Molecular cloning and characterization of a novel human kinase gene,PDIK1L

We isolated a 4301-bp cDNA from a human foetal brain cDNA library by high-throughput cDNA sequencing. It encodes a protein of 341 amino acids, which shows 69% identity with the human kinase CLIK1 (AAL99353), which was suggested to be the CLP-36 interacting kinase. Bioinformatics analysis suggests that the putative kinase may interact with PDZ and LIM domain proteins. Therefore the protein and its cDNA were named ’PDLIM1 interacting kinase 1 like’ (PDIK1L; nomenclature approved by the HUGO Gene Nomenclature Committee). Ensembl Genome Browser locatedPDIK1L to human chromosome 1p35.3. It spans about 13.7 kb and consists of four exons and three introns. Multiple-tissue cDNA panel PCR revealed that the gene is expressed widely in human tissues: liver, kidney, pancreas, spleen, thymus and prostate. The protein appears to be localized to the nucleus.     

Molecular cloning and expression of a novel adhesion molecule, SC1

SC1, an integral membrane glycoprotein of 100 kd, is uniquely and transiently expressed on spinal cord motoneurons early in development and appears in peripheral neurons and several other tissues during development. SC1 has been purified by immunoaffinity techniques, and SC1 cDNA clones have been obtained by screening an E4 chick embryo phage expression library with a rabbit polyclonal antibody produced against purified SC1. The deduced protein sequence of 588 amino acids consists of a signal peptide, five immunoglobulin-like domains, a transmembrane region, and a short cytoplasmic tail. The sequence is most similar to MUC18, reported as a tumor progression marker in human melanoma. Transfection of SC1 cDNA into mammalian cells leads to cell surface expression of SC1 antigen and a subsequent increase in cell-cell adhesion. SC1 molecules bind to each other via a homophilic adhesion mechanism, independently of calcium or magnesium ions. SC1 may have a role in lateral motor column formation or neurite growth or fasciculation.     

Molecular cloning and characterization of a novel cystatin-like molecule,CLM, from human bone marrow stromal cells

The cystatins are physiological cysteine proteinase inhibitors. Here we report the cloning of a novel human cystatin-like molecule (CLM) from human bone marrow stromal cell (BMSC) cDNA library. The putative CLM protein contained 159 residues with a 29-residue signal peptide. CLM protein was highly homologous to family 2 cystatins, especially mouse and human testatin. The CLM gene spanned two exons and was mapped on chromosome 20p11.2, among cystatin superfamily gene clusters. CLM mRNA was barely detected in most tumor cell lines except for breast adenocarcinoma MCF-7 cells and glioblastoma U251 cells, but after LPS or PMA stimulation, CLM expression was increased in myelogenous leukemia cell lines HL-60 and U-937. Northern blot analysis revealed CLM was ubiquitously expressed in normal tissues, which was clearly different from the testis-specific expression pattern of most family 2 cystatins. When overexpressed in 293 cells, GFP-fused CLM targeted extracellularly through secretory pathway by Golgi apparatus. The results indicated that the secreted CLM protein might play roles in hematopoietic differentiation or inflammation.     

Molecular cloning, expression and characterization of the zebrafish bram1 gene, a BMP receptor-associated molecule

Summary We have identified a cDNA clone encoding BMP receptor-associated molecule 1 (BRAM1) from the zebrafish expressed sequence tag (EST) database. The 2606 bp full-length bram1 cDNA was cloned, and further confirmed by nucleotide sequencing. The zebrafish sequence encodes a protein of 195 amino acids with an evolutionarily conserved MYND domain, which displays ∼ ∼98% homology with human and mouse BRAM1, and ∼ ∼64% homology with C. elegans BRA-1 and BRA-2. The bram1 gene, composed of five exons and four introns, spans ∼ ∼14 kb on linkage group 14 of the zebrafish genome. RT-PCR and whole mount in situ hybridization analyses disclosed that zebrafish BRAM1 is a maternal factor. The protein interacts directly with zebrafish BMP Receptor type IA, as observed from GST-pull down and co-immunoprecipitation assays. Furthermore, cotransfection of zebrafish BRAM1 with the corresponding BMP receptor resulted in down-regulation of BMP-mediated signaling. Our results collectively indicate that BRAM1 plays a biological role during zebrafish development.     

Molecular cloning and characterization of a novel cbl-family gene, cbl-c

We have cloned a novel gene, cbl-c, of mammalian cbl-family. The cbl-c gene is predicted to encode a protein of 52 kDa that has a phosphotyrosine-binding domain, a RING finger and a proline-rich region. Cbl-c shows 50% homology to the amino-terminal sequences of Cbl and Cbl-b, but a sequence corresponding to the carboxy-terminal half of Cbl and Cbl-b is largely missing in Cbl-c. The expression of cbl-c mRNA is distinct from that of cbl and cbl-b mRNAs, being high in the colon and small intestine, but undetectable in brain and lymphoid tissues. The cbl-c gene is mapped in 19q13.2-13.3. Finally, the 52 kDa Cbl-c protein binds to the EGF receptor and Fyn tyrosine kinase. We conclude that Cbl-c is a novel Cbl-family adaptor protein that would regulate intracellular signaling mediated by various tyrosine kinases.     

Molecular cloning and characterization of LKv1, a novel voltage-gated potassium channel in leech

We have cloned a novel voltage-gated K channel, LKv1, in two species of leech. The properties of LKv1 expressed in transiently transfected HEK293 cells is that of a delayed rectifier current. LKv1 may be a major modulator of excitability in leech neurons, since antibody localization studies show that LKv1 is expressed in the soma and axons of all neurons in both the central and peripheral nervous systems. Comparison of the biophysical and pharmacological properties of LKv1 with native voltage-gated conductances in leech neurons suggests that LKv1 may correspond to the previously characterized delayed rectifier current, I(K). Phylogenetic analysis of LKv1 shows that it is related to the Shaker subfamily of voltage-gated K channels although it occupies a separate branch from that of the monophyletic Shaker clade composed of the flatworm, Aplysia, Drosophila, and mammalian Shaker homologs as well as from that of two recently identified Shaker-related K channels in jellyfish. Thus, this analysis indicates that this group of voltage-gated K channels contains several evolutionarily divergent lineages.     

Molecular cloning and characterization of a cDNA for a novel ethylene receptor, NT-ERS1, of tobacco (Nicotiana tabacum L.)

The cDNA encoding a novel member (NT-ERS1) of ethylene receptor family of tobacco (Nicotiana tabacum L.) was obtained by a combination of RT-PCR and 5'-/3'-RACE cloning. The cDNA was 2,092 nucleotides long and had an open reading frame of 1,911 bp encoding 637 amino acids. The deduced polypeptide lacked a response regulator domain, indicating that the ethylene receptor belongs to an ERS-group. The amino acid sequence was similar to respective members of the tobacco ethylene receptor family: 67.8% to NT-ETR1, 39.1% to NTHK1 and 31.1% to NTHK2. Comparison of amino acid sequence suggested that NT-ERS1 is the counterpart of Nr in the ethylene receptor family of tomato, which belongs to Solanaceae as does tobacco. Northern blot analysis showed that mRNA of NT-ERS1 was present in leaf, shoot and root tissues, and accumulated in leaves treated with exogenous ethylene. A mutated NT-ERS1 cDNA transgene, obtained by introducing one nucleotide substitution into NT-ETR1 cDNA, conferred ethylene insensitivity in tobacco plants, indicating that the translation product of the cDNA actually functioned in the plants.     

Molecular cloning and characterization of a novel mammalian endo-apyrase (LALP1)

Here we describe the cloning, localization, and characterization of a novel mammalian endo-apyrase (LALP1) in human and mouse. The predicted human LALP1 gene encodes a 604-amino acid protein, whereas the mouse Lalp1 gene encodes a 606-amino acid protein. The human and mouse genes have 88% amino acid sequence identity. These genes share considerable homologies with hLALP70, a recently discovered mammalian lysosomal endo-apyrase. The human LALP1 gene resides on chromosome 10q23-q24 and contains 12 exons and 11 introns covering a genomic region of approximately 46 kilobase pairs. The subcellular localization and enzymatic activity of LALP1 indicated that LALP1 is indeed an endo-apyrase with substrate preference for nucleoside triphosphates UTP, GTP, and CTP.     

Molecular cloning and characterization of Kremen, a novel kringle-containing transmembrane protein

Kringle domain, a triple-disulfide-linked domain, is conserved in diverse proteins which play important roles in various biological processes. We cloned Kremen, a novel member of kringle-containing proteins, using a newly developed unique strategy, 'Kringle-SAGE (serial analysis of gene expression)', which enables comprehensive analysis of kringle-containing proteins. Kremen is likely to be a type-I transmembrane protein composed of 473 amino acid residues. Kremen has a kringle domain, a WSC domain, and CUB domains in the extracellular region, while the intracellular region has no conserved motif involved in signal transduction. In the mouse embryo, the Kremen mRNA level, which was increased during embryonic development, was localized in the apical ectodermal ridge of limb buds, myotome, and sensory organs (e.g. optic vesicle, otic vesicle, nasal pit). In the adult mouse, Kremen mRNA was expressed in a variety of tissues with a relatively strong expression in the lung, heart, and skeletal muscle. Kremen mRNA expression in C2C12 and NIE-115 cells increased during respective differentiation into muscular and neural cells. These results suggest a potential role for Kremen in the regulation of cellular responses upon extracellular stimulus or cell-cell interaction in neuronal and/or muscle cells. Kringle-SAGE is expected to facilitate further elucidation of structure and functions of kringle proteins.     

Molecular cloning and characterization of a novel anti-TLR9 intrabody

Toll-like receptor 9 (TLR9) is a component of the innate immune system, which recognizes the DNA of both pathogens and hosts. Thus, it can drive autoimmune diseases. Intracellular antibodies expressed inside the ER block transitory protein functions by inhibiting the translocation of the protein from the ER to its subcellular destination. Here, we describe the construction and characterization of an anti-TLR9 ER intrabody (αT9ib). The respective single-chain Fv comprises the variable domains of the heavy and light chain of a monoclonal antibody (mAb; 5G5) towards human and murine TLR9. Co-expression of αT9ib and mouse TLR9 in HEK293 cells resulted in co-localization of both molecules with the ER marker calnexin. Co-immunoprecipitation of mouse TLR9 with αT9ib indicated that αT9ib interacts with its cognate antigen. The expression of αT9ib inhibited NF-κB-driven reporter gene activation upon CpG DNA challenge but not the activation of TLR3 or TLR4. Consequently, TLR9-driven TNFα production was inhibited in RAW264.7 macrophages upon transfection with the αT9ib expression plasmid. The αT9ib-encoding open reading frame was integrated into an adenoviral cosmid vector to produce the recombinant adenovirus (AdV)-αT9ib. Transduction with AdVαT9ib specifically inhibited TLR9-driven cellular TNFα release. These data strongly indicate that αT9ib is a very promising experimental tool to block TLR9 signaling.     

宏基因组来源新过水解酶的分子克隆与酶学特性

旨在获得具有氯化功能的过水解酶,拓展过水解酶资源,为其工业应用奠定基础。以唐河某造纸厂污泥为材料构建宏基因组文库,通过活性筛选获得一个细菌过水解酶Per822。使用大肠杆菌异源表达Per822,研究纯化后的重组蛋白酶学性质并检测了生成过乙酸的能力。测序结果显示per822编码一个含273个氨基酸的蛋白。Per822是典型的多功能酶代表,分别具有过氧化物酶、卤代酶和酯酶的活性。Per822过水解氯化单氯二甲酮的最适反应pH为4.5,在pH3.5–8.0范围内酶活性稳定。最适反应温度是55℃,在70℃以下酶活性稳定且氯化活性能够被Fe2+激活。以乙酸乙酯为共底物Per822显示出较强的产过乙酸能力。重组Per822的高可溶性表达、催化多功能性、较强的产过乙酸能力使得Per822在有机合成、废水处理、杀菌、生物质预处理等方面有着潜在的应用价值。     

Molecular cloning and characterization of a novel mouse betaPix isoform

BetaPix, a Pak-interacting guanine nucleotide exchange factor is known to be involved in the regulation of Cdc42/Rac GTPases and Pak kinase activity. Currently, three 1Pix isoforms, betaPix-a, -b, and -c have been reported. In this study, the cDNA of a novel Pix splice variant was isolated from a mouse brain cDNA library. The cloned betaPix isoform, named betaPix-d, lacks leucine zipper domain that is present in other Pix isoforms, and has a 11 amino acid addition at carboxyl terminus and distinct 3'-UTR Analysis of the tissue distribution of betaPix-d using RT-PCR revealed that its message was present mainly in brain and testis but in lower levels in heart, spleen, lung, liver, skeletal muscle and kidney. In situ hybridization studies with the 13Pix-d specific probes in the rat embryo show that betaPix-d isoform is expressed mainly in the central nervous system. Moreover, temporal expression pattern of the isoform is correlated with the active neurogenesis period in the cerebral cortex and cerebellum during rat brain development. These findings suggest that betaPix-d isoform may be developmentally regulated.     

Molecular cloning and developmental expression of a zebrafish axonal glycoprotein similar to TAG-1

TAG-1 is a mammalian cell adhesion molecule of the immunoglobulin superfamily that is expressed transiently by a subset of neurons and serves as a fertile substrate for neurite outgrowth in vitro (Furley, A.H., Morton, S.B., Manalo, D., Karagogeos, S., Dodd, H., Jessell, T.M., 1990 The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth promoting activity. Cell 61, 157–170). In order to examine the in vivo function of this molecule, we have cloned a zebrafish tag1-like cDNA and analyzed its expression patterns. tag1 is expressed transiently by specific subsets of neurons when they are projecting their axons or when they are migrating. The specific and dynamic pattern of expression of zebrafish tag1 is consistent with its proposed role in axon guidance and cell migration.