Functional analysis of the human neurofilament light chain gene promoter.

We have carried out a structural and functional analysis on the human NF-L (H-NF-L) gene. It contains a methylation-free island, spanning the 5' flanking sequences and the first exon and a number of neuronal-specific DNase I hypersensitive sites have been identified in the upstream region as well as within the body of the gene. Analysis in cell lines and transgenic mice using a combination of these sites has revealed the presence of a conserved element(s) between -300bp and -190bp which is required for neuronal-specific expression.     

Role of the N-terminal region of the regulatory light chain in the dephosphorylation of myosin by myosin light chain phosphatase.

Myosin regulatory light chain (RLC) is phosphorylated at various sites at its N-terminal region, and heterotrimeric myosin light chain phosphatase (MLCP) has been assigned as a physiological phosphatase that dephosphorylates myosin in vivo. Specificity of MLCP toward the various phosphorylation sites of RLC was studied, as well as the role of the N-terminal region of RLC in the dephosphorylation of myosin by MLCP. MLCP dephosphorylated phosphoserine 19, phosphothreonine 18, and phosphothreonine 9 efficiently with almost identical rates, whereas it failed to dephosphorylate phosphorylated serine 1/serine 2. Deletion of the N-terminal seven amino acid residues of RLC markedly decreased the dephosphorylation rate of phosphoserine 19 of RLC incorporated in the myosin molecule, whereas this deletion did not significantly affect the dephosphorylation rate of isolated RLC. On the other hand, deletion of only four N-terminal amino acid residues showed no effect on dephosphorylation of phosphoserine 19 of incorporated RLC. The inhibition of dephosphorylation by deletion of the seven N-terminal residues was also found with the catalytic subunit of MLCP. Phosphorylation at serine 1/serine 2 and threonine 9 did not influence the dephosphorylation rate of serine 19 and threonine 18 by MLCP. These results suggest that the N-terminal region of RLC plays an important role in substrate recognition of MLCP.     

Disulfide formation within the regulatory light chain of skeletal muscle myosin

Thiol-disulfide exchange reactions between myosin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) lead to the formation of 5-thio-2-nitrobenzoic acid (TNB)-mixed disulfides as well as to protein disulfide bonds. After incubation with DTNB, myosin was treated with an excess of N-ethylmaleimide (NEM) before electrophoretic analysis of the protein subunits in sodium dodecyl sulfate (SDS) without prior reduction by dithiothreitol (DTT). Without NEM treatment, thiol-disulfide rearrangement reactions occurred in the presence of SDS between the residual free thiols and DTNB. In the absence of divalent metal ions at 25 degrees C, DTNB was shown to induce an intrachain disulfide bond between Cys-127 and Cys-156 of the RLC. This intrachain cross-link restricts partially the unfolding of the RLC in SDS and can be followed as a faster migrating species, RLC'. Densitometric evaluation of the electrophoretic gel patterns indicated that the stoichiometric relation of the light chains (including RLC and RLC') remained unchanged. The two cysteine residues of the fast migrating RLC' were no more available for reaction with [14C]NEM, but upon reduction with DTT, the electrophoretic mobility of the RLC' reverted to that of unmodified RLC and of the RLC modified with two TNB groups. Ca2+ or Mg2+ was able to prevent this disulfide formation in the RLC of myosin by 50% at a free ion concentration of 1.1 X 10(-8) and 4.0 X 10(-7) M, respectively, at 25 degrees C and pH 7.6. Intrachain disulfide formation of RLC never occurred in myosin at 0 degree C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and characterization of human atrial and ventricular myosin alkali light chain cDNA clones

We have isolated essentially full-length cDNA clones for atrial (ALC1) and ventricular (VLC1) myosin alkali light chains from a human fetal heart cDNA library. Comparison of overall nucleotide sequences of ALC1 and VLC1 cDNA clones has revealed that, while these two inserts show significant DNA sequence homology (78.4%) with respect to their coding regions, the 5'- and 3'-untranslated regions are highly divergent. Our statistical analysis suggests that human ALC1 and VLC1 diverged approximately 300 million years ago, during the time of separation of birds and mammals. RNA blot analysis shows that ALC1 mRNA is expressed in fetal ventricular and fetal skeletal muscles as well as fetal and adult atrial muscles and VLC1 mRNA is expressed in adult slow skeletal muscle as well as fetal and adult ventricular muscles. Southern blot analysis indicates that each protein is encoded by a single gene. Finally, we show that VLC1 mRNA is induced in pressure-overloaded human atrium.     

Conserved regulatory elements in the promoter region of the N-CAM gene.

Genomic clones containing 5'-flanking sequences, the first exon, and the entire first intron from the chicken N-CAM gene were characterized by restriction mapping and DNA sequencing. A > 600-bp segment that includes the first exon is very G + C-rich and contains a large proportion of CpG dinucleotides, suggesting that it represents a CpG island. SP-1 and AP-1 consensus elements are present, but no TATA- or CCAAT-like elements were found within 300 bp upstream of the first exon. Comparison of the chicken promoter region sequence with similar regions of the human, rat, and mouse N-CAM genes revealed that some potential regulatory elements including a "purine box" seen in mouse and rat N-CAM genes, one of two homeodomain binding regions seen in mammalian N-CAM genes, and several potential SP-1 sites are not conserved within this region. In contrast, high CpG content, a homeodomain binding sequence, an SP-1 element, an octomer element, and an AP-1 element are conserved in all four genes. The first intron of the chicken gene is 38 kb, substantially smaller than the corresponding intron from mammalian N-CAM genes. Together with previous studies, this work completes the cloning of the chicken N-CAM gene, which contains at least 26 exons distributed over 85 kb.     

拟南芥TCH4基因启动区转录调控元件的计算识别

TCH4基因在植物次生生长、疾病抵抗和逆境适应方面具有重要作用, 能被多种激素、环境和机械信号诱导表达。利用拟南芥TCH4的直系同源基因和芯片数据进行了启动子序列分析, 结果共识别出9个转录调控元件。它们均包含有已知元件序列, 并且在部分共表达基因和对应的直系同源基因启动子中排列顺序一致。根据已有TCH4基因启动子研究, 其中4个已被报道, 另5个为本研究新发现。根据预测结果进行知识整合, 构建了TCH4基因转录调控机制模型。     

Human ventricular/slow twitch myosin alkali light chain gene characterization, sequence, and chromosomal location

The gene coding for the human ventricular/slow twitch myosin alkali light chain isoform was isolated and sequenced. It was found to contain a total of seven exons, the last of which is completely 3'-untranslated sequence. Comparison of this gene sequence with that of the various fast twitch skeletal isoform gene sequences revealed that the exon-intron arrangement is conserved within the myosin alkali light chain gene family. In fact the introns are in exactly the same positions within analogous codons. Comparison of the derived amino acid sequence from the human ventricular/slow twitch isoform gene with that of other isoform protein sequences indicated that the protein encoded by this gene is more homologous to the chicken cardiac isoform protein sequence than to any of the other protein sequences. These results indicate that the gene duplication which gave rise to the ventricular/slow twitch and fast twitch isoform genes must have occurred prior to the divergence of mammals and avians. We have also localized the human ventricular/slow twitch isoform gene to the short arm of human chromosome 3. Interestingly the corresponding mouse gene has been mapped to the distal region of mouse chromosome 9 which contains a conserved syntenic group of genes that map to the short arm of human chromosome 3.     

The functional domains of human ventricular myosin light chain 1

The biological functions of the myosin light chain 1 (LC1) have not been clearly elucidated yet. In this work we cloned and expressed N- and C- terminal fragments of human ventricular LC1 (HVLC1) containing amino acid residues 1-98 and 99-195 and two parts, NN and NC of N fragment in GST-fusion forms, respectively. Using GST pull-down assay, the direct binding experiments of LC1 with rat cardiac G-actin, F-actin and thin filaments, as well as rat cardiac myosin heavy chain (RCMHC) have been performed. Furthermore, the recombinant complexes of rat myosin S1 with N- and C-fragments, as well as the whole molecular of HVLC1 were generated. The results suggested that both binding sites of HVLC1 for actin and myosin heavy chain are positioned in its N-terminal fragment, which may contain several actin-binding sites in tandem. The polymerization of G-actin, the tropomyosin and troponin molecules located in the thin filaments do not hinder the binding of N-terminal fragment of HVLC1 with actin and thin filaments in vitro. The recombinant complex of rat cardiac myosin S1 (RCMS1) with N fragment of HVLC1 greatly decreased actin-activated Mg(2+)-ATPase activity for lack of C fragment. We conclude that the N-fragment is the binding domain of human ventricular LC1, whereas the C-fragment serves as a functional domain, which may be more involved in the modulation of the actin-activated ATPase activity of myosin.     

Cardiac-specific gene expression facilitated by an enhanced myosin light chain promoter

BACKGROUND: Adenoviral gene transfer has been shown to be effective in cardiac myocytes in vitro and in vivo. A major limitation of myocardial gene therapy is the extracardiac transgene expression. METHODS: To minimize extracardiac gene expression, we have constructed a tissue-specific promoter for cardiac gene transfer, namely, the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene, which is known to be expressed in a tissue-specific manner in ventricular myocardium followed by a luciferase (luc) reporter gene (Ad.4 x MLC250.Luc). Rat cardiomyocytes, liver and kidney cells were infected with Ad.4 x MLC.Luc or control vectors. For in vivo testing, Ad.4 x MLC250.Luc was injected into the myocardium or in the liver of rats. Kinetics of promoter activity were monitored over 8 days using a cooled CCD camera. RESULTS: In vitro: By infecting hepatic versus cardiomyocyte cells, we found that the promoter specificity ratio (luc activity in cardiomyocytes per liver cells) was 20.4 versus 0.9 (Ad.4 x MLC250.Luc vs. Ad.CMV). In vivo: Ad.4 x MLC250.Luc significantly reduced luc activity in liver (38.4-fold), lung (16.1-fold), and kidney (21.8-fold) versus Ad.CMV (p =.01); whereas activity in the heart was only 3.8-fold decreased. The gene expression rate of cardiomyocytes versus hepatocytes was 7:1 (Ad.4 x MLC.Luc) versus 1:1.4 (Ad.CMV.Luc). DISCUSSION: This new vector may be useful to validate therapeutic approaches in animal disease models and offers the perspective for selective expression of therapeutic genes in the diseased heart.