Construction of an 800-kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta2 using a highly representative rice BAC library

We constructed a rice Bacterial Artificial Chromosome (BAC) library from green leaf protoplasts of the cultivar Shimokita harboring the rice blast resistance gene Pi-ta. The average insert size of 155 kb and the library size of seven genome equivalents make it one of the most comprehensive BAC libraries available, and larger than many plant YAC libraries. The library clones were plated on seven high density membranes of microplate size, enabling efficient colony identification in colony hybridization experiments. Seven percent of clones carried chloroplast DNA. By probing with markers close to the blast resistance genes Pi-ta2(closely linked to Pi-ta) and Pi-b, respectively located in the centromeric region of chromosome 12 and near the telomeric end of chromosome 2, on average 2.2 +/- 1.3 and 8.0 +/- 2.6 BAC clones/marker were isolated. Differences in chromosomal structures may contribute to this wide variation in yield. A contig of about 800 kb, consisting of 19 clones, was constructed in the Pi-ta2 region. This region had a high frequency of repetitive sequences. To circumvent this difficulty, we devised a "two-step walking" method. The contig spanned a 300 kb region between markers located at 0 cM and 0.3 cM from Pi-ta. The ratio of physical to genetic distances (> 1,000 kb/cM) was more than three times larger than the average of rice (300 kb/cM). The low recombination rate and high frequency of repetitive sequences may also be related to the near centromeric character of this region. Fluorescent in situ hybridization (FISH) with a BAC clone from the Pi-b region yielded very clear signals on the long arm of chromosome 2, while a clone from the Pi-ta2 region showed various cross-hybridizing signals near the centromeric regions of all chromosomes.     

A bacterial artificial chromosome-based framework contig map of human chromosome 22q

We have constructed a physical map of human chromosome 22q using bacterial artificial chromosome (BAC) clones. The map consists of 613 chromosome 22-specific BAC clones that have been localized and assembled into contigs using 452 landmarks, 346 of which were previously ordered and mapped to specific regions of the q arm of the chromosome by means of chromosome 22-specific yeast artificial chromosome clones. The BAC-based map provides immediate access to clones that are stable and convenient for direct genome analysis. The approach to rapidly developing marker-specific BAC contigs is relatively straightforward and can be extended to generate scaffold BAC contig maps of the rest of the chromosomes. These contigs will provide substrates for sequencing the entire human genome. We discuss how to efficiently close contig gaps using the end sequences of BAC clone inserts.     

Effect of prostaglandin F2 alpha treatment before norgestomet and estradiol valerate treatment on regression, formation, and function of corpora lutea in beef heifers

Despite the presence of several human disease genes on chromosome 11q13, few of them have been molecularly cloned. Here, we report the construction of a contig map encompassing 11q13.1-q13.3 using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC). The contig map comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and 1 YAC clone and spans a 3-Mb region from D11S480 to D11S913. The map encompasses all the candidate loci of Bardet-Biedle syndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5), one-third of the distal region for hereditary paraganglioma 2 (PGL2), and one-third of the central region for insulin-dependent diabetes mellitus 4 (IDDM4). In the process of map construction, 61 new sequence-tagged site (STS) markers were developed from the Not I linking clones and the termini of clone inserts. We have also mapped 30 ESTs on this map. This contig map will facilitate the isolation of polymorphic markers for a more refined analysis of the disease gene region and identification of candidate genes by direct cDNA selection, as well as prediction of gene function from sequence information of these bacterial clones.     

Physical mapping of the liguleless linkage group in Sorghum bicolor using rice RFLP-selected sorghum BACs

Physical mapping of BACs by fluorescent in situ hybridization (FISH) was used to analyze the liguleless (lg-1) linkage group in sorghum and compare it to the conserved region in rice and maize. Six liguleless-associated rice restriction fragment length polymorphism (RFLP) markers were used to select 16 homeologous sorghum BACs, which were in turn used to physically map the liguleless linkage group in sorghum. Results show a basic conservation of the liguleless region in sorghum relative to the linkage map of rice. One marker which is distal in rice is more medial in sorghum, and another marker which is found within the linkage group in rice is on a different chromosome in sorghum. BACs associated with linkage group I hybridize to chromosome It, which was identified by using FISH in a sorghum cytogenetic stock trisomic for chromosome I (denoted It), and a BAC associated with linkage group E hybridized to an unidentified chromosome. Selected BACs, representing RFLP loci, were end-cloned for RFLP mapping, and the relative linkage order of these clones was in full agreement with the physical data. Similarities in locus order and the association of RFLP-selected BAC markers with two different chromosomes were found to exist between the linkage map of the liguleless region in maize and the physical map of the liguleless region in sorghum.     

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.     

Bacterial contig map of the 21q11 region associated with Alzheimer's disease and abnormal myelopoiesis in Down syndrome

We present a high-resolution bacterial contig map of 3.4 Mb of genomic DNA in human chromosome 21q11-q21, encompassing the region of elevated disomic homozygosity in Down Syndrome-associated abnormal myelopoiesis and leukemia, as well as the markers, which has shown a strong association with Alzheimer's Disease that has never been explained. The map contains 89 overlapping PACs, BACs, or cosmids in three contigs (850, 850, and 1500 kb) with two gaps (one of 140-210 kb and the second <5 kb). To date, eight transcribed sequences derived by cDNA selection, exon trapping, and/or global EST sequencing have been positioned onto the map, and the only two genes so far mapped to this cytogenetic region, STCH and RIP140 have been precisely localized. This work converts a further 10% of chromosome 21q into a high-resolution bacterial contig map, which will be the physical basis for the long-range sequencing of this region. The map will also enable positional derivation of new transcribed sequences, as well as new polymorphic probes, that will help in elucidation of the role the genes in this region may play in abnormal myelopoiesis and leukemia associated with trisomy 21 and Alzheimer's Disease.     

The physical relationship of barley chromosome 5 (1H) to the linkage groups of rice chromosomes 5 and 10

Using a recently developed polymerase chain reaction (PCR)-mediated approach for physical mapping of single-copy DNA sequences on microisolated chromosomes of barley, sequence-tagged sites of DNA probes that reveal restriction fragment length polymorphisms (RFLP) localized on the linkage maps of rice chromosomes 5 and 10 were allocated to cytologically defined regions of barley chromosome 5 (1H). The rice map of linkage group 5, of about 135 cM in size, falls into two separate parts, which are related to the distal portions of both the short and long arms of the barley chromosome. The markers on the rice map of chromosome 5 were found to be located within regions of the barley chromosome which show high recombination rates. The map of rice chromosome 10, of about 75 cM in size, on the other hand, is related to an interstitial segment of the long arm of chromosome 5 (1H) which is highly suppressed in recombination activity. For positional cloning of genes of this homoeologous region from the barley genome, the small rice genome will probably prove to be a useful tool. No markers located on rice chromosomes were detected within the pericentric Giemsa-positive heterochromatin of the barley chromosome, indicating that these barley-specific sequences form a block which separates the linkage segments conserved in rice. By our estimate approximately half of the barley-specific sequences of chromosome 5 (1H) show a dispersed distribution, while the other half separates the conserved linkage segments.     

Exclusion of BMP6 as a candidate gene for cleidocranial dysplasia

Cleidocranial dysplasia (CCD) is an autosomal dominant, generalized skeletal dysplasia in humans that has been mapped to the short arm of chromosome 6. We report linkage of a CCD mutation to 6p21 in a large family and exclude the bone morphogenetic protein 6 gene (BMP6) as a candidate for the disease by cytogenetic localization and genetic recombination. CCD was linked with a maximal two-point LOD score of 7.22 with marker D6S452 at theta = 0. One relative with a recombination between D6S451 and D6S459 and another individual with a recombination between D6S465 and CCD places the mutation within a 7 cM region between D6S451 and D6S465 at 6p21. A phage P1 genomic clone spanning most of the BMP6 gene hybridized to chromosome 6 in band region p23-p24 using FISH analysis, placing this gene cytogenetically more distal than the region of linkage for CCD. We derived a new polymorphic marker from this same P1 clone and found recombinations between the marker and CCD in this family. The results confirm the map position of CCD on 6p21, further refine the CCD genetic interval by identifying a recombination between D6S451 and D6S459, and exclude BMP6 as a candidate gene.     

Assembly of a 1-Mb restriction-mapped cosmid contig spanning the candidate region for Finnish congenital nephrosis (NPHS1) in 19q13.1

We describe the assembly of a 1-Mb cosmid contig and restriction map spanning the candidate region for Finnish congenital nephrosis (NPHS1) in 19q13.1. The map was constructed from 16 smaller contigs assembled by fingerprinting, a BAC and a PAC clone, and 42 previously unmapped cosmids. In most cases, single-step cosmid walks were sufficient to join two previously assembled contigs, and all but one gap was filled from this cosmid contig library. The remaining gap of about 19 kb was spanned with a single BAC and a single PAC clone. EcoRI mapping of a dense set of overlapping clones validated the assembly of the map and indicated a length of 1040 kb for the contig. This high-resolution clone map provides an ideal resource for gene identification through cDNA selection, exon trapping, and DNA sequencing.     

Genomic organization and complete nucleotide sequence of the human PWP2 gene on chromosome 21

The human PWP2 gene is the human homologue of the yeast periodic tryptophan protein 2 (PWP2) gene and is a member of the gene family that contains tryptophan-aspartate (WD) repeats. Genomic sequencing revealed that the human PWP2 gene consists of 21 exons spanning approximately 24 kb and locates just between the two genes EHOC-1 and KNP-I and distal to a NotI site of LJ104 (D21S1460) on chromosome 21q22.3. Analysis of the 5'-flanking DNA sequence revealed that the upstream region of the PWP2 gene is associated with a CpG island containing the NotI site of LJ104. Since PWP2 is considered to be a candidate for genetic disorders mapped in the 21q22.3 region, the information including nucleotide sequence and genomic organization of the PWP2 gene should be invaluable for the mutation analysis of the corresponding genetic disorders.     

Development of physical dependence to lorazepam in rats: the effects of repeated short treatments, dose and age

A balanced t(1;11)(q42.1;q14.3) translocation segregates with schizophrenia and related mental illness in a single large Scottish pedigree. We have constructed a long-range restriction map covering at least 3 Mb of the chromosome 11 breakpoint region and conducted searches for genes whose expression could be altered by the translocation, resulting in schizophrenia. Novel transcribed sequences of unknown function clustered around putative CpG islands, located approximately 500 kb and 700 kb above the breakpoint, represent the only evidence to date for expressed genes within the mapped region.     

From amplification to gene in thyroid cancer: a high-resolution mapped bacterial-artificial-chromosome resource for cancer chromosome aberrations guides gene discovery after comparative genome hybridization

Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. We now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. We used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3-6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKCepsilon), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKCepsilon as a previously unmapped candidate gene involved in thyroid tumorigenesis.     

Application of the planning compass to the nursing curriculum: a tool for health promotion practice

Holoprosencephaly (HPE) is a common developmental defect involving the brain and face. HPE is extremely heterogeneous, some cases being associated with structural anomalies of the short arm of chromosome 3. For a detailed characterization of a t(3;19)(p14.1;p13.1) breakpoint associated with HPE, we performed fluorescence in situ hybridization (FISH) analysis using yeast artificial chromosomes (YACs) mapped to the short arm of chromosome 3 from the Le Centre d'Etude du Polymorphisme Humain (CEPH) library. Three YACs mapped proximal, and one was located distal to the described breakpoint on chromosome 3. One of the chromosome 3 'Mega-YACs' spanned the translocation breakpoint. From this chimeric YAC we generated a site specific probe of about 370 kb by digestion of the YAC-DNA, which will be assessed for gene alterations that could underlie HPE in this patient.     

Posttranscriptional regulation of connexin 32 expression in liver during acute inflammation

The gene for the stimulatory G protein-coupled human A2a adenosine receptor was isolated and sequence analysis revealed two exons that are interrupted by an intron of approximately 6.4 kb. An intron is located in the same region in the human A1 and A2b adenosine receptor genes. Comparison of the A2a genomic and cDNA sequences reveals two nucleotide differences in the coding region and the presence of an aberrant sequence in the 5'208 base pairs of the A2a cDNA including a polymorphism in the third base of codon Tyr-361 and Gly codon which was always detected at residue 392, indicated that the Arg codon present in the cDNA may be an artifact. Fluorescent in situ hybridization and PCR analysis of human-hamster hybrid cell panels shows that the A2a receptor gene is localized to chromosome 22q11.2. This is in contrast with previous reports (subsequently retracted) which mapped the A2a gene to chromosome 11q11-13.     

Minibrain (MNBH) is a single copy gene mapping to human chromosome 21q22.2

We have identified a human homologue of the Drosophila mnb gene (MNBH) on chromosome 21, while another study mapped an EST clone (R38268) with similarity to minibrain to human chromosome 1. This report describes the mapping of MNBH to a single locus on human chromosome 21q22.2 by FISH and Southern blotting. Comparison of the similarities between the two sequences and mnb demonstrates that MNBH on chromosome 21 is the true human homologue of Drosophila mnb.     

Cloning and structure of the gene encoding the human N-methyl-D-aspartate receptor (NMDAR1)

The complete gene encoding the human N-methyl-D-aspartate receptor subunit NR1 (NMDAR1) has been isolated on a single cosmid clone. The gene is composed of 21 exons distributed over a total length of about 31 kb. More than 24 kb were sequenced. Exons 4, 20 and 21 are identical in their amino-acid sequence to those exons that are subject to alternative splicing in rat, indicating that all eight NMDAR1 isoforms found in rat will also be expressed in the human brain. Computer analysis of the pre-mRNA sequence revealed no secondary structures stable enough to explain alternative splicing. We suggest that cell-specific factors control expression of different isoforms. The promoter region contains two perfect copies of the recognition sequence for the Drosophila even-skipped protein, indicating that the developmentally regulated expression of NMDAR1 is controlled by a homeobox protein. The complete cosmid clone covering NMDAR1 was mapped to chromosome 9q34.3-qter by fluorescent in situ hybridization (FISH). The telomeric location is supported by an imperfect (CA)n repeat homologous to a subtelomeric repeat on chromosome 16p.     

Patterns of radiological progression in early rheumatoid arthritis: results of an 8 year prospective study

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications.