A new 5′ terminal murine GAPDH exon identified using 5′RACE LaNe

In this work, a ligation-independent, fully gene-specific, nested polymerase chain reaction (PCR) method for the elucidation of 5′ cDNA sequence is described and demonstrated for the first time. Two manifestations of the method, rapid amplification of cDNA ends (RACE) by lariat-dependent nested PCR 5′ (RACE LaNe), at least as simple to perform as conventional RACE, were successfully applied to the murine housekeeping genes phosphoglycerate kinase 1 (PGK1), β-actin (β-ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the alpha thalassemia mental retardation Y homolog (ATRY) gene of the marsupial, Macropus eugenii. Significantly, a new murine GAPDH 5′ exon, separated by 365 kb of intronic sequence from previously annotated GAPDH sequence, was discovered using 5′RACE LaNe.     

Cloning and bioinformatics analysis of a novel acidophilic β-mannanase gene,Auman5A,from Aspergillus usamii YL-01-78

The full-length cDNA sequence, which encodes a novel acidophilic β-mannanase (abbreviated as AuMan5A) of Aspergillus usamii YL-01-78, was amplified by 3′ and 5′ rapid amplification of cDNA ends (RACE) using the total RNA as template. The cDNA sequence is 1,427 bp in length, including 5′ and 3′ non-coding regions and an open reading frame (ORF). The ORF encodes a 21-aa signal peptide, a 17-aa propeptide, and a 345-aa mature peptide (AuMan5A) with the calculated M.W. of 37,614 Da and pI of 4.09 and two putative N-glycosylation sites. Online analysis of amino acid sequence homology demonstrated that the AuMan5A belongs to the glycoside hydrolase (GH) family 5. Its three-dimensional structure was predicted using Pred3D Web Server 1.0 based on the crystal structure of the T. reesei RutC-30 β-mannanase (1QNO) from the GH family 5. Furthermore, the complete DNA sequence encoding the AuMan5A, designated as Auman5A, was cloned from the genomic DNA of A. usamii YL-01-78 by the conventional PCR and pUCm-T vector-mediated PCR techniques. The cloned Auman5A is 2,168 bp in length, harboring 5′ and 3′ flanking regulatory regions and the full-length cDNA sequence in which two short introns with 63 and 60 bp are inserted, respectively.     

Preferential β-sheet structuration of multiconformational poly(Glu-Leu) by metallic ions

Summary Alternating poly(Glu-Leu) exhibits a random coil structure in pure water at neutral pH. The addition of 0.5 equiv of Ca²⁺ induces a coil-to-β-sheet transition and the addition of 0.15 equiv of Fe³⁺ induces a coil-to-α-helix transition. Conformational competition between these two structures was studied by mixing preformed β-sheets and α-helices in different proportions. Circular dichroism spectra clearly show that β-sheets are favored at the expense of α-helices in β-sheet-rich mixtures.     

Cloning and sequence analysis of a novel xylanase gene, Auxyn10A, from Aspergillus usamii

A full-length cDNA sequence, encoding a novel endo-1,4-β-d-xylanase (AuXyn10A) of Aspergillus usamii, was obtained by using rapid amplification of cDNA ends (RACE) methods and cloned into the pUCm-T vector, followed by DNA sequencing. The cDNA gene, designated as Auxyn10A, is 1,235 bp in length harboring 5′- and 3′-non-encoding regions, as well as an ORF of 984 bp that encodes a 19-aa signal peptide, a 6-aa propeptide and a 302-aa mature peptide with a calculated MW of 32,756 Da. The AuXyn10A displays high similarity to the xylanases of Aspergillus niger, Aspergillus kawachii and Aspergillus niger, members of the glycoside hydrolase family 10. Its three-dimensional structure was predicted using programs based on the crystal structure of Penicillium simplicissimum xylanase (1B30_A) from the family 10. The complete DNA gene was cloned from the genomic DNA of A. usamii using conventional PCR and hairpin structure-mediated PCR techniques. The DNA gene is 2,255 bp in length, containing a 510 bp of 5′-flanking promoter region and a 1,745 bp of downstream fragment that consists of ten exons and nine short introns ranging from 52 to 62 bp.     

Cloning and characterization of β-catenin gene in early embryonic developmental stage of Artemia sinica

β-Catenin plays a crucial role in embryonic development and responds to the activation of several signal transduction pathways. In this paper, in order to understand the functions of β-catenin gene in early embryonic development of Artemia sinica, the complete cDNA sequence was cloned for the first time using RACE technology, then the sequence was analyzed by some bioinformatic methods. The expression of the β-catenin gene was investigated at various stages during the embryonic development using quantitative real-time PCR and immunohistochemistry assay. Through the investigation, the result of real-time PCR illustrated that β-catenin gene might relate to the response of A. sinica’s immune system and osmotic pressure system in early embryonic developmental stage. Meanwhile, Immunohistochemistry assay demonstrated that during embryonic development, β-catenin was mainly expressed in the cephalothorax. Besides, we discovered that β-catenin might not be a maternal gene in A. sinica, and this new phenomenon may explain a constitutive and regional expression during the early embryonic development of A. sinica.     

Cloning and sequence analysis of an acidophilic xylanase (XynI) gene from <Emphasis Type="Italic">Aspergillus usamii</Emphasis> E001

The full-length cDNA gene that encodes an acidophilic endo-1,4-β- xylanase XynI of Aspergillus usamii E001 was amplified by rapid amplification of cDNA 3′ and 5′ ends (RACE) using the total RNA as template and then cloned onto the pUCm-T vector, followed by sequencing. The cloned cDNA is 881 bp in length including 5′ and 3′ non-encoding regions, as well as a 678 bp of open reading frame (ORF) which encodes an E001 XynI of 188 amino acid residues together with a signal peptide of 37 amino acid residues. The homologies of E001 XynI with xylanases of Aspergillus niger, Aspergillus kawachii, Emericella nidulans and Penicillium funiculosum are 97.8, 92.0, 74.6 and 60.5%, respectively. From a BLAST search result, we concluded that E001 XynI belongs to the glycoside hydrolase family 11. Its three-dimensional structure was predicted using programs based on that of the P. funiculosum xylanase (1TE1B) from the family 11. In addition, the complete DNA gene xynI encoding E001 XynI was cloned from the genomic DNA of A. usamii E001 by conventional PCR and ligation-mediated PCR amplification. The cloned xynI is 1,206 bp in length, composed of a promoter region, a 68 bp of intron and two exons when compared with the cDNA of E001 XynI.     

Cloning of phytoene desaturase and expression analysis of carotenogenic genes in persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> L.) fruits

Persimmon is a commercially important fruit crop, and the fruit is rich in different kinds of bioactive compounds, among which carotenoids contribute significantly to its color and nutritional value. In this study, the cDNA of phytoene desaturase gene (PDS) was isolated by rapid amplification of cDNA ends (RACE) technique. Sequence analysis indicated that the full-length cDNA of PDS was 2064 bp, encoding 586 amino acids and containing one open reading frame (ORF) of 1761 bp. Homology analysis showed that DkPDS, which had been submitted in GenBank with accession number GU112527, shared high similarities of 80–86% with PDS cloned from other plants. Prediction of deduced proteins showed that there was no signal peptide and transmembrane topological structure in DkPDS. It was a hydrophilic and stable protein, and located in chloroplast. To examine the specific expression patterns of carotenogenic genes we had cloned from persimmon, including phytoene synthase (DkPSY), DkPDS, ζ-carotene desaturase (DkZDS), lycopene β-cyclase (DkLCYB) and β-carotene hydroxylase (DkBCH), real-time quantitative PCR (Q-PCR) was performed in flesh at five different developmental stages. The results revealed that the expression levels of DkPSY, DkPDS and DkZDS gradually increased. Nevertheless, the expression level of DkLCYB was very low and maintained relatively stable. The expression level of DkBCH was also at a low level from stage 1 to 4, and then reached the maximum at stage 5. In addition, the expression level of DkZDS was higher than that of other genes. Carotenoid detection demonstrated that both β-cryptoxanthin and total carotenoids increased with fruit development, and zeaxanthin had little change, but with a sudden increase in final stage.     

An improved method of protein isolation and proteome analysis with <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Laminariales) incubated under different pH conditions

The brown alga Saccharina japonica is abundant on rocky coasts of Far East Asia, including Korea, Japan, and China. S. japonica produces high levels of compounds used in the food, cosmetic, and pharmaceutical industries. Thus, many studies have focused on the biosynthesis, extraction, purification, and application of carbohydrates, as well as biochemical features that yield cellular proteins. However, total protein isolation has proved difficult, due to viscous polysaccharides on the surface of S. japonica. To extract total proteins cleanly from S. japonica, we examined various lysis buffers and detergents for effective cell lysis and removal of polysaccharide. Lysis solution D (7 M urea, 4% [3-(3-cholami-dopropyl dimethylammonio) propanesulfonate], 2 M thio-urea, 100 mM dithiothreitol, 4% pharmalyte, 4% polyvinylpyrrolidone) achieved a comparatively high yield of protein extraction, with 12 mg of proteins purified per 1 g of dry weight of S. japonica. Proteins isolated using lysis solution D and subjected to two-dimension polyacrylamide gel electrophoresis generated more than 200 protein spots. Of these, 60 spots were analyzed by matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and MALDI-TOF/MS/MS. A database search revealed that these proteins include glyceraldehyde-3-phosphate dehydrogenase, tryptophan synthase α chain, 6-phosphogluconate dehydrogenase (6PGD), actin, phosphoglycerate kinase, elongation factor Tu, kinesin, fucoxanthin-chlorophyll a–c binding protein F precursor and ATP synthase subunit β. Many protein spots were unidentified. When S. japonica was incubated at different pH, tryptophan synthase α chain and variant surface glycoprotein 7 precursor were highly expressed at pH 7.5 and 9.5, respectively, whereas 6PGD and kinesin showed low expression at pH 9.5.     

Molecular cloning,characterization and expression analysis of <Emphasis Type="Italic">Cm</Emphasis>APX

The RT PCR and RACE methods were used to obtain the cDNA sequence of an APX gene of muskmelon after the leaves were induced with powdery mildew. The cDNA length of the APX gene is 1,047 bp with a 750 bp ORF encoded a 249 amino acid and the molecular weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns. The identity of the amino acid sequence deduced from the cDNA with the APX family of other homologous members was about 74–97%. A Full-length of ORF was sub-cloned into prokaryotic expression vector pET24a. The recombinant proteins had high expression level in E. coli. Analysis of expression at mRNA level showed that CmAPX exhibited highly tissue-specific patterns of expression. The mRNA level and enzyme activities assays showed that CmAPX might play an important role in the pathogenesis of powdery mildew.     

Cloning,expression and enantioselective hydrolytic catalysis of a microsomal epoxide hydrolase from a marine fish, <Emphasis Type="Italic">Mugil cephalus</Emphasis>

The cDNA of a marine fish microsomal epoxide hydrolase (mEH) gene from Mugil cephalus was cloned by rapid amplification of cDNA ends (RACE) techniques. The homology model for the mEH of M. cephalus showed a characteristic structure of α/β-hydrolase-fold main domain with a lid domain over the active site. The characteristic catalytic triad, consisting of Asp(238), His(444), and Glu(417), was highly conserved. The cloned mEH gene was expressed in Escherichia coli and the recombinant mEH exhibited (R)-preferred hydrolysis activity toward racemic styrene oxide. We obtained enantiopure (S)-styrene oxide with a high enantiopurity of more than 99% enantiomeric excess and yield of 15.4% by batch kinetic resolution of 20 mM racemic styrene oxide.     

Characterization of Recombinant Terrelysin,a Hemolysin of <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

Fungal hemolysins are potential virulence factors. Some fungal hemolysins belong to the aegerolysin protein family that includes cytolysins capable of lysing erythrocytes and other cells. Here, we describe a hemolysin from Aspergillus terreus called terrelysin. We used the genome sequence database to identify the terrelysin sequence based on homology with other known aegerolysins. Aspergillus terreus mRNA was isolated, transcribed to cDNA and the open reading frame for terrelysin amplified by PCR using specific primers. Using the pASK-IBA6 cloning vector, we produced recombinant terrelysin (rTerrelysin) as a fusion product in Escherichia coli. The recombinant protein was purified and using MALDI-TOF MS determined to have a mass of 16,428 Da. Circular dichroism analysis suggests the secondary structure of the protein to be predominantly β-sheet. Results from thermal denaturation of rTerrelysin show that the protein maintained the β-sheet confirmation up to 65°C. Polyclonal antibody to rTerrelysin recognized a protein of approximately 16.5 kDa in mycelial extracts from A. terreus.     

Molecular characterization of growth differentiation factor 9 and its spatio-temporal expression pattern in gibel carp (<Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>)

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor β (TGF-β) superfamily with a key role in regulating follicle development. In this study, the GDF9 full-length genomic DNA and cDNA were isolated and characterized from the gibel carp ovary using rapid-amplification of cDNA ends (RACE) and LD-PCR. The full-length genomic DNA and cDNA sequences of GDF9 are 3979 and 2044 bp which code 428 amino acid residues with a specific RKKR protease cleavage site of TGF-β superfamily. Sequence analysis showed that gibel carp was similar to zebrafish and other fish species. Spatio-temporal expression analysis using real-time quantitative PCR revealed that GDF9 mRNA was largely expressed in ovary and testis. GDF9 is mainly present at stage I follicles indicating its important role in early follicles development. The same result was obtained in immunohistochemistry localization of GDF9 protein. Within the follicle, the follicle layer cells were barely expressed whereas GDF9 mRNA was mostly expressed in the oocytes. Supplemented with human chorionic gonadotropin (hCG) in isolated follicles, the expression of GDF9 mRNA was increased firstly and then decreased. The results of this study indicated that GDF9 gene played a role in fish during development of follicles, especially in the early stage follicles.     

Molecular characterization of gap region in 28S rRNA molecules in brine shrimp <Emphasis Type="Italic">Artemia parthenogenetica</Emphasis> and planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

In most insects and some other protostomes, a small stretch of nucleotides can be removed from mature 28S rRNA molecules, which could create two 28S rRNA subunits (28Sα and 28Sβ). Thus, during electrophoresis, the rRNA profiles of these organisms may differ significantly from the standard benchmark since the two subunits co-migrate with the 18S rRNA. To understand the structure and mechanism of the atypical 28S rRNA molecule, partial fragments of 28Sα and 28Sβ in brine shrimp Artemia parthenogenetica and planarian Dugesia japonica were cloned using a modified technology based on terminal transferase. Alignment with the corresponding sequences of 28S rDNAs indicates that there are 41 nucleotides in A. parthenogenetica and 42 nucleotides in D. japonica absent from the mature rRNAs. The AU content of the gap sequences of D. japonica and A. parthenogenetica is high. Both the gaps may form stem-loop structure. In D. japonica a UAAU cleavage signal is identified in the loop, but it is absent in A. parthenogenetica. Thus, it is proposed that the gap processing of 28S rRNA was a late enzyme-dependent cleavage event in the rRNA maturational process based on the AU rich gap sequence and the formation of the stem-loop structure to expose the processing segment, while the deletion of the gap region would not affect the structure and function of the 28S rRNA molecule.     

Comparative Protein Modeling, Prediction of Conserved Residue and Active Sites in Cold Resistant Protein Isolated from CRPF1, A Cold Tolerant Mutant of Pseudomonas fluorescens

Proteins interacting with the biological information molecules DNA and RNA play important cellular roles in all organisms. One widespread super family of proteins implicated in such function(s) is cold shock protein (CSP) that contains the cold shock domain (CSD). This work is planned to study the three-dimensional structure, conserved residues, and different active sites in the structure of cold resistant protein (CRP) from CRPF₁, cold tolerant mutant of Pseudomonas fluorescence by comparative homology modeling. Here we tried to identify crucial residues that are involved in active sites or functional sites of the protein. The study reveals that CRP represent the prototype of the CSD and share a highly similar overall fold consisting of five antiparallel β-sheets forming a β-barrel structure with surface exposed aromatic and basic residues that were responsible for nucleic acid binding properties of variable binding affinities and sequence selectivity and harbors the nucleic acid binding motifs RNP1 and RNP2 that is highly conserved in CSP family.