肌球蛋白轻链激酶活性片段对肌球蛋白轻链的非钙依赖性磷酸化作用特征

肌球蛋白轻链激酶 (MLCK)的活性片段 (MLCKF)能比完整的MLCK更有效地、以非钙依赖性的方式磷酸化肌球蛋白轻链 (MLC2 0 )。该片段是用胰蛋白酶水解MLCK ,再经DEAE 5 2柱层析分离而获得的 ,分子量约为 6 1kD。Western印迹已证实该MLCKF与完整的MLCK同源。MLCKF对肌球蛋白轻链的磷酸化作用及其作用特征通过甘油电泳及ScoinImage扫描软件检测 ,肌球蛋白ATP酶活性通过分光光度法检测。实验结果证实 ,MLCKF催化的MLC2 0 非钙依赖性磷酸化 (CIPM)比MLCK催化的CIPM效力高、耗能多 ,但比MLCK催化的MLC2 0 钙依赖性磷酸化 (CDPM)效力低、耗能少 ;MLCKF催化的CIPM与MLCK催化的CIPM均较MLCK催化的CDPM稳定 ,不易受温育温度、温育时间及离子浓度等变化的影响 ,且对MLCK抑制剂ML 9敏感性低。     

Molecular Cloning and Chromosomal Localization to 17q21 of the Human WNT3 Gene

In mouse mammary tumors, the Wnt-3 gene can be activated by proviral insertion. Here we report on the isolation of a human homolog, WNT3. A genomic clone was isolated by use of mouse Wnt-3 sequences as a probe, after which cDNA containing most of the protein-encoding domain of the human gene was obtained by PCR. Comparison between the deduced mouse and human WNT-3 protein sequences showed four changes in 333 amino acids. WNT3 is located on chromosome 17q21. The gene was not found to be amplified or rearranged in a collection of human breast tumors.     

Cloning of the Human Monocarboxylate Transporter MCT3 Gene: Localization to Chromosome 22q12.3–q13.2

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon–intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3–q13.2.     

翘嘴鳜肌球蛋白轻链3b基因(MLC3b)的分子克隆和特征分析

翘嘴鳜以其优良肉质性状近期已成为中国极具商品价值和大力推广的人工养殖名贵鱼类之一。为了解其控制优良肉质性状的遗传基础,本文采用同源克隆RT-PCR方法,获得该鱼肌球蛋白轻链MLC3b基因cDNA序列,该基因cDNA序列为453bp,编码150个氨基酸残基,通过PROSITE tools软件预测显示,该轻链具有两个保守的EF-手相结构和一个C-末端保守序列,且该MLC3轻链N端没有高等脊椎动物特有标志序列。MLC3b基因cDNA序列与MLC3a编码区的同源性达97.7%。本研究结果将为名贵鱼类肉质结构基因及功能的研究提供分子生物学基础。     

人层粘连蛋白α4链LG3组件的克隆、测序及在原核细胞中的表达

用Trizol法提取胎盘组织总RNA,通过RTPCR方法获得人层粘连蛋白α4链LG3组件的特异扩增产物。将PCR产物克隆入PMD18T载体中,PCR与酶切鉴定正确后进行序列测定。用测序正确的克隆片段与原核表达载体PET28a进行体外重组,构建了LG3的原核表达载体,并在BL21(DE3)菌株中得到了表达。     

Isolation of Myosin Light Chain Kinase Inhibitors from Microorganisms: Dehydroaltenusin,Altenusin, Atrovenetinone,and Cyclooctasulfur

Dehydroaltenusin, cyciooctasulfur, atrovenetinone, and altenusin were isolated from the culture broths of Penicillium verruculosum IAM-13756, Streptomyces verticil/us subsp. tsukushiensis ATCC-21633, Penicillium sp. SPC-16375, and Penicillium sp. SPC-16524, respectively, as new myosin light chain kinase (MLCK) inhibitors. These compounds inhibited the calmodulin-dependent activity of MLCK with IC₅₀ values of 0.69, 0.86, 3.7, and 340 μM, respectively. Among them, dehydroaltenusin was the best MLCK inhibitor in terms of potency and selectivity examined in the purified enzyme systems.     

Molecular Cloning, Expression Pattern, and Chromosomal Localization of the Human Na–Cl Thiazide-Sensitive Cotransporter (SLC12A3)

Electrolyte homeostasis is maintained by several ion transport systems. Na–(K)–Cl cotransporters promote the electrically silent movement of chloride across the membrane in absorptive and secretory epithelia. Two kidney-specific Na–(K)–Cl cotransporter isoforms are known, so far, according to their sensitivity to specific inhibitors. We have cloned the human cDNA coding for the renal Na–Cl cotransporter selectively inhibited by the thiazide class of diuretic agents. The predicted protein sequence of 1021 amino acids (112 kDa) shows a structure common to the other members of the Na–(K)–Cl cotransporter family: a central region harboring 12 transmembrane domains and the 2 intracellular hydrophilic amino and carboxyl termini. The ex- pression pattern of the human Na–Cl thiazide-sensitive cotransporter (hTSC, HGMW-approved symbol SLC12A3) confirms the kidney specificity. hTSC has been mapped to human chromosome 16q13 by fluorescencein situhybridization. The cloning and characterization of hTSC now render it possible to study the involvement of this cotransport system in the pathogenesis of tubulopathies such as Gitelman syndrome.     

Chromosomal Localization of the Human Urothelial “Tetraspan” Gene, UPK1B, to 3q13.3–q21 and Detection of aTaqI Polymorphism

The TI1/UPK1b gene codes for a protein of the “tetraspan” family and is expressed as a differentiation product of the mammalian urothelium. A partial genomic clone of the human homologue of the TI1/UPK1b gene was isolated and used as probe to localize the human gene to chromosome 3q13.3–q21 byin situhybridization. Using the same probe, aTaqI restriction fragment length polymorphism, with 29% heterozygosity, was identified by Southern analysis.     

Human Indolethylamine N-Methyltransferase: cDNA Cloning and Expression, Gene Cloning, and Chromosomal Localization

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K_m value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon–intron splice junctions conformed to the “GT-AG” rule, and no canonical TATA or CAAT sequences were present within the 5′-flanking region of the gene. Human INMT mapped to chromosome 7p15.2–p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.     

Localization of human c-mos to chromosome band 8q11 in leukemic cells with the t(8;21) (q22;q22)

Summary Chromosome in situ hybridization studies locate c-mos to chromosome band 8q11 in leukemic cells carrying the t(8;21) (q22;q22). This amends the previous assignment of c-mos to chromosome band 8q22 and conforms with its recent assignment to 8q11 in normal cells and in a cell line with a structurally abnormal chromosome 8. C-mos lies proximally to, and distant from, the breakpoint at 8q22 in the t(8;21) and is unlikely to have a role in the onset of acute myeloid leukemia characterized by this translocation.     

人心肌肌凝蛋白轻链(CMLC)的分离及临床应用

从人心肌提取了肌凝蛋白,并检测了其ATP酶活力的稳定性。用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)法纯化并鉴定了人心肌肌凝蛋白轻链(CMLC),结果表明CMLCI的分子量为27000;CMLCⅡ的分子量为20 000。它们的紫外吸收光谱显示A260>A280,表明其高含苯丙氨酸。制备了兔抗人CMLC抗血清,免疫双扩散的结果表明,纯化后的CMLC与肌凝蛋白有相同的抗原性。用阳性的兔抗人CMLC抗血清为对照作ELISA实验,不仅从免疫学角度进一步证实了人心肌CMLC的特性且为其在临床诊断应用提供了可靠的方法。     

人心肌肌球蛋白轻链1的克隆,表达纯化和单抗制备

报道了中国人心肌肌球蛋白轻链１ｃＤＮＡ的核苷酸序列,并由此推算的氨基酸序列。与国外发表的人心肌肌球蛋白轻链的氨基酸序列比较,发现有两处差异,即在２４位,由谷氨酸变为丙氨酸,则从９８位起至１０１位有４个氨基酸序列的连续差异,即由天冬酰胺－精氨酸－丝氨酸－赖氨酸变为赖氨酸－脯氨酸－精氨酸－谷氨酰妥,推测可能是由于人种差异而引起的。利用该ｃＤＮＡ在大肠杆菌内的表达产物,已获得一株高效的抗中国人心肌肌球蛋     

Cloning, Characterization, and Chromosomal Localization of Human Supervillin (SVIL)

Supervillin is a 205-kDa F-actin binding protein originally isolated from bovine neutrophils. This protein is tightly associated with both actin filaments and plasma membranes, suggesting that it forms a high-affinity link between the actin cytoskeleton and the membrane. Human supervillin cDNAs cloned from normal human kidney and from the cervical carcinoma HeLa S3 predict a bipartite structure with three potential nuclear localization signals in the NH₂-terminus and three potential actin-binding sequences in the COOH-terminus. In fact, throughout its length, the COOH-terminal half of supervillin is similar to segments 2–6 plus the COOH-terminal “headpiece” of villin, an actin-binding protein in intestinal microvilli. A comparison of the bovine and human sequences indicates that supervillin is highly conserved at the amino acid level, with 79.2% identity of the NH₂-terminus and conservation of three of the four nuclear localization signals found in bovine supervillin. The COOH-terminus is even more highly conserved, with 95.1% amino acid identity overall and 100% conservation of the villin-like headpiece. Supervillin mRNAs are expressed in all human tissues tested, but are most abundant in muscle, bone marrow, thyroid gland, and salivary gland; comparatively little message is found in brain. Human supervillin mRNA is ∼7.5 kb; this message is especially abundant in HeLa S3 cervical carcinoma, SW480 adenocarcinoma, and A549 lung carcinoma cell lines. The human supervillin gene (SVIL) is localized to a single chromosomal locus at 10p11.2, a region that is deleted in some prostate tumors.     

Translocation (3;12) (p21-pter; q24.1-qter) and phenylketonuria

Cytogenetic karyotyping in mental retardation associated with physical dysmorphism has been regarded as the primary key for the classification of syndromes and other genetic disorders for the predisposition of neoplasia and other fatal diseases. Giemsa-banding of metaphase chromosomes in lymphocytes is a traditional and routine process for the identification of the chromosomal counterpart which can provide a clue for molecular investigation in the subject. An 8-year-old girl showed a diploid karyotype 46, XX, t(3;12) (p21-pter, q24.1-qter) in peripheral blood lymphocyte culture. Biochemical examination of urine labelled her as a case of phenylketonuria. The maternal karyotyping was similar and confirmed the maternal transmission of the translocation.     

Cloning, Expression, and Physical Mapping of the 3β-Hydroxysteroid Dehydrogenase Gene Cluster (HSD3BP1–HSD3BP5) in Human

Seven members of the human 3β-hydroxysteroid dehydrogenase (3β-HSD) gene family (HGMW-approved symbols HSD3BP1–HSD3BP5) have been cloned and physically mapped. HSD3B1 and 2 express 3β-HSD enzymes; HSD3Bψ1–5 are unprocessed pseudogenes that are closely related to HSD3B1 and 2 but contain no corresponding open reading frames. mRNA is expressed from ψ4 and ψ5 in several tissues, but with altered splice sites that disrupt reading frames. A 0.5-Mb contig of 3 yeast artificial chromosome and 32 bacterial artificial chromosome genomic clones contained no additional members of the gene family. The seven genes and pseudogenes mapped within 230 kb in the order HSD3Bψ5–ψ4–ψ3–HSD3B1–ψ1–ψ2–HSD3B2. HSD3B1 and 2 are in direct repeat, 100 kb apart. Six HSD3B2 mutations involve substitutions that are present in several of the pseudogenes. In four cases, mutations arose in CpG sites that are conserved within the gene cluster. The tendency for CpG sites to mutate by transition provides an adequate explanation for these HSD3B2 mutations, which are unlikely to be due to recombination or conversion within the gene family.     

Cloning of the Human Homolog of Conductin (AXIN2), a Gene Mapping to Chromosome 17q23–q24

Conductin or Axil, an Axin homolog, plays an important role in the regulation of β-catenin stability in the Wnt signaling pathway. To facilitate the molecular analysis of the human gene, we isolated the human homolog, AXIN2. The cDNA contains a 2529-bp open reading frame and encodes a putative protein of 843 amino acids. Compared with rat and mouse homologs, AXIN2 shows an overall 89% amino acid identity. Several functional domains in this protein are highly conserved including the GRS (95.9%), GSK-3β (96.3%), Dsh (98%), and β-catenin (89.9%) domains. Radiation hybrid mapping localized the AXIN2 gene to human chromosome 17q23–q24, a region that shows frequent loss of heterozygosity in breast cancer, neuroblastoma, and other tumors. Human AXIN2 is thus a very strong candidate involved in multiple tumor types.