the polymerase chain reaction was employed. One thousand binucleated cells per individual were scored from cultures from twelve trisomy 21 patients aged 0.01-8.9 years (mean 4.3 years) and 20 normal children of similar age. Of trisomy 21 patients, increased frequencies of disomic cells in vivo (1.690+/-1.070%) and cells containing six signals with nondisjunction (0.822+/-0.554%) were found, compared with those of monosomic 21 cells in vivo (0.265+/-0.130%) and cells containing four signals with nondisjunction in normal children (0.369+/-0.250%; P=0.000 and P=0.000, respectively). These results show that malsegregation of chromosome 21 occurs more often in trisomic 21 cells than in disomic cells from normal children. The frequency of nondisjunction was significantly higher than the loss of chromosome 21 in both cultured trisomic (0.822+/-0.554% vs 0.043+/-0.049%, P=0.000) and disomic (0.369+/-0.250% vs 0.010+/-0.30%, P=0.000) cells. Comparisons of in vivo and in vitro data on aneuploidy indicate that a cell selection mechanism may exist in vivo. All these results show that FISH, with a chromosome-specific probe, on binucleated lymphocytes is a powerful tool for simultaneously detecting mosaic cell lines in vivo and malsegregation (loss and nondisjunction) of a corresponding chromosome in vitro in the same cell population.     

Assignment of human β-galactosidase-A gene to 3p21.33 by fluorescence in situ hybridization

GM₁ gangliosidosis and Morquio syndrome type B (MPS IVB) are inherited lyosomal storage disorders associated with deficiency of -galactosidase-A (GAL_A) activity. A recombinant plasmid containing a biotinylated cDNA (2.4-kb insert) encoding human GAL_A was used to localize the enzyme locus by fluorescence in situ hybridization (FISH). The human GAL_A gene was assigned to 3p21.33 by FISH.     

Fluorescence in situ hybridization reveals a break in the α-satellite DNA of chromosome 1 in a family with a balanced whole-arm translocation

We have characterized a whole-arm translocation involving chromosomes 1 and 19 by traditional cytogenetic methods and fluorescence in situ hybridization with chromosome-specific -satellite and whole-chromosome painting probes, and different satellite III DNA probes. We have identified a break in the -satellite DNA region of chromosome 1, with division of this material into two a-satellite DNA blocks. This leaves one translocation chromosome with truncated -satellite DNA from chromosome 1 and the other trranslocation chromosome with all the -satellite DNA from chromosome 19 and truncated -satellite DNA from chromosome 1. We speculate whether the recombination event observed has taken place in tetraplex structures of satellite III DNA interspersed between -satellite DNA.     

Localization of the β-globin gene to 11p15 by in situ hybridization: Utilization of chromosome 11 rearrangements

Summary Chromosome preparations from four subjects, one normal 46,XY male and three patients with different rearrangements of chromosome 11:46,XX,del(11)(p11.2p15.1), 46,XY,inv(11)(p13q24.2), and 46,XY,rec(11)inv(11)(p13q24.2) pat, were utilized for in situ hybridization studies with a tritium-labeled cDNA probe containing a -globin insert. Using the hybridization technique described by Harper and Saunders (1981), there were 1–2 grains over each labeled metaphase. Of 360 cells scored, 88 were labeled over chromosome 11, band p15 (24%). Approximately half of the chromosome 11s labeled from the abnormal patients were the del(11) or inv(11). These results exclude the -globin locus from 11p11p14, since these bands were not present in the recent 11, and assign it to 11p15. This is in agreement with the recent exclusion data of de Martiville and Francke (1984) and Junien (1984), and suggestive assignment data of Morton et al. (1984).     

Human high-affinity FcγRI (CD64) gene mapped to chromosome 1q21.2-q21.3 by fluorescence in situ hybridization

The human FcRI gene encodes for a highaffinity Fc receptor that plays pivotal roles in the immune response. We have used fluorescence in situ hybridization analysis to localize the FcRI gene to human chromosome 1. The human FcRI (CD64) gene has been assigned to human chromosome 1q21.2-q21.3 using R-banded human (pro)metaphase chromosomes.     

Prader-Willi syndrome: Methylation study or fluorescence in situ hybridization first?

Prader-Willi syndrome (PWS) is neurogenetic disorder involving the imprinting mechanism at 15q11-13 region. We report a 4-year-old girl who was referred to our laboratory to be investigated for clinical obesity, mental deficiency and respiratory problems. The patient was born for non-consanguineous and healthy biological parents. After normal pregnancy, the patient was delivered by cesarean section at full term, with a birth weight of 2500 g, and the height and head circumference were unknown. In neonatal stage, she presented severe hypotonia with feeding problems. Her developmental progress was delayed. She walked and developed speech at the age of 3 years. Since the age of 3 years, she presented severe dental problems. Methylation study had confirmed the diagnosis, and for detecting etiology, fluorescence in situ hybridization using probes for small nuclear ribonucleoprotein polypeptide N (SNRPN), which map inside the chromosomal region 15q11-15q13, was necessary to confirm the 15q11-15q13 deletion of paternal chromosome 15, which is the predominant genetic defect in PWS. In conclusion, we report this case with an objective to reinforce the necessity of analysis of DNA methylation within the 15q11-13 region, which is an important tool for the correct diagnosis among children presenting with neonatal hypotonia, mental deficiency and obesity.     

Mapping of18-26S rDNA loci in four species of the genus Paris by fluorescence in situ hybridization（FISH）

18-26S rDNA loci were mapped on chromosomes in four species of Par is,and the num-ber and position of rDNA sites in these species were compared f or analysis of the distribution of the sites. All the plants were diploids,and t he genome consisted of five chromosomes,A,B,C,D and E. （1）P. polyphylla var. yunnanensis,2n=10=6m+4t. Two18-26S rDNA loci were de-tected on the short arms o f C and D chromosomes;（2）P. forrestii,2n=10=6m+4t. One locus was detected on th e long arm of B chromosome,and also two loci on the short arms of C and D chromosomes;（3）P. axialis. 2n=10=6m（2sat）+4t（2sat）+1-2B. Two loci were detected o n the short arms of C and D chromosomes. One locus was detected in the cell with t wo B-chromosomes（B）,but none was detected in that with only one B chromosome, indicating that rRNA gene existed on B chromsome,and an unequal division occurr ed during mitotic cycle of B-chromosomes. （4）P. daliensis,2n=10=4m+2sm+2st+2t. O ne locus was detected on the short arm of D chromo-some. The signals of18-26S rD NA appeared not only in the second constriction but also in the other regions of chromosome. It is noteworthy that one locus was detected in the terminal region o n the short arm of C chromosome in all the four species studied.     

High-resolution mapping of YACs and the single-copy gene Hs1pro−1 on Beta vulgaris chromosomes by multi-colour fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a powerful approach for physical mapping of DNA sequences along plant chromosomes. Nematode-resistant sugar beets (Beta vulgaris) carrying aBeta procumbens translocation were investigated by FISH with two differentially labelled YACs originating from the translocation. At mitotic metaphases, the translocation was identified with both YACs in the terminal region on a pair of chromosomes. Meiotic chromosomes, representing a far more extended hybridization target, were used to determine the orientation of YACs with respect to chromosomal domains in combination with chromosomal landmark probes for telomeres and centromeres. The in situ detection of plant single-copy sequences is technically difficult, and the wild beet translocation was used to explore the potential resolution of the FISH approach and to introduce the chromosomal mapping of single-copy genes into genome analysis of Beta species. An internal fragment of the nematode resistance gene Hs1 ^pro–1, 684 bp long, was detected on both chromatids of different Beta chromosomes and represents one of the shortest unique DNA sequences localized on mitotic plant chromosomes so far. Comparative chromosomal mapping of the 684 bp Hs1 ^pro–1 probe in the translocation line, a monosomic addition line and in B. procumbens revealed the origin of the wild beet translocation leading to nematode-resistant sugar beets.     

Chromosomal localization of 5S and 18S–5.8S–25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was employed on mitotic metaphase chromosome preparations of five Asian Pinus species: Pinus tabuliformis, Pinus yunnanensis, Pinus densata, Pinus massoniana and Pinus merkusii, using simultaneously DNA probes of the 18S rRNA gene and the 5S rRNA gene including the non-transcribed spacer sequences. The number and location of 18S rDNA sites varied markedly (5-10 pairs of strong signals) among the five pines. A maximum of 20 major 18S rDNA sites was observed in the diploid genome (2n = 24) of P. massoniana. The 5S rDNA FISH pattern was less variable, with one major site and one minor site commonly observed in each species. The differentiation of rDNA sites on chromosomes among the five pines correlates well with their phylogenic positions in Pinus as reconstructed from other molecular data. P. densata, a species of hybrid origin, resembles its parents ( P. tabuliformis and P. yunnanensis), including some components characteristic of each parent in its pattern. However, the species is unique, showing new features resulting possibly from recombination and genome reorganization.     

Cellular localization of induced human interferon-β mRNA by non-radioactive in situ hybridization

Summary Induced interferon- (IFN-) mRNA was localized in human FS-4 fibroblasts by in situ hybridization using biotinylated probes. The hybridization sites were detected by incubation with a nick-translated genomic DNA probe (1.8 kb) via streptavidin-colloidal gold followed by silver contrast enhancement. The positive signals were observed by reflection-contrast light microscopy. IFN- mRNA was transiently induced by poly r(I):r(C) in fibroblasts 2–4 h after induction. Induction in the presence of cycloheximide and actinomycin D (superinduction conditions) exhibited an enhanced level of IFN- mRNA with a maximum at 4–8 h. The kinetics of the IFN- mRNA expression in the cytoplasm as revealed by in situ hybridization proved to be compatible with the results of Northern biotting experiments of total cellular RNA.     

Characteristics of βA chromosome haplotypes in Japanese

DNA polymorphism patterns linked to the ^A-globin gene were analyzed in healthy Japanese using four different restriction endonucleases. The chromosomes with the ^A-globin gene were mapped through an evaluation of the presence of seven different restriction sites (HincII 5 to ; HindIII in ^G and ^A; HincII in, and 3 to, ₁; AvaII in ; Bam-HI 3 to ). Among 36 chromosomes analyzed, 20 chromosomes had a haplotype of [+–––––+]. Among 55 individuals examined, 7 possessed a homozygous haplotye of [+–––––+]. All Japanese with the ^AT-globin gene had a subhaplotype of [–++–+] 5 to the -globin gene. Their major haplotypes were [–++–+–+] and [–++–++–]. It was expected that the presence of the ^AT-globin gene in Japanese may be deduced from subhaplotypes 5 to the -globin gene.     

Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization

Genomic in situ hybridization (GISH) was used to identify Festuca chromatin in mitotic chromosomes of Lolium multiflorum (Lm) × Festuca pratensis (Fp) hybrids and hybrid derivatives. In two inverse autoallotriploids LmLmFp and LmFpFp, in situ hybridization was able to discriminate between the Lolium and Festuca chromosomes. In a third triploid hybrid produced by crossing an amphiploid of L. multiflorum × F. pratensis (2n=4x=28) with L. multiflorum (2n=2x=14), the technique identified chromosomes with interspecific recombination. Also, in an introgressed line of L. multiflorum which was homozygous for the recessive sid (senescence induced degradation) allele from F. pratensis, a pair of chromosome segments carrying the sid gene could be identified, indicating the suitability of GISH in showing the presence and location of introgressed genes. By screening backcross progeny for the presence of critical alien segments and the absence of other segments the reconstitution of the genome of the recipient species can be accelerated.     

Localization of luteinizing hormone β-mRNA by in situ hybridization in the sheep pars tuberalis

Summary The localization of luteinizing hormone beta (LH)-mRNA was studied by in situ hybridization in the pars tuberalis of sheep using a homologous sheep double-stranded ³²P-or ³⁵S-cDNA. The labelled cDNA probe detected one mRNA sequence in the pars tuberalis by Northern blot analysis; this sequence was similar to that detected in the pituitary. In situ, the labelling of LH-mRNA in the horizontal and sagittal tissue sections was found throughout the pars tuberalis. This labelling was prevented by adding an excess of cold probe or treating the sections by ribonuclease before in situ hybridization. Controls showed a labelling in the pars distalis, but not in the median eminence, hypothalamus, cerebral cortex and liver sections. Double labelling by using a specific LH-antiserum indicated that the labelling of LH-mRNA appeared more intense in LH-containing cells that were found only in the ventral part of the pars tuberalis. These results suggest that the entire pars tuberalis is able to produce the LH subunit, but that the level of translation greatly varies according to the location of the cells.     

A populational survey of 45S rDNA polymorphism in the Jefferson salamander Ambystoma jeffersonianum revealed by fluorescence in situ hybridization （FISH）

The chromosomal localization of 45S ribosomal RNA genes in Ambystoma jeffersonianum was determined by fluorescence in situ hybridization with 18S rDNA fragment as a probe (FISH-rDNA). Our results revealed the presence of rDNA polymorphism among A.jeffersonianum populations in terms of number, location and FISH signal intensity on the chromosomes. Nine rDNA cytotypes were found in ten geographically isolated populations and most of them contained derivative rDNA sites. Our preliminary study provides strong indication of karyotypic diversification of A.jeffersonianum that is demonstrated by intraspecific variation of 45S rDNA cytotypes. rDNA cytotype polymorphism has been described in many other caudate amphibians. We predict that habitat isolation, low dispersal ability and decline of effective population size could facilitate the fixation and accumulation of variable rDNA cytotypes during their chromosome evolution [Current Zoology 55(2):145-149,2009].     

Regional mapping of the human gene for lysosomal α-glucosidase by in situ hybridization

Summary The current approach to the chromosomal localization of genes coding for lysosomal enzymes has been the correlation of enzymatic and karyotypic analyses of human-rodent somatic cell hybrids. The feasibility of regional mapping depends on the availability of human cells with informative chromosomal rearrangements. In this communication we report the first localization of a gene coding for a lysosomal enzyme by in situ hybridization. The application of an acid -glucosidase cDNA probe to normal human chromosomes allowed direct regional mapping of the -glucosidase locus (GAA) to the region q23q25 of chromosome 17.