Cloning and expression of a chicken α-amylase gene

We have isolated and sequenced a genomic clone for a pancreatic α-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY) of 512 aa that is 83% identical to the human pancreatic α-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the α-amylase produced by the yeast cells was identical to that of the native chicken enzyme.     

Robust computational reconstitution – a new method for the comparative analysis of gene expression in tissues and isolated cell fractions

Background  

Biological tissues consist of various cell types that differentially contribute to physiological and pathophysiological processes. Determining and analyzing cell type-specific gene expression under diverse conditions is therefore a central aim of biomedical research. The present study compares gene expression profiles in whole tissues and isolated cell fractions purified from these tissues in patients with rheumatoid arthritis and osteoarthritis.     

Cloning and expression of a β-galactosidase gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta(?). Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

Genome-wide classification and expression analysis of nucleobase–ascorbate transporter (NAT) gene family in tomato

Ascorbic acid or vitamin C is a wide spectrum antioxidant and plays a crucial role in a many metal-containing enzymes essential for humans, which are unable to synthesize the vitamin C and must obtain it from dietary sources. Ascorbic acid is transported by sodium-coupled ascorbic acid transporters or SVCTs in humans. However, little information is available about the nucleobase–ascorbate transporters (NATs) in tomato (Solanum lycopersicum). In the current study, we identified 12 NAT genes by screening SGN genome databases in tomato. A complete overview of this gene family in tomato is presented, including gene structures, chromosome distribution and localization, phylogenies, motif analysis and expression profiles. The SlNAT genes contained 14 exons, mostly, and dispersed on all the chromosomes except chromosome 8 and 9. All the SlNATs were located to plasma membrane, chloroplast thylakoid membrane, Golgi body, and endoplasmic reticulum (membrane). The phylogenetic tree showed that the plant NATs were divided into 4 clades, well-supported by the distribution of conserved motifs, and the SlNAT proteins shared higher similarity and clustered more closely with AtNAT proteins. Furthermore, the expression profiles of SlNAT genes in various organs showed 9 out of 12 SlNAT genes were constituently expression with differential expression levels under normal growth conditions. Our systematic analysis will provide a useful platform for molecular clone and functional identification of NAT genes in tomato and probably other Solanaceae plants.     

Cloning, identification and accurate normalization expression analysis of PPARα gene by GeNorm in Megalobrama amblycephala

Megalobrama amblycephala suffers from serious liver diseases recently and PPARα gene has been reported to play an important role in the immune system of animal liver. On the basis of these facts, we have cloned and identified full-length cDNA of PPARα and examined its expression patterns at different embryo developmental stages and in different tissues of adult and young fish in order to improve liver disease immunity of M. amblycephala. We also accurately normalized seven reference genes by GeNorm and calculated their gene expression normalization factors. The total length of PPARα cDNA was 2021 bp, comprising of 214-bp 5'-untranslated region; 1404-bp open reading frame (encoding 467-amino acids); and 403-bp 3'-untranslated region. PPARα peptide was predicted to consist of 4 domains, i.e. A/B, C, D, and E/F. PPARα mRNAs were detected in different tissues of adult and young fish including adipose tissue, gill, heart, liver, spleen, kidney, white muscle, intestine, brain and gonad. In adult fish, the expression of PPARα in white muscles was highest followed by liver and it was lowest in gonads. Its expression in male gonads was significantly higher than female gonads. In young fish, the expression of PPARα was highest in brain, followed by intestines and it was lowest in spleen. At different embryo developmental stages, the expression of PPARα was highest at 2 cells stage and it was lowest at gastrula stage, but it increased on first day after hatching. In unfertilized spermatozoa, the expression of PPARα was higher than unfertilized ovum.     

Cloning and expression of the α-amylase gene and oxytetracycline production in Streptomyces rimosus

An 8.4 kb Sau3AI DNA fragment containing the Streptomyces rimosus TM-55 -amylase gene (amy) was ligated to a vector pIJ702, named pCYL01, and cloned into amylase deficient mutant S. lividans M2 (amy ^–). Subcloning study showed that the amy gene was localized in 3.3 kbKpnI-PstI fragment. The molecular weight of the purified -amylases of S. lividans M2/pCYL01 and S. rimosus TM-55 were estimated to be 65.7 kDa. Different sizes of recombinant plasmids carrying the amy gene had been retransferred into the parental strain of S. rimosus TM-55. Among these S. rimosus transformants, TM-55/pCYL01, TM-55/pCYL12 and TM-55/pCYL36 showed amylase activity 1.36- to 2.05-fold at the seventh day (1.61 to 2.42 units vs 1.18 units), and oxytetracycline (OTC) production 2.00- to 2.50-fold at the ninth day (approximate 140 to 170 g ml^–1 vs 72 g ml^–1), higher than that of S. rimosus TM-55 alone, respectively. These results showed that industrial microorganisms could be improved by genetic and metabolic engineering.     

Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.     

Cloning, sequence analysis, and heterologous expression of a β-mannanase gene from Bacillus subtilis Z-2

Mannanase, an extracellular enzyme that catalyzes the hydrolysis of hemicelluloses to produce oligosaccharides, has potential to be applied in food industries. In this study a mannanase gene from B. subtilis Z-2 was isolated through PCR screening of a genomic DNA library. The nucleotide sequence of the mannanase gene, man, contained an open reading frame of 1080 bp, which codes for a deduced 26 amino-acid signal peptide and a mature protein with a deduced molecular mass of 38 kDa. The man gene can both be expressed heterologously into the periplasm from the plasmid pET22b(+) containing an intact signal peptide (pET-NdeI18) or the pelB signal peptide of the pET22b(+)vector (pET-NcoI3). Escherichia coli BL21 (DE3) containing pET-NcoI3 secreted about twice as much mannanase as that harboring pET-NdeI18. The E. coli DH5α expression of man was under the control of the lac promoter in the pRK415 vector; it was much more effective when the Shine Dalgarno (SD) sequence was changed from GGGGAG to AAGGAG and the start codon was changed from TTG to ATG, respectively. These results suggest that genetic modification of the SD sequence and start codon is practical for a high-level mannanase expression in different bacterial strains. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 3, pp. 418–424. This article was submitted by the authors in English.     

Cloning and expression in Escherichia coli of a Thermoanaerobacter cellulolyticus gene coding for heat-stable β-glucanase

Summary A 4.8 kb HindIII fragment of Thermoanaerobacter cellulolyticus DNA cloned in Escherichia coli was shown to direct the synthesis of -glucanase. The enzyme produced by the transformant was extremely heat-stable and the optimum temperature for the enzyme reaction was 80°C. The cloned enzyme could hydrolyse carboxymethyl cellulose and lichenan, but could not digest laminarin, xylan and cellobiose. Although T. cellulolyticus secreted cellulase(s) into the medium, most of the cloned enzyme activity was detected only in cytoplasm in the recombinant clone.     

Cloning and expression of β-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Summary The -galactosidase gene ofStreptococcus thermophilus was cloned into plasmid vector, pVT100-U, and used to transform a strain ofEscherichia coli andSaccharomyces cerevisiae. Transformants which expressed -galactosidase activity were obtained in bothE. coli andSaccharomyces cerevisiae, the highest activity found in a yeast recombinant. The expression and thermostability of the cloned -galactosidase genes from different plasmid constructions were compared with the streptococcal -galactosidase. The recombinant protein was equivalent to the specific activity and thermostability ofS. thermophilus.     

Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

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Transient expression of β-glucuronidase in plastids of various plant cells and tissues delivered by a pneumatic particle gun

Chloroplast expression plasmids pTRBCL-GUS (tobaccorbcL promoter-gusA-tobaccorbcL terminator) and pHHU3004 (spinach ‘x gene’ promoter-gusA-spinachrbcL terminator) and a control nuclear expression plasmid pBI221 (CaMV 35S promoter-gusA-NOS terminator) were introduced separately into cultured cells and tissues of tobacco andArabidopsis thaliana, as well as into cultured cells of the lower land plants liverwort and hornwort by a pneumatic particle gun. The pTRBCL-GUS and pHHU3004 plasmids produced many blue spots in the BY-2 cells and the roots ofArabidopsis thaliana, but not in any of the green cells or tissues. The results suggest that the pTRBCL-GUS and pHHU3004 plasmids are expressed more in proplastids and amyloplasts than in chloroplasts. GUS activities of the BY-2 cells bombarded with pTRBCL-GUS and pHHU3004 were insensitive to α-amanitin treatment (10 and 50 μg/ml), while that of the cells with pBI221 greatly decreased by the same treatment. Hence, it is likely that the pTRBCL-GUS and pHHU3004 plasmids were substantially expressed in the proplastids.     

Cloning and expression in Saccharomyces cerevisiae of an endo-β-glucanase gene from a thermophilic cellulolytic anaerobe

Summary The gene encoding heat-stable -glucanase from a thermophilic cellulolytic anaerobe was recloned in Saccharomyces cerevisiae. Yeast transformant expressed the heat-stable endo--glucanase and produced a level of enzyme activity similar to the Escherichia coli transformant.This work was supported in part by the Biomass Conversion Project of the Ministry of Agriculture, Forestry and Fisheries, Japan     

Appearence and localization of a β-glucosidase hydrolyzing coniferin in spruce (Picea abies) seedlings

-Glucosidase activity for coniferin (coniferyl alcohol -D-glucoside) is not present in spruce (Picea abies L. Karst.) seeds but appears in the young seedlings. Lignification starts at ca. day 9 of germination in the vascular bundles. An antiserum against glucosidase 1 isolated from spruce seedlings (Marcinowski and Grisebach, Eur J. Biochem. 1978) was employed for the localization of the enzyme in cross sections of hypocotyls using immunofluorescent technique. The results indicate that at this stage of development the glucosidase is localized at the inner layer of the secondary cell wall. Glucosidase activity was present in all cells of the investigated hypocotyl tissue.     

Cloning and expression of the 3′ portion of the T4 denV gene as a lacZ fusion gene

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage λgt11 T4 dC DNA library was screened for phage which produced a β-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25 % of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within λgt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native β-galactosidase.     

Cloning of α-amylase gene fromSchwanniomyces occidentalis and expression inSaccharomyces cerevisiae

The cloning of α-amylase gene ofS. occidentalis and the construction of starch digestible strain of yeast,S. cerevisiae AS. 2. 1364 with ethanol-tolerance and without auxotrophic markers used in fermentation industry were studied. The yeast/E.coli shuttle plasmid YCEp1 partial library ofS. occidentalis DNA was constructed and α-amylase gene was screened in S.cerevisiae by amylolytic activity. Several transformants with amylolysis were obtained and one of the fusion plasmids had an about 5.0 kb inserted DNA fragment, containing the upstream and downstream sequences of α-amylase gene fromS. occidentalis. It was further confirmed by PCR and sequence determination that this 5.0 kb DNA fragment contains the whole coding sequence of α-amylase. The amylolytic test showed that when this transformant was incubated on plate of YPDS medium containing 1 % glum and 1 % starch at 30°C for 48 h starch degradation zones could be visualized by staining with iodine vapour. α-amylase activity of the culture filtratate is 740–780 mU/mL and PAGE shows that the yeast harboring fusion plasmids efficiently secreted α-amylase into the medium, and the amount of the recombinant α-amylase is more than 12% of the total proteins in the culture filtrate. These results showed that α-amylase gene can be highly expressed and efficiently secreted inS. cerevisiae AS. 2.1364, and the promotor and the terminator of α-amylase gene fromS. occidentalis work well inS. cercvisiac AS. 2.1364.