Differential expression of three galaxin-related genes during settlement and metamorphosis in the scleractinian coral Acropora millepora

Background  

The coral skeleton consists of CaCO₃ deposited upon an organic matrix primarily as aragonite. Currently galaxin, from Galaxea fascicularis, is the only soluble protein component of the organic matrix that has been characterized from a coral. Three genes related to galaxin were identified in the coral Acropora millepora.     

Purification and Structural Determination of a Phosphorylated Peptide with Anti-calcification and Chitin-binding Activities in the Exoskeleton of the Crayfish,Procambarus clarkii

Organic matrices in calcified hard tissues have been considered to control calcification. A matrix peptide, designated CAP-1, was extracted and purified by anionexchange and reverse-phase high performance liquid chromatographies from the exoskeleton of the crayfish, Procambarus clarkii. The amino acid sequence of CAP-1 was determined by mass spectral and sequence analyses of the intact peptide and its enzymatically digested peptides. CAP-1 consisted of 78 amino acid residues, including a phosphoserine residue, and was rich in acidic amino acid residues. CAP-1 had a RebersRiddiford consensus sequence, which is conserved in cuticle proteins from many arthropods. CAP-1 inhibited precipitation of calcium carbonate in an in vitro anticalcification assay dose-dependently, and completelyinhibited it at 3×10^-7 M. CAP-1 also showed chitinbinding ability, indicating that this molecule was bifunctional and played an important role in formation of the exoskeleton.     

Studies on Two Closely Related Species of Octocorallians: Biochemical and Molecular Characteristics of the Organic Matrices of Endoskeletal Sclerites

Two species of alcyonarian corals, Lobophytum crassum and Sinularia polydactyla, are closely related to each other. It is reported that the calcified organic substances in the skeletons of both contain a protein–polysaccharide complex playing a key role in the regulation of biocalcification. However, information on the matrix proteins of endoskeletal sclerite has been lacking. Hence we studied the proteinaceous organic matrices of sclerites for both species, to analyze the sequences and the functional properties of the proteins present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed four bands of proteins with apparent molecular masses of 102, 67, 48, and 37 kDa for L. crassum and seven bands of 109, 83, 70, 63, 41, 30, and 22 kDa for S. polydactyla. A major protein band of about 67 kDa in L. crassum and two bands of proteins of about 70 and 63 kDa in S. polydactyla yielded N-terminal amino acid sequences. Periodic acid-Schiff staining indicated that the 67-kDa protein in L. crassum, and 83- and 63-kDa proteins in S. polydactyla were glycosylated. For detection of calcium binding proteins, a Ca²⁺ overlay analysis was conducted in the extract via ⁴⁵Ca autoradiography. The 102- and 67-kDa calcium binding proteins in L. crassum, and the 109- and 63-kDa Ca²⁺ binding proteins in S. polydactyla were found to be radioactive. An assay for carbonic anhydrase (CA), which is thought to play an important role in the process of calcification, revealed specific activities. Newly derived protein sequences were subjected to standard sequence analysis involving identification of similarities to other proteins in databases. The significantly different protein expressions and compositional analysis of sequences between two species were demonstrated.     

Biochemical analyses of the cement float of the goose barnacle Dosima fascicularis – a preliminary study

The goose barnacle Dosima fascicularis produces an excessive amount of adhesive (cement), which has a double function, being used for attachment to various substrata and also as a float (buoy). This paper focuses on the chemical composition of the cement, which has a water content of 92%. Scanning electron microscopy with EDX was used to measure the organic elements C, O and N in the foam-like cement. Vibrational spectroscopy (FTIR, Raman) provided further information about the overall secondary structure, which tended towards a β-sheet. Disulphide bonds could not be detected by Raman spectroscopy. The cystine, methionine, histidine and tryptophan contents were each below 1% in the cement. Analyses of the cement revealed a protein content of 84% and a total carbohydrate content of 1.5% in the dry cement. The amino acid composition, 1D/2D-PAGE and MS/MS sequence analysis revealed a de novo set of peptides/proteins with low homologies with other proteins such as the barnacle cement proteins, largely with an acidic pI between 3.5 and 6.0. The biochemical composition of the cement of D. fascicularis is similar to that of other barnacles, but it shows interesting variations.     

Characterization of proteins from the matrix of spicules from the alcyonarian, Lobophytum crassum

The organic matrix of spicules of the alcyonarian coral, Lobophytum crassum, was studied to investigate its molecular characteristics and functional properties. The shape of the spicules was identified using scanning electron microscopy. The soluble organic matrix comprised 0.03% of the spicule weight. The SDS-PAGE analysis of the preparation showed four protein bands with apparent molecular weights of 37, 48, 67 and 102 kDa. The 67- and 102-kDa proteins appeared to be calcium binding proteins, detected as radioactive bands by ⁴⁵Ca autoradiography. The 67-kDa protein appears to be glycosylated. The N-terminal amino acid sequence of the 67 kDa was determined; 7 of 20 residues were acidic. A database search for homologous proteins did not give a clear indication of the function of the 67-kDa protein. The isolated organic matrix possesses carbonic anhydrase activity which functions in calcium carbonate crystal formation, indicating that organic matrix is not only structural protein but also a catalyst. An interpretation of these results is that the spicule of alcyonarian corals has a proteinaceous organic matrix related to the calcification process.     

Soluble organic matrix of two Scleractinian corals: partial and comparative analysis

This study is a biochemical and molecular analysis of the soluble organic matrix (SOM) of two Scleractinian corals differing in their morphological characteristics: Stylophora pistillata, a branched robust coral and Pavona cactus, a leafy complex coral. Soluble organic matrix of both coral species were shown to contain high amounts of potentially acidic amino acids and glycine. However, proportions of glycosaminoglycans and SDS-PAGE analyses of soluble organic matrix proteins were very different. Three proteins of S. pistillata and at least five proteins of P. cactus were detected by silver staining, some of them being able to bind calcium. Internal peptide sequences of two matrix proteins (one from each species) were obtained. One sequence of S. pistillata is unusual because it contains a long poly-aspartate domain, as described in proteins belonging to the calsequestrin family and in proteins from molluscan species. This domain suggests an essential role for this protein in the control of mineralization.     

Molecular analysis of the phytochrome deficiency in an aurea mutant of tomato

Summary The au ^w mutant allele of the aurea locus in tomato has previously been shown to cause deficiency for the phytochrome polypeptide (Parks et al. 1987). We have begun to characterize the molecular basis and consequences of this deficiency. Genomic Southern blot analysis indicates that there are at least two and probably more phytochrome polypeptide structural genes in tomato. RNA blot analysis shows that the au ^w mutant contains normal levels of phytochrome mRNA and in vitro translation of au ^w poly(A)⁺ RNA yields a phytochrome apoprotein that is quantitatively and qualitatively indistinguishable on SDS-polyacrylamide gels from that synthesized from wild-type RNA. These results indicate that the phytochrome deficiency in aurea is not the result of lack of expression of phytochrome genes but is more likely due to instability of the phytochrome polypeptide in planta. Possible reasons for such instability are discussed. Analysis of the molecular phenotype of aurea indicates that the phytochrome-mediated increase in the abundance of the mRNA encoding chlorophyll a/b binding protein (cab) is severely restricted in the mutant as compared with wild-type tomato. Thus, the au w strain exhibits defective photoregulation of gene expression consistent with its very reduced level of the phytochrome photoreceptor.     

Purification and structural determination of a phosphorylated peptide with anti-calcification and chitin-binding activities in the exoskeleton of the crayfish, Procambarus clarkii

Organic matrices in calcified hard tissues have been considered to control calcification. A matrix peptide, designated CAP-1, was extracted and purified by anion-exchange and reverse-phase high performance liquid chromatographies from the exoskeleton of the crayfish, Procambarus clarkii. The amino acid sequence of CAP-1 was determined by mass spectral and sequence analyses of the intact peptide and its enzymatically digested peptides. CAP-1 consisted of 78 amino acid residues, including a phosphoserine residue, and was rich in acidic amino acid residues. CAP-1 had a Rebers-Riddiford consensus sequence, which is conserved in cuticle proteins from many arthropods. CAP-1 inhibited precipitation of calcium carbonate in an in vitro anticalcification assay dose-dependently, and completely inhibited it at 3 x 10(-7) M. CAP-1 also showed chitin-binding ability, indicating that this molecule was bifunctional and played an important role in formation of the exoskeleton.     

Analysis of expression and chitin‐binding activity of the wing disc cuticle protein BmWCP4 in the silkworm,Bombyx mori

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin‐binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic “R&R” chitin‐binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc‐containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full‐length BmWCP4 protein, “R&R” CBD peptide (CBD), non‐CBD peptide (BmWCP4‐CBD^?), four single site‐directed mutated peptides (M₁, M₂, M₃ and M₄) and four‐sites‐mutated peptide (M_F) were generated and purified, respectively, for in vitro chitin‐binding assay. The results indicated that both the full‐length protein and the “R&R” CBD peptide could bind with chitin, whereas the BmWCP4‐CBD^? could not bind with chitin. The single residue mutants M₁, M₂, M₃ and M₄ reduced but did not completely abolish the chitin‐binding activity, while four‐sites‐mutated protein M_F completely lost the chitin‐binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva‐to‐pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.     

不溶性基质中天冬氨酸丰富的蛋白在珊瑚的钙质骨片形成中的重要作用

一般认为, 酸性蛋白在控制矿物的形成和发展中发挥重要作用。因此, 在不溶性有机基质中鉴定酸性蛋白对于理解珊瑚中个体蛋白的功能是非常重要的一步。在短指多型软珊瑚(Sinularia polydactyla)的可溶性和不溶性基质层中分析蛋白组分表明, 在不溶性基质和可溶性基质层中天冬氨酸的含量分别是61%和29%。利用体外分析法发现, 基质蛋白诱导碳酸钙形成非晶态析出相先于其形成钙质的结晶态。利用X-射线衍射来鉴定骨片上结晶态的碳酸钙, 结果表明钙质的多晶态呈现强反射。傅利叶变换红外光谱分析表明珊瑚基质中富含天冬氨酸的蛋白和多醣的结构。在不溶性基质组分中用钙离子结合分析显示一个分子量为109 kD的蛋白质可以与形成骨片的钙离子结合, 这一过程对骨片形成非常重要。在对生物钙化过程中起重要作用的碳酸酐酶的分析中显示了此酶的新颖的活性。以上结果显示珊瑚中不溶性基质内的富含天冬氨酸的蛋白在生物矿化调控过程中起重要作用。     

Characterization and localization of repetitive DNA sequences in the ornamental Alstroemeria aurea Graham

 Three repetitive DNA sequences were isolated from a genomic DNA library of the ornamental Alstroemeria aurea Graham. Two repeats, A001-I and A001-II, were quite homologous and highly A. aurea-specific. A001-I was a 217-bp sequence with several telomeric TTTAGGG repeats at the 5′ end and a unique sequence of 98 bp at the other end. The third repeat, A001-IV, was a 840-bp sequence which contained two sub-sequences of 56 and 74 bp respectively, previously found in chloroplast (cp) DNA of tobacco and spinach and to a lesser extent in the cpDNA of maize and rice. Repeat A001-IV was not species-specific and its hybridization signal was weaker than the other repeats. Fluorescence in situ hybridization (FISH) revealed the A. aurea-specific repeats to be located in the heterochromatic regions of all A. aurea chromosomes. The differences in FISH pattern make them useful tools for karyotype analysis. The non-species-specific sequence A001-IV gave a dispersed signal over all the Alstroemeria chromosomes in an interspecific hybrid. The potential use of these repetitive DNA sequences for the study of phylogenetic relationships within the genus Alstroemeria is discussed. Received: 24 November 1996/Accepted: 20 December 1996     

The aurea mutant of tomato is deficient in spectrophotometrically and immunochemically detectable phytochrome

The aurea locus mutant (au ^w) of tomato contains less than 5% of the level of phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. Immunoblot analysis using antibodies directed against etiolated-oat phytochrome demonstrates that crude extracts of etiolated mutant tissue are deficient in a major immunodetectable protein (116 kDa) normally present in the parent wild type. Analyses of wild-type tissue extracts strongly indicate that the 116-kDa protein is phytochrome by showing that this protein: a) is degraded more rapidly in vitro after a brief far-red irradiation than after a brief red irradiation (Vierstra RD, Quail PH, Planta 156: 158–165, 1982); b) contains a covalently bound chromophore as detected by Zn-chromophore fluorescence on nitrocellulose blots; and c) has an apparent molecular mass comparable to phytochrome from other species on size exclusion chromatography under non-denaturing conditions. The demonstration that the aurea mutant is deficient in this 116-kDa phytochrome indicates that the lack of spectrally detectable phytochrome in this mutant is the result of a lesion which affects the abundance of the phytochrome molecule as opposed to its spectral integrity.     

Heat Shock Protein Produced by Helicobacter pylori

The cells of Helicobacter pylori were suspended in the medium containing³⁵S-methionine. After a heat shock of the cells at 42 C for 5, 10, and 30 min, the production of proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Out of many proteins produced by the cells, only 66 kDa protein production was dramatically increased by heat treatment. The N-terminal amino acid sequence of 66 kDa protein was quite similar to that of 62 kDa and 54 kDa proteins previously suggested as heat shock protein (HSP) of H. pylori based on the reaction with polyclonal and monoclonal antibodies against HSP 60 family proteins produced by other bacteria. Therefore, it was concluded that H. pylori produces the 66 kDa protein as its major heat shock protein which belongs to HSP 60 family.     

Characterization of a Matrix Protein in the Gastroliths of the Crayfish Procambarus clarkii

As a first step in understanding the calcification mechanism, a matrix protein in the gastrolith of the crayfish Procambarus clarkii was purified and sequenced. The protein was insoluble in acid, but after trypsin digestion, it dissolved in 6 m urea. The trypsin-digested protein dissolved in urea solution was purified by reversed-phase HPLC and designated gastrolith matrix protein fragment. The fragment had a molecular weight of 9658 and a blocked amino terminus. It had tandemly repeated units not reported before at the central part of the sequence, with each unit being Gly-Ser-X1-X2-Phe as the most typical sequence. This peptide was found associated with chitin, a main component of the organic matrix.     

A comparison of photolyase activity in three Australian tree frogs

Ultraviolet-B (UV-B) irradiation of DNA generates mutagenic photoproducts such as cyclobutane pyrimidine dimers (CPDs) which can affect the growth and development of amphibian embryos. Differential ability to repair UV-B-induced DNA damage may be␣responsible for differences in population stability between␣some amphibian species. Photoreactivation via the enzyme photolyase is a major mechanism used to remove CPDs from DNA. The aim of this study was to determine if photolyase activity differed in three sympatric Australian amphibian species, one of which has suffered marked population declines (Litoria aurea) and two whose populations do not appear to be in decline (L. dentata and L. peronii). The specific activity of photolyase was measured in each species and compared to the hatching success of their eggs under unfiltered summer sunlight. The mean specific activities of photolyase were 1.10 ± 0.18 × 10¹¹, 5.76 ± 1.01 × 10¹¹, and 2.66 ± 0.15 × 10¹¹ CPDs repaired per hour per microgram of egg protein extract, for L. aurea, L. dentata and L. peronii, respectively. When intrinsic differences in hatching success between species were controlled for, the relative percentage hatching success under unfiltered sunlight of L. aurea (77%) was lower than that of L.␣peronii (91%) and L. dentata (98%); however, these values did not differ significantly. L. aurea had the lowest photolyase activity of the three species and showed a non-significant trend of reduced hatching success under UV-B exposure. Received: 15 December 1997 / Accepted: 9 March 1998     

Dechlorination of Pesticides by a Specific Bacterial Glutathione S‐transferase,BphKLB400: Potential for Bioremediation

The bphK gene located in the bph operon of Burkholderia xenovorans LB400 encodes a protein, BphK^LB400, with significant sequence similarity to glutathione‐S‐transferases (GSTs). GSTs are a superfamily of enzymes involved in the detoxification of many endobiotic and xenobiotic substances. Recently, BphK^LB400 was shown to catalyze the dechlorination of a number of toxic chlorinated organic compounds. Comparison of the amino acid sequence of BphK^LB400 with GSTs from other bacteria that degrade polychlorinated biphenyls identified a number of highly conserved amino acids in the C‐terminal region of the protein thought to be associated with substrate specificity. Mutating the conserved amino acid at position 180 of BphK^LB400 from an alanine to a proline residue resulted in an increase in GST activity of bacterial cell extracts towards a number of chlorinated organic substrates tested including commonly used pesticides. Laboratory scale plant protection experiments suggested that E. coli expressing BphK^LB400 [wildtype and mutant (Ala180Pro)] could protect pea plants from the effects of chloromequat chloride. Therefore, BphK^LB400, identified as having dechlorination activity towards toxic chlorinated organic compounds used in the environment, could have potential in bioremediation.