Sequence of the fifth largest subunit of RNA polymerase II from plants

An affinity-purified antibody raised against the fifth largest subunit of cauliflower (Brassica oleracea) RNA polymerase II was used to screen an expression library and isolate an Arabidopsis thaliana cDNA clone. This cDNA clone was used to isolate a soybean (Glycine max) cDNA clone, and both clones were sequenced. The open reading frames contain 176 amino acids and predict polypeptides of 19.5 and 19.6 kDa for Arabidopsis and soybean, respectively. The amino acid sequences of the Arabidopsis and soybean polypeptides are 91.5% identical. The fifth largest subunit in plant RNA polymerase II is present at unit stoichiometry in purified enzyme and does not dissociate from the holoenzyme during nondenaturing polyacrylamide gel electrophoresis. The gene encoding the 19.5-kDa subunit has been isolated and sequenced from Arabidopsis. The gene is single copy and contains five introns. The size of the mRNA encoding this RNA polymerase II subunit in Arabidopsis and soybean is approximately 1 kilobase. None of the published yeast or animal RNA polymerase subunit sequences show similarity to the fifth largest subunit in plants.     

Cloning and characterisation of a novel P-glycoprotein homologue from barley

P-glycoproteins are members of a large superfamily of transport proteins (the 'traffic ATPases') that utilize ATP to translocate a wide range of substrates across biological membranes. Using a PCR-based approach, and degenerate oligonucleotides corresponding to conserved motifs, two 300-bp cDNA fragments (pBMDR1 and pBMDR2) with a significant sequence similarity to mammalian P-glycoproteins were amplified from barley (Hordeum vulgare) root poly A+ RNA and used as probes to screen a barley root cDNA library. A single full-length clone pHVMDR2 coding for a polypeptide of 1232 residues (c. 134 kDa) was isolated. Comparison of this barley sequence with Arabidopsis ATPGP1 and human MDR1 and MDR3 P-glycoprotein sequences showed that the barley cDNA has 44%, 37% and 38% amino acid (aa) identity, respectively, with these sequences, and conserved structural features. RNase protection analysis showed that HVMDR2 mRNA is expressed at low levels in both barley roots and leaves. Southern blot analyses indicated that there is a small multigene family related to P-glycoproteins in barley. Possible functions for these barley P-glycoproteins are discussed.     

Multiple loci for synapse protein SNAP-25 in the tetraploid goldfish

The common goldfish Carassius auratus is tetraploid and has 100 chromosomes. We describe here goldfish cDNA clones for SNAP-25, a 200-amino-acid synaptosome-associated protein that has remained highly conserved during evolution. SNAP-25 occurs as a single-copy gene in mouse, chicken, and Drosophila melanogaster. Sequences of six distinct goldfish cDNA clones and Southern hybridizations show that the goldfish has three, or possibly four, SNAP-25 loci rather than two as expected. A gene duplication early in actinopterygian fish evolution gave rise to the loci SnapA and SnapB. The proteins SNAP-A and SNAP-B are 94% and 91% identical to the mouse protein but are only 91% identical to each other. SNAP-B has a larger number of unique amino acid replacements than SNAP-A and also has more dramatic replacements. The tetraploidization resulted in two SnapB loci whose divergence from each other is consistent with a tetraploidization event 15-20 million years ago. The presence of duplicate SnapA loci has not yet been possible to confirm, possibly because they are still very similar to each other. Two of the SnapA cDNA clones and one SnapB cDNA clone have frameshift mutations. As these aberrant alleles otherwise display high sequence identity to the functional alleles, they probably became nonfunctional recently. The findings of allelic variability and aberrant alleles emphasize the importance of characterizing multiple DNA clones in tetraploid species.     

Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteine residue

We have determined the sequence of 23 peptides from bovine thioredoxin reductase covering 364 amino acid residues. The result was used to identify a rat cDNA clone (2.19 kilobase pairs), which contained an open reading frame of 1496 base pairs encoding a protein with 498 residues. The bovine and rat thioredoxin reductase sequences revealed a close homology to glutathione reductase including the conserved active site sequence (Cys-Val-Asn-Val-Gly-Cys). This also confirmed the identity of a previously published putative human thioredoxin reductase cDNA clone. Moreover, one peptide of the bovine enzyme contained a selenocysteine residue in the motif Gly-Cys-SeCys-Gly (where SeCys represents selenocysteine). This motif was conserved at the carboxyl terminus of the rat and human enzymes, provided that TGA in the sequence GGC TGC TGA GGT TAA, being identical in both cDNA clones, is translated as selenocysteine and that TAA confers termination of translation. The 3'-untranslated region of both cDNA clones contained a selenocysteine insertion sequence that may form potential stem loop structures typical of eukaryotic selenocysteine insertion sequence elements required for the decoding of UGA as selenocysteine. Carboxypeptidase Y treatment of bovine thioredoxin reductase after reduction by NADPH released selenocysteine from the enzyme with a concomitant loss of enzyme activity measured as reduction of thioredoxin or 5,5'-dithiobis(2-nitrobenzoic acid). This showed that the carboxyl-terminal motif was essential for the catalytic activity of the enzyme.     

The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants

A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the 3' end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated M(r) of 34866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other closteroviruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot.     

Nucleotide and deduced amino acid sequences of rat myosin binding protein H (MyBP-H)

The complete nucleotide sequence of the cDNA clone encoding rat skeletal muscle myosin-binding protein H (MyBP-H) was determined and amino acid sequence was deduced from the nucleotide sequence (GenBank accession number AF077338). The full-length cDNA of 1782 base pairs(bp) contains a single open reading frame of 1454 bp encoding a rat MyBP-H protein of the predicted molecular mass 52.7 kDa and includes the common consensus 'CA__TG' protein binding motif. The cDNA sequence of rat MyBP-H show 92%, 84% and 41% homology with those of mouse, human and chicken, respectively. The protein contains tandem internal motifs array (-FN III-Ig C2-FN III-Ig C2-) in the C-terminal region which resembles to the immunoglobulin superfamily C2 and fibronectin type III motifs. The amino acid sequence of the C-terminal Ig C2 was highly conserved among MyBPs family and other thick filament binding proteins, suggesting that the C-terminal Ig C2 might play an important role in its function. All proteins belonging to MyBP-H member contains 'RKPS' sequence which is assumed to be cAMP- and cGMP-dependent protein kinase A phosphorylation site. Computer analysis of the primary sequence of rat MyBP-H predicted 11 protein kinase C (PKC) phosphorylation site, 7 casein kinase II (CK2) phosphorylation site and 4 N-myristoylation site.     

Cloning, characterization, and tissue expression pattern of mouse tuftelin cDNA

Tuftelin is a protein that has been suggested to function during enamel crystal nucleation. Published sequences for bovine tuftelin cDNA and genomic clones proposed different reading frames that radically affected the derived amino acid sequence of the tuftelin carboxyl-terminus. We have isolated and characterized a full-length mouse cDNA clone and a partial porcine cDNA clone that include the region of the proposed frame-shift. The mouse tuftelin clone is 2572 nucleotides in length, exclusive of the poly(A+) tail. Translation from the 5'-most ATG yields a protein of 390 amino acids with an isotope-averaged molecular mass of 44.6 kDa and an isoelectric point of 5.9. Comparison of the bovine, mouse, and porcine cDNAs supports the revised bovine tuftelin amino acid sequence and suggests that the bovine tuftelin translation initiation codon be re-assigned to a more 5' ATG. Re-assigning the translation initiation codon lengthens the tuftelin protein by 52 amino acids, 51 of which are identical between bovine and mouse. At the carboxyl-terminus, the revised bovine and the mouse sequences match at 39 of the final 42 amino acid positions, compared with 2 identities with the originally published bovine reading frame. Northern blot analysis reveals that tuftelin is not ameloblast-specific but is expressed in multiple tissues, including kidney, lung, liver, and testis. Two tuftelin RNA messages, of 2.6 and 3.2 kb, were detected. DNA sequence characterization of an RT-PCR amplification product confirmed expression of tuftelin in kidney, and identified an alternatively spliced mouse tuftelin mRNA lacking exon 2.     

Identification and characterization of the murine homologue of CD22, a B lymphocyte-restricted adhesion molecule

The human B lymphocyte-specific Ag, CD22, is a cell adhesion molecule expressed on the surface during a narrow window of B cell development, coincident with surface IgD. A ligand for CD22 has recently been identified on human T cells as the low molecular mass isoform of the leukocyte common Ag, CD45RO. CD22 has been reported to function in the regulation of both T and B cell activation in vitro. In this study, we report the isolation and expression of a molecular cDNA clone encoding the murine homologue of CD22, mCD22. Within their predicted protein sequences, murine and human sequences overall have 62% identity, which includes 18 of 20 extracellular cysteines and six of six cytoplasmic tyrosines. BHK cells transfected with mCD22 cDNA specifically adhere to resting and activated T lymphocytes and in addition bound activated, but not resting, B cells. Five Th clones were analyzed for their ability to adhere to mCD22; two Th0 clones and one Th1 clone bound CD22+ BHK transfectants, but not all T cell clones bound CD22+ cells: another Th1 clone and a Th2 clone did not. mCD22+ BHK transfectants were also specifically bound by the B cell-specific mAb, NIM-R6, demonstrating that this mAb is specific for murine CD22. Human cell lines expressing the counter-receptors for human CD22 were also examined for adhesion to the murine CD22 homologue; the epitope responsible for B cell adhesion to CD22 is conserved, whereas the T cell epitope binding to CD22 is not. The cDNA and mAb to murine CD22 will be useful for defining the in vivo function of CD22.     

Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N, a hypoxia activated anthraquinone di-N-oxide prodrug

OBJECTIVE: The 65 kDa heat shock protein (HSP) chaperonin is a highly conserved intracellular protein. HSP are involved in the pathogenesis of arthritis, but are not able to induce experimental arthritis. T cell clones recognizing the 180-188 amino acid sequence of 65 kDa HSP are present in inflamed synovium of both adjuvant arthritis and rheumatoid arthritis (RA). Oral administration of bovine collagen II or co-chaperonin 10 kDa HSP has been shown to induce an immune tolerance state to collagen induced arthritis (CIA). We investigate the effect of oral gavage with 65 kDa HSP on CIA. METHODS: We immunized 6-8-week-old DBA1 male mice with bovine type II collagen. A group of 25 mice were given oral recombinant mycobacterial 65 kDa HSP before immunization (30 microg in 200 microl phosphate buffered saline (PBS) at Days -7, -5, -2) while PBS alone was administered in 27 controls. A 3rd group was fed 65 kDa HSP according to the same protocol but was not immunized with collagen II (n = 8). The clinical arthritis score was recorded 3 times/week until Day 60. Antibodies to collagen II were determined by ELISA. RESULTS: The incidence of arthritis was comparable in the 2 groups (72 vs 70%). The onset of arthritis was not delayed in mice fed HSP. However, the severity of arthritis was lower 10 days after arthritis onset in animals fed 65 kDa HSP (clinical score 1.83 +/- 0.79 vs 2.74 +/- 1.1; p < 0.0001). No animals in Group 3 had arthritis. Serum IgG anti-type II collagen levels were decreased in HSP treated mice (optical density 0.33 +/- 0.21 vs 0.46 +/- 0.21; p < 0.0001). However, the ratio of IgG1/IgG2a antitype II collagen antibody response remained unchanged in the mice fed 65 kDa HSP. CONCLUSION: These results suggest that oral administration of 65 kDa HSP may diminish collagen induced arthritis.     

Cloning, sequencing, and recombinant expression of the porcine inhibitor of carbonic anhydrase: a novel member of the transferrin family

The plasma from many vertebrates contains a component that specifically binds and inhibits carbonic anhydrase II with nanomolar affinity. Amino-terminal sequencing of pICA, the previously identified 79-kDa carbonic anhydrase inhibitor isolated from porcine plasma [Roush, E. D., & Fierke, C. A. (1992) Biochemistry 31, 12536-12542], and sequencing of four proteolytic fragments of pICA revealed that each of the partial sequences has 40-80% sequence identity with members of the transferrin protein family. We describe here the isolation of a full-length cDNA clone of pICA from a lambda gt11 porcine liver cDNA library. Heterologous expression of this cDNA clone in a Pichia pastoris expression system led to the secretion into the medium of 5 mg/L of a 79-kDa protein that specifically reacts with anti-pICA antibodies and binds tightly to a carbonic anhydrase-Sepharose affinity column. Pairwise sequential alignment of pICA with various transferrins reveals an amino acid identity as high as 64% and predicts that 16 transferrin disulfide bonds are conserved. However, despite these structural similarities, the properties of pICA are distinct from the properties of transferrin. pICA exhibits a significantly decreased affinity for iron that can be attributed to the loss of one of the eight amino acids that coordinate iron in the transferrins as well as both of the arginine residues responsible for anion binding. In addition, the antigenic determinants of pICA and the transferrins are not identical. These data imply that pICA, along with saxiphilin, is a member of a diverse superfamily of transferrin-like proteins with functions other than iron binding.     

Lack of correlation between apoptosis and DNA single-strand breaks in X-irradiated human peripheral blood mononuclear cells in the course of ageing

Inositol(1,3,4,5)tetrakisphosphate (InsP4) and phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) are two potential second messengers with a still largely unknown mode of action. We recently cloned the 42 kDa protein p42IP4 previously purified from pig cerebellum, which binds InsP4 (Kd approximately 2 nM) and PtdInsP3 with comparable affinities (Stricker et al., FEBS Lett. 405 (1997) 229). The protein p42IP4 (pig) is highly homologous to centaurin-alpha, a larger protein of 46 kDa, derived from a rat brain cDNA library clone (Hammonds-Odie et al., J. Biol. Chem. 271 (1996) 18859). Here we investigated whether also p42IP4 is expressed in rat brain and how it might be related to centaurin-alpha. When we carried out RT-PCR using mRNA from brain of rats of different ages we obtained several clones corresponding to p42IP4, but not to centaurin-alpha. The existence of p42IP4 in rat brain is supported by the following findings: (1) biochemical analysis of the purified rat brain protein shows inositol phosphate ligand affinities identical to those of the protein from other species; (2) Western blot analysis of rat brain membrane fractions using a peptide-specific antiserum revealed only the 42 kDa protein (p42IP4), but did not give evidence for the occurrence of a larger 46 kDa centaurin-alpha-like protein in rat brain; and (3) the amino acid sequences deduced from p42IP4 cDNA are highly homologous in several species and are confirmed by protein fragment microsequences. Thus, p42IP4 from rat brain which has two pleckstrin homology domains is a protein largely conserved between different species and most likely has an important function in inositol phosphate or inositol lipid signal transduction.     

Identification and mapping of two divergent, unlinked major histocompatibility complex class II B genes in Xiphophorus fishes

We have isolated two major histocompatibility complex (MHC) class II B genes from the inbred fish strain Xiphophorus maculatus Jp 163 A. We mapped one of these genes, designated here as DXB, to linkage group III, linked to a malic enzyme locus, also syntenic with human and mouse MHC. Comparison of genomic and cDNA clones shows the gene consists of six exons and five introns. The encoded beta1 domain has three amino acids deleted and a cytoplasmic tail nine amino acids longer than in other teleost class II beta chains, more similar to HLA-DRB, clawed frog Xela-F3, and nurse shark Gici-B. Key residues for disulfide bonds, glycosylation, and interaction with alpha chains are conserved. These same features are also present in a swordtail (Xiphophorus helleri) genomic DXB PCR clone. A second type of class II B clone was amplified by PCR from X. maculatus and found to be orthologous to class II genes identified in other fishes. This DAB-like gene is 63% identical to the X. maculatus DXB sequence in the conserved beta2-encoding exon and was mapped to new unassigned linkage group LG U24. The DXB gene, then, represents an unlinked duplicated locus not previously identified in teleosts.     

Immunochemical and biochemical identification of the rice seed protein encoded by cDNA clone A3-12

Previously isolated cDNA clone A3-12 that was expressed in E. coli as the fusion protein with Trp E showed immunoreactivity with the mouse antibody raised against isolated alpha-globulin from rice seed. The N-terminal amino acid sequences determined for the purified alpha-globulin and its tryptic peptides were identical with the deduced amino acid sequence reported, except for two residues at the protein N terminus. An error in the reported sequence was confirmed by re-sequencing the cDNA, the nucleotide sequence for the two N-terminal residues being shown to be CAGCTG and not CACGTG. Thus, the protein encoded by cDNA clone A3-12 was identified to be the major rice seed globulin, alpha-globulin, with an apparent molecular mass of 26kDa.     

A cuticular protein from the moulting stages of an insect

A 22 kDa peptide was purified from prepupal cuticles of 5th instar Calpodes ethlius caterpillars. It was absent earlier in the stadium and from the egg and adult, i.e. it is related to cuticle turnover rather than cuticle structure. It was present at larval and metamorphic moults, showing that it is related to moulting not just metamorphosis. The cDNA corresponding to the 22 kDa peptide was isolated by antibody screening of an epidermal cDNA expression library. Hybridization to Calpodes genomic DNA showed that the gene was present as a single copy. The deduced amino acid sequence is not like any of the sequences of cuticular structural proteins that have been published, but has a 47 amino acid sequence similar to bacteriophage T7 N-acetylmuramoyl-L-alanine amidase (34% identical, 51% similar). The amino acid sequence, the timing of expression in development, and the similarity between the substrate of the bacteriophage amidase and components of insect cuticle, all suggest that the 22 kDa protein may have a role in cleaving chitin-peptide bonds as a prerequisite for digestion of the cuticle by chitinases and proteases.