Some biocybernetic aspects in gene expression and regulatory control

In eucaryotes, gene expression and control is a complex nonlinear process, where there are many control mechanisms and ways, both physic, chemical and informational control. By the exploration from the angle of biocybernetics, the authors suggest that gene expression is a co-control process. In this process, physic, chemical and informational feedback controls are associated and influential each other, and are cross and co-functional. The physic, chemical and informational control ways composed an order non-linear feedback control system in eucaryotes.

Effects of exogenous double-stranded RNA on the basonuclin gene expression in mouse oocytes

In plants and less-advanced animal species, such as C.elegans, introduction of exogenous double-stranded RNA (dsRNA) into cells would trigger degradation of the mRNA with homologous sequence and interfere with the endogenous gene expression. It might represent an ancient anti-virus response which could prevent the mutation in the genome that was caused by virus infection or mobile DNA elements insertion. This phenomenon was named RNA interference, or RNAi. In this study, RNAi was used to investigate the function of basonuclin gene during oogenesis. Microinjection of dsRNA directed towards basonuclin into mouse germinal-vesicle-intact (GV) oocytes brought down the abundance of the cognate mRNA effectively in a time- and concentration-dependent manner. This reduction effect was sequence-specific and showed no negative effect on other non-homologous gene expression in oocytes, which indicated that dsRNA can recognize and cause the degradation of the transcriptional products of endogenous basonuclin gene in a sequence-specific manner. Immunofluorescence results showed that RNAi could reduce the concentration of basonuclin protein to some extent, but the effect was less efficient than the dsRNA targeting towards tPA and cMos which was also expressed in oocytes. This result might be due to the long half life of basonuclin protein in oocytes and the short reaction time which was posed by the limited life span of GV oocytes cultured in vitro. In summary, dsRNA could inhibit the expression of the cognate gene in oocytes at both mRNA and protein levels. The effect was similar to Knock-out technique which was based on homologous recombination. Furthermore, hairpin-style dsRNA targeting basonuclin gene could be produced by transcription from a recombinant plasmid and worked efficiently to deplete the cognate mRNA in oocytes. This finding offered a new way to study the function of basonuclin in the early stage of oogenesis by infection of primordial oocytes with the plasmid expressing hairpin-style basonuclin dsRNA.

Effect of beta-glucan on activity of antioxidant enzymes and Mx gene expression in virus infected grass carp

The effects of beta-glucan, an immunostimulatory agent, on the superoxide dismutase (SOD) and catalase (CAT) activities of erythrocytes and Mx gene expression were studied from grass carp that were challenged with grass carp hemorrhage virus (GCHV). The SOD and CAT activities in erythrocytes and Mx gene expression in spleen from the fish were detected by spectrophotometry and RT-PCR, respectively. Negative control fish were injected with PBS; positive control groups were injected with either P-glucan or GCHV only; and the experimental groups were pre-injected with beta-glucan 15 days prior to injection with GCHV. The results show that the SOD and CAT activities were higher in fish injected with beta-glucan for 15 days than the negative control group injected with PBS. The SOD and CATactivities significantly decreased when the fish were challenged with GCHV, but it was higher in the group pre-treated with beta-glucan than in infected fish not pre-treated, 15 days after GCHV infection. Mx gene expression levels increased during the early stages (at 12 h and 36 h) of GCHV infection, and it remained at higher levels from the 6th till the 10th day in the beta-glucan pre-treated group, but it was failing from the 6th day in the beta-glucan untreated group. The GCHV-infected group pre-treated with P-glucan had a higher survival rate (60%) than the group not pre-treated with P-glucan (20%), suggesting that beta-glucan possesses or enhances anti-viral responses. (C) 2009 Elsevier Ltd. All rights reserved.

Allelopathic mechanism of pyrogallol to Microcystis aeruginosa PCC7806 (Cyanobacteria): From views of gene expression and antioxidant system

Pyrogallol is a potent allelochemical on Microcystis aeruginosa, but its allelopathic mechanism is not fully known. In order to explore this mechanism, gene expressions for prx, mcyB, psbA, recA, grpE, fabZ under pyrogallol stress were studied, and activities of the main antioxidant enzymes were also measured. The results showed that expression of grpE and recA showed no significant change under pyrogallol stress, while psbA and mcyB were up-regulated at 4 mg L-1. Both prx and fabZ were up-regulated even under exposure to 1 mg L-1 pyrogallol concentration. The activities of superoxide dismutase (SOD) and catalase (CAT) were enhanced under pyrogallol stress. Levels of malodialdehyde (MDA) at 2 and 4 mg L-1 pyrogallol were significantly higher than those of the controls. It was concluded that oxidant damage is an important mechanism for the allelopathic effect of pyrogallol on M. aeruginosa. (c) 2009 Elsevier Ltd. All rights reserved.

The first study on the effects of microcystin-RR on gene expression profiles of antioxidant enzymes and heat shock protein-70 in Synechocystis sp PCC6803

Microcystins are heptapeptide toxins produced by cyanobacteria. Microcystin-RR(MC-RR) is a common variant among the 80 variants identified so far. There have been many investigations documenting the toxic effects of microcystins on animals and higher plants, but little is known on the toxic effects of microcystins on algae, especially at molecular level. We studied the effects of MC-RR on gene expression profile of a few antioxidant enzymes and heat shock protein-70 (Hsp70) in Synechocystis sp. PCC6803. After two days post-exposure, a high dose toxin (5 mg/l, about 4.8 x 10(-3) mM) significantly increased expression levels of the genes gpx1, sodB, katG, acnB, gamma-TMTand dnaK2, while a relatively low dose toxin (1 mg/l, about 9.63 x 10(-4) mM) induced a moderate and slow increase of gene expression. Our results indicate that MC-RR could induce the oxidative stress in Synechocystis sp. PCC6803 and the increase in gene expression of antioxidant enzymes and Hsp70 might protect the organism from the oxidative damage. in addition, cell aggregation was observed during the early period of exposure, which might be a specific oxidative stress reaction to MC-RR. (C) 2008 Elsevier Ltd. All rights reserved.

Cloning, characterization, and gene expression analysis of a novel cadmium metallothionein gene in Tetrahymena pigmentosa

A novel cadmium-inducible metallothionein (MT) gene (Tpig-MT1) was cloned and sequenced from the ciliate Tetrahymena pigmentosa. The number of deduced amino acids is 118. The polypeptide possesses CCC and CC clusters characteristic of typical Tetrahymena Cd-inducible MTs. The structure of Tpig-MT1 is different from the reported Cd-MT in T. pyriformis, T. thermophila and T. pigmentosa. Tpig-MT1 contains two intragenic tandem repeats with 72.9% identity described as Tpig-MT1 (repeat A1) and Tpig-MT1 (repeat A2). The transcriptional response of Tpig-MT1 gene to different heavy metals (Cd, Cu, Zn, Hg, Pb) and oxidative stress (H2O2) was measured using real-time quantitative PCR. The results showed that the gene was quickly induced (1 h) by the five heavy metals and the order of expression level was Hg>Pb>Cd>Cu>Zn. The induction effect of H2O2 was 5-fold after about 15 min, but soon decreased to a non-significant level (30 min). The genetic diversity of Tetrahymena MT genes is discussed in relation to the unique structure of the Tpig-MT1 gene and other reported Cd-MT isoforms. (C) 2008 Elsevier B.V. All rights reserved.

Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS

Perfluorooetanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship, were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure to PFOS concentrations of I mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development (hhex and pax8) and genes regulating the balance of androgens and estrogens (cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential mechanisms of developmental toxicity. (C) 2008 Elsevier Inc. All rights reserved.

Inhibition of no tail (ntl) gene expression in zebrafish by external guide sequence (EGS) technique

External guide sequence (EGS) technique, a branch of ribozyme strategy, can be enticed to cleave the target mRNA by forming a tRNA-like structure. In the present study, no tail gene (ntl), a key gene participating in the formation of normal tail, was used as a target for ribonuclease (RNase) P-mediated gene disruption in zebrafish in vivo. Transient expression of pH1-m3/4 ntl-EGS or pH1-3/4 ntl-EGS produced the full no tail phenotype at long-pec stage in proportion as 24 or 35%, respectively. As is expected that the full-length ntl mRNA of embryos at 50% epiboly stage decreased relative to control when injected the embryos with 3/4 EGS or m3/4 EGS RNA. Interestingly, ntl RNA transcripts, including the cleaved by EGS and the untouched, increased. Taken together, these results indicate that EGS strategy can work in zebrafish in vivo and becomes a potential tool for degradation of targeted mRNAs.

Gene expression profiles in liver of zebrafish treated with microcystin-LR

Microcystin-LR (MC-LR) is the most frequently studied cyclic heptatoxin produced by cyanobacteria, which has tremendous negative impacts on fish, while its molecular mechanism behind remained unclear at present. Here, Affymetrix Zebrafish GeneChip was used to identify alterations in gene expression of zebrafish (Danio rerio) after MC-LR exposure. Among the 14,900 transcripts in the microarray, 273 genes were differentially expressed, in which 243 genes were elevated and 30 were decreased. According to GOstat analysis, MC-LR mainly influenced the cell cycle and mitogen-activated protein kinases (MAPK) signaling pathways. In addition, many immune-related genes were also influenced. These data suggest that MC-LR could promote tumorigenesis and cause immunotoxicity in fish. (C) 2008 Elsevier B.V. All rights reserved.

Effects of age and dietary protein level on digestive enzyme activity and gene expression of Pelteobagrus fulvidraco larvae

The present research studied the effects of age and dietary protein level on pepsin, trypsin and amylase activity and their mRNA level in Petteobagrus fulvidraco larvae from 3 to 26 days after hatch (DAH). Three DAH larvae were fed three isoenergetic diets, containing 42.8% (CP 43), 47.3% (CP 47) and 52.8% (CP 53) crude protein. Live food (newly hatched Artemia, unenriched) was included as a control. The effects of age on enzyme activity and mRNA were as follows: pepsin and trypsin activity in all treatment groups showed a significant (P < 0.05) increase at the beginning and decrease later although the timing of decrease was not the same among treatment groups and between the digestive enzymes. Pepsin and trypsin mRNA level followed the pattern of their respective enzyme changes. Age significantly affected amylase activity (P < 0.05) while age had no effect on amylase mRNA during the experimental period. The four diets significantly (P < 0.05) affected activity and mRNA level of pepsin and trypsin. Diets did not affect amylase activity or mRNA level. These results suggest that the effects of age on pepsin and trypsin gene expressions are at the transcriptional level. Dietary protein level does affect pepsin and trypsin gene expression in the early life of P. fulvidraco. There were no transcriptional effects on amylase gene expression. (c) 2005 Elsevier B.V. All rights reserved.

Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps

Silver crucian carp (Carassius auratus gibelio) is a unique triploid bisexual species that can reproduce by gynogenesis. As all other gynogenetic animals, it keeps its chromosome integrity by inhibiting the first meiosis division (no extrusion of the first pole body). To understand the molecular events governing this reproduction mode, suppression subtractive hybridization was used to identify the genes differentially expressed in fully-grown oocytes of the gynogenetic and gonochoristic crucian carp (gyno-carp and gono-carp). From two specific subtractive cDNA libraries, the clones screened out by dot blots and virtual Northern blots were chosen to clone, full-length cDNA by RACE. Four differentially expressed genes were obtained. Two are novel genes and are expressed specifically in the oocytes. The gyno-carp stores much more mRNA of cyclin A2, a new member of the fish A-type cyclin gene, in its fully-grown oocyte than in the gono-carp. The last gene is histone H2A. The histone H2As of these two closely related crucian carps are quite different in the C-terminus. Preliminary characterization of the four genes has been analyzed by nucleotide and deduced amino acid sequence and Northern analysis. (C) 2001 Elsevier Science B.V. All rights reserved.

Differential gene expression of protein kinases in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp

A modified mRNA differential display method has been applied to studying differential expression of protein kinase genes in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp. Total RNA was reverse transcribed using downstream 3' primers T(12)MA, T(12)MG and T12MC respectively. Then the reverse transcription products were amplified using upstream 5' kinase-specific primer designed according to protein kinase conserved sequence. The PCR products had different patterns and numbers of: cDNA bands on polyacrylamide:gel. Totally 21 cDNAs fragments were recovered and cloned. Two of them were confirmed to be particularly expressed in oocytes of amphimictic crucian carp, and another was specific for gynogenetic silver crucian carp.

Cancer subtypes recognition and its gene expression profiles analysis based on geometrical learning

The accurate recognition of cancer subtypes is very significant in clinic. Especially, the DNA microarray gene expression technology is applied to diagnosing and recognizing cancer types. This paper proposed a method of that recognized cancer subtypes based on geometrical learning. Firstly, the cancer genes expression profiles data was pretreated and selected feature genes by conventional method; then the expression data of feature genes in the training samples was construed each convex hull in the high-dimensional space using training algorithm of geometrical learning, while the independent test set was tested by the recognition algorithm of geometrical learning. The method was applied to the human acute leukemia gene expression data. The accuracy rate reached to 100%. The experiments have proved its efficiency and feasibility.