Composite cDNA Research Articles

Isolation and molecular cloning of epidermal- and hair follicle-specific peptidylarginine deiminase (type III) from rat.

Peptidylarginine deiminase (PAD) is a post-translational modification enzyme that catalyzes deimination of arginine residues of proteins in the presence of calcium ions. There are three types of PAD in rodent tissues: PAD types I, II, and III [Terakawa et al. (1991) J. Biochem. 110, 661-666]. Type III enzyme was detected only in the epidermis and in hair follicles. In this study, we have purified PAD type III from 2-day-old rat epidermis by a four-step procedure that included soybean trypsin inhibitor-affinity chromatography. The enzyme was purified about 600-fold from the crude extract and the recovery was 23%. The final preparation of the enzyme gave only a single protein band on SDS-PAGE and showed an apparent molecular weight of 76,000. Subsequently, we cloned and sequenced the full-length cDNA encoding rat PAD type III by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers designed from the internal amino acid sequences and by the rapid amplification of the cDNA ends method. The composite cDNA sequence contained a 5' untranslated region of 42 bp, an open reading frame of 1,995 bases that encoded 664 amino acids (Mr=75,036), a 3' untranslated region of 1,063 bp, and part of a poly(A)+ tail. The entire reading frame sequence of rat PAD type III showed 51% homology with that of rat PAD type II, and the C-terminal region is highly conserved between the two types. The cloned gene was expressed in Escherichia coli cells to produce PAD type III, which had not only enzymatic activity, but also immunoreactivity against specific antibodies toward PAD type II. Furthermore, the specific expression of the enzyme in the epidermis and hair follicles was confirmed by RT-PCR assays of mRNAs from several tissues.

The Genomic Structure of the Gene Defective in Usher Syndrome Type Ib (MYO7A)

Usher syndrome type Ib is a recessive autosomal disorder manifested by congenital deafness, vestibular dysfunction, and progressive retinal degeneration. Mutations in the human myosin VIIa gene (MYO7A) have been reported to cause Usher type Ib. Here we report the genomic organization of MYO7A. An STS content map was determined to discover the YAC clones that would cover the critical region for Usher syndrome type Ib. Three of the YACs (802A5, 966D6, and 965F10) were subcloned into cosmids and used to assemble a preliminary cosmid contig of the critical region. Part of the gene encoding human myosin VIIa was found in the preliminary cosmid contig. A cosmid, P1, PAC, and long PCR contig that contained the entire MYO7A gene was assembled. Primers were designed from the composite cDNA sequence and used to detect intron–exon junctions by directly sequencing cosmid, P1, PAC, and genomic PCR DNA. Alternatively spliced products were transcribed from the MYO7A gene: the largest transcript (7.4 kb) contains 49 exons. The MYO7A gene is relatively large, spanning approximately 120 kb of genomic DNA on chromosome 11q13.

Molecular cloning and mapping of a human cDNA (PA2G4) that encodes a protein highly homologous to the mouse cell cycle protein p38-2G4.

We have identified a novel human gene with strong homology to the mouse Pa2g4 cell cycle gene. This novel gene (called PA2G4) belongs to a gene family with members in several chromosome regions: 3q24-q25, 6q22, 9q21, 12q13, 18q12, 20p12 and Xq25. A composite cDNA of 1697 nucleotides was isolated. The sequence of this cDNA predicts a protein of 394 amino acids. The deduced amino acid sequence of this human protein shows very strong homology to the mouse protein p38-2G4. The cDNA analyzed probably corresponds to a functional copy found at 12q13.

The Amino Acid Sequence of Rat Kidney 5-Oxo-L-Prolinase Determined by cDNA Cloning

5-Oxoprolinase (EC 3.5.2) catalyzes a reaction in which the endergonic cleavage of 5-oxo-L-proline to form L-glutamate is coupled to the exergonic hydrolysis of ATP to ADP and inorganic phosphate. Highly purified preparations of the enzyme have been obtained from rat kidney and Pseudomonas putida. The rat kidney enzyme is composed of two strongly interacting, apparently identical subunits (Mr = 142,000), whereas that from P. putida is composed of two functionally different protein components that can readily be dissociated. Here we report the cloning of rat kidney 5-oxoprolinase with preliminary expression studies. cDNA clones encoding the enzyme were isolated by screening a lambdagt11 cDNA library beginning with a degenerate oligonucleotide probe based on peptide sequence data obtained from the purified enzyme. The whole cDNA clone was completed by amplifying its 5' end from a premade library of rat kidney Marathon-ReadyTM cDNAs using polymerase chain reaction methodology. The composite cDNA (4,016 bases) revealed an uninterrupted open reading frame encoding 1,288 amino acid residues (Mr = 137,759). The deduced amino acid sequence contains all four of the peptide sequences that were independently found in peptide fragments derived from the enzyme. Expression of the full-length clone in Escherichia coli yielded a product of the same size as the rat kidney enzyme and which reacted with antibodies directed against the rat kidney enzyme. The predicted amino acid sequence is almost 50% identical throughout its entire length to that of a hypothetical yeast protein YKL215C. It is also 26% identical in half its length to the bacterial hydantoinase HyuA and 26% identical in the other half to the bacterial hydantoinase HyuB. The results suggest unexpected evolutionary relationships among the hydantoinases and rat kidney 5-oxoprolinase which share the common property of hydrolyzing the imide bond of 5-membered rings but which do not all require ATP.

Caprine β-Mannosidase: Sequencing and Characterization of the cDNA and Identification of the Molecular Defect of Caprine β-Mannosidosis

The complete sequence of the caprine β-mannosidase cDNA coding region has been determined, and a mutation that is associated with caprine β-mannosidosis has been identified. Reverse transcriptase–polymerase chain reactions were performed using primers based on bovine and, later, goat cDNA sequences to produce an overlapping series of amplicons covering the entire coding region. The composite cDNA codes for an 879-amino-acid peptide that has four potentialN-glycosylation sites. Comparison of the caprine and bovine cDNAs reveals that 96.3% of the nucleotides and 95.2% of the deduced amino acids are identical. A single-base deletion at position 1398 of the coding sequence was identified in the cDNA isolated from a goat affected with β-mannosidosis. This deletion results in a shift in the reading frame and a premature termination of translation, yielding a deduced peptide of 481 amino acids. An assay, developed to determine the presence or absence of this mutation, confirmed that animals affected with β-mannosidosis were homozygous for the mutation and that obligate carriers in a caprine β-mannosidosis colony were heterozygous. This assay accurately distinguished between mutation carrier and noncarrier goats and was used for prenatal diagnosis using DNA collected from fetal fluids. The assay also confirmed chimerism in a goat with an atypically mild β-mannosidosis phenotype. Thus, this application enables assessment of the efficacy of engraftment of hematopoietic stem cells after prenatal transfer from donor sources.

Cloning and Expression of Sialidase L, a NeuAcα2→3Gal-specific Sialidase from the Leech, Macrobdella decora

Sialidase L is a NeuAcalpha2-->3Gal linkage-specific sialidase that releases 2,7-anhydro-NeuAc instead of NeuAc from sialoglycoconjugates (Chou, M.-Y., Li, S.-C., Kiso, M., Hasegawa, A., and Li, Y.-T.(1994) J. Biol. Chem. 269, 18821-18826). A 2. 5-kilobase cDNA of sialidase L was cloned by a combination of methods based on polymerase chain reactions. The composite cDNA sequence reveals an open reading frame coding for 762 amino acids, including a putative 28-residue signal peptide at the N terminus that is similar to the signal sequence of the Clostridium septicum sialidase. The result suggests that sialidase L is a secretory enzyme. The coding sequence excluding the putative signal peptide of sialidase L was overexpressed in Escherichia coli. The purified recombinant enzyme was characterized to be as active as the enzyme isolated from the leech. It also possessed the strict NeuAcalpha2-->3Gal linkage specificity and released the unique cleavage product, 2,7-anhydro-NeuAc from sialoglycoconjugates. The deduced amino acid sequence of sialidase L exhibits little similarity with other reported sialidases. However, sialidase L contains a conserved "FRIP region" and four repeating "Asp box" motifs that align well with the corresponding positions of bacterial sialidases. The predicted beta-strand structures near the conserved motifs of sialidase L are similar to those of Salmonella typhimurium sialidase. Several conserved single amino acid residues of bacterial sialidases, including those known to be involved in the active site of Salmonella enzyme, are conserved in the deduced amino acid sequence of sialidase L. This observation suggests that part of the catalytic mechanism of sialidase L may be similar to the ordinary sialidase.

LPP,the Preferred Fusion Partner Gene ofHMGICin Lipomas, Is a Novel Member of the LIM Protein Gene Family

A major cytogenetic subgroup of lipomas is characterized by recurrent chromosome aberrations, mainly translocations, that involve chromosome segment 12q13–q15. Multiple chromosomes have been found as the translocation partners of chromosome 12 but 3q27–q28 is preferentially involved. In previous studies, it has been shown that the high mobility group (HMG) protein geneHMGICat 12q15 is consistently rearranged as a consequence of these translocations. Here, we report the identification and characterization of the chromosome 3-derived translocation partner gene, which we have designatedLPP(lipoma preferred partner gene). Using 3′-RACE analysis ofHMGICfusion transcripts in lipoma cell line Li-501/SV40, ectopic genetic sequences were obtained, which by CASH (chromosome assignment using somatic cell hybrids) and FISH (fluorescencein situhybridization) analysis were found to originate from chromosome segment 3q27–q28. In Northern blot analysis, an mRNA of over 10 kb was detected by these ectopic sequences in a variety of human tissues but not in brain and peripheral blood leukocytes. Upon partial cDNA cloning, features of the genetic organization ofLPPwere established. The gene was found to span a genomic region of over 400 kb. Nucleotide sequence analysis of a composite cDNA ofLPPrevealed an open reading frame of 1836 nucleotides encoding a proline-rich protein containing a leucine-zipper motif in its amino-terminal region and three LIM domains in its carboxy-terminal region. TheLPP-encoded protein should be classified as a novel member of the group 3 proteins of the LIM protein gene family. Using reverse transcriptase combined with polymerase chain reactions in the analysis of a number of lipoma cell lines and primary lipomas, it appeared thatLPPis frequently rearranged also in cases without a cytogenetically detectable involvement of 3q27–q28. Two alternativeHMGIC/LPPhybrid transcripts have been detected; the difference between them is mainly the presence of either two or three LIM domains in the predicted HMGI-C/LPP fusion proteins.

Isolation of Inositol 1,3,4-Trisphosphate 5/6-Kinase, cDNA Cloning, and Expression of the Recombinant Enzyme

Inositol 1,3,4-trisphosphate 5/6-kinase was purified 12,900-fold from calf brain using chromatography on heparin-agarose and affinity elution with inositol hexakisphosphate. The final preparation contained proteins of 48 and 36-38 kDa. All of these proteins had the same amino-terminal sequence and were enzymatically active. The smaller species represent proteolysis products with carboxyl-terminal truncation. The Km of the enzyme for inositol 1,3,4-trisphosphate was 80 nM with a Vmax of 60 nmol of product/min/mg of protein. The amino acid sequence of the tryptic peptide HSKLLARPAGGLVGERTCNAXP matched the protein sequence encoded by a human expressed sequence tag clone (GB T09063) at 16 of 22 residues. The expressed sequence tag clone was used to screen a human fetal brain cDNA library to obtain a cDNA clone of 1991 base pairs (bp) that predicts a protein of 46 kDa. The clone encodes the amino-terminal amino acid sequence obtained from the purified calf brain preparation, suggesting that it represents its human homologue. The cDNA was expressed as a fusion protein in Escherichia coli and was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity. Remarkably, both the purified calf brain and recombinant proteins produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 2.3-5:1. This finding proves that a single kinase phosphorylates inositol in both the D5 and D6 positions. Northern blot analysis identified a transcript of 3.6 kilobases in all tissues with the highest levels in brain. The composite cDNA isolated contains 3054 bp with a poly(A) tail, suggesting that 500-600 bp of 5' sequence remains to be identified.

CDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt).

We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.

Isolation and characterisation of full-length cDNA clones of the giant taro (Alocasia macrorrhiza) trypsin/chymotrypsin inhibitor

A full-length cDNA encoding the 206 amino acid open reading frame of a trypsin/chymotrypsin inhibitor abundant in the corms of giant taro (Alocasia macrorrhiza) was isolated. An internal fragment was cloned using degenerate primers corresponding to a region of the mature protein sequence and the 'rapid amplification of cDNA ends' (RACE) method used to generate a composite cDNA sequence. The length of the cDNA was close to the predicted size of the corresponding transcript deduced from northern blot analysis of corm mRNA. The inhibitor was expressed strongly in the mature corm, at low levels in leaf blades and petioles but not in roots. Southern blot analysis of the giant taro DNA indicated that this inhibitor is encoded by a small multigene family and this was further supported by the isolation of two different sequence classes from corm cDNA using primers to the ends of the composite sequence.

CDNA Sequence and Gene Locus of the Human Retinal Phosphoinositide-Specific Phospholipase-Cβ4 ( PLCB4)

Defects in the Drosophila norpA (no receptor potential A) gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4 , is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, we isolated several overlapping cDNA clones from a human retina library. The composite cDNA sequence predicts a human PLCβ4 polypeptide of 1022 amino acid residues (MW 117,000). This PLCβ4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLCβ4 monospecific antibody detected a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by fluorescence in situ hybridization.

Molecular Cloning and Characterization of Bovine β-Mannosidase

Deficiency of lysosomal beta-mannosidase activity results in a severe neurodegenerative disease in goats and cattle and a relatively milder phenotype in humans. A cDNA coding for the entire beta-mannosidase protein is described. Mixed oligonucleotides derived from bovine beta-mannosidase peptide sequences were used to screen a bovine thyroid cDNA library. Clones covering about 80% of the C-terminal region were recovered. The missing 5'-region was obtained using the technique of 5'-rapid amplification of cDNA ends. The composite cDNA contains 3852 nucleotides, encoding 879 amino acids. The N-terminal methionine is followed by 16 amino acids displaying the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrates a single 4.2-kilobase transcript in various tissues from both normal and affected goats and calves. The mRNA level is decreased in tissues of affected beta-mannosidosis animals. The gene encoding beta-mannosidase is localized to human chromosome 4 as shown by Southern analysis of rodent/human somatic cell hybrids. This is the first report of cloning of lysosomal beta-mannosidase.

Molecular Cloning, cDNA Sequence, and Localization of a Prohormone Convertase (PC2) from theAplysiaAtrial Gland

Neuropeptides and peptide hormones are synthesized as part of larger precursor proteins that are processed post-translationally by subtilisin-related calcium-dependent prohormone convertases (PCs), frequently at multiple basic sites, to generate biologically active peptides. The atrial gland of Aplysia californica produces large quantities of egg-laying hormone (ELH)-related peptides, providing a unique opportunity to study prohormone processing. We have screened an Aplysia atrial gland cDNA library using a Lymnaea stagnalis PC2 probe and have isolated an Aplysia PC2-related 4.6-kb cDNA partial clone that was truncated on the 5' end. The remaining 5' atrial gland PC2 nucleotide sequence was obtained by reverse transcription/polymerase chain reaction (RT-PCR). The composite cDNA structure (5.6 kb) was deduced from sequence analysis of the RT-PCR product combined with the sequence obtained from the cDNA clone. The deduced cDNA of Aplysia atrial gland PC2 encoded a putative preproendoprotease of 653 amino acids that was evolutionarily related to other eukaryotic PC2s, and showed the strongest sequence identity with recently reported Aplysia nervous tissue PC2 sequences. In situ hybridization demonstrated extensive expression of PC2 in atrial gland secretory cells. The cDNA clone contained a relatively long 3'untranslated region (3'-UTR) of 3,632 nucleotides. Strikingly, the 3'-UTR also contained several major nucleotide repeat sequences including the microsatellite repeats, (CA)n and (TG)n, and a TA-rich region comprised largely of the triplet repeat (TTA)n. The characterized Aplysia PC2 is a candidate endoprotease that may play an important role in the processing of ELH-related precursors in the atrial gland and represents the first example of PC2 expression in exocrine tissue.

Characterization of a cDNA encoding the 43-kDa membrane-associated inositol-polyphosphate 5-phosphatase.

Agonist stimulation of cells results in phosphatidylinositol turnover and the generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular calcium. The inositol-polyphosphate 5-phosphatase (5-phosphatase) enzymes hydrolyze Ins(1,4,5)P3 in a signal-terminating reaction. We have isolated a 2.7-kilobase (kb) composite cDNA, encoding the 43-kDa membrane-associated 5-phosphatase, by screening a human placental lambda gt11 library, using degenerate oligonucleotides. The 2.7-kb cDNA contains a 1.1-kb open reading frame, comprising 363 amino acids, which encodes a protein of a predicted molecular mass of 42 kDa. Amino acid sequence analysis demonstrates a number of potential sites for phosphorylation by protein kinase C and a CAAX motif in the COOH terminus, which may mediate membrane localization. The recombinant enzyme was expressed in COS-7 cells, resulting in a 50-fold increase in enzyme activity in the detergent-soluble membrane fraction of the cell (nanomole of Ins(1,4,5)P3 hydrolyzed per min/mg), but only a 2.5-fold increase in 5-phosphatase activity in the total cell homogenate. Sequence analysis demonstrated a 73-amino acid domain in the COOH terminus of the 43-kDa membrane-associated 5-phosphatase, which had 30% sequence identity and 67% similarity to a region in the 75-kDa 5-phosphatase and 34% identity and 70% similarity to a sequence in the protein that is encoded by the gene, defective in Lowe's oculocerebrorenal syndrome. As shown by RNA analysis the 43-kDa membrane-associated 5-phosphatase appears to be predominantly expressed in heart, brain, and skeletal muscle.

Nucleotide sequence, structure and developmental regulation of LHP82, a juvenile hormone-suppressible hexamerin gene from the waxmoth, Galleria mellonella

We cloned and sequenced a composite cDNA corresponding to the 2.6 kb last instar-specific, juvenile hormone-suppressible Lhp82 mRNA from Galleria mellonella. The identity of the cDNA was confirmed by N-terminal amino acid sequencing of the purified Lhp82 subunit. In addition, we sequenced all coding regions and most of the intronic DNA as well as 1300 nucleotides of 5′ flanking DNA from the Lhp82 gene. The eight exons of the Lhp82 gene specify a pre-protein of 706 residues, including the signal peptide of 18 amino acids. The deduced amino acid sequence of Lhp82 contains four potential N-glycosylation sites, and the calculated isoelectric point and molecular weight of secreted Lhp82 are pI = 6.43 and 79.9 kDa, respectively. Inspection of the 5′ flanking and intronic regions of Lhp82 DNA revealed a 301 nucleotide cassette in intron 6 that appears to be a recently inserted repetitive element. We also performed Northern blot and nuclear run-off transcription experiments in order to determine the basis for Lhp82 gene inactivity after day 2 of the pupal stage. The results of these studies indicate that Lhp82 transcription is permanently shut off by the ecdysteroid pulse which occurs in the absence of juvenile hormone beginning 24 h post-pupation.

Isolation and characterization of XE169, a novel human gene that escapes X-inactivation.

Overlapping cDNA clones for a novel human X-linked gene, XE169, have been isolated and characterized. The composite cDNA sequence comprises 5910 bp (or 5901 bp) plus a poly(A) tail, with a 531 bp 5' and 696 bp 3' untranslated regions. The sequence represents a full-length or near full-length cDNA for the gene since Northern blot analysis reveals only a single prominent band approximately 6 kb in size. Alternative splicing generates two distinct transcripts either containing or missing a stretch of nine nucleotides in the XE169 single large open reading frame, which in turn predict two XE169 protein isoforms composed of 1557 and 1560 amino acids, respectively. Southern hybridization analysis of a panel of human-mouse somatic cell hybrids containing various portions of translocated human X chromosomes has assigned XE169 to the proximal half of the X short arm between Xp21.1 and the centromere. XE169 is expressed in multiple human tissues tested and homologous sequences exist on the human Y chromosome and in the genomes of five other eutherian mammals examined. RT-PCR analysis of somatic cell hybrids containing either an active or an inactive human X chromosome on a rodent background demonstrated that XE169 escapes X-inactivation.

Nucleotide sequence encoding a novel member of the hydratase/dehydrogenase family

The nucleotide sequence encoding a novel member of the family of fatty acid oxidation enzymes has been determined. Clones were generated from porcine gastric corpus cDNA by application of the polymerase chain reaction (PCR) using oligodeoxyri-bonucleotides based on the amino acid sequence of a 78 kDa gastrin-binding protein isolated from porcine gastric mucosal membranes (Baldwin et al., J. Biol. Chem. 261 (1986) 12252–12257). Clones encoding the 3' and 5' ends of the cDNA were then isolated by conventional screening of a porcine liver cDNA library, and by application of anchored PCR, respectively. The composite cDNA of 2744 nucleotides encoded a protein of 763 amino acids, the sequence of which was related to a rat peroxisomal trifunctional enzyme, Δ 3, Δ 2-enoyl-CoA isomerase/enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, and to other members of the same enzyme family. Northern blots indicated that the 3-kb mRNA encoding the novel protein was abundant in gastric corpus and liver, with lower amounts also present in gastric antrum and forestomach.

Molecular cloning and expression of murine liver soluble epoxide hydrolase

A clofibrate-induced mouse liver cDNA library was prepared and used to isolate the coding sequence for soluble epoxide hydrolase. A 1668-base pair (bp) clone was isolated and found to contain a 1269-bp open reading frame coding for 423 amino acids. Subsequent RNA polymerase chain reaction resulted in the isolation of 396 bp of additional 5'-sequence. Translation of the resulting 1659-bp open reading frame produced a 553-residue protein (62,527 Da) containing deduced peptide segments that matched the amino acid sequences of six peptide fragments isolated previously from CNBr digests of pure murine soluble epoxide hydrolase. Neither the DNA nor the protein sequence showed significant similarity to other currently published sequences. Structural analysis of the soluble epoxide hydrolase coding region suggested at least one potential regulatory motif. Expression of the composite cDNA in COS-7 cells resulted in a 5-10-fold increase in soluble epoxide hydrolase activity and a similar increase in soluble epoxide hydrolase protein amount compared to mock-transfected or vector control-transfected cells. Treatment of C57BL/6J mice with clofibrate led to an approximately 4-fold increase in both soluble epoxide hydrolase enzyme activity and steady-state mRNA levels.

CDNA nucleotide sequence and primary structure of mouse uterine peptidylarginine deiminase. Detection of a 3'-untranslated nucleotide sequence common to the mRNA of transiently expressed genes and rapid turnover of this enzyme's mRNA in the estrous cycle.

Peptidylarginine deiminase is a protein-modulating enzyme which converts the arginine residues in proteins to citrulline residues. This study describes the complete primary structure of mouse peptidylarginine deiminase, which was deduced from nucleotide sequence analysis of cDNA clones plus proteochemical analysis of the purified enzyme. The composite cDNA sequence contained a 5' untranslated region of 7 bases, an open reading frame of 2019 bases that encoded 673 amino acids, a 3' untranslated region of 2662 bases, and part of a poly(A) tail. The N-terminal and C-terminal sequences of the enzyme matched the sequences deduced from nucleotide analysis. Furthermore, we determined that the N-terminal sequence was N alpha-acetyl-Met-Gln-, a sequence which has never previously been reported among N alpha-acetyl-Met proteins. The Arg 352 of the enzyme was converted to a citrulline residue and the potential Asn-linked glycosylation site (Asn542-Glu543-Ser544) had no carbohydrate moiety. Thus, mouse peptidylarginine deiminase consists of 673 amino acids with a molecular mass of 76,260. Mouse peptidylarginine deiminase mRNA has two AU-rich structures in the 3' untranslated region which exhibit a high degree of similarity to those in lymphokine, cytokine and proto-oncogene mRNA species. Since the rat enzyme (previously reported) does not possess these characteristic structures, we compared the levels of enzyme activity and mRNA in the mouse and rat uterus at four defined phases of the estrous cycle. The degradation of peptidylarginine deiminase and its mRNA proceeded significantly faster in the mouse than in the rat. We speculate that the unusual structure of the mouse enzyme and its mRNA be involved in this species-specific rapid degradation.

Molecular cloning and expression of RPE65, a novel retinal pigment epithelium-specific microsomal protein that is post-transcriptionally regulated in vitro

Studies reported previously from this laboratory have shown that microsomal membranes of the vertebrate retinal pigment epithelium (RPE) contain an RPE-specific 65-kDa protein, RPE65, which bears the determinant recognized by the strictly tissue-specific monoclonal antibody RPE9, and which is developmentally regulated (Hamel, C. P., Tsilou, E., Harris, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Neurosci. Res. 34, 414-425). Microsequencing of 17 tryptic and chymotryptic peptides obtained from the in situ digestion of the RPE65 blotted on nitrocellulose yielded primary sequences that were used to generate oligonucleotide probes. An 84-nucleotide guessmer was used to isolate two clones from a bovine RPE lambda Zap II cDNA library. Rapid amplification of cDNA ends was used to complete the 5' and 3' ends, resulting in a 3,115-base pair composite cDNA. The open reading frame encodes a novel protein of 533 amino acid residues with a computed molecular weight of 60,940. This protein does not match any other sequence in the data bases. The 231 amino acids obtained from peptide sequencing match 43% of the amino acid sequence deduced from the cDNA. The protein has a calculated pI of 6.41 and is not predicted to have any transmembrane segments. The open reading frame expressed in Escherichia coli has an apparent molecular weight identical to that of the native protein and is recognized by the monoclonal antibody RPE9, further corroborating its validity. Northern blot analysis detected a major mRNA species of 3.15 kilobases for RPE65, as well as shorter species, only in RPE and not in other tissues (including other ocular tissues). Cultured RPE cells (7 weeks in primary culture) contained RPE65 mRNA in amounts equivalent to fresh RPE. Such cells, however, contained no immunodetectable RPE65. The possible structure of this RPE-specific protein and hypotheses for the absence of translation in vitro are discussed.