scholarly journals Complete Thyroxine-Binding Globulin (TBG) Deficiency Produced by a Mutation in Acceptor Splice Site Causing Frameshift and Early Termination of Translation (TBG-Kankakee)12

1998 ◽  
Vol 83 (10) ◽  
pp. 3604-3608
Author(s):  
Gisah A. Carvalho ◽  
Roy E. Weiss ◽  
Samuel Refetoff
Keyword(s):  
Splice Site ◽  
Messenger Rna ◽  
Restriction Site ◽  
Splice Junction ◽  
Early Termination ◽  
Coding Region ◽  
Acceptor Splice Site ◽  
Exon 2 ◽  
Gene Level ◽  
Exon 1

Fourteen T4-binding globulin (TBG) variants have been identified at the gene level. They are all located in the coding region of the gene and 6 produce complete deficiency of TBG (TBG-CD). We now describe the first mutation in a noncoding region producing TBG-CD. The proband was treated for over 20 yr with L-T4 because of fatigue associated with a low concentration of serum total T4. Fifteen family members were studied showing low total T4 inherited as an X chromosome-linked trait, and affected males had undetectable TBG in serum. Sequencing of the entire coding region and promoter of the TBG gene revealed no abnormality. However, an A to G transition was found in the acceptor splice junction of intron II that produced a new HaeIII restriction site cosegregating with the TBG-CD phenotype. Sequencing exon 1 to exon 3 of TBG complementary DNA reverse transcribed from messenger RNA of skin fibroblasts from an affected male, confirmed a shift in the ag acceptor splice site. This results in the insertion of a G in exon 2 and causes a frameshift and a premature stop at codon 195. This early termination of translation predicts a truncated TBG lacking 201 amino acids.

Effect of 5' splice site mutations on splicing of the preceding intron

1990 ◽  
Vol 10 (12) ◽  
pp. 6299-6305
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factors ◽  
Exon 2 ◽  
Intron 1 ◽  
Direct Assembly ◽  
Internal Exon ◽  
Exon 1 ◽  
Lariat Rna

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

scholarly journals GEN-03 GENOTYPE-PHENOTYPE CORRELATION IN 111 FAMILIES OF VON HIPPEL-LINDAU DISEASE IN JAPAN

2019 ◽  
Vol 1 (Supplement_2) ◽  
pp. ii8-ii8
Author(s):  
Hiroshi Kanno ◽  
Tetsuya Yoshizumi ◽  
Masamichi Shinonaga ◽  
Masahiro Yao
Keyword(s):  
Amino Acids ◽  
Missense Mutation ◽  
Splice Site ◽  
Phenotype Correlation ◽  
Exon 2 ◽  
Endolymphatic Sac Tumor ◽  
Von Hippel Lindau ◽  
Genotype Phenotype Correlation ◽  
Exon 1 ◽  
Vhl Disease

Abstract BACKGROUND AND AIM von Hippel-Lindau (VHL) disease is a hereditary disease which manifest central nervous system (CNS) hemangioblastoma, retinal angioma, renal cell carcinoma (RCC), pheochromocytoma, endolymphatic sac tumor, and pancreas cyst. The VHL gene is located at 3p25.3 and is corresponding to 213 amino acids. Genotype-phenotype correlation analyses of VHL disease have been recently reported from several foreign countries, but the genotype-phenotype correlation has not been characterized since above 10 years ago. Therefore, this study aimed to evaluate the VHL mutation spectrum and genotype-phenotype correlations in Japanese VHL patients. METHODS Blood samples of 111 unrelated families of VHL disease were collected and DNAs were extracted. Direct sequencing and real-time PCR analysis were performed. Consequently, the clinical manifestations and family histories of the subjects were evaluated. RESULTS We identified VHL mutations as follows: missense 47; deletion 17; insertion 5; nonsense 8; splice-site 9; larger deletion 25. At hot-spot codon 167, 4 minsense mutations were identified, with Arg167Trp, 4 cases; Arg167Gln2, 2 cases. At codon 155, splice-site mutations were identified at 6 cases. Mutation sites were distributed in exon 1, 45; exon 2, 21; exon 3, 36. Large deletions were distributed in exon 1 & 2, 1; exon 2& 3, 1; all exons, 11. Genotype-phenotype correlation analysis revealed that age-specific risk and number of CNS hemangioblastoma were significantly higher in subjects carrying missense mutation within HIF-α binding site or non-missense mutation (P < 0.05). In addition, penetrance of RCC was significantly higher in subjects carrying non-missense mutation (P < 0.05). CONCLUSIONS The results of this study were similar to the previous foreign studies. This study provides insight into the genotype-phenotype correlation in that amino acids substitutions in the HIF- α binding and non-sense mutations may predispose VHL patients to age-related risk and number of CNS hemangioblastoma.

scholarly journals Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles.

1988 ◽  
Vol 8 (2) ◽  
pp. 814-821 ◽  
Author(s):  
M Zillmann ◽  
M L Zapp ◽  
S M Berget
Keyword(s):  
Gel Electrophoresis ◽  
Splice Junction ◽  
Rnase H ◽  
Exon 2 ◽  
Small Nuclear Ribonucleoprotein ◽  
Complementary Oligonucleotide ◽  
Exon 1 ◽  
Precursor Rna ◽  
Splice Junctions

Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.

scholarly journals Sanger sequencing as a first-line approach for molecular diagnosis of Andersen-Tawil syndrome

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1016 ◽  
Author(s):  
Armando Totomoch-Serra ◽  
Manlio F. Marquez ◽  
David E. Cervantes-Barragán
Keyword(s):  
Molecular Diagnosis ◽  
Sanger Sequencing ◽  
Heterozygous Mutation ◽  
Coding Region ◽  
First Line ◽  
Base Pairs ◽  
Exon 2 ◽  
Clinical Criteria ◽  
Exon 1 ◽  
Next Generation Sequencing Ngs

In 1977, Frederick Sanger developed a new method for DNA sequencing based on the chain termination method, now known as the Sanger sequencing method (SSM).  Recently, massive parallel sequencing, better known as next-generation sequencing (NGS),  is replacing the SSM for detecting mutations in cardiovascular diseases with a genetic background. The present opinion article wants to remark that “targeted” SSM is still effective as a first-line approach for the molecular diagnosis of some specific conditions, as is the case for Andersen-Tawil syndrome (ATS). ATS is described as a rare multisystemic autosomal dominant channelopathy syndrome caused mainly by a heterozygous mutation in the KCNJ2 gene. KCJN2 has particular characteristics that make it attractive for “directed” SSM. KCNJ2 has a sequence of 17,510 base pairs (bp), and a short coding region with two exons (exon 1=166 bp and exon 2=5220 bp), half of the mutations are located in the C-terminal cytosolic domain, a mutational hotspot has been described in residue Arg218, and this gene explains the phenotype in 60% of ATS cases that fulfill all the clinical criteria of the disease. In order to increase the diagnosis of ATS we urge cardiologists to search for facial and muscular abnormalities in subjects with frequent ventricular arrhythmias (especially bigeminy) and prominent U waves on the electrocardiogram.

scholarly journals Functional analysis of the AUG- and CUG-initiated forms of the c-Myc protein.

1994 ◽  
Vol 5 (5) ◽  
pp. 597-609 ◽  
Author(s):  
E M Blackwood ◽  
T G Lugo ◽  
L Kretzner ◽  
M W King ◽  
A J Street ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Chromosomal Translocation ◽  
Regulatory Elements ◽  
Genetic Alterations ◽  
Coding Region ◽  
Exon 2 ◽  
Translational Initiation ◽  
Selective Growth Advantage ◽  
Exon 1 ◽  
Normal Human

Activation of the c-myc proto-oncogene by chromosomal translocation or proviral insertion frequently results in the separation of the c-myc coding region from its normal regulatory elements. Such rearrangements are often accompanied by loss or mutation of c-myc exon 1 sequences. These genetic alterations do not affect synthesis of the major c-myc protein, p64, which is initiated from the first AUG codon in exon 2. However they can result in mutation or loss of the CUG codon located in exon 1 that normally serves as an alternative translational initiation codon for synthesis of an N-terminally extended form of c-Myc (p67). It has been hypothesized that p67 is a functionally distinct form of c-Myc whose specific loss during c-myc rearrangements confers a selective growth advantage. Here we describe experiments designed to test the functional properties of the two c-Myc protein forms. We introduced mutations within the translational initiation codons of a normal human c-myc cDNA that alter the pattern of Myc protein synthesis (p64 vs. p67). The functions of each of these proteins were experimentally addressed using co-transformation and transcriptional activation assays. Both the p64 and p67 c-Myc proteins were independently able to collaborate with bcr-abl in the transformation of Rat-1 fibroblasts. In addition, both the exon 1- and exon 2-initiated forms of the c-Myc protein stimulated transcription of a Myc/Max-responsive reporter construct to a similar level. Given the apparent absence of functional differences between p64 and p67, we conclude that the basis for c-Myc oncogenic activation lies primarily in the overall deregulation of its expression and not in alterations in the protein. The existence of the CUG translational initiator may reflect a mechanism for the continued synthesis of c-Myc protein under conditions where AUG initiation is inhibited.

scholarly journals Effect of 5' splice site mutations on splicing of the preceding intron.

1990 ◽  
Vol 10 (12) ◽  
pp. 6299-6305 ◽  
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factors ◽  
Exon 2 ◽  
Intron 1 ◽  
Direct Assembly ◽  
Internal Exon ◽  
Exon 1 ◽  
Lariat Rna

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

scholarly journals Characteristics of defensin1 gene and designing structure to create resistant transgenic corn lines to weevils

2016 ◽  
Vol 14 (2) ◽  
pp. 279-286
Author(s):  
Vì Thị Xuân Thủy ◽  
Lò Thị Mai Thu ◽  
Hồ Mạnh Tường ◽  
Lê Văn Sơn ◽  
Nguyễn Vũ Thanh Thanh ◽  
...  
Keyword(s):  
Amino Acid Sequences ◽  
Disulfide Bridges ◽  
Coding Region ◽  
Exon 2 ◽  
Transformation Vector ◽  
Plant Defensins ◽  
Maize Sample ◽  
Exon 1 ◽  
Hybrid Maize ◽  
Group 1

Plant defensins are multifunctional proteins, inhibiting the growth of fungal, anti-bacterial, altering membrane channels, inhibiting activity of trypsin and α-amylase. Plant defensin consists of 18 groups in which the group 1 includes  defensins to inhibit either α-amylase enzyme or trypsin. Defensins bind to the active site of α-amylase in the weevil gut, thus inhibit starch digestion in weevils. In this report, we present the results of cloning and determining the ZmDEF1 gene sequence isolated from mRNA and DNA of Sonla province local maize and LVN99 hybrid maize cultivar. The coding region of ZmDEF1 gene isolated from some maize samples had the size of 243 nucleotides, encoding 80 amino acids. Gen ZmDEF1 isolated from DNA had the size 345bp consists of two exons and one in tron (102 bp). The nucleotide sequences of ZmDEF1 gene (DNA) of the samples have 6 positions nucleotide difference, on exon 1 has two points difference (position 43, 53), on intron has a difference (position 150), on exon 2 has 3 nucleotide site difference (203, 263 and 297 position). Deduced amino acid sequences of defensin of the Sonla local maize sample has 8 cysteines to make 4 disulfide bridges, while LVN99 hybrid maize has 7 cysteines, which can formed only 3 disulfide bridges. Transformation vector pBetaPhaso-ZmDEF1 has been designed successfully, in which ZmDEF1 is controlled by seed specific Phasoline promoter. The correct insertion and expression of ZmDEF1 was examinated in transgenic tobacco plants throught PCR and RT-PCR, respectively. These results provide an firm evident for using the designed transformation vector to produce transgenic maỉze lines with an improved resistant ability  to weevils.

Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe

1989 ◽  
Vol 9 (4) ◽  
pp. 1526-1535
Author(s):  
K B Gatermann ◽  
A Hoffmann ◽  
G H Rosenberg ◽  
N F Käufer
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Splice Site ◽  
Base Pair ◽  
Point Mutations ◽  
Splice Junction ◽  
Simple Type ◽  
Coding Region ◽  
Synthetic Oligonucleotide ◽  
Functional Product ◽  
Splicing Efficiency

Insertion of a 36-base-pair (bp) synthetic oligonucleotide comprising the sequence 5'-GTAGGT(19N)CTAAT (4N)AG-3' into several different positions within the coding region of the naturally intronless ura4 gene of Schizosaccharomyces pombe leads to an efficiently spliced gene producing a functional product. This suggests that the proper signals within an intron are sufficient to initiate and complete a splicing event independent of the location of the intron in the gene. Point mutations in the 5' junction (5'-GTAGGT-3') and in the putative branch sequence (5'-CTAAT-3') affect splicing efficiency significantly. A G-to-A transition at the first nucleotide at the 5' splice junction (5'-ATAGGT-3') abolishes the use of the authentic splice junction and leads to the increased use of an alternative splice site. No functional product is produced from this transcript. An A-to-G transition of the second A in the putative branch sequence (5'-CTAGT-3') lowers the splicing efficiency drastically, but still results in a functional gene product. Furthermore, extension of the 36-bp intron to introns more than 180 bp in size abolishes splicing, suggesting that the splicing apparatus might be restricted to very short introns. We discuss the possibility that S. pombe introns represent a simple type of eucaryotic intron.

Analysis of the CC chemokine receptor 3 gene reveals a complex 5′ exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2346-2354 ◽  
Author(s):  
Nives Zimmermann ◽  
Bruce L. Daugherty ◽  
Jessica L. Kavanaugh ◽  
Faisal Y. El-Awar ◽  
Elizabeth A. Moulton ◽  
...  
Keyword(s):  
Chemokine Receptor ◽  
Messenger Rna ◽  
Direct Sequencing ◽  
Cell Types ◽  
Northern Analysis ◽  
Strong Activity ◽  
Exon 2 ◽  
Cc Chemokine ◽  
Untranslated Exon ◽  
Exon 1

Abstract To understand the regulation of CC chemokine receptor 3 (CCR3) expression, its gene structure and promoter have been characterized. The CCR3 gene contains 4 exons that give rise to multiple messenger RNA (mRNA) species by alternative splicing. Exon 1 is present in all transcripts, whereas exon 2 or 3 is present at low frequency (< 10%). Exon 4 contains the open reading frame and 11 bp of the 5′ untranslated region. Northern analysis revealed 4 species of CCR3 mRNA. Direct sequencing revealed that the first 1 kb of the promoter and exon 1 contained only one mutation in 19 individuals, indicating that the CCR3 promoter and exon 1 are conserved between individuals. The first 1.6 kb of the 5′ flanking region of exon 1 contained promoter elements including a TATA box and motifs for myeloid transcription factors and had strong promoter activity in eosinophilic, lymphoid, myeloid, and respiratory epithelial cell lines. Deletion analysis revealed differential regulation of the CCR3 promoter in eosinophilic and epithelial cells suggesting the presence of lineage-specific elements. Interestingly, exon 1 enhanced the activity of the promoter and this effect was especially prominent in eosinophilic cells. Thus, the humanCCR3 gene has a complex 5′ exon structure, a conserved promoter with strong activity in multiple cell types, and a functional 5′ untranslated exon.

scholarly journals 5′-Heterogeneity of Glucocorticoid Receptor Messenger RNA Is Tissue Specific: Differential Regulation of Variant Transcripts by Early-Life Events

2000 ◽  
Vol 14 (4) ◽  
pp. 506-517 ◽  
Author(s):  
J. A. McCormick ◽  
V. Lyons ◽  
M. D. Jacobson ◽  
J. Noble ◽  
J. Diorio ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Stress Responses ◽  
Early Life ◽  
Messenger Rna ◽  
Promoter Activity ◽  
Cpg Island ◽  
Rnase Protection ◽  
Exon 2 ◽  
Tissue Specific ◽  
Exon 1

Abstract Glucocorticoid receptor (GR) gene expression is regulated in a complex tissue-specific manner, notably by early-life environmental events that program tissue GR levels. We have identified and characterized several new rat GR mRNAs. All encode a common protein, but differ in their 5′-leader sequences as a consequence of alternate splicing of, potentially, 11 different exon 1 sequences. Most are located in a 3-kb CpG island, upstream of exon 2, that exhibits substantial promoter activity in transfected cells. Ribonuclease (RNase) protection analysis demonstrated significant levels of six alternate exons 1 in vivo in rat, with differences between liver, hippocampus, and thymus reflecting tissue-specific differences in promoter activity. Two of the alternate exons 1 (exons 16 and 110) were expressed in all tissues examined, together present in 77–87% of total GR mRNA. The remaining GR transcripts contained tissue-specific alternate first exons. Importantly, tissue-specific first exon usage was altered by perinatal environmental manipulations. Postnatal handling, which permanently increases GR in the hippocampus, causing attenuation of stress responses, selectively elevated GR mRNA containing the hippocampus-specific exon 17. Prenatal glucocorticoid exposure, which increases hepatic GR expression and produces adult hyperglycemia, decreased the proportion of hepatic GR mRNA containing the predomin-ant exon 110, suggesting an increase in a minor exon 1 variant. Such tissue specificity of promoter usage allows differential GR regulation and programming.

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