Seabream antiquitin: molecular cloning, tissue distribution, subcellular localization and functional expression

Subsequent to our earlier report on the first purification of antiquitin protein from seabream liver and demonstration of its enzymatic activity [FEBS Letters 516 (2002) 183-186], we report herein the cloning of its full-length cDNA sequence. The open reading frame encodes a protein of 511 amino acids. Results of RT-PCR indicate that antiquitin is highly expressed in both the seabream liver and kidney. Transfection studies in cultured eukaryotic cells provided further evidence that it is a cytosolic protein. Bacterial expression of the enzyme was also performed. The purified recombinant protein was demonstrated to exhibit similar kinetic properties as the native enzyme.     

Carp growth hormone: molecular cloning and sequencing of cDNA

cDNA clones of the fish Cyprinus carpio growth hormone (GH) mRNA have been isolated from a cDNA library prepared from carp pituitary gland poly(A)+RNA. The nucleotide sequence of one of the carp GH cDNA clones containing an insert of 1164 nucleotides (nt) was determined. The cDNA sequence was found to encode a polypeptide of 210 amino acids (aa) including a signal peptide of 22 aa and to contain 5' and 3' untranslated regions of the mRNA of 36 and 498 nt, respectively. The carp GH presents a 63% amino acid sequence homology with the salmon GH, has structural features common with other GH polypeptides of mammalian or avian origin and contains domains of conserved sequence near the N- and C-terminal regions. Southern blot hybridization of carp genomic DNA with GH cDNA probes shows the presence of at least two GH-coding sequences in the fish genome.     

Subcellular and developmental expression of alternatively spliced forms of fibroblast growth factor 14

Fibroblast growth factors (FGFs) 11–14 comprise a subfamily of FGFs with poorly defined biological function. Here we characterize two isoforms of FGF14 (FGF14-1a and FGF14-1b) that result from the alternative usage of two different first exons. We demonstrate that these isoforms have differential subcellular localization and that they are differentially expressed in various adult tissues. Using in situ hybridization we show that Fgf14 is widely expressed in brain, spinal cord, major arteries and thymus between 12.5 and 14.5 days of mouse embryonic development. We also show that during cerebellar development, Fgf14 is first observed at postnatal day 1 in post mitotic granule cells, and later in development, in migrating and post migratory granule cells. The developmental expression pattern of Fgf14 in the cerebellum is complementary to that of Math1, a marker for proliferating granule cells in the external germinal layer.     

Tissue-specific expression of two alternatively spliced insulin receptor mRNAs in man

Two previously reported insulin receptor cDNA sequences differ by 36 base pairs (bp) in the distal alpha-subunit, suggesting that alternative mRNA splicing within the coding region may occur (two insulin receptor isoforms). We developed a quantitative modification of the polymerase chain reaction technique in order to detect and characterize differential mRNA splicing at this site within the distal alpha-subunit. Using RNA derived from a variety of human cell types, we detected two polymerase chain reaction-amplified cDNA species reflecting the presence or absence of the above 36 nucleotides. Identity of the two cDNA species was confirmed by Southern blots, the use of a BANI restriction site present only in the 36 base pair segment and dideoxy sequencing. The relative expression of the two mRNA forms varied markedly in a tissue-specific manner. Buffy coat leukocytes and Epstein-Barr virus-transformed lymphocytes express only the shorter mRNA. Placenta expresses both species equally; muscle, isolated adipocytes and cultured fibroblasts express somewhat more of the longer mRNA (relative ratios of mRNA abundance of 1.51, 3.18, and 2.77, respectively); liver expresses mostly the longer mRNA (relative ratio of 9.8). In RNA derived from cultured and fresh cells from patients with several states of insulin resistance, the relative expression of the two mRNA species was similar to results obtained with comparable normal tissues. Although the functional significance of alternative splicing of the insulin receptor mRNA is unknown, differential expression of these two receptor mRNAs may provide a structural basis for previously observed tissue-specific differences in insulin binding and action.     

Molecular cloning and functional characterization of two alternatively spliced Ntcp isoforms from mouse liver1

To isolate the murine Na+/taurocholate cotransporting polypeptide (Ntcp), we screened a mouse liver cDNA library and identified Ntcp1, encoding a 362 amino acid protein and Ntcp2, encoding a 317 amino acid protein which had a shorter C-terminal end. Both isoforms mediated saturable Na+-dependent transport of taurocholate when expressed in Xenopus laevis oocytes. Analysis of the gene revealed that Ntcp2 is produced by alternative splicing where the last intron is retained.     

Porcine growth hormone: molecular cloning of cDNA and expression in bacterial and mammalian cells

Porcine growth hormone (PGH) precursor cDNAs were cloned from a pituitary cDNA library constructed in lambda gt11 by immunoscreening. One of the three clones characterized contained an entire nucleotide sequence for the 216-amino-acid precursor molecule. The deduced amino-acid sequence of PGH confirmed the sequence previously reported for that of the genomic DNA of PGH except for one base difference in the coding sequence. Expression of the full-length PGH cDNA was achieved in bacteria and mammalian cells. The mammalian cell line, COS-1, produced the GH molecule which processed the signal peptide and had the same molecular weight as standard PGH, in contrast to the higher molecular weight of the bacterial product. Radioimmunoassay of the recombinant PGH produced in COS-1 cells also revealed an inhibition curve similar to that of the standard PGH.     

尼罗罗非鱼生长激素及其受体的cDNA克隆与mRNA表达的雌雄差异

尼罗罗非鱼(Oreochromis niloticus)雌雄鱼生长差异明显,为了探讨其原因,本文采用RT-PCR方法克隆了尼罗罗非鱼生长激素(Growthhormone,GH)及其受体(Growth hormone receptor,GHR)的cDNA序列,并应用半定量RT-PCR方法比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达差异。序列分析表明:GH开放阅读框为615bp,共编码204个氨基酸;GHR开放阅读框为1908bp,共编码635个氨基酸。以RT-PCR方法研究了GH、GHR在各组织的分布情况,结果表明:GH仅在垂体中检测到有表达,而GHR在所检测的18种组织中均有表达,其中以肝脏、肌肉、性腺、下丘脑、胸腺表达量较高。以半定量RT-PCR方法进一步比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达量,结果表明:雄鱼垂体GHmRNA和肝脏GHRmRNA的表达量均显著高于雌鱼,肌肉GHRmRNA的表达量则无显著差异,推测垂体GHmRNA和肝脏GHRmRNA表达的雌雄差异是尼罗罗非鱼雌雄生长差异的主要原因之一。     

Follicle-stimulating hormone mediated calcium signaling by the alternatively spliced growth factor type I receptor

Ovarian granulosa cell and testicular Sertoli cell functions are regulated by the tropic action of the pituitary follicle-stimulating hormone (FSH), which may exert pleiotropic effects using a variety of signaling pathways. The effects of FSH on the mobilization of Ca(2+) into granulosa and Sertoli cells have been widely studied, but whether all the effects of the hormone are mediated by the single G-protein-coupled (G(s)) receptor with the seven-transmembrane structure (R1) has remained an enigma. With the object of resolving this mystery, we have compared the hormonal responses of HEK 293 cells transfected with three different cloned FSH receptor cDNAs of testis/ovary, designated R1 (G(s)), R2 (similar to R1 but having a shorter carboxyl terminus), and R3, a novel FSH receptor exhibiting a growth factor type I receptor motif. The latter two that use the same DNA segment for alternative splicing of the single large 80- to 100-kilobase gene create different structural motifs and carboxyl termini. Of the three receptors, only the FSH-R3 type induced a significant rise in intracellular free calcium concentration ([Ca(2+)](i)), as measured by single cell fluorescence digital imaging with the Ca(2+) sensitive dye fura-2AM. FSH induced a rapid [Ca(2+)](i) response that was concentration dependent. The response was hormone-specific, as neither its individual alpha/beta subunits nor the related glycoprotein hormone LH were effective. To determine whether the [Ca(2+)](i) response was due to Ca(2+) influx or to intracellular Ca(2+) mobilization, cells were exposed to Ca(2+)-free buffer and to the Ca(2+)-channel blocker diltiazem (10(-5) M). FSH-Induced [Ca(2+)](i) responses were inhibited in Ca(2+)-free buffer and abrogated in the presence of diltiazem. These novel data demonstrate that FSH can increase [Ca(2+)](i) through L-type voltage-dependent Ca(2+) channels via the growth factor type 1 receptor. Our findings support the concept that different receptor motifs act to integrate intracellular signaling events.     

Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA.

Melanocytes and melanoma cells are known to possess receptors for melanocyte stimulating hormone (MSH). A cDNA clone, designated 11D, has been isolated from human melanoma cells and encodes a MSH receptor. The cloned cDNA encodes a 317 amino acid protein with transmembrane topography characteristics of a G-protein-coupled receptor, but it does not show striking similarity to already published sequences of other G-protein-coupled receptors. When 11D cDNA is expressed in COS-7 cells, it binds an 125I-labelled MSH analogue (NDP-MSH) in a specific manner. The bound ligand could be displaced by melanotropic peptides, alpha-MSH, beta-MSH, gamma-MSH and ACTH (adrenocorticotropic hormone), but not by the non-melanotropic peptide, beta-endorphin. This is the first report of the cloning of the receptor gene of the melanotropin receptor family.     

Complete human NF1 cDNA sequence: two alternatively spliced mRNAs and absence of expression in a neuroblastoma line.

Neurofibromatosis type 1 (NF1) is caused by mutations in a large gene on chromosome 17q11.2. Previously described partial cDNAs for this gene predicted a protein related to yeast IRA1/IRA2 and the mammalian RAS GTPase activator protein GAP. To initiate a detailed study of the role of this gene in NF1, we have characterized a set of overlapping cDNAs that represent its complete coding sequence. Our results show that two differentially expressed human NF1 mRNAs differ by a 63-bp insertion in the GAP-related domain. These mRNAs predict two 2,818- and 2,839-amino acid proteins with calculated molecular masses of approximately 317 and 319 kD. Extensive similarity to IRA proteins is evident in a 1,450-amino-acid central segment, roughly between amino acids 900 and 2,350. However, the remainder of the NF1 protein is not significantly similar to other proteins. Interestingly, the SK-N-SH human neuroblastoma line expresses no detectable NF1 mRNA, indicating that expression of NF1 is not essential for viability of this neural crest-derived tumor cell line.     

Molecular identification and functional expression of mu 3, a novel alternatively spliced variant of the human mu opiate receptor gene

Studies from our laboratory have revealed a novel mu opiate receptor, mu 3, which is expressed in both vascular tissues and leukocytes. The mu 3 receptor is selective for opiate alkaloids and is insensitive to opioid peptides. We now identify the mu 3 receptor at the molecular level using a 441-bp conserved region of the mu 1 receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the mu opiate receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the mu 3 receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the mu 3 receptor cDNA exhibited dose-dependent release of NO following treatment with morphine, but not opioid peptides (i.e., Met-enkephalin). Naloxone was able to block the effect of morphine on COS-1 transfected cells. Nontransfected COS-1 cells did not produce NO in the presence of morphine or the opioid peptides at similar concentrations. Receptor binding analysis with [(3)H]dihydromorphine further supports the opiate alkaloid selectivity and opioid peptide insensitivity of this receptor. These data suggest that this new mu opiate receptor cDNA encodes the mu 3 opiate receptor, since it exhibits biochemical characteristics known to be unique to this receptor (opiate alkaloid selective and opioid peptide insensitive). Furthermore, using Northern blot, RT-PCR, and sequence analysis, we have demonstrated the expression of this new mu variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.