Lech Kirtiklis

Ribosomal genes in Coregonid fishes (Coregonus lavaretus, C. albula and C. peled) (Salmonidae): single and multiple nucleolus organizer regions

Major rDNA loci, i.e. nucleolus-organizing regions (NORs), were assigned using chromomycin-A3 (CMA3) staining followed by sequential silver (Ag) staining and in situ hybridization (ISH) with a rDNA probe to the chromosomes of the European whitefish (Coregonus lavaretus), the peled (Coregonus peled) and the vendace (Coregonus albula), three closely related coregonine salmonid fishes. One pair of NOR-bearing chromosomes was found in the peled karyotype. Multichromosomal, but stable, locations of rDNA sites on three pairs of chromosomes were observed in the European whitefish karyotype. Multichromosomal polymorphic locations, both in site and number, were observed in the karyotype of the vendace. Several Ag-, CMA3- and ISH-positive regions were found which defined up to seven cytotypes of five NOR-bearing chromosomes. All positive Ag-NORs detected corresponded both to rDNA-ISH- and CMA3-positive signals, which suggests extensive structural polymorphism in the locations of rDNA sites. Stable NOR sites were found at the same location on both homologous elements of the chromosome no. 9 in all individuals, while the remaining NORs were quite variable between individuals, and often present in heterozygous condition. The apparently similar and parallel evolutionary rDNA differentiation patterns in the subfamilies Coregoninae and Salmoninae (family Salmonidae) are observed and discussed.

Sexually dimorphic transcription of estrogen receptors in cod gonads throughout a reproductive cycle

The role of sex steroid regulation in gonadal maturation is a very complex process that is far from being fully understood. Hence, we have investigated seasonal changes in gonadal expression of estrogen receptors (ERs) in Atlantic cod (Gadus morhua L.), a batch spawner, throughout the annual reproductive cycle. Three nuclear ER partial cDNA sequences (esr1, esr2a, and esr2b) were cloned and all esr transcripts were detected mainly in liver and gonads of fish of both sexes. In situ hybridization of esrs along with germ cell (vasa) and gonadal somatic cell markers (gonadal soma-derived factor (gsdf), 3β-hydroxysteroid dehydrogenase (3βhsd), and anti-Müllerian hormone (amh) for testicular, or gsdf for ovarian somatic cells) showed that all three esrs were preferentially localized within interstitial fibroblasts composed of immature and mature Leydig cells in testis, whereas they were differentially expressed in both follicular cells and oocytes in ovary. Quantitative real-time PCR analysis revealed a sexually dimorphic expression pattern of the three esr paralogs in testis and ovary. A significant increase in esr2a expression was identified in testis and of esr2b in ovary, whereas esr1 transcripts were elevated in both testis and ovary in February and March before the spawning period. The localization and sexually dimorphic expression of esr genes in gonads indicate a direct function of estrogen via ERs in gonadal somatic cell growth and differentiation for Leydig cell in testis and follicular cells in ovary throughout the annual reproductive cycle in Atlantic cod.

Chromosomal characteristics and distribution of rDNA sequences in the brook trout Salvelinus fontinalis (Mitchill, 1814)

Brook trout Salvelinus fontinalis (Mitchill, 1814) chromosomes have been analyzed using conventional and molecular cytogenetic techniques enabling characteristics and chromosomal location of heterochromatin, nucleolus organizer regions (NORs), ribosomal RNA-encoding genes and telomeric DNA sequences. The C-banding and chromosome digestion with the restriction endonucleases demonstrated distribution and heterogeneity of the heterochromatin in the brook trout genome. DNA sequences of the ribosomal RNA genes, namely the nucleolus-forming 28S (major) and non-nucleolus-forming 5S (minor) rDNAs, were physically mapped using fluorescence in situ hybridization (FISH) and primed in situ labelling. The minor rDNA locus was located on the subtelo-acrocentric chromosome pair No. 9, whereas the major rDNA loci were dispersed on 14 chromosome pairs, showing a considerable inter-individual variation in the number and location. The major and minor rDNA loci were located at different chromosomes. Multichromosomal location (3–6 sites) of the NORs was demonstrated by silver nitrate (AgNO3) impregnation. All Ag-positive i.e. active NORs corresponded to the GC-rich blocks of heterochromatin. FISH with telomeric probe showed the presence of the interstitial telomeric site (ITS) adjacent to the NOR/28S rDNA site on the chromosome 11. This ITS was presumably remnant of the chromosome rearrangement(s) leading to the genomic redistribution of the rDNA sequences. Comparative analysis of the cytogenetic data among several related salmonid species confirmed huge variation in the number and the chromosomal location of rRNA gene clusters in the Salvelinus genome.

Molecular cytogenetic analysis of the crucian carp, Carassius carassius (Linnaeus, 1758) (Teleostei, Cyprinidae), using chromosome staining and fluorescence in situ hybridisation with rDNA probes

Abstract The crucian carp Carassius carassius (Linnaeus, 1758) is a species with restricted and decreasing distribution in Europe. Six males and six females of the species from the Baltic Sea basin in Poland were examined to show sequentially CMA3/AgNO3 staining pattern, DAPI staining, and, for the first time in literature, molecular cytogenetic analysis using double-colour fluorescence in situ hybridisation (FISH) with 28S and 5S rDNA probes. The karyotype consisted of 20 m, 36 sm and 44 sta chromosomes, NF=156. The AgNO3 stained NORs were most frequently located terminally in the short arms of two sm and two sta elements, and CMA3-positive sites were also observed suggesting abundant GC-rich repetitive DNA in the regions. Other CMA3-positive sites in the short arms of six to ten sm and sta chromosomes were detected. The results based on 28S rDNA FISH confirmed the location of rDNA sites. DAPI-negative staining of NORs suggested the scarcity of AT-rich DNA in the regions. FISH with 5S rDNA probe revealed 8–14 loci (ten and 12 in respectively 49 and 29% of metaphases). They were located in two sm and eight to ten sta chromosomes and six of them were larger than others. Simultaneously, mapping of the two rDNA families on the chromosomes of C. carassius revealed that both 28S and 5S rDNA probes were located in different chromosomes. Molecular cytogenetic data of C. carassius presented here for the first time give an important insight into the structure of chromosomes of this polyploid and declining species and may be useful in its systematics.

Chromosome study of peled (Coregonus peled, Salmoniformes).

Chromosomes of Coregonus peled were examined by Giemsa, CMA3, Ag-NOR and C-banding. The karyotype of peled had a diploid number 2n=76, arm number NF=96 and consisted of twenty meta-submeta chromosomes and 56 subtelo-acrocentric chromosomes. C-positive blocks of heterochromatin were observed on the telomeric regions of meta- and submetacentric chromosomes. Pairs no. 1 and 11 had short arms, completely heterochromatic. The NOR was observed at one acrocentric pair, no. 11. Arm length polymorphism was observed on the NOR-bearing pair.

An update to the chromosomal data of the European bitterling Rhodeus amarus (Teleostei: Cypriniformes: Acheilognathinae)

Chromosomal abnormalities in secondary bovine oocytes matured in vitro up to 48 hours Abstract Chromosomal abnormalities in secondary bovine oocytes matured in vitro up to 48 hours. 21st International Colloquium on Animal Cytogenetics and Gene Mapping Rubessa M., Pauciullo A., Peretti V., Iannuzzi L., Ramunno L., Di Berardino D.Edited by: L. Iannuzzi, A. Perucatti, A. Iannuzzi, A. Pauciullo, V. Genualdo, D. Incarnato, L. Keller (CNRISPAAM, Naples, Italy)Sister Chromatid exchange (SCE) test in river buffalo cells treated with Furocoumarins. / Iannuzzi A.; Perucatti A.; Genualdo V.; Pauciullo A.; Pucciarelli L.; Incarnato D.; Melis R.; Porqueddu C.; Marchetti M.; Iannuzzi L.. In: CHROMOSOME RESEARCH. ISSN 0967-3849. 22(2014), pp. 421-421. Original Citation: Sister Chromatid exchange (SCE) test in river buffalo cells treated with Furocoumarins.Comparative FISH-mapping of TNF, STAT5A and MNTR1A fecundity genes on river buffalo, cattle, sheep and goat. / Iannuzzi A.; Perucatti A.; Pauciullo A.; Genualdo V.; Incarnato D.; Pucciarelli L.; De Lorenzi L.; Parma P.; Iannuzzi L.. In: CHROMOSOME RESEARCH. ISSN 0967-3849. 22(2014), pp. 418-418. Original Citation: Comparative FISH-mapping of TNF, STAT5A and MNTR1A fecundity genes on river buffalo, cattle, sheep and goat.Multicolor FISH with 10 specific painting probes for the rapid identification of the sub-metacentric river buffalo autosomes (Bubalus bubalis, 2n=50) / Pauciullo A.; Perucatti A.; Iannuzzi A.; Incarnato D.; Genualdo V.; Pucciarelli L.; Di Berardino D.; Iannuzzi L.. In: CHROMOSOME RESEARCH. ISSN 0967-3849. 22(2014), pp. 410-410. Original Citation: Multicolor FISH with 10 specific painting probes for the rapid identification of the sub-metacentric river buffalo autosomes (Bubalus bubalis, 2n=50)

Gene mapping of 28S rDNA sites in allotriploid Cobitis females (Pisces: Cobitidae) from a diploid-polyploid population

We performed cytogenetic analysis on triploid Cobitis hybrid specimens from a natural diploid-polyploid population in the Bug River (Poland). Chromosomes were examined by fluorescence in situ hybridization method (FISH) with 28S rDNA as a probe. The rDNA sites were commonly identified in the terminal position of p arms of two small submetacentric chromosomes, in the centromere area of two acrocentric chromosomes and in the terminal position of q arm of one acrocentric chromosome. Some of the observed signals were stronger than others. In few cases, one small acrocentric chromosome showing rDNA sites located terminally on both p and a q arm was observed. This study brings useful information about polymorphism of 28S rDNA sites regarding their number, location and size in triploid specimens. Moreover, it gives preliminary data for comparison with similar researches on the parental species of Cobitis hybrid females, viz. C. taenia, C. taurica and C. tanaitica, and other related Cobitis taxa of different ploidy level. The present study increases our knowledge about ribosomal genes inheritance between Cobitis species and their polyploid hybrids.

Heterochromatin organization and chromosome mapping of rRNA genes and telomeric DNA sequences in the burbot Lota lota (Linnaeus, 1758) (Teleostei: Gadiformes: Lotidae)

Abstract The knowledge about the genome of the burbot Lota lota (Linnaeus, 1758), the only freshwater gadiform fish (Gadidae, Gadiformes), is limited to chromosome number (2n = 48, NF = 78). In this work analysis of burbot chromosomes by C-banding, DAPI staining and restriction endonuclease digestion proved both heterogeneity of the heterochromatin and interchromosomal differences in the vulnerability to the endonuclease treatment. Silver nitrate and chromomycine A3 staining and fluorescence in situ hybridization using 28S rDNA probes showed that nucleolus organizer regions appear in one pair of burbot chromosomes. There is also a single locus for 5S rDNA repeats exhibited in burbot; however, major and minor rRNA gene families are not linked. The single and separated location of major and minor rDNA families suggests that the burbot karyotype still retains some characteristics of the putative ancestral karyotype. Although the increased number of the chromosome arms in relation to the ancestral karyotype suggests that burbot karyotype experienced several inversions, we did not find any intercalary telomeric DNA sequences in their chromosomes.

Comparison of molecular and morphometric analysis in species discrimination of larvae among five cyprinids from the subfamily Leuciscinae: A tool for sustainable conservation of riverine ichthyofauna

Abstract Fish species from the subfamily Leuciscinae are one of the most abundant and ecologically valuable among riverine cyprinids of Central Europe. In order to support their recruitment or properly manage the restocking operations, there is a need to recognize the recruitment success which relies on the necessity of proper species discrimination among the larvae of those species caught on the spawning and/ or nursery grounds. In the present study a comparative analysis of the morphological and molecular (PCR-RFLP technique with two restriction endonucleases HinfI and TaqI based on mitochondrial DNA fragment encoding cytochrome b) techniques for identification of the species status among larvae of five cyprinid species, the ide (Leuciscus idus), common dace (Leuciscus leuciscus), asp (Leuciscus aspius), roach (Rutilus rutilus) and the European chub (Squalius cephalus) was performed. It was shown that the application of morphometric features solely could create many ambiguities in species discrimination. The use of applied molecular techniques was found to be a great support for the larvae identification, regardless the developmental stage of the fish. Moreover, this molecular method may also be used for the identification of the species even if the preserved fish (e.g., in ethanol), having usually deformed and/ or shrunk body caused by preservation, are about to be investigated.