Genomic structure of the gene encoding human 3-hydroxy-3-methyl-glutaryl coenzyme A reductase: comparison of exon/intron organization of sterol-sensing domains among four related genes

We determined the genomic structure of the human gene encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which catalyzes the conversion of HMG-CoA to mevalonate and is the rate-limiting and major regulatory enzyme in sterol biosynthesis. The gene is more than 21 kb long, about five times the size of its corresponding cDNA. It consists of 20 exons, ranging in size from 68 to 1809 bp. An amino-terminal hydrophobic membrane-bound domain is encoded by exons 2–10, a flexible linker domain by exons 10 and 11, and the catalytic domain by exons 11–20. Exons 3–7 encode a sterol-sensing domain. We compared its genomic structure in this region with the sterol-sensing domains of three related genes, sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP), Niemann-Pick type C1 protein (NPC1), and a morphogen receptor, Patched. Two of the five positions of introns in the sterol-sensing domain of the HMG-CoA reductase gene were identical to the exon/intron organization of this domain in the related human genes, but these positions of introns were not conserved in homologues from lower organisms, except in one instance. The data suggested that exon-shuffling may have occurred during relatively recent evolution; this would account for the structural similarity of this domain in four quite different human proteins. Received: April 25, 2000 / Accepted: June 12, 2000     

Differential expression of multiple isoforms of the ELKS mRNAs involved in a papillary thyroid carcinoma

A novel gene, ELKS, whose 5' portion was fused to the RET gene, was found in a papillary thyroid carcinoma. A cDNA of this gene obtained from a human-brain cDNA library revealed that it encoded a peptide of 948 amino acids, termed ELKSalpha. We identified four other isoforms, which encoded ELKSbeta, ELKSgamma, ELKSdelta, and ELKSepsilon proteins consisting, respectively, of 992, 720, 1088, and 1116 amino acid residues. Analysis of the gene structure revealed that the isoforms were generated by alternative splicing. Isoforms beta, gamma, delta, and epsilon all contain an optional exon (exon14a), but ELKSgamma, -delta, and -epsilon lack exon 1b. ELKSgamma lacks exons 3 to 6. ELKSdelta and -epsilon lack exons 12 and 17; ELKSepsilon contains an optional exon (exon 6a). Analysis by RT-PCR suggested that ELKSalpha and ELKSbeta mRNAs are abundant in the brain, ELKSdelta and ELKSepsilon mRNAs predominate in testis and thyroid, and ELKSepsilon mRNA predominates in other tissues. To prove whether the fusion of different ELKS isoforms to RET (between ELKS coiled-coil domains and the RET kinase domain) could produce chimeric proteins that could be autophosphorylated, we synthesized ELKSgamma-RET, ELKSdelta-RET, and ELKSepsilon-RET fusion proteins in vitro. Immunoblotting with anti-ELKS, anti-RET, and anti-phosphotyrosine antibodies demonstrated that the chimeric proteins were constitutively phosphorylated at tyrosine residues, whereas native RET protein was not. These results indicate that the ELKS gene is alternatively spliced, and that every type of ELKS-RET chimeric protein having oligomerization domains can activate RET's cytoplasmic tyrosine kinase.     

Fusion of a novel gene,ELKS, to RET due to translocation t(10;12)(q11;p13) in a papillary thyroid carcinoma

In papillary thyroid carcinomas, the genes for receptor‐type tyrosine kinase, RET or TRKA, are sometimes rearranged, resulting in fusion of its tyrosine kinase domain to 5′ portions of several activating genes. In a papillary thyroid carcinoma, we identified a novel gene (ELKS), the 5′ portion of which is fused to the RET gene by gene rearrangement due to the translocation t(10;12)(q11;p13). Subsequent cloning of the ELKS cDNA revealed that ELKS encodes a novel 948 amino acid peptide and is expressed ubiquitously in human tissues. The presence of multiple coiled‐coil domains in the ELKS product suggests that the ELKS protein forms dimers. Since the tyrosine kinase of RET is activated by dimerization that occurs when its ligands bind to the receptor, fusion of RET with the 5′ dimerization domains of ELKS would activate its cytoplasmic tyrosine kinase constitutively in papillary thyroid carcinomas. Genes Chromosomes Cancer 25:97–103, 1999. © 1999 Wiley‐Liss, Inc.     

Characterization of a genomic region encoding the 32-kDa dense granule antigen of Sarcocystis muris (Apicomplexa)

Two subgenomic libraries constructed from Sarcocystis muris total DNA were screened with a cDNA probe, specific for a 32-kDa protein associated with the dense granules. Two clones reacted positively and were isolated, gDG 32/1 and gDG 32/2. Genomic clone gDG32/1 and part of clone gDG 32/2 have been sequenced. The composite nucleotide sequence of these genomic clones comprises 4.34 kb. It contains a 5′ region of 2.14 kb, a first coding region (222 bp, exon I), a noncoding region (608 bp, intron), a second coding region (660 bp, exon II), and a 3′ region of 693 bp. The upstream region shows a eukaryotic promoter structure and a consensus sequence for the 5′ and 3′ splicing sites. Thus the open reading frame (ORF DG32) coding for the 32-kDa protein of the dense granules of S. muris cyst merozoites is interrupted by an intron. To our knowledge, dg32 is the first sarcosporidian mosaic gene to be characterized. Received: 27 April 1999 / Accepted: 11 May 1999     

The human ribosomal protein L6 gene in a critical region for Noonan syndrome

We have determined the genomic structure of the human ribosomal protein L6 gene (RPL6) and assigned it to the interval containing the Noonan syndrome locus. RPL6 spans 4415 bp and consists of seven exons and six introns. The first exon is only 19 bp in length, containing a 5′ non-coding region and a polypyrimidine tract. The second exon starts with the initiator ATG. Although the overall structure of the protein is highly conserved among mammalian species, there is significant variation in the N-terminal portion. We have refined the position of RPL6, using two different radiation hybrid panels. RPL6 was mapped to chromosome 12q24.1 between the markers D12S84 and D12S861, which is in the critical region for Noonan syndrome. Received: May 22, 2000 / Accepted: June 16, 2000     

Complex group-I introns in nuclear SSU rDNA of red and green algae: evidence of homing-endonuclease pseudogenes in the Bangiophyceae

 The green alga Scenedesmus pupukensis and the red alga Porphyra spiralis contain large group-IC1 introns in their nuclear small subunit ribosomal RNA genes due to the presence of open reading frames at the 5′ end of the introns. The putative 555 amino-acid Scenedesmus-encoded protein harbors a sequence motif resembling the bacterial S9 ribosomal proteins. The Porphyra intron self-splices in vitro, and generates both ligated exons and a full-length intron RNA circle. The Porphyra intron has an unusual structural organization by encoding a potential 149 amino-acid homing-endonuclease-like protein on the complementary strand. A comparison between related group-I introns in the Bangiophyceae revealed homing-endonuclease-like pseudogenes due to frame-shifts and deletions in Porphyra and Bangia. The Scenedesmus and Porphyra introns provide new insights into the evolution and possible novel functions of nuclear group-I intron proteins. Received: 26 May / 25 August 1999     

The trans-spliced intron 1 in the psaA gene of the Chlamydomonas chloroplast: a comparative analysis

In the secondary structure model that has been proposed for the trans-spliced intron 1 in the Chlamydomonas reinhardtii psaA gene, a third RNA species (tscA RNA) interacts with the 5 and 3 intron parts flanking the exons to reconstitute a composite structure with several features of group-II introns. To test the validity of this model, we undertook the sequencing and modelling of equivalent introns in the psaA gene from other unicellular green algae belonging to the highly diversified genus Chlamydomonas. Our comparative analysis supports the model reported for the C. reinhardtii psaA intron 1, and also indicates that the 5 end of the tscA RNA and the region downstream from the psaA exon 1 cannot be folded into a structure typical of domain I as described for most group-II introns. It is possible that a fourth RNA species, yet to be discovered, provides the parts of domain I which are apparently missing.     

Molecular basis of intermittent maple syrup urine disease: novel mutations in the E2 gene of the branched-chain α-keto acid dehydrogenase complex

The E2 gene of the branched-chain α-keto acid dehydrogenase (BCKDH) complex was studied at the molecular level in three patients with intermittent maple syrup urine disease (MSUD). All three patients had higher BCKDH activity than did those with the classical phenotype. In the first patient, a single base substitution from A to G in intron 8 created a new 5′ splice site and caused an insertion of 126 nucleotides between exons 8 and 9 by activating an upstream cryptic 3′ splice site in the same intron. The predicted mRNA encoded a truncated protein with 282 amino acids including 4 novel ones at the carboxyl terminus, compared with the normal protein with 421 amino acids. In vitro, the region from the patient but not from a normal control was recognized and was recovered as a novel exon, indicating that the single substitution was responsible for incorporation of the region into mRNA. This mutation probably supports an exon definition model in which the spliceosome recognizes a 3′ splice site and then scans downstream for an acceptable 5′ splice site, thereby defining an exon. The second patient was homozygous for a G to T transversion at nucleotide 1463 in exon 11, which predicted a substitution of the termination codon by a leucine residue and the addition of 7 extra amino acids at the carboxyl terminus. For each mutation, these two patients were homozygous and their parents were heterozygous. The third patient was a compound heterozygote for a C to G transversion at nucleotide 309 in exon 4 and a G to A transition at nucleotide 1165 in exon 9, causing an Ile-to-Met substitution at amino acid 37 and a Gly-to-Ser substitution at amino acid 323, respectively. Taken together, these results indicate that the molecular basis of intermittent phenotype MSUD in some patients can be due to mutations in the E2 gene, giving rise to a low but significant residual activity of the BCKDH complex. Received: October 29, 1997 / Accepted: November 27, 1997     

Non-homologous recombination between Alu and LINE-1 repeats caused a 430-kb deletion in the dystrophin gene: a novel source of genomic instability

Although large deletions in the dystrophin gene have been identified in more than two-thirds of Duchenne and Becker muscular dystrophy patients, the molecular mechanisms that lead to the generation of these deletions are largely unknown. Here, Alu and LINE-1 (L1) repetitive elements were shown to be present at one or other of the two ends, respectively, of a 430-kb deletion in the dystrophin gene. The breakpoint of the deletion, which stretches from exons 2 to 7, was defined more precisely by polymerase chain reaction (PCR) walking on introns 1 and 7. Finally, the region containing the breakpoint was amplified as a fragment of more than 10 kb. Sequencing of the deletion endpoint revealed the presence of an Alu sequence in intron 1, 25 kb downstream from the 3′ end of exon 1 that was joined directly to an L1 sequence in intron 7, 4.5 kb downstream from the 3′ end of exon 7. The deletion was calculated to be 430 kb. To our knowledge, this is a novel recombination event joining non-homologous Alu and L1 repeats, and is the largest known intrachromosomal deletion that is thought to involve repetitive genetic elements. Sequence characteristics around the breakpoint are discussed. Received: July 10, 2000 / Accepted: August 23, 2000     

Seventy genetic variations in human microsomal and soluble epoxide hydrolase genes (EPHX1 and EPHX2) in the Japanese population

Human microsomal and soluble epoxide hydrolases (mEH and sEH) are enzymes that metabolize xenobiotic molecules. We screened DNA from 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in both genes by direct sequencing of the entire genomic regions containing EPHX1 and EPHX2, except for repetitive elements. This approach identified 33 SNPs in the EPHX1 gene; 6 of them were located in the 5′ flanking region, 17 in introns, 8 in exons, and 2 in the 3′ flanking region. In the EPHX2 gene, we identified 36 SNPs, including 4 in the 5′ flanking region, 24 in introns, 5 in exons, and 3 in the 3′ flanking region, as well as one insertion/deletion polymorphism in the 5′ flanking region. These variants may contribute to a more precise understanding of the nature of correlations between genotypes and disease-susceptibility phenotypes that have been postulated in regard to human microsomal and soluble epoxide hydrolases. Received: January 25, 2001 / Accepted: February 21, 2001     

The structure of the avian fast skeletal muscle troponin T gene: seven novel tandem-arranged exons in the exon x region

To elucidate the mechanism that produces enormous molecular diversity in troponin T (TnT) of fast skeletal muscle, we determined the 5-half genomic sequence of the chicken fast muscle TnT gene. The sequence of ca. 16 kb included seven exons (exons 1, 2, 3, 4, w, 5, and 6), which have been reported previously and presumed by sequencing TnT cDNAs. Additionally we found six 15 nt and one 18 nt sequences in the region between exons 5 and 6 (i.e. the exon x region). They were encompassed by consensus splice donor and acceptor sites and preceded by putative branch sites, and designated herein as exons xa to xg. Our result shows that the sequence derived from exons x1, x2, and x3, the exons presumed previously by cDNA sequencing, is actually encoded by the seven exons xa to xg, establishing the precise gene structure in the exon x region. Based on our data, together with that on the 3-half genomic sequence of the quail fast muscle TnT gene, we conclude that the avian fast skeletal muscle TnT gene includes 27 exons, 16 of which are alternatively spliced.     

Homologous maturase-like proteins are encoded within the group I introns in different mitochondrial genes specifying Yarrowia lipolytica cytochrome c oxidase subunit 3 and Saccharomyces cerevisiae apocytochrome b

A mitochondrial cox3 gene in the alkane yeast, Yarrowia lipolytica, encodes a subunit-3 protein of cytochrome c oxidase, and contains a 1044 base-pair-long intron, as compared with the corresponding intronless gene in Saccharomyces cerevisiae. The intron belongs to a group I intron as determined by the cDNA sequence for the splicing sites as well as the predicted RNA secondary structure. Remarkably, this intron could code for a protein of 206 amino-acid residues which showed 63% similarity with an RNA maturase encoded by the second intron of the mitochondrial apocytochrome b gene in S. cerevisiae. Both introns occurred within the conserved exon sequence, 5-TT(G/C)AGGTGC-3, suggesting the possible transposition of a common ancestral intron.     

Genomic alterations in a case of alpha heavy chain disease leading to the generation of composite exons from the JH region

Human alpha heavy chain disease (HCD) is characterized by the presence in patient's serum of a short Ig alpha chain devoid of light chains. We analyzed the serum protein, the alpha HCD mRNA and the productive rearranged H chain gene from the leukemic cells of a new case (YAO) of alpha HCD. The abnormal YAO alpha 1 Ig was devoid of VH and CH1 domains and started at the beginning of the hinge region. The alpha HCD mRNA was shorter than normal alpha mRNA and the cDNA prepared from YAO mRNA encoded a leader sequence, an insert of 70 nucleotides and the CH2 and CH3 exons. The origin of the inserted sequence was assessed by cloning and sequence analysis of the alpha 1 productive gene. It started with a leader exon, a leader-VH intron and the first 11 bp of a VH exon. Then the VH region was deleted and replaced by a 19-nucleotide sequence that turned out to correspond to the 3' part of a modified JH5 exon. It was followed by a 221-bp sequence homologous to the JH5-psi JH3 intron and by an inserted sequence of unknown origin. The 3' part of this insertion and the remnant of a JH6 exon delineated a third exon that was followed by a relatively conserved JH6-C alpha intron. These two composite exons were flanked by splicing sites and accounted for the 70-nucleotide insert of the cDNA. The genomic nucleotide sequence also revealed a large deletion in the switch CH1 region which eliminated normal splicing sites and resulted in splicing of the third exon directly to the CH2 exon.     

The Human Complement Component C1R Gene: The Exon-intron Structure and the Molecular Basis of Allelic Diversity

Human C1r is a component of the complement system, which is a major mediator of innate immunity. In this study we investigated the exon‐intron organization of the human C1R gene, which spans 11 kb from the initiation codon to the stop codon, and is very similar in exon‐intron structure to the C1S gene. Six common and rare alleles, C1R*1, C1R*2, C1R*5, C1R*8, C1R*9, and C1R*13, were characterized by five mutations at amino acid positions 114, 135, 146, 167 and 244, in exons 4, 5 and 7 where the CUB1, EGF and CUB2 domains are encoded, respectively. A comparison with the cDNA of the mouse C1r gene showed that C1R*2is likely to be an ancestral allele. In addition, nine nucleotide substitutions and one length polymorphism were found in introns 2, 3, 4, 8 and 10.     

Distribution of mitochondrial introns in the species Schizosaccharomyces pombe and the origin of the group II intron in the gene encoding apocytochrome b

Summary The mitochondrial genome size of 26 different Schizosaccharomyces pombe strains varies between 17.6 and 24.6 kilobase pairs due to the presence or absence of introns. One of these is the group II intron in the gene encoding apocytochrome b (cob: intron cobI1 ). Partial DNA sequences of continuous cob genes from six strains (including strain EF1: Trinkl et al. 1985) revealed identical nucleotide sequence in the region where the group II intron is inserted in the mosaic form of the gene. In contrast, analysis of the mosaic cob, gene in strain UCD-Fstl revealed several base pair changes in the exon regions flanking the splice point, compared with the continuous genes and with the mosaic cob gene in strain 50 (Lang et al. 1985). The base pair differences between the exons of the two mosaic cob genes and the identity of exons in all continuous cob genes argue in favour of the two cob introns in strains 50 and UCD-FstI as independent later acquisitions of the genes, rather than loss of the intron from a common mosaic ancestor of all strains.Other introns present in some but not all strain include two group I introns without open reading frame in the gene encoding subunit 1 of cytochrome c oxidase (cox1: introns cox1I2a and cox1I3), and two group I introns with open reading frames in the same gene (introns cox1I1 and cox1I2b).     

Effect of +36T > C in intron 1 on the glutamine: fructose-6-phosphate amidotransferase 1 gene and its contribution to type 2 diabetes in different populations

Glutamine: fructose-6-phosphate amidotransferase 1 (GFPT1) acts as a rate-limiting enzyme in the hexosamine biosynthetic pathway, which is an alternative branch of glucose metabolism. To evaluate GFPT1 as a susceptibility gene to type 2 diabetes, we surveyed the polymorphisms related with the gene function of GFPT1 and assessed its contribution to type 2 diabetes with a case-control association study. Screening of the 5′-flanking and all coding regions of GFPT1 revealed eight polymorphisms, one in the 5′-flanking region, one synonymous polymorphism in exon 8, five in introns and one in 3′-UTR, but no mis-sense or non-sense polymorphism. With in silico simulation, a putative promoter region was apparently predicted between 1 kb upstream and 1 kb downstream of the start codon. In this region, +36T > C polymorphism was located on the GC box sequence in intron 1, and its functional effect on promoter activity was confirmed by luciferase reporter assay, introducing a new functional polymorphism of the GFPT1 gene. To examine its association with type 2 diabetes, we analyzed 2,763 Japanese (1,461 controls and 1,302 cases) and 330 Caucasians (190 controls and 140 cases). One possible association of +36T > C was observed in Caucasians, but no association of polymorphisms including +36T > C in intron 1 or haplotypes was observed in Japanese. Although we could not completely rule out a contribution to specific sub-groups or other populations, genetic variation of GFPT1 is unlikely to have a major role in the susceptibility to type 2 diabetes in Japanese.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Genomic organisation of rainbow trout, Oncorhynchus mykiss TGF-beta

The genomic organisation of Oncorhnchus mykiss TGF-beta has been determined through the generation of contiguous clones by PCR. The O. mykiss TGF-beta gene is approximately 3.4 Kb in length and consists of 7 coding exons with no introns in the 5'-UTR. Whilst this is the same number of exons found in TGF-beta genes of amphibians, birds and mammals, in the O. mykiss gene intron 2 of other vertebrates is absent and an additional intron is present at the 3' end of the molecule, splitting exon 7 of the other known TGF-beta genes into two exons (trout exons 6 and 7). Comparison of exon sizes in the coding region support the suggestion that the Xenopus TGF-beta5 and trout TGF-beta sequences are the forerunners of TGF-beta1. Conservation of exons coding for the mature TGF-beta peptide is relatively high (63-73% identity) but other exons show lower identities (37-58%). Comparison of the TGF-beta intron sequences reveals that in general the O. mykiss introns are considerably shorter than the avian homologs. The impact of the teleost TGF-beta gene organisation on theories of the gene evolution of this cytokine family are discussed.