Molecular cloning and characterization of rat estrogen receptor cDNA.

A cDNA clone of rat uterus estrogen receptor (ER) has been isolated and sequenced. This clone contains a complete open reading frame encoding 600 amino acid residues which is 5 and 11 amino acids larger than the corresponding molecules of human and chicken, respectively. The molecular weight of this protein is calculated to be 67,029. When this clone was ligated to the pSV2 vector and transfected into COS7 cells, a protein was produced that had the same affinity to estrogen as rat uterus ER. This sequence shows 88% homology with human ER; 528 amino acids are identical and 14 amino acids are conservative substitutions. The comparison of rat, human and chicken ER sequences indicate the presence of three highly conserved regions suggesting that these regions play important roles in ER function. The putative DNA-binding domain is completely identical in rat, human and chicken. The C-terminal half region which is thought to be the estrogen binding domain is also highly conserved and is rich in hydrophobic amino acid residues. Southern blot analysis of genomic DNA with ER cDNA as a probe has shown that related sequences are present in the genome.     

cDNA cloning and expression of a novel estrogen receptor beta-subtype in goldfish (Carassius auratus)

We have isolated a second goldfish estrogen receptor (ER) beta-subtype (gfER-beta2) cDNA which is distinct from the liver-derived ER-beta (gfER-beta1) cDNA reported previously. The 2650-bp cDNA, isolated from a goldfish pituitary and brain cDNA library, encodes a 610 amino acid (aa) protein which shows only a 53% aa sequence identity with gfER-beta1 in overall structure. RT-PCR analysis showed that mRNA of gfER-beta2, in contrast to that of gfER-beta1, was predominantly expressed in pituitary, telencephalon and hypothalamus as well as in liver of female goldfish. The existence of a second distinct ER-beta subtype opens new dimensions for studying tissue-specific regulation of gene expression by estrogen in the tetraploid goldfish.     

cDNA cloning and characterization of vinculin mRNA

Vinculin and meta-vinculin are related proteins that localize to adherens junctions. To facilitate studies on the biology of these proteins cDNA clones were isolated from a chick embryonic library made in lambda gt 11. Three clones, accounting for 1/2 of the vinculin message, were identified by immunological analyses of fusion proteins and by hybrid-arrested translation. Sequence analyses of the 3' ends of these clones reveal allelic variants. Northern blot analyses suggests the presence of multiple vinculin messages, the most prominent one at 6.2 kb, almost twice the size needed to encode vinculin.     

Rat KC cDNA cloning and mRNA expression in lung macrophages and fibroblasts.

We have isolated and sequenced overlapping cDNA clones for rat KC*. The 0.93 kb cDNA has a single open reading frame of 288 nucleotides, and substantial sequence identity with the platelet-factor 4 family members mouse KC, hamster gro, and human gro. Using cloned cDNA as a probe, expression of KC mRNA in lavaged rat alveolar macrophages (AMs) increased after lipopolysaccharide (LPS) treatment. We also studied expression in vitro by a rat fetal lung fibroblast cell line, RFL-6. Expression of KC mRNA in RFL-6 cells increased after treatment with interleukin 1 or with conditioned medium from rat AMs treated with LPS.     

Brain-specific expression of transthyretin mRNA as revealed by cDNA cloning from brain

cDNAs for rat transthyretin mRNA were cloned from a brain cDNA library. Sequencing analyses showed the presence of an additional 5' sequence that had not been reported for the liver mRNA corresponding to the flanking promoter region of the gene. This additional sequence was expressed only in the brain, suggesting the presence of a brain-specific promoter.     

Molecular cloning and expression of a cDNA encoding the secretin receptor.

Secretin is a 27 amino acid peptide which stimulates the secretion of bicarbonate, enzymes and potassium ion from the pancreas. A complementary DNA encoding the rat secretin receptor was isolated from a CDM8 expression library of NG108-15 cell line. The secretin receptor expressed in COS cells could specifically bind the iodinated secretin with high and low affinities. Co-expression of the secretin receptor with the alpha-subunit of rat Gs protein increased the concentration of the high affinity receptor in the membrane fraction of the transfected COS cells. Secretin could stimulate accumulation of cAMP in COS cells expressing the cloned secretin receptor. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor consists of 449 amino acids with a calculated Mr of 48,696. The secretin receptor contains seven putative transmembrane segments, and belongs to a family of the G protein-coupled receptor. However, the amino acid sequence of the secretin receptor has no significant similarity with that of other G protein-coupled receptors. A 2.5 kb mRNA coding for the secretin receptor could be detected in NG108-15 cells, and rat heart, stomach and pancreatic tissue.     

cDNA cloning and expression of acutin

Acutin, a thrombin-like enzyme was purified from Agkistrodon acutus venom in three steps by DEAE-Sepharose CL-6B, Superose 12 column on FPLC and Mono-Q column chromatographies. Its first 15 N-terminal amino acid residues sequence was then determined and the acutin cDNA was isolated from venom gland total RNA using RT-PCR. Determination of its nucleotide sequence allowed elucidation of the amino acid sequence of mature peptide for the first time. The mature acutin has 233 amino acids and its amino acid sequence exhibits significant homology with those of thrombin-like enzymes from crotaline snakes venoms. Based on the homology, the catalytic residues and disulfide bridges of acutin were deduced to be as follows: catalytic residues, His41, Asp84 and Ser179; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys231, Cys118-Cys185, Cys150-Cys164, Cys175-Cys200. The recombinant acutin has been expressed in E. coli and purified by affinity column. The renatured recombinant acutin is reported for the first time to have the activity of clotting fibrinogen and arginine-esterase.     

Hepatitis E virus: cDNA cloning and expression.

Viral hepatitis E is endemic, frequently provoking epidemic outbreaks in many developing countries. We have attempted to clone the viral genome and to develop an antibody assay system. A lambda gt11 cDNA library was constructed from the bile juice containing putative causative viruses and was immunoscreened by the antisera obtained from patients and monkeys infected with hepatitis E. Three virus-specific clones were isolated and were revealed to overlap one another in sequence, with 1,459 nucleotides in total length. These clones direct the synthesis of polypeptides probably having common immunological epitope(s). Immunoplaque assay revealed the occurrence of antibodies against this epitope in the sera from experimental monkeys with the convalescent phase and from patients of Myanmar, Nepal and India. The data indicate that the cDNA fragments are useful for immunodiagnosis of hepatitis E.     

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.     

cDNA cloning and mRNA expression of Transformer 2 (Tra 2) in chicken embryo

We report the cloning of a chicken Transformer 2 (Tra 2) cDNA that encodes a protein of 289 amino acids which are 97.9% identical to those of mammalian splicing factor, Tra 2. Tra 2 mRNA was expressed in chicken embryonic tissues and was observed as a band of 1.5 kb by Northern blot analysis. Whole mount in situ hybridization showed an mRNA expression of Tra 2 in telencephalon, mandible, hyoid arch, wing and leg buds as early as day 3.5 of incubation. These results suggest that the Tra 2 gene may play a role in organogenesis in the chicken embryo.     

cDNA cloning and mRNA expression of neuropeptide Y in orange spotted grouper, Epinephelus coioides

A full-length cDNA encoding the neuropeptide Y (NPY) was cloned from the hypothalamus of orange spotted grouper (Epinephelus coioides) by rapid amplification of cDNA ends approaches. The NPY cDNA sequence is 688 bp long and has an open reading frame of 300 bp encoding prepro-NPY with 99 amino acids. The deduced amino acid sequences contain a 28-amino-acids signal peptide followed by a 36-amino-acids mature NPY peptide. mRNA expression of NPY was determined using semi-quantitative RT-PCR followed by Southern blot analysis. NPY mRNA was expressed in olfactory bulb, telencephalon, pituitary, hypothalamus, optic tectum-thalamus, medulla oblongata, cerebellum and spinal cord. Low levels of NPY mRNA expression were found in retina, ovary and stomach, while much lower levels of expression were detected in liver, heart, gill, skin, anterior intestine, thymus and blood. No NPY mRNA expression was observed in unfertilized eggs, newly fertilized eggs, 16-cells stage and morula stage of the embryo and lower levels of expression were detected in the blastula, gastrula and neurula stages. It was highly expressed from lens formation stage to 52-day-old larval stage. NPY might be involved in the late embryonic and larval development of the orange spotted grouper.