Chiasma-based genetic map of the mouse X chromosome

The X chromosome pair was identified in diakinesis/metaphase I stage mouse oocytes using a repeat sequence DNA probe and fluorescence in situ hybridisation. Chiasma positions along the X bivalent were measured in 57 oocytes from 4 females. Overall, our observations showed that while there were no obvious hotspots for chiasma formation along the X chromosome, there was a tendency to favour the distal end. Minimum inter-chiasma distances were substantial indicating the occurrence of strong genetic interference. Estimates of both genetic distances and recombination fractions for any interval along the chromosome can be calculated from the chiasma data. The average chiasma frequency for the X bivalent was 1.37 giving an estimated total genetic map length of 68.5 cM. In general, the pattern of chiasma distribution along the X chromosome resembled that anticipated from recombination distances in published consensus linkage maps. There were, however, some intriguing differences between the two approaches. The reason for these discrepancies are unknown but may be related to lack of precision in cytogenetic mapping of loci, inter-strain and/or interspecies differences in the genetic controls over the distribution of crossover events. One advantage of the chiasma analysis approach is its suitability for investigating these problems.     

Male mouse recombination maps for each autosome identified by chromosome painting

Linkage maps constructed from genetic analysis of gene order and crossover frequency provide few clues to the basis of genomewide distribution of meiotic recombination, such as chromosome structure, that influences meiotic recombination. To bridge this gap, we have generated the first cytological recombination map that identifies individual autosomes in the male mouse. We prepared meiotic chromosome (synaptonemal complex [SC]) spreads from 110 mouse spermatocytes, identified each autosome by multicolor fluorescence in situ hybridization of chromosome-specific DNA libraries, and mapped >2,000 sites of recombination along individual autosomes, using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites. We show that SC length is strongly correlated with crossover frequency and distribution. Although the length of most SCs corresponds to that predicted from their mitotic chromosome length rank, several SCs are longer or shorter than expected, with corresponding increases and decreases in MLH1 frequency. Although all bivalents share certain general recombination features, such as few crossovers near the centromeres and a high rate of distal recombination, individual bivalents have unique patterns of crossover distribution along their length. In addition to SC length, other, as-yet-unidentified, factors influence crossover distribution leading to hot regions on individual chromosomes, with recombination frequencies as much as six times higher than average, as well as cold spots with no recombination. By reprobing the SC spreads with genetically mapped BACs, we demonstrate a robust strategy for integrating genetic linkage and physical contig maps with mitotic and meiotic chromosome structure.     

The mendelian inheritance of a human X chromosome-specific DNA sequence polymorphism and its use in linkage studies of genetic disease

Summary A recombinant DNA sequence, RB6, was isolated from a human X chromosome library and shown to be X-specific by hybridisation to DNA from a human-mouse somatic cell hybrid containing X as the only human chromosome. The cloned sequence was located on the long arm distal to Xq13 using a human-mouse somatic cell hybrid containing a partial human X chromosome. DNA samples isolated from control human females were digested with the restriction enzyme MspI, and analysed by blotting and hybridisation to the radioactive cloned DNA. Eight of 14 individuals from a random population showed a single hybridising band 7.5 kilobase pairs (kb) in length, but six showed an additional band 10.1 kb in length. DNA from 12 members of a family with X-linked thyroxine-binding globulin deficiency was analysed for the segregation of this polymorphism. The results show that the polymorphism is inherited in a Mendelian fashion, and that the disease locus is not closely linked to the polymorphic site. Such polymorphisms will be useful as markers for chromosome mapping and for the antenatal diagnosis of genetic diseases.     

The use of cloned Y chromosome-specific DNA probes for fetal sex determination in first trimester prenatal diagnosis

Summary Prenatal diagnosis by chorion biopsy in the first trimester of pregnancy has advantages over second trimester amniocentesis because diagnosis can be achieved at 9–12 weeks gestation, reducing prenatal anxiety and avoiding the trauma of late abortion. DNA can be prepared from chorionic villus biopsies in sufficient quantity and purity for use in prenatal diagnosis systems using specific DNA probes hybridised to restriction endonuclease digests.DNA probes derived from the Y chromosome have been used to determine fetal sex. The use of such probes means that the chromosomal sex of the fetus can be identified more quickly than by chromosome preparation and more accurately than by sex chromatin staining, and has the additional advantage that the same DNA preparation can be used for other diagnostic tests. A dot hybridisation method has been successfully used to provide even more rapid results than conventional hybridisation to Southern blots of restriction endonuclease digests.There is a risk that Y chromosome-specific DNA probes for sex determination may be subject to error if the parents have extreme Y chromosome variants such as a small or non-fluorescent Y or a Y autosome chromosome translocation. The precise extent to which such chromosome variants may lead to error has been investigated. Even extreme Y chromosome variants totally lacking fluorescence were identified as male by the cloned probes used. However, Y autosome translocations carried by females could cause error if not identified in the parents. The value of the probes has been confirmed provided that parental chromosomes and DNA are examined in parallel with the chorionic biopsy material     

Selection of human chromosome 21-specific DNA probes for genetic analysis in Alzheimer's dementia and Down syndrome

Summary We used a mouse-human somatic cell hybrid to construct a chromosome 21-enriched library in phage vector EMBL4. In all, 35 phage clones containing human inserts were identified by differential screening with total human and mouse DNA. Whole recombinant phages were regionally mapped on chromosome 21 by Southern blot analysis using competitive hybridisation conditions to block repetitive sequences. Ten phage clones mapped proximal to a translocation breakpoint in band 21q21.2, while 25 mapped distal to this point. Three of the phage clones identify restriction fragment length polymorphisms. Polymorphic chromosome 21 markers may be useful in the genetic analysis of Alzheimer's dementia and Down syndrome.     

Mini- and micro-satellites in the genome of rodent malaria parasites.

Higher eukaryotes contain within their DNA numerous arrays of repetitive DNA, many of which are known as satellite DNAs and display extensive variability. The presence of these repeats has been demonstrated for various species and they have been used for genetic identification and classification. Here, it is demonstrated that Southern hybridisation of DNA from rodent malaria parasites allows detection of micro- and minisatellite sequences in the genome of Plasmodium species. Closely related lines of malaria parasites exhibit a monomorphic hybridisation pattern, which is in contrast to the allelic variation observed in higher eukaryotes. Among different species, however, restriction-fragment length polymorphism was observed. Pulsed-field gel electrophoretic chromosome separation showed that the probes used in this study [33.15, 33.6, (CAC)n and (GT)n] detect several loci spread over different chromosomes.     

Use of restriction fragment length polymorphism to fingerprint beets at the genotype and species levels

Summary cDNA probes have been used to assess genetic variation in beet using hybridisation techniques that detect restriction fragment length polymorphism. Probes have been identified which differ in the levels of variation that they can detect (i) within closely related genetic material of sugar beet, and (ii) between sugar beet and a taxonomically distant Beta species.     

Progress towards construction of a total restriction fragment map of a human chromosome.

We present an approach to the construction of an overlapping restriction fragment map of a single human chromosome. A genomic cosmid library genome was constructed from a mouse-human hybrid cell line containing chromosome 17 as its only human genetic component. Cosmids containing human inserts were isolated by hybridisation to a human Alu sequence. DNAs from ninety-six randomly chosen cosmids were digested with either EcoRI or HindIII, end-labelled with 35S-dATP and analysed using agarose gel electrophoresis. Comparison of the restriction fragment patterns revealed two pairs of overlapping clones, that were confirmed by cross-hybridization of the overlapping fragments. The two pairs of cosmids both mapped to human chromosome 17, as shown by hybridization to a panel of somatic cell hybrids. These data demonstrate that the generation of an overlapping cosmid map along a human chromosome is feasible, representing an intermediate step towards the complete sequencing of a human chromosome.     

The genomic complexity of micro B chromosomes of Brachycome dichromosomatica

A major sequence component of the micro B chromosome of Brachycome dichromosomatica (2 n=4) is the tandem repeat Bdm29, which was found by in situ hybridisation to be distributed along the entire length of the chromosome. A high copy number of this sequence does not occur as a regular feature of the A chromosomes in this species but it was found in infrequent individuals in two wild populations that were analysed. In these instances Bdm29 is localised within heterochromatic, polymorphic segments on the long arm of chromosome 1. The origin of the micro B chromosomes was investigated by determining whether they are related to this A chromosome polymorphism by simple excision and/or integration. Results obtained by using Bdm29, together with a newly isolated repeat sequence, Bdm54, and a number of other sequences known to occur on the micro B chromosome, as probes in in situ hybridisation and Southern analysis demonstrated that the formation of micro B chromosomes is a complex multistep process. The observation that the genomic organisation of the micro B chromosome is unlike anything found on the A chromosomes precludes their origin by simple excision and also indicates that micro Bs do not integrate directly into the A complement to form polymorphic heterochomatic segments.     

Physical mapping of rDNA genes establishes the karyotype of almond

The localisation of ribosomal RNA genes on chromosomes of almond (Prunus amygdalus, 2n = 16) was studied by fluorescence in situ hybridisation. Simultaneous double-colour hybridisation with both 18S–5.8S–25S and 5S rDNA probes demonstrated that all chromosomes can be identified. In spite of the small size, differences in length between chromosomes that hybridised with the same rDNA probe as well as between chromosomes without hybridisation signal are apparent. Chromosomes were ordered in the karyotype according to their length. The 18S-5.8S-25S rDNA genes were detected in subdistal positions of chromosomes 2, 3, and 8. Sites located on chromosomes 2 and 3 carry a higher number of repeats than the site of chromosome 8. The 5S rDNA genes were found proximally located on chromosomes 5 and 7, the signal on chromosome 5 showing higher intensity than the signal on chromosome 7. Chromosomes 1, 4, and 6 show no hybridisation signal.     

Detection of interspecies hybridisation in Chondrichthyes: hybrids and hybrid offspring between Australian (Carcharhinus tilstoni) and common (C. limbatus) blacktip shark found in an Australian fishery

Interspecies hybridisation in nature is a well-studied phenomenon, but it has not been analysed using genetic markers in the class Chondrichthyes (sharks, rays and chimeras). Two black-tip whaler shark species (Australian, Carcharhinus tilstoni; Common, C. limbatus) have overlapping distributions in Australia, distinct mitochondrial DNA sequence (ND4, COI, control region) and distinct morphological features such as length at sexual maturity, length at birth and number of vertebrae. A mismatch was observed between species identification using mtDNA sequence and species identification using morphological characters. To test whether hybridisation between the two species was responsible, a nuclear gene with species-specific mutations was sequenced. Extensive interspecies hybridisation was found to be occurring. Hybrids were found from five locations on the eastern Australian coastline, spanning 2,000 km. If hybrid fitness is low and hybrids are common, then fisheries recruitment may be overestimated and the productivity of the black-tip shark fishery may be well below that required to support commercial exploitation. To guard against identification errors, the likelihood of hybridisation and subsequent introgression should be assessed prior to using mtDNA (e.g. barcoding) to identify shark species. The C. limbatus–C. tilstoni species complex provides a unique opportunity to investigate the ability of sharks to adapt to environmental change, in particular, the impact of hybridization on species distributions which favour C. tilstoni along the north and C. limbatus along the south eastern Australian coastline.     

Isolation of polymorphic DNA segments from human chromosome 21.

A somatic cell hybrid line containing only human chromosome 21 on a mouse background has been used as the source of DNA for construction of a recombinant phage library. Individual phages containing human inserts have been identified. Repeat-free human DNA subclones have been prepared and used to screen for restriction fragment length polymorphisms to provide genetic markers on chromosome 21. Nine independently isolated clones used as probes identified a total of 11 new RFLPs. Four of the DNA probes recovered from the library have been mapped unequivocally to chromosome 21 using a panel of somatic cell hybrid lines. A fifth probe detected an RFLP on chromosome 21 as well as sequences on other chromosomes. This set of RFLPs may now form the basis for construction of a genetic linkage map of human chromosome 21.     

Mapping of mouse carbonic anhydrase-3, Car-3: another locus in the homologous region of mouse chromosome 3 and human chromosome 8

At least six separate genes determining tissue- and organelle-specific isoforms of carbonic anhydrase are known. We have determined the chromosome location of one of these genes, carbonic anhydrase-3 (Car-3), in the mouse and carried out a linkage analysis of Car-1, Car-2, and Car-3. Car-3 has been assigned to band 3A2 by in situ hybridization. We identified a PstI restriction fragment length polymorphism between Mus spretus and Mus mus domesticus and, by using an interspecific backcross, showed that Car-3 is 2.4 +/- 1.7% SE from both Car-1 and Car-2, calculating genetic distance as percentage recombination. No recombinants were found between Car-1 and Car-2 in 100 backcross offspring, and when these data are combined with earlier results, these two loci are estimated to be 1.2 cM from each other at the 95% confidence interval. The three homologous carbonic anhydrase loci in man had earlier been assigned to 8q22, and the finding of linkage of Car-3 to Car-1 and Car-2 in the mouse adds another locus to the conserved segments on mouse chromosome 3 and human chromosome 8.     

Identification,characterisation and clinical applications of cosmids from the telomeric and centromeric regions of the long arm of chromosome 22

Using human telomeric repeats and centromeric alpha repeats, we have identified adjacent single copy cosmid clones from human chromosome 22 cosmid libraries. These single copy cosmids were mapped to chromosome 22 by fluorescence in situ hybridisation (FISH). Based on these cosmids, we established contigs that included part of the telomeric and subtelomeric regions, and part of the centromeric and pericentromeric regions of the long arm of human chromosome 22. Each of the two cosmid contigs consisted of five consecutive steps and spanned approximately 100–150 kb at both extreme ends of 22q. Moreover, highly informative polymorphic markers were identified in the telomeric region. Our results suggest that the telomere specific repeat (TTAGGG) _n encompasses a region that is larger than 40 kb. The cosmid contigs and restriction fragment length polymorphism markers described here are useful tools for physical and genetic mapping of chromosome 22, and constitute the basis of further studies of the structure of the subtelomeric and pericentromeric regions of 22q. We also demonstrate the use of these clones in clinical diagnosis of different chromosome 22 aberrations by FISH.     

The human aldose reductase gene maps to chromosome region 7q35

Summary The human aldose reductase (AR) gene has been mapped to chromosome 7 using the polymerase chain reaction to specifically amplify the human AR sequence in hamster/human hybrid DNA and also in mouse/ human monochromosome hybrids. The assignment to chromosome 7 was confirmed by in situ hybridisation to human metaphase chromosomes using a novel, rapid hybridisation, method giving a regional localisation at 7q35.     

A comparison of physical distribution of recombination in chromosome 1R in diploid rye and in hexaploid triticale

Summary Polymorphism for six C-bands on chromosome 1R was used to study the frequency and distribution of recombination along the chromosome in a diploid rye (Secale cereale L.) and in a hexaploid triticale (X Triticosecale Wittmack) derived from it. In rye, the total recombination frequency in five segments of chromosome 1R was 93.7%. Recombination was concentrated in the distal regions of both chromosome arms and was infrequent in the proximal regions. In hexaploid triticale the total recombination frequency in the same chromosome was reduced to 51.7%. In both backgrounds the distal half of the long arm showed similar recombination frequencies, 51.4% and 45.7% for rye and triticale, respectively. The remaining about two-thirds of the chromosome length showed 42.3% recombination in rye but only 6% recombination in triticale. The results demonstrate that the genetic background in which mapping is performed not only affects the total amount of recombination, but also its distribution along the chromosome length.     

Genetic analysis and FISH mapping of the Colourless non-ripening locus of tomato

Cnr (Colourless non-ripening) is a dominant pleiotropic ripening mutation of tomato (Lycopersicon esculentum) which has previously been mapped to the proximal region of tomato chromosome 2. We describe the fine mapping of the Cnr locus using both linkage analysis and fluorescence in situ hybridisation (FISH). Restriction fragment length polymorphism (RFLP)-, amplified restriction fragment polymorphism (AFLP)-, and cleaved amplified polymorphic sequence (CAPS)-based markers, linked to the Cnr locus were mapped onto the long arm of chromosome 2. Detailed linkage analysis indicated that the Cnr locus was likely to lie further away from the top of the long arm than previously thought. This was confirmed by FISH, which was applied to tomato pachytene chromosomes in order to gain an insight into the organisation of hetero- and euchromatin and its relationship to the physical and genetic distances in the Cnr region. Three molecular markers linked to Cnr were unambiguously located by FISH to the long arm of chromosome 2 using individual BAC probes containing these single-copy sequences. The physical order of the markers coincided with that established by genetic analysis. The two AFLP markers most-closely linked to the Cnr locus were located in the euchromatic region 2.7-cM apart. The physical distance between these markers was measured on the pachytene spreads and estimated to be approximately 900 kb, suggesting a bp:cM relationship in this region of chromosome 2 of about 330 kb/cM. This is less than half the average value of 750 kb/cM for the tomato genome. The relationship between genetic and physical distances on chromosome 2 is discussed. Received: 11 January 2001 / Accepted: 30 April 2001     

Genetic and cytogenetic localisation of the homeo box containing genes on mouse chromosome 6 and human chromosome 7.

Probes from the m6 homeo box cluster were mapped to mouse chromosome 6 by somatic cell genetics, in situ hybridisation, and by a Mus spretus--Mus musculus backcross mapping system. In addition, the testis-specific homeo box containing cDNA, clone, HBT-1, has been mapped using the same back-cross system and the B X D recombinant inbred strain set. Close genetic and physical linkage between the m6 cluster and HBT-1 was demonstrated, positioning these sequences to the same local cluster of homeo box containing genes. The map location of this cluster between IgK and Tcrb coincides with the morphological mutation hypodactyly (Hd). Synteny has been observed between a region of mouse chromosome 6 and the long arm of human chromosome 7 encompassing the markers Cpa, Tcrb and Try-1. Here we localise human sequences hybridising to the mouse m6 probes to the short arm of chromosome 7, breaking the region of synteny.     

Integration of the Physical and Genetic Linkage Map for Human Chromosome 13

We have used a panel of somatic cell hybrids containing different rearrangements of human chromosome 13 to integrate genetic and physical maps of this chromosome. The positions of 17 translocation/deletion breakpoints on human chromosome 13 have been determined relative to the microsatellite markers on the genetic linkage map compiled by Généthon. Because markers on maps from several other Consortium groups have also been analyzed using many of the same hybrids, it was possible to relate these with the Généthon map. The position of all of the chromosome breakpoints have been placed, wherever possible, between two adjacent markers on the genetic linkage maps using PCR analysis for the presence/absence of the markers in the somatic cell hybrids. The positions of the breakpoints have already been determined cytogenetically, and some of these breakpoints are located at landmark positions on the chromosome. The relative density of markers along the chromosome differs between independently derived maps, and, based on the known locations of certain breakpoints in the physical map, inconsistencies in the genetic maps have been identified.