Wolfgang Feichtinger

Simple repetitive sequences are associated with differentiation of the sex chromosomes in the guppy fish

SummaryHybridization of restriction enzymedigested genomic guppy (Poecilia reticulata, Poeciliidae) DNA with the oligonucleotide probe (GACA)4 revealed a male-specific simple tandem repeat locus, which defines the Y chromosome in outbred populations. The related (GATA)4 probe identifies certain males with the red color phenotype. In contrast only in two out of eight laboratory guppy strains was the typical (GACA)4 band observed. By specific staining of the constitutive heterochromatin one pair of chromosomes could also be identified as the sex chromosomes, confirming the XX/XY mechanism of sex determination. All males exhibit Y chromosomes with a large region of telomeric heterochromatin. Hybridization in situ with nonradioactively labeled oligonucleotide probes localized the (GACA)n repeats to this heterochromatic portion. Together these results may be regarded as a recent paradigm for the differentiation of heteromorphic sex chromosomes from a pair of autosomes during the course of evolution. According to the fish model system, this may have happened in several independent consecutive steps.

Distribution of telomeric (TTAGGG) (n) sequences in avian chromosomes

Abstract. The physical ends of mammalian and other vertebrate chromosomes consist of tandemly repeated (TTAGGG)n hexamers, nucleating a specialized telomeric structure. However, (TTAGGG)n sequences can also occur at non-telomeric sites, providing important insights into karyotypic evolution. By fluorescence in situ hybridization (FISH) we studied the chromosomal distribution of (TTAGGG)n sequences in 16 bird species, representing seven different orders. Many species, in particular the ratites, display (TTAGGG)n hybridization signals in interstitial and centromeric regions of their macrochromosomes in addition to the typical telomeric signals. In some but not all species these non-telomeric sites coincide with C-band-positive heterochromatin. The retention and/or amplification of telomeric (TTAGGG)n repeats at interstitial and centromeric sites may indicate the fusion of ancestral chromosomes. Compared with the macrochromosomes, the microchromosomes of most species are enriched with (TTAGGG)n sequences, displaying heterogeneous hybridization patterns. We propose that this high density of (TTAGGG)n repeats contributes to the exceptionally high meiotic recombination rate of avian microchromosomes.

Comparative chromosome painting of chicken autosomal paints 1–9 in nine different bird species

In a Zoo-FISH study chicken autosomal chromosome paints 1 to 9 (GGA1–GGA9) were hybridized to metaphase spreads of nine diverse birds belonging to primitive and modern orders. This comparative approach allows tracing of chromosomal rearrangements that occurred during bird evolution. Striking homologies in the chromosomes of the different species were noted, indicating a high degree of evolutionary conservation in avian karyotypes. In two species, the quail and the goose, all chicken paints specifically labeled their corresponding chromosomes. In three pheasant species as well as in the American rhea and blackbird, GGA4 hybridized to chromosome 4 and additionally to a single pair of microchromosomes. Furthermore, in the pheasants fission of the ancestral galliform chromosome 2 could be documented. Hybridization of various chicken probes to two different chromosomes or to only the short or long chromosome arm of one chromosome pair in the species representing the orders Passeriformes, Strigiformes, and Columbiformes revealed translocations and chromosome fissions during species radiation. Thus comparative analysis with chicken chromosome-specific painting probes proves to be a rapid and comprehensive approach to elucidate the chromosomal relationships of the extant birds.

Early stages of sex chromosome differentiation in fish as analysed by simple repetitive DNA sequences

Animal sex chromosome evolution has started on different occasions with a homologous pair of autosomes leading to morphologically differentiated gonosomes. In contrast to other vertebrate classes, among fishes cytologically demonstrable sex chromosomes are rare. In reptiles, certain motifs of simple tandemly repeated DNA sequences like (gata)n/(gaca)m are associated with the constitutive heterochromatin of sex chromosomes. In this study a panel of simple repetitive sequence probes was hybridized to restriction enzyme digested genomic DNA of poeciliid fishes. Apparent male heterogamety previously established by genetic experiments in Poecilia reticulata (guppy) was correlated with male-specific hybridization using the (GACA)4 probe. The (GATA)4 oligonucleotide identifies certain male guppies by a Y chromosomal polymorphism in the outbred population. In contrast none of the genetically defined heterogametic situations in Xiphophorus could be verified consistently using the collection of simple repetitive sequence probes. Only individuals from particular populations produced sex-specific patterns of hybridization with (GATA)4. Additional poeciliid species (P. sphenops, P. velifera) harbour different sex-specifically organized simple repeat motifs. The observed sex-specific hybridization patterns were substantiated by banding analyses of the karyotypes and by in situ hybridization using the (GACA)4 probe.

Spectral karyotyping of the human colon cancer cell lines SW480 and SW620

The cell lines SW480 and SW620, derived from different stages of colon carcinoma in the same patient, have been used for a number of biochemical, immunological, and genetic studies on colon cancer. A comparative analysis of their karyotypes may identify chromosomal aberrations that might represent markers for metastatic spread. In the present study spectral karyotyping (SKY) was applied to these two colon cancer cell lines. Compared to previously reported G-banded karyotypes, 9 (SW480) and 7 (SW620) markers were identical, 3 (SW480) and 3 (SW620) markers could be redefined, 5 (SW480) and 8 (SW620) markers were newly identified, and 4 (SW480) and 5 (SW620) of the previous described markers could not be confirmed. The redefined aberrations include very complex rearrangements, such as a der(16) t(3;16;1;16;8;16; 1;16;10) and a der(18)t(18;15;17)(q12; p11p13;??) in SW620 and a der(19)t(19;8;19;5) in SW480, that have not been identified by conventional banding techniques. The resulting chromosome gains (5q11→5q15, 7pter→q22, 11, 13q14→qter, 20pter→p12, X) and losses (8pter→p2, 18q12→qter, Y) found in both SW480 and SW620 were in good agreement with those frequently described in colorectal tumors as primary changes in the stem cell. Abnormalities found exclusively in SW620 cells only (gains of 5pter→5q11, 12q12→q23, 15p13→p11, and 16q21→q24 and losses of 2pter→2p24, 4q28→qter, and 6q25→qter) can be viewed as changes that occurred in a putative metastatic founder cell.

Chromosomal investigation of three Costa Rican frogs from the 30-chromosome radiation of Hyla with the description of a unique geographic variation in nucleolus organizer regions

A cytogenetic investigation of Hyla ebraccata Cope, H. microcephala Cope, and H. phlebodes Stejneger revealed that the karyotypes of these 30-chromosome Hyla are very conservative. With the exception of some structural rearrangements, only few differences in chromosomal morphology could be discerned. Based on our results, we hypothesize that the telomeric position of nucleolus organizer regions (NOR) on chromosome no. 10 may represent a derived condition in 30-chromosome Hyla. This cytotype was found only in the Caribbean population of H. ebraccata, Such within-species disparity has not been observed previously among amphibians. This phenomenon can most readily be explained by a translocation or insertion that rapidly drifted to high frequency in a small population.

Berenil-induced undercondensation in human heterochromatin

The aromatic diamidine berenil specifically inhibits the condensation of a subset of constitutive heterochromatin in human lymphocyte cultures. In the normal male chromosome complement, only the quinacrine-brilliant Y heterochromatin exhibits distinct undercondensation. The optimal culture conditions for inhibiting heterochromatin condensation are achieved when berenil is added at a final concentration of 150 micrograms/ml 24 h before cell harvest. Various examples of the use of berenil in the analysis of chromosome rearrangements involving quinacrine-brilliant heterochromatin are presented. A variant, giant-satellited chromosome 22 was found to respond to berenil treatment, although its enlarged and quinacrine-bright short-arm region did not contain Y heterochromatin. Southern blot analysis and chromosome in situ hybridization suggested that most chromosome 22 variants do not stem from Y; acrocentric translocations. The experimentally undercondensed Y heterochromatin is characterized by moderate C-band labeling, bright quinacrine fluorescence, and specific silver staining. At the ultrastructural level, undercondensation is associated with loosely packed, mutliply folded chromatin fibers with a diameter of approximately 250 A and organized probably as loops.

On the highest chromosome number in mammals

The mitotic and meiotic chromosomes of the semiaquatic rodent Ichthyomys pittieri (Rodentia, Cricetinae) from Venezuela were analyzed by means of conventional staining and several banding techniques. The diploid chromosome number of this rare species is 2n = 92, which is the highest value known for mammals. It is assumed that this exceptionally high chromosome number is the result of repeated centric fissions. The karyotype of I. pittieri was compared with that of Anotomys leander, for which a diploid number of 2n = 92 has also been reported. The karyological relationships existing within the Neotropical Cricetidae are summarized.

Terminal deletion of the long arm of chromosome 10: a new case with breakpoint in q25.3.

Since the first patient with partial deletion of the long arm of chromosome 10 was described in 1978, another 23 cases have been reported, with the breakpoint ranging from 10q23.3-26.2. To contribute further to the delineation of the monosomy 10qter syndrome, we describe a female child who, at age 3 6/12 years, was diagnosed with a de novo deletion of the long arm of chromosome 10, with a breakpoint in 10q25.3. The phenotypic manifestations in this child are compatible with those of previously reported cases. However, in contrast to most other patients, we found a moderate expression of the syndrome, with no genitourinary or cardiac malformations and with only mild retardation. Based on our observations and those of others, we conclude that a typical craniofacial appearance and varying degrees of psychomotor retardation are always found in patients with 10q- syndrome.

CHROMOSOME BANDING OF SIX DENDROBATID FROGS (COLOSTETHUS, MANNOPHRYNE)

We conducted a chromosome banding analysis (heterochromatin, nucleolus organizer regions, DAPI fluorescence, distamycin A/mithramycin fluorescence) of six phylogenetically basal dendrobatid frog species (Colostethus chalcopis, C. leopardalis, Mannophryne herminae, M. neblina, M. olmonae, M. trinitatis). With the exception of C. chalcopis (2n = 22), all examined species had a chromosome complement of 2n = 24 chromosomes. The C- and Q-band analyses showed that constitutive heterochromatin is present at the centromeres of all species, with Q−-regions occurring at the positions of the nucleolus organizer regions (NORs) of C. leopardalis, M. olmonae, and M. trinitatis. The C-band polymorphisms were detected in M. herminae and M. neblina on chromosome No. 6 and in M. herminae on chromosome No. 7. Silver-staining and distamycin A/mithramycin fluorescence resolved a single pair of NORs in each species. The DAPI fluorescence revealed pericentromeric bands on chromosome No. 1 and 5 in M. trinitatis and on chromosome No. 1 and 4 in C. chalcopis. Chromosome data clearly allow a distinction between M. olmonae and northern Trinidadian M. trinitatis, laying to rest arguments that M. olmonae may not be a good species. In conjunction with chromosome information found in the literature, our data confirm that chromosome complements of phylogenetically basal dendrobatids can be quite variable, with both 2n = 24 and 2n = 22 complements present. Discrepancies in the karyotypes of M. trinitatis from near Caracas, Venezuela, and from the Northern Range of Trinidad indicate the possibility of cryptic species within M. trinitatis. The notion that a reduction in chromosome number by up to three chromosome pairs has taken place among Dendrobatidae from a putative ancestral 2n = 24 karyotype is shown to be congruent with current molecular hypotheses of dendrobatid relationships.