Ekaterina Gornung

Classical and molecular cytogenetics of the zebrafish, Danio rerio (Cyprinidae, Cypriniformes): an overview.

The zebrafish, Danio rerio, has recently become the model system for the genetic analysis of vertebrate development. This paper reviews the advances in zebrafish cytogenetics, obtained through classical and molecular techniques, which will lead to the assignment of specific linkage groups to specific chromosome pairs in the zebrafish genome project. Several chromosome pairs of the 50-chromosome karyotype of D. rerio were differentially stained by classical staining techniques and additional information has been obtained by molecular cytogenetics. Indeed, the analysis of constitutive heterochromatin by C-banding and base-specific fluorochrome staining had suggested a differential composition of peri- and paracentromeric constitutive heterochromatin. The chromosome mapping of distinct AT- and GC-rich zebrafish satellite DNAs by means of PRINS (Primed in situ) and multicolor FISH (Fluorescence in situ Hybridization) has confirmed this hypothesis, which therefore provided the chromosome localization of 10% of the zebrafish genome. The analysis of nucleolus organizer regions (NORs) by silver staining and by FISH with 18S rDNA has also revealed the existence of variable and inactive NORs, in addition to those on the terminal regions of the long arms of the three NOR-bearing chromosome pairs. Other multicopy genes, such as minor ribosomal genes, or multicopy repeats, such as telomere specific sequences, have now been mapped on zebrafish chromosomes. The latest advancement in zebrafish molecular cytogenetics is the chromosome mapping of single locus genes. Single-copy genes from each of the 25 genetic linkage groups are now being mapped on zebrafish chromosomes by using PAC clones.

CMA3-banding pattern and fluorescence in situ hybridiz ation with 18S rRNA genes in zebrafish chromosomes

This study provides new data on zebrafish chromosomes, obtained from the chromomycin A3-banding pattern and mapping of18S rRNA genes by fluorescence in situ hybridization (FISH). C-banding and Ag-staining were also performed to analyse whether variation in heterochromatin and Ag-nucleolus organizer regions (NORs) exists among various commercially purchased strains. The results provide information on heterochromatin composition and on the existence of inter individual NOR polymorphism and contribute to the construction of an idiogram suitable for gene mapping.

Cytogenetic analysis of global populations of Mugil cephalus (striped mullet) by different staining techniques and fluorescent in situ hybridization

The present paper reports the results of cytogenetic analysis carried out on several scattered populations of the striped mullet, Mugil cephalus, the most widespead among mugilid species. The karyotype was investigated through Ag-staining, C-banding, fluorochrome-staining (chromomycin A3/DAPI) and fluorescent in situ hybridization with rDNA genes. All populations showed the same chromosome number and morphology and no changes were detected in heterochromatin and NORs. Therefore, neither population- nor sex-specific marker chromosomes were identified. In some of the specimens, NOR size heteromorphism was detected. Results are discussed with respect to karyotype and ribosomal cistrons organization and to cytotaxonomic implications.

Cytogenetic analysis of Liza ramada (Pisces, Perciformes) by different staining techniques and fluorescent in situ hybridization

A cytogenetic investigation was carried out on specimens of Liza ramada, a mugilid species common in the Mediterranean sea. The analysis of chromosomes was performed through Ag-staining, C-banding, chromomycin A3 and DAPI staining, and fluorescent in situ hybridization with ribosomal genes. The results obtained are discussed with respect to cytotaxonomic implications and to the features of NORs.

Cytogenetic analyses of chromosomal rearrangements in Mus minutoides/musculoides from North-West Zambia through mapping of the telomeric sequence (TTAGGG)n and banding techniques

Three specimens of M. minutoides/musculoides from Zambia were cytogenetically studied through G- and C-banding, DAPI staining and fluorescence in-situ hybridization (FISH) with a (TTAGGG)n telomeric sequence. Biarmed chromosomes were identified according to the current nomenclature as follows: Rb(2.7), Rb(3.12), Rb(4.5), Rb(6.8), Rb(9.16), and the sex chromosomes Rb(1.X), Rb(1.Y) and Rb(1.Xd), originated from the deleted X chromosome. One female showed the diploid number 2n=24; in the two other individuals, the Rb(9.16) occurred in a heteromorphic condition, and, accordingly, the diploid number was 2n=25. FISH showed the sites of telomeric sequences at telomeres of all the chromosomes, and in an interstitial position at the centromeres of all Robertsonian metacentrics, except one – the Rb(6.8), though the patterns of hybridization varied between chromosomes. Sex chromosome pairs, in the male and females, showed a similar C-banding pattern, but revealed clear differences after FISH. Traces of telomeric sequences were found dispersed in the whole-heterochromatic arm of the Rb(1.Xd). No visible bond between C-positive heterochromatin and telomeric sequences were detected in the other either bi- or uniarmed chromosomes, indicating that they may actually represent retained telomeres in the Robertsonian metacentrics.

FISH-mapping of 18S ribosomal RNA genes and telomeric sequences in the Japanese bitterlings Rhodeus ocellatus kurumeus and Tanakia limbata (Pisces, Cyprinidae) reveals significant cytogenetic differences in morphologically similar karyotypes.

The Japanese rose bitterling, Rhodeus ocellatus kurumeus, and the oily bitterling, Tanakia limbata, were cytogenetically studied by silver (Ag)- and chromomycin A3 (CMA3)-staining, by C-banding and by mapping of the 18S ribosomal genes and of the (TTAGGG)n telomeric sequence. These two representative species of related genera of the subfamily Acheilognathinae show very similar chromosome complements. Nevertheless, significant differences in the chromosomal distribution of nucleolus organizer regions (NORs) and interstitial telomeric sequences were observed. Whereas R. ocellatus kurumeus shows a single NOR-bearing chromosome pair, T. limbata is characterized by a higher number of variable NORs. Multiple telomeric sequence sites were found at the pericentromeric regions of several chromosomes in the rose bitterling. No telomeric sequence sites were detected near centromeres, but they were found to be scattered along the NORs in the oily bitterling. Two karyoevolutive trends might have been identified in the subfamily.

Zebrafish 5S rRNA genes map to the long arms of chromosome 3.

The zebra¢sh, Danio rerio, is a model species in genetic screening for embryonic mutations. In this study, we have localised the 5S ribosomal RNA (5S rDNA) genes. The 5S rDNA was ampli¢ed by polymerase chain reaction using the sequences from the rainbow trout 5S coding region as primers [1]. Two PCR ampli¢cation products of approximately 180 and 500 bp were obtained from four individuals. Two representative clones of different size, pDR-5S19-2 and pDR-5S17-1, were sequenced (Genbank AF213516-213517). Each clone contains two separate regions which form an almost complete 5S coding sequence, joined by a non-transcribed spacer (NTS). Fluorescence in-situ hybridization (FISH) with either puri¢ed PCR ampli¢cation products or the two cloned genes as probes localized the 5S rDNA scattered along most of the long arms of the largest submetacentric chromosome pair (Figure 1), classi¢ed as number 3 [2,3]. The long arms of this chromosome are slightly C [4] and slightly chromomycin A3 (CMA3)-positive [3], as well as late replicating [2]. Present data suggest that the NTS variants, averaging 56% AT, are not responsible for the CMA3-staining pattern. Instead, this could be due to the slight prevalence of GC (around 60%) in the 5S rRNA coding regions and to their high redundancy and interspersion. The 5S rRNA genes constitute the ¢rst singlechromosome marker DNA sequences mapped in the zebra¢sh karyotype. We are grateful to Dr. Alberto Penda© s and Dr. Giovanni Destro-Bisol for their advice in setting up experiments.

Comparative molecular cytogenetic analysis of two congeneric species, Mugil curema and M. liza (Pisces, Mugiliformes), characterized by significant karyotype diversity

Two congeneric mullet species, Mugil liza and M. curema, respectively with an all-uniarmed and an all-biarmed karyotype, were cytogenetically studied by base-specific fluorochrome staining and FISH-mapping of 45S and 5S ribosomal RNA genes (rDNA) and the (TTAGGG)n telomeric repeats. Whereas 45S rDNA sites might be homeologus in the two species, 5S rDNA sites are not, as they are localized on chromosome arms of different size. In both species, the (TTAGGG)n telomeric probe hybridized to natural telomeres and was found scattered along the NORs. In metacentric chromosomes of M. curema, no pericentromeric signals of the telomeric probe were detected. Data are discussed in relation to the karyotype evolution in Mugilidae and to the mechanisms and the evolutionary implications of Robertsonian rearrangements in M. curema.

Comparative cytogenetic study of two sister species of Iberian ground voles, Microtus (Terricola) duodecimcostatus and M. (T.) lusitanicus (rodentia, cricetidae).

The two Iberian species of pine voles, Microtus (Terricola) duodecimcostatus and M. (T.) lusitanicus of the subfamily Arvicolinae (Cricetidae, Rodentia), were compared after G- and C-banding and chromosomal mapping of ribosomal RNA genes (rDNA), telomeric repeats, and satellite DNA Msat-160. Notwithstanding their close relationship (one sister group in phylogenetic analyses) and sharing of the diploid and fundamental chromosome numbers, the 2 species show notable differences in the sex chromosome morphology, the number and distribution of rDNA sites, constitutive heterochromatin and satDNA patterns. The only telomeric repeats showed normal, all-telomeric, distribution in karyotypes of both species. The data are discussed with regard to interspecific and intrageneric variation of the analyzed characters and the chromosomal evolution in the genus Microtus.

Chromosomal localization of zebrafish AluI repeats by primed in situ (PRINS) labeling

Two zebrafish AluI repeats were localized in metaphase chromosomes by means of the primed in situ (PRINS) labeling technique, using oligonucleotide primers based on published sequences. An AT-rich, tandemly repeated, long AluI restriction fragment (RFAL1) labeled the (peri)centromeric regions of all chromosomes. The GC-rich short frag- ment (RFAS) was found to be localized in the paracentromeric regions of 17 chromosome pairs, which were mostly subtelocentric. The RFAS labeling pattern generally fits the previously described chromomycin A3 (CMA3) staining pattern. The differential composition of heterochromatin in zebrafish chromosomes is discussed.