Identification of four major hornet silk genes with a complex of alanine-rich and serine-rich sequences in Vespa simillima xanthoptera Cameron

Hornet silk, a fibrous protein in the cocoon produced by the larva of the vespa, is composed of four major proteins. In this study, we constructed silk-gland cDNA libraries from larvae of the hornet Vespa simillima xanthoptera Cameron and deduced the full amino acid sequences of the four hornet silk proteins, which were named Vssilk 1-4 in increasing order of molecular size. Portions of the amino acid sequences of the four proteins were confirmed by Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and N-terminal protein sequencing. The primary sequences of the four Vssilk proteins (1-4) were highly divergent, but the four proteins had some common properties: (i) the amino acid compositions of all four proteins were similar to each other in that the well-defined and characteristic repetitive patterns present in most of the known silk proteins were absent; and (ii) the characteristics of the amino acid sequences of the four proteins were also similar in that Ser-rich structures such as sericin were localized at both ends of the chains and Ala-rich structures such as fibroin were found in the center. These characteristic primary structures might be responsible for the coexisting alpha-helix and beta-sheet conformations that make up the unique secondary structure of hornet silk proteins in the native state. Because heptad repeat sequences of hydrophobic residue are present in the Ala-rich region, we believe that the Ala-rich region of hornet silk predominantly forms a coiled coil with an alpha-helix conformation.     

Amino acid analysis of spider dragline silk using H NMR

The amino acid composition of Nephila clavipes dragline silk fiber was determined by conducting ¹H nuclear magnetic resonance (NMR) spectroscopy experiments on acid-hydrolyzed material. N. clavipes dragline silk was found to consist of 43.0 ± 0.6% Gly, 29.3 ± 0.2% Ala, 9.1 ± 0.1% Glx, 4.0 ± 0.1% Leu, 3.3 ± 0.1% Tyr, 3.4 ± 0.2% Ser, 2.7 ± 0.1% Pro, 2.1 ± 0.1% Arg, 1.07 ± 0.05% Asx, 0.96 ± 0.05% Val, 0.48 ± 0.03% Thr, 0.35 ± 0.03% Phe, and 0.28 ± 0.03% Ile. Compared with standard chromatography-based amino acid analysis (AAA), the chemical resolution of NMR allows for an amino acid solution to be characterized without separation and is shown to provide considerably higher precision. This allows for more accurate statistics on the variability of amino acids in spider dragline silk. In general, this ¹H NMR AAA technique is applicable to a large range of proteins and peptides for precise composition characterization, especially when the precise content of a minor component is critical and relatively large amounts of sample are available (microgram to milligram quantities).     

Identification of a gene sequence encoding a putative pyruvate oxidoreductase in Serpulina pilosicoli

Serpulina pilosicoli is a recently described species of intestinal spirochaete which can be identified using a species-specific monoclonal antibody BJL/AC1 reactive with a 29-kDa protein located in the cell envelope. A genomic library of the type strain of S. pilosicoli P43/6/78^T was created in λ zap express™ and screened using BJL/AC1. Single positive clones were isolated and excised into the phagemid vector pBK-CMV. Phagemid DNA was purified and a single clone was selected for sequencing. The size of spirochaetal DNA insert was determined by digestion with restriction endonucleases EcoRI and PstI as being approximately 2.6 kb. The nucleotide sequence of the gene encoding the protein with which the antibody reacted was determined by cycle sequencing. The insert contained an open reading frame of 825 nucleotides. Translation of the nucleotide sequence into amino acid (aa) residues showed a sequence of 275 aa. Comparison of this sequence with databases revealed homology to pyruvate oxidoreductases from various organisms found in the gastrointestinal tract. These included the pyruvate ferredoxin oxidoreductase (POR) α subunit of Helicobacter pylori (38.8% identity in 250 aa), pyruvate-flavodoxin oxidoreductase of Escherichia coli (28.7% identity in 258 aa) and Giardia intestinalis (25.1% identity in 251 aa). A significant level of homology was also observed with hyperthermophilic bacteria such as the POR of Thermatoga maritima (38.6% in 254 aa) and the 2-ketovalerate-ferredoxin oxidoreductase of Pyrococcus furiosus (34% in 262 aa).     

脂肪酸对中华哲水蚤摄食两种海洋微藻的指示作用

在室内以饥饿培养为对照,以海洋原甲藻(Prorocentrum micans)和中肋骨条藻(Skeletonema costatum)培养中华哲水蚤(Calanus sinicus),研究了各脂肪酸标记对中华哲水蚤摄食不同饵料的指示作用。结果显示,海洋原甲藻中18 ∶ 4ω3、22 ∶ 6ω3含量较高,中肋骨条藻中16 ∶ 1ω7、20 ∶ 5ω3的含量较高。二者分别表现出典型的甲藻门和硅藻门的脂肪酸组成特征。中华哲水蚤的脂肪酸组成有两个特点:(1)20 ∶ 5ω3和22 ∶ 6ω3的含量均较高;(2)其体内表征桡足类浮游植物食性、由桡足类自身合成的20 ∶ 1和22 ∶ 1脂肪酸占有相当的比例。虽然中华哲水蚤对不同脂肪酸的吸收和转化效率不同,但以脂肪酸作为标记还是成功的指示了中华哲水蚤对微藻的摄食。在饥饿培养中,首先消耗的是那些浮游动物自身不能合成的多不饱和脂肪酸,而结构脂肪酸都表现出了较高的保守性。结合各脂肪酸标记变化趋势和Pearson相关性分析的结果认为,18 ∶ 4ω3、18 ∶ 4ω3/16 ∶ 1ω7、∑18/∑16能较好的指示中华哲水蚤对海洋原甲藻的摄食,仅16 ∶ 1ω7/18 ∶ 4ω3能指示中华哲水蚤对中肋骨条藻的摄食。     

Meiotic transmission of a hypomethylated repetitive DNA family in tobacco

We have recently shown that hypomethylation of cytosine residues in the HRS60 family of repetitive DNA sequences can be induced with 5-azacytidine (5-azaC) in tobacco tissue cultures. We have also proven that such a DNA methylation status is maintained during the recovery of protoplasts, plant regeneration, and vegetative development. In the present paper we follow meiotic transmission of hypomethylated HRS60 DNA. Plants obtained from seeds treated with 5-azaC were either self pollinated or crossed with a non-treated control in a reciprocal way. Analysis of the methylation status of the HRS60 DNA revealed that these sequences were hypomethylated in the progenies up to the extent found in the parental 5-azaC-treated plant. Since no parent-of-origin effect was observed, we presume that both male and female gametes transmit an artificial methylation imprint to a similar extent. This result is supported by methylcytosine evaluation in the total genomic DNA samples. A temporal analysis of 5-azaC effects on germinating seeds and a phenotypic evaluation of 5-azaC-treated tobacco plants are also presented.On leave from the School of Biology and Biochemistry, University of Bath, England     

Identification of a novel adrenomedullin gene family in teleost fish

Adrenomedullin (AM) is a multifunctional peptide known to form a hormone family with calcitonin gene-related peptide (CGRP) and amylin. We have cloned five distinct AM cDNAs from the pufferfish, Takifugu rubripes, and named them TrAM-1, -2, -3, -4, and -5. Judging from the deduced precursor sequences and processing pattern of the C-terminal mature peptides, TrAMs may be divided into at least two groups; AM-2 and -3, and AM-1, -4, and possibly -5. Phylogenetic analysis of the mature peptides, exon-intron structure of their genes, and tissue distribution of their mRNA also support this classification. TrAM-1 and -4 were ubiquitously expressed in various tissues including the kidney and interrenal (adrenal homolog) as in the case of mammalian AM, while TrAM-2 and -3 were expressed most abundantly in the brain followed by the vascular tissues. Synteny of the genes around AM gene showed that TrAM-1 is the ortholog of mammalian AM. The presence of a PAMP-like sequence in the prosegment of TrAM-1 also supports this notion. Multiple AMs were also detected in another pufferfish, Tetraodon nigroviridis, and in zebrafish, Danio rerio. The present study shows for the first time the presence of a novel AM family in teleost fish that is independent from CGRP and amylin, which further suggests the possible existence of multiple AMs in mammals.     

Purification, amino acid sequence and characterization of the class IIa bacteriocin weissellin A, produced by Weissella paramesenteroides DX

Weissella paramesenteroides DX has been shown to produce a 4450-Da class IIa bacteriocin, weissellin A, composed of 43 amino acids with the sequence KNYGNGVYCNKHKCSVDWATFSANIANNSVAMAGLTGGNAGN. The bacteriocin shares 68% similarity with leucocin C from Leuconostoc mesenteroides. Computational analyses predict that the bacteriocin is a hydrophobic molecule with a beta-sheet type conformation. Weissellin A exhibited various levels of activity against all gram-positive bacteria tested, but was not active against Salmonella enterica Enteritidis. The antimicrobial activity was not associated with target-cell lysis. The bacteriocin retained activity after exposure to 121 °C for 60 min or to −20 °C for 6 months, and to pH 2.0-10.0. It was not sensitive to trypsin, α-chymotrypsin, pepsin and papain, but was inactivated by proteinase K. At a dissolved oxygen concentration of 50%, weissellin A was produced with growth-associated kinetics. The properties of weissellin A make this bacteriocin a potentially suitable agent for food and feed preservation.     

Modeling a hox gene network in silico using a stochastic simulation algorithm

The amount of molecular information that has been gathered about Hox cis-regulatory mechanisms allows us to take the next important step: integrating the results and constructing a higher-level model for the interaction and regulation of the Hox genes. Here, we present the results of our investigation into a cis-regulatory network for the early Hox genes. Instead of using conventional differential equation approaches for analyzing the system, we have adopted the use of a stochastic simulation algorithm (SSA) to model the network. The model allows us to track in detail the behavior of each component of a biochemical pathway and to produce computerized movies of the time evolution of the system that is a result of the dynamic interplay of these various components. The simulation is able to reproduce key features of the wild-type pattern of gene expression, and in silico experiments yield results similar to their corresponding in vivo experiments. This analysis shows the utility of using stochastic methods to model biochemical networks. In addition, the model has suggested several intriguing new results that are currently being investigated in vivo.     

Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917(T)

The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.     

A new member of the Balbiani ring multigene family in the dipteran Chironomus tentans consists of a single-copy version of a unit repeated in other gene family members

The known Balbiani ring (BR) multigene family members in the dipteran Chironomus tentans encode salivary gland secretory proteins in the size range between 38 and 1,000 kDa. The proteins interact to form protein fibers used by the aquatic larvae to spin feeding and protective larval tubes or pupation tubes. Here, we describe a new BR multigene family member, the spl7 gene, which codes for an 89-amino-acid-long protein with a relative mobility of 17k. The gene has a high content of charged amino acid residues and consists of two structurally different halves. Five regularly spaced cysteine codons are present in the 5 half while the 3 half contains five proline codons. These two different halves exhibit similarities to the C and SR regions, respectively, which form the tandemly repeated units in the about 40-kb-long BR genes and which also, in different versions, are the building blocks of all genes in the BR multigene family.In this multigene family, encoding interacting structural proteins, the long BR genes with their 125–150 tandemly arranged repeat units as well as the short sp17 gene with its single-copy version of such a repeat unit, have therefore evolved from a common ancestor.Correspondence to: L. Wieslander     

Identification and characterization of a novel spermidine/spermine acetyltransferase encoded by gene ste26 from Streptomyces sp. 139

Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which can bind IL-1R specifically and exhibits anti-rheumatic arthritis activity in vivo. With the Ebosin biosynthesis gene cluster (ste) consisting of 27 ORFs identified previously the focus of this study was to characterize the protein encoded by ste26 gene. After cloning and expressing ste26 in Escherichia coli BL21, we purified the recombinant Ste26 protein and revealed its ability of transferring the acetyl group from AcCoA to spermidine and spermine, with spermine being the preferred substrate. Therefore Ste26 has been determined to be a spermidine/spermine acetyltransferase which can use spermine (Km of 72.1 ± 7.4 μM), spermidine (Km of 147.2 ± 11 μM), AcCoA (Km of 45.7 ± 2.5 μM) and poly-l-lysine (Km of 99.7 ± 11 μM) as substrates. The optimum pH, temperature and time for the activity have been shown to be 7.5, 37°C and 10 min, respectively. This is the first spermidine/spermine acetyltransferase characterized in Streptomyces and its function in Ebosin biosynthesis is discussed.