Middle Of Intron Research Articles

Splicing regulation in spinal muscular atrophy by an RNA structure formed by long‐distance interactions

Humans carry two copies of the survival motor neuron gene: SMN1 and SMN2. Loss of SMN1 coupled with skipping of SMN2 exon 7 causes spinal muscular atrophy (SMA), a leading genetic disease associated with infant mortality. Our discovery of intronic splicing silencer N1 (ISS-N1) is a promising target, currently in a phase III clinical trial, for an antisense oligonucleotide-mediated splicing correction in SMA. We have recently shown that the first residue of ISS-N1 is locked in a unique RNA structure that we term ISTL1 (internal stem through long-distance interaction-1). Complementary strands of ISTL1 are separated from each other by 279 nucleotides. Using site-specific mutations and chemical structure probing, we confirmed the formation and functional significance of ISTL1. Located in the middle of intron 7, the 3' strand of ISTL1 falls within an inhibitory region that we term ISS-N2. We demonstrate that an antisense oligonucleotide-mediated sequestration of ISS-N2 fully corrects SMN2 exon 7 splicing and restores high levels of SMN in SMA patient cells. These results underscore the therapeutic potential of the regulatory information present in a secondary and high-order RNA structure of a human intron.

Mutation Analysis of TP53 Tumor Suppressor Gene in Colorectal Cancer in Patients from Iran (Kerman Province).

P53 is an important tumor suppressor, which is mutated in later stages of many cancers and leads to resistance to chemotherapy. The aim of this study was to reveal mutations of TP53 in colorectal cancer in Kerman province. A total of Forty-three colon cancer specimens as paraffin block or fresh tissues, which passed stage IIIA, were selected. Three exons 5, 7 and 8 of P53 were amplified by PCR technique and sequenced directly. The results showed two deletions at codon 140 and 142 in one tumor sample. GAT→AAT mutation at codon 184, and CGG→TGG mutation at codon 248 were seen in some tumor samples. Some mutations were also observed in middle of intron 7 in some tumor or normal tissues. Some of those patients with mutation in P53 gene had metastasis in other organs. Therefore, genetic test before chemotherapy is helpful for successful treatment.

A splice-supporting intronic mutation in the last bp position of a cryptic exon within intron 6 of the CYBB gene induces its incorporation into the mRNA causing chronic granulomatous disease (CGD)

Chronic granulomatous disease (CGD) is caused by a defect in both the host's defenses and its regulation of inflammation normally provided by phagocytes and other leukocytes. As in the case described here, it is not uncommon that CGD patients are diagnosed late, only after organ-damaging manifestations have already progressed. In this patient, we found that CGD arose due to a splice-supporting mutation in the last position of a cryptic exon towards the middle of intron 6 of the CYBB (gp91-phox) gene. The mutation led to the insertion of 56 bp into most of the CYBB mRNA of leukocytes causing a frame shift and a premature stop codon. The normal cryptic exon was also found to be mildly active in some tissues other than leukocytes in healthy donors, to be conserved in many primates, and to a lesser degree in other mammals. Some sequence similarity suggests that the cryptic exon may have originated from a mammalian interspersed repetitive (MIR) element. Taken together, we clarify an unusual disease-causing mutation, indicate its evolutionary background and emphasize the importance of a timely diagnosis of CGD.

Juxtaposition of the HPFH2 Enhancer Is Not Sufficient To Reactivate the g-Globin Gene in Adult Erythropoiesis in bYAC Transgenic Mice.

Hereditary persistence of fetal hemoglobin (HPFH) is characterized by high-level expression of the g globin gene in adult patients. HPFH2 contains a genetic deletion of approximately 83.5 kb from the middle of intron 2 of the yb gene to the region 66 kb 3′ to the b gene. The deletion juxtaposes an enhancer located downstream to the 3′ breakpoint to the vicinity of the g gene. To understand the reactivation mechanism of this fetal stage specific globin, we introduced the deletion into a 213 kb b-YAC. Four transgenic lines bearing 1–3 intact copies of the YAC construct were established. Globin gene expression at different developmental stages was measured by RNase protection assays. In the HPFH2 transgenic mice the e and g genes were expressed at high levels similar to the wild type YAC mice in embryonic and fetal stages. Unexpectedly, the g gene was completely silenced in the adult mice. The failure of g gene reactivation by juxtaposition of the HPFH2 enhancer contradicts the results reported by the Forget lab (Mol. Cell. Biol. 17:2076–2089, 1997) and the Strouboulis lab (Blood 102:3412–3419, 2003). The discrepancy could be due to the differences between the constructs used in the different labs. The construct used in the Forget lab was a cosmid containing the mLCR linked a 13 kb Gg and Ag fragment. The cosmid used by the Strouboulis lab contained the 22 kb LCR and a 5 kb Ag fragment. The bYAC construct we used in this study contains the whole b globin locus and spanned from about 40 kb 5′ to the e gene to 100 kb 3′ to the b gene. The failure of g gene reactivation in the HPFH2 YAC mice suggests that additional elements, which are missing in the 213 kb bYAC construct, are involved in reactivation of the g gene in the HPFH patients. Based on data from literature (Bender et al., Mol. Cell 5:387–393, 2000; Forrester et al., Genes & Dev. 4:1637–1649, 1990) and our own studies (Ping et al., this meeting) we speculate that the 200 kb upstream region is the candidate for harboring this activity.

Ethylnitrosourea-induced base pair substitution affects splicing of the mouse gammaE-crystallin encoding gene leading to the expression of a hybrid protein and to a cataract.

A novel ENU-induced mutation in the mouse leading to a nuclear and cortical opacity of the eye lens (ENU418) was mapped to proximal chromosome 1 by a genome-wide mapping approach. It suggests that the cluster of gamma-crystallin encoding genes (Cryg) and the betaA2-crystallin encoding gene Cryba2 are excellent candidate genes. An A --> G exchange in the middle of intron 1 of the Cryge gene was found as the only alteration cosegregating with the cataractous phenotype. The mutation was confirmed by the presence of a novel restriction site for ApaI in the corresponding genomic DNA fragment. The mutation represses splicing of intron 1; the additional 92 bp in the corresponding cDNA leads to a frameshift and the expression of a novel hybrid protein containing 3 amino acids of the gammaE-crystallin at the N terminus, but 153 novel amino acids. The Cryge(ENU418) protein has a calculated molecular mass of approximately 15.6 kD and an alkaline isoelectric point (pH 10.1) and is predicted to have two hydrophobic domains. Western blot analysis using a polyclonal antibody against the hydrophilic C-terminal part of the Cryge(ENU418)-specific protein demonstrated its stable expression in the cataractous lenses; it was not found in the wild types. Histological analysis of the cataractous lenses indicated that the expression of the new protein disrupts the cellular structure of the eye lens.

An unusual intronic mutation in the CYBB gene giving rise to chronic granulomatous disease

The most common, X-linked, form of chronic granulomatous disease (CGD) is caused by mutations in the CYBB gene located at Xp21.1. The product of this gene is the large subunit of flavocytochrome b 558, gp91 phox , which forms the catalytic core of the antimicrobial superoxide-generating enzyme, NADPH oxidase. In the overwhelming majority of cases, mutations are family-specific and occur in the exonic regions of the gene, or more rarely at the intron/exon borders. Alternatively, they are large (often multi-gene) deletions. In addition, four mutations have been found in the promoter region. In contrast, very few intronic mutations have been reported. Here we describe an intronic mutation that causes X-linked CGD. A single nucleotide substitution in the middle of intron V creates a novel 5′ splice site and results in multiple abnormal mRNA products.

CYP2A6 genetic polymorphisms and liver microsomal coumarin and nicotine oxidation activities in Japanese and Caucasians.

Genotypes of CYP2A6, namely CYP2A6(*)1 (wild-type), CYP2A6(*)2, and CYP2A6(*)3, were examined in liver DNA of 39 Japanese and 43 Caucasians using two-step polymerase chain reaction (PCR) methods. We first amplified a DNA fragment (1725 bp) located between near middle of exon 1 and end of exon 4 of the CYP2A6 gene and further amplified using a forward primer 't' or 'mut' (middle of exon 3) and a reverse primer 'E3R' (middle of intron 3) for the detection of CYP2A6(*)2-genetic polymorphism. The 1725 bp fragment was also used for the amplification between exon 3 and near middle of intron 3 of the CYP2A6 gene and the fragment thus obtained digested with XcmI or DdeI to detect and confirm the CYP2A6(*)2- and CYP2A6(*)3-types, respectively. Only one DNA sample from a Japanese origin (J18) was not amplified by CYP2A6-specific primers; liver microsomes from this individual had very low activity of coumarin 7-hydroxylation and were devoid of protein(s) immunoreactive to anti-CYP2A6 antibody. Thus, this individual was suggested to be due to the gene deletion in CYP2A6. By analyzing the remaining 38 Japanese and 43 Caucasians, we found that there were no cases of CYP2A6(*)3-type polymorphism in the samples examined in this study, and no cases of CYP2A6(*)2-type polymorphism in the Japanese samples. Of Caucasians studied two individuals were classified into heterozygous CYP2A6(*)1/(*)2-type. Liver microsomal coumarin 7-hydroxylation activities in these two Caucasians were found to be lower than those of the other 41 Caucasians. Kinetic analysis showed that two CYP2A6(*)1/(*)2 individuals had a very low ratio of V(max) to K(m) for nicotine C-oxidation as well as coumarin 7-hydroxylation in liver microsomes, compared with those of homozygous CYP2A6(*)1-type. These results suggest that among 39 Japanese and 43 Caucasians examined one Japanese is classified to be CYP2A6 gene deletion and two Caucasians are heterozygous CYP2A6(*)1/(*)2-genotype. Thus the race-related differences in the occurrence of CYP2A6 genetic polymorphisms were supported.

Elements in the distal 5'-flanking sequence and the first intron function cooperatively to regulate glutamine synthetase transcription during adipocyte differentiation.

Glutamine synthetase (GS) expression increases dramatically during adipocyte differentiation of confluent 3T3-L1 cells. To identify differentiation-responsive cis-acting elements in the GS gene, several GSfusion genes were prepared and analyzed in stably transfected 3T3-L1 cells under conditions that trigger adipocyte differentiation. We find that the GS proximal 5'-flanking sequence lacks the regulatory elements required for differentiation-responsive expression. In contrast, a 2 kb intron 1 restriction fragment fused upstream of a heterologous promoter does drive reporter gene expression during hormone-triggered differentiation. The enhancer activity was localized to a 310 bp sequence near the middle of intron 1. Expression of fusion genes that include this 310 bp sequence does not temporally coincide with native gene expression. However, a composite gene that includes a far upstream GS sequence and the 2 kb intron 1 sequence yields a qualitatively different pattern of expression that closely resembles that of the native GS gene. The far upstream sequence alone exhibits no enhancer activity. Electrophoretic mobility shift analyses indicate that a 32 bp sequence within the 310 bp functional enhancer specifically binds differentiation-associated nuclear proteins. Although a C/EBP consensus sequence occurs in the 32 bp fragment, supershift analyses exclude C/EBP isoforms as the binding factor. In contrast, mutational analysis of the putative enhancer suggests that an HNF-3 isoform is involved. Thus our data indicate that elements in the distal 5'-flanking sequence and the first intron function cooperatively to regulate GS transcription and that HNF-3 may participate in that regulation.

Cloning of a human homologue of the Xenopus laevis APX gene from the ocular albinism type 1 critical region.

Ocular albinism type 1 (OA1) is an X-linked recessive disorder characterized by a major impairment of visual acuity, nystagmus, strabismus, photophobia and retinal hypopigmentation. From the analysis of patients carrying deletions and translocations involving the distal short arm of the X chromosome (Xp22.3) we have identified a region of approximately 110 kb in which the OA1 gene must lie. We have extensively searched for genes in this region using a variety of techniques which included exon amplification, cDNA selection and direct hybridization of cosmid inserts to cDNA libraries. Putative exons identified by exon amplification were used to screen a human retina cDNA library and several cDNA clones corresponding to an approximately 7.5 kb transcript were isolated and characterized. Transcripts of this newly identified gene were found to be abundant in retina and melanoma and could also be detected in brain, placenta, lung, kidney and pancreas. Interestingly, sequence analysis revealed that this new gene encodes a 1616 amino acid protein sharing significant similarities with the Apical Protein from Xenopus laevis (APX) which is implicated in amiloride-sensitive sodium channel activity. The gene, termed APXL (APX-Like), spans approximately 160 kb, contains 10 exons and covers over 70% of the 110 kb critical region for OA1. A truncated pseudogene sharing very high levels of homology with the rat eIF-5 gene, a eukaryotic translation initiation factor, was found to lie in the middle of intron 1. APXL was found deleted in two patients with contiguous gene syndromes including OA1 and in one patient with isolated OA1. Mapping, expression and patient analysis data led us to consider the APXL gene a strong candidate for the OA1 gene. DNA from 57 unrelated patients with OA1 was, therefore, scanned for mutations in the coding region, using both SSCP analysis and direct sequencing. No functionally significant mutation was identified, suggesting that APXL is not directly involved in OA1. Further studies are needed to clarify the physiologic role of this highly conserved gene.

DNA sequence analysis of spontaneous hprt mutations arising in vivo in cynomolgus monkey T-lymphocytes.

To study the mechanisms of mutagenesis in vivo, we analyzed mutations at the hypoxanthine phosphoribosyl transferase (hprt) locus using cDNA from cynomolgus monkey T-lymphocytes. In the present study, the spectrum of spontaneous hprt mutations arising in vivo in wild-caught cynomolgus monkey peripheral T-lymphocytes is described. Cells were isolated from peripheral blood, and mutant clones were selected in 6-thioguanine, propagated, and stored frozen. cDNA was copied from hprt mRNA from a lysate of 7,000 to 20,000 cells. A 780-base-pairs (bp) region including the coding region was amplified by polymerase chain reaction and directly sequenced. We sequenced 40 spontaneous mutants from 11 monkeys. Of these 40 clones, 23 (57%) had base-pair substitutions, 11 (28%) had small (< 20 bp) deletions and/or insertions, and 6 (15%) had large (> 20 bp) deletions and/or insertions. Of the 23 base substitutions, 13 were transitions (11 G:C-->A:T, 1 A:T-->G:C, and 1 tandem TT-->CC) and 10 were transversions (3 G:C-->T:A, 3 G:C-->C:G, 2 A:T-->T:A, 2 A:T-->C:G). Bases 209 and 617 were apparent substitution hotspots, which have also been observed as hotspots in human hprt. In 2 clones with large insertions, the inserted bases were of intronic origin. One of these lost 272 bp from exons 2-3 and contained a 93-bp insertion from the middle of intron 3. Two clones with small deletions and 5 clones with large deletions or insertions (7/40 or 17.5%) could be splice mutants.

Overlapping palindromic sequences associated with somatic deletion and meiotic recombination of MHC class I genes

H-2L-null variants were immunoselected from a transfected murine fibroblast cell line carrying a single copy H-2L gene, and were characterized to determine the basis for the loss of this MHC class I cell surface product. Molecular analysis indicated that inactivation of H-2L expression in nearly every null clone resulted from an apparent deletion or rearrangement of 5'-flanking and 5'-coding H-2L sequences, with breakpoints consistently mapping to within a 550 bp GC-rich region between exon 1 and the middle of intron 2. Notably, this region of the H-2L gene contains a large number of overlapping, inverted repeat sequences as well as potential topoisomerase I cleavage sites. Examination of several in vivo mutant class I genes, believed to have been generated by recombination, has revealed that each of these genes bears similar palindromic structures overlapping or adjacent to the regions of sequence exchange. These findings suggest that inverted repeat sequences may play a role in recombination and deletion within the MHC class I multigene family.