Sequential Q- and Acridine orange-marker technique.

A standardized Q- and acridine orange (AO)-fluorescence dual marker technique was described. It involved preservation of unstained chromosome slides in a vacuum desiccator up to 18 months, Q-staining, destaining, and treatment in Hanks' solution, pH 5.1, at 85 degrees C for 13 min, and acridine orange staining. Q-markers were found at the paracentromeric regions of chromosomes 3 and 4, the short arms and the satellites of the acrocentric chromosomes, while AO-marker spots were on the satellite-stalks of the acrocentrics. The advantage of the dual marker technique was illustrated by the determination of the origin of trisomy 22 in a spontaneous abortus.     

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.     

Supernumerary chromosomal elements in lymphocytes cultured from phenotypically normal human adult males

In a group of phenotypically normal men there were approximately 0.24% of metaphase lymphocytes with extra chromosomal elements along with the regular 46 chromosomes. They ranged in size from small acrocentric-acentric elements to elements longer than any chromosome arm. These elements have been referred to as supernumerary chromosomal elements. No significant effects due to donor's age, smoking history, season, storage of blood samples prior to culture, or culture medium, were found either in the frequency of supernumerary elements per cell or in the frequency of cells with supernumerary elements. Furthermore, the same subject did not consistently exhibit supernumerary elements. Furthermore, the same subject did not consistently exhibit supernumerary elements when sampled during four successive quarters of the year. Some of these elements in pairs were identified by G-banding technique as translocation chromosomes bearing long arms of chromosome number 2 and presumptive short arms of chromosome 8, acentric long arms of chromosome 4, and iso-acentric chromosomes for the long arms of chromosome 5. Presumably, more than one type of cytogenetic event occurred in their formation. Circumstantial evidence has been presented to show that the means of elimination of these supernumerary elements is a process of chromosomal disintegration.     

Sequential Q- and acridine orange-marker technique

Summary A standardized Q- and acridine orange (AO)-fluorescence dual marker technique was described. It involved preservation of unstained chromosome slides in a vacuum desiccator up to 18 months, Q-staining, destaining, and treatment in Hanks' solution, pH 5.1, at 85°C for 13 min, and acridine organe staining. Q-markers were found at the paracentromeric regions of chromosomes 3 and 4, the short arms and the satellites of the acrocentric chromosomes, while AO-marker spots were on the satellite-stalks of the acrocentrics. The advantage of the dual marker technique was illustrated by the determination of the origin of trisomy 22 in a spontaneous abortus.This work was supported by grants from the Ford Foundation Population Program No. 640-0411 B), the World Health Organization, and Fonds National Suisse (No. 3.424-0.74).     

Chromosome organisation in the Australian plague locust,Chortoicetes terminifera

In Chortoicetes terminifera, G-banding, produced by the trypsin treatment of air-dried slides followed by Giemsa staining, leads to light staining gaps at the secondary constrictions on autosomal pair 6 and regions proximal to the centromere on the long arms of pair 4. The variable short arms of two of the three smallest pairs were usually flared and lightly stained after treatment. In contrast to the relatively minor response of the normal chromosome set to G-banding, the large supernumerary chromosomes of C. terminifera show a spectacular series of dark bands alternating with lightly stained gaps. Two G-band variants of the B-chromosome were found in a laboratory stock. These patterns of G-banding are discernable both at mitosis in adults and embryos of both sexes and at all stages of male meiosis. Some regions which are gaps after G-banding appear as dark bands after C-banding. Consequently the supernumerary chromosome is mainly darkly stained with C-banding. In addition the centromeres and some telomeres are C-banded along with narrow interstitial bands and polymorphic heterochromatic blocks. — C-banding was not always successful, the technique often yields a mixture of G- and C-banding. The disparity of banding between the normal complement and the B-chromosome implies that whatever the source of origin of the B it has undergone spectacular changes in organisation since its origin.     

Nucleolar-organiser polymorphism in natural populations of Allium schoenopmsum

Allium schoenoprasum, 2n=16, has a single pair of highly acrocentric chromosomes which always have nucleolar-organiser regions on the short arms. In some British populations one or both homologues of submetacentric chromosome 7 may also carry a nucleolar-organiser region terminal on the long arm. These supernumerary satellites are simply inherited. Of the 13 populations examined, six were polymorphic for chromosome 7s. The frequency of chromosome 7s ranges from 0.130 to 0.825 and the distribution of the three karyotypes in all these populations corresponds to a HardyWeinberg equilibrium.     

QFH banding and DNA distribution in the macrochromosomes of the chicken Gallus domesticus

Chromosomes of Gallus domesticus were stained with rivanolum-SO2 (fluorescence Feulgen reaction) and by Hoechst 33258. Fluorescence photography was performed on a 35 mm film KN-3(KN-3) "Svema". The negatives were analyzed with the microdensitometer. The Feulgen (Fr--) and the Hoechst 3358 (H--) densitometric profiles of chromosomes showed light and dark segments along the metaphase chromosomes. The amount of DNA, as determined by the fluorescence Feulgen reaction, is not constant along the chromosome arms. Consequently, the base composition is not the only factor influencing the fluorescence of Hoechst 33258 along the chromosomes. The comparative analysis of densitometric profiles of the Hoechst 33258 and rivanolum-SO2 stained chromosomes shows that the Iq telomere band consists of GC-rich DNA (about 70% GC). The variation of DNA contents along the metaphase chromosome arms can be realized at both chromoneme and/or subchromoneme levels.     

Complete and precise characterization of marker chromosomes by application of microdissection in prenatal diagnosis

A straightforward and extremely efficient reverse chromosome painting technique is described which allows the rapid and unequivocal identification of any cytogenetically unclassifiable chromosome rearrangement. This procedure is used to determine the origin of unknown marker chromosomes found at prenatal diagnosis. After microdissection of the marker chromosome and amplification of the dissected fragment by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), fluorescence in situ hybridization (FISH) to aberrant and normal metaphase chromosomes with the marker-derived probe pool is performed. With this strategy, marker chromosomes present in amniotic fluid samples were successfully identified in three cases. The origin of the supernumerary markers was ascertained as deriving from 3p(p12-cen), 18p(pter-cen) and 9p(p12-cen), respectively. Since a specific FISH signal on chromosomes can be obtained within 2 working days using a probe generated without any pretreatment from one chromosomal fragment only and without additional image processing devices, this technique is considered to be highly suitable for routine application in pre- and postnatal cytogenetic analysis.     

An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes

Summary A reliable technique for staining human chromosomal nucleolar organizers (NOR's) with silver solutions is described. The NOR's can be selectively stained dark brown by silver solutions leaving the chromosome arms unstained and available for counterstaining with orcein or Giemsa dyes. Unequivocal identification of chromosome pairs bearing NOR's can be achieved using fluorescent banding techniques followed by silver staining. The silver staining procedure for NOR's was simplified and standardized through control of the chemical and physical conditions during silver impregnation and developing.     

Characterization of a familial small supernumerary marker chromosome in a patient with adult-onset tongue cancer

Cytogenetic analysis in peripheral blood lymphocytes of a 50-year-old female with tongue cancer showed the presence of one to three copies of a small supernumerary marker chromosome (sSMC) in a mosaic state. Family studies also revealed the marker in mosaic form in four (age <29 years) of eleven clinically normal individuals studied from her family of 16 individuals spanning three generations. Due to the extremely small size of the marker chromosome, identification by classical cytogenetics was not informative. Multicolor FISH followed by whole chromosome painting identified the marker as a derivative of chromosome 21. This is the first report of sSMC21 in an adult-onset tongue cancer patient and some of her family members with no clinical symptoms.     

Reassessment of presumed Y/22 and Y/15 translocations in man using a new technique.

A new chromosome banding technique, distamycin A plus DAPI, has been used to reexamine cases of presumed Y/autosome translocations. In contrast with the results obtained with quinacrine fluorescence (Q-banding), the satellites of acrocentric chromosomes do not fluoresce brightly with this new (DA-DAPI) method, making it more specific for the long arm of the Y chromosome. Previous cases with intensely Q-fluorescent and abnormally long short arms on a chromosome 22 were considered as presumptive 22/Y translocations: The new technique clearly shows that, in these cases, the additional material on 22p is not derived from Yq. In contrast, in other cases the Yq nature of additional material on 15p, in conjunction with the presence of an extra Y-body in interphase nuclei and the presence of a male-specific DNA, supports the previous diagnosis of a presumptive 15/Y translocation.     

Characterization of the first supernumerary tricentric ring chromosome 1 mosaicism by conventional and molecular cytogenetic techniques

We report on the conventional cytogenetic and fluorescence in situ hybridization (FISH) results obtained for a 3.5-year-old girl with developmental and language delay and a supernumerary ring chromosome mosaicism in 8% of T-lymphocytes analyzed. Using different conventional and molecular cytogenetic techniques as YAC hybridization and comparative genomic hybridization, we could show that the extra tricentric ring chromosome consists of three heterochromatic blocks with inserted euchromatic material. Additionally, chromosome microdissection followed by FISH analysis demonstrated that the small tricentric ring chromosome consisted of material from the pericentromeric region of chromosome 1q21. Thus, the patient has a mosaic of normal cells and cells with partial pentasomy of the pericentromeric region of chromosome 1. So far, 19 cases with single supernumerary marker chromosome 1 have been published, but no tricentric ring chromosome 1 is, to our knowledge, reviewed in the literature. In this study, we compare the clinical features of our patient with cytogenetically comparable cases described in the literature. We introduce a hypothesis for the formation of a tricentric ring chromosome: starting with a monocentric ring, sister chromatid exchange leading to the formation of a tetracentric ring, which underwent intrastrand recombination generating the tricentric ring.     

Construction and uses of new compound B-A-A maize chromosome translocations

Maize B-A translocations result from reciprocal interchanges between a supernumerary B chromosome and an arm of an essential A chromosome. Because of the frequent nondisjunction of the B centromere at the second pollen mitosis, B-A translocations have been used to locate genes to chromosome arms and to study the dosage effects of specific A segments. Compound B-A translocations (B-A-A translocations) are created by bringing together a simple B-A translocation with an A-A translocation in which breakpoints in the A-A and B-A translocations are in the same arm. Recombination in the region of shared homology of these A chromosome segments creates a B-A-A translocation. Success in creating and testing for a new B-A-A translocation requires that the B-A translocation be proximal to the A-A translocation and that the A-A translocation be proximal to the tester locus. The breakpoints of most of the A-A translocations have been cytologically defined by earlier investigators. Previous investigators have produced 16 B-A-A translocations and one B-A-A-A translocation, which collectively define 35 A chromosome breakpoints. We have enlarged this group by creating 64 new B-A-A translocations. We present a summary of the total of 81 B-A-A translocations showing their distribution among the chromosome arms and the 163 cytologically defined chromosome segments delimited by them. We also illustrate the method of construction of these B-A-A stocks and their uses.     

A stably transmitted pair of translocated supernumerary chromosomes in maize.

A pair of stably transmitted supernumerary chromosomes of unknown source has been found in a maize stock carrying a desynaptic mutant. The presence of the supernumerary chromosome appears to be unrelated to the meiotic mutant, but is believed to have been derived from a translocated B chromosome contaminant. The supernumerary chromosomes carry a segment of a A chromosome in this stock where there appear to be two normal copies of each of the 10 A chromosomes. Thus, this A chromosome segment is present in quadruplicate. Surprisingly, a quadrivalent configuration is formed in most microsporocytes, which involves not only synapsis but also chiasma formation in the A chromosome segments involved in the quadrivalent. This represents a strong preferential pairing of supernumeraries with the normal A chromosome segments. Such nonrandom association and crossing over might provide information on the nature of early homologue alignment at meiosis.     

The origin and behavior of two isodicentric bisatellited chromosomes.

Karyotyping revealed three cell lines in a boy with mental retardation and few other abnormalities. Thirty cells exhibited a normal karyotype, and 54 had an extra acrocentric chromosome of E group size with satellites on the long and short arms. The remaining 20 cells each had, in addition to the first marker (M1), a second tiny bisatellited chromosome (M2). C-banding demonstrated that both markers were dicentric. G-, C-, and Q-banding and satellite association data were consistent with the markers having originated from chromosome 15 material. We propose that M1 was formed from a meiotic breakage and a chromatid fusion in the proximal long arms of an acrocentric pair. This would have produced a symmetrical isodicentric chromosomes, plus one or two acentric fragments. M2 then could have resulted from a dicentric bridge-break-synthesis-reunion phenomenon. This model of abnormal meiotic exchange can be generalized to encompass the formation of other dicentric isochromosome cases of isochromosome X.     

Phenotypic variability of the cat eye syndrome. Case report and review of the literature.

We present a male infant with preauricular skin tags and pits, downslanting palpebral fissures, hypertelorism, ectopic anus, hypospadias, and hypoplastic left heart syndrome. The clinical features in our patient show phenotypic overlap with the cat eye syndrome, as illustrated by the review of 105 reported cases. Cytogenetic analysis revealed a supernumerary marker chromosome, which was identified by microdissection and fluorescence in situ hybridization as an isodicentric chromosome 22(pter --> q11.2::q11.2 --> pter). It was proved with probes specific for the cat eye syndrome critical region that this region was present in quadruplicate in the propositus. We conclude that CES is characterized by large phenotypic variability, ranging from near normal to severe malformations, as reflected in the neurodevelopmental outcome. Preauricular skin tags and/or pits are the most consistent features, and suggest the presence of a supernumerary bisatellited marker chromosome 22 derived from duplication of the CES critical region.     

Recombination within extra segments: evidence from the grasshopper Chorthippus jucundus

A detailed analysis, using a modified silver staining technique which permits the visualization of the chromatid cores in bivalents at metaphase I, shows that some so-called chromosome associations are indeed true chiasmata. This study employs a supernumerary segment on the longest of the autosome pairs in the grasshopper Chorthippus jucundus that produces clear asymmetric bivalents in heterozygotes. Clear evidence of crossing over within the supernumerary segment explains part of the polymorphism of this segment, since unequal crossing over can produce different-sized segments. The origin of this segment and its mode of inheritance are also considered in the light of these results.     

De novo origin of multiple small supernumerary marker chromosomes (sSMCs) in a child with intellectual disability and dysmorphic features

Small supernumerary marker chromosomes (sSMCs) are a heterogeneous group with regards to their clinical effects as well as their chromosomal origin and their shape. The sSMCs are associated with mental retardation and dysmorphic features. Multiple sSMCs are rarely reported. We report four sSMCs in a case of dysmorphic features and intellectual disabilities. Among the four sSMCs, one sSMC confirmed to be chromosome 5 derived sSMC using fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). The sSMCs were de novo originated as parental chromosomal analysis revealed normal karyotypes. The sSMC derived from chromosome 5 might be associated with mental retardation and dysmorphic features in the present case. However the remaining three sSMCs might have originated from repetitive sequences of chromosomes.     

Heterochromatic chromosomes and satellite DNAs of Drosophila nasutoides

Drosophila nasutoides is distinguished from other Drosophila species in that the metaphase karyotype shows a pair of very large V-shaped chromosomes. With Giemsa, a distinctive C-banding pattern is revealed along the arms of this large chromosome, indicating a largely heterochromatic nature. Furthermore, the banding patterns of the arms are symmetrical, indicating that it is an iso-chromosome. A comparison between the metaphase karyotype and polytene chromosomes suggests that the large V chromosome appears as the dot chromosome in polytene squash. One autosome has twice the arm length of typical Drosophila polytene chromosomes and arose either by centric fusion and a pericentric inversion, or by translocation connecting distal ends with a subsequent loss of one centromere. This chromosome appears to have a short arm which ectopically pairs with the proximal region of the long arm, representing a duplication of about ten bands. When the nuclear DNA is examined by neutral CsCl gradient, four satellites are observed. As much as sixty percent of the total DNA appears as satellites in the lysate of larval brains. No satellite was detectable in the lysate of salivary glands. These observations led us to suggest that the heterochromatic nature of the large V chromosome is due to the presence of all four satellites in this chromosome and that this large chromosome appears as the dot because of the under-reduplication of the satellites during polytenization.     

De novo supernumerary marker chromosome originating from chromosome 17 resulting in a normal pregnancy outcome

We report here a prenatal case with de novo supernumerary marker chromosome originating from chromosome 17 in non-mosaic form resulting in normal pregnancy outcome. In this case, a 26-year-old pregnant woman was referred for amniocenthesis and microdeletion Fluorescence In Situ Hybridization (FISH) testing at 18 weeks of gestation due to history of a previous child with Angelman Syndrome. PWS/AS region deletion was excluded by FISH. A de novo supernumerary, non-satellited, monocentric marker chromosome was detected during conventional cytogenetic analysis. With the use of FISH testing, it was found that the marker chromosome originated from chromosome 17. Additionally, the marker chromosome was found not to contain the Smith-Magenis and Miller Dieker syndrome regions. After detailed review of the literature, genetic counseling was given to the family, and the family decided to continue the pregnancy to term. A female child was born at term without any phenotypical abnormalities and clinical complications. Follow-up at 15 months-of-age revealed no developmental abnormalities. To our knowledge, our patient is the first reported prenatal case with a de novo monocentric, supernumerary marker chromosome derived from chromosome 17 in a non-mosaic form that resulting in normal pregnancy outcome.