Cloning and localization of hFP(S): a six-transmembrane mRNA splice variant of the human FP prostanoid receptor

FP prostanoid receptors are G-protein coupled receptors that mediate the actions of prostaglandin F2alpha. Two isoforms, designated FP(A) and FP(B), have been previously described. We now report the cloning of a FP receptor mRNA alternative splice variant from human heart and placenta cDNA, named hFP(S). The cDNA encoding hFP(S) has a 71 bp insert that produces a frame shift resulting in a truncated receptor lacking transmembrane-7 and the intracellular carboxyl tail. This 71 bp sequence has been identified as a distinct exon localized in the human FP receptor gene on chromosome one. Northern blot analysis suggests that hFPs is expressed in skeletal muscle as well as human heart and placenta. Immunohistochemical microscopy showed positive immunoreactivity on vascular endothelial, trophoblast, and decidual cells from human placenta. hFPs represents the first confirmed alternative splice variant of the human FP prostanoid receptor gene, however, its function is presently unknown.     

Molecular cloning and characterization of a novel human protein phosphatase 2C cDNA (PP2C ε)

We have isolated a novel cDNA from the human fetal brain cDNA library with homology to the Mg2+ -dependent serine/threonine protein phosphatase 2C (PP2C) family. The cDNA is 3055 bp in length, and the predicted coding region encodes a 360-amino-acid protein, which shows 99% identity to the PP2C epsilon from rat and mouse. Then we term it human PP2C epsilon gene. The gene is mapped to chromosome 3q26.1 and contains 4 exons. RT-PCR analysis shows that the PP2C epsilon is widely expressed in human tissues and the expression levels in heart, placenta, lung, liver, kidney, and pancreas are relatively high.     

Expression of CEA-related genes in the first trimester human placenta

Eight cDNA clones, closely related to the carcino-embryonic antigen gene family, have been isolated from a cDNA library representing genes expressed in the first trimester human placenta. Sequence analysis of one clone shows it to be a pregnancy-specific beta 1-glycoprotein (PS beta G) closely related to three other PS beta G cDNA recently characterised from a term placenta library. The protein encoded by the cDNA is predicted to be less high glycosylated than those reported previously and differs markedly in the C-terminal sequence. The 3' untranslated region of the cDNA is very similar to the equivalent region of beta 1-glycoprotein PS beta G E except that it contains the 12bp repeat sequence found flanking the Alu sequence in CEa and an additional 67bp of sequence that appears to be derived from CEA.     

人类核糖体S6激酶RPS6KA5的cDNA克隆及组织表达谱分析

Human ribosomal protein S6 kinase includes two protein families: P90RSK and P70S6K, they participate in two different signaling pathways. When the two kinases were inhibited by their antibodies or rapamycin, the proliferation of cells was arrested. However, their analog, the immunosupressant FK-506, can inhibit the proliferation of fibroblast PBL1 without interfering with the activities of P90RSK, P70S6K and MAPK. We take the tactics of "homolog screening" to demonstrate whether there are some novel proteins which can substitute for the known P90RSK and P70S6K or other pathways without interfering with the known P90RSK and P70S6K. With the conserved sequence of mouse p90RSK as a probe, we screened the homologous sequence in NCBI EST database and got three human EST fragments. With the assembled contig as a probe to screen human brain cDNA library, a full-length cDNA of 3833 bp was attained. It contains a completed open reading frame from 165 bp to 2570 bp encoding 802 amino acids. The putative protein has higher homology with other members of p90RSK family. The gene was named RPS6KA5, the accession number in GenBank is AF090421. Northern hybridization showed the gene expressed in 16 human tissues tested, and the gene was localized in 14q31-32.1 by RH mapping. Another novel P70S6K gene has also been cloned. Thus, our initial presumption that there is an analog of known P90RSK and P70S6K in human beings was proved.     

人类核糖体S6激酶RPS6KA5的cDNA克隆及组织表达谱分析

人类核糖体S6激酶包括两个蛋白家族P90RSK和P70S6K,它们分别介导着两条细胞信号传导通路。当这些激酶活性被它们的抗体或纳巴霉素抑制时,细胞的增殖随之停止。但当纳巴霉素的结构类似物－免疫抑制剂FK－506作用于细胞时,虽可抑制成纤维细胞PBLl的增殖,但却不能抑制P90RSK、P70S6K和MAPK的活性。这提示体内还存在着已知P90RSK和P70S6K蛋白的替代者或还存在着不涉及已知P90RSK和70S6K的信号通路。为此,本文采用“同源筛选”策略,试图证实上述推测。我们以小鼠P90RSK基因的保守性序列为探针,在NCBIEST数据库中进行同源筛选,得到三个人体同源EST片段。以EST片段的整合序列为探针,在人脑组织cDNA文库中进行杂交筛选,最终获得3833bp的全长cDNA序列,其中第165-2570bp为一完整的开放阅读框,编码了802个氨基酸。这个推导蛋白质与人P90RSK家族成员具有较高氨基酸同源性,并被命名为RPS6KA5,它在国际GenBank的登录号为AF090421,Northern杂交显示该基因在人各组织中广泛表达,RH定位将该基因定于14号染色体长臂31-32．1的范围内,另一新的P70S6K基因(GenBank注册登记号为AF037447)也已被克隆,从而证实了人体内存在着已知P90RSK及P70S6K家族基因替代者的最初设想。     

Molecular cloning and nucleotide sequence of the cDNA for sperm-specific lactate dehydrogenase-C from mouse.

Mouse sperm-specific lactate dehydrogenase-C (LDH-C) cDNA was cloned and sequenced from lambda gt11 expression library. The LDH-C cDNA insert of 1236 bp consists of the protein-coding sequence (999 bp), the 5' (54 bp) and 3' (113 bp) non-coding regions, and the poly(A) tail (70 bp). The Northern blot analysis of poly(A)-containing RNAs from mouse testes and liver indicates that the LDH-C gene is expressed in testes but not in liver, and that its mRNA is approx. 1400 nucleotides in length. The nucleotide and amino acid sequences of the mouse LDH-C cDNA show 73% and 72% homologies, respectively, with those of the mouse LDH-A. The Southern blot analysis of genomic DNAs from mouse liver and human placenta indicates the presence of multiple LDH-C gene-related sequences.     

Molecular cloning and characterization of the human S100A14 gene encoding a novel member of the S100 family

S100 proteins form a growing subfamily of proteins related by Ca2+-binding motifs to the Efhand Ca2+-binding protein superfamily. By analyzing a human lung cancer cell line subtraction cDNA library, we have identified and characterized a new member of the human S100 family that we named S100A14 (GenBank acc. no. NM_020672). It encodes a mRNA present in several normal human tissues of epithelial origin, with the highest level of expression in colon. The full-length cDNA is 1067 nt in length, with a coding region predicting a protein of 104 amino acids that is 68% homologous to the S100A13 protein. The deduced amino acid sequence of the human S100A14 and its mouse homolog (identified as GenBank entry) contains two EF-hand Ca2+-binding domains, a myristoylation motif, a glycosylation site, and several potential protein kinase phosphorylation sites. We have mapped this gene to human chromosome 1q21, within a region where at least 15 other S100 genes are tightly clustered. A 3.2-kb genomic fragment containing the entire S100A14 was cloned and sequenced. The gene is split into four exons and three introns spanning a total of 2165 bp of genomic sequence. We examined the intracellular distribution of the epitope-tagged S100A14 protein in two human lung carcinoma cell lines and one immortalized monkey cell line. Pronounced staining was observed in the cytoplasm, suggesting an association with the plasma membrane and in the perinuclear area. We also provide evidence for heterogenic expression of S100A14 in tumors, demonstrating its overexpression in ovary, breast, and uterus tumors and underexpression in kidney, rectum, and colon tumors, a pattern suggesting distinct regulation with potentially important functions in malignant transformation.     

S100P, a novel Ca(2+)-binding protein from human placenta. cDNA cloning, recombinant protein expression and Ca2+ binding properties.

A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/polypeptide chain. The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence. The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85). Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan). The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target. In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension.     

Molecular characterization and tissue distribution of a novel member of the S100 family of EF-hand proteins.

We have isolated from a human prostate cDNA library a cDNA encoding a novel member of the S100 family of EF-hand proteins. The encoded 99-amino acid protein, designated S100Z, is capable of interacting with another member of the family, S100P. S100Z cDNA was cloned into a bacterial expression system, and the S100Z protein was purified to homogeneity from bacterial lysates by a combination of hydrophobic column and gel-filtration chromatography. Direct amino acid sequencing of the 20 N-terminal amino acids confirmed that the sequence of the recombinant protein is identical to the sequence deduced from the cDNA. Low-resolution structural data have been obtained using circular dichroism and fluorescence spectroscopies, and equilibrium analytical centrifugation. These results show that S100Z is a dimeric, predominantly alpha-helical protein. Addition of calcium to a solution of S100Z changes the fluorescence intensity of the protein, indicating that S100Z is capable of binding calcium ions. Analysis of the calcium-binding isotherm indicates the existence of two calcium-binding sites with apparent affinities on the order of 5 x 10(6) and 10(2) M(-1). Binding of calcium results in conformational changes and exposure of hydrophobic surfaces on the protein. Using a PCR-based assay, we have detected differences in the expression level of S100Z mRNA in various tissues. The highest levels were found in spleen and leukocytes. S100Z gene expression appears to be deregulated in some tumor tissues, compared to expression in their normal counterparts.