A high-resolution cytogenetic map of 168 cosmid DNA markers for human chromosome 11.

We have constructed a high-resolution cytogenetic map with 168 DNA markers, including 90 RFLP markers for human chromosome 11. The cosmid clones were mapped by fluorescence in situ suppression hybridization, in which discrete fluorescent signals can be detected directly on prometaphase R-banded chromosomes. Although these cosmid clones were distributed throughout the chromosome, they had some tendency to localize in the regions of R-positive band, such as 11p15, 11p11.2, 11q13, 11q23, and 11q25. Since these regions of chromosome 11 are considered to contain genes responsible for certain genetic diseases, cancer breakpoints involved in chromosome rearrangements, and tumor-suppressor genes, this high-resolution cytogenetic map will contribute to the molecular characterization of such genes. This map will also provide many landmarks essential for construction of the complete physical map with contigs of cosmid and YAC clones.     

A high-resolution cytogenetic map of human chromosome 3: localization of 291 new cosmid markers by direct R-banding fluorescence in situ hybridization.

We localized 291 new cosmid markers (including 65 RFLPs) on human chromosome 3 by direct R-banding fluorescence in situ hybridization. This system, which is based on fluorescence in situ hybridization combined with replicated prometaphase R-bands, allows the direct visualization of signals on R-banded prometaphases stained with propidium iodide and provides a more rapid and efficient method for genome mapping of cosmid clones. The signals of 291 markers examined here were localized preferentially to R-positive bands throughout chromosome 3. The detailed map positions of 366 clones and the characterization of 142 RFLPs, including the preliminary data reported by Yamakawa et al. (1991, Genomics 9: 536-543; and 11: 565-572), are summarized. This high-resolution cytogenetic map (average distance of 0.58 Mb), in conjunction with a genetic linkage map, can facilitate the analysis of chromosomal and molecular aberrations in genetic diseases and cancers. Furthermore, these mapping data will provide many useful landmarks for the construction of contig maps of chromosome 3.     

Integration of cytogenetic with recombinational and physical maps of mouse chromosome 16.

To link the cytogenetic map for mouse chromosome 16 (Chr 16) to the more detailed recombinational and physical maps, multiple probes were mapped by fluorescence in situ hybridization (FISH). Sixteen large insert clones (YACs, BACs, and PACs) containing markers that have been assigned to the Chr 16 recombinational map were localized to a cytogenetic band or subband by high-resolution FISH. This linkage of the cytogenetic and recombinational maps provides a useful tool for assigning new probe locations and for defining breakpoints in mice with chromosomal rearrangements. A direct application of these probes is demonstrated by identifying mice trisomic for distal Chr 16 using FISH analysis of interphase nuclei.     

Cytogenetic and molecular cytogenetic investigations in relapse of B-cell chronic lymphocytic leukemia

Сhromosomal abnormalities have been analyzed in bone marrow cells of 61 patients with relapse of B-cell chronic lymphocytic leukemia. The cytogenetic results have allowed the structural stratification of the obtained karyotypes into ten groups of clones: normal, normal/near tetraploid, abnormal/normal, abnormal/ near tetraploid/normal, evolution of clonal chromosome abnormalities; evolution of clonal chromosome abnormalities/normal, evolution of clonal chromosome abnormalities/near tetraploid/normal, independent clones, independent/normal clones; and independent/near tetraploid/normal clones. The identified structural rearrangements included translocations, deletions, insertions, and duplications; however, deletions with the involvement of bands 17p12, 13q12–q14, 11q14, and 11q23 dominated (63.8%). The application of i-FISH helped to show the presence of one to four abnormalities per karyotype. The identified cytogenetic and molecular cytogenetic rearrangements may signify a multilevel nature of the process underlying the development of resistant karyotypes. The results obtained under both methods have revealed the presence of a heterogenic cell population with possibly different levels of chemotherapy resistance.     

An Integrated Genetic and Cytogenetic Map for Zhikong Scallop,Chlamys farreri,Based on Microsatellite Markers

The reliability of genome analysis and proficiency of genetic manipulation requires knowledge of the correspondence between the genetic and cytogenetic maps. In the present study, we integrated cytogenetic and microsatellite-based linkage maps for Zhikong scallop, Chlamys farreri. Thirty-eight marker-anchored BAC clones standing for the 19 linkage groups were used to be FISH probes. Of 38 BAC clones, 30 were successfully located on single chromosome by FISH and used to integrate the genetic and cytogenetic map. Among the 19 linkage groups, 12 linkage groups were physically anchored by 2 markers, 6 linkage groups were anchored by 1 marker, and one linkage group was not anchored any makers by FISH. In addition, using two-color FISH, six linkage groups were distinguished by different chromosomal location; linkage groups LG6 and LG16 were placed on chromosome 10, LG8 and LG18 on chromosome 14. As a result, 18 of 19 linkage groups were localized to 17 pairs of chromosomes of C. farreri. We first integrated genetic and cytogenetic map for C. farreri. These 30 chromosome specific BAC clones in the cytogenetic map could be used to identify chromosomes of C. farreri. The integrated map will greatly facilitate molecular genetic studies that will be helpful for breeding applications in C. farreri and the upcoming genome projects of this species.     

Regional assignment of human genes TPI1, GAPDH, LDHB, SHMT, and PEPB on chromosome 12.

Karyological analysis was performed on a series of human-Chinese hamster cell hybrids containing deletions of human chromosome 12. Chromosome breakage was produced by treatment of the cells with either X-rays or 5-bromodeoxyuridine and near-visible light. The hybrid clones were analyzed for the presence or absence of the following five human gene markers known to be located on chromosome 12: triosephosphate isomerase-1 (TPI1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase-B (LDHB), serine hydroxymethyltransferase (SHMT), and peptidase-B (PEPB). Based on the correlation between the isozyme markers and karyological analysis of these clones, a regional map of the five human genes on chromosome 12 was established. The linear order for these genes is: pter-TPI1-GAPDH-LDHB-centromere-SHMT-PEPB-qter. The locations of these genes are: TPI1, GAPDH, LDHB: pter leads to p12; SHMT: q12 leads to q14; PEPB: q14 leads to qter. Statistical analysis similar to that of Goss and Harris (1975, 1977a, b) has been performed on the segregation data in the hybrid clones. The statistical map, in general, agrees with the cytogenetic map and further localizes PEPB to 12q21.     

Isolation and mapping of 62 new RFLP markers on human chromosome 11.

To obtain new RFLP markers on human chromosome 11 for a high-resolution map, we constructed a cosmid library from a Chinese hamster x human somatic hybrid cell line that retains only human chromosome 11 in a Chinese hamster genomic background. A total of 3,500 cosmids were isolated by colony hybridization with labeled human genomic DNA. DNA was prepared from 130 of these cosmid clones and examined for RFLP. In 62 of them, polymorphism was detected with one or more enzymes; four RFLPs were VNTR systems. All polymorphic clones were assigned to one of 22 intervals obtained by mapping on a deletion panel of 15 somatic hybrid cell lines containing parts of chromosome 11; 11 clones were finely mapped by in situ hybridization. Although RFLP markers were scattered on the whole chromosome, they were found predominantly in the regions of R-banding. These DNA markers will contribute to fine mapping of genes causing inherited disorders and tumor-suppressor genes that reside on chromosome 11. Furthermore, as one-third of the cosmid clones revealed a band or bands in Chinese hamster DNA, indicating sequence conservation, this subset of clones may be useful for isolating biologically important genes on chromosome 11.     

Identification and characterization of three genes and two pseudogenes on chromosome 13

A study was conducted on the feasibility of isolating genes and pseudogenes that map to chromosome 13 by a hybridization-based approach using a 13-specific library and pools of repeat-free cDNA clones. Five pairs of cDNA and chromosome 13 genomic clones were identified and characterized. Partial or full-length sequence was derived from all cDNAs, and database searches were performed for putative gene identification. Partial sequence was also obtained from the chromosome 13 genomic clones for comparison with those of the hybridizing cDNAs. As a result of these analyses we identified three genes, a putative homologue of a porcine mRNA encoding an unidentified hepatic protein, a putative homologue of a yeast integral membrane protein, and a gene for a translationally controlled tumor protein, and two processed pseudogenes, ribosomal proteins L23a and S3a. The latter was formerly identified as the v-fos transformation effector gene, Fte-1, and recently cited as a possible candidate for the BRCA2 gene on chromosome 13. All genes and pseudogenes were localized to cytogenetic bands by in situ hybridization of metaphase chromosomes with probes derived from the chromosome 13 genomic clones.     

Cosmid linking clones localized to the long arm of human chromosome 11.

Molecular probes that contain DNA flanking CpG-rich restriction sites are extremely valuable in the construction of physical maps of chromosomes and in the identification of genes associated with hypomethylated HTF (HpaII tiny fragment) islands. We describe a new approach to the isolation and characterization of linking clones in arrayed chromosome-specific cosmid libraries through the large-scale semiautomated restriction mapping of cosmid clones. We utilized a cosmid library representing human chromosome 11q12-11qter and carried out automated restriction enzyme analysis, followed by regional localization to chromosome 11q using high-resolution in situ suppression hybridization. Using this approach, 165 cosmid linking clones containing one or more NotI, BssHII, SfiI, or SacII sites were identified among 960 chromosome-specific cosmids. Furthermore, this analysis allowed clones containing a single site to be distinguished from those containing clusters of two or more rare sites. This analysis demonstrated that more than 75% of cosmids containing a rare restriction site also contained a second rare restriction site, suggesting a high degree of CpG-rich restriction site clustering. Thirty chromosome 11q-specific cosmids containing rare CpG-rich restriction sites were regionally localized by high-resolution fluorescence in situ suppression hybridization, demonstrating that all of the CpG-rich sites detected by this method were located in bands 11q13 and 11q23. In addition, the distribution of (CA)n repetitive sequences was determined by hybridization of the arrayed cosmid library with oligonucleotide probes, confirming a random distribution of microsatellites among CpG-rich cosmid clones. This set of reagent cosmid clones will be useful for physical linking of large restriction fragments detected by pulsed-field gel electrophoresis and will provide a new and highly efficient approach to the construction of a physical map of human chromosome 11q.     

The Cytogenetic Map of the Poncirus trifoliata (L.) Raf.��A Nomenclature System for Chromosomes of All Citric Species

A cytogenetic map was established for the trifoliate orange, Poncirus trifoliata. Chromosome size, centromere position and CMA⁺ heterochromatin content were determined for each chromosome pair, together with the position of BAC clones previously described as chromosome-specific markers. Although P. trifoliata is a true biological species, heteromorphisms for the size of the heterochromatic bands in two chromosome pairs were observed. A minimum set of four BACs was proposed for chromosome identification in P. trifoliata, as well as a chromosome nomenclature that can be applied to all citrus species. The Ctv locus was assigned to chromosome 9 and the remaining linkage groups can now be integrated to this physical map. This standard nomenclature for trifoliate orange will be valuable for genome-sequencing projects in all citrus species.     

Chromosomal localization of sixty autosomal loci in sheep (OVIS ARIES, 2n = 54) by fluorescence in situ hybridization and R-banding

Sixty autosomal loci (5 type I and 55 type II) from 24 bovine syntenic groups, and previously FISH-mapped to goat and river buffalo chromosomes, were localized by fluorescence in situ on sheep (OVIS ARIES, 2n = 54) chromosomes, thereby notably extending the cytogenetic map of this economically important species. Caprine BAC clones were hybridized to R-banded chromosome preparations. FITC-signals and RBPI- banding (R-banding by late BrdU-incorporation and propidium iodide staining) were simultaneously visualized and captured by a colour CCD-camera. All mapped loci were localized on homoeologous chromosomes and chromosome regions (bands) of sheep, goat and river buffalo, further supporting chromosome and genetic (loci) homoeologies among bovids.     

An integrated map of Oryza sativa L. chromosome 5

The developments of molecular marker-based genetic linkage maps are now routine. Physical maps based on contigs of large insert genomic clones have been established in several plant species. However, integration of genetic, physical, and cytological maps is still a challenge for most plant species. Here we present an integrated map of rice (Oryza sativa L.) chromosome 5, developed by fluorescence in situ hybridization mapping of 18 bacterial artificial chromosome (BAC) clones or PI-derived artificial chromosome (PAC) clones on meiotic pachytene chromosomes. Each BAC/PAC clone was anchored by a restriction fragment length polymorphism marker mapped to the rice genetic linkage map. This molecular cytogenetic map shows the genetic recombination and sequence information of a physical map, correlated to the cytological features of rice chromosome 5. Detailed comparisons of the distances between markers on genetic, cytological, and physical maps, revealed the distributions of recombination events and molecular organization of the chromosomal features of rice chromosome 5 at the pachytene stage. Discordance of distances between the markers was found among the different maps. Our results revealed that neither the recombination events nor the degree of chromatin condensation were evenly distributed along the entire length of chromosome 5. Detailed comparisons of the correlative positions of markers on the genetic, cytological, and physical maps of rice chromosome 5 provide insight into the molecular architecture of rice chromosome 5, in relation to its cytological features and recombination events on the genetic map. The prospective applications of such an integrated cytogenetic map are discussed.     

An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2

Background

Integration of molecular, genetic and cytological maps is still a challenge for most plant species. Recent progress in molecular and cytogenetic studies created a basis for developing integrated maps in cucumber (Cucumis sativus L.).

Results

In this study, eleven fosmid clones and three plasmids containing 45S rDNA, the centromeric satellite repeat Type III and the pericentriomeric repeat CsRP1 sequences respectively were hybridized to cucumber metaphase chromosomes to assign their cytological location on chromosome 2. Moreover, an integrated molecular cytogenetic map of cucumber chromosomes 2 was constructed by fluorescence in situ hybridization (FISH) mapping of 11 fosmid clones together with the cucumber centromere-specific Type III sequence on meiotic pachytene chromosomes. The cytogenetic map was fully integrated with genetic linkage map since each fosmid clone was anchored by a genetically mapped simple sequence repeat marker (SSR). The relationship between the genetic and physical distances along chromosome was analyzed.

Conclusions

Recombination was not evenly distributed along the physical length of chromosome 2. Suppression of recombination was found in centromeric and pericentromeric regions. Our results also indicated that the molecular markers composing the linkage map for chromosome 2 provided excellent coverage of the chromosome.     

Index, Comprehensive Microsatellite, and Unified Linkage Maps of Human Chromosome 14 with Cytogenetic Tie Points and a Telomere Microsatellite Marker

Three sets of linkage maps (index, comprehensive microsatellite, and unified) have been constructed for human chromosome 14 based on genotypes from the CEPH reference pedigrees. The index maps consist of 18 microsatellite markers, with heterozygosities of at least 68% and intermarker spacing no greater than 11 cM. The sex-average comprehensive microsatellite map is 125 cM in length and includes 115 markers with 54 loci uniquely placed with odds for marker order of at least 1000:1. The sex-average index map length is 121 cM, and the female- and male-specific maps are 143 and 101 cM, respectively. A unified map was also constructed from 147 loci (162 marker systems), which includes 32 RFLP markers in addition to the 115 microsatellites. The sex-average length of the unified map is 128 cM with 69 loci uniquely placed. Our maps are anchored by a microsatellite telomere marker sCAW1 (D14S826), developed from a telomere YAC clone TYAC196, which extends the linkage map to the physical terminus of the long arm of chromosome 14. Furthermore, we have also physically mapped seven of the loci by fluorescencein situhybridization of cosmid clones orAlu-PCR products amplified from YACs containing the marker sequences. Together with previously established cytogenetic map designations for other loci, our maps display links between genetic markers for 10 of 13 cytogenetic bands of chromosome 14 at the 550 genome band resolution.     

Quantitative characteristics of the differential banding pattern prometaphase chromosomes of the domestic horse (Equus caballus)

A high-resolution cytogenetic map (670 bands per haploid set) of RBA-banded chromosomes has been constructed in the domestic horse Equus caballus. The size and distribution of the replication-based R(G)-bands were analyzed using the computer program VideoTest-Karyo. The obtained data were compared to the results of cytogenetic mapping in other mammalian species and human.     

Physical assignment of six type I anchor loci to bovine chromosome 19 by fluorescence in situ hybridization

A bovine bacterial artificial chromosome (BAC) library was screened for the presence of eight type I anchor loci previously used within hybrid somatic cells and an interspecies hybrid backcross to construct a genome map of bovine chromosome 19 (BTA19). Six out of eight loci were identified in the BAC library ( NF1, CRYB1, CHRNB1, TP53, GH1 and P4HB ). The BACs were then used in single-colour fluorescence in situ hybridization (FISH) to assign these genes to BTA19 band locations. Gene order was determined by single-colour FISH, and was confirmed by dual-colour FISH to mitotic and meiotic chromosomes. The order, centromere- NF1-CRYB1-CHRNB1-TP53-GH1-P4HB , was in agreement with the order determined by linkage analyses. In addition, the order of CHRNB1 and TP53 , previously unresolved by linkage analyses, was established. These data provide high-resolution cytogenetic anchorage of the BTA19 genome map from chromosome bands 14–22.     

Defining the orientation of the tandem fusions that occurred during the evolution of Indian muntjac chromosomes by BAC mapping

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n=6 in the female and 7 in the male, the karyotypic evolution of which through extensive tandem fusions and several centric fusions has been well-documented by recent molecular cytogenetic studies. In an attempt to define the fusion orientations of conserved chromosomal segments and the molecular mechanisms underlying the tandem fusions, we have constructed a highly redundant (more than six times of whole genome coverage) bacterial artificial chromosome (BAC) library of Indian muntjac. The BAC library contains 124,800 clones with no chromosome bias and has an average insert DNA size of 120 kb. A total of 223 clones have been mapped by fluorescent in situ hybridization onto the chromosomes of both Indian muntjac and Chinese muntjac and a high-resolution comparative map has been established. Our mapping results demonstrate that all tandem fusions that occurred during the evolution of Indian muntjac karyotype from the acrocentric 2n=70 hypothetical ancestral karyotype are centromere–telomere (head–tail) fusions.     

A comparative cytogenetic study of chromosome homology between chicken and Japanese quail

In order to construct a chicken (Gallus gallus) cytogenetic map, we isolated 134 genomic DNA clones as new cytogenetic markers from a chicken cosmid DNA library, and mapped these clones to chicken chromosomes by fluorescence in situ hybridization. Forty-five and 89 out of 134 clones were localized to macrochromosomes and microchromosomes, respectively. The 45 clones, which localized to chicken macrochromosomes (Chromosomes 1-8 and the Z chromosome) were used for comparative mapping of Japanese quail (Coturnix japonica). The chromosome locations of the DNA clones and their gene orders in Japanese quail were quite similar to those of chicken, while Japanese quail differed from chicken in chromosomes 1, 2, 4 and 8. We specified the breakpoints of pericentric inversions in chromosomes 1 and 2 by adding mapping data of 13 functional genes using chicken cDNA clones. The presence of a pericentric inversion was also confirmed in chromosome 8. We speculate that more than two rearrangements are contained in the centromeric region of chromosome 4. All 30 clones that mapped to chicken microchromosomes also localized to Japanese quail microchromosomes, suggesting that chromosome homology is highly conserved between chicken and Japanese quail and that few chromosome rearrangements occurred in the evolution of the two species.     

Chromosome 16-specific repetitive DNA sequences that map to chromosomal regions known to undergo breakage/rearrangement in leukemia cells.

Human chromosome 16-specific low-abundance repetitive (CH16LAR) DNA sequences have been identified during the course of constructing a physical map of this chromosome. At least three CH16LAR sequences exist and they are interspersed, in small clusters, over four regions that constitute more than 5% of the chromosome. CH16LAR sequences were observed in one unusually large cosmid contig (number 55), where the ordering of clones was difficult because these sequences led to false overlaps between noncontiguous clones. Contig 55 contains 78 clones, or approximately 2% of all the clones contained within the present cosmid contig physical map. Fluorescent in situ hybridization of multiple clones, including cosmid and YAC contig 55 clones, mapped the four CH16LAR-rich regions to bands p13, p12, p11, and q22. These regions are of biological interest since the pericentric inversion and the interhomologue translocation breakpoints commonly found in acute nonlymphocytic leukemia (ANLL) subtype M4 fall within these bands. Sequence analysis of a 2.2-kb HindIII fragment from a cosmid containing a CH16LAR sequence indicated that one of the CH16LAR elements is similar to a minisatellite sequence in that the core repeat is only 40 bp in length. Additional characterization of other repetitive elements is in progress.     

Integrated maps of the Drosophila genome: progress and prospects

A physical map of the Drosophila melanogaster genome is being assembled, consisting of ordered overlapping cosmid clones. The map is constructed in steps, separately for each chromosomal division. Gaps in this map are to be bridged with yeast artificial chromosome clones. Hybridization to previously cloned genes and extensive use of in situ hybridization to polytene chromosomes ensure that the cosmid map is firmly anchored to the wealth of available genetic and cytogenetic information. The intention is to make the physical map widely available as part of an overall, integrated genetic resource for the Drosophila research community.