Comparative FISH mapping of bovine cosmids to reindeer chromosomes demonstrates conservation of the X-chromosome

Three X chromosome-specific bovine cosmids were used for fluorescencein situ hybridization mapping on reindeer (Rangifer tarandus) chromosomes, to test whether such large genomic clones could be used for comparative mapping across distantly related species. All three cosmids showed distinct unique hybridization sites on the reindeer X. Comparative map locations of these cosmids, together with the relative C-banding and genome size data on the X chromosomes of the two species, provide preliminary indications that the short and long arms of bovine X correspond, respectively, to the long and short arms of the reindeer X. The study also demonstrates that cosmid clones can be used successfully for comparative mapping across species that diverged 35 million years ago.accepted for publication by M. Schmid     

Possible origin of a B chromosome deduced from its DNA composition using double FISH technique

Double fluorescentin situ hybridization (FISH) with two DNA probes (a 180 bp tandemly repeated DNA and ribosomal DNA) was performed in embryo cells of the grasshopperEyprepocnemis plorans. Repetitive DNA was present in most standard chromosomes (excepting 7, 8 and 10) and in the proximal two-thirds of the B chromosome, which was its major location in the complement. Ribosomal DNA was present distally on the B, and in the active nucleolar organizer regions (NORs) of the X, 9, 10 and 11 chromosomes. A small number of rRNA gene clusters was also observed in the pericentromeric regions of chromosomes 1–8. The double FISH technique showed that the B chromosome (B₂ type) is mainly composed of a 180 bp tandem repeat and ribosomal DNA, the minute short arm being the only region that does not hybridize with them. The location and order of the centromere and both the DNA sequences on the B chromosome coincide only with those in the X chromosome, indicating that the B most probably derives from the X.     

Characterization of marker chromosomes by fish using microdissected probes from old carnoy-fixed cells: Report of two cases

Summary We reported on two patients with ade novo marker chromosome of which the origins were successfully identified by FISH using microdissected probes. These probes were established by microdis-sections of extra chromosomal segments from Carnoy-fixed cells stored at −20°C for several years. Using these probes, we could verify partial 1q32 trisomy in a patient with 17p+ as well as partial 16q2 trisomy in another patient with 4p+.     

Reciprocal chromosome painting between a New World primate, the woolly monkey, and humans

We employed fluorescence-activated chromosome sorting (FACS) to construct chromosome paint sets for the woolly monkey (Lagothrix lagotricha) and then FISH to reciprocally paint human and woolly monkey metaphases. Reciprocal chromosome painting between humans and the woolly monkey allowed us to assign subchromosomal homologies between these species. The reciprocal painting data between humans and the woolly monkey also allow a better interpretation of the chromosomal difference between humans and platyrrhines, and refine hypotheses about the genomic rearrangements that gave origin to the genome of New World monkeys. Paints of woolly monkey chromosomes were used to paint human metaphases and forty-five clear signals were detected. Paints specific to each human chromosome were used to paint woolly monkey metaphases. The 23 human paints gave 39 clear signals on the woolly monkey karyotype. The woolly monkey chromosomes painted by human paints produced 7 associations of segments homologous to human chromosomes or human chromosome segments: 2/16, 3/21, 4sol;15, 5/7, 8/18, 10/16 and 14/15. A derived translocation between segments homologous to human chromosomes 4 and 15 is a synapomorphic marker linking all Atelines. These species may also be linked by fragmentation of homologs to human 1, 4, and 15.     

Comparison of ribosomal DNA sites inLolium species by fluorescencein situ hybridization

The position of the 18S-5.8S-26S and 5S rRNA genes have been physically mapped on the chromosomes of sevenLolium taxa. 18S-5.8S-26S sites were seen on two pairs of chromosomes in the inbreeding taxa. In the outbreeding taxa six sites were found in theL. multiflorum, seven inL. perenne and nine inL. rigidum var.rigidum. Two 5S sites were found in each of the taxa. In the inbreeders, the 5S sites were found adjacent to the 18S-5.8S-26S sites on chromosome 2. InL. multiflorum andL. perenne the 5S sites were on the short arm of chromosome 3. However, inL. rigidum var.rigidum the 5S rDNA site was found in either of the two positions.accepted by J.S. (Pat) Heslop-Harrison     

High chromosome conservation detected by comparative chromosome painting in chicken, pigeon and passerine birds

Chicken chromosome paints for macrochromosomes 1-10, Z, and the nine largest microchromosomes (Griffin et al. 1999) were used to analyze chromosome homologies between chicken (Gallus gallus domesticus: Galliformes), domestic pigeon (Columba livia: Columbiformes), chaffinch (Fringilla coelebs Passeriformes), and redwing (Turdus iliacus: Passeriformes). High conservation of syntenies was revealed. In general, both macro- and microchromosomes in these birds showed very low levels of interchromosomal rearrangements. Only two cases of rearrangements were found. Chicken chromosome 1 corresponds to chromosome 1 in pigeon, but to chromosomes 3 and 4 in chaffinch and chromosomes 2 and 5 in redwing. Chicken chromosome 4 was shown to be homologous to two pairs of chromosomes in the karyotypes of pigeon and both passerine species. Comparative analysis of chromosome painting data and the results of FISH with (TTAGGG)n probe did not reveal any correlation between the distribution of interstitial telomere sites (ITSs) and chromosome rearrangements in pigeon, chaffinch and redwing. In chaffinch, ITSs were found to co-localize with a tandem repeat GS (Liangouzov et al. 2002), monomers of which contain an internal TTAGGG motif.     

Microdissection of the Y chromosome and fluorescencein situ hybridization analysis of the sex chromosomes of lake trout,Salvelinus namaycush

Lake trout,Salvelinus namaycush, is one of the few salmonids with morphologically differentiated sex chromosomes. Genetic analysis suggested that the sex-determining region of this species lies on the short arm of the Y chromosome. The differential arm of the Y chromosome was microdissected and the resulting DNA amplified in a sequence-independent manner. Amplified DNA was biotin labeled as a probe for fluorescencein situ hybridization (FISH). Strong hybridization signals were seen covering defined regions of both the Y and X chromosomes. Homeologous chromosomes of the ancestrally tetraploid genome were not identified by FISH with the Y probe, indicating diploidization of this region of the genome.     

Application of synthetic DNA probes of human alpha satellite consensus monomer for detection of centromereinvolved chromosome abnormalities

We have synthesized the alphoid monomer of 171 bp based on the consensus sequence of human alpha satellite DNA and constructed a clone of dimeric or tetrameric sequence unit. Southern blot analysis using the clone as a probe showed restriction site periodicities in human DNA digested byEcoRI orBamHI. The synthetic consensus unit could detect the alpha repeated centromeric regions of all human chromosomes by fluorescencein situ hybridization. Using the cells having a dicentric X chromosome, we showed that the two centromeric regions were stained with fluorescent alpha satellite DNA probes. Thus the probe would be useful to detect chromosomal abnormalities such as dicentrics.     

Multi-chromosomal location of ribosomal RNA genes and heterochromatin association in brown trout

The ribosomal rRNA genes have been mapped by fluorescentin situ hybridization (FISH) to brown trout chromosomes. One major NOR chromosome pair and 8 novel minor NOR chromosome pairs have been found. Both major and minor NORs were closely related to polymorphic heterochromatin, as revealed by FISH and C-banding. These results are discussed with respect to NOR expression, the relationship between rDNA and heterochromatin, and evolutionary aspects.     

Direct detection of repetitive,whole chromosome paint and telomere DNA probes by immunogold electron microscopy

Biotinylated repetitive, whole chromosome paint and telomere DNA probes were investigated at the electron microscope level after non-isotopicin situ hybridization and direct immunogold detection. The protocol described allowed the visualization of a biotinylated chromosome 1 specific satellite DNA probe in the light microscope without silver intensification. This sensitive method was exploited to analyse factors contributing to signal strength in immunogold chromosome painting. Furthermore, it allowed us to investigate the distribution of (TTAGGG)n telomere repeats in human metaphase chromosomes and interphase nuclei. Telomeric and internal (TTAGGG)n repeats were detected at high spatial resolution in human metaphase chromosomes. In the periphery of lymphocyte interphase nuclei, two types of telomere hybridization signals were observed. They differed remarkably in compactness, indicating two types of chromatin conformation present at the interphase telomeresin situ.     

Identification of iso(18p) marker chromosome by fluorescencein situ hybridization with single-copy DNA probe

Summary The patient displayed the clinical features consistent with tetrasomy (18p) syndrome, who had an extra small metacentric iso(18p) chromosome in otherwise normal karyotype. Identification of the marker chromosome used the chromosome 18 band-specific fluorescencein situ hybridization strategy.     

Meiotic instability of chicken ultra-long telomeres and mapping of a 2.8 megabase array to the W-sex chromosome

The objective of this research was to study the meiotic stability of a subset of chicken telomere arrays, which are the largest reported for any vertebrate species. Inheritance of these ultra-long telomere arrays (200 kb to 3 mb) was studied in a highly homozygous inbred line, UCD 003 (F ≥ 99.9). Analysis of array transmission in four families indicated unexpected heterogeneity and non-Mendelian segregation including high-frequency-generation of novel arrays. Additionally, the largest array detected (2.8 Mb) was female-specific and correlated to the most intense telomeric DNA signal on the W-sex chromosome by fluorescence in situ hybridization (FISH). These results are discussed in regard to the potential functions of the ultra-long telomere arrays in the chicken genome including generation of genetic variation through enhanced recombination, protection against erosion by providing a buffer for gene-dense regions, and sex-chromosome organization.     

Autosomal location of a new subtype of 1.688 satellite DNA ofDrosophila melanogaster

During the screening of aDrosophila melanogaster YAC library with DNA from the minichromosomeDp(1;f)1187 we isolated a clone, yw20D5, which contains a new subtype of 1.688 satellite DNA. Although the sequences of several monomers subcloned from the YAC show a considerable variation in length, the derived consensus sequence is 356-bp long. This new subtype and the one constituted by the 353-bp repeats are both located on the left arm heterochromatin of chromosome 3, arranged in separate arrays. Despite their autosomal location, phylogenetic relationships among 1.688 satellite sequences suggest that they may have originated from the 359-bp repeats of the X chromosome heterochromatin. We have used the new 356-bp repeats to investigate whether sequences related to the 1.688 satellite are dispersed along the euchromatic arms of the autosomes in a similar way to that in which they are found along the X chromosome euchromatin.accepted for publication by D. Ward     

Human type II collagen gene (COL2A1) assigned to chromosome 12q13.1-q13.2 byin situ hybridization with biotinylated DNA probe

We have made a regional assignment of the type II collagen gene (COL2A1) on human chromosome 12 by means of anin situ hybridization technique with a biotinylated DNA probe. The precise localization of the signal was mapped to the band 12q13.1-q13.2. This result was in agreement with the previous mapping by isotopicin situ hybridization technique (12q13.1-q13.2), but not with the result of Southern hybridization analysis using somatic cell hybrids (12q14.3).     

Characterization of DNA sequences constituting the terminal heterochromatin of the chicken Z chromosome

Two clones, pCZTH5-8 and pCZTH12-8, were isolated from a female chicken genomic library by screening with sequences obtained from genomic libraries which had been constructed from a terminal region of a single Z chromosome of chicken utilizing laser microbeam irradiation and PCR amplification. Fluorescencein situ hybridization to the mitotic Z chromosome and the lampbrush ZW bivalent of chicken demonstrated that both the cloned sequences are located in the heterochromatic region of the Z chromosome at the end opposite to the pairing region with the W chromosome. The sequences pCZTH5-8 and pCZTH12-8 are distributed widely on both the telomeric bow-like loops (TBL) and the region I (short loops region) of the Z lampbrush chromosome. These clones, pCZTH12-8 particularly notably, hybridized also to the TBLs of lampbrush bivalents 1–4 of chicken. Both sequence are transcribed in the lampbrush stage oocytes on the Z chromosome and on other macrobivalents. The subfragment of pCZTH5-8 which hybridizes to the TBLs and the insert of pCZTH12-8 contain regions that are closely similar in sequence. The pCZTH5-8 sequence has no internal repeats and may be part of the 24-kb macrosatellite repeating unit that is evident afterNhel digestion of the genomic DNA. A cloned 24-kb unit, pFN-1, does not show significant DNA curvature, but cytosines of its CpG dinucleotides may be highly methylatedin vivo. This contrasts with the repeat sequences of the W heterochromatin which not only have highly methylated CpG but are also strongly curved. The 24-kb unit is repeated about 830 times in the diploid genome of a female chicken, suggesting that nearly the entire terminal heterochromatin on the Z chromosome consists of this macrosatellite family. Sequences of the greater part of the pCZTH5-8 are restricted to the genusGallus but the sequence of one subregion which hybridizes to TBLs is present in the genomes of the order Galliformes.accepted for publication by M. Schmid     

A heritable folate-sensitive fragile site on chromosome 2p11.2 (FRA2L)

Mouse synaptonemal complexes (SCs) were hybridized with a telomeric DNA probe. The biotinylated probe was detected with antibodies conjugated to 5-nm colloidal gold and observed in the transmission electron microscope. The distribution of the probe was clearly marked by clusters of the gold particles and the ultrastructure of the SC was not adversely affected by the procedure.accepted for publication by G. Sutherland     

Assignment of the linkage group EAM-TYRP2-TPP2 to chromosome 11 in pigs byin situ hybridization mapping of the TPP2 gene

Restriction fragment length polymorphisms are described for the genes coding for tripeptidyl peptidase II (TPP2) and tyrosinase related protein II (TYRP2) in pigs. A linkage group comprising these loci and the locus for blood group M (EAM) was established by two-point lod score analysis in a three-generation pedigree. Multipoint analysis indicated the linear order EAM-1.1-TYRP2-8.4-TPP2 (recombination distances are given as Kosambi cM). The linkage group was assigned to porcine chromosome 11—the first on this chromosome—throughin situ hybridization mapping of the TPP2 gene. TPP2 is the first gene localized on this chromosome usingin situ hybridization.     

Distribution and linkage of repetitive clusters from the heterochromatic region of human chromosome 22

The pericentric regions of eukaryotic chromosomes consist of several types of repetitive DNA families. In human chromosome 22, the organization of such families was studied in more detail. In addition to the known families of alpha and beta repeats, an additional repeat with a 48-bp motif was previously assigned to 22pter-q11. Here, we report in more detail the distribution of these repeat families, applying pulsed-field gel electrophoresis, fluorescencein situ hybridization and physical linkage on cosmid recombinants. At least two clusters of 48-bp repeats are localized on chromosome 22, one on the distal p-arm and one in the region 22cen-q11. Cosmids from a chromosome 22 library, containing both 48-bp and -repeats, link both arrays on 22p and define their maximum distances to less than 44 kb. Loss of 48-bp repeat sequences in a Di-George cell line carrying a deletion in 22q11 suggests the presence of a second cluster in 22q11, a distribution supported by (fluorescenein situ hybridization)-FISH signal analysis. As additional members of the 48-bp repeat family can be found on all acrocentric chromosomes it remains to be determined whether the distribution seen on chromosome 22 is also common in other human acrocentric chromosomes.accepted for publication by M. Schmid     

Comparative chromosome mapping of sex-linked genes and identification of sex chromosomal rearrangements in the Japanese wrinkled frog (Rana rugosa, Ranidae) with ZW and XY sex chromosome systems

There are regional variations of sex chromosome morphologies in the Japanese wrinkled frog, Rana rugosa (2n = 26): heterogametic ZZ/ZW-type and XX/XY-type sex chromosomes, and two different types of homomorphic sex chromosomes. To search for homology between the ZW and XY sex chromosomes and the chromosome rearrangements that have occurred during sex chromosomal differentiation in R. rugosa, we performed chromosome mapping of sexual differentiation genes for R. rugosa by FISH. Three genes, AR, SF-1/Ad4BP and Sox3, were localized to both the ZW and XY chromosomes, and their locations were all different between the Z and W and between the X and Y. AR and SF-1/Ad4BP were located on the short arms of the W and X and the long arms of Z and Y, and Sox3 was mapped to the different locations on the long arms between the Z and W and between the X and Y, probably as a result of multiple rearrangements that occurred during the process of sex chromosome differentiation. However, the chromosomal locations of three genes were almost consistent between the Z and Y and between the W and X, indicating that the Z and Y chromosomes and the W and X chromosomes were respectively derived from the same origins. Dmrt1, which is located on avian sex chromosomes, was localized to autosomes in R. rugosa with both the ZW and XY sex chromosomes, suggesting that Dmrt1 might not be related to sex determination in this species.