In this study, a full-length heat shock protein88 complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening of P. tenuipesJocheon-1 Uni-Zap cDNA library and 5' RACE polymerase chain reaction. The Paecilomyces tenuipes Jocheon-1 heat shock protein88 cDNA contains an open reading frame of 2,139 bp encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipes Jocheon-1 HSP88 cDNA showed 77% identity to N. haematococca HSP88 and 45-76% identity to other fungi HSP88. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipes Jocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade and P. tenuipes Jocheon-1 HSP88 also contains the conserved ATPase domain at the N-terminal. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as a 88 kDa polypeptide in baculovirus-infected insect Sf9 cells. Under different stress conditions, mRNA expression of P. tenuipes Jocheon-1 HSP88 were quantified by real-time PCR and the result showed that heat shock stress affected the mRNA expression levels of P. tenuipes Jocheon-1 HSP88.

Presently, We have constructed an olig-d(T) primed directional cDNA library from the silkworm Dongchunghacho, an entomopathogenic fungus, of which species is belonging to Paecilomyces tenuipes Jocheon-1. To isolate and screen genes in the fungus, 626 expressed sequence tags(ESTs) were generated by a partial sequencing from the cDNA library. Paecilomyces tenuipes Jocheon-1 cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase(Pt-GAPDH) of Paecilomyces tenuipes Jocheon-1 was cloned from the above cDNA library. The complete cDNA sequence of Pt-GAPDHis comprised of 1,014bp encoding 338 amino acid residues. The deduced protein sequence of Pt-GAPDH showed higher homology with Beauberia bassiana-GAPDH(93% amino acid identity). Hydropathy analysis revealed that Pt-GAPDH protein is hydrophilic. The major three amino acids in its composition of amino acid residues were alanine(11.54%), valine(9.47%) and glycine(8.88%). The cDNA encoding Pt-GAPDH was expressed as a 37 kDa polypeptide in baculovirus-infected insect Sf9 cells. The Pt-GAPDH gene of Paecilomyces tenuipes entomopathogenic fungus consisted of three exons and two introns coding for 338 amino acid residues, and the genomic DNA length of the gene spans 1302bp. The accession number of the gene in GenBank are GU997099 for Pt-GAPDH cDNA and GU997102 for Pt-GAPDH genomic DNA.

Scavenger receptors (SRs) are transmembrane cell surface molecules recognized in apophotic cells, bacteria and lipopolysaccharide. With no physiological information on SRs in insects except SR-CI of Drosophila melanogaster, a putative SR gene was cloned and characterized in Spodoptera exigua. A partial S. exigua SR gene was obtained from hemocyte transcripts and exhibited high homology with type C. Its expression was confirmed in all developmental stages. Among different tissues, S. exigua SR was expressed highly in hemocytes. To confirm change in SR expression by infection, Escherichia coli was injected to fifth instar and RNA was extracted after 10 hours. SR expression in hemocytes of E. coli injected larva was not significantly different from the control but SR expression in fat body of E. coli injected larva was higher than the control. It is expected that SRs of S. exigua are related with immune responses against bacteria such as E. coli. To address its function, S. exigua SR expression was suppressed by double-stranded RNA (dsRNA).

Early cleavage is a reliable prognostic tool for successful embryo transfer in assisted reproduction because early cleaved embryo show better pregnancy rate after transfer. There for, preparation of good embryo recipient is important factor to optimize efficiency of pig cloning. The present study was performed to evaluate the effect of early cleavage on the in vivo development of cloned embryos and to analyze breed, parity and estrous synchrony to optimize recipient for pig cloning. In vitro matured porcine oocytes derived from local slaughterhouse and fibroblasts derived from miniature pig fetuses were used for somatic cell nuclear transfer (SCNT). Reconstructed embryos were transferred to recipient pigs on the same day of SCNT or after 1~2 days of in vitro culture for selecting early cleaved embryos. Breed, parity and date of standing estrous of recipients were recorded for analysis. After 25~35 days after embryo transfer pregnancy was diagnosed using ultrasonography, and pregnant recipients were monitored till delivery. Between purebred and crossbred, no significant difference was founded in both pregnancy and delivery rates. However, early cleaved embryos showed significantly higher pregnancy (46.2%) and delivery (12.8%) rates compared to non-selectively transferred group (24.8% and 4.5%, respectively). The results also showed that the recipients showing standing estrous on the same day of SCNT and less than 4 parities were most suitable for pig cloning.

We have purified and characterized of metalloprotease metalloprotease from Nomuraea atypicola. N. atypicola was cultured in Sabouraud medium supplemented with powdered pupae. The metalloprotease from culture supernatant was purified to electrophoretically homogeneous state. The molecular mass of metalloprotease from N. atypicola was 50 kDa. The enzyme was most active at pH 8.5 and 40oC and stable at pH 5.0-7.0 and up to 40oC. The activity was inhibited by o-phenanthroline and EDTA. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases (Metallo peptidase M36 family (Fungalysin)) from Coccidioides posadasii and Aspergillus fumigatus. The enzyme was found to be Fungalysin-like metalloprotease. cDNA encoding metalloprotease from N. atypicola was amplified by PCR using oligonucleotides deduced from the N-terminal endo peptide sequence, 5’- and 3’-RACE. Predicted enzyme structure consists of 637 amino acids with pro- and signal sequences. The mature enzyme had 391 amino acids and its deduced amino acid sequence coincided completely with the N- terminal amino end (20 amino acids) of metalloprotease purified from N. atypicola. We are studying on expression of the metalloprotease gene in Escherichia coli.

Lipoxygenase (LOX) is considered to be a key enzyme in the biosynthetic pathways of the most important mushroom aroma, 1-octen-3-ol. In previous work, we purified and characterized a LOX from Pleurotus ostreatus (probably H1 strain) fruit bodies [1] and also determined its partial amino acid sequence. In this study, to clarify the biosynthetic mechanism of 1-octen-3-ol, we isolated cDNA and genomic DNA corresponding to a LOX (Polox1) gene of P. ostreatus H1, and analyzed the expression of the gene in the fruit bodies. A commercial P. ostreatus H1 strain (Onuki kinjin, Utsunomiya, Japan) was used in this study. To isolate the Polox1 cDNA, RT-PCR was done using degenerate primers designed from the partial amino acid sequence. This approach generated a single DNA band of approximately 1.1 kbp, which was cloned and sequenced. The deduced amino acid sequence showed high similarity to LOXs of some ascomycetes fungi. To obtain the full-length cDNA of Polox1, clones corresponding to the Polox1 gene were isolated by plaque hybridization from a cDNA library of the P. ostreatus H1 fruit body. DNA sequences of all clones were determined. The 5’ end of the Polox1 cDNA was amplified by the 5’ RACE method and cloned. The full-length cDNA of Polox1 is 2,031 bp long and contains 640 amino acid residues. The deduced amino acid sequence contains LOX iron-binding catalytic domain signature sequences. Next, to determine the genomic DNA sequence of the Polox1 gene, inverse PCR and PCR was done with P. ostreatus H1 genomic DNA. After inverse PCR and PCR, 3.3 and 1.9 kbp DNA fragments, respectively, were amplified and sequenced. Sequence comparison between cDNA and genomic DNA showed that Polox1 gene contained one intron. To investigate expression of the Polox1 gene, northern blot analysis and measurement of LOX activity were performed. P. ostreatus fruit bodies were produced in a sawdust medium containing beech sawdust and rice bran and separated into pileus and stipe. Two transcripts were detected by northern blot analysis in both pileus and stipe. The band intensities were relatively higher in the stipe than in the pileus. The level of LOX activity in the stipe was 3.8 times higher than that in the pileus. By Southern blot analysis, several major bands were detected after the digestion of 4 restriction enzymes. These blot analyses suggest that the Polox1 gene is probably a member of a small gene family. [1] T. Kuribayashi et al., J. Agric. Food Chem., 50, 1247 (2002).

The epigenetic therapy of cancers is emerging as an effective and valuable approach to both chemotherapy and the chemoprevention of cancer. The utilization of epigenetic targets that include histone methyltransferase (HMTase), Histone deacetylatase, and DNA methyltransferase, are emerging as key therapeutic targets. SET containing proteins such as the HMTase Setd1b has been found significantly amplified in cancerous cells. In order to shed some light on the histone methyl transferase family, we cloned the Setd1b gene from Mus musculus and build a collection of vectors for recombinant protein expression in E.coli that will pave the way for further structural biology studies. We prospect the role of the Setd1b pathway in cancer therapy and detail its unique value for designing novel anti-cancer epigenetic-drugs.

In the previous molecular cloning study from human salivary gland cDNA l ibrary a novel clone (C77-091) was known as a candidate gene for antimicrobial protein by GenBank database search and RNA in situ hybridization. This study is aimed to identify the molecular characteristics of C77-091 protein, which showed an antimicrobial activity on E.coli, thereby named as salivary antimicrobial protein (SAMP). SAMP consisted of a typical hydrophobic amino acid rich domain in the N-terminus, a cluster of basic amino acids, carbohydrate attachment site, a possible transglutaminase catalyzed cross-linking site, and multiple consensus sequences of phosphorylation site in the C-terminus. Western blot analysis of human organs and tissue with the monospecific antibody to the synthetic SAMP peptide showed strong interacting protein from the extracts from submandibular gland and parotid saliva but absent in the mixed saliva, and the immunohistochemical staining detected a strong positive regions in the secretory granules in the luminal cytoplasm of interlobular ductal cells of salivary gland. The SAMP was also distributed in the human sebaceous gland and prostate. These data suggest that C77-091 named SAMP gene is a novel antimicrobial protein in human salivary gland, which may play a role for the innate immunity by protecting and stabilizing the mucosal epithelium to maintain homeostasis of oral mucosa.

Glutathione S-transferases (GSTs) are multifunctional enzymes that are mainlyinvolved in the xenobiotic metabolism and protection against oxidative damage. Most studies of GSTs in insects have been focused on their role in detoxifying exogenous compounds in particular insecticides. Here, we show the expression profiles of GSTs of the bumblebee Bombus ignitus in response to oxidative stress. We identified a sigma-class GST from B. ignitus (BiGSTS). The BiGSTSgene consists of 4 exons that encode 201 amino acids. Comparative analysis indicates that the predicted amino acid sequence of BiGSTS shares a high identity with the sigma-class GSTs of hymenopteran insects such as Apis mellifera (70% protein sequence identity) and Solenopsis invicta (59% protein sequence identity). Tissue distribution analyses showed the presence of BiGSTS in all tissues examined, including the fat body, midgut, muscle and epidermis. The oxidative stress responses analyzed by quantitative real-time PCR showed that under H2O2 overload, BiGSTS and BiGSTD (identified in our previous study) were upregulated in all tissues examined, including the fat body and midgut of B. ignitus worker bees. Under uniform conditions of H2O2 overload, the expression profile of GSTs and other antioxidant enzyme genes, such as phospholipid-hydroperoxide glutathione peroxidase (Bi-PHGPx) and peroxiredoxins (BiPrx1 and BiTPx1), showed that other antioxidant enzyme genes are acutely induced at 3 h after H2O2 exposure, whereas BiGSTS and BiGSTD are highly induced at 9 h after H2O2 exposure in the fat body of B. ignitus worker bees. These findings indicate that GSTs and other antioxidant enzyme genes in B. ignitusare differentially expressed in response to oxidative stress. Taken together, our findings indicate that BiGSTS and BiGSTD are oxidative stress-inducible antioxidant enzymes that may play a role in oxidative stress response.

The black soldier fly (BSF), Hermetia illucens, is known as a beneficial insect and feeds on organic materials derived from animals and human, resulting in reduction of food waste and conversion of organic materials. Despite of many studies on the BSF, there have been no reports of cloned genes encoding serine proteases in the BSF. Thus, the primary objective of this study is to clone and to investigate expression pattern of genes encoding serine proteases released from the midgut of the BSF larvae in order to gain a better understanding of expression mechanism of serine proteases. We cloned two serine proteases from the BSF larva. Based on phylogenetic tree analysis, one was chymotrypsin, the other was trypsin. The open reading frame (ORF) of chymotrypsin was 804bp, which encoded a polypeptide of 267 amino acids. In case of trypsin, the ORF was 744bp, which encoded a polypeptide of 247 amino acids. To investigate expression pattern of two serine proteases, we conducted semi-quantitative RT-PCR at different tissues and different developmental stages. A chymotrypsin and trypsin transcripts were revealed strongly in mid gut. Especially, a chymotrypsin was detected largely at feeding stage more than molting stage, while trypsin was expressed similarly between feeding stage and molting stage

In order to unravel unidentified genes from human salivary gland, a cDNA library of human submandibular gland was constructed in the Uni‐ZAP XR vector by use of mRNA from human submandibular gland and ZAP‐cDNA® Gigapack® III Gold Cloning Kit. cDNA of salivary gland was subtracted with cDNA of immortalized human keratinocyte cell line, Rhim Human Epithelial Keratinocyte cell line. The phage cDNA library was converted into a pBluescript phagemid cDNA library, which was subsequently plated on LB plates with ampicillin, IPTG, and X‐gal, and white colonies were selected for sequencing. Among 200 clones analyzed, four clones containing C77‐091, C75‐014, C76‐022, and C76‐012 designated orphan genes that are intensely expressed in the interlobular ductal and serous acinar cells of human submandibular gland. Particularly C77‐091 gene expresses 46 amino acids peptide (pI=9.45). C75‐014 and C76‐022 genes were characterized as those expressing excretory basic proteins primarily consist of alanine, proline, and leucine residues, mimicking a basic proline‐rich protein (bPRP) showing helical structures and having multiple consensus sequences of phosphorylation sites. The strong expression of C76‐012 mRNA in the nuclei of salivary ductal and acinar cells suggests a role of C76‐012 gene as a DNA binding RNA/protein. These data suggest that the identification of four orphan genes from the human salivary glands may add further understanding of greater role of salivary proteins providing innate immunity by protecting and stabilizing the mucosal epithelium in the maintaining homeostasis of oral mucosa.

Fungi belonging to the Paecilomyces spp. have recently been used as food and herbal medicines in Korea and are greatly popular as commercially available powdered supplement or dried fruiting body. Despite this acceptance and its use, little is known of the genes related to its reactive agents. Presently, We have constructed an olig-d(T) primed directional cDNA library from the silkworm Dongchunghacho, an entomopathogenic fungus, of which species is belonging to Paecilomyces spp. based on the previous identification of ITS1 and ITS2 at the molecular level and collected from Jocheon Miryang, Korea. To isolate and screen genes in the fungus, 626 expressed sequence tags(ESTs) were generated by a partial sequencing from the cDNA library. cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase(Pt-GAPDH) of Paecilomyces tenuipes- Jocheon was cloned from the above cDNA library. The complete cDNA sequence of Pt-GAPDH is comprised of 1,014bp encoding 338 amino acid residues. The deduced protein sequence of Pt-GAPDH showed higher homology with Beauberia bassiana-GAPDH(93% amino acid identity). Hydropathy analysis revealed that Pt-GAPDH protein is hydrophilic. The major three amino acids in its composition of amino acid residues were alanine(11.54%), valine(9.47%) and glycine(8.88%). The Pt-GAPDH gene of Paecilomyces tenuipes entomopathogenic fungus consisted of three exons and two introns coding for 338 amino acid residues, and the genomic DNA length of the gene spans 1302bp. The accession number of the gene in GenBank are GU997099 for Pt-GAPDH cDNA and GU997102 for Pt-GAPDH genomic DNA. More investigation works including gene expression, immunological analysis etc. will be carried continuously without hesitation after this presentation.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of the innate immune system that recognize peptidoglycan, a unique cell wall component of bacteria. Here we cloned and characterized PGRP-S from the bumblebee Bombus ignitus (BiPGRP-S). The BiPGRP-S gene consists of four exons encoding 194 amino acid residues. Comparative analysis indicates that the predicted amino acid sequence of BiPGRP-S shares high identity with enzymatically active PGRP-S proteins and contains the amino acids required for amidase activity. BiPGRP-S in B. ignitus worker bees is constitutively expressed in boththe fat body and epidermis, and it is secreted into the hemolymph. Quantitative real-time PCR assays revealed that in both the fat body and epidermis, the BiPGRP-S gene is highly induced by an injection of Bacillus thuringiensis. In addition, recombinant BiPGRP-S expressed as a 19-kDa protein in baculovirus-infected insect cells can bind to B. megaterium and B. thuringiensis but not to Staphylococcus aureus, Escherichia coli or Beauveria bassiana. Consistent with these data, BiPGRP-S shows antibacterial activity against B. megaterium and B. thuringiensis. These results indicate that BiPGRP-S is an inducible protein that may be involved in the immune response against bacterial infection of the genus Bacillus as an amidase-type PGRP-S.