Autosomal localization of interstitial telomeric sites (ITS) in brook trout, Salvelinus fontinalis (Pisces, Salmonidae)

Cytogenetic analysis of brook trout performed with molecular and conventional methods led to identification of interstitial telomeric sites on one or two subtelocentric chromosomes within the same pair. Morphology and specific patterns of these chromosomes using fluorochromes associated with A/T- or G/C-rich DNA proved that these chromosomes are not sex related. The chromomycin-positive region was located on the short arms of the ITS bearing chromosome pair and flanked by telomeric sequences, suggesting that this part of the chromosome had been translocated from another one. Our observations confirm that GC-rich regions are highly mobile genetic structures, and led to ITS formation on brook trout chromosomes.     

Detection of interstitial telomeric sequences in the Arctic charr (Salvelinus alpinus) (Teleostei: Salmonidae)

Highly polymorphic Arctic charr ( Salvelinus alpinus Linnaeus, 1758) chromosomes were studied using conventional and molecular methods. The diploid chromosome number in the studied individuals was 2n = 81 or 2n = 82, with a fundamental arm number (NF) = 100. These differences are due to Robertsonian fusions. Interindividual variation in the number and size of DAPI and CMA(3) positively stained chromatin sites was observed in studied specimens. In the case of two individuals, the subtelomeric region of the long arm (q) of the largest acrocentric chromosome (chromosome number 10) was positively stained by CMA(3) fluorochrome. Both primed in situ labelling (PRINS) and fluorescence in situ hybridization (FISH) revealed that this CMA(3)-positive region was flanked by telomeric sequences. Previously, the subterminal position of interstitial telomeric sequences located in the vicinity of the CMA(3)-positive guanine-rich chromatin have been described in two other Salvelinus species, brook trout ( Salvelinus fontinalis ) and lake trout ( Salvelinus namaycush ). Moreover, multichromosomal location and variation in size of CMA(3) bands have been observed in various Salvelinus taxa, including fishes with internally located telomeric sequences. These results suggest that relocation of CMA(3)-positive chromatin segments in these species may be facilitated by flanking interstitial telomeric sequences (ITSs).     

Chromosome rearrangements and survival of androgenetic rainbow trout (Oncorhynchus mykiss)

The purpose of this work was to quantify the impact of spontaneous and X-radiation-induced chromosome rearrangements on survival rate of androgenetic rainbow trout (Oncorhynchus mykiss). Various doses of X irradiation (50, 150, 250, 350 Gy) were used for inactivation of nuclear DNA in oocytes. After the irradiation, eggs were inseminated with normal sperm from 4 males derived from a strain characterized by Robertsonian rearrangements and length polymorphism of the Y chromosome. The haploid zygotes were exposed to a high hydrostatic pressure (7000 psi) to duplicate the paternal DNA. Neither Robertsonian chromosome polymorphism nor the Y chromosome morphology impaired the viability of the androgenetic embryos and alevins. Moreover, survival of eyed embryos of the androgenetic rainbow trout increased significantly with increasing doses of oocyte X irradiation. After 6 months of rearing, only specimens from the 250 and 350 Gy variants survived. The number of fingerlings with remnants of the maternal genome in the forms of chromosome fragments was higher in the 250 Gy group. Intraindividual variation of chromosome fragment number was observed, and some individuals exhibited haploid/diploid mosaicism and body malformations. Individuals irradiated with less than 250 Gy died, presumably because of the conflict between intact paternally derived chromosomes and the residues of maternal genome in the form of chromosome fragments.     

Cytogenetic identification of genomic elements of brook trout (Salvelinus fontinalis) in the karyotype of Arctic char (Salvelinus alpinus) from the aquaculture broodstock

Growing interest of Arctic char (Salvelinus alpinus) aquaculture in Europe, and the fact that it can easily hybridize with brook trout (Salvelinus fontinalis) resulting in fertile progeny, led us to investigate fish from the farmed stocks. Chromosomes of sampled Arctic char were examined using conventional and molecular cytogenetic (FISH) techniques in order to determine possible contamination of genomic elements of brook trout. Investigated fish possessed karyotypes composed of 80–82 chromosomes and up to three chromosome fragments. Using staining methods and FISH approach enabled identification of the brook trout chromosomes in the eight out of twenty‐two examined Arctic char. Specific location of AT‐, GC‐ positive and NOR sites observed on chromosomes as well as chromosome fragments in the karyotypes of several individuals points on past chromosomal rearrangements in fish from examined broodstock. Based on our results, it may be assumed that individuals with the brook trout genomic elements, although phenotypically identified as Arctic chars, were hybrids. Our results highlights that special care should be taken to protect gene pools of brook trout and Arctic char in farms where both species are cultured.     

Cytological and molecular analysis of centromere misdivision in maize.

B chromosome derivatives suffering from breaks within their centromere were examined cytologically and molecularly. We showed by high resolution FISH that misdivision of the centromere of a univalent chromosome can occur during meiosis. The breaks divide the centromere repeat sequence cluster. A telocentric chromosome formed by misdivision was found to have the addition of telomeric repeats to the broken centromere. A ring chromosome formed after misdivision occurred by fusion of the broken centromere to the telomere. Pulsed-field electrophoresis analyses were performed on the telocentric and ring chromosomes to identify fragments that hybridize to both the telomeric repeat and the B-specific centromeric repeat. We conclude that healing of broken maize centromeres can be achieved through the mechanisms of addition or fusion of telomeric repeat sequences to the broken centromere.     

Construction of midget chromosomes in wheat.

To test the usefulness of breakage-fusion-bridge (BFB) cycles in generating new chromosome aberrations in bread wheat (Triticum aestivum L.) and to extend the range of aberrations available, a series of midget chromosomes was produced from the long arm of chromosome 1B. Using a reverse tandem duplication initiated chromatid type BFB cycle, the 1BL arm was broken and fused with centromeres of either chromosome 5BL or 1RS to form dicentric chromosomes. The 1R and 5B centromeres were broken by centric misdivision. Among the progenies of plants with dicentric chromosomes, two classes of monocentric chromosomes were selected: deficient chromosomes 1B and chromosomes that had 1RS or 5BL for one arm and various fragments of 1BL for the other arm. Following centric misdivision of these monocentrics, midget chromosomes 1BL were isolated: deficient and deletion telocentrics and telocentrics derived from interstitial regions of 1BL. By chance, one deficient chromosome 1BS and one deletion chromosome 1BS were identified in unrelated lines of the same wheat. Following centric misdivision of these chromosomes, two midget chromosomes covering the whole of 1BS were added to the set.     

Analysis of radiation-induced micronuclei by fluorescence in situ hybridization (FISH) simultaneously using telomeric and centromeric DNA probes.

Fluorescence in situ hybridization using simultaneously a combination of DNA probes for the telomeric hexamer repeat (TTAGGG) and the centromerically repeated murine gamma-satellite DNA was applied to analyze the nature of radiation-induced micronuclei in mouse NIH 3T3 fibroblasts. After subtraction of spontaneously occurring micronuclei independent from the dose and time after irradiation, approximately 22% of the radiation-induced micronuclei did not reveal any hybridization signal. Approximately 17% showed one centromeric hybridization signal and about four telomeric signals, suggesting their origin from whole chromosomes. Almost 60% of radiation-induced micronuclei had telomeric signals only, suggesting their origin from acentric fragments. A fraction of micronuclei were found to contain two or more acentric fragments. Micronuclei derived from whole chromosomes or from multiple acentric fragments might, together with DNA synthesis in micronuclei, explain the occurrence of radiation-induced micronuclei with DNA contents greater than the largest chromosome arm.     

Cytogenetic analyses of a Salvelinus hybrid reveal an evolutionary relationship between the parental species

Mitotic analyses of the brook trout (Salvelinus fontinalis) x arctic char (S. alpinus) hybrids (sparctic trout) revealed a mode of 2n = 82 with 18 metacentric and 64 acrocentric chromosomes. The brook trout had 2n = 84 with 16 metacentric chromosomes and the arctic char had 2n = 80 with 20 metacentric chromosomes; both species are derivatives of a single tetraploid event. Variable multivalent-like configurations that may be centromeric associations of bivalents were observed in C-banded pachytene figures of female sparctic trout. Metaphase I analyses of sparctic trout males indicated that two fusions of nonhomologous acrocentric chromosomes representing two duplicated chromosome sets must have occurred in the arctic char after its evolutionary divergence from the brook trout. A mode of seven tetravalent rods per cell suggests that preferential multivalent pairing occurs in the sparctic hybrid; metaphase I analyses of S. alpinus males revealed a mode of only five tetravalent rods per cell. The presence of multivalents implies that the arctic char, like the brook trout, is still undergoing diploidization. Cytochemical detection of the nucleolar organizer region (NOR) revealed intra- and interspecific as well as intraindividual variability in the numbers and types of chromosomes (metacentric or acrocentric) on which NORs appeared in arctic char and sparctic trout. Brook trout only had NORs on acrocentric chromosomes. This may indicate that different chromosomal fusions occurred in the evolution of brook trout from arctic char.     

Silver-banded karyotypes of the rainbow trout and the brook trout and their hybrids: disappearance in allotriploids of Ag-NORs originated from the brook trout

Silver-banded karyotypes of the rainbow trout Salmo gairdneri, the brook trout Salvelinus fontinalis, and their hybrids were described. Diploid-type and triploid-type hybrids were obtained. Triploid-type hybrids had two maternal genomes of the rainbow trout and one paternal genome of the brook trout. The rainbow trout had one pair of M or SM with Ag-NORs near the satellite. In the brook trout, Ag-NORs were observed in four pairs of chromosomes, but each cell had different number of chromosomes with Ag-NORs. In all observed cells of triploid-type hybrids, Ag-NORs originated from the brook trout were not recognized, whereas those from the rainbow trout were found.     

Rye chromosome translocations in hexaploid wheat: a re-evaluation of the loss of heterochromatin from rye chromosomes

Summary Using in situ hybridization techniques, we have been able to identify the translocated chromosomes resulting from whole arm interchanges between homoeologous chromosomes of wheat and rye. This was possible because radioactive probes are available which recognize specific sites of highly repeated sequence DNA in either rye or wheat chromosomes. The translocated chromosomes analysed in detail were found in plants from a breeding programme designed to substitute chromosome 2R of rye into commercial wheat cultivars. The distribution of rye highly repeated DNA sequences showed modified chromosomes in which (a) most of the telomeric heterochromatin of the short arm and (b) all of the telomeric heterochromatin of the long arm, had disappeared. Subsequent analyses of these chromosomes assaying for wheat highly repeated DNA sequences showed that in type (a), the entire short arm of 2R had been replaced by the short arm of wheat chromosome 2B and in (b), the long arm of 2R had been replaced by the long arm of 2B. The use of these probes has also allowed us to show that rye heterochromatin has little effect on the pairing of the translocated wheat arm to its wheat homologue during meiosis. We have also characterized the chromosomes resulting from a 1B-1R translocation event.From these results, we suggest that the observed loss of telomeric heterochromatin from rye chromosomes in wheat is commonly due to wheat-rye chromosome translocations.     

Chromosomal localization of the 18S-28S and 5S rRNA genes and (TTAGGG)n sequences of butterfly lizards (Leiolepis belliana belliana and Leiolepis boehmei, Agamidae, Squamata)

Chromosomal mapping of the butterfly lizards Leiolepis belliana belliana and L. boehmei was done using the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences. The karyotype of L. b. belliana was 2n = 36, whereas that of L. boehmei was 2n = 34. The 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, while the 5S rRNA genes were found in the pericentromeric region of chromosome 6 in both species. Hybridization signals for the (TTAGGG)n sequence were observed at the telomeric ends of all chromosomes, as well as interstitially at the same position as the 18S-28S rRNA genes in L. boehmei. This finding suggests that in L. boehmei telomere-to-telomere fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located or, alternatively, at the secondary constriction of chromosome 1. The absence of telomeric sequence signals in chromosome 1 of L. b. belliana suggested that its chromosomes may have only a few copies of the (TTAGGG)n sequence or that there may have been a gradual loss of the repeat sequences during chromosomal evolution.     

Telomeric signals in robertsonian fusion and fission chromosomes: implications for the origin of pseudoaneuploidy.

In situ hybridization with synthetic plant telomeric sequences resulted in labeling of all broad bean (Vicia faba) chromosomes at their ends only. Telocentric chromosomes derived by fission of the metacentric satellite chromosome of V. faba also showed signals at both of their ends, whereas the ancestral metacentric did not display signals at its primary constriction, the point of fission. As in V. faba, all acrocentric mouse chromosomes were labeled by in situ hybridization with a vertebrate telomeric probe at both ends of each chromatid exclusively. However, different metacentric Robertsonian chromosomes derived by fusion of defined acrocentrics did not show signals at their primary constrictions. The mechanism of Robertsonian rearrangement leading to a pseudoaneuploid increase or decrease in chromosome number therefore cannot consist solely of a simple fission or fusion of chromosomes without a concomitant gain or loss of chromatin material. The additional assumption of a subdetectable deletion of telomeric sequences after fusion and amplification of these sequences following fission is necessary to explain the present observations.     

Telomeric repeat organization--a comparative in situ study between man and rodent

Human, hamster, and mouse chromosomes show both similarities and differences in telomeric organization, detectable with a novel version of the PRINS technique. The differences allowed us to investigate the fate of the telomeres on a chromosome from one species when this chromosome was introduced into the cells of another species. For this purpose, we tested telomeres in cell lines of somatic cell hybrids containing human chromosomes on a rodent background, finding that the telomeres on human chromosomes could not be discriminated from the telomeres on rodent chromosomes. All telomeres in the cell lines were much shorter than the telomeres in normal cells. In the mouse-derived cell lines, half of the mouse chromosomes were fused to other mouse chromosomes at the ends of their short arms. At the points of fusion we were generally unable to detect telomeric signals. In these cell lines, we also found a fraction of chromosomes ends with only one telomeric signal. In chromosomes where both ends showed only one signal, the relative orientation of the signals appeared to be nonrandom with respect to sister chromatids.     

Chromosomal localization of telomeric sequences in three species of Akodon (Rodentia, Sigmodontinae)

The distribution of the vertebrate telomeric sequence T2AG3 in three species of the rodent genus Akodon was examined by FISH with a peptide nucleic acid probe. In addition to the expected telomeric hybridization, non-telomeric signals were observed in the three species. In A. dolores, centromeric signals were visible in two of the four biarmed autosome pairs featuring Robertsonian polymorphism, indicating the retention of at least part of the telomeric sequences during the fusion process, and an interstitial signal of lower intensity was observed in the short arm of another. In A. boliviensis, a strong signal was observed near the centromeric end of the first chromosome pair. The first pair of A. azarae (homologous to the first pair of A. boliviensis) showed a similar but markedly amplified signal, and a subcentromeric signal in the X chromosome corresponding to a heterochromatic region; additionally, interstitial signals of lower intensity were present in one to four chromosomes in the majority of cells examined.     

Molecular cytogenetic characterization of telomere-specific repetitive DNA sequences in Rana rugosa

We performed a molecular cloning of the glutamic oxaloacetic transaminase (GOT1) gene from R. rugosa, and determined its chromosomal location. This gene was reportedly localized near the sex-determining region of the ZW sex chromosomes in the frog Buergeria buergeri; however, the GOT1 gene was mapped to the distal end of chromosome 9 in R. rugosa using a GOT1 cDNA FISH probe. This was also the case when a 46.3?kb genomic clone containing exon 8 and 9 and the 3'-neighboring region of the GOT1 gene, designated clone B, was used as probe. However, weak signals were also detected at the telomeric ends of other autosomes and the Z sex chromosome, and near the centromeric region of the W sex chromosome. To intensify the signals, we used eight internal fragments in clone B and applied them to chromosome mapping. Consequently, only two fragments containing repeated sequence blocks produced hybridization signals; those signals were observed on autosomes and ZW sex chromosomes. The 3'-neighboring region contained two types of repeated sequence elements: a 41?bp element, designated 41-REL, localized to telomeric ends of autosomes and a 31?bp element, designated 31-REL, localized to telomeric ends of all autosomes and the ZW sex chromosomes, and also near the centromere on the W long arm. The results collectively suggest that the two repeated sequence elements were independently amplified around the chromosomal telomeres in R. rugosa, indicating that they will be useful cytogenetic markers for studying karyotypic evolution-especially the W chromosome differentiation-in this species.     

The sockeye salmon neo-Y chromosome is a fusion between linkage groups orthologous to the coho Y chromosome and the long arm of rainbow trout chromosome 2

Unlike other Pacific salmon, sockeye salmon (Oncorhynchus nerka) have an X(1)X(2)Y sex chromosome system, with females having a diploid chromosome number of 2n = 58 and males 2n = 57 in all populations examined. To determine the origin of the sockeye Y chromosome, we mapped microsatellite loci from the rainbow trout (O. mykiss; OMY) genetic map, including those found on the Y chromosomes of related species, in kokanee (i.e. non-anadromous sockeye) crosses. Results showed that 3 microsatellite loci from the long arm of rainbow trout chromosome 8 (OMY8q), linked to SEX (the sex-determining locus) in coho salmon (O. kisutch), are also closely linked to SEX in the kokanee crosses. We also found that 3 microsatellite loci from OMY2q are linked to those markers from OMY8q and SEX in kokanee, with both linkage groups fused to form the neo-Y. These results were confirmed by physical mapping of BAC clones containing microsatellite loci from OMY8q and OMY2q to kokanee chromosomes using fluorescence in situ hybridization. The fusion of OMY2q to the ancestral Y may have resolved sexual conflict and, in turn, may have played a large role in the divergence of sockeye from a shared ancestor with coho.     

Identification of sex chromosomes in lake trout (Salvelinus namaycush)

In the male trout there is a difference in the quinacrine banding and C-banding patterns between the two homologs of the second largest chromosome pair. This chromosome is the only large submetacentric in the karyotype, making it easy to identify and suggesting that the sex chromosomes have become differentiated since the time of tetraploidization. In males one homolog has a medium-to-large quinacrine bright heterochromatic band on the end of the short arm, while the other lacks it completely. In females both homologs have medium-to-large quinacrine bright heterochromatic bands. Approximately half the progeny from every lake trout cross studied and half the eggs from every lake trout population examined were heteromorphic for a difference in this chromosome band. Results from sexed fish, reciprocal F1 hybrids between brook trout and lake trout, and gynogenetic haploids are all consistent with the interpretation that chromosome 2 is the sex chromosome. These results suggest that the addition of heterochromatin to the X can be the first step in the inhibition of crossing over between the X and Y chromosomes required for sex chromosome differentiation.     

Failure of interspecies androgenesis in salmonids

Androgenetic development of salmonid embryos was induced in recipient oocytes from the same or other species (intra- or interspecies androgenesis). Parameters for induced androgenesis were investigated in brown trout Salmo trutta and brook trout Salvelinus fontinalis . Reciprocal androgenetic and control crosses were conducted among fishes from three genera: Oncorhynchus (rainbow trout, O. mykiss ), Salmo (brown trout) and Salvelinus (brook trout), and within two genera: Salmo (brown trout and Atlantic salmon, S. salar ) and Salvelinus (brook trout and Arctic charr, S. alpinus ). Live hatched androgenetic progenies were obtained in all intraspecies variants, where oocytes and sperm originated from the same species. Interspecies androgenesis resulted in no viable larvae, despite the fact that most hybrid controls and intraspecies androgenetic individuals were viable. When recipient oocytes originated from other genera (interspecific intergeneric androgenesis), embryonic development ceased in early developmental stages, except for haploid controls of brook trout produced in eggs of brown trout. Survival of embryos to the eyed-egg stage was relatively high in the intrageneric androgenesis experiment. Nevertheless, none of these embryos survived to hatching. Some of the presumed Atlantic salmon individuals developing in brown trout eggs contained maternal DNA, questioning the accuracy of enucleation using irradiation. The inability to induce interspecific androgenesis among the examined salmonid species may have been the result of substantial kariotypical and developmental differences between spermatozoal donors and oocyte recipients, causing an incompatibility between maternal cytoplasmic regulatory factors and the paternal nuclear genome.     

Nucleolar organizing regions of normal Chinese hamster and CHW cell line chromosomes

The chromosome preparations from fibroblasts of normal male and female Chinese hamsters and the cell line CHW were stained with AgNO3. The silver stain was usually localized at the telomeres of autosomes. The marker chromosome M1 in the CHW cell line has Ag-NOR near the centre of the long arm, which indicates that either the long arms of two number 5 chromosomes fused at the telomeres or the intact telomeric region of one chromosome fused with one with a deleted telomere. The variation of Ag-NORs' number per cell and Ag-heteromorphism in chromosome number 4 were observed. The Ag-NORs of chromosome number 4 and 5 are in approximately the same position as the positive C-bands and these may play a role in the preservation of heterochromatin.     

Occurrence and evolution of homogeneously staining regions may be due to breakage-fusion-bridge cycles following telomere loss

Chromosomes with homogeneously staining regions (HSR) were analysed in a subclone of the H4 rat hepatoma cell line, where they represent amplification of the ribosomal RNA (rRNA) genes. Detailed G-band analysis of the subclone revealed that an HSR on the short arm of chromosome 3 became unstable and changed its position within the chromosome. The evolution of this marker chromosome was associated with the terminal deletion of the normal long arm of the HSR-bearing chromosome 3 and may have involved ring formation as a result of fusion between the HSR on the short arm and the broken end of the long arm. Evidence was obtained for breakage at different sites within the ring, producing chromosomes with HSRs located terminally on either the long arms or both arms. The terminally located HSR underwent elongation in some cells presumably as a result of a breakage-fusion-bridge cycle characteristic of instability due to telomeric loss. It is suggested that terminally located HSRs may generally occur this way.