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Exon trap cloning: Using PCR to rapidly detect and clone exons from genomic DNA fragments

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... Several groups have developed alternative strategies for isolating mammalian genes from genomic DNA which involve using a retroviral construct to trap exons by RNA splicing (Reilly et al, 1990;Buckler et al, 1991;Auch and Reth, 1990;Duyk et al, 1990). The procedure involves the "shotgun" cloning of fragments of genomic DNA or cloned genomic DNA into a specially constructed retroviral vector. ...
... The procedure involves the "shotgun" cloning of fragments of genomic DNA or cloned genomic DNA into a specially constructed retroviral vector. The cloning site is situated either between a viral donor and acceptor site from the HIV-1 tat gene (Buckler et al, 1991), or between a donor and acceptor site from the rat preproinsulin gene (Auch and Reth, 1990). Alternatively the clone is inserted downstream of a human beta globin donor site (Duyk et al, 1990). ...
Thesis
This thesis describes experiments to test the hypothesis that it is possible to detect coding regions in genomic DNA by using short synthetic oligonucleotides. Two types of sequences were targeted. The first consisted of sequences complementary to sites for rare-cutter restriction enzymes, which are often clustered in the CpG rich islands located adjacent to, or within, coding regions. The second were sequences with complementarity to consensus sites for control and regulation within or around coding regions, such as splice sites and transcription factor binding sites. Southern hybridization experiments were first carried out to test the hypothesis that short oligonucleotides, based on G/C rich regions and on consensus splice site sequences, could be used as hybridization probes to detect cosmids or fragments of cosmids containing CpG islands or splice site junctions. The cosmid vector pWE15 which contains two Notl sites and several clones containing the genes for human proteolipid protein, calcitonin/calcitonin gene related peptide-a (CGRP), glutathione-S-transferase and NADH-ubiquinone oxidoreductase were used as model systems for testing this hypothesis. Secondly, the polymerase chain reaction was used to test the hypothesis that short oligonucleotides based on rare cutter sites could be used as specific PCR primers using subclones of the cosmid vector pWE15 and phagemid pSL1180 as model systems, or in a less specific manner to amplify DNA in well characterised clones such as those containing the PLP, calcitonin/CGRP, glutathione-S-transferase and NADH-ubiquinone oxidoreductase genes. In addition the hypothesis that this method could also be used on total human genomic DNA, so that clones enriched for either CpG islands or for coding regions could be obtained, using rare cutter site, splice site and transcription factor site oligonucleotides, was also tested. Several methods including a "TA" cloning strategy were employed in order to generate mini-libraries of the amplification product for subsequent evaluation. Degenerate oligonucleotides with just their 3' ends based on the splice site and translation start site consensus sequences and with cloning sites at their 5' ends were also used on the model clones to test whether coding regions could be identified. Thirdly, experiments were carried out to demonstrate that short oligonucleotides based on rare cutter sites could be used as PCR primers for sequencing directly into CpG islands/coding regions in cloned DNA. Subclones of the four genes mentioned above were used as models to test this. Finally, experiments were carried out to test the hypothesis that the ligation of primer/linkers to rare-cutter restricted cosmids followed by direct PCR-sequencing could be used to obtain direct sequence from putative CpG islands in cloned genes. Several cosmid clones including the glutathione-S-transferase and NADH-ubiquinone oxidoreductase genes were used for this. The experiments showed that some of the hypotheses concerning G/C rich sequence detection could be confirmed. When genomic DNA was used as template, G/C rich oligonucleotides as short as 8-mers could prime PCR amplification and enable "TA" clones to be produced which were enriched 66-fold for CpG rich sequences. In addition, conditions necessary for direct and specific amplification using G/C rich oligonucleotides as short as 7-mers with vector constructs as model target DNA were identified. However, PCR using G/C rich oligonucleotides was not capable of identifying CpG islands within cosmid clones. G/C rich 8-mer oligonucleotides may also be used in certain situations for directed sequencing within cloned genes and could thus be used as random or directed primers in a large volume sequencing project. Primer/linkers for rare-cutter restriction sites may also be used for sequencing into CpG islands within cosmids. Oligonucleotides that have their 3' ends complementary to the splice site consensus sequence can also prime amplification (Degenerate Oligonucleotide Primed-PCR) from some splice sites in some cloned genes, although with low success rate. In conclusion, the development of methods to enrich for CpG islands in genomic DNA was successful, but identifying CpG islands in cosmid clones was not. However a degree of success was achieved in the direct sequencing of CpG islands within clones. The work with splice site sequences was less successful, and it must be concluded that other methods should be employed for the identification of coding regions within cloned DNA.
... The techniques described by Auch and Reth, (1990), Hamaguchi et al. (1992) and Ozawa et al. (1993) follow broadly similar principles to exon amplification (Buckler et al., 1991) but use different exon trap vectors. Auch and Reth, (1990) described vector pL531n which contained the LTR of Rous Sarcoma Virus as a strong promoter, the origin of SV40 for plasmid amplification in COS cells, a unique Kpnl cloning site and 5' splice donor site, 3' splice acceptor site and poly adénylation site of the rat preproinsulin gene. ...
... The techniques described by Auch and Reth, (1990), Hamaguchi et al. (1992) and Ozawa et al. (1993) follow broadly similar principles to exon amplification (Buckler et al., 1991) but use different exon trap vectors. Auch and Reth, (1990) described vector pL531n which contained the LTR of Rous Sarcoma Virus as a strong promoter, the origin of SV40 for plasmid amplification in COS cells, a unique Kpnl cloning site and 5' splice donor site, 3' splice acceptor site and poly adénylation site of the rat preproinsulin gene. Hamaguchi et al. (1992) constructed vector pMHC2 and genomic DNA was partially digested and cloned into the Bglil site within intron 10 of the p53 gene. ...
Thesis
Tuberous sclerosis (TSC) is an autosomal dominant disorder with clinical symptoms ranging from minor skin lesions to more serious manifestations such as mental retardation and seizures. Linkage analysis established heterogeneity and assigned genes to 9q34 (TSC1) and 16p13.3 (TSC2). Detection of TSC-associated deletions on 16p13.3 refined the localisation of the gene and TSC2 has now been identified. Meiotic recombination events in TSC families have defined a consensus candidate region for TSC1 between D9S149 and D9S114, but further refinement gives conflicting positions for the gene. A positional cloning strategy has been applied to characterise the TSC 1 candidate region and identify the gene. Radiation hybrids retaining markers from the target interval were characterised and used as a resource for region specific DNA. The clones isolated plus ones from other sources were confirmed to map to 9q34 by FISH. Localisations were refined by using three lymphoblastoid cell lines with translocations breakpoints within 9q34, thereby subdividing the band into 4 intervals. A total of 32 loci from 28 contigs, and 43 anonymous contigs were mapped enabling a FISH map to be constructed. Cosmids mapping to the TSC1 candidate region were subsequently screened for transcribed sequences by exon amplification. The initial study led to the identification VAV2, a good candidate for TSC1. It was followed by more extensive analysis of 6 contigs mapping to the same FISH interval as D9S149 and D9S114. A total of 171 putative exons were cloned, 48 potentially different products were sequenced and 34 were inferred to be authentic exons. A subset from 4 contigs were confirmed to be conserved sequences by cross-species hybridisation and cDNA clones were isolated and partially characterised. The exons and cDNAs isolated are important resources for construction of a 9q34 transcription map and may facilitate identification of the TSC1 gene.
... I have outlined the steps that I consider a priority to take above, however there are several additional or alternative strategies which could be taken, including exon trapping. This technique makes it possible to screen large stretches of DNA for sequences which are transcribed using an "exon trapping" construct, which detects splice donor sites in tens of kb of DNA at a time (Duyk et al., 1990;Auch and Reth, 1990). Thus it may be viable to screen DXS178 ...
Thesis
X-linked agammaglobulinemia is a primary immunodeficiency which arises as a result of a block in B cell differentiation and the subsequent prevention of antibody production by these cells. The disease locus has been previously mapped to Xq21.3-q22 by linkage studies. Pulsed field gel electrophoresis and radiation hybrid mapping were used to construct a physical map of Xq22. The map incorporated ten probes arranged into three unlinked submaps, and spanned 6 Mb of DNA. The physical map was consistent with genetic recombination frequency data for this part of the genome. PFGE was also used to screen unrelated XLA patients for deletions. The probe 212/9 (DXS178), which is closely linked to XLA, was found to be flanked by several restriction sites for infrequently cutting restriction enzymes which impaired attempts to span the distance separating the markers which flank the locus. A high resolution physical map was made around this marker which showed that the restriction sites were clustered. This indicated the presence of CpG islands, which are frequently associated with expressed sequences. A chromosome walk towards the CpG islands was instigated by screening a cosmid library with a subfragment of 212/9. Several clones were isolated and two were restriction mapped using infrequently cutting restriction enzymes. Comparison of the cosmid restriction maps with the genomic map around DXS178 indicated that the cosmids were a maximum of 20 kb from the nearest CpG island.
... Exontrap analysis. Exontrap is a vector system designed to selectively clone exons from large genomic fragments (Auch & Reth, 1990). We used this system to investigate the processing of transcripts carrying a single base deletion in exon 2 detected in a thrombasthenic phenotype. ...
Article
This work reports the molecular genetic analysis of two patients who suffer mucocutaneous haemorrhages, prolonged bleeding time and failure of platelets to aggregate, either spontaneously or in response to agonists. The absence of platelet surface glycoprotein (GP)IIb–IIIa complexes confirmed the clinical diagnosis of Glanzmann's thrombasthenia (GT). Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of exon 2 of GPIIb showed polymorphic bands caused by the homozygous deletion of a cytosine at position 288 relative to the translation start site, causing a shifting of the reading frame and appearance of a premature termination codon. The heterozygous relatives showed a reduced platelet content of GPIIb–IIIa, and a correlation was found between the levels of GPIIb mRNA and surface expression of GPIIb–IIIa complexes. Unlike other mRNAs carrying a nonsense mutation, (288Cdel)GPIIb does not force alternative splicing of GPIIb mRNA. As expected, co-transfection of Chinese hamster ovary (CHO) cells with cDNAs encoding GPIIIa and (288delC)GPIIb failed to enhance the surface exposure of GPIIIa. It is concluded that the (288delC)GPIIb mutation is responsible for the thrombasthenic phenotype of the patients. In addition, it has also been determined that heterodimerization of GPIIb–IIIa requires the integrity of exons 2 and 3 of GPIIb.
... The cells were harvested, and total cellular RNA was prepared (9). The RNA was used to generate an Fgf-8 cDNA, using an exon trap protocol (2). ...
Article
Full-text available
We have used mouse mammary tumor virus (MMTV) infection of Wnt-1 transgenic mice to accelerate mammary tumorigenesis and to molecularly tag insertionally activated proto-oncogenes that cooperate oncogenically with Wnt-1 (G. M. Shackleford, C. A. MacArthur, H. C. Kwan, and H. E. Varmus, Proc. Natl. Acad. Sci. USA 90:740-744, 1993). Here we report the identification and characterization of a 31-kb genomic locus that contains clonal MMTV integrations in 8 of 80 mammary tumors from MMTV-infected Wnt-1 transgenic mice. Two genes were identified within this locus, one of which was transcriptionally activated by MMTV insertions. This activated gene is identical to androgen-induced growth factor (AIGF/Fgf-8) (A. Tanaka, K. Miyamoto, N. Minamino, M. Takeda, B. Sato, H. Matsuo, and K. Matsumoto, Proc. Natl. Acad. Sci. USA 89:8928-8932, 1992), the eighth member of the fibroblast growth factor (FGF) family. Transcriptional activation of Fgf-8 was found in all tumors with MMTV insertions in this locus. Fgf-8 mRNA was absent in normal mammary glands and was detected only in adult testis and ovary and in midgestational embryos. The sequences of Fgf-8 genomic and cDNA clones revealed five coding exons, in contrast to the three coding exons found in other FGF genes. cDNAs encoding three isoforms of the FGF-8 protein were isolated. The three corresponding mRNAs resulted from the alternative use of two 5' splice sites and two 3' splice sites for the second and third exons, respectively. These results implicate Fgf-8 as the third FGF gene found to cooperate with Wnt-1 in MMTV-induced murine mammary tumorigenesis, suggesting that FGFs and Wnts are strong collaborators in this process.
... Several approaches towards the identification of coding sequences within large regions of non-transcribed DNA have been described (18)(19)(20)(21). However, the direct isolation of expressed sequences from ordered genomic libraries by exonamplification (22)(23)(24)(25)(26)(27) or direct cDNA selection (28)(29)(30)(31) is a more appropriate strategy for integrating physical and transcriptional data. cDNA selection is a hybridisation-based method dependent on the presence of the transcript in the cDNA population whereas exon-amplification takes advantage of the splicing mechanism and theoretically does not depend on the pattern of gene expression. ...
Article
The construction of a transcriptional map for human chromosome 21 requires the generation of a specific catalogue of genes, together with corresponding mapping information. Towards this goal, we conducted a pilot study on a pool of random chromosome 21 cosmids representing 2 Mb of non-contiguous DNA. Exon-amplification and cDNA selection methods were used in combination to extract the coding content from these cosmids, and to derive expressed sequences libraries. These libraries and the source cosmid library were arrayed at high density for hybridisation screening. A strategy was used which related data obtained by multiple hybridisations of clones originating from one library, screened against the other libraries. In this way, it was possible to integrate the information with the physical map and to compare the gene recovery rate of each technique. cDNAs and exons were grouped into bins delineated by EcoRI cosmid fragments, and a subset of 91 cDNAs and 29 exons have been sequenced. These sequences defined 79 non-overlapping potential coding segments distributed in 24 transcriptional units, which were mapped along 21q. Northern blot analysis performed for a subset of cDNAs indicated the existence of a cognate transcript. Comparison to databases indicated three segments matching to known chromosome 21 genes: PFKL, COL6A1 and S100B and six segments matching to unmapped anonymous expressed sequence tags (ESTs). At the translated nucleotide level, strong homologies to known proteins were found with ATP-binding transporters of the ABC family and the dihydroorotase domain of pyrimidine synthetases. These data strongly suggest that bona fide partial genes have been isolated. Several of the newly isolated transcriptional units map to clinically important regions, in particular those involved in Down's syndrome, progressive myoclonus epilepsia and auto-immune polyglandular disease. The study presented here illustrates the complementarity of exon-amplification and cDNA selection techniques for generating a large resource of new expressed landmarks, which contribute to the construction of a chromosome 21 transcript map.
... A prerequisite for the SDL (as well as SOE) application is information on the sequences of exon termini usually resulted' from sequencing of genes in genomic DNA. The highly interesting approach to cloning (and, potentially, sequencing) of exons recently described (28) can be an efficient source of that information. It is a combination of the latter method with SDL, capable of the precise joining of exons provided primary structure of their termini is known, that appears to be paricularly promising in synthesis of spliced eukaryotic genes from genomic DNA. ...
Article
Full-text available
An approach to directed genetic recombinationin vitro has been devised, which allows for joining together, in a predetermined way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA splicing by directed ligation, SDL). The approach makes use of amplification, by means of several polymerase chain reactions (PCR), of a chosen set of DNA segments. Primers for the amplifications contain recognition sites of the class IIS restriction endonucleases, which transform blunt ends of the amplification products into protruding ends of unique primary structures, the ends to be used for joining segments together being mutually complementary. Ligation of the mixture of the segments so synthesized gives the desired sequence in an unambiguous way. The suggested approach has been exemplified by the synthesis of a totally processed (intronless) gene encoding human mature interleukin-1α.
... To obtain subclones of the YAC, total yeast DNA in agarose blocks from one of the YAC clones was digested for 2 min with 1 unit of Taq I per jig of DNA. After phenol/ chloroform extraction, the fragments were cloned into the unique Cla I site of pL53In (22) and used to transform Escherichia coli DH5a cells. Clones containing CD44 were identified by colony hybridization to 32P-labeled CD44 variant E (17) and intron PCR products (this paper). ...
Article
Full-text available
The CD44 molecule is known to display extensive size heterogeneity, which has been attributed both to alternative splicing and to differential glycosylation within the extracellular domain. Although the presence of several alternative exons has been partly inferred from cDNA sequencing, the precise intron-exon organization of the CD44 gene has not been described to date to our knowledge. In the present study we describe the structure of the human CD44 gene, which contains at least 19 exons spanning some 50 kilobases of DNA. We have identified 10 alternatively spliced exons within the extracellular domain, including 1 exon that has not been previously reported. In addition to the inclusion or exclusion of whole exons, more diversity is generated through the utilization of internal splice donor and acceptor sites within 2 of the individual exons. The variation previously reported for the cytoplasmic domain is shown to result from the alternative splicing of 2 exons. The genomic structure of CD44 reveals a remarkable degree of complexity, and we confirm the role of alternative splicing as the basis of the structural and functional diversity seen in the CD44 molecule.
... Exon 3 C is recognized by the splicing machinery only if the "A" polymorphism occurs. This could be shown when the alternative SLP-65 exons 3A, 3B and 3C were tested for their ability to be recognized as exons by the splicing machinery using the "Exon Trap" vector system (Auch and Reth, 1990). With this assay the splicing of potential exons can be analyzed ( Fig. 11 and Figure legend for details). ...
Article
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SLP-65 is an adaptor protein preferentially expressed in B cells with a central role in BCR and pre-BCR mediated signal transduction. By providing multiple binding sites for other cellular effector molecules it assembles various proteins into one signaling complex. Therefore SLP-65 is involved in the control of different signaling pathways important for B cell development, survival and activation. SLP-65 KO mice demonstrated the critical role of SLP-65 in B cell development. Based on the fact that 5-10 of the SLP-65 KO mice develop pre-B cell leukemia most similar to childhood precursor B cell acute lymphoblastic leukemia, SLP-65 was analyzed in human tumor samples. SLP-65 expression was investigated by RT-PCR, immunoblotting and flow cytometry in 38 samples of precursor B acute lymphoblastic leukemia. In half of the cases SLP-65 protein was reduced or absent. Interestingly, aberrant SLP-65 mRNA splicing was detected in the tumor samples. Alternative exons containing pre-mature stop codons were included in the aberrantly spliced SLP-65 mRNA. These exons were shown to contain pre-mature stop codons leading to nonsense mediated decay of the aberrantly spliced SLP-65 mRNA. In addition, a genetic polymorphism involved in mediating exon recognition was identified. Three of ten patients were deficient for the transcription factor pax-5 involved in regulating SLP-65 expression and one of those patients had aberrant pax-5 mRNA’s lacking the sequences for the transactivation domain. Thus, loss of pax-5 expression could be another possibility to explain the SLP-65 deficiency in addition to aberrant splicing. Our data indicate that a block in pre-B cell differentiation, due to a somatic deficiency in key signaling elements combined with proliferation capacity and active RAG 1/2 enzymes may be the primary cause of pre-B ALL.
... The DNA from a tertiary nude mice tumor (section 1.2) was cloned into EMBL-3 phage and screened with a human specific repetitive Alu-probe. Alu-positive phage clones were isolated and submitted to exon-trap analysis (Auch and Reth, 1990). Isolated exon fragments were used to screen a cDNA library from RNA of a tertiary nude mice tumor and a novel putative oncogene, designated myeov (myeloma overexpressed gene), was isolated. ...
Article
The myeov gene was identified using the tumorigenicity assay with DNA from a patient suffering a gastric carcinoma. The Myeov gene is localized at chromosome band 11q13, a frequent site for chromosomal rearrangements in various carcinomas and B-cell neoplasms. The gene was shown to be involved in cases of multiple myeloma harboring the t(11;14)(q13;q32). In addition, myeov is coamplified with cyclin D1 and overexpressed in carcinomas of the breast, lung, bladder, esophageal squamous cell carcinomas and oral squamous cell carcinomas. Myeov DNA amplification and overexpression was detected in several carcinoma cell lines, however, hardly any MYEOV protein could be detected using specific antibodies in Western blot analysis. The 5 untranslated region (5UTR) of the myeov gene is long, encompasses four upstream AUG codons (uAUGs) and is predicted to fold in a strong secondary structure. These features are common among mRNAs regulated by their 5UTR and suggest that MYEOV protein synthesis might be regulated at a posttranscriptional level. These findings prompted us to investigate the possible role of the myeov 5UTR in controlling its protein level, and the possibility of MYEOV protein synthesis to be mediated by an internal ribosome entry site (IRES). Here we show that initial experiments using mono- and bicistronic reporter constructs supported this view. However, further examination by in vitro transcription/translation assays, Northern blot analysis and the application of promoterless constructs revealed promoter activity in the myeov 5UTR. Despite this strong promoter activity, we did not find any translation products. Our experiments showed that this was due to the presence of uAUGs codons present in the myeov 5UTR. DNA and RNA transfection of the wild type and mutated 5UTR, where the uAUGs were mutated to AAG, confirmed that these uAUGs abrogate translation of the reporter gene as well as the myeov gene. Alternative splicing mechanisms in specific cell types and/or developmental stages may be a way to evade this translation control. Das Myeov Gen wurde mittels eines Tumorigenizitätsassays aus der DNA eines Patienten mit einem Magentumor isoliert. Das Gen ist auf der Chromosomenbande 11q13 lokalisiert worden, einem häufig betroffenen Abschnitt für chromosomale Rekombinationen in verschiedenen Karzinomen und B-Zell-Neoplasien. Es ist nachweislich an Fällen multipler Myelome beteiligt, die eine t(11;14)(q13;q32) zeigen. Auch findet man Myeov zusammen mit Cyclin D1 koamplifiziert und überexprimiert in Karzinomen der Brust, der Lunge, der Blase, sowie in Epithelzellkarzinomen des Ösophagus und der Mundhöhle. Amplifikation und Überexpression von MYEOV wurden zwar in mehreren Karzinom-Zelllinien gezeigt, jedoch konnte in der Western-Blot-Analyse mit myeov-spezifischen Antikörpern kein Protein nachgewiesen werden. Die 5 nichttranslatierte Region (5UTR) des Myeov-Gens ist relativ lang, sie enthält vier vorgeschaltete AUG Kodons (uAUGs) und ist laut Prognose eines Computerprogramms in der Lage, eine starke Sekundärstruktur zu bilden. Diese Eigenschaften kommen häufig bei mRNAs vor, die durch ihre 5UTR reguliert werden und deuteten auch in unserem Fall darauf hin, daß die MYEOV-Proteinsynthese auf postranskriptionaler Ebene gesteuert sein könnte. Diese Entdeckungen veranlassten uns dazu, einerseits die mögliche Rolle der Myeov-5UTR bei der Regulation der Proteinexpression näher zu untersuchen, sowie andererseits zunächst die Möglichkeit zu überprüfen, ob die Proteinsynthese durch eine �internal ribosome entry site� (IRES) ermöglicht werden könnte. Erste Experimente mit mono- und bicistronischen Reporterkonstrukten schienen diese Ansicht zu stützen. Jedoch wies die weitere Prüfung durch in vitro-transcription/translation assays, Northern-Blot-Analyse und die Verwendung promotorloser Konstrukte auf eine Promotorfunktion der Myeov 5UTR hin. Doch trotz deutlicher Promotoraktivität fanden wir keine translatierten Produkte. Unsere Experimente zeigten schließlich, daß die Ursache in den uAUG Kodons der Myeov 5UTR zu suchen war. Die Transfektion verschiedener DNA- und RNA- Luciferase- Reporterkonstrukte mit Myeov 5UTR und parallel dazu einer mutierten Form, bei der die uAUGs zu AAG verändert worden waren, bestätigte, daß diese uAUGs die Translation des Reportergens sowie des Myeov-Gens verhindern. Alternatives Splicing in bestimmten Zell-Arten und/oder Entwicklungsstadien könnten eine Möglichkeit sein, diese Translationskontrolle zu umgehen.
... The methods based on genomic DNA, or expression independent methods, include CpG island identification (Bird 1986; Larsen et al. 1992), exon trapping (Auch and Reth 1990; Duyk et al. 1990), zoo-blots (Claudio et al. 1994), and genomic sequencing. CpG islands, unmethylated GC-rich sequences, are typically found near the 5' ends of vertebrate genes, and can serve as landmarks in gene identification (Larsen et al. 1992). ...
... Exon trapping detects the presence of exons flanked by functional splice sites in a clone, via an artificial RNA splicing assay (Auch and Reth, 1990;Buckler et al, 1991). This approach was successful in the identification of the gene for Opitz G/BBB syndrome (Quaderi et al, 1997). ...
Thesis
The aim of this work on the molecular genetics of deafness was to study the molecular basis of deafness in several families with non-syndromic and syndromic forms of deafness, including X-linked deafness and autosomal recessive Jervell and Lange-Nielsen syndrome (JLNS). From one of the first studies on mapping of genes for X-linked deafness, it was shown that X-linked deafness, although very rare, is genetically heterogeneous. Linkage analysis of a single large family identified a novel locus for X-linked deafness, DFN2. Several candidate genes, including COL4A5, DDP and Diaphanous X, were screened. A major locus for X-linked deafness, DFN3, has previously been identified at Xq13-q21. Deafness in most of the families mapping to this region is accounted for by mutation in the transcription factor gene, POU3F4. However, the remaining families have microdeletions proximal to, but not encompassing, the POU3F4 coding region, leaving the precise reason for their deafness unexplained. POU3F4 expression was studied in these individuals to determine whether there is a position effect on the POU3F4 gene, or a second gene proximal to it. Homozygosity mapping in conjunction with screening of candidate loci identified a new locus for the cardioauditory syndrome, JLNS. A positional candidate approach identified IsK as the causative gene. An IsK mutant construct has been created to study the mechanism of action of this mutation. During the course of this work, the potassium channel gene KVLQT1 was implicated in JLNS. Haplotype analysis of 12 additional JLNS families in this study was consistent with mutation in KVLQT1. Exons of the KVLQT1 gene were identified and primers designed for use with genomic DNA. Novel mutations were identified, confirming the role of KVLQT1 in JLNS and demonstrating that KVLQT1 is the major locus for this syndrome.
... Complete overlapping Y AC contigs have already been constructed covering some chromosomes such as chromosome 21 (Chumakov I, 1992 (Auch D, 1990;Buckler AJ, 1991). The technique was used in the identification of MIDI as the causative gene for Opitz/BBB syndrome (Quaderi NA, 1997). ...
Thesis
Craniosynostosis, the premature fusion of the cranial sutures, is a common disorder resulting in craniofacial malformations. It occurs in syndromes, which are distinguishable by other dysmorphic features. Causative mutations have been found in three members of the Fibroblast Growth Factor Receptor family (FGFR1, -2 and -3), with the majority of the mutations occurring within the Immunoglobulin-like loop HI (IgIII) of FGFR2. Over 50% of cases do not have an identified causative mutation in this region. By screening the extracellular domain of FGFR2 a number of novel and recurrent mutations were identified in both syndromic and non-syndromic cases, broadening the sites and the range of phenotypes associated with FGFR2 mutations. Mutations at an acceptor splice site were shown to cause exon skipping, which would result in premature termination of the protein. This is currently uncharacteristic of the FGFR mutations. A conserved serine-proline dipeptide in the IgII-III linker region of the FGFR molecules, which is a site of mutations in FGFR1, -2 and -3 in a variety of phenotypes was screened for changes in all four FGFRs in a large sample set. No mutations or polymorphisms were revealed, emphasizing the specificity of the mutations at the site. At other sites in IgIII, different syndromic phenotypes can arise from identical mutations in FGFR2. These samples were tested for an association between the phenotype and a polymorphic variant in relevant genes. To identify additional genes involved in craniosynostosis, candidate genes were analysed in non-syndromic families, and also in families with craniofrontonasal dysplasia (CFND). Two CFND families were shown to be consistent with linkage to Xp22, although in one family this included an apparently unaffected male with a normal clinical phenotype sharing the affected marker allele haplotype, emphasizing the variability of the male phenotype in the condition.
Chapter
A novel strategy for rapid identification of unknown coding sequences from large genomic regions has been developed. It is based on selective in vitro recombination of genomic DNA and cDNA followed by Polymerase Chain Reaction. The technique involves generation of cDNA primers, by restriction digestion of cDNA libraries, that hybridize to their cognate genomic DNA sequences. These hybrids are chain elongated using the free 3’ end of the cDNA fragment as a primer, then PCR amplified using primers previously attached to genomic DNA and cDNA. Unknown coding sequences are clearly discernable as genomic cDNA chimeras.
Article
Hairless nude mice are immunodeficient because they lack a thymus. The nude gene has now been identified; it encodes a winged-helix transcription factor that is expressed specifically in skin and thymus.
Article
We have developed a method, exon amplification, for fast and efficient isolation of coding sequences from complex mammalian genomic DNA. This method is based on the selection of RNA sequences, exons, which are flanked by functional 5' and 3' splice sites. Fragments of cloned genomic DNA are inserted into an intron, which is flanked by 5' and 3' splice sites of the human immunodeficiency virus 1 tat gene contained within the plasmid pSPL1. COS-7 cells are transfected with these constructs, and the resulting RNA transcripts are processed in vivo. Splice sites of exons contained within the inserted genomic fragment are paired with splice sites of the flanking tat intron. The resulting mature RNA contains the previously unidentified exons, which can then be amplified via RNA-based PCR and cloned. Using this method, we have isolated exon sequences from cloned genomic fragments of the murine Na,K-ATPase alpha 1-subunit gene. We have also screened randomly selected genomic clones known to be derived from a segment of human chromosome 19 and have isolated exon sequences of the DNA repair gene ERCC1. The sensitivity and ease of the exon amplification method permit screening of 20-40 kilobase pairs of genomic DNA in a single transfection. This approach will be extremely useful for rapid identification of mammalian exons and the genes from which they are derived as well as for the generation of chromosomal transcription maps.
Thesis
Tuberous sclerosis is an inherited disease typically characterised by a facial rash, seizures and mental handicap. Patients suffer from benign tumours in many organs including the brain, heart, skin, kidneys, lungs and eyes. Genetic linkage studies show that the disease may be caused by a defect in one of two genes; TSC1 on chromosome 9q34 or TSC2 on chromosome 16p13. TSC2 had previously been identified and encodes a 180kD protein, tuberin, which may function as a GTPase activating protein (GAP). Previous genetic mapping studies delineated a candidate region for TSC1 of about 1.2Mb and physical mapping efforts had provided a complete cosmid contig of the region. This thesis describes exon trapping experiments performed in order to identify candidate genes in the region. Statistical analysis was subsequently performed in an attempt to determine why only a subset of exons were successfully trapped, but no significant factors were found. Exon trapping and EST mapping experiments in the candidate region allowed the identification and characterisation of several genes, including a gene which may encode a G protein subunit. Genomic sequence obtained from the cosmid contig as part of the Human Genome Project was analysed using a combination of database searching and gene prediction programs, resulting in the identification of several genes including one which appears to encode a sugar transporter. As part of a large collaborative effort, the TSC1 gene was identified. Its protein product, hamartin, has a coiled-coil domain but its function is unknown. Work described here includes mutation analysis of the gene and identification of neighbouring transcripts. The mutation screen in 79 tuberous sclerosis patients showed that most, if not all mutations in TSC1 are predicted to truncate the protein product prematurely, that TSC1 mutations are underrepresented in sporadic cases of tuberous sclerosis as compared to familial tuberous sclerosis, that non-penetrance is unlikely to exist in tuberous sclerosis and that a single ungual fibroma is not diagnostic of the disease.
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We report an extension of 3'-terminal exon trapping technology to the identification of transcribed sequences from yeast artificial chromosomes (YACs). A 350-kb YAC containing mouse genomic DNA was gel-purified and used as the target DNA for the 3'-terminal exon trapping strategy. A novel direct ligation/transfection approach was employed to increase the efficiency of trapping 3'-terminal exons from recombinant vector-derived chimeric mRNA. The resulting RT-PCR product was then used to generate a plasmid library. Randomly chosen individual subclones from this library were sequenced, and the results indicate that 86% met sequence criteria characteristic of 3'-terminal exons, whereas 14% were background from identified sources. PCR mapping efforts suggest eight putative last exons present within this YAC, whereas RT-PCR studies demonstrate that three reside within valid expressed sequences.
Article
Characterization of the structure, multiplicity, organization, and cell lineage-specific expression of T-cell receptor (TCR) genes of nonmammalian vertebrate species is central to the understanding of the evolutionary origins of rearranging genes of the vertebrate immune system. We recently described a polymerase chain reaction (PCR) strategy that relies on short sequence similarities shared by nearly all vertebrate TCR and immunoglobulin (Ig) variable (V) regions and have used this approach to isolate a TCR beta (TCRB) homolog from a cartilaginous fish. Using these short PCR products as probes in spleen cDNA and genomic libraries, we were able to isolate a variety of unique TCR and TCR-like genes. Here we report the identification and characterization of a chicken TCR gamma (TCRG) homolog, apparent Xenopus and pufferfish TCR alpha (TCRA) homologs, and two horned shark TCR delta (TCRD)-like genes. In addition, we have identified what could be a novel representative of the Ig gene superfamily in the pufferfish. This method of using short, minimally degenerate PCR primers should speed progress in the phylogenetic investigations of the TCR and related genes and lend important insights into both the origins and functions of these unique gene systems.
Article
Exon trapping allows for the rapid identification and cloning of coding regions from cloned eukaryotic DNA. In preliminary experiments, we observed two phenomena which limited the exon-trapping efficiency of pSPL3-based systems. The first factor that affected performance was revealed when we found that up to 50% of the putative trapped exons contained sequences derived from the intron of the pSPL3 trapping vector. Removal of the DNA sequences responsible for the cryptic splice event from the original splicing vector resulted in a new vector, pSPL3B. We demonstrate that pSPL3B virtually eliminates pSPL3-only spliced products while maximizing the proportion of exon traps containing genomic DNA (> 98%). The other step which impacted performance was our observation that a majority of the ampicillin-resistant (APR) clones produced after shotgun subcloning from ApR cosmids into pSPL3 were untrappable, pSPL3-deficient, recircularized cosmid vector fragments. Replacement of the pSPL3 ApR gene with the CmR cassette encoding chloramphenicol (Cm) acetyltransferase enabled selection for only pSPL3-containing CmR clones. We show a 30-40-fold increase in the initial subcloning efficiency of cosmid-derived fragments with pSPL3-CAM, when compared to pSPL3. The collective vector alterations described improve the overall exon-trapping efficiency of the pSPL3-based trapping system.
Article
We have developed an exon-trapping system with a newly constructed trapping vector containing multiple cloning sites (designated pEXT2). The system revealed high sensitivity for trapping a control exon from several hundred kbp of DNA. We have applied the system to the cosmid clones located on human chromosome 21p11-q21, and identified two fragments highly homologous to neurofibromatosis 1 (NF1) gene and a clearly transcribed fragment hybridized with approximately 1.6 kb RNA from human brain and human glioblastoma A172 cell.
Article
The immunoglobulin kappa locus (Ig kappa) is active only in the B-lymphocyte cell lineage. By exon-trapping we found a gene situated downstream from the murine Ig kappa locus. This gene encodes a protein with 53% sequence identity to the ribose 5-phosphate isomerase A (RPI-A) of Escherichia coli and is therefore likely to be the murine homologue (mRPI) of this enzyme. We confirmed this assumption by showing that a glutathione S-transferase (GST)::mRPI fusion protein has enzymatic activity and that an anti-mRPI antibody detects a protein of the predicted mass of RPI (33 kDa). Cloning and sequencing of the human counterpart show that the RPI gene is evolutionarily conserved. The expression of mRPI is not influenced by the rearrangement status of the Ig kappa locus in B cells and mRPI is expressed in all tissues. We thus show that two genes with very different expression patterns, a housekeeping gene and a gene expressed in a tissue-specific manner, can be located on a chromosome in close proximity to each other.
Article
A central issue in genome analysis is the identification and characterization of coding regions. Estimating the coding complexity of vertebrate genomes by measuring the kinetic complexity of mRNA populations and by sequence analysis of cDNAs is limited by the fact that any given source of mRNA represents a very biased sample of all genes. Exon trapping is a method that enables the identification of genes irrespective of their transcriptional status. Exons were trapped from the entire mouse genome, and the resulting fragments cloned. About 7% of a random sample of exons taken from this library have significant structural homology or sequence similarity to previously sequenced genes. Using cDNAs derived from several stages of mouse development, evidence for expression of about 62% of this sample of exons was found. These data suggest that the great majority of 'exons' in the library are derived from genes. We estimate that the fraction of the genome contained in trapped exons is 2.4%; this corresponds to a sequence complexity of about 72 megabases. The library of exons trapped from the entire mouse genome probably represents one of the least biased and most comprehensive libraries of mouse coding regions, and should therefore prove very useful for finding genes during genome mapping and sequencing.
Article
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We have reported the cloning from mouse genomic DNA of a fragment encoding a G-protein-coupled receptor related to the receptor for the blood clotting enzyme thrombin. Like the thrombin receptor this receptor is activated by proteolytic cleavage of its extracellular amino terminus. Because the physiological agonist at the receptor was unknown, we provisionally named it proteinase-activated receptor 2 (PAR-2). Here we present a PAR-2 cDNA of 2729 nucleotides that differs from the published genomic sequence at the 5' end, including a part of the protein coding region. The differences do not affect the peptide sequence of the activating proteinase cleavage site proper, but may include amino acid residues important for enzyme-substrate recognition. Analysis of the PAR-2 gene structure showed that the cDNA 5' end is derived from a separate exon located about 10 kilobases away from the 3' exon. Results from a primer extension experiment indicate that transcription starts at a unique site around nucleotide -203 respective to the translation initiation ATG. Chinese hamster ovary cells transfected with either the PAR-2 cDNA or a construct made from the published PAR-2 genomic sequence responded with intracellular calcium mobilization to stimulation with 1 nM trypsin, 10 microM PAR-2-activating peptide (SLIGRL), or 1 microM thrombin receptor-activating peptide (SFLLRN). Untransfected cells responded only to stimulation with thrombin receptor activating peptide. Only transcripts corresponding to the PAR-2 cDNA could be detected in three mouse tissues examined.
Article
Full-text available
Exon trapping is a method to functionally clone expressed sequences from genomic DNA. We have previously developed the vector system pETV-SD2, which contains only a splice donor site (SD) followed by a LacZ gene, allowing trapping of internal exons of human genes by blue-white selection. We now describe the adaptation of the same system for the efficient trapping of 3′-terminal exons, by using different RT-PCR primers in a 3′ RACE reaction. The addition of a T7 promoter to the RT-PCR products derived from pETV-SD2 allows their amplification in an isothermic amplification reaction called NASBA (nucleic acid sequence-based amplification reaction) and results in a strong signal from amplified 3′ exons in addition to a great reduction of non-specific background. As a test for the system, 3′ exon trapping was performed using a cosmid containing the Α-globin gene cluster on chromosome 16. The 3′-terminal exons of the human Α1, Ζ2. and θx-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3′ flanking region of the a-globin gene. This exon appears to belong to a previously unidentified gene within the α-globin gene cluster. This 3′ exon trapping strategy should facilitate the cloning of genes from large genomic regions.
Article
Exon amplification is an increasingly popular approach to the identification of transcribed sequences and will complement other strategies to isolate genes. We have used this system to amplify candidate exons from 32 cosmids, including 8 cosmids which span a well characterized 185-kb region of the human major histocompatibility class II region on Chromosome (Chr) 6. We have examined the efficiency, specificity, and reproducibility of the system in isolating exons from genes known to be present on particular cosmids and have determined the nature and frequency of artefact amplifications in routine cosmid screening. We were able to clone at least one exon from 88% (7/8) of all known genes tested (including exons which are differentially spliced) and obtained artefacts from 19% (6/32) of the cosmids tested. Such artefacts generally arise from the amplification of noncoding sequences flanked by regions with high homology to acceptor and donor splice junctions. We show that the exon amplification procedure can be used successfully with a wide variety of cosmids which have different numbers of genes and gene structures and describe several approaches to the characterization of novel exons cloned in this study.
Article
Modifications to exon amplification have been instituted that increase its speed, efficiency and reliability. Exons were isolated from target human or mouse genomic DNA sources ranging from 30 kilobases (kb) to 3 megabases (Mb) in complexity. The efficiency was dependent upon the amount of input DNA, and ranged from isolation of an exon for every 20 kb to an exon for every 80 kb of target genomic DNA. In these studies, several novel genes and a smaller number of genes isolated previously that reside on human chromosome 9 have been identified. These results indicate that exon amplification is presently adaptable to large scale isolation of exons from complex sources of genomic DNA.
Article
Identification of expressed sequences within genomic DNA is a hurdle in the characterization of complex genomes. We developed an exon trapping scheme that provides a positive selection for vertebrate 3′-terminal exons. A copyof the trapped exon sequence is obtained by RT/PCR amplification. The technique detects valid terminal exons without interference from partial exons or non-specific sequences, including simple human repeated sequences. Application to random human cosmids yielded one unique trapped terminal exon per cosmid on average. Because vertebrate terminal exons average 600–700 nucleotides in length, the technique provides transcribed sequences of sufficient length to assist further mapping efforts.
Article
The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies--examining the critical parameters and variations and their widespread applications--giving the strengths and limitations of these methodologies.
Article
We constructed cosmid libraries from human-hamster somatic cell hybrids that possess all or part of the short arm of chromosome 11 as their only human complement and isolated 129 human 11p clones. These cosmids map to 22 of 25 intervals distinguished by a hybrid panel for chromosome 11p. Forty-eight single-copy sequences were subcloned from 25 cosmids. Six of 17 (35%) single-copy sequences tested identify 11 new polymorphisms. Restriction endonuclease analysis identified CpG islands in 16 of 68 cosmids (23.5%). Analysis of the distribution of restriction endonuclease sites recognizing CpG dinucleotides showed that clusters of these sites, including those associated with the 5' region of an 11p13 Wilms' tumor gene, WT1, can span greater distances than generally recognized. The cosmids reported here should contribute to the construction of long-range physical maps and the isolation of additional genes on the short arm of chromosome 11.
Chapter
We present here a relational mapping strategy to establish integrated physical and transcriptional maps of large DNA regions. In this study, human chromosome 21 is used as a model because a physical map spanning 40 megabases on the 21q arm has already been achieved. The first step of this global analysis is to extract most of the genic content encoded by this chromosome. For this purpose, exon-amplification and cDNA selection methods are applied in conjunction to derive expressed sequence libraries from chromosome 21-specific cosmids. These resources will constitute one of the tools necessary for the relational analysis, allowing cross-screenings between genomic and coding sequence libraries. Eventually, the new genes will represent a resource to hunt for potential candidate genes implicated in the determinism of severe diseases associated to chromosome 21.
Chapter
Coincident Sequence Cloning (CSC) is a strategy for genome analysis which involves the selective isolation of sequences on the basis of their shared existence in a pair of otherwise distinct DNA mixtures. Two related methodologies, Hybrid Fishing CSC (HF-CSC) and End Ligation CSC (EL-CSC), have been developed which are able to handle highly complex DNAs and can enrich for coincident elements by 103–105 fold and 106–107 fold respectively. Using these methods to ‘integrate’ genomic DNA with cDNA provides an effective way of identifying transcribed sequences within defined genomic domains. The immense enrichment power of the EL-CSC procedure permits target genes to be directly purified to homogeneity.
Chapter
Exon amplification is an increasingly popular approach to the identification of transcribed sequences and complements other strategies to rapidly isolate coding sequence. The following chapter describes application of this system to amplify candidate exons from cosmids and YACs, including 8 cosmids which span a well characterised 185 kb region of the human major histocompatibility class II region on chromosome 6. We have examined the efficiency, specificity and reproducibility of the system in isolating exons from genes on cosmids and YACs and describe the nature and frequency of artefact amplifications in routine screening. We show that the exon amplification procedure can be used successfully with a wide variety of cosmids and YACs which have different numbers of genes and gene structures and describe an efficient approach to the isolation of full length cDNA sequences by the integration of exon amplification and cDNA enrichment technology.
Chapter
A protocol has been established which allows obtaining growth-factor-independent cell mutants by retroviral insertion mutagenesis in vitro. In order to identify gene alterations possibly leading to growth-factor independency, two techniques were established for the cloning of retroviral insertion sites. One technique makes use of the amplification of retroviral flanking fragments by inverse polymerase chain reaction (IPCR). The other strategy involves complementation of a truncated kanamycin gene present in a bacterial plasmid vector by a neomycin gene fragment originating from the retroviral vector, which allows direct selection for kanamycin resistant bacterial cell clones. Using these techniques flanking fragments with several putative genes have been obtained. One flanking fragment shows high homology to the rat ionotrophic glutamate receptor, i.e. GluR5.
Chapter
Linkage analysis in cancer families and the observation of rare cytogenetic abnormalities in the germline DNA of affected individuals can be used to identify the chromosomal location of the genetic defect associated with a familial cancer trait, as described in Chapter 2. The resolution of genetic mapping involving large consortium studies and multiple highly polymorphic marker probes is of the order of 1–2 cM, which corresponds to approximately 1–2 megabases (Mb). This compares to the size of whole chromosomes, which is of the order of 50 to 300 Mb.
Article
Exon Trapping is a powerful method developed recently for isolating transcribed sequences from genomic DNA. Two novel exons, A91 and D12, were isolated from YAC209G4 spanning FRAXA site using the exon trapping system, with pSPL3 as splicing vector. A91 was amplified strongly in fetal liver and skeletal muscle cDNA library and mildly in liver, kidney and bone marrow library, respectively. While D12 was failed to be detected from the 8 libraries. A fragment of 315 bp containing A91 has been cloned (AM4470) from the skeletal muscle cDNA library. A single transcript of 2.8 kb was detected from mRNA samples of human heart and skeletal muscle on multiple tissues Northern blot using AM4470 probe.
Chapter
A variety of techniques now exist for the identification and isolation of gene sequences from cloned genomic DNA. We report the use of exon amplification to isolate candidate exons of genes in the chromosome 17q21 region associated with familial breast and ovarian cancer. We have used the second generation splicing vector pSPL3, which provides greater flexibility for cloning genomic fragments and which reduces the frequency of the major classes of false positive clones. In two experiments, exon amplification was performed using DNAs of approximately 170 cosmids spanning 1–2 Mb of this region. Cosmid DNAs were pooled in groups of 6–10 each. More than 2000 candidate exon clones from these experiments have been arrayed in microtiter dishes. The average size determined for nearly 400 cloned inserts was approximately 200 base pairs. Ongoing efforts to identify and eliminate clone redundancy have thus far yielded more than 100 unique exon clones. Less than 10% of the clones were found to be repetitive or to be artifacts resulting from cryptic splicing involving sequences present in the splicing vector. Thus the great majority of clones were found to be single copy and to derive from the correct chromosomal location. These exons have been used as hybridization probes to isolate cDNA clones derived from normal breast tissue. The cloned exons and corresponding cDNAs are being localized within developing cosmid contigs in order to assemble a transcription map of the region, and to position transcribed sequences with respect to critical recombinants in breast/ovarian cancer families. While database searches suggest that many of the exon sequences are unique, these searches have also identified several genes which were either mapped previously to proximal 17q or which appear to be homologs of genes in other species. Exon amplification represents a rapid and efficient means for isolating candidate gene sequences from genomic clones, facilitating efforts to identify specific genes associated with disease using positional cloning strategies. Utilization of this technique to survey large genomic regions will also assist in efforts to construct transcription maps of chromosomes.
Article
Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes, which lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that ESE evolution led to the fortuitous acquisition of IGF2 binding by M6P/IGF2R that drew IGF2R into parental conflict; subsequent imprinting may then have accelerated affinity maturation.
Article
This work was aimed at elucidating the molecular genetic lesion(s) responsible for the thrombasthenic phenotype of a patient whose low platelet content of glycoprotein (GP) IIb-IIIa indicated that it was a case of type II Glanzmann’s thrombasthenia (GT). The parents did not admit consanguinity and showed a reduced platelet content of GPIIb-IIIa. Polymerase chain reaction (PCR)–single-stranded conformational polymorphism analysis of genomic DNA showed no mutations in the patient’s GPIIIa and two novel mutations in the GPIIb gene: one of them was a heterozygous splice junction mutation, a C→A transversion, at position +2 of the exon 5-intron 5 boundary [IVS5(+2)C→A] inherited from the father. The predicted effect of this mutation, insertion of intron 5 (76 bp) into the GPIIb-mRNA, was confirmed by reverse transcription-PCR analysis of platelet mRNA. The almost complete absence of this mutated form of GPIIb-mRNA suggests that it is very unstable. Virtually all of the proband’s GPIIb-mRNA was accounted for by the allele inherited from the mother showing a T2113→C transition that changes Cys674→Arg674 disrupting the 674-687 intramolecular disulfide bridge. The proband showed a platelet accumulation of proGPIIb and minute amounts of GPIIb and GPIIIa. Moreover, transfection and immunoprecipitation analysis demonstrated that [Arg674]GPIIb is capable of forming a heterodimer complex with GPIIIa, but the rate of subunit maturation and the surface exposure of GPIIb-IIIa are strongly reduced. Thus, the intramolecular 674-687 disulfide bridge in GPIIb is essential for the normal processing of GPIIb-IIIa complexes. The additive effect of these two GPIIb mutations provides the molecular basis for the thrombasthenic phenotype of the proband.
Chapter
Since their mtroductlon in 1987, yeast artificial chromosomes (commonly referred to as YACs) have had a tremendous impact on the field of molecular genetics The ability of YACs to carry and propagate DNA fragments of up to -2 mllhon base pairs in size has been instrumental in the construction of the first- and second-generation physical maps of the human genome (1,2) YACs have assisted in the generation of detailed maps of discrete chromosomal regions and slgmficantly speeded up posltlonal cloning projects leading to the ldentlficatlon and cloning of a variety of genes involved in inherited diseases (3,4) YACs have also been used for studies on gene function via complementatlon of mutations as well as for the transfer of complete genomlc gene sequences for the generation of transgemc animals Widespread use of this broad range of appllcatlons has been facilitated by the avallabrhty of a number of high-quality genomlc YAC libraries via centralized screening centers This clearly illustrates the scientific advances that can be made when tools and technology are shared in the spirit of cooperation.
Chapter
The technology of exon trapping, sometimes called exon amplification, strives to exploit the phenomenon of mRNA splicing to discover genes directly from genomic DNA. There are three distinct exon trapping methodologies that differ simply in the genomic target of interest. The original experimental design was to capture isolated 3′-splice sites residing within fragments of genomic DNA (1), whereas later approaches focused on either complete internal exons (2–5) or entire 3′-terminal exons (6). The requirement of complete, intact exons as targets has proven absolutely essential, and only the trapping of complete internal or 3′-terminal exons is practical.
Article
Linkage analysis in families containing affected individuals can be used to identify the location of disease susceptibility genes. The aim of this chapter is to provide an overview of the molecular methods employed to clone these susceptibility genes on the basis of linkage data.
Chapter
Exon trapping is a method to functionally clone expressed sequences from genomic DNA. We have developed an exon trapping procedure based on the use of vector pETVSD2. Cosmid DNA is partially digested and cloned in pETV-SD2. DNA of an entire library of subclones is introduced into COS-1 cells and transiently expressed. RNA is isolated and vector-derived transcripts are amplified by RT-PCR. Cloning of the RT-PCR products, which contain a ColEI-origin of replication and a supF marker, is established by NotI digestion and intramolecular circularisation. Due to their shorter length, spliced clones are preferentially amplified and cloned. Using this approach, we have been able to trap several exons from test cosmids of the DMD-gene, including the single 176 by exon 45 present in 40 kb of intronic sequences. We have applied this system in the search for coding sequences in various genomic regions, including the candidate region for the gene involved in facioscapulohumeral muscular dystrophy (FSHD) on 4q35. We have developed a screening procedure which allows more efficient identification of potential exon containing clones, thus discarding the high background of false positives.
Chapter
The technology of exon trapping, sometimes called exon amplification, strives to exploit the phenomenon of mRNA splicing to discover genes directly from genomic DNA. There are three distinct exon trapping methodologies that differ simply in the genomic target of interest. The original experimental design was to capture isolated 3′-splice sites residing within fragments of genomic DNA (1), whereas later approaches focused on either complete internal exons (2–5) or entire 3′-terminal exons (6). The requirement of complete, intact exons as targets has proven absolutely essential, and only the trapping of complete internal or 3′-terminal exons is practical.
Article
Full-text available
Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971, rs17615, rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs 32.6% in controls, P=0.016, OR=0.90 (0.82-0.98)). Two of these SNPs are in exon 10, directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon. Effects of these SNPs and a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.
Article
Flanking recombination events have defined the Huntington Disease gene candidate region to between D4S10 and D4S98, about 2.2Mb. Because of the large size of the candidate region and the likely large number of genes within it we decided to screen cDNA libraries with probes generated from whole Yeast Artificial Chromosomes (YACs) containing parts of this region. We have thus far used 4 YACs ranging in size from 180kb to 600kb covering 880kb and have isolate 13 cDNA clones, 7 of which are unique. Three of the 13 clones contain parts of the 3' untranslated region of the alpha-adducin gene. One YAC of 600kb could not be purified from two yeast chromosomes, therefore this YAC probe had a net complexity of 1.8Mb. Even so this probe identified a cDNA from the HD candidate region indicating that very large YACs may be used as probes.
Article
The quality of a cDNA library depends on the integrity of the messenger RNA and the fidelity with which it can be reverse transcribed. RNA cannot be cloned directly; in a reaction catalyzed by reverse transcriptase, the RNA, together with a suitable primer and a supply of deoxyribonucleoside triphosphates (dNTPs), must be converted to a double-stranded molecule. The product contains a complementary strand (first, antisense, or minus-strand cDNA) that is hybridized to what remains of the original RNA template. Such DNA-RNA hybrids can be cloned albeit often with lower efficiency than their double-stranded DNA counterparts. Usually the hybrid molecules are treated as intermediates in a scheme aimed at replacing the fragmented RNA with continuous DNA to form a double-stranded cDNA molecule. From this brief summary of cDNA cloning, it should be obvious that, regardless of the strategy, reverse transcriptase does and how it does it in vitro is discussed.
Article
We have examined the level of immunoglobulin gene V(D)J recombination activity in a number of cell lines derived from lymphoid or nonlymphoid lineages. The assay we employed uses extrachromosomal DNA as substrate and thereby avoids difficulties associated with the use of chromosomally integrated substrates. The recombination activity decreases during B-lymphoid development. It is highest at the earliest stages of committed B-cell differentiation and then falls progressively, reaching undetectable levels at the mature B-cell stage. The activity is also present in multipotential progenitors of myeloid cells and in pre-T cells but not mature T cells. No activity was found in several nonhematopoietic cell lines. Recombination was seen only among substrate molecules which had replicated in the eukaryotic cells. Several possible interpretations of this result are discussed.
Article
A novel, highly quantitative transient expression assay based on the human interleukin-2 (IL-2) gene was used to examine the trans-activation of the Human Immunodeficiency Virus (HIV/HTLV-III/LAV/ARV) long terminal repeat (LTR) in a range of eukaryotic cell lines. In the absence of the trans-activating viral gene product, tat-III, IL-2 transcripts specific for the HIV LTR were present in low abundance in transfected cells and showed a low translational efficiency, when compared with IL-2 mRNAs transcribed from other viral promoters. Coexpression of tat-III resulted in a marked increase in the steady state level of IL-2 mRNAs transcribed from the HIV LTR, and these mRNAs also demonstrated a specific enhancement of their translational efficiency. These results suggest a bimodal mechanism of action for tat-III in the trans-activation of HIV-specific gene expression.
  • M Ringwald
  • H Baribault
  • S Mayer
  • R Kemler
Ringwald,M., Baribault,H., Mayer,S. and Kemler,R. (1989) Eur. J. Cell Biol. 48, Supplement Abstract 156.