Comparative mapping of bovine chromosome 13 by fluorescence in situ hybridization

We present chromosomal fluorescence in situ hybridization (FISH) results that both extend the HSA20/BTA13 comparative map as well as cytogenetically anchor two microsatellite markers. A bovine bacterial artificial chromosome (BAC) library was screened for conserved genes (type I loci) previously assigned to HSA10 or HSA20 and BTA13, and for microsatellites selected from two published BTA13 linkage maps. Clones from six out of nine comparative loci and both microsatellites were found represented in the BAC library. These BAC clones were used as probes in single colour FISH to determine the chromosome band position of each locus. As predicted by the human/bovine comparative map, all type I loci mapped to BTA13. Because single colour FISH analysis revealed that the loci were clustered within the distal half of BTA13, dual colour FISH was used to confirm the locus order. Established order was centromere- PRNP-(SODIL/AVP/OXT)-(BL42/GNAS1)-HCK-CSSM30 . The findings confirm the presence of a conserved HSA20 homologous synteny group on BTA13 distal of a HSA10 homologous segment.     

Digital mapping of bacterial artificial chromosomes by fluorescence in situ hybridization

The bacterial artificial chromosome (BAC) has become the most popular tool for cloning large DNA fragments. The inserts of most BAC clones average 100-200 kilobases (kb) and molecular characterization of such large DNA fragments is a major challenge. Here we report a simple and expedient technique for physical mapping of BAC inserts. Individual BAC molecules were immobilized on glass slides coated with Poly-L-lysine. The intact circular BAC molecules were visualized by fluorescence in situ hybridization using BAC DNA as a probe. The 7.4 kb BAC vector was extended to approximately 2.44 kb per micrometer. Digitally measured linear distances can be transformed into kilobases of DNA using the extension of BAC vector as a standard calibration. We mapped DNA fragments as small as 2 kb directly on circular BAC molecules. A rice BAC clone containing both tandem and dispersed repeats was analyzed using this technique. The distribution and organization of the different repeats within the BAC insert were efficiently determined. The results showed that this technique will be especially valuable for characterizing BAC clones that contain complex repetitive DNA sequences.     

Deletion mapping of the medulloblastoma locus on chromosome 17p

Isochromosome 17q has previously been observed consistently in cytogenetic studies of medulloblastoma, the most common posterior fossa neoplasm in children. We performed a restriction fragment length polymorphism (RFLP) investigation of medulloblastoma which showed a loss of chromosome 17p sequences in 45% of these tumors. This finding was predictive of a poor clinical response to treatment. A contiguous panel of markers permitted mapping of the deletion to 17p12-p13.1, the same chromosomal region for which loss of alleles has been shown in tumor specimens from patients with colon cancer, and the same region to which the p53 gene has been mapped. This suggests that medulloblastoma is associated with a recessive oncogene on chromosome 17p that may be involved in the genesis of several embryologically unrelated neoplasms and that the absence of this gene in tumor tissue has prognostic significance.     

Evolution of chromosome 6 of Solanum species revealed by comparative fluorescence in situ hybridization mapping

Comparative genetic linkage mapping using a common set of DNA markers in related species is an important methodology in plant genome research. Here, we demonstrate a comparative fluorescence in situ hybridization (FISH) mapping strategy in plants. A set of 13 bacterial artificial chromosome clones spanning the entire length of potato chromosome 6 was used for pachytene chromosome-based FISH mapping in seven distantly related Solanum species including potato, tomato, and eggplant. We discovered one paracentric inversion and one pericentric inversion within specific lineages of these species. The comparative FISH mapping data revealed the ancestral structure of this chromosome. We demonstrate that comparative FISH mapping is an efficient and powerful methodology to study chromosomal evolution among plant species diverged for up to 12 million years.     

Localization of the murine methylmalonyl CoA mutase (Mut) locus on chromosome 17 by in situ hybridization

Murine methylmalonyl CoA mutase (Mut) has been localized to chromosome 17C-D by in situ hybridization in cell line containing a 2.17 Robertsonian translocation. This locus, which was mapped with the help of a murine methylmalonyl CoA mutase cDNA probe, and others on murine chromosome 17 are syntenic, though not necessarily colinear, with loci on human chromosome 6.     

Subregional mapping of 13 single-copy genes on the long arm of chromosome 12 by fluorescence in situ hybridization.

Subregional localization of 13 single-copy DNA sequences previously assigned to the long arm of chromosome 12 has been performed using the fluorescence in situ hybridization (FISH) technique. The following order is suggested for the 13 mapped genes: cen-->COL2A1-->(VDR-D12S15)-->(D12S17-D12S4++ +-D12S14-D12S6)-->D12S8-->(IAPP-MGF- D12S7-D12S12)-->IGF1-->qter. Eight of the mapped genes clustered at two regions, one at 12q13 (D12S17-D12S4-D12S14-D12S6) and the other at 12q22 (IAPP-MGF-D12S7-D12S12). Our results show that single-copy DNA sequences as small as 500 bp can be successfully mapped by FISH.     

Linkage mapping and fluorescence in situ hybridization of TCTE1 on human chromosome 6p: analysis of dinucleotide polymorphisms on native gels

Highly informative dinucleotide repeat polymorphisms were identified at the T-complex-associated-testes-expressed-1 (TCTE1) locus on human chromosome 6p. Electrophoresis of single-stranded DNA on native gels facilitated the analysis of the dinucleotide polymorphisms. Linkage mapping positions this marker midway between the centromere and HLA with recombination fractions as follows: D6Z1-0.21-TCTE1-0.24-HLA. Two-color fluorescence in situ hybridization places TCTE1 proximal to CRIL171 (D6S19). Together, linkage and in situ hybridization indicate that the order of the loci is D6Z1-D6S4-D6S90-TCTE1-D6S19-D6S29-HL A-telomere. A sequence tagged site (STS) was established, and three yeast artificial chromosome (YAC) clones were identified for the TCTE1 locus.     

Regional mapping of cKi-ras proto-oncogene on mouse chromosome 6 by in situ hybridization

We have used in situ hybridization techniques to determine the subchromosomal location of the mouse cKi-ras proto-oncogene. The data confirm the assignment of cKi-ras to mouse chromosome 6 and provide evidence that the gene maps to the distal portion of this chromosome, in bands 6F3----G3.     

Detection of chromosome 17- and X-bearing human spermatozoa using fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with DNA probes specific to chromosomes 17 and the X has been applied to human ejaculated sperm. After sperm nuclei were decondensed with EDTA and DTT, biotinylated alpha satellite DNA probes TR17 and TRX were separately used on preparations from thirteen healthy donors. After hybridization 96% of sperm were labelled with the TR17 probe and 48% of sperm were labelled with the TRX probe. Frequencies of 0.33% disomic 17 and 0.29% disomic X sperm were found. The frequencies of diploid sperm were assessed as 0.37% using the TR17 probe and 0.20% using the TRX probe which labelled only one half of the sperm; after correcting the result from the X-probe to 0.40% the two frequencies are very similar.     

Rapid mapping of cosmid clones on pig chromosomes by fluorescence in situ hybridization

Nineteen cosmids have been mapped to pig chromosomes by fluorescence in situ hybridization. Two kinds of cosmid clones were isolated as potential physical and genetic markers for the pig genome. Anonymous cosmids were obtained by screening a commercial cosmid library and were localized to Chromosomes (Chrs) 1, 2, 6, 7, 8, 10, 11, 12, 13, and 14. Some of these cosmids were found to reveal RFLP type DNA polymorphism. Microsatellite-containing cosmid clones were isolated by screening a pig cosmid library with a (CA)₁₀ probe and were regionally mapped to Chrs 2, 6, 7, 13, and 14. Ten of the 19 chromosomes in the pig were labeled with these probes. Two-color fluorescence in situ hybridization was used to increase the efficiency of the cosmid localizations.     

The mapping of transgenes by fluorescence in situ hybridization on G-banded mouse chromosomes

A highly sensitive method for the mapping of transgenes and other genes in the mouse genome is described. This technique combines high-resolution G-banding and fluorescence in situ hybridization (FISH) with either biotin/avidin-FITC or digoxigenin-anti-digoxigenin-FITC, the latter being the more sensitive. Banding patterns are obtained with trypsin/Geimsa-treated slides, and sensitivity is greatly increased by the use of mouse Cot-1 DNA. With this protocol, four different 14.5-kb human Cu/Zn-superoxide dismutase transgene insertions ranging in copy number from 2 to 8 have been localized to four different mouse chromosomes. The utility and sensitivity of this procedure were verified with a Chromosome (Chr) 16-specific cosmid probe, H22, as well as with the mapping of a high-copy-number human -amyloid/A4 transgene.     

Comparative mapping of mouse and rat chromosomes by fluorescence in situ hybridization

Comparative fluorescence in situ hybridization mapping using DNA libraries from flow-sorted mouse chromosomes and region-specific mouse BAC clones on rat chromosomes reveals chromosomal homologies between mouse (Mus musculus, MMU) and rat (Rattus norvegicus, RNO). Each of the MMU 2, 3, 4, 6, 7, 9, 12, 14, 15, 16, 18, 19, and X chromosomes paints only a single rat chromosome or chromosome segment and, thus, the chromosomes are largely conserved between the two species. In contrast, the painting probes for MMU chromosomes 1, 5, 8, 10, 11, 13, and 17 produce split hybridization signals in the rat, disclosing evolutionary chromosome rearrangements. Comparative mapping data delineate several large linkage groups on RNO 1, 2, 4, 7, and 14 that are conserved in human but diverged in the mouse. On the other hand, there are linkage groups in the mouse, i.e., on MMU 1, 8, 10, and 11, that are disrupted in both rat and human. In addition, we have hybridized probes for Nap2, p57, Igf2, H19, and Sh3d2c from MMU 7 to RNO 1q and found the orientation of the imprinting gene cluster and Sh3d2c to be the same in mouse and rat. Hybridization of rat genomic DNA shows blocks of (rat-specific) repetitive sequences in the pericentromeric region of RNO chromosomes 3-5, 7-13, and 20; on the short arms of RNO chromosomes 3, 12, and 13; and on the entire Y chromosome.     

Localization of the B-hordein locus on barley chromosomes using fluorescence in situ hybridization

The B-hordein gene family consists of at least 13 genes that appear to be clustered in two regions on the barley chromosome 1H (5). Using fluorescence in situ hybridization techniques, we have localized the B-hordein locus (Hor2) to the distal end of the short arm of chromosome 1H (5), corresponding to the genetically determined position, 91% distal on the short arm. This result is based on appropriate barley lines (i.e. the two cultivars Igri and Betzes, the telotrisomic and ditelotetrasomic lines 1HS and the translocation line T21), on two experimental approaches and on a signal frequency of between 20% and 60% on metaphase chromosomes.     

Completely distinguishing individual A-genome chromosomes and their karyotyping analysis by multiple bacterial artificial chromosome - fluorescence in situ hybridization

Twenty bacterial artificial chromosome (BAC) clones that could produce bright signals and no or very low fluorescence in situ hybridization (FISH) background were identified from Gossypium arboreum cv. JLZM, and G. hirsutum accession (acc.) TM-1 and 0-613-2R. Combining with 45S and 5S rDNA, a 22-probe cocktail that could identify all 13 G. arboreum chromosomes simultaneously was developed. According to their homology with tetraploid cotton, the G. arboreum chromosomes were designated as A1-A13, and a standard karyotype analysis of G. arboreum was presented. These results demonstrated an application for multiple BAC-FISH in cotton cytogenetic studies and a technique to overcome the problem of simultaneous chromosome recognition in mitotic cotton cells.     

Genetic mapping of the Adh locus in the repleta group of Drosophila by in situ hybridization

A biotinylated probe of the Adh (alcohol dehydrogenase) gene of Drosophila melanogaster was used for in situ hybridization on polytene chromosomes of D. mojavensis and D. buzzatii, two species of the repleta group of the genus Drosophila. Hybridization showed that the Adh gene maps at the G1a band of the third chromosome. This is in accordance with a previous result obtained through the use of interspecific hybrid asynapsis as a cytological marker and establishes the limits of the precision of this method.     

Malaria liver stage susceptibility locus identified on mouse chromosome 17 by congenic mapping

Host genetic variants are known to confer resistance to Plasmodium blood stage infection and to control malaria severity both in humans and mice. This work describes the genetic mapping of a locus for resistance to liver stage parasite in the mouse. First, we show that decreased susceptibility to the liver stage of Plasmodium berghei in the BALB/c mouse strain is attributable to intra-hepatic factors and impacts on the initial phase of blood stage infection. We used QTL mapping techniques to identify a locus controlling this susceptibility phenotype (LOD score 4.2) on mouse chromosome 17 (belr1 locus). Furthermore, analysis of congenic mouse strains delimited the belr1 locus boundaries distally to the H2 region. Quantification of parasites in the liver of infected congenic mice strongly suggested that the belr1 locus represents a genetic factor controlling the expansion of P. berghei in the hepatic tissue. The mapping of belr1 locus raises the hypothesis that host gene variation is able to control the progression of Plasmodium liver stage infection and opens the possibility that the human genomic region orthologue to belr1 may contain genes that confer resistance to the human malaria liver stage.