Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus

We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at approximately 2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales > 100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation.     

Efficient isolation of X chromosome-specific single-copy probes from a cosmid library of a human X/hamster hybrid-cell line: mapping of new probes close to the locus for X-linked mental retardation.

We isolated X-chromosomal DNA probes from a cosmid library constructed from a single human X/hamster hybrid-cell line (C12D). One hundred human clones were isolated and used to construct a pool of X-chromosomal DNA. This DNA was digested into 0.15-2-kb fragments and subcloned into plasmids allowing the rapid characterization of new single-copy probes. These were regionally mapped and used for the detection of restriction-site polymorphisms. Together with a series of subcloned probes from individually isolated cosmids, we found seven polymorphic probes among 53 tested. Thirty-one of the probes were physically localized to different regions of the X chromosome. Four polymorphic probes map to Xq27-Xq28: DXS102 (cX38.1), DXS105(cX55.7), DXS107(cpX234), and DXS134(cpX67). These were genetically mapped by multipoint analysis relative to previously characterized loci, a mapping that resulted in the following order: DXYS1, DXS107, DXS51/DXS102, F9, DXS105, Fra-X, F8/DXS52, DXS15, DXS134. The mapping of DXS105 between F9 and Fra-X makes this probe useful for Fra-X analysis. For the linkage between FraX and DXS105, a maximum lod score of 5.01 at 4 cMorgans has been obtained in one large Dutch pedigree.     

Isolation of new probes from Xq12-->q13: an example of the screening of reference libraries with Alu-PCR products from radiation hybrids.

In order to isolate new probes from the juxtacentromeric region of the long arm of the human X chromosome, we used Alu-mediated polymerase chain reaction (Alu-PCR) products as probes to directly screen a chromosome X-specific gridded cosmid library. These Alu-PCR products were synthesized from radiation hybrids containing the loci DXS159, PGK1, and PGK1P1. This approach allowed us to select 18 cosmids capable of hybridizing with at least two Alu-PCR products. Four cosmids hybridized to more than three Alu-PCR products. Three of these four cosmids were contiguous, and the fourth was independent. Two cosmids that hybridized with two Alu-PCR products were further characterized. Physical mapping indicated that all of these clones are located in the expected region on Xq, confirming the validity of our approach.     

Toward a physical map of the Xq28 region in man: Linking color vision, G6PD, and coagulation factor VIII genes to an X-Y homology region

We are using pulsed-field gel electrophoresis (PFGE) to establish a physical map of the human Xq28 region. We have identified a new probe 35.239 (DXYS64), localized in Xq28 by somatic hybrid mapping and belonging to a region of greater than 99% homology between the X and the Y chromosomes. PFGE data show that probes 35.239 and the polymorphic locus DXS115 (probe 767) map within a common 300-kb BssHII fragment. Both probes, in addition, hybridize to 575-kb BssHII and 590-kb ClaI fragments that contain the gene coding for coagulation factor VIII (F8C). The order F8C-DXS115-DXYS64 could be determined. Our results also provide evidence for linkage between the red/green color vision locus (RCP,GCP) and probes MD13 and T1.7 (GdX, DXS254) within a 750-kb ClaI fragment. Although the latter two probes are located within 50 kb of the 3' end of the G6PD gene, a G6PD cDNA probe did not hybridize to this fragment. G6PD, on the other hand, could be linked to F8C on a 290-kb BssHII fragment. All these data allow us to propose the order (RCP,GCP)-MD13-GdX-G6PD-F8C-DXS115-DXYS 64. We also linked probes St14 (DXS52), MN12 (DXS33), and DX13 (DXS15) to a member of a small family of X-linked dispersed sequences (DNF22S3) within a 575-kb BssHII fragment. The preliminary physical map presented here should be useful for further fine mapping of disease genes in the Xq28 region and should be helpful in orientating efforts toward the cloning of sequences close to the fragile X syndrome.     

Isolation and mapping of discrete DXS101 loci in Xq22 near the X-linked agammaglobulinaemia gene locus

The X-linked agammaglobulinaemia (XLA) gene locus has previously been mapped to Xq22 in genetic linkage studies. The DXS101 locus has shown no recombinations with XLA in the ten informative meioses investigated so far. The DXS101 sequence, recognised by the cX52.5 plasmid, is moderately repeated in Xq22. We have isolated cosmids which contain this sequence; two copies of which have been found to lie near DXS178 and XLA, and a third copy which lies near the PLP gene, distal to these loci. We have used the cosmids to generate probes which should be of use for RFLP analysis, and thus in both prenatal diagnosis and carrier testing for XLA, and in constructing a genetic map of this region. These probes will also be used to complement the genetic map in the construction of a complete physical map of Xq22.     

The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs

The physical distance between DNA sequences in interphase nuclei was determined using eight cosmids containing fragments of the Chinese hamster genome that span 273 kb surrounding the dihydrofolate reductase (DHFR) gene. The distance between these sequences at the molecular level has been determined previously by restriction enzyme mapping (J.E. Looney and J.L. Hamlin, 1987, Mol. Cell Biol. 7: 569-577; C. Ma et al., 1988, Mol. Cell Biol. 8: 2316-2327). Fluorescence in situ hybridization was used to localize the DNA sequences in interphase nuclei of cells bearing only one copy of this genomic region. The distance between DNA sequences in interphase nuclei was correlated to molecular distance over a range of 25 to at least 250 kb. The observed relationship was such that genomic distance could be predicted to within 40 kb from interphase distance. The correct order of seven probes was derived from interphase distances measured for 19 pair-wise combinations of the probes. Measured distances between sequences approximately 200 kb apart indicate that the DNA is condensed 70- to 100-fold in hybridized nuclei relative to a linear DNA helix molecule. Cell lines with chromosome inversions were used to show that interphase distance increases with genomic distance in the 50-90 Mb range, but less steeply than in the 25-250 kb range.     

Physical mapping of the factor VIII gene proximal to two polymorphic DNA probes in human chromosome band Xq28: implications for factor VIII gene segregation analysis

Genomic DNA segments for the coagulation factor VIIIc gene (F8C), which exhibits only limited restriction length polymorphism, map to the proximal region of band Xq28 by somatic cell hybridization analysis and in situ hybridization. Using somatic cell hybrids, we have obtained data which place probes DX13 (used to detect locus DXS15) and St14 (used to detect DXS52) distal to F8C, within band Xq28. Previous studies have mapped the factor IX gene (F9) and probe 52A (used to detect DXS51) proximal to F8C, in Xq26----q27 and Xq27, respectively (Camerino et al., 1984; Drayna et al., 1984; Mattei et al., 1985). Thus, the relative order of genetic marker loci in the Xq27----qter region is most likely cen-F9-DXS51-F8C-(DXS15, DXS52)-Xqter. The collection of these molecular probes is thus potentially useful in three-factor crosses of factor VIII gene segregation.     

A 10-megabase physical map of human Xp21, including the Duchenne muscular dystrophy gene

Using pulsed-field gel electrophoresis and 12 Xp21-derived DNA probes, we have constructed a continuous restriction map spanning more than 4 million base pairs (4 Mbp), including the Duchenne muscular dystrophy gene of more than 2 Mbp. This detailed map is part of a less detailed map spanning 10 Mbp, also spanning the genes for glycerol kinase and congenital adrenal hypoplasia, constructed under electrophoresis conditions which separated DNA fragments in the range 200 to 4000 kbp. DNA from three different tissues was analyzed, and differential methylation was observed.     

Genomic analysis of a region encompassing QRfs1 and QRfs2: genes that underlie soybean resistance to sudden death syndrome.

Candidate genes were identified for two loci, QRfs2 providing resistance to the leaf scorch called soybean (Glycine max (L.) Merr.) sudden death syndrome (SDS) and QRfs1 providing resistance to root infection by the causal pathogen Fusarium solani f.sp. glycines. The 7.5 +/- 0.5 cM region of chromosome 18 (linkage group G) was shown to encompass a cluster of resistance loci using recombination events from 4 near-isogenic line populations and 9 DNA markers. The DNA markers anchored 9 physical map contigs (7 are shown on the soybean Gbrowse, 2 are unpublished), 45 BAC end sequences (41 in Gbrowse), and contiguous DNA sequences of 315, 127, and 110 kbp. Gene density was high at 1 gene per 7 kbp only around the already sequenced regions. Three to 4 gene-rich islands were inferred to be distributed across the entire 7.5 cM or 3.5 Mbp showing that genes are clustered in the soybean genome. Candidate resistance genes were identified and a molecular basis for interactions among the disease resistance genes in the cluster inferred.     

High-resolution FISH on super-stretched flow-sorted plant chromosomes

A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched flow-sorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA]n microsatellite, the [TTTAGGG]n telomeric repeat and a bacterial artificial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5-10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.     

Physical Map of the Saccharomyces Cerevisiae Genome at 110-Kilobase Resolution

A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.     

A physical map of the human salivary proline-rich protein gene cluster covers over 700 kbp of DNA

By using a linking library, we have experimentally linked, ordered, and spaced four of the six loci that constitute the human salivary proline-rich protein (PRP) multigene family. The methods used for mapping these four PRP genes may be useful in other multigene systems in which no probes unique to each member of genes are available, but in which some enzyme site that occurs only once in each member of the family can be found. The remaining two PRP loci have been provisionally mapped and linked within the gene cluster primarily on the basis of the resulting order giving a simple map. The order of the six loci that most simply accounts for our data is PRB2, PRB1, PRB4, PRH2, PRB3, and PRH1. The PRP gene cluster spans at least 700 kbp on chromosome 12 at p13.2. A scheme for the evolution of the cluster that requires an initial gene duplication followed by three unequal but homologous crossovers is given.     

Large-scale isolation of human 1p36-specific P1 clones and their use for fluorescence in situ hybridization

A series of 80 microclone probes derived from the chromosomal region 1p36 was used to isolate corresponding clones from the ICRF human P1 library (see Francis et al., this issue). Hybridization screenings were performed using probe pools on high-density filter grids. A total of 87 P1 clones specific for 1p36 were isolated. This large-scale approach allowed a detailed evaluation of the complexity, quality, and utility of this library. The isolated P1 clones were used both for size determination by pulsed-field gel electrophoresis and as probes for fluorescence in situ hybridization (FISH) analysis. FISH of P1 clones is shown to be both easy and efficient to perform on metaphase chromosomes and interphase nuclei. This observation is expected to reveal new avenues for diagnosis of disease-related chromosomal changes. The use of P1 clones as a tool in clinical and tumor interphase cytogenetics is discussed and compared with FISH data of other long insert clones such as cosmids and YAC clones.     

A 5.5-Mb High-Resolution Integrated Map of Distal 11q13

The distal part of 11q13, which contains several genes relevant to human diseases, has been poorly mapped as part of genome-wide mapping efforts. In the prospect of drawing a fine-scale integrated map of the area containingKRN1andOMP,we have established a framework of markers by hybridization to DNA of somatic cell hybrids and by fluorescencein situhybridization (FISH) on metaphase chromosomes. The probes studied were used to isolate 27 YACs and 16 cosmids that could be organized in three contigs covering approximately 6 Mb. These contigs were separated by two gaps that are likely to contain sequences underrepresented in YAC libraries. They were then integrated based on long-range restriction mapping and DNA-fiber FISH into a high-resolution physical map, which covers a 5.5-Mb region and includes 36 anonymous markers and 10 genes. This map will be used to search for genes within the 2/3 of this region where none have been localized as yet. It will also lay the ground for the characterization of an amplicon surroundingGARPin breast cancer and for the search of disease genes within this region.     

Ornithine aminotransferase-related sequences map to two nonadjacent intervals on the human X chromosome short arm.

Using a panel of human/rodent somatic cell hybrids segregating human X/autosome translocations and deletions, we have refined the localization of the X-linked sequences homologous to ornithine-delta-aminotransferase (OAT), the structural locus for which (OAT) maps to chromosome 10. OAT-related ("-like") (OATL) sequences mapped to two nonadjacent intervals: OATL1 mapped to Xp11.3-p11.23, while OATL2 mapped to Xp11.22-p11.21. X-linked OATL1 sequences polymorphic for ScaI and StuI map to the more distal interval in Xp11.3-p11.23. These results should help guide long-range cloning and mapping studies, as well as refine the genetic linkage map in this region of the X chromosome.     

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.     

The gene for Aarskog syndrome is located between DXS255 and DXS566 (Xp11.2-Xq13).

Aarskog syndrome has been mapped to Xq13 on the basis of a patient carrying an Xq13:8p21.2 translocation. We have identified a new microsatellite marker in a clone mapping to this region (HX60;DXS566). Using primers flanking this microsatellite along with primers detecting a microsatellite at PGK1P1 and DXS255, and DXS72, we have performed a multipoint analysis in a large kindred with Aarskog syndrome. Our results suggest that the Aarskog locus lies proximal to Xq13. This is supported by the recent redefining of the breakpoint of the original translocation as between DXS14 (Xp11.21-p11.1) and DXS146 (Xp11.23-p11.22).