cDNAs of aminopeptidase-like protein genes from Plodia interpunctella strains with different susceptibilities to Bacillus thuringiensis toxins

Aminopeptidase N has been reported to be a Bacillus thuringiensis (Bt) Cry1A toxin-binding protein in several lepidopteran insects. cDNAs of aminopeptidase-like proteins from both Bt-susceptible RC688s and Bt-resistant HD198r strains of the Indianmeal moth, Plodia interpunctella, were cloned and sequenced. They contain 3345 and 3358 nucleotides, respectively, and each has a 3048 bp open reading frame that encodes 1016 amino acids. Putative protein sequences include 10 potential glycosylation sites and a zinc metal binding site motif of HEXXH, which is typical of the active site of zinc-dependent metallopeptidases. Sequence analysis indicated that the deduced protein sequences are most similar to an aminopeptidase from Heliothis virescens with 62% sequence identity and highly similar to three other lepidopteran aminopeptidases from Plutella xylostella, Manduca sexta, Bombyx mori with sequence identities of 51-52%. Four nucleotide differences were observed in the open reading frames that translated into two amino acid differences in the putative protein sequences. Polymerase chain reaction (PCR) confirmed an aminopeptidase gene coding difference between RC688s and HD198r strains of P. interpunctella in the PCR amplification of a specific allele (PASA) using preferential primers designed from a single base substitution. The gene mutation for Asp185-->Glu185 was also confirmed in two additional Bt-resistant P. interpunctella strains. This mutation is located within a region homologous to the conserved Cry1Aa toxin binding regions from Bombyx mori and Plutella xylostella. The aminopeptidase-like mRNA expression levels in the Bt-resistant strain were slightly higher than those in the Bt-susceptible strain. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF034483 for susceptible strain RC688s and AF034484 for resistant strain HD198r).     

Complete cDNA and gene sequence of the developmentally regulated arylphorin of Calliphora vicina and its homology to insect hemolymph proteins and arthropod hemocyanins.

Two cDNA libraries were prepared from poly(A)+ RNA isolated from fat bodies of last instar larvae of the blowfly Calliphora vicina. The libraries were probed with a genomic clone containing the coding sequence for an arylphorin subunit. Two cDNA clones as well as the genomic clone were mapped and their nucleotide sequences were determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 759 amino acid residues. The deduced primary structure of Calliphora arylphorin and hemolymph proteins of other insect species and arthropod hemocyanine show nearly 30% identity. Highly conserved regions could be also identified.     

Sequence analysis and comparison of avocado fruit and bean abscission cellulases

A 1700 nucleotide cDNA clone for a bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase (endo-(1,4)-β-d-glucanase) has been identified and sequenced. This cDNA clone contains a 1485 nucleotide open reading frame which includes coding sequences for a putative signal peptide and mature protein. The nucleotide and deduced amino acid sequences for the bean abscission cellulase are compared to the previously reported sequences of an avocado fruit ripening cellulase. Optimal alignment of these sequences shows 64% and 50% identically matched nucleotides and amino acids, respectively. Analysis of the deduced amino acid sequences for the mature bean and avocado cellulases indicates that these two proteins share similar molecular weights, position of cysteine residues, and hydropathic character, but have very different isoelectric points and glycosylation. Genomic blot data suggest that the avocado fruit cellulase belongs to a small gene family, whereas the bean abscission cellulase appears to be encoded by a single gene or a few very closely related genes.     

Molecular cloning of ribosomal protein L26 (RPL26) cDNA from Ailuropoda melanoleuca and its potential value in phylogenetic study

The cDNA fragment of ribosomal protein L26 (RPL26) was cloned from Ailuropoda melanoleuca using RT-PCR method. The cDNA fragment is composed of 475 bp, containing an open reading frame of 145 amino acids. Alignment analyses indicated that the nucleotide sequence and the deduced amino acid sequence showed high identity to other known RPL26 sequences from vertebrates and invertebrates. The cDNA sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate RPL26 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of RPL26 gene in phylogenetic analysis.     

Molecular cloning of rabbit cytochrome b5 genes: evidence for the occurrence of two separate genes encoding the soluble and microsomal forms.

The rabbit genomic segments for the soluble cytochrome b5 (b5) and microsomal b5 were amplified and isolated, respectively, by means of the polymerase chain reaction using primers corresponding to various portions of the open reading frame of microsomal b5 cDNA. The DNA sequence analysis revealed that the soluble b5 gene has an extra 24 nucleotide long insert which encodes a C-terminal amino acid and a termination codon which are specific to the soluble b5. Except for the insert, the sequences of the soluble and microsomal b5 genes are identical with each other from the 5' end to the 3' end of the open reading frame of the microsomal b5 cDNA. Comparison of the genomic sequences with the cDNA sequences suggested that the soluble and microsomal genes are intronless within their open reading frames. These data indicate that rabbit soluble and microsomal b5 mRNAs are encoded by two highly conserved but separate genes.     

Identification of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodiesterase family

Alkaline sphingomyelinase (alk-SMase) hydrolyzes dietary sphingomyelin and generates sphingolipid messengers in the gut. In the present study, we purified the enzyme, identified a part of the amino acid sequence, and found a cDNA in the GenBank coding for the protein. The cDNA contains 1841 bp, and the open reading frame encodes 458 amino acids. Transient expression of the cDNA linked to a Myc tag in COS-7 cells increased alk-SMase activity in the cell extract by 689-fold and in the medium by 27-fold. High activity was also identified in the anti-Myc immunoprecipitated proteins and the proteins cross-reacted with anti-human alk-SMase. Northern blotting of human intestinal tissues found high levels of alk-SMase mRNA in the intestine and liver. The amino acid sequence shared no similarity with acid and neutral SMases but was related to the ecto-nucleotide phosphodiesterase (NPP) family with 30-36% identity to human NPPs. Alk-SMase has a predicted signal peptide domain at the N terminus and a signal anchor domain at the C terminus. The ion-binding sites and the catalytic residue of NPPs were conserved, but the substrate specificity domain was modified. Alk-SMase had no detectable nucleotidase activity, but its activity against sphingomyelin could be inhibited by orthovanadate, imidazole, and ATP. In contrast to NPPs, alk-SMase activity was not stimulated by divalent metal ions but inhibited by Zn2+. Differing from NPP2, the alk-SMase cleaved phosphocholine but not choline from lysophosphatidylcholine. Phylogenetic tree indicated that the enzyme is a new branch derived from the NPP family. Two cDNA sequences of mouse and rat that shared 83% identity to human alk-SMase were identified in the GenBank. In conclusion, we identified the amino acid and cDNA sequences of human intestinal alk-SMase, and found that it is a novel ecto-enzyme related to the NPP family with specific features essential for its SMase activity.     

Molecular cloning of rabbit CAP-50, a calcyclin-associated annexin protein.

CAP-50 is a member of annexin family proteins which binds specifically to calcyclin in a Ca2+ dependent manner (Tokumitsu. H., Mizutani. A., Minami. H., Kobayashi. R., and Hidaka. H. (1992) J. Biol. Chem. 267,8919-8924). The cDNA representing the rabbit form of this protein has been cloned from rabbit lung cDNA library. Sequence analysis of two overlapping clones revealed a 81-nucleotides 5'-nontranslated region, 1512-nucleotides of open reading frame, a 672-nucleotides 3'-nontranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of portions of the deduced amino acid sequence with eight sequences of proteolytic peptides obtained from rabbit lung protein. CAP-50 cDNA encodes a 503 residue protein with a calculated M(r) of 54,043 and shows that the protein is composed of four imperfect repeats and hydrophobic N-terminal region. C-terminal region including four imperfect repeats shows 58.1% identity with human synexin (annexin VII), 48.0% identity with annexin I, 47.4% identity with annexin II, 60.1% identity with annexin IV, 54.5% identity with annexin V. Hydrophobic N-terminal region composed of 202 amino acid residues is not homologous with other annexin proteins suggesting that CAP-50 is a novel member of annexin family proteins.     

Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex

We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.     

The C1orf9 gene encodes a putative transmembrane member of a novel protein family

Here we report the characterization of a human mRNA encoding a novel protein denoted C1orf9 (chromosome 1 open reading frame 9). The cDNA sequence, derived from a testis cDNA library, contains 5700 bp which encodes an open reading frame of 1254 amino acids. The deduced protein contains a putative N-terminal signal peptide and one putative transmembrane region, indicating membrane localization. No significant homology was found with known characterized proteins. However, a 150 amino acid region has significant homology to deduced protein sequences from other organisms, including Caenorhabditis elegans (43% identity), Saccharomyces cerevisiae (47% identity), Schizosaccharomyces pombe (48% identity), and two proteins from Arabidopsis thaliana (42% and 40% identity), suggesting a novel family of conserved domains. The C1orf9 gene was assigned to chromosome 1q24. The gene spans approximately 78.7 kb and is organized into at least 24 exons. Expression analysis revealed a single C1orf9 mRNA species of approximately 6.0 kb with a predominant expression in pancreas and testis, and only low levels of expression in other tissues examined.     

Different mechanisms of resistance to Bacillus thuringiensis toxins in the indianmeal moth

Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of (125)I-labeled Cry1Ab and Cry1Ac has been examined in three Plodia interpunctella colonies, one susceptible (688(s)) and two resistant (198(r) and Dpl(r)) to Bacillus thuringiensis. Toxicological studies showed that the 198(r) colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dpl(r) colony was 4-fold more resistant to protoxin versus toxin. Binding results with (125)I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac. Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways. Compared with the susceptible colony, insects from the Dpl(r) colony showed a drastic reduction in binding affinity (60-fold higher K(d)), although they had similar concentrations of binding sites. Insects from the 198(r) colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higher K(d) and ca. three-fold-lower R(t) compared with the 688(s) colony). No major difference in Cry1Ac binding was found among the three colonies. The fact that the 198(r) colony also has a protease-mediated mechanism of resistance (B. Oppert, R. Hammel, J. E. Throne, and K. J. Kramer, J. Biol. Chem. 272:23473-23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab. It is noteworthy that the three colonies used in this work derived originally from ca. 100 insects, which reflects the high variability and high frequency of B. thuringiensis resistance genes occurring in natural populations.     

The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins.

60% of the peripheral membrane skeleton of Euglena gracilis consists of equimolar amounts of two proteins (articulins) with M(r)s in SDS gels of 80 and 86 kD. To understand eventually how these proteins assemble and function in maintaining cell form and membrane integrity we have undertaken a molecular characterization of articulins. A lambda gt11 expression library constructed from Euglena gracilis mRNAs was screened with antibodies against both articulins. Two sets of cDNAs were recovered, and evidence from three independent assays confirmed that both sets encoded articulins: (a) Anti-articulin antibodies recognized a high molecular weight beta-galactosidase (beta-gal) fusion protein expressed in bacteria infected with lambda gt11 cDNA clones. (b) Antibodies generated against the bacterially expressed beta-gal fusion protein identified one or the other articulin in Western blots of Euglena proteins. These antibodies also localized to the membrane skeletal region in thin sections of Euglena. (c) Peptide maps of the beta-gal fusion protein were similar to peptide maps of Euglena articulins. From the nucleotide sequence of the two sets of cDNAs an open reading frame for each articulin was deduced. In addition to 37% amino acid identity and overall structural similarity, both articulins exhibited a long core domain consisting of over 30 12-amino acid repeats with the consensus VPVPV--V--. Homology plots comparing the same or different articulins revealed larger, less regular repeats in the core domain that coincided with predicted turns in extended beta-sheets. Outside the core domain a short hydrophobic region containing four seven-amino acid repeats (consensus: APVTYGA) was identified near the carboxy terminus of the 80-kD articulin, but near the amino terminus of the 86-kD articulin. No extensive sequence similarities were found between articulins and other protein sequences in various databanks. We conclude that the two articulins are related members of a new class of membrane cytoskeletal proteins.     

Cloning and sequence analysis of the human liver rhodanese: comparison with the bovine and chicken enzymes

The cDNA for the human rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), a nuclearly encoded protein of the mitochondrial matrix, was isolated from a human fetal liver cDNA library. Nucleotide sequence revealed an open reading frame coding for a polypeptide of 295 amino acids, which presented a 57% and 58% identity with the bovine and avian rhodanese, respectively. The analysis of the 5'-ends of the coding region gave no evidence for the presence of a cleavable signal sequence as found in other mitochondrial proteins. A comparison with two available amino acid sequences (cow and chicken) showed that sequence similarity is not restricted to the alpha-helices and beta-structures motifs which are remarkably superimposable in the two halves of bovine rhodanese, but extends to adjacent regions.     

Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae)

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites (His109, Asp156, Ser257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp251, Gly274, Gly284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).     

Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell line MCF-7.

We present the complete sequence of an mRNA which is induced by estrogen in the human breast cancer cell line MCF-7 [pS2 mRNA, Masiakowski et al., Nucleic Acids Res. 10, 7895-7903 (1982)]. Primer extension and cloning of double-stranded cDNA (ds-cDNA) into a vector designed to make full-length cDNA were used to determine the sequence of the fifteen 5'-terminal nucleotides which were not present in the original pS2 ds-cDNA clone. The mRNA sequence has a major open reading frame encoding 84 amino-acids, flanked by a 40 nucleotide 5'-untranslated region and a 198 nucleotide 3'-untranslated region preceding the polyA tail. The 3'-untranslated region contains a polyadenylation signal, AUUAAA, 14 nucleotides upstream from the polyA tail. The derived protein sequence contains a putative signal peptide region suggesting that the protein may be secreted. The nucleotide and derived amino-acid sequences were compared to previously determined sequences, particularly to those of hormone-regulated proteins and growth factors, and no obvious similarities were observed.     

CHARACTERIZATION OF A GENE ENCODING THE LIGHT-HARVESTING VIOLAXANTHIN-CHLOROPHYLL PROTEIN OF NANNOCHLOROPSIS SP. (EUSTIGMATOPHYCEAE)

In contrast to vascular plants, green algae, and diatoms, the major light-harvesting complex of the marine eustigmatophyte genus Nannochloropsis is a violaxanthin–chlorophyll a protein complex that lacks chlorophylls b and c . The isolation of a single polypeptide from the light-harvesting complex of Nannochloropsis sp. (IOLR strain) was previously reported ( Sukenik et al. 1992 ). The NH₂-terminal amino acid sequence of this polypeptide was significantly similar to NH₂-terminal sequences of the light-harvesting fucoxanthin, chlorophyll a/c polypeptides from the diatom Phaeodactylum tricornutum Bohlin. Using polyclonal antibodies raised to the Nannochloropsis light-harvesting polypeptide, a gene encoding this polypeptide was isolated from a cDNA expression library. The deduced amino acid sequence of the Nannochloropsis violaxanthin–chlorophyll a polypeptide reveals a 36 amino acid presequence followed by 173 amino acids that constitute the mature polypeptide. The mature polypeptide has 30%–40% sequence identity to the diatom fucoxanthin–chlorophyll a/c polypeptides and less then 27% identity to the green algal and vascular plant light-harvesting chlorophyll polypeptides that bind both chlorophylls a and b . Its molecular mass, as deduced from the gene sequence, is 18.4 kDa with three putative transmembrane helices and several residues that may be involved in chlorophyll binding. The cDNA encoding the violaxanthin–chlorophyll a polypeptide was used to isolate and characterize a 10 kb genomic fragment containing the entire gene. The open reading frame was interrupted by five introns ranging in size from 123 to 449 bp. The intron borders have typical eukaryotic GT … AG sequences.     

Cloning of type 1 cannabinoid receptor in Rana esculenta reveals differences between genomic sequence and cDNA

The endocannabinoid system is a conserved system involved in the modulation of several physiologic processes, from the activity of the central nervous system to reproduction. Type 1 cannabinoid receptor (CNR1) cDNA was cloned from the brain and testis of the anuran amphibian, the frog Rana esculenta. Nucleotide identity ranging from 62.6% to 81.9% is observed among vertebrates. The reading frame encoded a protein of 462 amino acids (FCNR1) with all the properties of a membrane G-coupled receptor. Alignments of FCNR1 with those of other vertebrates revealed amino acid identity ranging from 61.9% to 88.1%; critical domains for CNR1 functionality were conserved in the frog. As nucleotide differences of cnr1 cDNA were observed in brain and testis, the genomic sequence of the cnr1 gene was also determined in the same tissue preparations. Nucleotide changes in codons 5, 30, 70, 186, 252 and 408 were observed when cDNA and genomic DNA were compared; the nucleotide differences did not affect the predicted amino acid sequences, except for changes in codons 70 and 408. Interestingly, the predicted RNA folding was strongly affected by different nucleotide sequences. Comparison of cnr1 mRNA sequences available in GenBank with the corresponding genomic sequences revealed that also in human, rat, zebrafish and pufferfish, nucleotide changes between mRNA and genomic sequences occurred. Furthermore, amino acid sequences deduced from both mRNA and the genome were compared among vertebrates, and also in pufferfish the nucleotide changes corresponded to modifications in the amino acid sequence. The present results indicate for the first time that changes in nucleotides may occur in cnr1 mRNA maturation and that this phenomenon might not be restricted to the frog.