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Fine mapping of the polled locus to a 1-Mb region on bovine chromosome 1q12.

The absence of horns in Bos taurus is under genetic control of the autosomal dominant polled locus which has been genetically mapped to the centromeric region of cattle Chromosome 1. Recently a 4-Mb BAC contig of this chromosomal region has been constructed. Toward positional cloning of the bovine polled locus, we identified 20 additional microsatellite markers spread over the contig map by random sequencing of bacterial artificial chromosome (BAC) subclones. A total of 26 markers were genotyped in 30 two-generation half-sib families of six different German cattle breeds segregating for the hornless phenotype including 336 informative meioses for the polled character. Our fine-mapping study involving 19 recombinant haplotypes allowed us to narrow the critical region for the bovine polled locus to a 1-Mb segment with a centromeric boundary at RP42-218J17_MS1 and a telomeric boundary at BM6438. For marker-assisted selection purposes, the first evidence of informative flanking markers helps to predict polled genotypes with a higher degree of accuracy within families until testing of the causative mutation is available.

Animals↗

Mapping analysis of the Xylella fastidiosa genome.

A cosmid library was made of the 2.7 Mb genome of the Gram-negative plant pathogenic bacterium Xylella fastidiosa and analysed by hybridisation mapping. Clones taken from the library as well as genomic restriction fragments of rarely cutting enzymes were used as probes. The latter served as a backbone for ordering the initial map contigs and thus facilitated gap closure. Also, the co-linearity of the cosmid map, and thus the eventual sequence, could be confirmed by this process. A subset of the eventual clone coverage was distributed to the Brazilian X.FASTIDIOSA: sequencing network. Data from this effort confirmed more quantitatively initial results from the hybridisation mapping that the redundancy of clone coverage ranged between 0 and 45-fold across the genome, while the average was 15-fold by experimental design. Reasons for this not unexpected fluctuation and the actual gaps are being discussed, as is the use of this effect for functional studies.

Brazil↗

Characterization of four yeast artificial chromosome clones mapped to human chromosome 21q22.1 with eight markers.

Yeast artificial chromosome (YAC) clones have been successfully utilized to generate a YAC contig map of the long arm of human chromosome 21 (Hu21q). The chromosome subband of 21q22.1 where five genetic loci (IFNAR1, IFNAR2, CRFB4, AF-1, and GART) are mapped is a gene-rich region and needs to be characterized in further detail. YAC D142H8 and YAC F136C5, which were characterized previously by a functional YAC expression procedure, and two new YAC clones, YAC 872B5 and YAC 876D4 located at 21q22.1 whose YAC sizes are 800 kb and 1,500 kb, respectively, were used in this study. To obtain more markers useful for making a detailed physical map of the region, a purified yeast artificial chromosome (YAC D142H8) was used to screen the 3 x 1 S cDNA library. As a result three anonymous cDNA clones (Kmy1, Kmy2, and Qorf4) were obtained, and the nucleotide sequences of Kmy1 and Kmy2 were determined. In an attempt to make a detailed physical map of the region, the locations of five known genes as well as the three new markers were determined on the four YACs by Southern blot analysis. YAC 872B5 contained all markers except GART while YAC F136C5, YAC D142H8, and YAC 876D4 contained three markers (CRFB4, IFNAR1, and IFNAR2), four markers (Kmy1, Kmy2, Qorf4 and AF-1), and four markers (Kmy1, Kmy2, Qorf4 and GART), respectively. YAC 872B5 may represent 1,500 kb of the 21q22.1 subband and half of the 3 x 1 S region, so it should be very useful for studying the relevent region of the human chromosome functionally and physically.

Base Sequence↗

A 2.8-Mb clone contig of the multiple endocrine neoplasia type 1 (MEN1) region at 11q13.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder that results in parathyroid, anterior pituitary, and pancreatic and duodenal endocrine tumors in affected individuals. The MEN1 locus is tightly linked to the marker PYGM on chromosome 11q13, and linkage analysis has placed the MEN1 gene within a 2-Mb interval flanked by D11S1883 and D11S449. As a step toward cloning the MEN1 gene, we have constructed a 2.8-Mb clone contig consisting of YAC and bacterial clones (PAC, BAC, and P1) for the D11S480 to D11S913 region. The bacterial clones alone represent nearly all of the 2.8-Mb contig. The contig was assembled based on a high-density STS-content analysis of 79 genomic clones (YAC, PAC, BAC, and P1) with 118 STSs. The STSs included 22 polymorphic markers and 20 transcripts, with the remainder primarily derived from the end sequences of the genomic clones. An independent cosmid contig for the 1-Mb PYGM-SEA region was also generated. Support for correctness of the 2.8-Mb contig map comes from an independent ordering of the clones by fiber-FISH. This sequence-ready contig will be a useful resource for positional cloning of MEN1 and other disease genes whose loci fall within this region.

Chromosomes, Artificial, Yeast↗

A physical map of chromosome 7 of Candida albicans.

As part of the ongoing Candida albicans Genome Project, we have constructed a complete sequence-tagged site contig map of chromosome 7, using a library of 3840 clones made in fosmids to promote the stability of repeated DNA. The map was constructed by hybridizing markers to the library, to a blot of the electrophoretic karyotype, and to a blot of the pulsed-field separation of the SfiI restriction fragments of the genome. The map includes 149 fosmids and was constructed using 79 markers, of which 34 were shown to be genes via determination of function or comparison of the DNA sequence to the public databases. Twenty-five of these genes were identified for the first time. The absolute position of several markers was determined using random breakage mapping. Each of the homologues of chromosome 7 is approximately 1 Mb long; the two differ by about 20 kb. Each contains two major repeat sequences, oriented so that they form an inverted repeat separated by 370 kb of unique DNA. The repeated sequence CARE2/Rel2 is a subtelomeric repeat on chromosome 7 and possibly on the other chromosomes as well. Genes located on chromosome 7 in Candida are found on 12 different chromosomes in Saccharomyces cerevisiae.

Candida albicans↗

Seeding of YACs over regions 1q41-q42.3 and 11q14.3-q23 with microdissection clones.

We describe the use of pooled, region-specific hybridisation probes to screen high-density replica filters of a human genome YAC library. The probes were derived by microdissection of an approximately 30-Mbp region subtending the translocation breakpoint on a der(1)(1;11)(q42.1;q14.3) chromosome. Of 70 microdissection clones used in pools of 4-10, 47 identified a total of 77 YAC recombinants, representing over 50% of the microdissected region. This strategy can easily be adapted to other poorly mapped subchromosomal regions of the human or other mammalian genomes and will provide a solid framework for detailed contig map constructions.

Chromosomes, Artificial, Yeast↗

Construction of two YAC contigs in human Xp11.23-p11.22, one encompassing the loci OATL1, GATA, TFE3, and SYP, the other linking DXS255 to DXS146.

We have constructed two YAC contigs in the Xp11.23-p11.22 interval of the human X chromosome, a region that was previously poorly characterized. One contig, of at least 1.4 Mb, links the pseudogene OATL1 to the genes GATA1, TFE3, and SYP and also contains loci implicated in Wiskott-Aldrich syndrome and synovial sarcoma. A second contig, mapping proximal to the first, is estimated to be over 2.1 Mb and links the hypervariable locus DXS255 to DXS146, and also contains a chloride channel gene that is responsible for hereditary nephrolithiasis. We have used plasmid rescue, inverse PCR, and Alu-PCR to generate 20 novel markers from this region, 1 of which is polymorphic, and have positioned these relative to one another on the basis of YAC analysis. The order of previously known markers within our contigs, Xpter-OATL1-GATA-TFE3-SYP-DXS255146- Xcen, agrees with genomic pulsed-field maps of the region. In addition, we have constructed a rare-cutter restriction map for a 710-kb region of the DXS255-DXS146 contig and have identified three CPG islands. These contigs and new markers will provide a useful resource for more detailed analysis of Xp11.23-p11.22, a region implicated in several genetic diseases.

Animals↗

Genetic and physical mapping of Pi36(t), a novel rice blast resistance gene located on rice chromosome 8.

Blast resistance in the indica cultivar (cv.) Q61 was inherited as a single dominant gene in two F2 populations, F2-1 and F2-2, derived from crosses between the donor cv. and two susceptible japonica cvs. Aichi Asahi and Lijiangxintuanheigu (LTH), respectively. To rapidly determine the chromosomal location of the resistance (R) gene detected in Q61, random amplified polymorphic DNA (RAPD) analysis was performed in the F2-1 population using bulked-segregant analysis (BSA) in combination with recessive-class analysis (RCA). One of the three linked markers identified, BA1126(550), was cloned and sequenced. The R gene locus was roughly mapped on rice chromosome 8 by comparison of the BA1126(550) sequence with rice sequences in the databases (chromosome landing). To confirm this finding, seven known markers, including four sequence-tagged-site (STS) markers and three simple-sequence repeat (SSR) markers flanking BA1126(550) on chromosome 8, were subjected to linkage analysis in the two F2 populations. The locus was mapped to a 5.8 cM interval bounded by RM5647 and RM8018 on the short arm of chromosome 8. This novel R gene is therefore tentatively designated as Pi36(t). For fine mapping of the Pi36(t) locus, five additional markers including one STS marker and four candidate resistance gene (CRG) markers were developed in the target region, based on the genomic sequence of the corresponding region of the reference japonica cv. Nipponbare. The Pi36(t) locus was finally localized to an interval of about 0.6 cM flanked by the markers RM5647 and CRG2, and co-segregated with the markers CRG3 and CRG4. To physically map this locus, the Pi36(t)-linked markers were mapped by electronic hybridization to bacterial artificial chromosome (BAC) or P1 artificial chromosome (PAC) clones of Nipponbare, and a contig map was constructed in silico through Pairwise BLAST analysis. The Pi36(t) locus was physically delimited to an interval of about 17.0 kb, based on the genomic sequence of Nipponbare.

Alleles↗

Genetic and physical mapping of Pi37(t), a new gene conferring resistance to rice blast in the famous cultivar St. No. 1.

The famous rice cultivar (cv.), St. No. 1, confers complete resistance to many isolates collected from the South China region. To effectively utilize the resistance, a linkage assay using microsatellite markers (SSR) was performed in the three F2 populations derived from crosses between the donor cv. St. No. 1 and each of the three susceptible cvs. C101PKT, CO39 and AS20-1, which segregated into 3R:1S (resistant/susceptible) ratio, respectively. A total of 180 SSR markers selected from each chromosome equally were screened. The result showed that the two markers RM128 and RM486 located on chromosome 1 were linked to the resistance gene in the respective populations above. This result is not consistent with those previously reported, in which a well-known resistance gene Pif in the St. No. 1 is located on chromosome 11. To confirm this result, additional four SSR markers, which located in the region lanked by RM128 and RM486, were tested. The results showed that markers RM543 and RM319 were closer to, and RM302 and RM212 completely co-segregated with the resistance locus detected in the present study. These results indicated that another resistance gene involved in the St. No. 1, which is located on chromosome 1, and therefore tentatively designated as Pi37(t). To narrow down genomic region of the Pi37(t) locus, eight markers were newly developed in the target region through bioinformatics analysis (BIA) using the publicly available sequences. The linkage analysis with these markers showed that the Pi37(t) locus was mapped to a approximately 0.8 centimorgans (cM) interval flanked by RM543 and FPSM1, where a total of seven markers co-segregated with it. To physically map the locus, the Pi37(t)-linked markers were landed on the reference sequence of cv. Nipponbare through BIA. A contig map corresponding to the locus was constructed based on the reference sequence aligned by the Pi37(t)-linked markers. Consequently, the Pi37(t) locus was defined to 374 kb interval flanking markers RM543 and FPSM1, where only four candidate genes with the resistance gene conserved structure (NBS-LRR) were further identified to a DNA fragment of 60 kb in length by BIA.

China↗

[Radiation mapping as one of main methods of creating high resolution maps of human and animal genomes].

Radiation hybrid mapping (RH mapping) is considered as one of the main methods of constructing physical maps of mammalian genomes. In introduction, theoretical prerequisites of developing of the RH mapping and statistical methods of data analysis are discussed. Comparative characteristics of universal commercial panels of the radiation hybrid somatic cells (RH panels) are shown. In experimental part of the work, RH mapping is used to localise nucleotide sequences adjacent to NotI sites of human chromosome 3 with the aim to integrate contig map of NotI clones to comprehensive maps of human genome. Five nucleotide sequences adjacent to the sites of integration of papilloma virus in human genome and expressed in the cells of cervical cancer were localised. It was demonstrated that the region 13q14.3-q21.1 was enriched with nucleotide sequences involved in the processes of oncogenesis. RH mapping can be considered as one of the most perspective applications of the modern radiation biology in the field of molecular genetics, that is, in constructing physical maps of mammalian genomes with high resolution level.

Animals↗

Comparative mapping of human Chromosome 19 with the chicken shows conserved synteny and gives an insight into chromosomal evolution.

Human Chromosome 19 (HSA19) is virtually completely sequenced. A complete physical contig map made up of BACs and cosmids is also available for this chromosome. It is, therefore, a rich source of information that we have used as the basis for a comparative mapping study with the chicken. Various orthologs of genes known to map to HSA19 have been mapped in the chicken. Five chicken microchromosomes (two of which were previously undefined) are seen to show conserved synteny with this chromosome, along with individual gene homologs on Chr 1 and another tiny microchromosome. Compared with the mouse, which has 12 chromosomal regions homologous to HSA19, the chicken genotype displays fewer evolutionary rearrangements. The ancestral nature of the chicken karyotype is demonstrated and may prove to be an excellent tool for studying genome evolution.

Animals↗

A chromosome bin map of 16,000 expressed sequence tag loci and distribution of genes among the three genomes of polyploid wheat.

Because of the huge size of the common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) genome of 17,300 Mb, sequencing and mapping of the expressed portion is a logical first step for gene discovery. Here we report mapping of 7104 expressed sequence tag (EST) unigenes by Southern hybridization into a chromosome bin map using a set of wheat aneuploids and deletion stocks. Each EST detected a mean of 4.8 restriction fragments and 2.8 loci. More loci were mapped in the B genome (5774) than in the A (5173) or D (5146) genomes. The EST density was significantly higher for the D genome than for the A or B. In general, EST density increased relative to the physical distance from the centromere. The majority of EST-dense regions are in the distal parts of chromosomes. Most of the agronomically important genes are located in EST-dense regions. The chromosome bin map of ESTs is a unique resource for SNP analysis, comparative mapping, structural and functional analysis, and polyploid evolution, as well as providing a framework for constructing a sequence-ready, BAC-contig map of the wheat genome.

Chromosome Mapping↗

Localization of the L-glutamine synthetase gene to chromosome 1q23.

Glutamine synthetase (E.C. 6.3.1.2) is expressed throughout the body and plays an important role in controlling body pH and in removing ammonia from the circulation. The enzyme clears L-glutamate, the major neurotransmitter in the central nervous system, from neuronal synapses. The enzyme is a very sensitive marker of many disease and aging processes, especially those involving reactive oxygen species. This report describes the localization of the enzyme to chromosome 1 by PCR analysis of a human/rodent somatic cell hybrid panel. We also describe the localization of a recently described pseudogene to chromosome 9. Further localization of the glutamine synthetase gene locus to 1q23 was accomplished by fluorescence in situ hybridization. The glutamine synthetase gene was mapped to five CEPH megaYACs between the polymorphic PCR markers D1S117 and D1S466 by analysis of the Whitehead Institute's recently described chromosome 1 contig map.

Animals↗

Chicken genomics charts a path to the genome sequence.

In this paper, the current status of chicken genomics is reviewed. This is timely given the current intense activity centred on sequencing the complete genome of this model species. The genome project is based on a decade of map building by genetic linkage and cytogenetic methods, which are now being replaced by high-resolution radiation hybrid and bacterial artificial chromosome (BAC) contig maps. Markers for map building have generally depended on labour-intensive screening procedures, but in recent years this has changed with the availability of almost 500,000 chicken expressed sequence tags (ESTs). These resources and tools will be critical in the coming months when the chicken genome sequence is being assembled (eg cross-checked with other maps) and annotated (eg gene structures based on ESTs). The future for chicken genome and biological research is an exciting one, through the integration of these resources. For example, through the proposed chicken Ensembl database, it will be possible to solve challenging scientific questions by exploiting the power of a chicken model. One area of interest is the study of developmental mechanisms and the discovery of regulatory networks throughout the genome. Another is the study of the molecular nature of quantitative genetic variation. No other animal species have been phenotyped and selected so intensively as agricultural animals and thus there is much to be learned in basic and medical biology from this research.

Animals↗

Mapping human chromosomes by walking with sequence-tagged sites from end fragments of yeast artificial chromosome inserts.

Sequence-tagged sites (STSs) derived from end fragments of chromosome-specific yeast artificial chromosomes (YACs) can facilitate the assembly of an overlapping YAC/STS map. Contigs form rapidly by iteratively screening YAC collections with end-fragment STSs from YACs that have not yet been detected by any previous STS. The map is rendered rapidly useful during its assembly by incorporating supplementary STSs from genes and genetic linkage probes with known locations. Methods for the systematic development and testing of the end-fragments STSs are given here, and a group of 100 STSs is presented for the X chromosome. The mapping strategy is shown to be successful in simulations with portions of the X chromosome already largely mapped into overlapping YACs by other means.

Base Sequence↗

Genome mapping by fluorescent fingerprinting.

The construction of sequence-ready maps of overlapping genomic clones is central to large-scale genome sequencing. We have implemented a method for fluorescent fingerprinting of bacterial clones to assemble contig maps. The method utilizes three spectrally distinct fluorescently tagged dideoxy ATPs to specifically label the HindIII termini in HindIII and Sau3AI restriction digests of clones that are multiplexed prior to electrophoresis and data collection. There is excellent reproducibility of raw data, improved resolution of large fragments, and concordance between the results obtained using this and the equivalent radioactive protocol. This method also allows detection of smaller overlaps between clones when compared to the analysis of restriction digests on nondenaturing agarose gels.

Chromosome Mapping↗

Physical mapping of a commonly deleted region, the site of a candidate tumor suppressor gene, at 12q22 in human male germ cell tumors.

A candidate tumor suppressor gene (TSG) site at 12q22 characterized by a high frequency of loss of heterozygosity (LOH) and a homozygous deletion has previously been reported in human male germ cell tumors (GCTs). In a detailed deletion mapping analysis of 67 normal-tumor DNAs utilizing 20 polymorphic markers mapped to 12q22-q24, we identified the limits of the minimal region of deletion at 12q22 between D12S377 (proximal) and D12S296 (distal). We have constructed a YAC contig map of a 3-cM region of this band between the proximal marker D12S101 and the distal marker D12S346, which contained the minimal region of deletion in GCTs. The map is composed of 53 overlapping YACs and 3 cosmids onto which 25 polymorphic and nonpolymorphic sequence-tagged sites (STSs) were placed in a unique order. The size of the minimal region of deletion was approximately 2 Mb from overlapping, nonchimeric YACs that spanned the region. We also developed a radiation hybrid (RH) map of the region between D12S101 and D12S346 containing 17 loci. The consensus order developed by RH mapping is in good agreement with the YAC STS-content map order. The RH map estimated the distance between D12S101 and D12S346 to be 246 cR8000 and the minimal region of deletion to be 141 cR8000. In addition, four genes that were previously mapped to 12q22 have been excluded as candidate genes. The leads gained from the deletion mapping and physical maps should expedite the isolation and characterization of the TSG at 12q22.

Chromosomes, Human, Pair 12↗

Localization and genomic structure of human deoxyhypusine synthase gene on chromosome 19p13.2-distal 19p13.1.

The amino acid hypusine is formed post-translationally in a single cellular protein, the eukaryotic translation initiation factor 5A, by two enzymes, namely deoxyhypusine synthase and deoxyhypusine hydroxylase. Hypusine is found in all eukaryotes and in some archaebacteria, but not in eubacteria. The deoxyhypusine synthase cDNA was cloned and mapped by fluorescence in situ hybridization on chromosome 19p13.11-p13.12. Rare cDNAs containing internal deletions were also found. We localized the deoxyhypusine synthase gene on a high resolution cosmid/BAC contig map of chromosome 19 to a region in 19p13.2-distal 19p13.1 between MANB and JUNB. Analysis of the genomic exon/intron structure of the gene coding region showed that it consists of nine exons and spans a length of 6.6kb. From observation of the genomic structure, it seems likely that the internally deleted forms of mature RNA are the result of alternative splicing, rather than of artifacts.

Alternative Splicing↗