Low-molecular-mass polypeptide components of a photosystem II preparation from the thermophilic cyanobacterium Thermosynechococcus vulcanus

Using a recently introduced electrophoresis system [Kashino et al. (2001) Electrophoresis 22: 1004], components of low-molecular-mass polypeptides were analyzed in detail in photosystem II (PSII) complexes isolated from a thermophilic cyanobacterium, Thermosynechococcus vulcanus (formerly, Synechococcus vulcanus). PsbE, the large subunit polypeptide of cytochrome b(559), showed an apparent molecular mass much lower than the expected one. The unusually large mobility could be attributed to the large intrinsic net electronic charge. All other Coomassie-stained polypeptides were identified by N-terminal sequencing. In addition to the well-known cyanobacterial PSII polypeptides, such as PsbE, F, H, I, L, M, U, V and X, the presence of PsbY, PsbZ and Psb27 was also confirmed in the isolated PSII complexes. Furthermore, the whole amino acid sequence was determined for the polypeptide which was known as PsbN. The whole amino acid sequence revealed that this polypeptide was identical to PsbTc which has been found in higher plants and green algae. These results strongly suggest that PsbN is not a member of the PSII complex. It is also shown that cyanobacteria have cytochrome b(559) in the high potential form as in higher plants.     

Sequences of six genes and several open reading frames in the kinetoplast maxicircle DNA of Leishmania tarentolae

The DNA sequence of approximately 80% of the transcribed region of the kinetoplast maxicircle DNA of Leishmania tarentolae was obtained, and structural genes were localized by comparison of the translated amino acid sequences with those of known mitochondrial genes from other organisms. By this method, the genes for cytochrome oxidase subunits I, II, and III, cytochrome b, and human mitochondrial unidentified reading frames 4 and 5 were identified. By comparing the amino acid sequences of the putative L. tarentolae genes with those of known genes, we conclude that TGA codes for tryptophan, as in most other mitochondrial systems. This is the only apparent change from the universal genetic code. The six identified structural genes show various degrees of divergence from the homologous genes in other species, with cytochrome oxidase subunit I being the most conserved and cytochrome oxidase subunit III being the least conserved. A comparison of the cytochrome b genes from L. tarentolae and Trypanosoma brucei showed that the ratio of transversions to transitions is 1:1, suggesting that these species diverged from each other more than 80 X 10(6) years ago. Several as yet unidentified open reading frames were also present in the maxicircle sequence. These data confirm that maxicircle DNA has a coding potential which typifies other mitochondrial systems.     

Differential binding of a CCAAT DNA binding factor to the promoters of the mouse alpha 2(I) and alpha 1(III) collagen genes

An exonuclease III assay (Wu, C. (1985) Nature 317, 84-87) was used to identify in nuclear extracts of NIH 3T3 cells a factor which binds to the CCAAT segment of the alpha 2(I) collagen promoter between -80 and -84. This sequence is located on the coding strand in the alpha 2(I) collagen promoter. Binding is specific since only promoter fragments which contain the CCAAT box sequences on one or the other DNA strand inhibit binding to the alpha 2(I) collagen CCAAT box. The CCAAT binding factor protects approximately 26 base pairs of the alpha 2(I) collagen promoter from exonuclease III digestion. Binding to the alpha 2(I) collagen promoter CCAAT box is not inhibited by a fragment of the alpha 1(III) collagen promoter (from -396 to +16), which does not contain a CCAAT sequence on either one or the other strand. Our data suggest that two genes such as the alpha 2(I) and alpha 1(III) collagen genes, which are coordinately expressed in many tissues, are not necessarily regulated by the same trans-acting DNA binding proteins.     

Mechanism of expression of the overlapping genes of Bacillus subtilis aspartokinase II

The mechanism of expression of the overlapping genes that encode the alpha and beta subunits of aspartokinase II of Bacillus subtilis was studied by specific mutagenesis of the cloned coding sequence. Escherichia coli or B. subtilis VB31 (aspartokinase II-deficient), transformed with plasmids carrying either a deletion of the translation start site and about one-half of the coding region for the larger alpha subunit or a frameshift mutation early in the alpha subunit coding region, produced the smaller beta subunit in the absence of alpha subunit synthesis, indicating that beta subunit is not derived from alpha subunit and that its synthesis does not depend on the alpha subunit translation initiation site. The beta subunit translation start site was identified by oligonucleotide-directed mutagenesis of the putative translation start codon. Modification of the nucleotide sequence encoding methionine residue 247 of the alpha subunit from ATG to either TTA or AAT (but not GTG) abolished beta subunit synthesis but had no effect on the production of alpha subunit. This observation is consistent with peptide chain initiation by N-formylmethionine, which specifically requires an ATG or GTG sequence, and indicates that translation of the beta subunit starts at a site corresponding to Met247 of the alpha subunit. Initial studies on the function of the aspartokinase II subunits, using E. coli as a heterologous host, showed that beta subunit was not essential for the expression of the catalytic function of aspartokinase, measured in vitro and in vivo, nor for its allosteric regulation by L-lysine. Whether the beta subunit has a function specific to B. subtilis needs to be explored in a homologous expression system.     

Cloning and structure of the gene for the subunits of aspartokinase II from Bacillus subtilis

A library of Bacillus subtilis DNA in lambda Charon 4A (Ferrari, E., Henner, D.J., and Hoch, J.A. (1981) J. Bacteriol. 146, 430-432) was screened by an immunological procedure for DNA sequences encoding aspartokinase II of B. subtilis, an enzyme composed of two nonidentical subunits arranged in an alpha 2 beta 2 structure (Moir, D., and Paulus, H. (1977a) J. Biol. Chem. 252, 4648-4654). A recombinant bacteriophage was identified that harbored an 18-kilobase B. subtilis DNA fragment containing the coding sequences for both aspartokinase subunits. The coding sequence for aspartokinase II was subcloned into bacterial plasmids. In response to transformation with the recombinant plasmids, Escherichia coli produced two polypeptides immunologically related to B. subtilis aspartokinase II with molecular weights (43,000 and 17,000) indistinguishable from those found in enzyme produced in B. subtilis. Peptide mapping by partial proteolysis confirmed the identity of the polypeptides produced by the transformed E. coli cells with the B. subtilis aspartokinase II subunits. The size of the cloned B. subtilis DNA fragment could be reduced to 2.9 kilobases by cleavage with PstI restriction endonuclease without affecting its ability to direct the synthesis of complete aspartokinase II subunits, irrespective of its orientation in the plasmid vector. Further subdivision by cleavage with BamHI restriction endonuclease resulted in the production of truncated aspartokinase subunits, each shortened by the same extent. This suggested that a single DNA sequence encoded both aspartokinase subunits and provided an explanation for the earlier observation that the smaller beta subunit of aspartokinase II was highly homologous or identical with the carboxyl-terminal portion of the alpha subunit (Moir, D., and Paulus, H. (1977b) J. Biol. Chem. 252, 4655-4661). A map of the gene for B. subtilis aspartokinase II is proposed in which the coding sequence for the smaller beta subunit overlaps in the same reading frame the promoter-distal portion of the coding sequence for the alpha subunit.     

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.     

Isolation and analysis of the genes for cytochrome c oxidase in Paracoccus denitrificans

Synthetic oligonucleotide probes were used to clone two loci from the chromosomal DNA of Paracoccus denitrificans that contain the genes for cytochrome c oxidase (cytochrome aa₃). One locus seems to contain four or five genes probably forming an operon. Two of these code for the oxidase subunits II and III. Three open reading frames are found between the COII and COIII genes. The other locus codes for the subunit I. A short open reading frame is found upstream of this gene. All three subunits of the Paracoccus enzyme show remarkable homology to the corresponding subunits of the mitochondrial cytochrome oxidase. Possible protein products of the open reading frames have not yet been identified.     

The elusive third subunit IIa of the bacterial B-type oxidases: the enzyme from the hyperthermophile Aquifex aeolicus

The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba(3)-type two-subunit (subunits I and II) enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix "subunit IIa", which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome) and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA(2)) of 59000 Da, subunit II (encoded by coxB(2)) of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba(3) cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes.     

Identities of four low-molecular-mass subunits of the photosystem I complex from Anabaena variabilis ATCC 29413. Evidence for the presence of the psaI gene product in a cyanobacterial complex

Photosystem I (PSI) complex of Anabaena variabilis ATCC 29413 consists of at least 11 subunits, 9 of which are resolved by high resolution gel electrophoresis. N-terminal amino acid sequences of the four subunits with molecular masses of 6.8, 5.2, 4.8 and 3.5 kDa were determined. Based on the sequence homology, the 3.5 kDa subunit was revealed to correspond to PSI-I (the gene product of psaI), which had so far been detected only in higher plant PSI complexes. The 6.8 kDa protein and 4.8 kDa protein were identified as gene products of psaK and psaJ, respectively. The 5.2 kDa protein was homologous to a 4.8 kDa subunit of PSI of the thermophilic cyanobacterium Synechococcus vulcanus, suggesting that this protein is a component of PSI in cyanobacteria.     

The gene encoding cytochrome c oxidase subunit II from Rhodobacter sphaeroides; comparison of the deduced amino acid sequence with sequences of corresponding peptides from other species.

The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa3) has been isolated by screening a genomic DNA library in phage lambda with a probe derived from coxII of Paracoccus denitrificans. A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined. The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon. The deduced amino acid (aa) sequence of subunit II of R. sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63% overall) and P. denitrificans oxidase (68% overall). The postulated redox-active copper ion (CuA) binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer. Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R. sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P. denitrificans.     

Chloroplast ATPase genes in the diatom Odontella sinensis reflect cyanobacterial characters in structure and arrangement.

We have cloned and sequenced a 5200 base restriction fragment and an overlapping 3100 base fragment of the large single copy region of the chloroplast genome of the diatom Odontella sinensis, which hybridized to several ATPase gene probes. These fragments contain six closely linked reading frames that were identified as atpI, atpH, atpG, atpF, atpD, and atpA, coding for subunits IV, III, II, I, delta, and alpha, respectively. Remarkably, the genes atpG and atpD, which are nucleus-encoded in chlorophyll a + b plants, are present in the Odontella chloroplast gene cluster. They map at the same positions as in cyanobacteria. The genes atpD and atpF overlap by four base-pairs as in certain photosynthetic and heterotrophic eubacteria. Upstream from the atpA gene cluster an open reading frame coding for 251 amino acid residues was found, which shows sequence similarity to ATP-binding subunits of periplasmic prokaryotic and eukaryotic transport systems. No similar reading frame is present in the land plant chloroplast genomes analysed so far. Sequences and arrangement of the genes are discussed with respect to the peculiar evolution of the chlorophyll a + c-containing chromophytic plastids.     

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.     

Sequence of the 18S-5S ribosomal gene region and the cytochrome oxidase II gene from mtDNA of Zea diploperennis

Summary The coding and flanking sequences of the 18S-5S ribosomal RNA genes and the cytochrome oxidase subunit II gene of Zea diploperennis mitochondrial DNA have been determined and compared to the corresponding sequences of normal maize (Zea mays L.) Both length and substitution mutations are found in the coding region of the 18S rRNA gene, whereas only one substitution mutation is found in the coding region of cytochrome oxidase II. Sequence divergence between maize and Zea diploperennis is about one-tenth of that between wheat and maize. The rate of nucleotide divergence by base substitution is less for plant mitochrondrial genes than for comparable genes in animal mitochondria.     

Archaeal DNA replication: identifying the pieces to solve a puzzle.

Archaeal organisms are currently recognized as very exciting and useful experimental materials. A major challenge to molecular biologists studying the biology of Archaea is their DNA replication mechanism. Undoubtedly, a full understanding of DNA replication in Archaea requires the identification of all the proteins involved. In each of four completely sequenced genomes, only one DNA polymerase (Pol BI proposed in this review from family B enzyme) was reported. This observation suggested that either a single DNA polymerase performs the task of replicating the genome and repairing the mutations or these genomes contain other DNA polymerases that cannot be identified by amino acid sequence. Recently, a heterodimeric DNA polymerase (Pol II, or Pol D as proposed in this review) was discovered in the hyperthermophilic archaeon, Pyrococcus furiosus. The genes coding for DP1 and DP2, the subunits of this DNA polymerase, are highly conserved in the Euryarchaeota. Euryarchaeotic DP1, the small subunit of Pol II (Pol D), has sequence similarity with the small subunit of eukaryotic DNA polymerase delta. DP2 protein, the large subunit of Pol II (Pol D), seems to be a catalytic subunit. Despite possessing an excellent primer extension ability in vitro, Pol II (Pol D) may yet require accessory proteins to perform all of its functions in euryarchaeotic cells. This review summarizes our present knowledge about archaeal DNA polymerases and their relationship with those accessory proteins, which were predicted from the genome sequences.