Mapping of the Pim-1 oncogene in mouse t-haplotypes and its use to define the relative map positions of the tcl loci t0(t6) and tw12 and the marker tf (tufted).

Pim-1 is an oncogene activated in mouse T-cell lymphomas induced by Moloney and AKR mink cell focus (MCF) viruses. Pim-1 was previously mapped to chromosome 17 by somatic cell hybrids, and subsequently to the region between the hemoglobin alpha-chain pseudogene 4 (Hba-4ps) and the alpha-crystalline gene (Crya-1) by Southern blot analysis of DNA obtained from panels of recombinant inbred strains. We have now mapped Pim-1 more accurately in t-haplotypes by analysis of recombinant t-chromosomes. The recombinants were derived from Tts6tf/t12 parents backcrossed to + tf/ + tf, and scored for recombination between the loci of T and tf. For simplicity all t-complex lethal genes properly named tcl-tx are shortened to tx. The Pim-1 gene was localized 0.6 cM proximal to the tw12 lethal gene, thus placing the Pim-1 gene 5.2 cM distal to the H-2 region in t-haplotypes. Once mapped, the Pim-1 gene was used as a marker for further genetic analysis of t-haplotypes. tw12 is so close to tf that even with a large number of recombinants it was not possible to determine whether it is proximal or distal to tf. Southern blot analysis of DNA from T-tf recombinants with a separation of tw12 and tf indicated that tw12 is proximal to tf. The mapping of two allelic t-lethals, t0 and t6 with respect to tw12 and tf has also been a problem.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic and Molecular Analysis of the Proximal Region of the Mouse T-Complex Using New Molecular Probes and Partial T-Haplotypes

The t-complex is located on the proximal third of chromosome 17 in the house mouse. Naturally occurring variant forms of the t-complex, known as complete t-haplotypes, are found in wild mouse populations. The t-haplotypes contain at least four nonoverlapping inversions that suppress recombination with the wild-type chromosome, and lock into strong linkage disequilibrium loci affecting normal transmission of the chromosome, male gametogenesis and embryonic development. Partial t-haplotypes derived through rare recombination between t-haplotypes and wild-type homologs have been critical in the analysis of these properties. Utilizing two new DNA probes. Au3 and Au9, and several previously described probes, we have analyzed the genetic structure of several partial t-haplotypes that have arisen in our laboratory, as well as several wild-type chromosomes deleted for loci in this region. With this approach we have been able to further our understanding of the structural and dynamic characteristics of the proximal region of the t-complex. Specifically, we have localized the D17Tul locus as most proximal known in t-haplotypes, achieved a better structural analysis of the partial t-haplotype t6, and defined the structure and lethal gene content of partial t-haplotypes derived from the lethal tw73 haplotype.     

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.     

Genetic analysis of fertility of male mice with various t-haplotypes

Fertility of 47 mouse males carrying various combinations of lethal, t-haplotypes (t6/tw18, t12/tw18, Tw73/tw12, tw5/tw18, t6/dt5, t12/tw12, tw5/twPa-1, tw18/twPa-1, tw5/tw12) was studied in crosses with females of different genotypes. The t-haplotypes studied belong to 7 main groups of complementation. The presence of at least two factors of fertility in the t-complex was revealed. The influence of female genotype on the degree of male fertility was also demonstrated. The data presented confirm that different combinations of lethal complete t-haplotypes exhibit sterility, with the exception of t8/tw18 compound.     

Fine structure DNA mapping studies of the chromosomal region harboring the genetic defect in neurofibromatosis type 1

To better map the location of the von Recklinghausen neurofibromatosis (NF1) gene, we have characterized a somatic cell hybrid designated 7AE-11. This microcell-mediated, chromosome-transfer construct harbors a centromeric segment and a neo-marked segment from the distal long arm of human chromosome 17. We have identified 269 cosmid clones with human sequences from a 7AE-11 library and, using a panel of somatic cell hybrids with a total of six chromosome 17q breakpoints, have mapped 240 of these clones on chromosome 17q. The panel included a hybrid (NF13) carrying a der(22) chromosome that was isolated from an NF1 patient with a balanced translocation, t(17;22) (q11.2;q11.2). Fifty-three of the cosmids map into a region spanning the NF13 breakpoint, as defined by the two closest flanking breakpoints (17q11.2 and 17q11.2-q12). RFLP clones from a subset of these cosmids have been mapped by linkage analysis in normal reference families, to localize the NF1 gene more precisely and to enhance the potential for genetic diagnosis of this disorder. The cosmids in the NF1 region will be an important resource for testing DNA blots of large-fragment restriction-enzyme digests from NF1 patient cell lines, to detect rearrangements in patients' DNA and to identify the 17;22 NF1 translocation breakpoint.     

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.     

Mapping of 50 cosmid clones isolated from a flow-sorted human X chromosome library by fluorescence in situ hybridization.

Fifty cosmids have been mapped to metaphase chromosomes by fluorescence in situ hybridization under conditions that suppress signals from repetitive DNA sequences. The cosmid clones were isolated from a flow-sorted human X chromosome library. Thirty-eight of the clones were localized to chromosome X and 12 to autosomes such as chromosomes 3, 7, 8, 14, and 17. Although most of the cosmids mapped to the X chromosome appeared to be scattered along both the short and long arms, 10 cosmids were localized to the centromeric region of the chromosome. Southern blot analysis revealed that only two of these clones hybridized to probe pXBR-1, which detects the DXZ1 locus. In addition, 4 out of 5 cosmids mapped on chromosome 8 also localized on the centromeric region. While localization of X-specific cosmids will facilitate the physical mapping of the human X chromosome, cosmids mapped to the centromeric regions of chromosomes X and 8 should be especially useful for studying the structure and organization of these regions.     

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.     

Gene for lymphoid enhancer-binding factor 1 (LEF1) mapped to human chromosome 4 (q23-q25) and mouse chromosome 3 near Egf.

LEF-1 is a 54-kDa nuclear protein that is expressed specifically in pre-B and T-cells. It binds to a functionally important site in the T-cell receptor alpha enhancer and contributes to maximal enhancer activity. LEF-1 is a member of a family of regulatory proteins that share homology with the high mobility group protein 1 (HMG1). The location of the LEF1 gene on human and mouse chromosomes was determined by Southern blot analysis of DNA from panels of interspecies somatic cell hybrids using a murine cDNA probe. Human-specific DNA fragments were detected in all somatic cell hybrids that retained the human chromosomal region 4cen-q31.2. Fluorescent in situ hybridization with two biotin-labeled overlapping human genomic cosmids revealed a specific hybridization signal at 4q23-q25. The homologous locus in the mouse was mapped to chromosome 3 by Southern analysis of rodent x mouse hybrid cell DNA. This chromosomal location was confirmed by the use of a restriction fragment length polymorphism (RFLP) in recombinant inbred mouse strains. The results of this RFLP analysis indicated that the mouse Lef-1 gene was closely linked to Pmv-39 and Egf and was likely placed between these loci, both of which were previously mapped to distal mouse chromosome 3. Our mapping results did not suggest involvement of this gene in previously mapped genetic disorders or in known neoplasia-associated translocation breakpoints.     

The molecular organization of the H-2K region of two t-haplotypes: implications for the evolution of genetic diversity.

The genetic diversity between the t12 and tw5 haplotype chromosomes was studied by analyzing the molecular organization of the H-2K region. Twenty-one cosmid clones spanning over 150 kb of the H-2K region of both t-haplotypes were defined, and high resolution restriction maps were determined. Detailed comparison of the t12 and tw5 restriction maps revealed the following. (i) The H-2K regions of both t-haplotypes retain a very similar molecular organization to that reported for B10, BALB/c and AKR. The nucleotide sequence diversity estimated from restriction site polymorphism is 0.68% between the t12 and tw5 haplotypes; these two t-haplotypes are no more similar to one another than BALB/c is to AKR. (ii) Genetic recombination is strongly implicated in generating H-2 polymorphism. (iii) Genetic polymorphisms, defined as small restriction fragment size differences, are observed at multiple sites along the H-2K region. An Alu-like B2 sequence and BAM5-R homologous sequence were identified as the inserted/deleted DNA segments of two of these sites, suggesting that insertion/deletion of mobile elements is a general mechanism for generating genetic diversity.     

Towards a physical map of the Drosophila melanogaster genome: mapping of cosmid clones within defined genomic divisions.

A physical map of the D. melanogaster genome is being constructed, in the form of overlapping cosmid clones that are assigned to specific polytene chromosome sites. A master library of ca. 20,000 cosmids is screened with probes that correspond to numbered chromosomal divisions (ca. 1% of the genome); these probes are prepared by microdissection and PCR-amplification of individual chromosomes. The 120 to 250 cosmids selected by each probe are fingerprinted by Hinfl digestion and gel electrophoresis, and overlaps are detected by computer analysis of the fingerprints, permitting us to assemble sets of contiguous clones (contigs). Selected cosmids, both from contigs and unattached, are then localized by in situ hybridization to polytene chromosomes. Crosshybridization analysis using end probes links some contigs, and hybridization to previously cloned genes relates the physical to the genetic map. This approach has been used to construct a physical map of the 3.8 megabase DNA in the three distal divisions of the x chromosome. The map is represented by 181 canonical cosmids, of which 108 clones in contigs and 32 unattached clones have been mapped individually by in situ hybridization to chromosomes. Our current database of in situ hybridization results also includes the beginning of a physical map for the rest of the genome: 162 cosmids have been assigned by in situ hybridization to 129 chromosomal subdivisions elsewhere in the genome, representing 5 to 6 megabases of additional mapped DNA.     

Evaluation of a cosmid contig physical map of human chromosome 16.

A cosmid contig physical map of human chromosome 16 has been developed by repetitive sequence finger-printing of approximately 4000 cosmid clones obtained from a chromosome 16-specific cosmid library. The arrangement of clones in contigs is determined by (1) estimating cosmid length and determining the likelihoods for all possible pairwise clone overlaps, using the fingerprint data, and (2) using an optimization technique to fit contig maps to these estimates. Two important questions concerning this contig map are how much of chromosome 16 is covered and how accurate are the assembled contigs. Both questions can be addressed by hybridization of single-copy sequence probes to gridded arrays of the cosmids. All of the fingerprinted clones have been arrayed on nylon membranes so that any region of interest can be identified by hybridization. The hybridization experiments indicate that approximately 84% of the euchromatic arms of chromosome 16 are covered by contigs and singleton cosmids. Both grid hybridization (26 contigs) and pulsed-field gel electrophoresis experiments (11 contigs) confirmed the assembled contigs, indicating that false positive overlaps occur infrequently in the present map. Furthermore, regional localization of 93 contigs and singleton cosmids to a somatic cell hybrid mapping panel indicates that there is no bias in the coverage of the euchromatic arms.     

Integration of the Aedes aegypti mosquito genetic linkage and physical maps

Two approaches were used to correlate the Aedes aegypti genetic linkage map to the physical map. STS markers were developed for previously mapped RFLP-based genetic markers so that large genomic clones from cosmid libraries could be found and placed to the metaphase chromosome physical maps using standard FISH methods. Eight cosmids were identified that contained eight RFLP marker sequences, and these cosmids were located on the metaphase chromosomes. Twenty-one cDNAs were mapped directly to metaphase chromosomes using a FISH amplification procedure. The chromosome numbering schemes of the genetic linkage and physical maps corresponded directly and the orientations of the genetic linkage maps for chromosomes 2 and 3 were inverted relative to the physical maps. While the chromosome 2 linkage map represented essentially 100% of chromosome 2, approximately 65% of the chromosome 1 linkage map mapped to only 36% of the short p-arm and 83% of the chromosome 3 physical map contained the complete genetic linkage map. Since the genetic linkage map is a RFLP cDNA-based map, these data also provide a minimal estimate for the size of the euchromatic regions. The implications of these findings on positional cloning in A. aegypti are discussed.     

Four restriction fragment length polymorphisms revealed by probes from a single cosmid map to human chromosome 12q

Summary Human gene mapping would be greatly facilitated if marker loci with sufficient polymorphism information content were generally available. As a source of such markers, we have used cosmids from a human genomic library. We have used a rapid method for screening random cosmids to identify those homologous to genomic regions especially rich in restriction fragment length polymophisms (Litt and White 1985). This method allows whole cosmids to be used as probes against Southern transfers of genomic DNA; regions of cosmid probes homologous to repeated genomic sequences are rendered unable to anneal with Southern transfers by prerendered of the probes with a vast excess of non-radioactive genomic DNA. From one cosmid (C1-11) identified by this procedure, we have isolated four single-copy probes, each of which identifies a polymorphic locus. Despite the existence of some linkage disequilibrium in this system, the polymorthism information content was computed as 0.73. Using a somatic cell hybrid mapping panel, we have mapped probes from cosmid 1–11 to human chromosome 12q. Additionally, in situ hybridization of the whole cosmid to metaphase spreads allowed more precise assignment of the locus to the region 12cenq13. The locus revealed by probes from cosmid 1–11 has been designated D12S6.     

Characterization of a group of transposed human V kappa genes

A genomic region with three V kappa pseudogenes which has been transposed to chromosome 22 is characterized by detailed restriction mapping. A number of subclones are described one of which proved useful to establish an allelic restriction fragment length polymorphism (RFLP) in the region. Allelic and duplication-derived restriction site differences in cosmid clones are discussed with respect to possible problems in genomic walking experiments.     

Characterization of Two Chromosome 12 Cosmid Libraries and Development of STSs from Cosmids Mapped by FISH

We have constructed and characterized two related human chromosome 12-specific cosmid libraries. DNA from flow-sorted chromosomes from a somatic cell hybrid was cloned into a cosmid vector. Approximately 61% of the cosmids in the nearly 26,200 member arrayed libraries (LLt2NC01 and LLt2NC02) contain human DNA inserts, and 31% of the cosmids derived from human DNA contain CA repeats. One hundred and fifty-two cosmids isolated from the libraries have been mapped by fluorescence in situ hybridization (FISH). Cosmids containing human DNA inserts were localized by FISH exclusively to chromosome 12, confirming the chromosomal specificity of the libraries. The cosmids have been localized to all parts of this chromosome, although some regions are more highly represented than others. Partial sequence information was obtained from 44 mapped cosmids, and oligonucleotide primer pairs were synthesized that define unique sequence tagged sites (STSs). These mapped cosmids, and unique STSs derived from them, provide a set of useful clones and primer pairs for screening YAC libraries and developing contigs centered on regions of interest within chromosome 12. In addition, 120 of the mapped cosmids contain CA repeats, and thus they also provide a useful resource for defining highly polymorphic simple tandem repeat elements that serve as genetic markers for linkage analysis and disease gene localization.     

Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes

Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.     

A large inverted duplication allows homologous recombination between chromosomes heterozygous for the proximal t complex inversion

We have examined the molecular organization of a region of mouse chromosome 17 that allows homologous recombination between wild-type and t haplotype chromosomes across a large inversion. We have used a combination of genetic mapping of restriction fragment length polymorphisms, molecular characterization of cloned regions isolated on overlapping cosmids, and subchromosomal restriction mapping using the pulsed field gel technique. Our analyses show that the wild-type form of chromosome 17 contains an inverted duplication of an element of at least 650 kb that is present in only one copy in the t haplotype form. Two chromosomes, th45 and tAE5, arose by homologous recombination across the element that is present in both chromosomal variants in the same orientation.     

Integrating the porcine physical and linkage map using cosmid-derived markers

An essential part in the development of informative linkage maps is to include genetic markers that have been anchored by physical mapping. Here a set of 18 porcine cosmid-derived genetic markers are reported that have been mapped by linkge analysis, and that also have been physically localized by fluorescence in situ hybridization (FISH). Three different strategies were used to establish polymorphic markers from the cosmid clones. Firstly, dinucleotide microsatellite loci were derived by sequencing cosmid subclones containing (CA), repeats. Secondly, variable SINE 3′ poly(A) tracts (SINEVA) were identified by direct SINE-PCR amplification of cosmid clones. Thirdly, the cosmids were used in Southern blot hybridization to detect restriction fragment length polymorphisms (RFLPs). Compared with the most recent consensus compilation of the porcine gene map, the present assignment of markers to chromosomes Zp, 3, 4, 10, 12q, and 16 represents the first loci mapped to these chromosomes, for which linkage as well as in situ data are now available.