Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants

Messenger RNAs encoding the nitrate reductase apoenzyme from tobacco can be translated in a cell-free system. Poly(A)+ mRNA fractions from the 23-32 S area of a sucrose gradient were used to build a cDNA library in the expression vector gt11 with an efficiency of cloning of approximately 10(4) recombinants/ng mRNA. Recombinant clones were screened with a rabbit polyclonal antibody directed against the corn nitrate reductase, which cross reacts specifically with the nitrate reductases from dicotyledons. Among 240000 recombinant plaques, eight clones were isolated containing inserts of sizes ranging from 1.6 kb to 2.1 kb and sharing sequence homologies. Seven of these clones contained a common internal 1.6 kb EcoRI fragment. The identity of these clones was confirmed as follows. A fusion protein of 170 kDa inducible by IPTG and recognized by the rabbit nitrate reductase antibody was expressed by a lysogen derived from one of the recombinants. The antibodies binding the fused protein were eluted and shown to be inhibitory to the catalytic activity of tobacco nitrate reductase. Two monoclonal antibodies directed against nitrate reductase were also able to bind the hybrid protein. The 1.6 kb EcoRI fragment was sequenced by the method of Sanger. The open reading frame corresponding to a translational fusion with the -galactosidase coding sequence of the vector shared strong homology at the amino acid level with the heme-binding domain of proteins of the cytochrome b5 superfamily and with human erythrocyte cytochrome b5 reductase. When the 1.6 kb EcoRI fragment was used as a probe for Northern blot experiments a signal corresponding to a 3.5 kb RNA was detected in tobacco and in Nicotiana plumbaginifolia mRNA preparations but no cross-hybridization with corn mRNAs was detected. The probe hybridized with low copy number sequences in genomic blots of tobacco DNA.     

The light-harvesting complex of photosystem I in Chlamydomonas reinhardtii: protein composition, gene structures and phylogenic implications

Light-harvesting chlorophyll a/b-binding proteins (LHCI) associated with photosystem I (PSI) and the genes encoding these proteins have been characterized in the unicellular green alga Chlamydomonas reinhardtii, extending previous studies of the PSII-LHCII [Teramoto et al. (2001) Plant Cell Physiol. 42: 849]. In order to assign LHCI proteins in the thylakoid membranes, the PSI-LHCI supercomplex that retains all of the major LHCI proteins was purified. Seven distinct LHCI proteins were resolved from the purified supercomplex by a high-resolution SDS polyacrylamide gel electrophoresis, and their N-terminal amino acid sequences were determined. One LHCI protein (band e) was newly found, although the other six LHCI proteins corresponded to those previously reported. Genomic clones encoding these seven LHCI proteins were newly isolated and the nucleotide sequences were determined. A comprehensive characterization of all members of Lhc gene family in this alga revealed that LHCI proteins are more highly diverged than LHCII, suggesting functional differentiation of the protein components in LHCI. Neighbor joining trees were constructed for LHC proteins from C. reinhardtii and those of Arabidopsis thaliana or Galdieria sulphuraria to assess evolutionary relationships. Phylogenetic analysis revealed that (1). green algal LHCI and LHCII proteins are more closely related to one another than to LHCI proteins in red algae, (2). green algae and higher plants possess seven common lineages of LHC proteins, and (3). Type I and III LHCI proteins are conserved between green algae and higher plants, while Type II and IV are not. These findings are discussed in the context of evolution of multiple diverse antenna complexes.     

Immunological cross-reactivity between the light-harvesting chlorophyll a/b-proteins of a marine green alga and spinach

Immunoblotting was used to probe the reactivity of rabbit polyclonal antibodies against PS1I and PSI light-harvesting chlorophyll a/b-proteins of spinach ( Spinacea oleracea L.) with the light-harvesting complexes of a siphonaceous marine alga, Codium , that have more chlorophyll b, siphonaxanthin and siphonein instead of the lutein. The spinach LHCII antibodies cross-reacted only with the apoproteins of Cod-ium LHCII. Antisera against the spinach LHCI apoproteins showed strong affinity for the apoproteins of Codium LHCI, and also reacted with the polypeptides of spinach LHCII and Codium LHCII. Our results indicate some similarities in the amino acid sequences between the Codium siphonaxanthin-Chl a/fe-proteins of LHCII and LHCI and the corresponding spinach lutein-chlorophyll a/b-proteins.     

Identification of the polypeptides of the major light-harvesting complex of photosystem II (LHCII) with their genes in tomato.

Using an improved SDS-PAGE system, the polypeptides of the major chlorophyll a/b light-harvesting complex of PSII (LHCII) from tomato leaves were resolved into five polypeptide bands. All the polypeptides were matched with the genes encoding them by comparing amino acid sequences of tryptic peptides with gene sequences. The two major LHCII bands (usually comigrating as a '27 kDa' polypeptide) were encoded by cab1 and cab3 (Type I LHCII) genes. A third strong band of about 25 kDa was encoded by cab4 (Type II) genes. Polypeptides from two minor bands of 23-24 kDa were not N-terminally blocked; their N-terminal sequences showed they were Type III LHCII proteins. One complete cDNA clone and several incomplete clones for Type III polypeptides were sequenced. Combined with the peptide sequences, the results indicate that there are at least four different Type III genes in tomato, encoding four almost identical polypeptides. Thus, all the LHCII CAB polypeptides have been identified, and each type of LHCII polypeptide is encoded by distinct gene or genes in tomato.     

Immunological characterization of chlorophyll a/b-binding proteins of barley thylakoids

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCI) using a photosystem (PS) I preparation (PSI-200) wild-type from barley (Hordeum vulgare L. cv. Svaløf's Bonus) as the antigen. These antibodies cross-reacted with a minor light-harvesting chlorophyll a/b-protein of PSII (Chla/b-P1=CP29), but not with the major one, LHCII (=Chla/b-P2^**). Similarly, a monoclonal antibody to Chla/b-P1, elicited by a PSII preparation as the antigen, cross-reacted with LHCI, but not LHCII. This explains why an antigen consisting of LHCII, free of LHCI, but contaminated with Chla/b-P1, can elicit antibodies which cross-react with LHCI. Immunoblot assays showed that LHCI and Chla/b-P1 have at least two epitopes in common. Immunogold labelling of thin-sectioned wild-type thylakoids confirmed a preferential localisation of Chla/b-P1 in grana partition membranes and LHCI in stroma lamellae. The presence of LHCI was demonstrated in barley mutants lacking the PSI reaction centre (viridis-zb ⁶³) and chlorophyll b (chlorina-f2), and was correlated with the presence of long-wavelength (730 nm) fluorescence emission at 77 K. The mutant viridis-k ²³, which has a 77 K long-wavelength fluorescence peak at 720 nm, was shown by immune-blot assay to lack LHCI, although Chla/b-P1 was present.Abbreviations Chl-P chlorophyll-protein - CM Carlsberg Monoclonal - Da dalton - LHC light-harvesting complex - PAGE polyacrylamide gel electrophoresis - PSI, II photosystem I, II - PSI-200 PSI containing LHCI polypeptides - SDS sodium dodecyl sulphate     

Proteomic approach to characterize the supramolecular organization of photosystems in higher plants

A project to investigate the supramolecular structure of photosystems was initiated, which is based on protein solubilizations by digitonin, protein separations by Blue native (BN)-polyacrylamide gel electrophoresis (PAGE) and protein identifications by mass spectrometry (MS). Under the conditions applied, nine photosystem supercomplexes could be described for chloroplasts of Arabidopsis, which have apparent molecular masses between 600 and 3200 kDa on BN gels. Identities of the supercomplexes were determined on the basis of their subunit compositions as documented by 2D BN/SDS-PAGE and BN/BN-PAGE. Two supercomplexes of 1060 and approximately 1600 kDa represent dimeric and trimeric forms of photosystem I (PSI), which include tightly bound LHCI proteins. Compared to monomeric PSI, these protein complexes are of low abundance. In contrast, photosystem II mainly forms part of dominant supercomplexes of 850, 1000, 1050 and 1300 kDa. According to our interpretation, these supercomplexes contain dimeric PSII, 1-4 LHCII trimers and additionally monomeric LHCII proteins. The 1300-kDa PSII supercomplex (containing four LHCII trimers) is partially converted into the 1000-kDa PSII supercomplex (containing two LHCII trimers) in the presence of dodecylmaltoside on 2D BN/BN gels. Analyses of peptides of the trypsinated 1300-kDa PSII supercomplex by mass spectrometry allowed to identify known subunits of the PSII core complex and additionally LHCII proteins encoded by eight different genes in Arabidopsis. Further application of this experimental approach will allow new insights into the supermolecular organization of photosystems in plants.     

Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1.

Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65KDBP) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65KDBP. Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65KDBP, was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65KDBP. The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65KDBP, thus confirming the gene assignment.     

Tissue-specific expression and androgen regulation of different genes encoding rat prostatic 22-kilodalton glycoproteins homologous to human and rat cystatin

22-Kilodalton (kDa) protein cDNA clones were isolated from a rat prostatic library. Nucleotide sequence analysis revealed three different cDNA sequences encoding two somewhat different open reading frames of 176 amino acids. The N-terminal 24 amino acids of these sequences show the typical characteristics of signal peptides of secretory proteins. The C-terminal end of the derived protein sequences displays sequence similarity to a number of cysteine proteinase inhibitors, called cystatins, suggesting a common physiological function. Upon Northern blotting with a labeled cDNA fragment, three different 22-kDa protein mRNAs, i.e. 950 nucleotides (nt), 920 nt and 860 nt, could be detected in the rat ventral prostate and the lacrymal gland. In both tissues these messengers were regulated by androgens showing the most rapid androgen response for the 950 nt mRNA form. Administration of cycloheximide nearly completely abolished the observed androgen effect suggesting that a short-living protein is required for the full induction of the 22-kDa protein genes. Hybridization experiments with specific oligonucleotides which distinguish between the mRNAs encoding both 22-kDa protein variants indicate that one protein form is less androgen dependent in the ventral prostate and not expressed in the lacrymal gland.     

Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence

Lysyl oxidase cDNA clones were identified by their reactivity with anti-bovine lysyl oxidase in a neonatal rat aorta cDNA lambda gt11 expression library. A 500-bp cDNA sequence encoding four of six peptides derived from proteolytic digests of bovine aorta lysyl oxidase was found from the overlapping cDNA sequences of two positive clones. The library was rescreened with a radiolabeled cDNA probe made from one of these clones, thus identifying an additional 13 positive clones. Sequencing of the largest two of these overlapping clones resulted in 2672 bp of cDNA sequence containing partial 5'- and 3'-untranslated sequences of 286 and 1159 nucleotides, respectively, and a complete open reading frame of 1227 bp encoding a polypeptide of 409 amino acids (46 kDa), consistent with the 48 +/- 3 kDa cell-free translation product of rat smooth muscle cell RNA that was immunoprecipitated by anti-bovine lysyl oxidase. The rat aorta cDNA-derived amino acid sequence contains the sequence of each of the six peptides isolated and sequenced from the 32-kDa bovine aorta enzyme, including the C-terminal peptide with sequence identity of 96%. Northern blots screened with lysyl oxidase cDNA probes identified hybridizing species of 5.8 and 4.5 kb in mRNA of rat aorta and lung, while dot blot analyses were negative for lysyl oxidase mRNA in preparations of rat brain, liver, kidney, and heart. A 258-bp segment of the 3'-untranslated region of lysyl oxidase cDNA is 93% identical with a highly conserved region of the 3'-untranslated sequence of rat elastin cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Calvin cycle enzyme pentose-5-phosphate 3-epimerase is encoded within the cfx operons of the chemoautotroph Alcaligenes eutrophus.

Several genes (cfx genes) encoding Calvin cycle enzymes in Alcaligenes eutrophus are organized in two highly homologous operons comprising at least 11 kb. One cfx operon is located on the chromosome; the other is located on megaplasmid pHG1 of the organism (B. Bowien, U. Windh?vel, J.-G. Yoo, R. Bednarski, and B. Kusian, FEMS Microbiol. Rev. 87:445-450, 1990). Corresponding regions of about 2.7 kb from within the operons were sequenced. Three open reading frames, designated cfxX (954 bp), cfxY (765 bp), and cfxE (726 bp), were detected at equivalent positions in the two sequences. The nucleotide identity of the sequences amounted to 94%. Heterologous expression of the subcloned pHG1-encoded open reading frames in Escherichia coli suggested that they were functional genes. The observed sizes of the gene products CfxX (35 kDa), CfxY (27 kDa), and CfxE (25.5 kDa) closely corresponded to the values calculated on the basis of the sequence information. E. coli clones harboring the cfxE gene showed up to about 19-fold-higher activities of pentose-5-phosphate 3-epimerase (PPE; EC 5.1.3.1) than did reference clones, suggesting that cfxE encodes PPE, another Calvin cycle enzyme. These data agree with the finding that in A. eutrophus, PPE activity is significantly enhanced under autotrophic growth conditions which lead to a derepression of the cfx operons. No functions could be assigned to CfxX and CfxY.     

Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.     

DNA sequence preferences for the anti-cancer drug mitoxanthrone and related anthraquinones revealed by DNase I footprinting

Human T-cell leukemia virus type II (HTLV-II) isolated from a T-cell variant of hairy cell leukemia contains gag, pol and env genes as well as a fourth gene termed X, which can code three major open reading frames Xa, Xb and Xc. Proteins with molecular masses of 26 kDa (p26Xb) and 24 kDa (p24Xb) encoded by the Xb open reading frame were identified with antisera directed against synthetic peptides corresponding to the N-terminal and C-terminal amino acid sequences deduced from the structure of the Xb open reading frame. More than half the Xb products were found to be located in the nuclear fraction of HTLV-II-infected cells.     

Molecular cloning and partial characterization of a novel collagen chain, alpha 1(XVI), consisting of repetitive collagenous domains and cysteine-containing non-collagenous segments.

In an effort to identify new members of the collagen family, we screened a human placenta cDNA library with a collagenous probe. A novel 3.7 kb cDNA was identified encoding an open reading frame of 1,186 amino acids and containing a termination codon. The predicted polypeptide consists of 9 repetitive collagenous (stretches of Gly-X-Y) and several non-collagenous segments. Two cysteinyl residues separated by two amino acid residues (Cys-X-X-Cys) are regularly located in the N-terminal region of each non-collagenous segment. The deduced amino acid sequence described above is distinct from those of known types of collagen. Therefore, this novel collagen chain is designated alpha 1(XVI). Northern blot analysis revealed an alpha 1(XVI) mRNA of 5.2 kb, indicating that the overlapping cDNA clones isolated in this study covered nearly three-fourths of the mRNA. As a tool for further study on the expression of type XVI collagen, we prepared an antibody against the nonadecapeptide CFLSLERPRAEEARGDNSE, derived from the putative translation product of the cDNA. In immunoblot analysis, the antibody recognized a 160 kDa protein, which was bacterial collagenase-sensitive. Immunohistochemical stainings of human placental tissues with anti-peptide antibody revealed a positive reaction with amnion, the membranous tissue lining the amniotic cavity. The gene of alpha 1(XVI) chain, COL16A1, is mapped on the short arm of human chromosome 1 (1p13-p34).