Induced reciprocal translocation in transgenic mice near sites of transgene integration

Transgenic mice (JCP₀ #18), heterozygous for an insertion of approximately 50 copies of the rat peripheral myelin (P₀) protein cDNA, displayed a pattern of reduced litter size that suggested a chromosome rearrangement. Chromosome banding studies of fetal cells disclosed the presence of an apparently balanced translocation between a Chromosome (Chr) 1 and 14 with breakpoints at bands 1H3 and 14C3. In situ hybridization of biotin-labeled P₀ rat cDNA probe to chromosome spreads and detection of specific signal with fluorescein isothiocyanate-conjugated avidin revealed a strong signal on the 1¹⁴ translocation chromosome at the site of the breakpoint. A weaker signal was present near the breakpoint on the 14¹ derivative chromosome. These results suggest an etiologic relationship between the insertion of the transgene and the origin of the translocation. To further elucidate possible mechanisms, we first mapped the endogenous P₀ gene (gene symbol Mpp). As previously reported (You et al., Genomics 9: 751, 1991), we found that Mpp is located on Chr 1 in the region of the translocation breakpoint in JCP₀ mice. Subsequently, we have carried out pulsed-field gel and standard Southern analyses with P₀ gene probes, but found no evidence for a direct involvement of the endogenous P₀ gene in the process that generated the balanced reciprocal translocation. Thus, we favor the hypothesis that, during repair of DNA strand breakage—possibly induced by the microinjection procedure—the transgene copies were ligated to broken ends of Chrs 1 and 14. According to convention, this translocation is designated T(1;14) 1Po. Homozygotes are phenotypically normal and breed well; they will be useful for genetic and physical mapping of Chrs 1 and 14.     

Ts65Dn -- localization of the translocation breakpoint and trisomic gene content in a mouse model for Down syndrome

Fluorescent in situ hybridization (FISH) -- using mouse chromosome paints, probes for the mouse major centromeric satellite DNA, and probes for genes on chromosomes (Chr) 16 and 17 -- was employed to locate the breakpoint in a translocation used to produce a mouse model for Down syndrome. The Ts65Dn trisomy is derived from the reciprocal translocation T(16;17)65Dn. The Ts65Dn mouse carries a marker chromosome containing the distal segment of Chr 16, a region that shows linkage conservation with human Chr 21, and the proximal end of Chr 17. This chromosome confers trisomy for most of the genes in the Chr 16 segment and Ts65Dn mice show many of the phenotypic features characteristic of Down syndrome. We used FISH on metaphase chromosomes from translocation T65Dn/+ heterozygotes and Ts65Dn mice to show that the Chr 17 breakpoint is distal to the heterochromatin of Chr 17, that the Ts65Dn marker chromosome contains a small portion of Chr 17 euchromatin, that the Chr 16 breakpoint lies between the Ncam2 and Gabpa/App genes, and that the Ts65Dn chromosome contains >80% of the human Chr 21 homologs. The significance of this finding is discussed in terms of the utility of this mouse model.     

Nucleolus formation in structurally reconstructed barley karyotypes with six satellite chromosomes

Two structurally reconstructed karyotypes of Hordeum vulgare which, due to appropriate reciprocal translocations, contain three chromosome pairs with nucleolus organizing activity (chromosomes 5⁷, 6 and 7⁷ in translocation line T 21, chromosomes 3⁶, 6³, and 7 in translocation line T 627) have been studied with respect to the position and function of rDNA as inferrred from the pattern of nucleolus formation. The results obtained reveal quantitative relationships between the size of the secondary constriction (the amount of rRNA cistrons) and the number and size of nucleoli being formed. Quantitative and qualitative aspects of rDNA location and expression are being discussed.     

Light microscope autoradiography of ribosomal DNA amplification in Xenopus laevis

A technique for the isolation of very high molecular weight rDNA¹ from the ovary of Xenopus laevis is described. Tritiated rDNA was prepared by this method from ovaries at the amplification stage, and spread on slides for light microscope autoradiography. The average molecular weight of the spread DNA was greater than 180×10⁶ daltons. Unlike chromosomal DNA grain tracks, rDNA tracks after 2 or 4 hours of labelling were not tandemly arranged. By allowing ovaries to equilibrate gradually with exogenous precursors, tracks showing a single gradient of grain density were produced, indicating that replication was proceeding in one direction at these sites. Bidirectional initiations, if they occur at all during amplification, are rare. The rate of rDNA chain growth is 10.5 μ/hour at 23° C, which is the same as the rate for chromosomal DNA synthesis in X. laevis. After 24 hours some tracks are over 200 μ long, suggesting that replication at a site may be continuous for at least this period. Although they do not distinguish between several alternative mechanisms, the results are compatible with a rolling circle mechanism for gene amplification.     

Cytogenetics for the model system Arabidopsis thaliana

A detailed karyotype of Arabidopsis thaliana is presented using meiotic pachytene cells in combination with fluorescence in situ hybridization. The lengths of the five pachytene bivalents varied between 50 and 80 μm, which is 20–25 times longer than mitotic metaphase chromosomes. The analysis confirms that the two longest chromosomes (1 and 5) are metacentric and the two shortest chromosomes (2 and 4) are acrocentric and carry NORs subterminally in their short arms, while chromosome 3 is submetacentric and medium sized. Detailed mapping of the centromere position further revealed that the length variation between the pachytene bivalents comes from the short arms. Individual chromosomes were unambiguously identified by their combinations of relative lengths, arm-ratios, presence of NOR knobs and FISH signals with a 5S rDNA probe and chromosome specific DNA probes. Polymorphisms were found among six ecotypes with respect to the number and map positions of 5S rDNA loci. All ecotypes contain 5S rDNA in the short arms of chromosomes 4 and 5. Three different patterns were observed regarding the presence and position of a 5S rDNA locus on chromosome 3. Repetitive DNA clones enabled us to subdivide the pericentromeric heterochromatin into a central domain, characterized by pAL1 and 106B repeats, which accommodate the functional centromere and two flanking domains, characterized by the 17 A20 repeat sequences. The upper flanking domains of chromosomes 4 and 5, and in some ecotypes also chromosome 3, contain a 5S rDNA locus. The detection of unique cosmids and YAC sequences demonstrates that detailed physical mapping of Arabidopsis chromosomes by cytogenetic techniques is feasible. Together with the presented karyotype this makes Arabidopsis a model system for detailed cytogenetic mapping.     

Localization of rDNA in the chimpanzee (Pan troglodytes) chromosome complement

In situ hybridization was used to identify the sites of rDNA in the chromosome complement of the chimpanzee (Pan troglodytes). The rDNA was present in the satellite regions of chimpanzee chromosomes 14, 15, 17, 22 and 23. Four of these (14, 15, 22, 23) are homologous to human chromosomes carrying rDNA: 13, 14, 21 and 22.     

The genes for ribosomal RNA in diploid and polytene chromosomes of Drosophila melanogaster

The proportion of the Drosophila genome coding for ribosomal RNA was examined in DNA from both diploid and polytene tissues of Drosophila melanogaster by rRNA-DNA hybridization. Measurements were made on larvae with one, two, three and four nucleolus organizer regions per genome. In DNA from diploid tissues the percent rDNA (coding for 28S and 18S ribosomal DNA) was found to be in proportion to the number of nucleolus organizers present. The number of rRNA genes within a nucleolus organizer therefore does not vary in response to changes in the number of nucleolus organizers. On the other hand, in DNA from cells with polytene chromosomes the percent rDNA remained at a level of about 0.1% (two to six times lower than the diploid values), regardless of either the number of nucleolus organizers per genome or whether the nucleolus organizers were carried by the X or Y chromosomes. This independence of polytene rDNA content from the number of nucleolus organizers is presumably due to the autonomous polytenization of this region of the chromosome. When the rDNA content of DNA from whole flies is examined, both the rDNA additivity of the diploid cells and the rDNA independence of polytene cells will affect the results. This is a possible explanation for the relative rDNA increase known to occur in X0 flies, but probably not for the phenomenon of rDNA magnification. — In further studies on DNA from larval diploid tissues, the following findings were made: 1) the Ybb-chromosome carries no rDNA; 2) flies carrying four nucleolus organizers do not tend to lose rDNA, even after eleven generations, and 3) the nucleolus organizer on the wild type Y chromosome may have significantly less rDNA than does that on the corresponding X chromosome.     

Mammalian Ino80 mediates double-strand break repair through its role in DNA end strand resection

Chromatin modifications/remodeling are important mechanisms by which cells regulate various functions through providing accessibility to chromatin DNA. Recent studies implicated INO80, a conserved chromatin-remodeling complex, in the process of DNA repair. However, the precise underlying mechanism by which this complex mediates repair in mammalian cells remains enigmatic. Here, we studied the effect of silencing of the Ino80 subunit of the complex on double-strand break repair in mammalian cells. Comet assay and homologous recombination repair reporter system analyses indicated that Ino80 is required for efficient double-strand break repair. Ino80 association with chromatin surrounding double-strand breaks suggested the direct involvement of INO80 in the repair process. Ino80 depletion impaired focal recruitment of 53BP1 but did not impede Rad51 focus formation, suggesting that Ino80 is required for the early steps of repair. Further analysis by using bromodeoxyuridine (BrdU)-labeled single-stranded DNA and replication protein A (RPA) immunofluorescent staining showed that INO80 mediates 5'-3' resection of double-strand break ends.     

DNA variants with telomere probe enable genetic mapping of ends of mouse chromosomes

Dde I-digested DNA fragments from 11 inbred mouse strains were separated by electrophoresis, blotted and probed with a labeled oligomer, TELO, containing five repeats of the consensus mammalian telomere sequence, TTAGGG. Each strain produced a unique set of hybridizing fragments. Segregation analysis of TELO-hybridizing fragments from the BXD RI strains indicated that each fragment segregated as expected for a single gene. One fragment from strain DBA/2J was genetically linked to locusXmv-9, previously mapped near the distal end of the map of chromosome (Chr) 4 and three fragments toCck, near the distal end of Chr 9, suggesting that these fragments are telomeric and represent the ends of the chromosome maps. Confirmation of these map positions was obtained from a backcross. Fragments associated with the short arm of the Y Chr were found in DNA from strains C57BL/6J and DBA/2J. TELO-hybridizing fragments from DBA/2J were digested by the exonuclease Bal 31, under conditions in which fragments hybridizing to a cDNA probe for themetallothioneine locus, located at the middle of mouse Chr 8, remained intact. Thus both biochemical and genetic tests indicate that several TELO-hybridizing fragments fromDde I-digested DNA are at the ends of chromosomes and probably derive from mouse telomeres. Using this approach should allow the mapping of genes relative to the ends of other mouse chromosomes.     

Telomeric Repeats Facilitate CENP-ACnp1 Incorporation via Telomere Binding Proteins

The histone H3 variant, CENP-A, is normally assembled upon canonical centromeric sequences, but there is no apparent obligate coupling of sequence and assembly, suggesting that centromere location can be epigenetically determined. To explore the tolerances and constraints on CENP-A deposition we investigated whether certain locations are favoured when additional CENP-A^Cnp1 is present in fission yeast cells. Our analyses show that additional CENP-A^Cnp1 accumulates within and close to heterochromatic centromeric outer repeats, and over regions adjacent to rDNA and telomeres. The use of minichromosome derivatives with unique DNA sequences internal to chromosome ends shows that telomeres are sufficient to direct CENP-A^Cnp1 deposition. However, chromosome ends are not required as CENP-A^Cnp1 deposition also occurs at telomere repeats inserted at an internal locus and correlates with the presence of H3K9 methylation near these repeats. The Ccq1 protein, which is known to bind telomere repeats and recruit telomerase, was found to be required to induce H3K9 methylation and thus promote the incorporation of CENP-A^Cnp1 near telomere repeats. These analyses demonstrate that at non-centromeric chromosomal locations the presence of heterochromatin influences the sites at which CENP-A is incorporated into chromatin and, thus, potentially the location of centromeres.     

Visualization by atomic force microscopy and FISH of the 45S rDNA gaps in mitotic chromosomes of Lolium perenne

The mitotic chromosome structure of 45S rDNA site gaps in Lolium perenne was studied by atomic force microscope (AFM) combining with fluorescence in situ hybridization (FISH) analysis in the present study. FISH on the mitotic chromosomes showed that 45S rDNA gaps were completely broken or local despiralizations of the chromatid which had the appearance of one or a few thin DNA fiber threads. Topography imaging using AFM confirmed these observations. In addition, AFM imaging showed that the broken end of the chromosome fragment lacking the 45S rDNA was sharper, suggesting high condensation. In contrast, the broken ends containing the 45S rDNA or thin 45S rDNA fibers exhibited lower density and were uncompacted. Higher magnification visualization by AFM of the terminals of decondensed 45S rDNA chromatin indicated that both ends containing the 45S rDNA also exhibited lower density zones. The measured height of a decondensed 45S rDNA chromatin as obtained from the AFM image was about 55–65 nm, composed of just two 30-nm single fibers of chromatin. FISH in flow-sorted G2 interphase nuclei showed that 45S rDNA was highly decondensed in more than 90% of the G2/M nuclei. Our results suggested that a failure of the complex folding of the chromatin fibers occurred at 45S rDNA sites, resulting in gap formation or break.     

Variation in size and location of the Ag-NOR in the Atlantic halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

The distribution of differentially stained chromatin was studied in the Atlantic halibut (Hippoglossus hippoglossus) chromosomes (2n = 48). Four pairs of homologous chromosomes were identified using a combination of traditional cytogenetic staining techniques (Giemsa/DAPI/CMA3/Ag-NO3). Chromosome 1 showed a length polymorphism (1^S-short, 1^L-long isoforms of the chromosome 1) which was related to the variation of the size of the Ag-NORs. In one specimen the Ag-NOR was translocated from chromosome 1 into the telomeric region on the q-arm of the chromosome 2 forming a derivative chromosome der(2)t(1^S;2)(q?;q?). Four Ag-NOR genotypes have been shown: 1^S1^S, 1^S1^L, 1^L1^L and 1^S der(2)t(1^S;2)(q?;q?). The chromosome rearrangements did not leave any interstitially located telomeric sequences and the telomeres were confined to the ends of the chromosomes. A single chromosomal location of 5S rDNA clusters was found using the PRINS technique. In the extended metaphase spreads two adjacent clusters of 5S rDNA could be seen on one chromosome while condensed chromatin gave a single hybridization signal. Double 5S rDNA signals on the same chromosome arm suggested paracentric inversion of the minor rDNA site. 5S rDNA clusters were not co-localized with Ag-NORs. Although female and male karyotypes were compared no sex related cytogenetic markers were found.     

The chromosomal location of ribosomal DNA in the mouse

In situ hybridization of I¹²⁵-labelled ribosomal RNA to mouse chromosomes was used to determine the location of the rDNA loci. The results demonstrate the presence of rDNA sites on chromosomes 15, 18 and 19.     

Ribosomal DNA Is a Site of Chromosome Breakage in Aneuploid Strains of Neurospora

In wild-type strains of Neurospora crassa, the rDNA is located at a single site in the genome called the nucleolus organizer region (NOR), which forms a terminal segment on linkage group (LG) V. In the quasiterminal translocation strain T(I;V)AR190, most of the right arm of LG I moved to the distal tip of the NOR, and one or a few rDNA repeat units are moved to the truncated right arm of LG I. I report here that, in partial diploid strains derived from T(I;V)AR190, large terminal deletions result from chromosome breakage in the NOR. In most of these partial diploids, chromosome breakage is apparently frequent and the breakpoints occur in many parts of the NOR. The rDNA ends resulting from chromosome breakage are "healed" by the addition of new telomeres. Significantly, the presence of ectopic rDNA creates a new site of chromosome breakage in the genome of partial diploids. These results raise the possibility that, under certain conditions, rDNA is a region of fragility in eukaryotic chromosomes.     

Classical and molecular cytogenetics and DNA content in Maihuenia and Pereskia (Cactaceae)

We studied cacti species of the subfamilies Pereskioideae (five species of the southern clade) and both species of Maihuenioideae using molecular cytogenetic techniques and DNA content. Mitotic chromosomes were analyzed for Pereskia aculeata, P. bahiensis, P. grandifolia, P. nemorosa, P. sacharosa, Maihuenia poeppigii, and M. patagonica, using the Feulgen stain, CMA/DAPI fluorescent chromosome banding, fluorescence in situ hybridization (FISH, probes of 5S rDNA and pTa71 for 18-5.8-26S rDNA), and DNA content by flow cytometry technique. The karyotypes were highly symmetrical, most of the pairs being metacentric (m). CMA/DAPI banding revealed the presence of CMA⁺/DAPI^? bands associated with NORs in the first m pair of all species. The co-localization of 18-5.8-26S rDNA loci with CMA⁺/DAPI^?/NORs blocks allowed the identification of homeologous chromosome pairs between species of both subfamilies. FISH using probe 5S rDNA was applied for the first time in both subfamilies. Diploid species had always one m pair carrying 5S rDNA genes, with pericentromeric location in different chromosome pairs. In the tetraploid cytotype of M. patagonica, the 5S rDNA probe hybridized to two pairs. The 2C DNA content obtained by FC varied twofold (from 1.85 to 2.52 pg), with significant differences between species. Mean chromosome length, karyotype formula, percentage of heterochromatin position of 5S rDNA locus, and nuclear Cx DNA content vary among Maihuenia and Pereskia species and allowed to differentiate them. Both genera are closely related and that the differences found are not strong enough to separate Maihuenioideae from Pereskioideae.     

Localization of rDNA in the chromosome complement of the Rhesus (Macaca mulatta)

In situ hybridization was used to identify the region of the Rhesus genome complementary to ribosomal RNA. The rDNA sites have been assigned to the achromatic region of chromosome 20.     

Cytogenetic characterization and mapping of rDNAs,core histone genes and telomeric sequences in <Emphasis Type="Italic">Venerupis aurea</Emphasis> and <Emphasis Type="Italic">Tapes rhomboides</Emphasis> (Bivalvia: Veneridae)

We describe the chromosomal location of GC-rich regions, 28S and 5S rDNA, core histone genes, and telomeric sequences in the veneroid bivalve species Venerupis aurea and Tapes (Venerupis) rhomboides, using fluorochrome staining with propidium iodide, DAPI and chromomycin A3 (CMA) and fluorescent in situ hybridization (FISH). DAPI dull/CMA bright bands were coincident with the chromosomal location of 28S rDNA in both species. The major rDNA was interstitially clustered at a single locus on the short arms of the metacentric chromosome pair 5 in V. aurea, whereas in T. rhomboides it was subtelomerically clustered on the long arms of the subtelocentric chromosome pair 17. 5S rDNA also was a single subtelomeric cluster on the long arms of subtelocentric pair 17 in V. aurea and on the short arms of the metacentric pair 9 in T. rhomboides. Furthermore, V. aurea showed four telomeric histone gene clusters on three metacentric pairs, at both ends of chromosome 2 and on the long arms of chromosomes 3 and 8, whereas histone genes in T. rhomboides clustered interstitially on the long arms of the metacentric pair 5 and proximally on the long arms of the subtelocentric pair 12. Double and triple FISH experiments demonstrated that rDNA and H3 histone genes localized on different chromosome pairs in the two clam species. Telomeric signals were found at both ends of every single chromosome in both species. Chromosomal location of these three gene families in two species of Veneridae provides a clue to karyotype evolution in this commercially important bivalve family.     

Position-dependent NOR activity in barley

Two types of intraspecific nucleolar dominance/suppression are described for barley,Hordeum vulgare L. When the nucleolus organizing regions (NORs) originally belonging to chromosomes 6 and 7 are combined by translocation in one chromosome, NOR 6 is dominant over NOR 7. Neither significant loss of rDNA nor its hypermethylation is the reason for the reduced nucleolus forming activity of NOR 7. Intrachromosomal NOR suppression probably does not occur in isochromosome 6s, which has two NORs 6 in one chromosome. Meiotic and somatic pairing of the homologous arms might be the reason for early fusion of their nucleoli and thus for the lower than expected maximum number of interphase nucleoli. Variable suppression of a partial NOR (6³) is described for descendants of crosses between translocation lines with split NORs 6 and 7. In these cases also, the reduced activity of the partial NOR 6³ is not due to deletion of rDNA as shown by in situ hybridization. Unstable methylation of NOR 6³ in heterozygous F₁ individuals is probably the cause of this phenomenon.     

Mouse chromosome-specific painting probes generated from microdissected chromosomes

Using degenerate primer amplification of chromosomes microdissected from banded cytogenetic preparations, we constructed both whole chromosome painting probes for mouse Chromosomes (Chrs) 1, 2, 3, and 11 and a centromere probe that strongly paints most mouse centromeres. We also amplified a Robertsonian translocation chromosome microdissected from unstained preparations to construct a painting probe for Chrs 9 and 19. The chromosomes probes uniformly painted the respective chromosomes of origin. We demonstrated the utility of the Chr 11 probe in aberration analysis by staining mutants that we had previously identified as containing a Chr 11 translocation, and in some mutant cell lines we observed chromosome rearrangements not previously detected in stained cytogenetic preparations. The technology of microdissection and amplification applies to all mouse chromosomes or to specific subchromosomal regions and will be useful in mouse genetics, in aberration analysis, and for chromosome identification.