Exon Trapping Research Articles

Cloning, genomic organization and expression of a putative human transmembrane protein related to the Caenorhabditis elegans M01F1.4 gene.

A novel human transcript CG-2 (C9ORF5), was isolated from the familial dysautonomia candidate region on 9q31 using a combination of cDNA selection and exon trapping. CG-2 was detected as a relatively abundant 8kb transcript in all adult and fetal tissues with the exception of adult thymus. Genomic analysis of CG-2 identified 18 exons that span more than 110kb. The gene encodes a 911-amino-acid protein with a predicted molecular weight of 101kDa and a hypothetical pI of 9.03. Sequence analysis of CG-2 indicates that it is likely to encode a transmembrane protein. Here, we assess CG-2 as a candidate for familial dysautonomia.

HREC2, a RAD51-like Gene, is Disrupted by t(12;14)(q15;q24.1) in a Uterine Leiomyoma

A balanced translocation between chromosomes 12 and 14 is commonly seen in uterine leiomyoma (UL). We have previously cloned and characterized a 2 Mb segment of human chromosomal subband 14q24.1, and have shown that the t(12;14)(q15;q24.1) breakpoints from several ULs map within this region. Exon trapping of DNA clones spanning one such breakpoint revealed coding sequences from hREC2, a gene that shows significant amino acid sequence identity to the double-strand break repair enzyme RAD51. We report here that this breakpoint is located within a 19 kb intron of the hREC2 gene and that the translocation results in the premature truncation of the major hREC2 transcript. Mapping and sequence analyses show that alternative transcripts of the hREC2 gene, including novel isoforms identified in testis and uterus, are not interrupted by the translocation.

Structure of tau exon 10 splicing regulatory element RNA and destabilization by mutations of frontotemporal dementia and parkinsonism linked to chromosome 17.

Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Intronic mutations and some missense mutations increase splicing in of exon 10, leading to an increased ratio of four-repeat to three-repeat tau isoforms. Secondary structure predictions have led to the proposal that intronic mutations and one missense mutation destabilize a putative RNA stem-loop structure located close to the splice-donor site of the intron after exon 10. We have determined the three-dimensional structure of this tau exon 10 splicing regulatory element RNA by NMR spectroscopy. We show that it forms a stable, folded stem-loop structure whose thermodynamic stability is reduced by frontotemporal dementia and parkinsonism linked to chromosome 17 mutations and increased by compensatory mutations. By exon trapping, the reduction in thermodynamic stability is correlated with increased splicing in of exon 10.

Campomelic Dysplasia Translocation Breakpoints Are Scattered over 1 Mb Proximal to SOX9: Evidence for an Extended Control Region

Campomelic dysplasia (CD), a skeletal malformation syndrome with or without XY sex reversal, is usually caused by mutations within the SOX9 gene on distal 17q. Several CD translocation and inversion cases have been described with breakpoints outside the coding region, mapping to locations >130 kb proximal to SOX9. Such cases are generally less severely affected than cases with SOX9 coding-region mutations, as is borne out by three new translocation cases that we present. We have cloned the region extending 1.2 Mb upstream of the SOX9 gene in overlapping bacterial-artificial-chromosome and P1-artificial-chromosome clones and have established a restriction map with rare-cutter enzymes. With sequence-tagged-site-content mapping in somatic-cell hybrids, as well as with FISH, we have precisely mapped the breakpoints of the three new and of three previously described CD cases. The six CD breakpoints map to an interval that is 140-950 kb proximal to the SOX9 gene. With exon trapping, we could isolate five potential exons from the YAC 946E12 that spans the region, four of which could be placed in the contig in the vicinity of the breakpoints. They show the same transcriptional orientation, but only two have an open reading frame (ORF). We failed to detect expression of these fragments in several human and mouse cDNA libraries, as well as on northern blots. Genomic sequence totaling 1,063 kb from the SOX9 5'-flanking region was determined and was analyzed by the gene-prediction program GENSCAN and by a search of dbEST and other databases. No genes or transcripts could be identified. Together, these data suggest that the chromosomal rearrangements most likely remove one or more cis-regulatory elements from an extended SOX9 control region.

Vertebrate exon trapping methods: implications for transcript mapping with mosquito DNA

Exon trapping methods have played an important role in the development of transcript maps. In one in vivo vertebrate method, exons in a genomic DNA clone are transcribed, and they are recovered without any a priori information on the nature of the expressed transcript. The only requirement is that the genomic DNA clone contains exons separated by intervening introns that are removed by splicing during mRNA transcription and that the splice donor and acceptor site sequences follow those used by vertebrates. It is not known whether invertebrate splice donor and acceptor sites from genes that contain short introns will be processed correctly using an in vivo vertebrate exon trapping method. In this report, an analysis of mosquito splice sites using software designed to identify exons in genomic DNA sequence suggested that the vertebrate exon trapping method could recognize mosquito introns and exons. When a mosquito genomic DNA clone containing the D7 gene was tested experimentally, this method failed to recognize and process small introns (<63 bp) faithfully. In spite of this failure, exons and exon fragments were recovered. The implications of these findings and their application to map-based positional cloning in mosquito genomics is discussed.

The mouse p52 subunit of the transcription/DNA repair factor TFIIH is located in the class III region of the H2 complex: cloning and sequence polymorphism.

Loci controlling susceptibility to a number of diseases, including cortisone-induced cleft palate, experimental allergic orchitis, and chemically-induced transplacental lung tumors have been mapped to a 27 kilobase (kb) region within the class III region of the mouse major histocompatibility complex (H2). This region, contains three genes G7e, which resembles a viral envelope gene, Bat6 (G7a), which encodes a valyl-tRNA synthetase, and G7c, which has no known function. We cloned a set of overlapping cosmid clones containing 115 kb of DNA surrounding Bat6. Exon trapping has identified a new gene located telomeric of Bat6. Northern blot analysis detected a transcript of 1.7 kb with highest expression in the testis. DNA sequence analysis identified this gene as the mouse homologue of the human gene encoding the p52 subunit of the TFIIH transcription/DNA repair factor. Nucleotide sequence identity was 91% between mouse and human, and the protein sequence was 98% identical. Sequence analysis of p52 cDNA from congenic mouse strains detected an amino acid polymorphism at position 209, which results in the substitution of a threonine in the H2b haplotype to a methionine in the H2a,d haplotypes.

Detailed transcript map of a 810-kb region at 11p14 involving identification of 10 novel human 3' exons.

A limited number of genes, including the human brain-derived neutrotrophic factor (BDNF) gene, have been identified in the human chromosome 11p14 region. Since this area is involved in a genetic disorder (WAGR syndrome) and because of interest in studying the regulation of the human BDNF gene, we have established a detailed transcript map of a 810-kb region clone in a yeast artificial chromosome (YAC), corresponding to a portion of this genomic locus. A set of nested deletion mutants has been generated to map genes at a mean resolution of 75kb. Four genic markers from available mapping databases have been mapped on the YAC. Ten potential novel human exons have been isolated by a 3' terminal exon trapping procedure directly applied to purified YAC DNA. Most of these exons display polyadenylation signals and they all yield positive signals in RT-PCR experiments, confirming their status of transcribed sequences. The BDNF gene is now co-localised with three other genes on a 120 kb DNA fragment.

Localization of a Human Nucleoporin 155 Gene (NUP155) to the 5p13 Region and Cloning of Its cDNA

Nucleoporins are the main components of nuclear pore complexes (NPCs) involved in nucleo-cytoplasmic transport. Starting with an expressed DNA fragment retrieved by exon trapping from pooled human BAC clones mapped to the short arm of chromosome 5, we identified a human nucleoporin cDNA sequence by PCR from a human testis cDNA library. The coding sequence showed high homology to that of the rat nucleoporin 155 (Nup155) cDNA. FISH analysis with the human BAC clone as probe localized the human NUP155 gene to chromosome band 5p13. Northern analysis showed that the human NUP155 gene was expressed at different levels in all tissues tested. Two species of transcripts were observed with estimated lengths of 5.4 and 4.7 kb, respectively, in concordance with the finding of two alternative polyadenylation sites in the cDNA. The genomic location of the human NUP155 gene suggests a possible role in the mental and developmental retardation associated with hemizygous deletions of the 5p13 region.

Genomic Organization and Biological Characterization of the Novel Human CC Chemokine DC-CK-1/PARC/MIP-4/SCYA18

The chemokines are a group of chemotactic molecules that appear to regulate the directed movement of white blood cellsin vitroandin vivoand may therefore play important roles in inflammation and immunity. The genes encoding the chemokines are clustered in close physical proximity to each other. A large cluster of human CC chemokine genes resides on chromosome 17. We have used this information in a positional cloning approach to identify novel chemokine genes within this cluster. We constructed a YAC contig encompassing the MIP-1α (HGMW-approved symbol SCYA3) gene region and used exon trapping and sequence analysis to isolate novel chemokine genes. Using this approach, a gene encoding a chemokine named MIP-4, based on its homology with MIP-1α (49.5% identity at the nucleotide level and 59.6% at the predicted amino acid level), was found. The MIP-4 gene (HGMW-approved symbol SCYA18) consists of three exons spread over 7.1 kb and is separated from the MIP-1α gene by 16 kb. The MIP-4 gene encodes a 750-bp mRNA that is expressed in lung and macrophages but not in brain or muscle. The mRNA encodes an 89-amino-acid protein and includes a predicted signal peptide of 21 amino acids. Recombinant or synthetic MIP-4 induced calcium mobilization in naive and activated T lymphocyte subpopulationsin vitro.Injection of synthetic MIP-4 into the peritoneal cavity of mice led to the accumulation of both CD4+and CD8+T lymphocytes, but not monocytes or granulocytes. These observations provide new information concerning the arrangement of the CC chemokine gene cluster on human chromosome 17 and indicate that the MIP-4 gene product is chemotacticin vivofor both CD4+and CD8+T lymphocytes and may therefore be implicated in both humoral and cell-mediated immunity.

Transcription Mapping in a Medulloblastoma Breakpoint Interval and Smith–Magenis Syndrome Candidate Region: Identification of 53 Transcriptional Units and New Candidate Genes

The chromosomal band 17p11.2 is associated with a number of neurological disorders and malignant diseases. This region is also characterized by the presence of complex repeat elements that are probably responsible for the frequent occurrence of interstitial deletions, duplications, and isochromosome formation. In the course of the molecular analysis of this interval, an integrated map with YACs, PACs, and cosmids covering approximately 6 Mb was established. Focusing on the 1.4-Mb interval containing the Smith–Magenis syndrome critical region and the breakpoint region for medulloblastomas, we constructed a detailed transcript map between the marker PS2 and the proximal CMT1A repeat. FISH analysis of the PACs allowed determination of the position of the transcripts with respect to the SMS critical region and the presumptive chromosomal breakpoint in medulloblastomas. One PAC (G21100) provided evidence for the presence of a novel complex repeat unit, indicating that there are at least three independent repeat elements within 2 Mb. Five genes were mapped to clone G21100 and are likely to form part of this novel complex sequence repeat. In summary, 53 new transcripts were isolated by using cDNA selection and exon trapping. This included 8 known but previously unmapped genes and 45 novel transcripts. The expression profile of 21 transcripts was determined by RT-PCR. Based on their homologies to known genes or proteins, some of the novel genes are considered candidate genes either for malignant diseases or for the Smith–Magenis syndrome.

7] Cosmid-based exon trapping

Publisher Summary Exon trapping is a widely used gene identification method. In contrast to RNA-based methods, such as screening of cDNA libraries and cDNA selection, exon trapping is independent of gene expression. Exon trapping provides a functional assay in which cloned DNA sequences are selectively isolated based on the presence of functional splice sites. Consequently, sequences are isolated directly from the clone under analysis without knowledge or availability of tissues expressing the unknown gene. This chapter discusses cosmid based exon trapping that was developed to bypass problems related to plasmid-based exon trapping. It discusses the principle of cosmid-based exon trapping. The chapter also discusses reverse transcription (RT) and amplification by polymerase chain reaction (PCR), which is the most critical step of the entire procedure.

22] Targeted display: A new technique for the analysis of differential gene expression

The phenotype of tissues within an organism is determined by their patterns of gene expression. Several powerful molecular approaches have been developed allowing the simultaneous assessment of gene expression profiles between two populations of cells or tissues under study. This chapter describes a new technique called Targeted Display (TD); this is an extension of a published technique. Targeted display utilizes the surprising finding that there is a nonrandom distribution frequency for octanucleotide sequences throughout randomly selected protein-coding regions of DNA. Statistically designed octanucleotides were adapted for polymerase chain reaction (PCR) and used to investigate the differential expression of genes involved in the acquisition of the differentiated neuronal phenotype in rat PC12 cells. The principle of TD remains, as with similar approaches, to identify and isolate genes exhibiting differential patterns of gene expression between two populations of cells. However, this technique offers additional prospects for the specific and highly selective targeting of genomic subsets, for DNA fingerprinting, phylogenetic analyses, exon trapping, and the elucidation of translocations, insertions, or deletions within genetic lesions.

10] Saturation identification of coding sequences in genomic DNA

Publisher Summary This chapter provides an overview of comprehensive transcriptional mapping, as it would be applied to the genomes of mammals or other higher vertebrates. In this context, the chapter discusses four techniques for gene identification that have demonstrated their utility for reliable analysis of large genomic regions: dbEST (database of expressed sequence tags) analysis, cDNA selection, exon trapping, and genomic sequence analysis. Generally developed for analysis of mammalian genomes, these techniques are applicable to any genome where the presence of introns, large intergenic distances, and/or repetitive DNA significantly reduces the proportion of coding to noncoding sequence. The chapter describes the general advantages and disadvantages of each technique. The chapter discusses the preliminary considerations for defining the magnitude and scope of the gene search and the necessary resources for beginning the gene search.

An exon map of the AZFc male infertility region of the human Y chromosome.

STS markers spanning the AZFc region of human Y Chromosome (Chr) were used to isolate a series of 14 P1 artificial chromosome (PAC) clones covering at least 560 kb of DNA. End clone analysis of these PAC clones was used to derive 10 new STS markers. Together with other markers mapped to the region, an STS content analysis was used to assemble these PAC clones into three distinct contigs. A minimum tiling path of ten PAC clones was subjected to exon trapping to identify potentially new genes mapping to the AZFs region. In all, 39 potential exons were isolated, including 2 exons from the DAZ gene, 3 exons from the BPY2 gene, 2 exons from the PRY gene, and 1 exon from a member of the RBM II gene family; all these genes have been shown previously to map to the AZFc region. One further exon was found that shows homology to the DFFRY gene, which maps to Yq11. 2, indicating that there may be a further copy of this gene or a pseudogene in the distal Yq euchromatin. The majority of the remaining potential exons appear to be novel, suggesting that additional genes lie in the AZFc region. Mapping of these exons by PCR analysis of somatic cell hybrids has shown that six of these exons are homologous to autosomal sequences, and five to sequences on the X Chr. RT-PCR analysis of primary cDNA from adult testis, brain, liver, and skeletal muscle mRNA has shown that 11 of the novel exons are expressed in one or a combination of these tissues, indicating that they form parts of genuine transcripts.

A 500-kb YAC and BAC Contig Encompassing the High-Growth Deletion in Mouse Chromosome 10 and Identification of the MurineRaidd/CraddGene in the Candidate Region

The mousehigh growth(hg) gene was identified in a selection experiment for rapid growth. It produces a 30–50% increase in weight gain of homozygous individuals without resulting in obesity. Recently,hgwas mapped to a deletion around markerD10Mit69.Here we report a map of yeast artificial chromosome and bacterial artificial chromosome clones spanning the entire region deleted in high-growth mice. The size of the deletion estimated from the clone lengths is approximately 500 kb. Using exon trapping, the murineRaidd/Craddgene was identified in thehgregion, and its cDNA sequence and expression pattern were determined. The human RAIDD/CRADD was mapped using radiation hybrid mapping to human chromosome 12, 2.9 cR distal to marker AFMB311WC5. The identifiedRaidd/Craddgene, which is deleted in high-growth mice, presents a potential candidate forhg.

Comparative Mapping of Distal Murine Chromosome 11 and Human 17q21.3 in a Region Containing a Modifying Locus for Murine Plasma von Willebrand Factor Level

Type 1 von Willebrand disease (VWD) is a common inherited disorder characterized by mild to moderate bleeding and reduced levels of von Willebrand factor (VWF). An animal model for human type 1 VWD, the RIIIS/J mouse strain, exhibits a prolonged bleeding time and reduced plasma VWF levels. We have previously mapped the defect in RIIIS/J to distal mouse Chr 11, distinct from theVwflocus on Chr 6. This locus,Mvwf,was localized to an ∼0.5-cM interval, tightly linked toGip,distal toNgfr,and proximal toHoxb.We have now used these genetic markers to construct a contig of yeast and bacterial artificial chromosomes and bacteriophage P1 clones spanning the ∼300-kbMvwfnonrecombinant interval. In a comparative mapping approach, mouse homologues of mapped human expressed sequence tags (ESTs) were localized relative to the candidate interval. Twenty-one sequence-tagged sites and ESTs from the corresponding human syntenic region 17q21.3 were ordered using the high-resolution Stanford TNG3 radiation hybrid panel. Based on the resulting radiation hybrid map and our mouse genetic and physical maps, the order of human and mouse genes in a >0.7-cM region appears to be conserved. Six genes localized to theMvwfnonrecombinant interval by comparative mapping included orthologs ofGNGT2, ATP6N1,and a nuclear domain protein. Seven other genes or ESTs were excluded from the candidate interval, including orthologs ofPHB, PDK2,a speckle-type protein, and a UDP-galactose transporter. Using exon trapping, 10 additional putative expressed sequences were identified within theMvwfnonrecombinant interval, including a previously cloned murine glycosyltransferase as well as exons showing sequence similarity to genes forCaenorhabditis elegansandSaccharomyces cerevisiaepredicted proteins, anArabidopsis thalianaubiquitin-conjugating enzyme, and aGallus gallusmRNA zipcode-binding protein. Further characterization of these putative genes could identify the dominant mutation responsible for low plasma VWF levels in RIIIS/J mice. These data may also aid in the localization of other disease loci mapped to this region, including the gene for tricho-dento-osseous syndrome and a murine locus for susceptibility to ozone-induced acute lung injury.

Generation of a transcription map distal to HLA-F.

We have constructed a transcription map covering a 2 Mb region beginning approximately 1 Mb distal to HLA-F. Cosmids isolated from a chromsome 6 library were positioned by YAC hybridisation, STS and fingerprint analysis. Using direct cDNA selection, exon trapping, and direct genomic sequence analysis, we identified 42 potential exonic fragments in this region. Six fragments corresponded to previously characterised genes, four previously broadly mapped to this region. Five fragments were similar to known genes, eight fragments matched ESTs and 10 of the remaining 23 novel fragments, gave a positive signal on northern analysis. All cDNA fragments were mapped to the YAC and cosmid contig covering the region and with respect to other known genes and STS in this area. The distribution of the cDNA fragments indicated their organisation in three clusters around CpG islands.

Genetic and Physical Mapping of theLpsLocus: Identification of the Toll-4 Receptor as a Candidate Gene in the Critical Region

ABSTRACT: On the basis of 2093 meioses analyzed in two separate intraspecific backcrosses, the location of the mouseLpsdmutation was circumscribed to a genetic interval 0.9 cM in size. A total of 19 genetic markers that lie in close proximity to the mutation were examined in mapping. Most of these were previously unpublished polymorphic microsatellites, identified by fragmentation of YAC and BAC clones spanning the region of interest.Lpsdwas found to be inseparable from the microsatellite marker D4MIT178, and from three novel polymorphic microsatellites identified near D4MIT178. The mutation was confined between two novel microsatellite markers, herein designated “B” and “83.3.” B lies centromeric to the mutation, and was separated by four crossovers in a panel of 1600 mice; 83.3 lies distal to the mutation and was separated by three crossovers in a panel of 493 mice. 66 BAC clones and one YAC clone were assembled to cover >95% of the critical region. Estimates based on pulsed field gel electrophoresis and fluorescence in situ hybridization indicate that the The B→ 83.3 interval is about 3.2 Mb in length. A minimal area of zero recombinational distance fromLpsdwas also assigned, and found to occupy approximately 1.2 Mb of physical size. To identify gene candidates, nearly 40,000 sequencing runs were performed across the critical region. Selective hybridization and exon trapping were also employed to identify genes throughout the “zero” region. Only a single intact gene was identified within the entire critical region. This gene encodes the Toll-4 receptor, a member of the IL-1 receptor family.

Comparison of Methods for Identifying Transcription Units and Transcription Map of the Werner Syndrome Gene Region

To isolate a human disease gene by positional cloning, a critical step is the identification of candidate genes from a targeted genomic region. We used cDNA selection, exon trapping, and genomic sequencing to identify 12 transcription units from a 1.4-Mb genomic region containing the Werner syndrome gene (WRN). This included sequencing of 650 kb in the region of the WRN gene, to date, the most DNA sequenced as part of a positional cloning effort. The result of this combined method was significant overlap among the transcription units identified by each method; yet, no one method identified all of the transcription units. We present here a comparison of the effectiveness and efficiency of these methods and present a transcription map of the Werner syndrome gene region.

Consistent Fusion of ZNF198 to the Fibroblast Growth Factor Receptor-1 in the t(8;13)(p11;q12) Myeloproliferative Syndrome

The 8p11 myeloproliferative syndrome is a rare, aggressive condition associated with reciprocal translocations of chromosome band 8p11, most commonly the t(8;13)(p11;q12). To identify the genes involved in this translocation, we used fluorescence in situ hybridization (FISH) analysis to show that the chromosome 8 breakpoints fell within YAC 899e2 and that the chromosome 13 breakpoints are clustered in a region flanked by YACs 929f11 and 911h8. FISH using chromosome 13 PAC clones indicated that the t(8;13) is not simply a reciprocal translocation but also involves an inversion of 13q11-12. Exon trapping of a PAC that spanned the chromosome 13 translocation breakpoints led to the identification of a gene, ZNF198, that detected rearranged bands when used as a probe against Southern blots of patient DNA. Conceptual translation of the full-length ZNF198 cDNA sequence predicts a protein of 1377 amino acids that shows significant homology to the DXS6673E/KIAA0385 and KIAA0425 proteins. Alignment of these three proteins revealed a novel, conserved Zn-finger–related motif (MYM domain) of the general form CX2C19-22CX3CX13-19CX2CX19-25FCX3CX3F/Y that is repeated five times in each protein. To identify the translocation partner gene on chromosome 8, 5′ and 3′ RACE polymerase chain reactions (PCRs) were performed on patient RNA with several combinations of ZNF198 primers. Clones were identified in which the ZNF198 was fused to exon 9 of the fibroblast growth factor receptor-1 (FGFR1), a gene known to map to 8p11. An identical ZNF198-FGFR1 fusion was detected in the three patients with a t(8;13) for whom RNA was available; reciprocal FGFR1-ZNF198 transcripts were not detected. Rearrangements of both ZNF198 and FGFR1 were found in two further patients by Southern blotting. ZNF198-FGFR1 includes the five MYM domains of ZNF198 and the intracellular tyrosine kinase domain of FGFR1. We hypothesize that this fusion leads to constitutive activation of the FGFR1 tyrosine kinase in a manner analogous to the activation of ABL by BCR in chronic myeloid leukemia.© 1998 by The American Society of Hematology.