Stimulation of cell growth ofTetrahymena pyriformis andChlamydomonas reinhardtii by trace elements

Tetrahymena pyriformis, S1 andBJ11, andChlamydomonas reinhardtii CR were employed to study the stimulation of cell proliferation induced by a number of elements. It was observed that traces of Ga, As, Se, Rb, Sr, Ag, Sn, Cs, Yb, Re, and Ir promote the population growth ofT. pyriformis in peptone-glucose culture media or chemically defined media. Inhibition effects onT. pyriformis were observed in media containing traces of In, Te, ba, T1, and Pb. UsingCh. reinhardtii CR, the stimulation effects induced by trace amounts of Ga, Ge, As, Cs, La, Ce, Re, and Ir, respectively, were also determined. Concentration ranges of trace elements promoting cell proliferation are given.     

Detection and characterization of phosphatidylcholine in various strains of the genus Chlamydomonas (Volvocales,Chlorophyceae)

The laboratory strains of Chlamydomonas reinhardtii have been reported to contain no phosphatidylcholine (PC), which is considered to be replaced by another zwitterionic lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). According to the recently published classification, the strains belonged to the subgroup Reinhardtinia. Screening for PC in 13 selected strains of Chlamydomonas in the NIES Algal Collection, which are different in habitats and belong to different phylogenetic subgroups in the genus, revealed the presence of PC in four strains: a strain in the subgroup Polytominia, and three strains in Reinhardtinia. PC was not detected in three other strains of Reinhardtinia analyzed. The presence/absence of PC was not related to the phylogenetic relationship based on 18S rRNA. DGTS was detected in all the strains analyzed. The rare isomer of linolenic acid, 18:3(5,9,12), which has been found in the DGTS of C. reinhardtii, was found in the PC of the two strains and in the DGTS of the five strains. The occurrence of this fatty acid seems limited to a branch of Reinhardtinia. Acquisition and loss of PC in various strains of Chlamydomonas are discussed from the viewpoint of evolution of PC biosynthetic pathway.     

Growth relationships of a lipid-producing Chlorella-alga with common microalgae in laboratory co-cultures

Co-existence growth relationships were studied in communities consisting of a lipid-producing alga Chlorella sp. HQ, another green alga and one cyanobacterium: I Scenedesmus obliquus and Microcystis aeruginosa; II Chlamydomonas reinhardtii and Anabaena flos-aquae; III Selenastrum capricornutum and Microcystis wesenbergii. The cyanobacteria and green algae except for Chlorella sp. HQ were commonly detected in Chinese reservoir and wastewater. The rate of increase of apparent cell number difference with other algae (k _app), inhibition/stimulation ratio (ISR) and the parameters of logistic model and co-existence model were determined for Chlorella. Chlorella strains were the most competitive in Combination I, and were stimulated during 75% of the cultivation time for all three combinations. Anabaena growth exceeded those of Chlorella and Chlamydomonas on the 5th cultivation day under 1 : 1 : 1 inoculum ratio. Scenedesmus colonies consisted of fewer cells, whose average length significantly shortened after the 5th cultivation day under 1 : 1 : 1 inoculum ratio. The developed co-existence model can identify the concrete growth inhibitor or stimulator among three species compared with the single method of cell number monitoring. Good correlation was found between transformed and non-transformed co-existence model through a _mn and b _mn values. Allelopathy and nutrient competition are both possible mechanisms in the above growth relationships.     

Isolation and physiological and biochemical characterization of the Chlamydomonas reinhardtii C-41 mutant,a superproducer of ζ-carotene

The composition of the carotenes and xanthophylls of Chlamydomonas reinhardtii Dang. C-41, a mutant of a unicellular green alga and a superproducer of ζ-carotene, was studied. The light-harvesting complexes and a complex of the PS-II reaction center were established to be disrupted in the C-41 mutant. However, the mutant retained a high (up to 46%) photosynthetic activity and the capacity to accumulate chlorophylls and carotenoids (up to 50%). The composition of carotenes was studied, and it was shown that, in contrast to wild-type K(+) cells, which accumulate up to 95% of β-carotene and 5% α-carotene, cells of the C-41 mutant contained 43% β-carotene, 19% β-zeacarotene, and 38% ζ-carotene. The high level of C-41 mutant biomass accumulation made it possible to recommend the mutant as a superproducer of ζ-carotene in phytobiotechnology.     

Structural-functional organization of the cells of Brc-1 mutant of Chlamydomonas reinhardtii accumulating protoporphyrin IX in the dark

The structural-functional characteristics of the cells of wild type CC-124 and Brc-1 mutant of the unicellular green algae Chlamydomonas reinhardtii grown in the dark and in the light were studied. The cells of the wild type in heterotrophic and mixotrophic conditions had a well developed structure and high functional activity due to the ability of the cells to synthesize chlorophyll both in the light and in the dark. The cells of Brc-1 mutant lost their ability to synthesize chlorophyll in the dark and the cell color was orange due to brc-1 mutation in the nuclear gene LTS3 that regulated the activity of Mg-chelatase enzyme. In the dark the mutant cells accumulated protoporphyrin IX and had weakly developed structure with low functional activity. Because of the high content of protoporphyrin IX, even a short-term exposure of the Brc-1 mutant cells to the light was accompanied by very strong destructive changes in all the membranes in a cell: plasmalemma, chloroplast, mitochondrion, envelopes of the nucleus and vacuoles. The causes of significant impairment of the membrane components and O₂-gas exchange in the Brc-1 mutant cells are discussed.     

Production of IgM monoclonal antibodies in DG44 cells

Two-bicistronic vectors for the production of recombinant IgM monoclonal antibodies in the DG44 DHFR-negative cell line have been designed. We used tandem vectors, in which one bicistronic unit encoded the immunoglobulin light chain and DHFR and the other encoded the heavy chain and EGFP. The construct structure presumes that green cells surviving selection would be capable of producing both immunoglobulin chains. We found that the agglutinating IgM antibodies could be secreted in the absence of J-peptide. It was shown that the germinal leader peptide plays a key role in the expression of the genes for the light and heavy chains. A comparison of the chromatin regulatory elements demonstrated that construct-flanking 2xHS4 insulators stabilized the biosynthesis of the recombinant antibodies, whereas the 5′-MARLyz matrix attachment region proved to be less efficient. The strategy for obtaining a DG44-based producer cell line should include the following consecutive steps: selection on the medium without nucleoside → amplification of the inserted gene → cloning of transfectants → selection of high-productive clones. An attempt to clone before amplification and to amplify individual clones failed to result in effective producers. Cloning on a medium without selection pressure allows a more adequate assessment of the stability of the antibody production.     

Structural studies of a human hybrid immunoglobulin

The structure of an hypothesized hybrid or “Lepore-type” IgG contained in the serum of donor 2904 was investigated. Previous immunological studies suggested that this serum contained IgG with aγ3-γ1 hybrid heavy chain consisting of aγ3 Fd portion and aγ1 Fc. Structural studies have now shown that the carboxyl terminal end of the 2904 hybrid IgG, including much of the Fc fragment, isγ1 in character. However, the fingerprints of the Fc tryptic peptides at both pH 3.6 and pH 6.4 included a peptide, possibly from the hinge region, in peptide maps of Fc from aγG3 Gm (5) myeloma protein, but absent from maps of Fc peptides fromγG1 proteins. Gel filtration of the CNBr fragments of Fc from 2904 suggested that the hinge region isγ3-like. Papain cleavage experiments indicated an elevated level of resistant IgG, which agrees with immunological findings of an increasedγG2 subclass level. Our data confirm previous reports that theγ chain C-terminal octadecapeptides fromγG3 proteins have a subclass specific residue of arginine and indicate that within this subclass there is an allotypic variation related to the Gm type of the protein.     

Functional identification of ELO-like genes involved in very long chain fatty acid synthesis in Arabidopsis thaliana

Very long chain fatty acids (VLCFAs) are essential lipid components in many plants. 3-Ketoacyl-CoA synthase (KCS) catalyzes the condensation reaction to form 3-ketoacyl-CoA in VLCFA synthesis. AtELO4 has been reported to be involved in VLCFA synthesis, functioning as a KCS in Arabidopsis. However, no studies on other three AtELO members have been reported. Here, we initially found by real-time PCR in Arabidopsis thaliana (L.) Heynh. that AtELO1, AtELO3, and AtELO4 displayed characteristic expression patterns, but AtELO2 was nearly expressed in any organ. Then the transient expression of ELO-like-eGFP fusions in Arabidopsis green leaf protoplasts showed that AtELO1, AtELO3, and AtELO4 were localized in the endoplasmic reticulum (ER), where VLCFA synthesis took place. Finally, we found that the contents of all fatty acids were decreased by 10–20% in seeds of atelo1 T-DNA insertion mutants. In seeds of Pro35S:AtELO1 plants, the levels of all remaining components, except C20:0 and C20:3, were significantly increased. Taken together, our study revealed biological functions of AtELO members and might lay the foundation for further genetic manipulations to generate oil crops with the high oil content.     

A reliable and efficient protocol for induction of hairy roots in Agastache foeniculum

Hairy root culture system is a valuable tool to study the characteristics of gene expression, gene function, root biology, biochemical properties and biosynthesis pathways of secondary metabolites. In the present study, hairy roots were established in Anise hyssop (Agastache foeniculum) via Agrobacterium rhizogenes. Three strains of Agrobacterium rhizogenes (A4, A7 and 9435), were used for induction of hairy roots in four various explants (hypocotyl, cotyledon, one-month-old leaf and five-month-old leaf) of Anise hyssop. The highest frequency of transformation was achieved using A4 strain in one-month-old leaves (51.1%). The transgenic states of hairy root lines were confirmed by PCR (Polymerase chain reaction) method. High performance liquid chromatography analysis revealed that the production of rosmarinic acid (RA) in transformed roots of A. foeniculum was almost 4-fold higher than that of the non-transformed roots. In a separate experiment, hairy roots obtained from one-month-old leaves inoculated with A4 strain, were grown in liquid medium and the effects of different concentrations of salicylic acid (0.0, 0.01, 0.1 and 1 mM) and chitosan (0, 50, 100 and 150 mg L^?1) (as elicitor) and sucrose (20, 30, 40 and 50 g L^?1) on the growth of hairy roots were evaluated. The results showed that, 30 g L^?1 sucrose and 100 mg L^?1 chitosan increased the biomass of hairy root cultures and application of salicylic acid reduced the growth of hairy roots compared with control roots.     

Nucleotide sequence of 5S RNA ofMycobacterium smegmatis

³²P labelled 5S RNA isolated fromMycobacterium smegmatis was digested withT ₁ and pancreatic ribonucleases separately and fingerprinted by two dimensional high voltage electrophoresis on thin-layer DEAE-cellulose plates. The radioactive spots were sequenced and their molar yields were determined. The chain length of the 5S RNA was found to be 120. It showed resemblances to both prokaryotic and eukaryotic 5S RNAs.     

Eight SNPs of the Myf5 gene and diplotypes associated with growth and reproductive traits in Jinghai yellow chicken

The objective of this study was to analyze possible associations between single nucleotide polymorphisms (SNPs) in the Myf5 gene with chicken growth and reproductive traits. SNPs in Myf5 of the Jinghai yellow chicken were detected by the polymerase chain reaction single-strand conformation polymorphism method and the haplotypes were analyzed. Eight SNPs were identified in the exons of Myf5. Nine haplotypes were established in a group of 379 Jinghai yellow chickens. In terms of growth traits, least square analysis showed that haplotype H1H5 had significant effects on weight at weeks 8 and 12 (P < 0.05). Haplotype H2H6 had significant effects on weight at weeks 12 and 14 (P < 0.05). For reproductive traits, H1H5 had higher body weight for the first egg than H1H4 and H2H4 (P < 0.05), and H1H3 (P < 0.01). H1H3 had a poor performance in average egg weight at 300 days. On the other hand, H1H3 had an advantage in egg number at 300 days. The results showed that SNPs of Myf5 have certain effects on growth and reproductive traits in Jinghai yellow chickens, which can be used in marker-assisted selection to accelerate chicken genetic progress.     

The electron transport system of the anaerobic Propionibacterium shermanii

1. Electron transport particles obtained from cellfree extracts of Propionibacterium shermanii by centrifugation at 105000xg for 3 hrs oxidized NADH, d,l-lactate, l-glycerol-3-phosphate and succinate with oxygen and, except for succinate, with fumarate, too.
2. Spectral investigation of the electron transport particles revealed the presence of cytochromes b, d and o, and traces of cytochrome a ₁ and a c-type cytochrome. Cytochrome b was reduced by succinate to about 50%, and by NADH, lactate or glycerol-3-phosphate to 80–90.
3. The inhibitory effects of amytal and rotenone on NADH oxidation, but not on the oxidation of the other substrates, indicated the presence of the NADH dehydrogenase complex, or “site I region”, in the electron transport system of P. shermanii.
4. NQNO inhibited substrate oxidations by oxygen and fumarate, as well as equilibration of the flavoproteins of the substrate dehydrogenases by way of menaquinone. The inhibition occurred at low concentrations of the inhibitor, and reached 80–100%, depending on the substrate tested. The site of inhibition of the respiratory activity was located between menaquinone and cytochrome b. In addition, inhibition of flavoprotein equilibration suggested that NQNO acted upon the electron transfer directed from menaquinol towards the acceptor to be reduced, either cytochrome b or the flavoproteins, which would include fumarate reductase.
5. In NQNO-inhibited particles, cytochrome b was not oxidized by oxygen-free fumarate, but readily oxidized by oxygen. It was concluded from this and the above evidence that the branching-point of the electron transport chain towards fumarate reductase was located at the menaquinone in P. shermanii. It was further concluded that all cytochromes were situated in the oxygen-linked branch of the chain, which formed a dead end of the system under anaerobic conditions.
6. Antimycin A inhibited only oxygen-linked reactions of the particles to about 50% at high concentrations of the inhibitor. Inhibitors of terminal oxidases were inactive, except for carbon monoxide.