Linkage mapping of gene-associated SNPs to pig chromosome 11

Single nucleotide polymorphisms (SNPs) were discovered in porcine expressed sequence tags (ESTs) orthologous to genes from human chromosome 13 (HSA13) and predicted to be located on pig chromosome 11 (SSC11). The SNPs were identified as sequence variants in clusters of EST sequences from pig cDNA libraries constructed in the Sino-Danish pig genome project. In total, 312 human gene sequences from HSA13 were used for similarity searches in our pig EST database. Pig ESTs showing significant similarity with HSA13 genes were clustered and candidate SNPs were identified. Allele frequencies for 26 SNPs were estimated in a group of 80 unrelated pigs from Danish commercial pig breeds: Duroc, Hampshire, Landrace and Large White. Eighteen of the 26 SNPs genotyped in the PiGMaP Reference Families were mapped by linkage analysis to SSC11. The EST-based SNPs published here are new genetic markers useful for linkage and association studies in commercial and experimental pig populations. This study represents the first gene-associated SNP linkage map of pig chromosome 11 and adds new comparative mapping information between SSC11 and HSA13. Furthermore, our data facilitate future studies aimed at the identification of interesting regions on pig chromosome 11, positional cloning and fine mapping of quantitative trait loci in pig.     

Localization of retina/pineal-expressed sequences: identification of novel candidate genes for inherited retinal disorders.

More than 100 genes causing inherited retinal diseases have been mapped to chromosomal locations, but less than half of these genes have been cloned. Mutations in many retina/pineal-specific genes are known to cause inherited retinal diseases. Examples include mutations in arrestin, rhodopsin kinase, and the cone-rod homeobox gene, CRX. To identify additional candidate genes for inherited retinal disorders, novel retina/pineal-expressed EST clusters were identified from the TIGR Human Gene Index database and mapped to specific chromosomal sites. After known human gene sequences were excluded, and repeat sequences were masked, 26 novel retina and pineal gland cDNA clusters were identified. The retinal expression of each novel EST cluster was confirmed by PCR assay of a retinal cDNA library, and each cluster was localized in the genome using the GeneBridge 4.0 radiation hybrid panel. In silico expression data from the TIGR database suggest that these EST clusters are retina/pineal-specific or predominantly expressed in these tissues. This combination of database analysis and laboratory investigation has localized several EST clusters that are potential candidates for genes causing inherited retinopathy.     

From identification of genomic polymorphism to diagnostic and prognostic markers of human epithelial tumors

The review considers the results obtained by several groups in the fields of identification of polymorphic loci in the human genome, localization and analysis of genes associated with epithelial tumors of various origins, and generation of molecular markers of socially important oncological diseases. In the first two cases, work was initiated and supported by the Russian program Human Genome. To find new polymorphic loci in the human genome, di-, tri-, and tetranucleotide repeats were searched for in an ordered cosmid library of chromosome 13, NotI and cosmid clones of chromosome 3, and in brain EST. In total, nine polymorphisms and almost 200 STS were identified. Markers of NotI clones of chromosome 3 were associated with particular genes. Polymorphic loci NL1-024, NL2-007, and EST04896 were employed in analysis of deletions from chromosome 3p in tumor DNA. Deletion mapping of 3p in epithelial tumors of five types revealed six critical regions containing potential tumor suppressor genes. Of these, two were in the distal region of chromosome 3p and four, in region 3p21.3. A significant correlation was observed for the frequency of allelic deletions and the stage and the grade of tumors (P < 0.05). On the strength of these findings, genes of region 3p were associated with both tumor development and progression, and proposed as prognostic markers. Regions LUCA and AP20 (3p21.3) showed a high (90%) frequency of aberrations, including homozygous deletions in almost 20% cases. The peak of allelic deletions from region D3S2409-D3S3667 (600 kb) was statistically valid (P = 10(-3)). Regions AP20 and D3S2409-D3S3667 (3p21.3) were for the first time associated with tumorigenesis. Clusters of tumor suppressor genes were identified in regions LUCA, AP20, and D3S2409-D3S3667. Methylation of RASSF1A and RARbeta2 (3p) was associated with early carcinogenesis, and that of SEMA3B, with tumor progression. These findings are useful for early diagnostics and post-surgery prognosis of tumors.     

In silico screening for tumour-specific expressed sequences in human genome.

A computer-based differential display tool named HsAnalyst has been developed and successfully used for the comparison of expression patterns in a set of tumours versus a set of normal tissues. A list of EST clusters highly represented in tumours and rarely observed in normal tissues has been developed as a resulting output file of the program. These differentially expressed EST clusters (genes) can be useful for developing new tumour markers and prognostic indicators for a wide set of human malignancies. Tumour-specific protein-coding genes may be considered a manifestation of tumour-specific gene expression.     

From Identification of Genomic Polymorphisms to Diagnostic and Prognostic Markers of Human Epithelial Tumors

The review considers the results obtained by several groups in the fields of identification of polymorphic loci in the human genome, localization and analysis of genes associated with epithelial tumors of various origins, and generation of molecular markers of socially important oncological diseases. In the first two cases, work was initiated and supported by the Russian program Human Genome. To find new polymorphic loci in the human genome, di-, tri-, and tetranucleotide repeats were searched for in an ordered cosmid library of chromosome 13, NotI and cosmid clones of chromosome 3, and in brain EST. In total, nine polymorphisms and almost 200 STS were identified. Markers of NotI clones of chromosome 3 were associated with particular genes. Polymorphic loci NL1-024, NL2-007, and EST04896 were employed in analysis of deletions from chromosome 3p in tumor DNA. Deletion mapping of 3p in epithelial tumors of five types revealed six critical regions containing potential tumor suppressor genes. Of these, two were in the distal region of chromosome 3p and four, in region 3p21.3. A significant correlation was observed for the frequency of allelic deletions and the stage and the grade of tumors (P < 0.05). On the strength of these findings, genes of region 3p were associated with both tumor development and progression, and proposed as prognostic markers. Regions LUCA and AP20 (3p21.3) showed a high (90%) frequency of aberrations, including homozygous deletions in almost 20% cases. The peak of allelic deletions from region D3S2409–D3S3667 (600 kb) was statistically valid (P = 10^–3). Regions AP20 and D3S2409–D3S3667 (3p21.3) were for the first time associated with tumorigenesis. Clusters of tumor suppressor genes were identified in regions LUCA, AP20, and D3S2409–D3S3667. Methylation of RASSF1A and RAR-beta2 (3p) was associated with early carcinogenesis, and that of SEMA3B, with tumor progression. These findings are useful for early diagnostics and post-surgery prognosis of tumors.     

An 800-kb region of deletion at 13q14 in human prostate and other carcinomas

Deletions of regions at 13q14 have been detected by various genetic approaches in human cancers including prostate cancer. Several studies have defined one region of loss of heterozygosity (LOH) at 13q14 that seems to reside in a DNA segment of 7.1 cM between genetic markers D13S263 and D13S153. To define the smallest region of overlap (SRO) for deletion at 13q14, we first applied tissue microdissection and multiplex PCR to detect homozygous deletion and/or hemizygous deletion at 13q14 in 134 prostate cancer specimens from 114 patients. We detected deletions at markers D13S1227, D13S1272, and A005O48 in 13 (10%) of these tumor specimens. Of the 13 tumors with deletions, 12 were either poorly differentiated primary tumors or metastases of prostate cancer. To fine-map the deletion region, we then constructed a high-resolution YAC/BAC/STS/EST physical map based on experimental and database analyses. Several markers encompassing the deletion region were analyzed for homozygous deletion and/or hemizygous deletion in 61 cell lines/xenografts derived from human cancers of the prostate, breast, ovary, endometrium, cervix, and bladder, and a region of deletion was defined by duplex PCR assay between markers A005X38 and WI-7773. We also analyzed LOH at 13q14 in the 61 cell lines/xenografts using the homozygosity mapping of deletion approach and 26 microsatellite markers. We found 24 (39%) of the cell lines/xenografts to show LOH at 13q14 and defined a region of LOH by markers M1 and M5. Combination of homozygous or hemizygous deletion and LOH results defined the SRO for deletion to be an 800-kb DNA interval between A005X38 and M5. There are six known genes located in or close to the SRO for deletion. This region of deletion is at least 2 Mb centromeric to the RB1 tumor-suppressor gene and the leukemia-associated genes 1 and 2, each of which is located at 13q14. These data suggest that the 800-kb DNA segment with deletion contains a gene whose deletion may be important for the development of prostate and other cancers. This study also provides a framework for the fine-mapping, cloning, and identification of a novel tumor-suppressor gene at 13q14.     

Characterization of a novel gene, C21orf6, mapping to a critical region of chromosome 21q22.1 involved in the monosomy 21 phenotype and of its murine ortholog, orf5

Phenotypic and molecular analyses of patients with partial chromosome 21 monosomy enabled us to define a region, spanning 2.4 Mb between D21S190 and D21S226, associated with arthrogryposis, mental retardation, hypertonia, and several facial anomalies. The markers of the region were used to screen a total human PAC library (Ioannou, RZPD). We isolated 57 PACs, which formed primary contigs. EST clusters (UNIGENE collection) located in a 6-Mb interval, between D21S260 and D21S263, were mapped in individual bacterial clones. We mapped the WI-17843 cluster to the PAC clone J12100, which contains the two anchor markers LB10T and LA329. The open reading frame extends over 960 bp, with three putative start codons. The 1695-bp cDNA containing a polyadenylation signal should correspond to the full-length cDNA. From the genomic sequence, we deduced that the gene contained five exons and that there was a putative promoter sequence upstream from exon 1. In silico screening of DNA databases revealed similarity with a murine EST. The corresponding cDNA (1757 bp) sequence was very similar (>85%) to the human cDNA and had an open reading frame of 876 nucleotides. Somatic hybrid mapping localized the cDNA to mouse chromosome 16. EST analyses and RT-PCR indicated that the third exon in the human gene (exon 2 in the mouse) undergoes alternative splicing. Northern blot hybridization showed that the gene was ubiquitously expressed in humans and mice. The longest mouse clone was used to generate riboprobes, which were hybridized to murine embryos at stages E-9.5, E-10.5, E-12.5, E-13.5, and E-14.5-15, to study the pattern of expression during development. Ubiquitous labeling was observed, with strong signals restricted to limited areas of the telencephalon, the mesencephalon, and the interrhombomeric regions in the central nervous system, and other regions of the body such as the limb buds, branchial arches, and somites.     

Allelotype of human thyroid tumors: loss of chromosome 11q13 sequences in follicular neoplasms.

Two classes of genes are the targets of mutations involved in human tumorigenesis: oncogenes, the activation of which leads to growth stimulation, and tumor suppressor genes, which become tumorigenic through loss of function, often through allelic deletion. To obtain evidence for a role for tumor suppressor genes in thyroid tumorigenesis, we examined DNA from 80 thyroid neoplasms for loss of heterozygosity in multiple chromosomal loci using 19 polymorphic genomic probes. None of the informative thyroid tumors studied had allelic loss detected with probes for chromosome 2q (D2S44), 3p (D3F15S2, D3S32), 3q (D3S46), 4p (D4S125), 6p (D6S40), 8q (D8S39), 9q (D9S7), 12p (D12S14), 13q (D13S52), 17p (D17S30), or 18q (D18S10). One of eight of the follicular adenomas had a 10q deletion detected with marker D10S15, and one of 26 had a 10q deletion detected with D10S25. One of two of the follicular carcinomas had an 11p deletion in the H-ras locus. The most significant findings were on chromosome 11q13, the site containing the putative gene predisposing to multiple endocrine neoplasia type I. Four of 27 follicular adenomas had loss of heterozygosity for probes in this region. Allelic deletions were detected with the following probes: D11S149, PYGM, D11S146, and INT2. None of 13 informative papillary carcinomas and none of two follicular carcinomas had loss of heterozygosity detectable with these 11q13 markers. Allelic loss is a relatively infrequent event in human thyroid tumors. Deletions of chromosome 11q13 are present in about 14% of follicular, but not papillary, neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS)     

一种新的EST聚类方法

该研究发展了一种EST（expressed sequence tag）聚类方法（ESTClustering）,用于分析大规模EST测序中所产生的大量数据,以获得高质量,非重复表达序列,该方法在聚类过程中采用MEGABLAST工具对一致序列进行序列同源比较,并用phrap程序对每一EST簇进行拼接检验。这一聚类策略能降低测序错误带来的影响,有效识别基因家族成员,并避免选择性剪接的干扰,与NCB（National Center for Biotechnology Information）的UniGene clustering）方法相比,ESTClustering的聚类结果可以更好地反映表达序列的多样性,用ESTClustering对112256条拟南芥EST聚类测试,产生23581个EST簇,其中13597个EST簇有对应拟南芥基因组编码序列,与该基因组中有EST作为依据的预测基因数目接近。应用该方法对收集的147191条水稻EST序列进行聚类,形成33896个EST簇。     

Census of orthologous genes and self-organizing maps of biologically relevant transcriptional patterns in chickens (Gallus gallus)

The launch of large-scale chicken expressed sequence tags (EST) projects has placed the chicken in the lead for the number of EST sequences in agriculturally important animals. More than 451,000 chicken ESTs derived from over 158 libraries have been deposited in the NCBI dbEST database as of December 2003. But how many genes these ESTs represent and how they are expressed in different chicken tissues/organs remain undetermined. In the present research, we developed a human gene-based strategy for census of chicken orthologous genes and identification of their expression patterns. Among 34,157 human coding genes used in the study, BLAST analysis revealed that 11,066 genes provisionally matched 248,628 chicken ESTs. Based on the average EST abundance of the orthologous genes, the current public repository of chicken ESTs could represent 20,000 provisional genes. Analysis of gene expression in 14 single tissues/organs showed that approximately 15% of genes were expressed exclusively in single tissue/organ whereas the remaining 85% of genes were co-expressed in two or more tissues/organs. A majority (91.15%) of genes expressed in chicken embryos were also expressed at post-hatch stages, indicating that most genes activated in chicken embryos could serve housekeeping functions. Self-organizing maps (SOM) analysis organized 8807 provisional genes in selected chicken tissues into 98 clusters with each cluster being indicative of common regulatory factors and pathways. A total of 969 provisional orthologous genes were identified as preferentially expressed genes (PEGs) in various chicken tissues/organs (LOD>3.0). No doubt, the present study on gene expression patterns will provide insight into dynamics of metabolic pathways and tissue/organ programming and reprogramming in chickens.     

New genes in the class II region of the human major histocompatibility complex

A detailed map of the class II region of the human major histocompatibility complex has been constructed by pulsed-field gel electrophoresis. This map revealed clusters of sites for enzymes that cut preferentially in unmethylated CpG-rich DNA often found at the 5' ends of genes. Three of these clusters have been cloned by cosmid walking and chromosome jumping. Analysis of the clones encompassing these regions through the use of zoo blots, Northern blots, and cDNA libraries resulted in the discovery of four novel genes. The D6S111E and D6S112E genes are centromeric to the HLA-DPB2 gene, while D6S113E and D6S114E are between HLA-DNA and HLA-DOB. Preliminary characterization of the new genes indicates that they are unrelated to the class II genes themselves, although D6S114E expression, like class II expression, is inducible with interferon. In addition, the HLA-DNA gene has been accurately positioned and oriented for the first time.     

Genomic distribution and characterization of EST-derived resistance gene analogs (RGAs) in sugarcane

A large sugarcane EST (expressed sequence tag) project recently gave us access to 261,609 EST sequences from sugarcane, assembled into 81,223 clusters. Among these, we identified 88 resistance gene analogs (RGAs) based on their homology to typical pathogen resistance genes, using a stringent BLAST search with a threshold e-value of e(-50). They included representatives of the three major groups of resistance genes with NBS/LRR, LRR or S/T KINASE domains. Fifty RGAs showed a total of 148 single-dose polymorphic RFLP markers, which could be located on the sugarcane reference genetic map (constructed in cultivar R570, 2n=approximately 115). Fifty-five SSR loci corresponding to 134 markers in R570 were also mapped to enable the classification of the various haplotypes into homology groups. Several RGA clusters were found. One cluster of two LRR-like loci mapped close to the only disease resistance gene known so far in sugarcane, which confers resistance to common rust. Detailed sequence comparison between two NBS/LRR RGA clusters in relation to their orthologs in rice and maize suggests their polyphyletic origins, and indicates that the degree of divergence between paralogous RGAs in sugarcane can be larger than that from an ortholog in a distant species.     

Gene expression profile of the mouse organ of Corti at the onset of hearing

We describe the generation of an expressed sequence tag (EST) database of the mouse organ of Corti (OC). Over 20,000 independent clones were isolated, analyzed, and grouped into 8690 unique gene clusters. A large pool of novel genes unique to the OC was identified. Sequence alignments frequently revealed alternatively spliced forms of known genes potentially relevant in the OC function. We have also electronically mapped a subset of OC mouse ESTs to several syntenic regions associated with human autosomal and recessive deafness, which may prove useful for the identification of new positional candidates for these human diseases. The EST dataset is available as an interactive Web-based public database at. This resource provides both a view of the profile of gene expression in the OC at the onset of hearing and a tool to identify novel genes of importance in hearing.     

Physical mapping of two loci (D9S5 and D9S15) tightly linked to Friedreich ataxia locus (FRDA) and identification of nearby CpG islands by pulse-field gel electrophoresis

The Friedreich's ataxia locus (FRDA) has recently been mapped to 9q13-q21 by tight linkage to D9S15 and D9S5 loci. The present lack of recombination between these loci precludes further genetic mapping and suggests that the distances involved are in the megabase range. We have established a 1-Mb map around loci D9S15 (defined by probe MCT112) and D9S5 (defined by probe DR47) and found that they are at most 260 apart. Six rare cutting site clusters were found in a 450-kb segment containing both loci. Three clusters were completely unmethylated in two cell lines tested and might correspond to CpG islands flanking transcribed sequences. Cosmid mapping of a 52-kb region around D9S5 and pulse-field gel electrophoresis analysis showed the presence of three other CpG clusters that were partially or completely methylated. Two of them were present in the cosmid clones available and were associated with sequences conserved in other vertebrate species. The CpG islands and conserved sequences presented here can be used to search for genes defective in Friedreich's ataxia.     

Tomato SNP Discovery by EST Mining and Resequencing

Many economically important crop species are relatively depauparate in genetic diversity (e.g., soybean, peanut, tomato). DNA polymorphism within cultivated tomato has been estimated to be low based on molecular markers. Through mining of more than 148,000 public tomato expressed sequence tags (ESTs) and full-length cDNAs, we identified 764 EST clusters with potential single nucleotide polymorphisms (SNPs) among more than 15 tomato lines. By sequencing regions from 53 of these clusters in two to three lines, we discovered a wealth of nucleotide polymorphism (62 SNPs and 12 indels in 21 Unigenes), resulting in a verification rate of 27.2% (28 of 103 SNPs predicted in EST clusters were verified). We hypothesize that five regions with 1.6–13-fold more diversity relative to other tested regions are associated with introgressions from wild relatives. Identifying polymorphic, expressed genes in the tomato genome will be useful for both tomato improvement and germplasm conservation.