A protein with antimicrobial activity in the skin of Schlegel's green tree frog Rhacophorus schlegelii (Rhacophoridae) identified as histone H2B

An extract of the skin of Schlegel's green tree frog, Rhacophorus schlegelii (Anura: Rhacophoridae), contained a protein that inhibited the growth of the Gram-negative bacterium Escherichia coli but was inactive against the Gram-positive bacterium Staphylococcus aureus. The protein was purified to near homogeneity by reverse-phase HPLC and amino acid sequence analysis of the products of an endoproteinase Glu-C digest identified the protein as histone H2B. The complete primary structure of the 125 amino acid residue Rhacophorus histone H2B was determined by nucleotide sequence analysis of a cloned cDNA encoding the protein. Mass spectrometry demonstrated that the protein isolated from the skin was not post-translationally modified. Histone fragments with antimicrobial activity were not identified in the Rhacophorus skin extract nor were cationic, alpha-helical antimicrobial peptides of the kind isolated from the skins of several other frog families. The data provide further evidence that histones play a role in the defense against microorganisms.     

Tetrahymena HMG nonhistone chromosomal protein. Isolation and amino acid sequence lacking the N- and C-terminal domains of vertebrate HMG 1

The complete amino acid sequence of a high mobility group (HMG) nonhistone chromosomal protein of the ciliated protozoan Tetrahymena pyriformis (GL strain) was determined. This protein was extracted with 0.5 M HClO4 together with histone H1 (molar ratio 1:1) from the whole histone extract, then purified by gel filtration and reverse-phase HPLC. The HMG protein showed a single electrophoretic band on SDS gel electrophoresis. The amino acid sequence was determined by Edman degradation of intact protein, BrCN fragments, and their staphylococcal protease and tryptic peptides. Thus the total sequence, consisting of 99 amino acid residues and having a molecular weight of 11,626, was completely determined. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that this HMG protein was phosphorylated at two positions, each 6-7%, and contained 0.15 mol phosphate/mol protein. This Tetrahymena HMG is rather similar to the central part of vertebrate HMG 1 in terms of the amino acid sequence and the hydropathy profile.     

Identification of the phosphoserine residue in histone H1 phosphorylated by protein kinase C

The site-specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N-bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71-122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97-Thr-Gly-Ala-Ser-Gly-Ser(PO4)-Phe-Lys105, supports the contention that basic amino acid residues C-terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.     

Structure of a cluster of mouse histone genes.

The four mouse histone genes (2 H3 genes, an H2b gene and an H2a gene) present in a cloned 12.9 kilobase fragment of DNA have been completely sequenced including both 5' and 3' flanking regions. These genes are expressed in cultured mouse cells and the 3' and 5' ends of the mRNA have been determined by S1 nuclease mapping. These genes code for a minor fraction of the histone mRNAs expressed in cultured mouse cells. They comprise at most 5-8% of the total histone mRNA of each type. The two H3 genes code for H3.2 and H3.1 histone proteins, while the H2b gene codes for an H2b.1 protein with a single amino acid change (val-leu) at position 18. Only the 3' portion of the H2a gene is contained in the clone and there is an amino acid change (alanine-proline) at position 126. Comparison of the 5' and 3' flanking sequences reveals a conserved sequence at the 3' end of the mRNA which forms a hairpin loop structure. The codon usage in the genes is non-random and there has been no discrimination against CG doublets in the coding region of the genes.     

Purification of Chlamydomonas 28-kDa ubiquitinated protein and its identification as ubiquitinated histone H2B.

One of the most predominantly ubiquitinated protein species in Chlamydomonas, of which the apparent molecular mass in SDS-PAGE was 28 kDa, was found to exist abundantly in nuclei. The 28-kDa ubiquitinated protein was purified to homogeneity from the isolated nuclei of Chlamydomonas, and its partial amino acid sequence was determined. The N-terminal peptide sequence was identical with that of ubiquitin. Sequences homologous to those Chlamydomonas ubiquitin [corrected] and wheat histone H2B, and paired sequences of both of them were found in arginylendopeptidase-digested or protease V8-digested polypeptide fragments of the 28-kDa ubiquitinated protein. Based on these results, it was concluded that Chlamydomonas 28-kDa ubiquitinated protein is monoubiquitinated histone H2B.     

Pterocarpan phytoalexin biosynthesis in elicitor-challenged chickpea (Cicer arietinum L.) cell cultures. Purification, characterization and cDNA cloning of NADPH:isoflavone oxidoreductase

NADPH:isoflavone oxidoreductase (IFR) is the first soluble enzyme of the pterocarpan-specific part of phytoalexin biosynthesis in chickpea (Cicer arietinum L.). The enzyme was purified to apparent homogeneity by a five-step procedure from chickpea cell cultures treated with yeast extract as elicitor. Analysis by gel filtration and SDS/PAGE showed that the enzyme consists of a single polypeptide with a molecular mass of 36 kDa. Km values for the substrates 2'-hydroxyformononetin, 2'-hydroxypseudobaptigenin and NADPH were 6, 6 and 20 microM, respectively. The IFR showed pronounced specificity for the substitution pattern of isoflavones. We found a 2'-hydroxy group and a 4',5'-methylenedioxy or 4'-methoxy function to be essential for acceptance as substrate. The isoelectric point of the protein was determined as 6.3 by IEF and there is no evidence for the existence of isoenzymes. Partial amino acid sequences of IFR were determined from internal peptides obtained by tryptic digestion of the protein and corresponding oligonucleotides were synthesized. A lambda gt10 cDNA library was constructed using poly(A)-rich RNA isolated from chickpea cell cultures treated with Ascochyta rabiei elicitor. 150 positive clones were obtained by screening 2 x 10(5) clones with an IFR-specific oligonucleotide. The identity of sequenced clones was confirmed by comparison of the deduced amino acid sequence with the internal peptide sequences of purified IFR. The sequence of a 1183-bp clone contained a continuous open reading frame of 954 bases encoding a polypeptide of 318 amino acids with a calculated molecular mass of 35.4 kDa, indicating that a full-length cDNA coding for IFR was isolated.     

Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha.

A gene library from the methanol utilizing yeast Hansenula polymorpha, constructed in a lambda Charon4A vector, was used to clone the gene encoding a key methanol assimilating enzyme, dihydroxyacetone synthase (DHAS) by differential plaque hybridization. The nucleotide sequence of the 2106 bp structural gene and the 5' and 3' non-coding regions was determined. The deduced amino acid sequence of the protein is in agreement with the apparent molecular weight and amino acid composition of the purified protein. The codon bias is not so pronounced as in some Saccharomyces cerevisiae genes.     

The amino acid sequence of nucleoside diphosphate kinase I from spinach leaves, as deduced from the cDNA sequence.

The primary structure of nucleoside diphosphate (NDP) kinase from spinach leaves has been deduced from its cDNA sequence. A lambda gt 11 cDNA library derived from spinach leaves was screened using an antibody against NDP kinase I, which we previously purified to electrophoretic homogeneity (T. Nomura, T. Fukui, and A. Ichikawa, 1991, Biochim. Biophys. Acta 1077, 47-55). The cDNA sequences of positive clones contained the amino acid coding region (444 base pairs) for NDP kinase I as well as 5' and 3' noncoding regions of 33 and 361 base pairs, respectively. The cDNAs hybridized to a 1.1-kb mRNA. NDP kinase I contains 148 amino acid residues with a molecular mass of 16,305, which is in excellent agreement with that of the purified enzyme (16 kDa). Homology was found between the sequence of spinach NDP kinase I and those of the rat, Myxococcus xanthus, and Dictyostelium discoideum NDP kinases, as well as the human Nm23-gene product and the awd protein of Drosophila melanogaster.     

The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins

Ch21, a developmentally regulated low molecular weight protein observed in chick embryo skeletal tissues, is expressed "in vitro" by differentiating chondrocytes at a late stage of development. Here we report the complete amino acid sequence of the protein. 86% of the total amino acid sequence was deduced by sequences of 17 high performance liquid chromatography-separated proteolytic fragments and 33 amino acid residues at the amino-terminal end of protein purified from spent culture medium of hypertrophic chondrocytes. Furthermore we isolated by molecular cloning the corresponding cDNA and determined its nucleotide sequence. By combining protein and nucleotide sequence data we determined the primary structure of the entire Ch21. It consists of 158 amino acids and has a molecular mass of 18.065 kDa. Computer-assisted analysis showed that the Ch21 belongs to the superfamily of low molecular weight proteins sharing a basic framework for binding and transport of small hydrophobic molecules.     

Purification and characterization of histone H1 from meiotic cells of a monocotyledonous plant, Lilium longiflorum.

To identify molecules involved in regulating meiotic chromatin structure, nuclear proteins from meiocytes of Lilium longiflorum were chromatographed on hydroxylapatite and the bound and unbound proteins were examined. An abundant nuclear protein was purified from the unbound fraction and by the following criteria was identified as a histone H1 molecule. The protein is soluble in acidic (perchloric and sulfuric acid) solutions, and its amino acid composition and the sequence of its amino terminus are similar to that of known histone H1s. An antiserum was produced against the protein to facilitate subsequent studies. Immunoblotting experiments demonstrated that histone H1 immunostaining declines in the developmental interval spanning the diplotene to tetrads stages. Concommitant with this decline is the appearance of several lower molecular mass, cross-reacting proteins. Neither the identity nor roles of these proteins is known. Immunoblotting experiments also demonstrate that, while the level of the protein is relatively constant in nuclei prepared from meiotic and vegetative cells, histone H1 is apparently enriched in total cellular extracts of meiotic cells compared with vegetative cells. This difference was found to be at least 16-fold. I conclude that in meiotic cells, histone H1 accounts for more of the total cellular protein than it does in vegetative cells. The difference in its relative abundance as a percent of the total cellular protein is probably in part due to differences in the ratio of nuclear to cytoplasmic volume in the different cell types, or the purging of sporophytic proteins from the cytoplasm of the meiocytes, or both.     

巴西橡胶树43 kD橡胶粒子膜蛋白基因的cDNA克隆及表达

对43 kD的橡胶粒子膜蛋白进行了分离纯化和其N端氨基酸序列分析,根据N端氨基酸序列,设计一简并引物,通过3'RACE(Rapid Amplification ofcDNA Ends)的方法,获得了43 kD的橡胶粒子膜蛋白的cDNA.该cDNA含有1 385个核苷酸,含有完整的阅读框架,编码381个氨基酸.在终止密码子下游,包含有一个239bp的3'非编码区.该cDNA由5个首尾相连的重复单元组成,每个单元编码76个氨基酸组成的泛素(ubiquitin)单体.编码43 kD橡胶粒子蛋白的基因具有多个拷贝,在胶乳、叶片和树皮都表达.     

The structural organization and DNA sequence of a wheat histone H4 gene.

Some wheat histone H4 genes have been cloned from a Charon 4 wheat genomic DNA library using sea urchin histone H4 DNA as a probe. DNA sequence analysis of a cloned gene showed that the deduced amino acid sequence of wheat histone H4 protein was identical to that of pea. The 5' end of wheat histone H4 mRNA was mapped on the cloned gene by the S1-procedure. Southern blotting analysis of the genomic DNA indicated that histone H4 genes were reiterated 100 to 125 times per hexaploid wheat genome.     

PL-I of Spisula solidissima, a highly elongated sperm-specific histone H1

The major chromosomal protein of the mature sperm of the surf clam, Spisula solidissima, is a histone H1-related protamine-like (PL-I) protein of low electrophoretic mobility. We report here the complete sequence of two isoforms of its encoding genes. These genes encode a protein of 453 and 454 amino acids, respectively. The predicted mass of the larger isoforms (51,437 Da) was confirmed using electrospray ionization mass spectrometry. The amino-terminal tail of the S. solidissima PL-I is greatly elongated because of the presence of 39 tandem hexapeptide repeats of the motif (K/R)KRSAS with a few semiconservative amino acid substitutions. These repeats are very closely mirrored by their encoding DNA sequence, which indicates that an expansion because of sequence duplication most likely occurred. The C-terminal domain consists of a histone H1-related core with a predicted winged-helix tertiary structure, which is followed by an unstructured lysine-rich tail. This information provides additional molecular support for the classification and underlying evolution of sperm nuclear basic proteins in bivalve molluscs.     

Isolation, amino acid sequence, and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin

A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.     

Primary structure of rat liver 5'-nucleotidase deduced from the cDNA. Presence of the COOH-terminal hydrophobic domain for possible post-translational modification by glycophospholipid

Rat liver 5'-nucleotidase was purified from a crude microsomal fraction, and its molecular mass was estimated to be 73 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein was subjected to cleavage with CNBr or lysyl endopeptidase, and the resulting 21 peptides as well as the NH2 terminus of the native protein were sequenced by Edman degradation. For further information on the molecular structure, we constructed a lambda gt11 liver cDNA library and isolated two cDNA clones for 5'-nucleotidase, lambda cNTP6 and lambda cNT34. The 3.2-kilobase cDNA insert of lambda cNTP6 contains an open reading frame that encodes a 576-residue polypeptide with a calculated size of 63,965 Da, which is in reasonable agreement with that of 5'-nucleotidase (62 kDa) immunoprecipitated from cell-free translation products. The NH2-terminal 28 residues comprise a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. The predicted structure contains all the other peptide sequences determined by Edman degradation. Five potential N-linked glycosylation sites are found in the molecule, accounting for the difference in mass between the precursor and mature forms. Another characteristic feature is that the primary structure contains a highly hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophospholipid. In fact, labeling experiments of rat hepatocytes demonstrated that 3H-labeled compounds such as ethanolamine, myo-inositol, and palmitic acid, components of the glycolipid anchor, were incorporated into 5'-nucleotidase. Phosphatidylinositol-specific phospholipase C released 5'-nucleotidase from the cell surface, and the released protein no longer contained the radioactivity of [3H]palmitic acid incorporated.     

Purification, characterization, and molecular cloning of tyrosinase from Pholiota nameko

Tyrosinase (monophenol, 3,4-dihydroxy L-phenylalanine (L-DOPA):oxygen oxidoreductase, EC 1.14.18.1) was isolated from fruit bodies of Pholiota nameko and purified to homogeneity. The purified enzyme was a monomer with a molecular weight of 42,000 and contained 1.9 copper atoms per molecule. The N-terminal of the purified enzyme could not be detected by Edman degradation, probably due to blocking, while the C-terminal sequence of the enzyme was determined to be -Ala-Ser-Val-Phe-OH. The amino acid sequence deduced by cDNA cloning was made up of 625 amino acid residues and contained two putative copper-binding sites highly conserved in tyrosinases from various organisms. The C-terminal sequence of the purified enzyme did not correspond to that of the deduced sequence, but agreed with Ala384-Ser385-Val386-Phe387 in sequence. When the encoded protein was truncated at Phe387, the molecular weight of the residual protein was calculated to be approximately 42,000. These results suggest that P. nameko tyrosinase is expressed as a proenzyme followed by specific cleavage to produce a mature enzyme.     

Amino acid sequence of rat thymus histone H2B and identification of the in vitro phosphorylation sites.

The amino acid sequence of rat thymus histone obtained in highly purified form by preparative electrophoresis, was determined. This sequence is identical to the sequence of calf thymus histone H2B. The in vitro phosphorylation of the rat histone with a cyclic AMP-dependent protein kinase isolated from rat pancreas led to the identification of four sites of phosphorylation: two major ones, at serine residues 32 and 36, and two minor ones, specific of the rat protein kinase, at serine residues 87 and 91.     

Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator.

A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred.