Isolation and characterization of viruses from fetal calf serum

Summary Ten lots of specially procured fetal calf serum collected under sterile conditions and not filtered and 16 lots of commercial fetal calf serum were tested for both human and bovine viral contamination. The presence of viruses was evaluated by observing for cytopathogenic effect (CPE), hemadsorption with guinea pig erythrocytes, and interference with cytopathogenic challenge viruses in both embryonic bovine trachea (EBTr) and human diploid lung (HDL) cells. Isolates were characterized by their cytopathogenicity, morphology, serology, and ability to propagate and produce cPE in a variety of bovine and nonbovine cells. One isolate was unequivocally identified as bovine herpes virus 1, and the other was presumptively identified as a bovine virus-diarrhea virus. This study was supported by The National Institutes of Health under Contract PH 43-66-539.     

Isolation and characterization of PERV-C env from domestic pig in Korea

Clone PERV-C (A3) env was isolated from the genomic DNA of domestic pig (Sus scrofa domesticus) in Korea to investigate the molecular properties of PERV-C. The nucleic acid homologies between the PERV-MSL (type C) reference and the PERV-C(A3) clone was 99% for env, but a single base pair deletion was found in the transmembrane (TM) region of the env open reading frame. To examine the functional characteristics of truncated PERV-C env, we constructed a replication-incompetent retroviral vector by replacing the env gene of the pCL-Eco retrovirus vector with PERV-C env. A retroviral vector bearing PERV-C/A chimeric envelopes was also created to complement the TM defect. Our results indicated that truncated PERV-C env was not infectious in human cells as expected. Interestingly, however, the vector with the PERV-C/A envelope was able to infect 293 cells. This observation suggests that recombination within PERV-C TM could render PERV-C infectious in humans. To further characterize PERV-C/A envelopes, we constructed an infectious molecular clone by using a PCR-based technique. This infectious molecular clone will be useful to examine more specific regions that are critical for human cell tropism.     

Isolation and characterization of thiosulfate reductases from the green alga Chlorella fusca

Thiosulfate-reductase activity (TSR) measured as sulfide release from thiosulfate was detected in crude extracts of Chlorella using dithioerythritol (DTE) as electron donor. Purification of this activity by ammonium-sulfate precipitation between 35% and 80% followed by Sephadex G-50 gel filtration, diethylaminoethyl-cellulose chromatography, and gel filtration on Biogel A 1.5 M led to four distinct proteins having molecular weights of: TSR I, 28000; TSR II, 26500; TSR III_a, 55000; TSR III_b, 24000 daltons. These thiosulfate reductases were most active with DTE; the monothiols glutathione, l-cysteine, and -mercaptoethanol had little activity towards this system. The following pH optima were obtained: for TSR I and TSR II, 9.0; for TSR III_a, 8.5; and for TSR III_b, 9.5. The apparent-K_m data for DTE and thiosulfate were determined to: TSR I, 0.164 mmol·l^-1 and TSR II, 0.156 mmol·l^-1; K_mDTE TSR I, 1.54 mmol·l^-1 and TSR II 1.54 mmol·l^-1. The thiosulfate reductases III_a and III_b were further stimulated by addition of thioredoxin. All TSR fractions catalyzed SCN formation from thiosulfate and cyanate and thus had rhodanese activity; this activity, however, could only be detected in the presence of thiols.Abbreviations DTE dithioerythritol - TSR thiosulfate reductase Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Isolation and characterization of soybean Bowman-Birk inhibitor from different sources

A chromatographic procedure for isolation of different isoforms of Bowman--Birk soybean trypsin inhibitors was developed. The number of isoforms was shown to depend on soybean cultivar. The amount of the classical Bowman--Birk inhibitor (BBI) in different soybean cultivars, commercial flour, and processing products was analyzed. BBI reaches its highest concentration in freshly milled seeds. Storage conditions optimum for preservation of maximum inhibitory activity in soybean raw material were developed. The use of indirect enzyme immunoassay for BBI detection during its isolation from different sources was demonstrated.     

Isolation and characterization of proteorhodopsin homologue from Yellow Sea of Korea

Many organisms use proton pump to earn energy for living. Some proton pumps start to work by light and one of the famous proteins are called proteorhodopsin (PR). From recent study it used not only protons but also mono-valent cations, divalent cations, or mono-valent anions during pumping activity. The goal of this study is to find new types of proton pumping proteins in the surface of the ocean. Metagenome samples were collected from the beach in Taean-gun and Incheon (Kkotji beach (36°30′0′′N, 126°19′56′′E), Kkotji mud (36°30′8′′N, 126°19′60′′E), Duegi beach (36°31′6′′N, 126°19′39′′E), Sorae salt pond (37°24′25′′N, 126°44′41′′E), swamp (37°24′59′′N, 126°44′54′′E) and reservoir (37°24′39′′N, 126°45′5′′E) in West Sea of Korea. Genomic DNA of each sample was isolated and used for PCR with specific primers for PR and sodium pumping rhodopsin. As a result, we obtained an unidentified PR in Duegi beach sample. The unidentified PR was expressed with chimeric expression system. It has 528 nm absorption maximum at pH 7. By the light differential spectrum measurement, putative M and O photo-intermediates were detected at around 400 and 600 nm, respectively. Similar to GPR, it has light driven outward proton transfer activity.     

Isolation of magnetotactic bacterium WM-1 from freshwater sediment and phylogenetic characterization

The magnetotactic bacterium was isolated from freshwater sediment from North Lake of Wuhan. The isolate, designated WM-1, was Gram-negative, helical shaped, and studied by means of electron microscopy. The strain WM-1 was 0.2-0.4 μm in diameter and 3–4 μm in length. The DNA G + C content was found to be 65.7 mol%. Phylogenetic analysis of the 16S rDNA gene (Accession number DQ899734 in GeneBank) revealed that this isolate was a member ofαsubdivision of the Proteobacteria. Strain WM-1 was closely related (97.7%) to Magnetospirillum sp. AMB-1. Randomly amplified polymorphic DNA analysis showed that these two strains were in fact different strains. Electron diffraction patterns of WM-1 magnetosomes indicated that the magnetosomes were composed of magnetite. The magnetosomes from WM-1 were cuboidal in shape as observed by electron microscopy. Statistical analysis of magnetite crystals from WM-1 showed narrow asymmetric size distribution. The average number of magnetosomes in each WM-1 bacterium was 8 ± 3.4. The average length of magnetosomes in WM-1 was 54 ± 12.3 nm and the average width is 43 ± 10.9 nm. These data showed that the grains in WM-1 were single-domain crystals.     

Isolation and characterization of a carotenoid oxygenase gene from Chlorella zofingiensis (Chlorophyta)

The green alga Chlorella zofingiensis produces large amounts of the valuable ketocarotenoid astaxanthin under dark, heterotrophic growth conditions, making it potentially employable for commercial production of astaxanthin as feed additives, colorants, and health products. Here, we report the identification and characterization of a β-carotene oxygenase (CRTO) gene that is directly involved in the biosynthesis of ketocarotenoids in C. zofingiensis. The open reading frame of the crtO gene, which is interrupted by three introns of 243, 318, and 351 bp, respectively, encodes a polypeptide of 312 amino acid residues. Only one crtO gene was detected in the genome of C. zofingiensis. Furthermore, the expression of the crtO gene was transiently up-regulated upon glucose treatment. Functional complementation in Escherichia coli showed that the coding protein of the crtO gene not only exhibits normal CRTO activity by converting β-carotene to canthaxanthin via echinenone, but also displays a high enzymatic activity of converting zeaxanthin to astaxanthin via adonixanthin. Based on the bifunctional CRTO, a predicted pathway for astaxanthin biosynthesis in C. zofingiensis is described, and the CRTO is termed as carotenoid 4,4′-β-ionone ring oxygenase.     

Chlorella viruses isolated in China

Plaque-forming viruses of the unicellular, eucaryotic, exsymbiotic, Chlorella-like green algae strain NC64A, which are common in the United States, were also present in fresh water collected in the People's Republic of China. Seven of the Chinese viruses were examined in detail and compared with the Chlorella viruses previously isolated in the United States. Like the American viruses, the Chinese viruses were large polyhedra and sensitive to chloroform. They contained numerous structural proteins and large double-stranded DNA genomes of at least 300 kilobase pairs. Each of the DNAs from the Chinese viruses contained 5-methyldeoxycytosine, which varied from 12.6 to 46.7% of the deoxycytosine, and N6-methyldeoxyadenosine, which varied from 2.2 to 28.3% of the deoxyadenosine. Four of the Chinese virus DNAs hybridized extensively with DNA from the American virus PBCV-1, and three hybridized poorly.     

Isolation and characterization of a unicellular manganese-oxidizing bacterium from a freshwater lake in northwestern Russia

A unicellular manganese-oxidizing bacterium (strain L7), isolated from Lake Ladoga, is identified as "Siderocapsa" sp. according to its morphology. However, this bacterium belongs to the phylogenetic cluster of Pseudomonas putida. The physiological characteristics (utilization of sugars, polyatomic alcohols, organic acids, and phenolic substrates as carbon and energy sources) also indicate the similarity of strain L7 to representatives of the genus Pseudomonas. The growing culture oxidizes Mn(II); the rate of oxidation depends on the type of added organic substrate. Carbonate requirement for this process indicates mixotrophic metabolism. The relatedness of the isolated bacterium to the representatives of the genus Pseudonomas and their phenotypic similarity provide a basis for considering strain L7 not as "Siderocapsa" sp., but as a new species, Pseudomonas siderocapsa sp. nov., of the P. putida cluster.     

Isolation and characterization of mitochondria-rich cell types from the gill of freshwater rainbow trout

A magnetic cell separation technique (MACS) was developed for isolating and characterizing peanut lectin agglutinin positive (PNA(+)) cells from rainbow trout gills. Percoll density separated mitochondria-rich (MR) cells were serially labeled with PNA-FITC and an anti-FITC antibody covalently coupled to a 50-nm iron particle and then applied to a magnetic column. PNA(+) MR cells were enriched to >95% purity. Transmission electron microscopy analysis of both the PNA(+) and PNA negative (PNA(-)) fraction showed that PNA binds to MR chloride cells while the PNA(-) cell fraction is comprised of MR cells with features characteristic of pavement cells. Western blotting demonstrated that both PNA(+) and PNA(-) fractions had high levels of Na(+)-K(+)-ATPase and Sco1 expression; however, relative expression of H(+)-ATPase in PNA(+) and PNA(-) cells demonstrated that untreated fish had twofold higher H(+)-ATPase levels in PNA(-) cells relative to the PNA(+) cells. Furthermore, hypercapnic acidosis significantly increased the relative H(+)-ATPase expression on PNA(-) cells only, whereas metabolic alkalosis had no significant effect.     

Isolation and characterization of a unicellular manganese-oxidizing bacterium from a freshwater lake in northwestern Russia

A unicellular manganese-oxidizing bacterium (strain L7), isolated from Lake Ladoga, is identified as “Siderocapsa” sp. according to its morphology. However, this bacterium belongs to the phylogenetic cluster of Pseudomonas putida. The physiological characteristics (utilization of sugars, polyols, organic acids, and phenolic substrates as carbon and energy sources) also indicate the similarity of strain L7 to representatives of the genus Pseudomonas. The growing culture oxidizes Mn(II); the rate of oxidation depends on the type of added organic substrate. Carbonate requirement for this process indicates mixotrophic metabolism. The relatedness of the isolated bacterium to the representatives of the genus Pseudomonas and their phenotypic similarity provide a basis for considering strain L7 not as “Siderocapsa” sp., but as a new species, Pseudomonas siderocapsa sp. nov., of the P. putida cluster.     

Isolation and characterization of microsatellites in giant freshwater prawn Macrobrachium rosenbergii

Microsatellite loci were characterized in a freshwater prawn from enriched genomic library using six biotinylated probes: (AG)₁₀, (TG)₁₀, (CAA)₁₀, (CAG)₁₀, (GAT)₁₀ and (TAC)₁₀. Primers for DNA amplification were designed and synthesized for 20 loci. Ten loci were polymorphic with the number of alleles ranging from five to 17 alleles per locus and the observed heterozygosity ranging from 0.27 to 0.83 per locus. Developed microsatellite primers should prove useful for selective breeding programs and population genetic studies of freshwater prawn.     

Chlorella viruses isolated in China.

Plaque-forming viruses of the unicellular, eucaryotic, exsymbiotic, Chlorella-like green algae strain NC64A, which are common in the United States, were also present in fresh water collected in the People's Republic of China. Seven of the Chinese viruses were examined in detail and compared with the Chlorella viruses previously isolated in the United States. Like the American viruses, the Chinese viruses were large polyhedra and sensitive to chloroform. They contained numerous structural proteins and large double-stranded DNA genomes of at least 300 kilobase pairs. Each of the DNAs from the Chinese viruses contained 5-methyldeoxycytosine, which varied from 12.6 to 46.7% of the deoxycytosine, and N6-methyldeoxyadenosine, which varied from 2.2 to 28.3% of the deoxyadenosine. Four of the Chinese virus DNAs hybridized extensively with DNA from the American virus PBCV-1, and three hybridized poorly.     

Isolation and Characterization of Chloroplast DNA from Chlorella ellipsoidea

A circular DNA molecule was isolated from chloroplasts of Chorella ellipsoidea. The DNA had a buoyant density of 1.695 grams per cubic centimeter (36% GC) and a contour length of 56 micrometers (175 kilobase pairs). The restriction endonuclease analysis gave the same size. Agarose gel electrophoretic patterns of chloroplast DNA digested by several restriction endonucleases were also presented. The digestion by the restriction enzymes, HpaII, MspI, SmaI, and XmaI revealed no appreciable methylation at CG sites in chloroplast DNA.     

Isolation and characterization of diazotrophic growth promoting bacteria from rhizosphere of agricultural crops of Korea

Free-living nitrogen fixing bacteria were isolated from rhizosphere of seven different plant namely sesame, maize, wheat, soybean, lettuce, pepper and rice grown in Chungbuk Province, Korea. Five isolates with nitrogenase activity above 150nmol(-1) mg(-1) protein were identified based on, phenotypic and 16S rDNA sequences analysis. The strains were identified as Stenotrophomonas maltophilia (PM-1, PM-26), Bacillus fusiformis (PM-5, PM-24) and Pseudomonas fluorescens (PM-13), respectively. All the isolates produced indole-3-acetic acid (IAA), in the presence of tryptophan, ranging from 100.4 microg ml(-1) (PM-13) to 255 microg ml(-1) (PM-24). The isolate PM-24 (Bacillus fusiformis) exhibiting highest nitrogenase activity (3677.81 nmol h(-1) mg(-1) protein) and IAA production (255microg ml(-1)) has a promising potential for developing as a plant growth promoting rhizobacteria.     

Isolation and molecular characterization of the [Fe]-hydrogenase from the unicellular green alga Chlorella fusca

[Fe]-hydrogenases are redoxenzymes that catalyze the reversible reduction of protons to hydrogen. Hydrogenase activity was observed in a culture of the unicellular green alga Chlorella fusca after an anaerobic incubation, but not in the related species Chlorella vulgaris. Specific polymerase chain reaction (PCR) techniques lead to the isolation of the cDNA and the genomic DNA of a special type of [Fe]-hydrogenase in C. fusca. The functional [Fe]-hydrogenase was purified to homogeneity and its N-terminus was sequenced. The polypeptide sequence shows a high degree of identity with the amino acid sequence deduced from the respective cDNA region. Structural and biochemical analyses indicate that ferredoxin is the main physiological electron donor.     

Isolation and characterization of a Chlorella mutant producing high amounts of chlorophyll and carotenoids

Mutants of Chlorella that form dark green colonies under heterotrophic conditions were generated from the parental Chlorella regularisS-88 by MNNG mutagenesis. The mutant Y-21 was the highest in chlorophyll and carotenoids contents among these isolates. Y-21 grew a little slower than its parental S-88 under heterotrophic and photoautotrophic conditions. Although chloroplasts were generated in both S-88 and Y-21 during glucose starvation and under illumination, the chlorophyll and carotenoid contents of Y-21 were approximately 1.5–1.8 times those of S-88 under both growth conditions, suggesting that relative size of chloroplast is larger in Y-21 than in S-88. Consistent with these results, photosynthesis genes on chloroplast DNA were more highly up-regulated in Y-21 than in S-88 during glucose starvation and under illumination. These results lead us a model in which chloroplast generation is sugar-repressed and photoinduced in Chlorella. Y-21 appears to be a mutant in which sugar repression of chloroplast generation is weak.     

Isolation and characterization of microsatellite loci in giant freshwater prawn, Macrobrachium rosenbergii

This paper reports on isolation and characterization of 8 microsatellite loci from a partial genomic library of Macrobrachium rosenbergii using DIG labeled dinucleotides, (GT)₁₅ and (CT)₁₅ as probes. Primers were designed and PCR conditions optimized for repeat arrays. Polymorphism was studied using 24 individuals collected from the wild. All the loci were polymorphic with number of alleles ranging from 3 to 5 and observed heterozygosity 0.50–0.85. All the loci except one were in agreement with Hardy–Weinberg equilibrium. No significant pair wise linkage disequilibrium was found among the loci. These markers should prove useful for characterization of natural population as well as brood stock management of this species.