Identification Of Exons Research Articles

Erratum: Computational identification of promoters and first exons in the human genome

Nature Genet. 29, 412–417 (2001). Published online 26 November 2001; doi:10.1038/ng780 On page 413, the last line of the caption of Fig. 2 should read “... the CpG window overlapped with the region ranging from −200 bp of the transcription start site to the splice-donor site.” On page 414, the last sentence of the caption of Table 2 should read “Approximately 70% of those 58 promoters are CpG-related.

Low expression of interferon regulatory factor-1 and identification of novel exons skipping in patients with chronic myeloid leukaemia.

Chronic myeloid leukaemia (CML) is a malignant clonal disorder of the haematopoietic stem cell. Treatment of CML patients with interferon alpha (IFN-alpha) has induced haematological and cytogenetic remission. Interferons transcriptionally activate target genes through the JAK-STAT and interferon regulated factors (IRFs) family pathways. Interferon regulated factor-1 (IRF-1) is a transcriptional activator of genes critical for cell growth, differentiation and apoptosis. The skipping of exons 2 or 2 and 3 of IRF-1 in patients with myelodysplastic syndromes and acute myelogenous leukaemia suggests that this factor may have a critical role in leukaemogenesis. The role of IRF-1 in CML is currently unknown. Therefore, mutational analysis of IRF-1 was performed and its expression pattern was also studied in CML patients. We studied IRF-1 in peripheral blood mononuclear cells of 21 patients in chronic phase CML. No point mutations were identified at the cDNA level. Surprisingly, fourfold reduction of full-length IRF-1 mRNA expression was established in 17/21 patients compared with normal individuals. Low expression of full-length IRF-1 was observed in conjunction with high levels of aberrantly spliced mRNAs, reported for the first time. In three patients who were also analysed during blastic transformation, further reduction of full-length IRF-1 mRNA was observed. These findings demonstrate that, in CML patients, IRF-1 can produce high levels of aberrant spliced mRNAs with subsequent reduction in the levels of full-length IRF-1 mRNA. This observation is consistent with the notion that exon skipping may constitute another mechanism of tumour suppressor gene inactivation in this disease.

Use of hybridization kinetics for differentiating specific from non-specific binding to oligonucleotide microarrays.

Hybridization kinetics were found to be significantly different for specific and non-specific binding of labeled cRNA to surface-bound oligonucleotides on microarrays. We show direct evidence that in a complex sample specific binding takes longer to reach hybridization equilibrium than the non- specific binding. We find that this property can be used to estimate and to correct for the hybridization contributed by non-specific binding. Useful applications are illustrated including the selection of superior oligonucleotides, and the reduction of false positives in exon identification.

Computational identification of promoters and first exons in the human genome.

The identification of promoters and first exons has been one of the most difficult problems in gene-finding. We present a set of discriminant functions that can recognize structural and compositional features such as CpG islands, promoter regions and first splice-donor sites. We explain the implementation of the discriminant functions into a decision tree that constitutes a new program called FirstEF. By using different models to predict CpG-related and non-CpG-related first exons, we showed by cross-validation that the program could predict 86% of the first exons with 17% false positives. We also demonstrated the prediction accuracy of FirstEF at the genome level by applying it to the finished sequences of human chromosomes 21 and 22 as well as by comparing the predictions with the locations of the experimentally verified first exons. Finally, we present the analysis of the predicted first exons for all of the 24 chromosomes of the human genome.

Where is the New Biology Taking Us?The Genetic Revolution and Human Rights. Justine Burley

A review of THE GENETIC REVOLUTION AND HUMAN RIGHTS. The Oxford Amnesty Lectures 1998. Edited byJustine Burley. Oxford and New York: Oxford University Press. $14.95 (paper). xxviii + 220 p; index. ISBN: 019-286201-4. 1999. As the twentieth century ends, we have hardly taken stock of what has happened during this century of biology. We began the new century in 1900 (just as we begin, erroneously, the new century and millennium in 2000) with the rediscovery of Gregor Mendel's laws by Hugo De Vries, Carl Correns, and Ernst von Tschermak (the latter despite Curt Stern's efforts to demote him as a rediscoverer). William Bateson quickly took up the cause of Mendelism and named the field of genetics (1906), winning the battle against the biometricians who erroneously (then) thought that Mendelism had nothing to do with either evolution or heredity. In 1909 WilhelmJohannsen gave us a name-the gene-to replace contending verbal monstrosities such as character-unit, unit factor, and other laden terms that misled early investigators. Classical genetics emerged from the fusion of cytology (Walter Sutton's chromosome theory of heredity in 1902) and breeding analysis, especially in the able hands of Edmund Beecher Wilson and Thomas Hunt Morgan at Columbia University. The Drosophila Group (or Fly Lab) established linkage, chromosome maps, X-linked inheritance, the correlation of linkage groups to chromosome number, assorted chromosomal rearrangements, nondisjunction, dosage compensation, complex association of gene and character through chief genes and genetic modifiers, construction of genetic stocks, and a variety of new, wonderful insights and tools for genetic analysis. The studies of plant geneticists (especially by E M East and D FJones) provided the agricultural revolution that liberated, at least for the twentieth century, the world from the gloomy predictions by Malthus of world hunger on a massive scale as human population soared in response to the practical benefits of the germ theory and the reduction of infant mortality. By the 1920s many insights into gene number and function had been established, and mutation of the gene was isolated from other varieties of chromosomal disturbances, allowing geneticists to focus on mutation rates and the search for agents that induce mutations. HermannJoseph Muller was the first to successfully tackle these problems, and his induction of mutations by x-rays in 1927 set off a wave of repetitions in plants, animals, and microbes that created both the field of radiation genetics and the practical application of radiation (later chemical mutagens) to produce desired mutations, including the greatly augmented yields of penicillin from mold. The late 1930s saw the first rumblings of biochemical genetics and the conception of molecular genetics. Virus genetics (Max Delbruick's school), sex in bacteria Joshua Lederberg's findings), and the hints that DNA may be of genetic significance after all (so E B Wilson thought as early as the 1890s) led to the explosion of new knowledge that followed the end of the second World War. DNA was the genetic material (as the Rockefeller and Cold Spring Harbor laboratories reported), its structure (as worked out by Watson and Crick and confirmed by Wilkins and Franklin) was a double helix, and its implications rolled forth: the genetic code, semiconservative replication, molecular basis of mutagenesis, genetic fine structure associated with pseudoallelism and nests of cognate genes, and genetic regulation, all by 1965. In the next thirty-five years came the sequencing of genes, enzymology of DNA replication, the PCR tool of copying genes, identification of introns and exons in eukaryotic genes, directed mutation, and recombinant DNA technology. Genetics suddenly became fused with engineering, in addition to physics, mathematics, physiology, cell biology, embryology, breeding analysis, cytology, chemistry, biochemistry, and evolution. At the same time as genetics shifted in the middle of the century from the classical to molecular approaches, human genetics expanded greatly, purged of eugenics, and found its way into medical schools, infiltrating departments of pathology, obstetrics, and pediatrics.

Identification and characterization of the human homologue of the short PDE4A cAMP-specific phosphodiesterase RD1 (PDE4A1) by analysis of the human HSPDE4A gene locus located at chromosome 19p13.2.

The HSPDE4A gene spans 50 kb, consists of at least 17 exons and is orientated 5'-3', telomere to centromere. It is located at chromosome 19p13.2, being 350 kb proximal to the gene encoding TYK2 and 850 kb distal to the gene encoding the low-density lipoprotein receptor. Its structure is consistent with the production of active 'long' and 'short' isoenzymes as the result of alternative mRNA splicing at two splice junctions. Identified is the single alternatively spliced 5' exon encoding the unique N-terminal region of the long isoenzyme HSPDE4A4B (pde46). The upstream conserved regions, UCR1 and UCR2, which form characteristic domains of PDE4 long forms are each encoded by three exons. The PDE4A-subfamily-specific linker region LR1, which joins UCR1 and UCR2, is encoded by two exons, whereas LR2, which joins UCR2 to the catalytic unit, is encoded by a single exon. Identification of exons encoding an enzymically inactive product of this gene, HSPDE4A8A (2el), indicates that this is an authentic gene product. The 5' exon encoding the unique N-terminal region of the human homologue of the rodent isoform RNPDE4A1A (RD1) was located, and the splice junction used to produce this short PDE4A isoform shown to occur at a different position from that seen in both the rat PDE4B and PDE4D genes. Reverse transcriptase PCR analysis indicates that RD1 homologues are conserved across species, having a conserved membrane-targeting region and a hypervariable LR2 region. Human RD1 was expressed transiently in COS-7 cells and detected as an 83 kDa species primarily associated with the high-speed membrane fraction. Human RD1 exhibited a Km for cAMP of about 3 microM, an IC50 value for inhibition by the PDE4-selective inhibitor rolipram of about 0.3 microM and was considerably more thermostable than rat RD1. Human RD1 was generated as a mature 80 kDa species in an in vitro transcription-translation system and shown to be capable of binding to membranes. Knowledge of the gene structure and the associated sequence information should facilitate analysis of the involvement of PDE4A in hereditary disorders that may result from alterations in enzyme expression, activity, regulation and intracellular targeting and serve as a resource for determining authenticity of cloned PDE4A species.

Identification of Novel Exons 3′ to the HumanSNRPNGene

The gene for the small nuclear ribonucleoprotein N (SNRPN) maps to human chromosome 15 and has 10 exons. Using cDNA cloning, direct cDNA selection, and exon-connection reverse transcriptase (RT)–PCR, we have identified three novel 3′ exons ofSNRPN,which have no protein coding potential. Like the otherSNRPNexons, the novel exons are expressed from the paternal allele only. In contrast to several cDNA clones and RT-PCR products, however, the 3.4-kb transcript detected by Northern blot hybridization with a probe for the novel exons does not containSNRPN.It is possible that the steady-state RNA observed in fetal tissues and in adult testis, ovary, brain, and muscle is initiated at an as yet unidentified transcription start site downstream ofSNRPNor is generated by endonucleolytic cleavage of a precursor transcript, as has been shown for another imprinted gene, the insulin-like growth factor II gene.

Identification of exons in a novel embryonal carcinoma locus using the GRAIL program

Embryonal carcinoma (EC) cells have proven to be of particular value in studies of both oncogenesis and mammalian development, as well as in evaluating the relationship between these two phenomena. Infection of the EC cell line, NR1-0, with a defective retrovirus containing a neomycin resistance cassette (Neo(r)), produced a mutant cell line: NR1-6. Genetic analysis of this variant cell line indicates that there is only a single insertion site. Interestingly, however, the NR1-6 cell line is unique in its morphology, tumorigenicity, and differentiative potential (1). We have sequenced over 18 kb from the regions flanking the retroviral insertion which we then analyzed using the computer programs GCG and BLAST. Although homology was found to 4 B1 repeat elements (approximately 150 bp long) and a novel CA/GT dinucleotide repeat, no homology was found to any known genes (2). Furthermore, attempts to identify potential exons or transcripts using various molecular techniques and the above mentioned computer programs were all negative. Most recently we employed the GRAIL (Gene Recognition and Analysis Internet Link) computer program which was specifically designed to identify potential exons (3). Analysis with this program identified 5 exon candidates: two characterized as excellent (>90% probability) and three as marginal (>60% probability). Using reverse transcriptase (RT)-PCR we have demonstrated that the two 'excellent' and one of the 'marginal' exon candidates identified by GRAIL are expressed as mRNA in the mutant cells. Sequencing of these PCR products indicates that the mRNA is identical to the genomic DNA sequence. Thus, we have found that GRAIL provides an efficient, reliable means of identifying real exons within long regions of novel genomic DNA.

The Identification of Exons from the MED/PSACH Region of Human Chromosome 19

We have used exon amplification to identify putative transcribed sequences from an 823-kb contig consisting of 28 cosmids that form a minimum tiling path from the interval 19p12–p13.1. This region contains the genes responsible for multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). We have trapped 66 exons (an average of 2.4 exons per cosmid) from pools of 2 or 3 cosmids. The majority of exons (51.5%) show only weak similarity or no similarity (36.3%) to sequences in current databases. Six of 8 exons examined from these groups, however, show cross-species sequence conservation, indicating that many of them probably represent authentic exons. Eight exons show identity or significant similarity to ESTs or known genes, including the human TNF receptor 3′-flanking region gene, human epoxide hydrolase (EPHX), human growth/differentiation factor (GOF-1), human myocyte-specific enhancer factor 2, the rat neurocan gene, and the human cartilage oligomeric matrix protein gene (COMP). Mutations in this latter gene have recently been shown to be responsible for MED and PSACH.

Identification of exons in a region of human chromosome 6q known to contain tumour suppressor genes.

An important objective in human genomic mapping is the isolation of expressed sequences from defined chromosomal regions. The 6q27 region of the long arm of chromosome 6 is an important target in this endeavour because a number of tumour suppressor genes have been mapped to this region. We have used exon trapping and amplification in conjunction with a chromosome specific genomic library to generate ESTs in 6q27. Nine novel expressed sequences have been identified and these provide additional markers and potential candidate genes for the tumour suppressor genes known to be in 6q27.

The pregnancy-specific glycoprotein family of the immunoglobulin superfamily: Identification of new members and estimation of family size

The members of the carcinoembryonic antigen (CEA)/pregnancy-specific glycoprotein (PSG) gene family have a characteristic N-terminal domain that is homologous to the immunoglobulin variable region. We have estimated the size of the PSG subfamily by identification of N-domain exons from isolated genomic clones and from total genomic DNA through PCR amplification and DNA sequence determination. The PSG subfamily contains at least 11 different genes. For 7 of these, two DNA sequences differing from each other in 1 to 4 nucleotides were detected. Most likely, they represent different alleles. They are PSG1, PSG2, PSG3, PSG4, PSG5, PSG6, PSG7, PSG8, PSG11, PSG12, and PSG13. Six of the N-domain sequences described here are new. All of the PSGs except PSG1, PSG4, and PSG8 contained the arginine-glycine-aspartic acid sequence at position 93–95 corresponding to the complementarity determining region 3 of immunoglobulin. Parsimony analysis of 24 CEA and PSG sequences using 12 members of the immunoglobulin gene superfamily as out-groups to root the family free shows that the N-domain of the CEA group genes evolved in one major branch and the PSG group genes in the other.

Exon amplification: a strategy to isolate mammalian genes based on RNA splicing.

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.

Exon cloning: immunoenzymatic identification of exons of the chicken lysozyme gene.

A 10-kilobase DNA fragment containing exons 1 and 2 of the chicken lysozyme gene has been randomly cleaved with DNase I. After tailing and cloning into the plasmid pUK230, Lac+ colonies were selected. Colonies harboring expressed fragments of the exons could be detected by an immunoenzymatic assay using antibodies against lysozyme. The smallest fragment coded for 10 amino acids and the largest coded for almost all residues of exon 2. These results suggest that any gene of any genome cloned in this way can be detected if antibodies against the gene product are available.