Complete coding sequences of cDNAs of four variants of rabbit skeletal muscle troponin T

Summary Four variants of troponin T (TnT) cDNAs have been isolated and sequenced. These cDNAs have been derived from rabbit skeletal muscle, the most widely studied source of troponin, of a 11-day-old animal. One variant (TnT-1) contains the complete coding sequence, while in three variants the coding sequences are truncated at the 5 termini. The previously published amino acid sequence differs from the present cDNA-derived sequences at three locations. At least two, possibly all, of them are probably accounted for by errors in peptide sequencing. The present results are consistent with the two types of alternative splicing of TnT genes, both being first reported on the rat gene. (1) Highly variable sequences in the amino-terminal region are accounted for by the alternative splicing of exons 4–8 in an interchangeable but not mutually exclusive manner. (2) In the carboxyl-terminal region, the alternative splicing of two exons 17 (-type) or 16 (-type) in mutually exclusive manner is consistent with the difference between all the four cDNAs, which express exon 17, and the previously published peptide sequence (derived from the adult muscle) in which exon 16 is present. This variation also corresponds to the finding in chicken skeletal muscle that the choice of exon 16 or 17 may be dependent on developmental stages. Finally, a sequence is observed corresponding to an extra exon or exons between exons 5 and 6. This sequence is shorter than that of the chicken skeletal muscle gene and is not detected in the rat skeletal muscle gene.     

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.     

The gradual expression of troponin T isoforms in chicken wing muscles

Troponin T (TnT) is one of the muscle regulatory proteins and is thought to be related to unique contractile properties in diverse muscles and also to myogenesis. The expression of TnT isoforms in the chicken wing muscles was investigated by two-dimensional gel electrophoresis and immunoblotting with an antiserum against fast skeletal muscle TnT. The upper arm muscles, M. biceps brachii and M. triceps brachii, showed striking differential expression of high molecular weight B type and low molecular weight L type TnT isoforms in the proximal, middle and distal portions of each muscle. The ratio of the total quantity of B type TnT isoforms to that of L type TnT isoforms decreased gradually along the proximo-distal axis of the wing. The upper arm muscles, M. deltoideus and M. tensor patagii longus, and most of the lower arm muscles examined in this study did not show such differential expression. The lower arm muscles, M. flexor carpi ulnaris and M. extensor carpi radialis longus, showed some gradual expression of TnT isoforms, but the B/L ratio in the former slightly increased along the proximo-distal axis. The gradual expression in M. biceps brachii was not found in the 1-day-old chick, but was established by 30 days post-hatching. The biological significance of differential expression of TnT isoforms in different muscles and even in single muscles was speculated upon with respect to muscle contractile regulation and myogenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA

The rat beta-tropomyosin gene encodes two isoforms, termed skeletal muscle beta-tropomyosin and fibroblast last tropomyosim 1 (TM-1), via an alternative RNA processing mechanism. The gene contains 11 exons. Exons 1-5 and exons 8 and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts, as well as smooth muscle, whereas exons 7 and 10 are used only in skeletal muscle. In the present studies we focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). We have identified two distinct elements in the intron, upstream of exon 7, involved in splice site selection. The first element is comprised of a polypyrimidine tract located 89-143 nucleotides upstream of the 3' splice site, which specifies the location of the lariat branchpoints used, 144-153 nucleotides upstream of exon 7. The 3' splice site AG dinucleotide has no role in selection of these branchpoints. The second element is comprised of intron sequences located between the polypyrimidine tract and the 3' splice site of exon 7. It contains an important determinant in alternative splice site selection, because deletion of these sequences results in the use of the skeletal muscle-specific exon in nonmuscle cells. We propose that the use of lariat branchpoints located far upstream from a 3' splice site may be a general feature of some alternatively excised introns, reflecting the presence of regulatory sequences located between the lariat branch site and the 3' splice site. The data also indicate that alternative splicing of the rat beta-tropomyosin gene is regulated by a somewhat different mechanism from that described for rat alpha-tropomyosin gene and the transformer-2 gene of Drosophila melanogaster.     

Porcine T-cell receptor beta-chain: a genomic sequence covering Dbeta1.1 to Cbeta2 gene segments and the diversity of cDNA expressed in piglets including novel alternative splicing products

Porcine TCRbeta-chain cDNA clones were isolated from thymic and peripheral blood lymphocytes of piglets. Using these nucleotide sequences, a genomic 18kbp sequence stretch covering Dbeta1 to Cbeta2 gene segments was identified, which revealed that the porcine TCRbeta-chain locus consists of two sets of Dbeta-Jbeta-Cbeta gene groups with each set having a Dbeta gene segment, seven Jbeta gene segments and a down stream Cbeta gene segment composed of four exons. This structure is consistent with other known mammalian TCRbeta-chain loci. With this genomic information, TCRbeta-chain clones from cDNA libraries were analyzed. Sixteen Vbeta gene segments were obtained accompanied by either Dbeta1 or Dbeta2 and by one of the nine Jbeta gene segments. Five different Cbeta cDNA sequences were obtained including four types of Cbeta1 sequences and one type of Cbeta2 sequence. The differences among the Cbeta1 sequences are either allelic polymorphisms or two splice variants, one being a product of exon1 splicing to exon3 (exon2 skipping), and another being an alternative splicing using a splice acceptor site newly discovered inside Cbeta1 exon4. The latter splice acceptor site was also found in human, mouse and horse all giving short cytoplasmic domain with Phe at their C-terminal ends. Other splicing products included trans-splicing of Jbeta2 to Cbeta1, non-functional splicing of two Jbeta gene segments in tandem and a part of Jbeta2.7-Cbeta2 intron to Cbeta2 exon1. Numerous examples of splice variants may suggest the involvement of splicing in generating TCRbeta-chain functional diversity.     

人CAPN3基因在骨骼肌和白细胞中存在不同的剪切

目的 比较CAPN3基因在外周血白细胞和骨骼肌组织中的剪切变异,探讨用外周血白细胞CAPN3 mRNA进行基因诊断的可行性.方法 抽提正常人外周血和骨骼肌组织中总RNA,通过逆转录-聚合酶链反应和DNA测序确定CAPN3基因的cDNA序列,比较外周血白细胞和骨骼肌组织中CAPN3cDNA序列.结果 由骨骼肌组织抽提的RNA进行逆转录合成cDNA为CAPN3基因全长cDNA,包含全部24个外显子;而由外周血白细胞抽提的RNA进行逆转录合成cDNA为CAPN3基因非全长cDNA,包含23个外显子,缺失了第15外显子.结论 人的CAPN3基因在骨骼肌和外周血自细胞中存在着不同的剪切方式,若用外周血抽提RNA进行CAPN3基因的编码序列分析时会漏检第15外显子的突变.这提示从cDNA水平分析CAPN3基因突变时应采用患者肌肉组织,而非外周血白细胞.     

Alternative splicing of exon 14 determines nuclear or cytoplasmic localisation of fmr1 protein isoforms

Impaired expression of the FMR1 gene is responsible for the fragile X mental retardation syndrome. The FMR1 gene encodes a cytoplasmic protein with RNA-binding properties. Its complex alternative splicing leads to several isoforms, whose abundance and specific functions in the cell are not known. We have cloned in expression vectors, cDNAs corresponding to several isoforms. Western blot comparison of the pattern of endogenous FMR1 proteins with these transfected isoforms allowed the tentative identification of the major endogenous isoform as ISO 7 and of a minor band as an isoform lacking exon 14 sequences (ISO 6 or ISO 12), while some other isoforms (ISO 4, ISO 5) were not expressed at detectable levels. Surprisingly, in immunofluorescence studies, the transfected splice variants that exclude exon 14 sequences (and have alternate C-terminal regions) were shown to be nuclear. Such differential localisation was however not seen in subcellular fractionation studies. Analysis of various deletion mutants suggests the presence of a cytoplasmic retention domain encoded in exon 14 and of a nuclear association domain encoded within the first eight exons that appear however to lack a typical nuclear localisation signal.      

Alpha-tropomyosin mutually exclusive exon selection: competition between branchpoint/polypyrimidine tracts determines default exon choice

We have used exons 2 and 3 of the rat alpha-tropomyosin gene to analyze the basis of mutually exclusive exon selection. The basis of the strict mutually exclusive behavior of this exon pair is enforced by the proximity of the exon 3 branchpoint to the 5' splice site of exon 2. With the exception of smooth muscle cells, exon 3 rather than exon 2 is incorporated into mRNA in all cell types. We show here, using both in vivo and in vitro cell-free systems, that this alternative exon selection is a consequence of general principles that govern 3' splice site selection. In the absence of exon 3, exon 2 is utilized efficiently in all cells. Selection of exon 3 is therefore the default result of a competition between exons 2 and 3 for the flanking constitutive splice sites. The basis of this competition is the relative strength of the polypyrimidine tract/branchpoint elements of the two exons. The major determinant of this splice site strength is the pyrimidine content adjacent to the branchpoint, and this involves no other sequence specificity. The branchpoint elements play an important but secondary role. The functional strengths of the different polypyrimidine tract/branchpoint combinations, as determined in cis competition assays, showed a perfect correlation with their binding affinities to a spliceosome component that interacts with the pre-mRNA in an ATP-independent manner. Selection of exon 3 in most cell types therefore reflects the preferential interaction of these splice site elements with constitutive splicing factors early in spliceosome assembly. The aspects of splice site selection analyzed here are likely to be of general applicability to constitutive and alternative pre-mRNA splicing.     

Molecular analysis of mutations affecting hprt mRNA splicing in human T-lymphocytes in vivo.

Molecular analysis of hypoxanthine-guanine phosphoribosyltransferase (hprt) cDNA from 6-thioguanine-resistant T-lymphocytes cloned from smoking and non-smoking adult donors showed that 35% of these mutants were defective in splicing of hprt mRNA. Among a set of 42 hprt splice mutants, we observed i) complete loss of one or more exons, ii) partial loss of one exon, or iii) inclusion of part of an intron sequence between adjacent exons. Loss of exon 4 was significantly more frequent than of the other exons, suggesting that the sequences that regulate splicing of this exon are either larger than those of the other exons or especially prone to mutation. In order to identify the molecular nature of DNA alterations causing aberrant splicing of hprt mRNA, 17 splice mutants were analyzed in more detail by sequencing the genomic regions flanking the mis-spliced exon. Base pair substitutions or small deletions causing defective splicing were either detected in exon sequences or in splice site consensus sequences of introns. Furthermore, genomic deletions encompassing entire exons were found. In some mutants, the alteration responsible for incorrect splicing could not be identified, suggesting that the target sequence for splice mutations is larger than merely the splice junctions. Molecular characterization of hprt splice mutations will lead to the identification of specific sequences regulating splicing of hprt mRNA and will reveal whether the mutational spectrum in splice mutants is similar to that found in the hprt coding region.     

Evaluation of the ability to make non-invasive estimation of muscle contractile properties on the basis of the muscle belly response

The histochemical and biomechanical relationships of limb muscles are examined in two groups of 15 men aged between 17 and 40 years. Seven muscles are chosen: biceps brachii, triceps brachii (TB), flexor digitorum superficialis, extensor digitorum, biceps femoris, tibialis anterior and gastrocnemius caput mediale (GCM). The aim of the preliminary study is to evaluate an alternative method based on a tensiomyographic (TMG) non-invasive measurement technique. The percentage of type I muscle fibres obtained with the histochemical method is 2.2 times higher for the slowest measured muscle (GCM) than for the fastest (TB). The contraction time of a muscle belly twitch response measured by TMG is 1.9 times higher for GCM than for TB. Statistical analysis of the data obtained by tensiomyographic and histochemical techniques shows a significant correlation between the contraction time of muscle response measured by TMG and the percentage of type I muscle fibres (correlation coefficient equals 0.93). Results of the study suggest using the TMG measuring technique as a basis for the estimation of the percentage of type I muscle fibres.     

Changes in exon-intron structure during vertebrate evolution affect the splicing pattern of exons

Exon-intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon-intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3' and 5' splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery.     

Alternative splicing of the human luteal LH receptor during luteolysis and maternal recognition of pregnancy

Deletion of exon 10 of the human LH receptor impairs LH but not hCG action. Other splice variants of the LH receptor impair both LH and hCG action in other species. We hypothesized that alternatively spliced LH receptors are involved in luteolysis and luteal rescue with hCG in women. mRNA was extracted from human luteinized granulosa cells and from corpora lutea from across the luteal phase and after luteal rescue in vivo with exogenous hCG. Splice variants were detected by RT-PCR using carefully designed primer pairs. Products were visualized on agarose gels, extracted, purified and sequenced. Three splice variants of the human LH receptor were detected and characterized. These demonstrate a region of multiple splicing between exons 8 and 11 of the receptor. A naturally occurring splice variant with exon 10 alone removed was not identified. There was no obvious change in the pattern of splice variants across the luteal phase in the presence or absence of hCG. These data do not support the hypothesis that qualitative changes in LH receptor splicing have a role in luteolysis or that a naturally occurring LH receptor lacking exon 10 has a role in maternal recognition of pregnancy.     

白细胞介素-7选择性剪接变异体的研究

目的　寻找人肿瘤细胞选择性的白细胞介素 7(Interleukin 7,IL 7)剪接变异体。方法　运用自行设计的IL 7引物 ,采用逆转录聚合酶链反应 (RT PCR)技术 ,分离正常肝组织、原发肝癌组织、正常胃组织和胃癌组织IL 7mRNA ,再对电泳所获条带进行克隆、测序。结果　正常肝组织、原发肝癌组织、正常胃组织和胃癌组织都能检测到IL 7mRNA ,而且从肝癌组织、胃癌组织中各分离出一条新带 ,经亚克隆及测序 ,证实这两条带分别是人IL 7基因cDNA第 4外显子 (肝癌组织 )和第 5外显子 (胃癌组织 )缺乏所致。结论　肝癌组织和胃癌组织存在不同选择性IL 7剪接变异体。这些变异体的发现 ,为肿瘤病人的免疫调节紊乱以及肿瘤病人的临床免疫治疗提供了新的研究方向。     

白细胞介素7(IL-7)选择性剪接变异体的筛选和序列测定

目的：寻找人肿瘤细胞选择性的白细胞介素7（Interleukin-7）剪接变异体。方法：运用自行设计的IL-7引物，采用逆转录聚合酶链反应（RT-PCR）技术，分离正常肝组织、原发肝癌组织、正常胃组织和胃癌组织IL-7mRNA，再对电泳所获条带进行克隆、测序。结果：正常肝组织、原发肝癌组织、正常胃组织和胃癌组织都能检测到IL-7mRNA，而且从肝癌组织、胃癌组织中各分离出一条新带，经亚克隆及测序，证实这两条带分别是人IL-7基因eDNA第四外显子（肝癌组织）和第五外显子（胃癌组织）缺乏所致。结论：肝癌组织和胃癌组织存在不同选择性IL-7剪接变异体。这些变异体的发现，为肿瘤病人的免疫调节紊乱以及肿瘤病人的临床免疫治疗提供了新的研究方向。