Physical mapping of unique nucleotide sequences on identified rice chromosomes

A physical mapping method for unique nucleotide sequences on specific chromosomal regions was developed combining objective chromosome identification and highly sensitive fluorescence in situ hybridisation (FISH). Four unique nucleotide sequences cloned from rice genomic DNAs, varying in size from 1.3 to 400 kb, were mapped on a rice chromosome map. A yeast artificial chromosome (YAC) clone with a 399 kb insert of rice genomic DNA was localised at the distal end of the long arm of rice chromosome (1q2.1) and a bacterial artificial chromosome (BAC) clone (180 kb) containing the rice leaf blast-resistant gene (Pi-b) was shown to occur at the distal end of the long arm of chromosome 2 (2q2.1). A cosmid (35 kb) with the resistance gene (Xa-21) against bacterial leaf blight was mapped on the interstitial region of the long arm on chromosome 11 (11q1.3). Furthermore a single RFLP marker, 1.29 kb in size, was mapped successfully to the distal region of the long arm of rice chromosome 4 (4q2.1). For precise localisation of the nucleotide sequences within the chromosome region, image analyses were effective. The BAC clone was localised to the specific region, 2q2.1:96.16, by image analysis. The result was compared with the known location of the BAC clone on the genetic map and the consistency was confirmed. The effectiveness and reliability in physically mapping nucleotide sequences on small plant chromosomes achieved by the FISH method using a variety of probes was unequivocally demonstrated.     

An optimized set of human telomere clones for studying telomere integrity and architecture

Telomere-specific clones are a valuable resource for the characterization of chromosomal rearrangements. We previously reported a first-generation set of human telomere probes consisting of 34 genomic clones, which were a known distance from the end of the chromosome ( approximately 300 kb), and 7 clones corresponding to the most distal markers on the integrated genetic/physical map (1p, 5p, 6p, 9p, 12p, 15q, and 20q). Subsequently, this resource has been optimized and completed: the size of the genomic clones has been expanded to a target size of 100-200 kb, which is optimal for use in genome-scanning methodologies, and additional probes for the remaining seven telomeres have been identified. For each clone we give an associated mapped sequence-tagged site and provide distances from the telomere estimated using a combination of fiberFISH, interphase FISH, sequence analysis, and radiation-hybrid mapping. This updated set of telomeric clones is an invaluable resource for clinical diagnosis and represents an important contribution to genetic and physical mapping efforts aimed at telomeric regions.     

A sequence-ready map of the human chromosome 17p telomere.

A half-YAC clone derived from human chromosome 17p was mapped at high resolution using cosmid subclone fingerprint analysis. Colinearity of the half-YAC with the telomeric human genomic DNA fragment was ascertained by RecA-assisted restriction endonuclease cleavage mapping. Previously isolated and radiation hybrid-mapped markers TEL17P37, TEL17P49, and TEL17P80 mapped 30-60 kb from the 17p terminus. This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 17p, and will provide the cloned DNA required for ascertaining the nucleotide sequence of this subtelomeric region.     

A sequence-ready map of the human chromosome 1q telomere

A 260-kb half-YAC clone derived from human chromosome 1q was mapped at high resolution using cosmid subclone fingerprint analysis and was integrated with overlapping clones from the telomeric end of a separately derived 1q44 BAC contig to create a sequence-ready map extending to the molecular telomere of 1q. Analysis of 100 kb of sample sequences from across the 260-kb region encompassed by the half-YAC revealed the presence of EST sequence matches corresponding to 12 separate Unigene clusters and to 12 separate unclustered EST sequences. Low-copy subtelomeric repeats typical of many human telomere regions are present within the distal-most 30 kb of 1q. The previously isolated and radiation hybrid-mapped markers Bda84F03, 1QTEL019, and WI11861 localized at distances approximately 32, 88, and 99 kb, respectively, from the 1q terminus. This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 1q and will enable telomeric closure of the human chromosome 1q DNA reference sequence by connecting the molecular 1q telomere to an internal BAC contig.     

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.     

Genetic and physical mapping of telomeres and macrosatellites of rice

Telomeres and telomere-associated satellites of rice were genetically and physically analyzed by pulsed-field gel electrophoresis (PFGE) using Arabidopsis telomeric DNA and rice satellite sequences as probes. We demonstrate that Arabidopsis telomeric sequences hybridize to rice telomeres under the conditions of high stringency. Using the Arabidopsis probe, multiple, discrete telomeric fragments could be identified on pulsed-field gel blots of rice DNAs digested with rare-cutting restriction enzymes. Most of the telomeric bands larger than 300 kb are physically linked with satellite bands as revealed by PFGE. Some of the telomeric and satellite bands segregate in a Mendelian fashion and are highly reproducible. Three such telomeric bands have been mapped to the distal ends of RFLP linkage groups: Telsm-1 on chromosome 8, Telsa-1 on chromosome 9 and Telsm-3 on chromosome 11. One segregating satellite band was mapped to an internal region of chromosome 10. Telomeric fragments were shown not only to be genetically linked to but also physically linked (based on PFGE) to the terminal RFLP markers. The physical distance from telomeric sequences to a distal RFLP marker, r45s gene, on chromosome 9, is 200 kb while the distance from telomeric sequences to RG98, a terminal RFLP marker on chromosome 11, is 260 kb. Physical maps of the telomere regions of chromosome 9 and chromosome 11 are presented.     

Physical and Genetic Mapping of Chromosome 9s in Maize Using Mutations with Terminal Deficiencies

Deletion mapping was employed to determine the physical order of five morphological variants, pyd1, yg2, wd1, v28 and v31, with respect to restriction fragment length polymorphism (RFLP) markers located at the distal end of chromosome 9S in maize. The genetic materials used were a series of terminal-deficiency mutants, newly derived with MCCLINTOCK's original stocks developed in the 1940s, via break-age-fusion-bridge cycles. A combined physical map and genetic map has been constructed based on data gathered from both genetic complementation tests and RFLP analysis. The location of v31 in relation to RFLP markers was further determined by interval mapping. The physical distance between the healed telomeric end and the most distal RFLP marker in two terminal-deficiency lines was established by using pulsed field gel electrophoresis and verified by Bal31 digestion. The results from this study set a foundation for studies on the mechanism of healing of broken chromosome ends in higher plants.     

Characterization of the promoter controlling Mona/Gads expression in the megakaryocytic lineage

The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a 'mirror' inverted duplication of the 'right' end of chromosome 1a (approximately 25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over approximately 105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb 'switch' region) was found. By contrast, XC155 contains two large (approximately 13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp 'satellite' DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after >150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp 'satellite' DNA bound by telomeric repeats.     

The Saccharomyces cerevisiae chromosome III left telomere has a type X, but not a type Y'', ARS region.

A yeast Saccharomyces cerevisiae telomeric region was isolated by chromosome walking from HML alpha, the most distal known gene on the chromosome III left (IIIL) end. The terminal heterodisperse 3.3-kilobase (kb) SalI fragment on chromosome IIIL, 8.6 kb distal to HML alpha, was cloned in a circular vector to generate a telomeric probe. Southern hybridization and DNA sequencing analyses indicated that 0.6 kb (+/- 200 base pairs) of 5'-C1-3A-3' simple tandem repeat sequence, adjacent to a 1.2-kb type X ARS region, constitutes the telomere on the chromosome IIIL end, and no type Y' ARS region homologies exist between HML alpha and the IIIL terminus.     

Cloning a fragment from the telomere of the long arm of human chromosome 9 in a YAC vector

The construction of a yeast artificial chromosome containing a human DNA insert is reported. This molecule of about 200 kb behaves as a native yeast chromosome since it has a very high mitotic stability and is present in the yeast transformant clone at a copy number similar to that of the resident chromosomes. Hybridization with the TTAGGG sequence demonstrates that this chromosome contains human telomeric sequences. In situ hybridization of the biotin-labelled artificial chromosome to metaphase human chromosomes shows that the insert occupies a telomeric position on the long arm of chromosome 9. Since the fragment was cloned as an EcoRI insert and not as a telomere, it is situated medially to the telomeric sequences and harbours telomere-associated sequences, that have been shown to contain the TTAGGG sequence. The fragment represents the end of the genetic map of chromosome 9 and thus can be used to characterize the sequence and the structure of the chromosomal region that runs from the end of the chromosome to the first gene.     

Genetic Polymorphism and Recombination in the Subtelomeric Region of Chromosome 14q

Subtelomeric regions of human chromosomes are the sites of increased meiotic recombination and have a male-to-female recombination ratio that is higher than elsewhere in the genome. We isolated two novel, polymorphic CA repeat markers from the distal part of the immunoglobulin heavy chain gene cluster, approximately 90 and 200 kb from the telomere of chromosome 14q. The 14q telomere was unambiguously located by physical mapping of telomeric YACs andBal31 exonuclease digestion of genomic DNA. We then constructed haplotypes using genotype data from these markers and data from sCAW1 (D14S826) for use as a highly polymorphic genetic marker. Linkage analysis using the 40 pedigree CEPH reference panel and genotype data from these and other loci physically mapped to the terminal 1.5 Mb of chromosome 14q revealed an apparent increase in meiotic recombination within this region, relative to the average rate for the genome. Further, we found that recombination was higher in females than in males, indicating that the subtelomeric region of 14q differs from other human subtelomeric regions.     

Direct cloning of a long restriction fragment aided with a jumping clone.

Long-range physical mapping with rare-cutting restriction enzymes (rare cutters) is an important step for structural analysis of complex genomes. Combination of two types of DNA clones bearing the rare-cutter sites, linking clones and jumping clones (Fig. 1a), facilitates the physical mapping [Poustka et al., Nature 325 (1987) 353-355]. A step followed by the physical mapping is the cloning of the large (rare-cutter-generated) restriction fragment of interest. For facilitating this step, we devised a method to directly clone a long restriction fragment without constructing the whole genomic DNA library using the jumping clone as starting material. The short DNA segments of a jumping clone, which are derived from the 5' and 3' terminal regions of the large restriction fragment, are inserted into the yeast artificial chromosome plasmid (pYAC) vector, and then converted into single strands with T7 gene 6-encoded 5'----3' exonuclease. The total genomic DNA digested with the restriction enzyme is also treated with the exonuclease to convert the terminal regions of the restriction fragments into single strands. In the resulting products, only the fragment corresponding to the jumping clone can form hybrids with the just-mentioned, single-stranded DNAs, which are connected to the pYAC, and only this fragment is cloned in yeast. We describe the protocol of this method with Escherichia coli DNA as a model experiment. Judging from the cloning efficiency, this method could be applied to cloning single-copy regions of the human genome, provided a jumping clone is available. The instability of inserts in the pYAC vector is also discussed.     

Molecular genetic analysis of theripening-inhibitor andnon-ripening loci of tomato: A first step in genetic map-based cloning of fruit ripening genes

Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato,ripening-inhibitor (rin), andnon-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning therin andnor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning therin locus is estimated to be 200–300 kb/cM, while thenor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked torin andnor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains thenot locus.     

Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS).

The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1).     

Long-range restriction map of a region of human chromosome 19 containing the apolipoprotein genes,a CLL-associated translocation breakpoint,and two polymorphic MluI sites

Summary The apolipoprotein gene cluster on human chromosome 19 (APOC1, APOC2, APOE) has been localised by pulsed-field gel electrophoresis to within 200 kb of a chronic lymphocytic leukemia-associated translocation breakpoint. A restriction map covering 1300 kb around these loci has been constructed and contains two polymorphic MluI sites, which appear to show Mendelian inheritance. The orientation of the map on the chromosome has been established as 19cen CLL breakpoint-APOC2-19qter. Pedigree analysis using APOC2, a probe derived from the CLL breakpoint, and other localised markers on 19q suggests that the myotonic dystrophy locus is distal to APOC2 on 19q.     

Fidelity of a YAC clone in the region of human MCF-2 gene.

A cosmid library was constructed from a YAC clone (XY311) carrying an insert of 650 kb from the F IX/mcf-2 region on human X chromosome. A contig of 200 kb that includes the mcf-2 gene and the genomic region downstream was assembled. Eighty kb of this contig represents a chromosome fragment already cloned and analyzed in detail with conventional restriction enzymes: comparison with this published map suggests that this region was correctly maintained during the procedure of YAC cloning. A discrepancy between the published map and the cloned YAC material was identified, but it resulted to be an EcoRI polymorphism present in the X3000.11 from which the YAC library was derived. The 3' portion of this contig, representing the telomeric end of the YAC XY311, provides new cosmid material for further analysis of the region downstream of the mcf 2 locus.     

The human dopamine D2 receptor gene is located on chromosome 11 at q22-q23 and identifies a TaqI RFLP.

Human dopaminergic neurons are involved in the control of hormone secretion, voluntary movement, and emotional behavior. Mediating these effects are the dopamine D1 and D2 receptors. These macromolecules belong to a large family of related sequences known as the G protein-coupled receptors. The D2 receptors have been of special interest because they bind, with high affinity and specificity, many of the commonly prescribed antipsychotic drugs. We previously isolated a full-length cDNA clone of the rat D2 receptor. When a chromosome mapping panel was probed with the rat D2 receptor cDNA a 15-kb EcoRI restriction fragment was identified and localized to human chromosome 11. The rat cDNA was also used to clone a human genomic fragment, lambda hD2G1, which contains the last coding exon of the D2 receptor gene (DRD2) and 16.5 kb of 3' flanking sequence. Hybridization of lambda hD2G1 to a chromosome 11 regional mapping panel localized DRD2 to 11q. In situ hybridization of lambda hD2G1 to metaphase chromosomes refined this assignment to the q22-q23 junction of chromosome 11. A search for RFLPs associated with D2DR identified a frequent two-allele TaqI RFLP.     

RXRA and HSPA5 map to the telomeric end of dog chromosome 9

Previous results showed that loci from human chromosome 17q (HSA17q) map to the centromeric two-thirds of dog chromosome 9 (CFA9). In these studies fluorescence in situ hybridization (FISH) using a human total chromosome 17 painting probe, indicated that the telomeric one-third of CFA9 must have homology to one or more human chromosomes other than HSA17. Here we report that this distal part of CFA9 contains a segment syntenic to the telomeric end of HSA9q and mouse chromosome 2 (MMU2). The gene loci encoding retinoid X receptor, alpha (RXRA) and heat shock protein 5 (HSPA5 or GRP78), which are found on HSA9q34 and MMU2, occupy a region on CFA9 distal to NF1 and CRYBA1. FISH of a canine specific genomic cosmid clone for RXRA demonstrated the more telomeric localization of this locus to NF1 on CFA9. A linkage map developed for the distal region of CFA9 included: NF1-(2·7 CM )-CRYBA1-(6·5 CM )-RXRA-(22 CM )-HSPA5. The next best order, RXRA-NF1-CRYBA1-HSPA5 with a difference in the log odds of 1·43 does not correspond to our findings with FISH. The most probable map order places HSPA5 distal to RXRA on CFA9 whereas in humans it lies centromeric of RXRA on HSA9q34.     

Map-based cloning in crop plants: tomato as a model system II. Isolation and characterization of a set of overlapping yeast artificial chromosomes encompassing the jointless locus

A map-based cloning technique for crop plants is being developed using tomato as a model system. The target gene jointless is a recessive mutation that completely suppresses the formation of flower and fruit pedicel abscission zones. Previously, the jointless locus was mapped to a 3 cM interval between the two molecular markers TG523 and RPD158. Physical mapping of the jointless region by pulsed-field gel electrophoresis demonstrated that TG523 and RPD158 reside on a 600 kb SmaI fragment. In this study, TG523 was used as a probe to screen a tomato yeast artificial chromosome (YAC) library. Six tomato YAC (TY) clones were isolated, ranging from 220 to 380 kb in size. Genetic mapping of YAC ends demonstrated that this set of overlapping YACs encompasses the jointless locus. Two YAC ends, TY159L (L indicates left end) and TY143R (R indicates right end), cosegregate with the jointless locus. Only one of the six YACs (TY142) contained single-copy DNA sequences at both ends that could be mapped. The two ends of TY142 were mapped to either side of the jointless locus, indicating that TY142 contains a contiguous 285 kb tomato DNA fragment that probably includes the jointless locus. Physical mapping of the TY142 clone revealed that TY159L and TY143R reside on a 55 kb SalI fragment. Southern blot hybridization analysis of the DNAs of tomato lines nearly isogenic for the jointless mutation has allowed localization of the target locus to a region of less than 50 kb within the TY142 clone.Communicated by H. Saedler     

Cloning and identification of the H-2D p gene

We have cloned six different class I genes from a B10.P sperm library. After cotransfection with the herpes simplex tk gene, one L-cell line was found to react with six H-2D^p-specific monoclonal antibodies. The cell line L12a did not react with K^p-specific monoclonal antibodies. This identification was confirmed by mapping a 2.5 kb Bam H 1 restriction fragment present in the 12a DNA clone to the D-TL region of H-2 ^p. Only a single 8.8 kb Barn H1 fragment can be assigned to K ^p by restriction fragment length polymorphism, while many others map to the D-TL interval. A restriction map of 12a is presented.