Two-domain hemoglobin from the blood clam,Barbatia lima. The cDNA-derived amino acid sequence

The blood clam,Barbatia lima, from Kochi, Japan, expresses a tetrameric (α ₂ β ₂) and a polymeric hemoglobin in erythrocytes. The latter hemoglobin is composed of unusual 34-kDa hemoglobin with a two-domain structure, and its molecular mass (about 430 kDa) is exceptionally large for an intracellular hemoglobin. The 3′ and 5′ parts of the cDNA ofB. lima two-domain globin have been amplified separately by polymerase chain reaction and the complete nucleotide sequence of 1147 bp was determined. The open reading frame is 930 nucleotides in length and encodes a protein with 309 amino acid residues, of which 73 amino acids were identified directly by protein sequencing. The mature protein begins with the acetylated Ser, and thus the N-terminus Met is cleaved. The molecular mass for the protein was calculated to be 35,244 Da. The cDNA-derived amino acid sequence ofB. lima two-domain globin shows 89% homology with that of two-domain globin fromB. reeveana, a North American species. The sequence homology between the two domains is 75%, suggesting that the two-domain globin resulted from the gene duplication of an ancestral 17-kDa globin.     

The cDNA-derived amino acid sequence of indoleamine dioxygenase like-myoglobin from the gastropod molluscOmphalius pfeifferi

Myoglobin was isolated from the radular muscle of the archaegastropod molluscOmphalius pfeifferi (Trochidae). The molecular mass was estimated by SDS-PAGE to be about 40 kDa, 2.5 times larger than that of usual myoglobin. The cDNA forOmphalius myoglobin was amplified by polymerase chain reaction, and the cDNA-derived amino acid sequence of 375 residues was determined, of which 73 residues were identified directly by the chemical sequencing of internal peptides. The amino acid sequence ofOmphalius myoglobin showed no significant homology with any other usual 16-kDa globins, but showed 84% and 36% identities with indoleamine dioxygenase-like myoglobins fromBattilus (Turbinidae) andSulculus (Haliotiidae), respectively. It also shows significant homology (26% identity) with human indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme containing heme. The distribution of indoleamine dioxygenase-like myoglobins suggests that they must have arisen exclusively along the specified lineage including the three families Haliotiidae, Turbinidae, and Trochidae of Archaegastropoda in molluscan evolution.     

The hemoglobin gene of the deep-sea clam Calyptogena soyoae has a novel intron in A-helix

The cDNAs encoding two dimeric hemoglobins, Hbs I and II, of the deep-sea clam Calyptogena soyoae were amplified by PCR and the complete nucleotide sequences determined. The cDNA-derived amino acid sequences agreed completely with those determined chemically. Many of the molluscan intracellular globin genes have a characteristic four-exon/three-intron structure, with the precoding and two conventional introns conserved widely in animal globin genes. In this work we have determined the exon/intron organization of two hemoglobin genes of the deep-sea clam C. soyoae. Surprisingly, this gene has no precoding intron but instead contains an additional intron in the A-helix (A3.1), together with the two conventional introns (B12.2 and G6.3). This observation suggests that the precoding intron has been lost and the insertion of intron in A-helix occurred in the genes of Calyptogena. Alternatively, the sliding of intron from precoding to A-helix might have occurred.     

The amino acid sequence of hemoglobin III from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clamLucina pectinata consists of 152 amino acid residues, has a calculated M_m of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66.Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnsonet al., J. Prot. Chem. 10, 609–622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 Å of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the twoLucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the twoLucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36–49% when compared to the two groups of mollusc globins. The highest homology appears to exist between theLucina globins and the cytoplasmic hemoglobin ofBusycon canaliculatum.     

The cDNA-derived amino acid sequence of indoleamine dioxygenase like-myoglobin from the gastropod molluscOmphalius pfeifferi

Myoglobin was isolated from the radular muscle of the archaegastropod molluscOmphalius pfeifferi (Trochidae). The molecular mass was estimated by SDS-PAGE to be about 40 kDa, 2.5 times larger than that of usual myoglobin. The cDNA forOmphalius myoglobin was amplified by polymerase chain reaction, and the cDNA-derived amino acid sequence of 375 residues was determined, of which 73 residues were identified directly by the chemical sequencing of internal peptides. The amino acid sequence ofOmphalius myoglobin showed no significant homology with any other usual 16-kDa globins, but showed 84% and 36% identities with indoleamine dioxygenase-like myoglobins fromBattilus (Turbinidae) andSulculus (Haliotiidae), respectively. It also shows significant homology (26% identity) with human indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme containing heme. The distribution of indoleamine dioxygenase-like myoglobins suggests that they must have arisen exclusively along the specified lineage including the three families Haliotiidae, Turbinidae, and Trochidae of Archaegastropoda in molluscan evolution.     

The amino acid sequence of hemoglobin II from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin II from the gill of the clamLucina pectinata consists of 150 amino acid residues, has a calculatedM _m of 17,476, including heme and an acetylated N-terminal residue. It retains the invariant residues Phe 44 at position CD1 and His 65 at the proximal position F8, as well as the highly conserved Trp 15 at position A12 and Pro 38 at position C2. The most likely candidate for the distal residue at position E7, based on the alignment with other globins, is Gln 65. However, optical and EPR spectroscopic studies of the ferri Hb II (Kraus, D. W., Wittenberg, J. B., Lu, J. F., and Peisach, J.,J. Biol. Chem. 265, 16054–16059, 1990) have implicated a tyrosinate oxygen as the distal ligand. Modeling of theLucina Hb II sequence, using the crystal structure of sperm whale aquometmyoglobin, showed that Tyr 30 substituting for the Leu located at position B10 can place its oxygen within 2.8 Å of the water molecule occupying the distal ligand position. This structural alteration is facilitated by the coordinate mutation of the residue at position CD4, from Phe 46 in the sperm whale myoglobin sequence to Leu 47 inLucina Hb II.     

The Vitreoscilla hemoglobin gene: Molecular cloning, nucleotide sequence and genetic expression in Escherichia coli

Summary Vitreoscilla hemoglobin is involved in oxygen metabolism of this bacterium, possibly in an unusual role for a microbe. We have isolated the Vitreoscilla hemoglobin structural gene from a pUC19 genomic library using mixed oligodeoxy-nucleotide probes based on the reported amino acid sequence of the protein. The gene is expressed in Escherichia coli from its natural promoter as a major cellular protein. The nucleotide sequence, which is in complete agrecment with the known amino acid sequence of the protein, suggests the existence of promoter and ribosome binding sites with a high degree of homology to consensus E. coli upstream sequences. In the case of at least some amino acids, a codon usage bias can be detected which is different from the biased codon usage pattern in E. coli. The down-stream sequence exhibits homology with the 3 end sequences of several plant leghemoglobin genes. E. coli cells expressing the gene contain greater than fivefold more heme than controls.     

Molecular cloning and the cDNA-derived amino acid sequence of Narcissus tazetta isolectins

Recently several complete cDNAs encoding the Narcissus tazetta lectins (NTL) were cloned. The sequence analyses of the cloned DNAs reveal that there are at least three unidentical positive clones for NTLs. The primary structure of the three NTL clones contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension beyond the C-terminal amino acids Thr-Gly. There are two fixed-position cysteines within the protein domain (amino acids 29 and 52), which are probably involved in the disulfide-bond linkage within the molecules to confer the secondary structure of the mature lectin. One third of the deduced amino acid composition consisted of glycine, leucine, and asparagine. From the cDNA-derived amino acid sequences the three NTL clones are not identical and are suggested to be isolectins present in N. tazetta var. chinensis. This study further confirms the previous isolation of mannose-specific isolectins from Chinese daffodil leaves [Ooi et al. (2000), J. Protein Chem. 19, 163-168].     

Abalone myoglobins evolved from indoleamine dioxygenase: The cDNA-derived amino acid sequence of myoglobin fromNordotis madaka

The cDNA for the unusual 41 kD myoglobin of the abaloneNordotis madaka was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 378 residues was determined. As with the myoglobin of the related abaloneSulculus diversicolor (Suzuki and Takagi,J. Mol. Biol. 228, 698–700, 1992), the sequence ofNordotis myoglobin showed no significant homology with any other globins, but showed high homology (35% identity) with vertebrate indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. The amino acid sequence homology betweenNordotis andSulculus myoglobins was 87%. These results support our previous idea that the abalone myoglobins evolved from a gene for indoleamine dioxygenase, but not from a globin gene, and therefore all of the hemoglobins and myoglobins are not homologous. Thus, abalone myoglobins appear to be a typical case of convergent evolution.     

Complete amino acid sequence of theGlycera dibranchiata monomer hemoglobin Component IV: Structural implications

The globin derived from the monomer Component IV hemoglobin of the marine annelid,Glycera dibranchiata, has been completely sequenced, and the resulting information has been used to create a structural model of the protein. The most important result is that the consensus sequence of Component IV differs by 3 amino acids from a cDNA-predicted amino acid sequence thought earlier to encode the Component IV hemoglobin. This work reveals that the histidine (E7), typical of most heme-containing globins, is replaced by leucine in Component IV. Also significant is that this sequence is not identical to any of the previously reportedGlycera dibranchiata monomer hemoglobin sequences, including the sequence from a previously reported crystal structure, but has high identity to all. A three-dimensional structual model for monomer Component IV hemoglobin was constructed using the published 1.5 å crystal structure of a monomer hemoglobin fromGlycera dibranchiata as a template. The model shows several interesting features: (1) a Phe31 (B10) that is positioned in the active site; (2) a His39 occurs in an interhelical region occupied by Pro in 98.2% of reported globin sequences; and (3) a Met41 is found at a position that emerges from this work as a previously unrecognized heme contact.Abbreviations used GMHX the holo-protein (including b-type heme, Glycera dibranchiata monomer hemoglobin Component X (X=2, 3, or 4) - GMGX the apo-protein, or globin, Glycera dibranchiata monomer globin derived from Component X (X=2, 3, or 4) - rec-gmg the globin derived from a recombinant holoprotein of a Glycera dibranchiata monomer hemoglobin, rec-gmh, whose sequence has been inferred from an isolated cDNA insert - CB label refers to peptides generated from cyanogen bromide cleavage of GMG4 - HPLC high-performance liquid chromatography - T label refers to peptides generated from trypsin digests of GMG4 - Mb myoglobin - MCS monomer hemoglobin crystal structure from Glycera dibranchiata. H, N-terminal sequence of GMG4 - SWMb sperm whale myoglobin     

沙田柚无机焦磷酸酶基因的cDNA克隆及序列分析

植物自交不亲和性是植物生殖过程中普遍存在的一种现象,是植物特异性识别并拒绝自身花粉或亲缘关系很相近的花粉的一种遗传机制。无机焦磷酸酶(inorganic pyrophosphatase,IPPase)在植物生长发育方面起重要作用。该研究根据沙田柚花柱消减文库中EST序列(无机焦磷酸酶基因内部片段),设计了2对特异引物5'-GSP1,5'-n GSP1,3'-GSP2 and 3'-n GSP2,通过SMART-RACE PCR技术从所构建的沙田柚花柱抑制性消减文库中克隆了沙田柚无机焦磷酸酶基因的c DNA全长序列,利用Blastn、DNAman和Expasy软件对所克隆的基因进行同源性分析,以及基因编码的氨基酸的分子量、等电点、疏水性等理化性质分析。结果表明:IPPase基因c DNA全长为1 136 bp(Gen Bank登录号为KF990474),开放阅读框(ORF)全长为654 bp,共编码217个氨基酸,包括170 bp 5'UTR和312 bp的3'UTR;编码的蛋白质的分子量为24.4 k Da,等电点为5.96;蛋白结构域分析显示沙田柚IPPase与焦磷酸酶具有相同的保守结构域;对沙田柚IPPase蛋白质序列进行疏水性分析,结果表明沙田柚IPPase基因编码的肽链中疏水性最大值约为3.21,最小值约为-2.98,属于亲水性蛋白,无跨膜区域;Blastn搜索的结果显示,沙田柚IPPase基因序列与多种植物的IPP基因高度同源;序列分析表明,沙田柚IPPase基因核苷酸的同源性与毛果杨(Populus trichocarpa)和橡胶树(Hevea brasiliensis)IPPase基因均为87%;氨基酸序列与克莱门柚(Citrus clementina)无机焦磷酸酶完全一致。该研究结果可为深入研究无机焦磷酸酶在沙田柚自交不亲和中的作用机理提供基础。     

Gastropod arginine kinases from Cellana grata and Aplysia kurodai. Isolation and cDNA-derived amino acid sequences

Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a K_m value of 0.6 mM for arginine and a V_max value of 13 μmole Pi min⁻¹ mg protein⁻¹ for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1608 and 1239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66–73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63–71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55–57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered.     

The complete amino acid sequence of ribosomal protein S8 fromThermus thermophilus

Protein S8 fromThermus thermophilus consists of 138 amino acids ofM, 15,840. Its primary structure was established using peptide sequences from two different digests. Protein S8 fromT. thermophilus shares a high percentage of identity with protein S8 fromThermus aquaticus. There are some consensus sequences between proteins S8 from eubacteria, archebacteria, chloroplasts, and cyanelles.     

The amino acid sequence of glutamate dehydrogenase fromPyrococcus furiosus, a hyperthermophilic archaebacterium

The complete amino acid sequence of glutamate dehydrogenase from the archaebacteriumPyrococcus furiosus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage with cyanogen bromide, Asp-N endoproteinase, trypsin, or pepsin. The enzyme subunit is composed of 420 amino acid residues yielding a molecular mass of 47,122 D. In the recently determined primary structure of glutamate dehydrogenase from another thermophilic archaebacterium,Sulfolobus solfataricus, the presence of some methylated lysines was detected and the possible role of this posttranslational modification in enhancing the thermostability of the enzyme was discussed (Maras, B., Consalvi, V., Chiaraluce, R., Politi, L., De Rosa, M., Bossa, F., Scandurra, R., and Barra, D. (1992),Eur. J. Biochem. 203, 81–87). In the primary structure reported here, such posttranslational modification has not been found, indicating that the role of lysine methylation should be revisited. Comparison of the sequence of glutamate dehydrogenase fromPyrococcus furiosus with that ofS. solfataricus shows a 43.7% similarity, thus indicating a common evolutionary pathway.     

Prorocentrum lima (Dinophyceae) in northeastern USA coastal waters: II: Toxin load in the epibiota and in shellfish

The seasonal variation in diarrhetic shellfish poisoning (DSP)-type toxins was followed in the epibiotic community and in shellfish between 41° and 44°N in coastal waters of the northwest Atlantic during a 2-year period. Low levels of okadaic-acid equivalents were detected at all stations in the <90 μm fraction of the collected epibiota as measured by the protein phosphatase inhibition assay, but only 3.5% of the samples had values greater than 100 ng (g dry weight of epibiota)⁻¹. No seasonal pattern could be detected due to differences in intensity, duration and timing of toxin content in the epibiota between the 2 years and between stations. Nevertheless, the concentration of DSP-type toxins in the epibiota correlated weakly but significantly with the abundance of Prorocentrum lima, when data from all stations were considered. A very limited toxin uptake by shellfish was measured at only one station in October and November 2001 and in June and July 2002 at times of maximum cell concentration of P. lima in the epibiota. Toxin levels in shellfish remained well below regulatory limits that would have required quarantine or bans on harvesting. Results from our 2-year survey suggest that, at this time, the threat of DSP events appears minimal. However, the presence of a known toxin producer and its demonstrated ingestion by shellfish would argue for further studies to better understand conditions leading to DSP outbreaks generated by an epiphytic dinoflagellate.     

Epiphytic abundance and toxicity of Prorocentrum lima populations in the Fleet Lagoon, UK

Planktonic Dinophysis spp. and epiphytic Prorocentrum lima (Ehrenberg) Dodge are known dinoflagellate producers of okadaic acid (OA) and dinophysistoxins (DTX), causative phycotoxins of diarrhetic shellfish poisoning (DSP). Underestimation of toxic dinoflagellates associated with a toxic event may be due to the lack of sampling of species with epiphytic and epibenthic strategies, such as P. lima. As Dinophysis spp. is not found in the Fleet Lagoon, Dorset, but previous DSP events have closed the Crassostrea gigas oyster farm, P. lima is the most likely causative organism. A field assay for separating microalgal epiphytes and concentrating wild cells on to filters was successfully applied to sub-samples of a variety of macroalgae and macrophytes (seagrass) collected from the Fleet during summer 2002. P. lima was present in increasing cell densities on most substratum species, over the sampling period, from 10² to 10³ cells g⁻¹ fresh weight (FW) plant biomass. LC–MS analysis detected OA and DTX-1 in extracts of wild P. lima cells, in ratios characteristic of P. lima strains previously isolated from the Fleet. No toxins, however, were detected in oyster flesh.     

Amino acid sequence of myoglobin from the molluscBursatella leachii

The complete amino acid sequence of myoglobin from the triturative stomach of gastropodic molluscBursatella leachii has been determined. It is composed of 146 amino acid residues, is acetylated at the N-terminus, and contains a single histidine residue at position 95 which corresponds to the heme-binding proximal histidine. The E7 distal histidine, which is conserved widely in myoglobins and hemoglobins, is replaced by valine inBursatella myoglobin. The amino acid sequence ofBursatella myoglobin shows strong homology (73–84%) with those ofAplysia andDolabella myoglobins.     

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO

Summary The gene braB, encoding the Na^–-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na^–-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated M_r of 45279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with thebrnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.