Insect chitin synthase cDNA sequence, gene organization and expression.

Chitin is a major component of the cuticle of arthropods. However, the synthesis of chitin is poorly understood. Feeding larvae of the insect Lucilia cuprina on the fungal chitin synthase competitive inhibitor, nikkomycin Z resulted in strong concentration-dependent mortality of the larvae (LD50 = 280 nM). This result demonstrates that chitin is an essential component of this insect. The complete cDNA and deduced amino-acid sequences of the first arthropod chitin synthase-like protein, LcCS-1, from the larvae of the insect L. cuprina have been determined. The cDNA sequence is 5757 bp in length and codes for a large complex protein containing 1592 amino acids (Mr = 180 717). Analysis of the whole protein sequence reveals low, but significant, similarity to yeast chitin synthases with stronger areas of conservation centred on local regions implicated in the active sites of the yeast enzymes. Strikingly, LcCS-1 contains 15-18 potential transmembrane segments, indicating that the protein is an integral membrane protein. Two alternative topographical models of LcCS-1 are described, which involve its association with either the plasma membrane or the membrane of intracellular vesicles. LcCS-1 mRNA is produced in all life stages of the insect with expression in the larval stage limited to the integument and trachea. In a third instar larva the mRNA was localized to a single layer of epidermal cells immediately underlying the procuticle region of the integument. cDNA or genomic sequences that are highly related to fragments of LcCS-1 were demonstrated in three insect orders, one arachnid and Caenorhabditis elegans, thereby attesting to the importance of this enzyme in these chitin-producing organisms. Bioinformatics has been used to deduce the gene sequence and organization of the highly homologous Drosophila melanogaster orthologue of LcCS-1, DmCS-1.     

Structure-function studies of murine epidermal growth factor: expression and site-directed mutagenesis of epidermal growth factor gene

Wild-type murine epidermal growth factor (mEGF) and mutants with Leu47 replaced by serine and valine, respectively, have been produced by recombinant DNA methodology. A synthetic gene for mEGF was fused to the coding sequence for the signal peptide of the outer membrane protein A (ompA) of Escherichia coli in the secretion vector pIN-III-ompA3, and the recombinant plasmid was used to transform E. coli. Upon induction of gene expression, mEGF and the mutants was expressed and secreted into the periplasmic space. Purification of the wild-type Leu47-mEGF and the mutants was carried out by reversed-phase and anion-exchange high-performance liquid chromatography (HPLC). Amino acid analysis and Western blot analysis further confirmed the identities of the proteins. Specific activities for wild-type and mutant proteins were measured in both mEGF receptor binding and autophosphorylation assays. The recombinant mEGF has specific activities identical with that of mEGF purified from mouse submaxillary glands, while both mutants have reduced specific activities in both bioassays. The data demonstrate the importance of the highly conserved Leu47 residue in mEGF for full biological activity.     

Human tissue factor: cDNA sequence and chromosome localization of the gene

A human placenta cDNA library in lambda gt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, lambda HTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of lambda HTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of approximately 3.2 kilobases in poly(A)+ RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased severalfold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of lambda HTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(A) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 amino acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of epidermal growth factor receptor gene expression

Synthesis and metabolism of the epidermal growth factor (EGF) receptor are extensively regulated to modulate cellular responses to ligand. To study regulation of EGF receptor gene expression, the 5' region of the gene was isolated from a human placental genomic library. A 5' proximal 1.1-kilobase fragment (-1100 to -19 relative to the ATG translation start site) and subfragments of this were subcloned in both forward and reverse orientations into the luciferase expression vector pSVOAL delta 5' and transfected into human cell lines. Luciferase activity was stimulated by treatment of transfected HeLa cells with EGF, 12-O-tetradecanoylphorbol 13-acetate (TPA), (Bu)2 cAMP, retinoic acid, and dexamethasone. Deletion analysis indicated full retention of activity after removal of the -1100 to -485 region (-485 to -19 fragment), but a 5-fold reduction in activity on removal of the -485 to -153 region (-153 to -19 fragment). Despite a reduction in basal activity, the proximal 134-basepair fragment retained responses to all inducers. Additivity was observed in response to maximal concentrations of TPA plus retinoic acid and of TPA plus (Bu)2 cAMP; the response to a combination of four inducers exceeded that to the RSV-LTR strong promoter. Differences in stimulated responses were observed in various recipients, with hepatoma HepG2 cells lacking responses to (Bu)2 cAMP and glioblastoma T98G cells lacking responses to EGF and TPA. These results indicate that a 134-basepair DNA fragment closely adjacent to the translation start site contains elements responsible for directing basal and stimulated expression of the EGF receptor gene.     

Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization.

We have isolated a 1918-bp cDNA from a human adrenal cDNA library which encodes a novel thioredoxin reductase (TrxR2) of 521 amino acid residues with a calculated molecular mass of 56.2 kDa. It is highly homologous to the previously described cytosolic enzyme (TrxR1), including the conserved active site CVNVGC and the FAD-binding and NADPH-binding domains. However, human TrxR2 differs from human TrxR1 by the presence of a 33-amino acid extension at the N-terminus which has properties characteristic of a mitochondrial translocation signal. Northern-blot analysis identified one mRNA species of 2.2 kb with highest expression in prostate, testis and liver. We expressed human TrxR2 as a fusion protein with green fluorescent protein and showed that in vivo it is localized in mitochondria. Removal of the mitochondrial targeting sequence abolishes the mitochondrial translocation. Finally, we determined the genomic organization of the human TrxR2 gene, which consists of 18 exons spanning about 67 kb, and its chromosomal localization at position 22q11.2.     

Identification and expression of epidermal growth factor gene in mouse testis

INTRODUCTIONEpidermalgrowthfactor(EGF)wasinitiallyisolatedandpurifiedfromthesubmaxillarygland(SMG)ofmalemouse[1].Itisapolypeptidecomposedof53aminoacidresidues[2].Itinfluencescellproliferationanddifferentiationandmodulatesthegrowthanddevelopmentofmammalianorgans[3--7].AnoteworthyfindingisthatextirpationofmouseSMGresultsinamarkedreductionofserumEGFconcentrationassociatedwithanimpairedspermatogenesis[3].ThisfindingsuggeststhatEGFmayregulatespermproductionanddifferentiation.Inhumantest…     

Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.     

Regulated expression of the tyrosine hydroxylase gene by epidermal growth factor.

The addition of epidermal growth factor (EGF) to cultures of the rat PCG2 pheochromocytoma cell line increased the level of RNA coding for tyrosine hydroxylase (TH). A region of DNA containing 5'-flanking sequences of the TH gene was fused to a heterologous gene and transfected into a rat anterior pituitary cell line, GH4. The TH gene sequences from +27 to -272 contained information sufficient for the induction of TH by EGF. Two regions within this TH DNA were extensively homologous to the EGF regulatory element of the rat prolactin gene.     

Complete cDNA sequence,expression, alternative splicing,and genomic organization of the mouse Nfat5 gene

Members of the NFAT (nuclear factors of activated T cells) gene family have been investigated in numerous organisms, including man and mouse. All NFATs may be synthesized in several isoforms differing in amino or carboxy termini due to 5' and 3' alternative splicing of the corresponding mRNA. Recently, we mapped the murine Nfat5 gene to chromosome 8D. In the present paper we describe for the first time the complete sequence and primary structure of murine Nfat5, two new spliced isoforms, and the expression of murine Nfat5 in embryonic and adult mouse tissues.