First Base Of Exon Research Articles

The correlation between DNA methylation and transcriptional expression of human dopamine transporter in cell lines

This study aims to investigate the relationship between DNA methylation and expression of human dopamine transporter (hDAT). We examined methylation status of hDAT in cells with various hDAT expression levels, including two dopaminergic neural cell lines (SK-N-AS and SH-SY-5Y) and one non-dopaminergic cell line (HEK293) by bisulfite sequencing PCR(BSP). The effects of DNA methyltransferase inhibitor 5-aza-dC or/and histone deacetylase inhibitor (HDACi, sodium butyrate, NaB) on the DNA methylation status and mRNA expression levels of hDAT were examined. The results revealed marked hypomethylation of the two promoter regions (-1214 to -856bp and -48 to 439bp, the first base of exon 1 was taken as +1 bp)of hDAT in SK-N-AS (4.7%±2.0mC and 3.5%±1.0mC, respectively) compared with SH-SY-5Y (88.0%±4.4%mC and 81.1%±8.8%mC) and HEK293 (90.7%±2.4mC and 84.4%±8.6% mC) cell lines, indicating a cell-specific methylation regulation of hDAT. 5-aza-dC and NaB decreased hypermethylation,while increase hDAT expression in SH-SY-5Y cells and recovered hDAT mRNA expression in HEK293 cells. DNA methylation enabled the cell-specific differential expression of the hDAT gene. hDAT silencing was reversed by the introduction of DNA hypomethylation via 5-aza-dC or/and NaB.

A novel PRKAG2 mutation in a Chinese family with cardiac hypertrophy and ventricular pre-excitation

PRKAG2 syndrome is a rare autosomal dominant inherited disorder that is characterized by cardiac hypertrophy, ventricular pre-excitation and conduction system abnormalities. There is little knowledge in cardiovascular magnetic resonance (CMR) characteristics of PRKAG2 cardiomyopathy. This study investigated the genetic defect in a three-generation Chinese family with cardiac hypertrophy and ventricular pre-excitation using whole-exome sequencing. A novel missense mutation, c.1006 G > T (p.V336L), was identified in PRKAG2. This mutation had not been identified in the ExAC database, and the prediction result of MutationTaster indicated a deleterious effect. Furthermore, it cosegregated with the disease in the present family and was absent in unrelated 300 healthy controls. cDNA analysis did not detect any splicing defects, although the variant occurred in the first base of exon 9. CMR evaluation in five affected members showed diffuse hypertrophy in a concentric pattern, with markedly increased left ventricular mass above age and gender limits (median 151.3 g/m2, range 108.4–233.4 g/m2). Two patients in progressive stage and one patient with sudden cardiac death exhibited extensive subendocardial late gadolinium enhancement. In conclusion, molecular screening for PRKAG2 mutations should be considered in patients who exhibit cardiac hypertrophy coexisting with ventricular pre-excitation. CMR offers promising advantages for evaluation of PRKAG2 cardiomyopathy.

EYA1 and SIX1 gene mutations in Japanese patients with branchio-oto-renal (BOR) syndrome and related conditions

We isolated genomic DNA from 15 patients with branchio-oto-renal (BOR) syndrome or BOR-related conditions. Seven patients had BOR syndrome (two familial and five sporadic), and eight had deafness and renal malformations without branchial fistula (BOR-related conditions). We analyzed all exons and exon-intron boundaries of EYA1 and SIX1 using the polymerase chain reaction (PCR) direct sequencing, and characterized their mutations. In some patients, analysis of mRNA by reverse transcription (RT)-PCR was performed to examine whether the mutation affects the mRNA splicing. We identified five novel disease-causing heterozygous EYA1 mutations in five patients with BOR syndrome (two familial and three sporadic, 5/7=71%), but EYA1 and SIX1 mutations were not detected in the other two patients with BOR syndrome or any of the patients with BOR-related conditions. The detected EYA1 mutations were two nonsense mutations, two splicing acceptor-site mutations, and a point mutation (G>T) of the first base of exon 10. Analysis of mRNA by RT-PCR direct sequencing revealed that the latter point mutation led to the skipping of exon 10. In conclusion, (1) EYA1 mutations are a major cause of BOR syndrome in Japanese, (2) EYA1 and SIX1 mutations were not a major cause of BOR-related conditions, (3) we demonstrated for the first time that the point mutation (G>T) of the first base of the exon in EYA1 gene induced exon skipping.

Analysis of Distinct Tartrate-resistant Acid Phosphatase Promoter Regions in Transgenic Mice

The tartrate-resistant acid phosphatase (TRAP) is present in multiple tissues, including kidney, liver, lung, spleen, and bone. Recent study of (TRAP) gene expression has provided evidence for distinct promoters within the (TRAP) gene, suggesting that the gene has alternative, tissue-preferred mRNA transcripts. Examination of endogenous (TRAP) exon 1B and 1C mRNA transcripts revealed tissue-preferred transcript abundance with increased exon 1B transcripts detected in liver and kidney and increased exon 1C transcripts detected in bone and spleen. In this investigation, we have made transgenic mice that express a marker gene driven by two candidate promoters, designated BC and C, within the (TRAP) gene. The BC and C promoters are 2.2 and 1.6 kb, respectively, measured from the translation initiation site. Evaluation of BC transgenic lines demonstrated robust expression in multiple tissues. In contrast, significant transgene expression was not detected in C transgenic lines. Evaluation of transgene mRNAs in BC transgenic lines revealed that virtually all expression was in the form of B transcripts, suggesting that the tissue-preferred pattern of endogenous (TRAP) was not replicated in the BC transgenic line. Likewise, osteoclastogenic cultures from BC, but not C, transgenic bone marrow cells expressed the transgene following receptor activator of NFkappaB ligand/macrophage colony-stimulating factor stimulation. In conclusion, when compared with the 2.2-kb BC portion of the (TRAP) promoter region, the 1.6-kb C portion does not account for significant gene expression in vivo or in vitro; production of the bone- and spleen-preferred (TRAP) C transcript must depend on regulatory elements outside of the 2.2-kb promoter. As the majority of currently investigated transcription factors that influence transcriptional regulation of osteoclast gene expression bind within the 1.6-kb C portion of the (TRAP) promoter, it is likely that transcription binding sites outside of the 2.2-kb region will have profound effects on regulation of the gene in vivo and in vitro.

Leiomiosarcoma de la vena renal izquierda

Waardenburg syndrome is an autosomal dominant disorder characterized by sensorineural deafness and pigmentary disturbances. Previous work has linked the disease to PAX3 on chromosome 2, and several mutations within the highly conserved paired-box and octapeptide motifs, but not the homeobox, have been reported. In this report, we have used the published cDNA sequence to further define the genomic structure of PAX3, using inverse PCR. We have identified exon/intron boundaries between exons 5 and 6 and between exons 6 and 7. Further, we have identified the first two mutations within the homeobox in two different families with type 1 Waardenburg syndrome. The first is a point mutation (G-->T) at the first base of exon 6, which substitutes phenylalanine for valine. In another family, we have identified a point mutation (C-->G) within the homebox, in exon 6, which substitutes a glycine for arginine at a highly conserved site. The homeodomain is important in binding of DNA and in effecting transcriptional control. These mutations likely result in structural change within the homeodomain that either change the DNA-binding specificity of the homedomain or reduce the affinity of the PAX3 protein for DNA. These homeodomain mutations should aid in elucidating the role of the homeodomain in the function of the PAX3 protein.

A novel exon in the cystic fibrosis transmembrane conductance regulator gene activated by the nonsense mutation E92X in airway epithelial cells of patients with cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We report on a novel nonsense mutation that leads to exon skipping and the activation of a cryptic exon. Screening of genomic DNA from 700 German patients with CF uncovered four cases with the nonsense mutation E92X, a G-->T transversion that creates a termination codon and affects the first base of exon 4 of the CFTR gene. Lymphocyte RNA of two CF patients heterozygous for E92X was found to contain the wild type sequence and a differentially spliced isoform lacking exon 4. In RNA derived from nasal epithelial cells of E92X patients, a third fragment of longer size was observed. Sequencing revealed the presence of E92X and an additional 183-bp fragment, inserted between exons 3 and 4. The 183-bp sequence was mapped to intron 3 of the CFTR gene. It is flanked by acceptor and donor splice sites. We conclude that the 183-bp fragment in intron 3 is a cryptic CFTR exon that can be activated in epithelial cells by the presence of the E92X mutation. E92X abolishes correctly spliced CFTR mRNA and leads to severe cystic fibrosis.

A reply: the human proacrosin gene

Vazquez-Levin et al. have published, in this edition of your journal, a paper on the molecular cloning, sequencing and restriction mapping of the genomic sequence encoding human proacrosin [I]. The gene was isolated by using our human proacrosin cDNA probe [2] which we sent to Dr Gordon in 1990. The results obtained by the authors confirm the nucleotide sequence and genomic organization of the proacrosin gene which we published in 1990 [3]. Vazquez-Levin et al. report two differences between their nucleotide sequence and ours, namely in 22 bases of intron 1 and in the localisation of a cytosine residue in exons 3 and 4. We would like to comment on these differences. 1. Vazquez-Levin et al. [l] report in the abstract of their paper that the 500 bases sequenced distal to the initial 54 bases at the 5’ end of intron 1 shows no similarity to our published sequence [3]. Such a conclusion is not justified because we have published only 76 bases of the 5’ end of intron 1. We have obtained this 76-bp sequence by sequencing the 1.2-kb PstIEcoRI genomic fragment containing exon 1, intron 1 and exon 2 from both ends (Fig. 1 in [3]). Therefore, we believe the explanation that an inversion is the reason for the 22-bp difference between our sequence and that of Vazquez-Levin et al. to be unlikely. 2. Vazquez-Levin et al. state that the boundaries of the third intron of the human proacrosin gene differed such that a cytosine residue reported by us in exon 3 [3] was found to be the first base of exon 4. This situation is true in the proacrosin gene of mouse [4], rat [5] and boar (unpublished results), but not in the human proacrosin gene. To exclude possible sequencing errors, we confirmed the results in the human proacrosin gene several times and the cytosine residue was always found in exon 3. Vazquez-Levin et al. [l] report that they have sequenced more than 500 bases of the promoter region of the human

Molecular cloning, sequencing and restriction mapping of the genomic sequence encoding human proacrosin.

In the present study, molecular cloning, sequencing and restriction mapping of the genomic sequence encoding human proacrosin is described. The full-length cDNA encoding human proacrosin was utilized to recover a 17-kb human genomic clone which was sequenced without further subcloning. The nucleotide sequences of the exons agree with the sequence of the cDNA reported previously. More than 500 bases of the promoter region were sequenced and found to be highly GC rich but devoid of an identifiable TATA box. These findings are generally consistent with a recently published report [Keime, S., Adham, I. M. & Engel, W. (1990) Eur. J. Biochem. 190, 195-200]. However, further sequence analysis revealed discrepancies between our clone and that previously reported. Sequencing of the first intron showed similarity with the published data for 54 bases of the 5' region, beginning with the donor splice site, and for 114 bases at the 3' end. However, 500 bases sequenced distal to the initial 54 bases at the 5' end of intron 1 showed no similarity with the published sequence. In addition, the boundaries of intron 3 differed such that a cytosine residue previously reported to be in exon 3 was found to be the first base of exon 4. Detailed studies were undertaken to confirm that our clone constitutes the authentic sequence of human proacrosin. Cloning and characterization of the human proacrosin gene may allow for informative studies of its regulation, and for a more detailed examination of its role in fertilization.

Expression of the gene for the beta subunit of mouse thyrotropin results in multiple mRNAs differing in their 5′-untranslated regions.

The gene encoding the beta subunit of mouse thyrotropin (TSH beta) has been isolated from a mouse genomic library, and its nucleotide sequence has been determined. Blot hybridization analysis of restriction enzyme digests of mouse DNA indicates that there is a single mouse TSH beta gene. The gene is 4.8 kilobases in length and contains five exons, which are 27, 47, 41, 163, and 328 base pairs long. Exons 1, 2, and 3 encode only 5'-untranslated mRNA sequences and are separated by introns that are 150 and 380 base pairs long. The protein-coding mRNA sequences are found in exons 4 and 5 and are interrupted by a 460-base pair intron. The position of this intron, between the codons for amino acids 34 and 35, has been conserved in all the known glycoprotein hormone beta subunit genes. Exons 3 and 4 are separated by a large 3.2-kilobase intron. When primer extension analysis, using an oligonucleotide primer complementary to exon 4 sequences, was employed to locate the transcription start site, four products were obtained. Nucleotide sequencing of these products showed that they were derived from separate TSH beta mRNAs that differed in the lengths of their 5'-untranslated regions. These 5'-untranslated mRNA sequences are derived from different combinations of exons 1, 2, and 3, each spliced to exons 4 and 5. The longest 5'-untranslated sequence, 116 nucleotides long, includes exons 1, 2, and 3 and the first base of exon 4; the shorter 5'-untranslated regions, 75, 69, and 28 nucleotides long, arise by splicing out the second and/or the third exon sequences. In contrast to the mouse TSH beta gene, transcription of the rat TSH beta gene from the analogous start site has been reported to give only a single mRNA, with a 5'-untranslated region of 28 nucleotides. Divergence of the mouse and rat TSH beta gene sequences at RNA splice sites can account for the absence of exon 2, but not exon 3, sequences in rat TSH beta mRNA. Primer extension and RNase protection analyses also showed that the mouse TSH beta gene contains a second transcription start site, located 43 base pairs upstream of the first start site, in a position corresponding to that in the rat TSH beta gene. Each start site in the mouse gene is flanked by characteristic TATAA box and CAAT box sequences.(ABSTRACT TRUNCATED AT 400 WORDS)