Effect of integration loci of genome on heterologous gene expression in Saccharomyces cerevisiae / 生物工程学报

Chromosomal integration of heterologous genes or pathways is preferred over the use of episomal plasmids for its inherently stability and thus more desirable in the industrial setting. However, the position of integration of heterologous genes in the genome influences the expression levels. In combination of high throughput transformation of the Yeast Knock-out Collection (YKO) and FACS analysis, the position effect on heterologous reporter gene gfp was identified across the whole genome in yeast. In total 428 high-expressed sites and 444 low-expressed sites were spotted, providing massive data to analyze patterns and reasons for region dependency of gene expression on the genome-wide scale.

Progress in omics research of Aspergillus niger / 生物工程学报

Aspergillus niger, as an important industrial fermentation strain, is widely applied in the production of organic acids and industrial enzymes. With the development of diverse omics technologies, the data of genome, transcriptome, proteome and metabolome of A. niger are increasing continuously, which declared the coming era of big data for the research in fermentation process of A. niger. The data analysis from single omics and the comparison of multi-omics, to the integrations of multi-omics based on the genome-scale metabolic network model largely extends the intensive and systematic understanding of the efficient production mechanism of A. niger. It also provides possibilities for the reasonable global optimization of strain performance by genetic modification and process regulation. We reviewed and summarized progress in omics research of A. niger, and proposed the development direction of omics research on this cell factory.

Associação entre resiliência e qualidade de vida relacionada à saúde bucal em idosos / Association between resilience and quality of life related to oral health in the elderly

OBJETIVO: Avaliar a associação entre resiliência e qualidade de vida relacionada à saúde bucal, por meio de uma abordagem hierárquica baseada em um modelo teórico conceitual em uma coorte de idosos do Rio Grande do Sul. MÉTODOS: Foi conduzido um estudo transversal aninhado a um estudo de coorte, em 2008. Foram avaliados 498 idosos de Carlos Barbosa, Rio Grande do Sul. As medidas avaliadas foram sociodemográficas, comportamentos de saúde, qualidade de vida relacionada à saúde bucal, medida pelo Oral Health Impact Profile (OHIP-14), Escala de Resiliência e CPOD. A associação entre o potencial de resiliência e os impactos na percepção de saúde bucal relacionados à qualidade de vida foi verificada por meio de regressão binomial negativa. Razões das médias (RM) são apresentadas com seus intervalos de confiança de 95% (IC95%). RESULTADOS: Maiores médias do OHIP foram encontradas entre mulheres (6,7 ± 6,3; p = 0,011), moradores da zona rural (7,3 ± 6,7; p = 0,004) e solteiros (8,0 ± 6,3; p = 0,032). O modelo final da análise multivariada mostrou que ser morador da zona rural (RM = 1,32; IC95% 1,06 - 1,65) e casado (RM = 1,36; IC95% 1,07 - 1,72) foram variáveis independentemente associadas à qualidade de vida relacionada à saúde bucal. Não houve associação entre resiliência e qualidade de vida relacionada à saúde bucal. CONCLUSÃO: Os resultados sugerem que variáveis sociodemográficas estão associados à qualidade de vida relacionada à saúde bucal. A hipótese de que a resiliência pudesse exercer um papel importante no desfecho não foi confirmada. .

OBJECTIVE: To evaluate the association between psychological resilience and oral health related to quality of life through a hierarchical approach based on a conceptual theoretical model in a cohort of elderly residents in Rio Grande do Sul, Brazil. METHODS: We conducted a cross-sectional study nested in a cohort study in 2008. We evaluated 498 elderly residents in Carlos Barbosa, Rio Grande do Sul. The measures included sociodemographic questionnaire, health behavior, quality of life related to oral health (OHRQOL), measured by the Oral Health Impact Profile (OHIP-14), Resilience Scale, and DMFT. The association between resilience and potential impacts on perceptions of oral health-related quality of life was assessed through negative binomial regression. Mean ratios (MR) are presented with 95% confidence intervals (95%CI). RESULTS: Higher means of OHIP were found in women 6.7 ± 6.3; p = 0.011), in rural dwellers (7.3 ± 6.7; p = 0.004) and singles (8.0 ± 6.3; p = 0.032). The final model of multivariate analysis showed that being a rural dweller (MR = 1.32; 95%CI 1.06 - 1.65) and being married (MR = 1.36; 95%CI 1.07 - 1.72) were independently associated with OHRQOL. There was no association between resilience and OHRQOL. CONCLUSION: The results suggest that factors such as sociodemographic variables are associated with OHRQOL. The hypothesis that resilience might play a role in the outcome has not been confirmed. .

In Silico Sequence Analysis Reveals New Characteristics of Fungal NADPH Oxidase Genes

NADPH oxidases (Noxes), transmembrane proteins found in most eukaryotic species, generate reactive oxygen species and are thereby involved in essential biological processes. However, the fact that genes encoding ferric reductases and ferric-chelate reductases share high sequence similarities and domains with Nox genes represents a challenge for bioinformatic approaches used to identify Nox-encoding genes. Further, most studies on fungal Nox genes have focused mainly on functionality, rather than sequence properties, and consequently clear differentiation among the various Nox isoforms has not been achieved. We conducted an extensive sequence analysis to identify putative Nox genes among 34 eukaryotes, including 28 fungal genomes and one Oomycota genome. Analyses were performed with respect to phylogeny, transmembrane helices, di-histidine distance and glycosylation. Our analyses indicate that the sequence properties of fungal Nox genes are different from those of human and plant Nox genes, thus providing novel insight that will enable more accurate identification and characterization of fungal Nox genes.

Comparative studies on codon usage bias of Ganoderma lucidum based on analysis of genomic and transcriptomic data / 药学学报

Codon usage bias is an important characteristic of genetic information transfer in organisms. Analysis of codon usage bias of different species is important for understanding the rules on genetic information transfer. The previous method for analysis of codon usage bias is mainly based on genomic data. However, this method is greatly limited, because the genome sequences of higher organisms are still not available up to now. In this study, we found that we could obtain the same optimal codons of Ganoderma lucidum (Curtis: Fr.) P. Karst based on its whole genomic data or large-scale transcriptomic data from its liquid-cultured hyphae, primordium and fruiting body, separately. This result indicated the feasibility to understand the codon usage bias based on the large-scale transcriptomic data. By calculating the proportion of rare codons of Escherichia coli and Saccharomyces cerevisiae in 26 terpene synthases (TS) of G. lucidum, we found that the rare codons of S. cerevisiae have a higher proportion in TS genes, while the rare codons of E. coli have relatively lower, suggesting that the TS genes of G. lucidum are possibly more difficult to be expressed in S. cerevisiae than in E. coli. Chemical synthesis of TS genes according to the yeast optimal codons will be an effective way to solve the problem on the mismatch of gene codon bias between the foreign genes and the host strain.

Study of EST-SSR marker system of Cordyceps / 中国中药杂志

<p><b>OBJECTIVE</b>To establish the EST-SSR marker system for Cordyceps by using ESTs of C. bassiana and C. militaris.</p><p><b>METHOD</b>The ESTs of Cordyceps were downloaded from the public database of NCBI, and the redundant ESTs with low quality were removed. The EST-SSR primers were designed by Sequece Seiner 1. 2. And the primers were screened through PAGE-Electrophoresis.</p><p><b>RESULT</b>The 4 556 non-redundant ESTs which from C. bassiana with total length of 2 953 173 bp were selected. 718 EST-SSRs distributed in 616 ESTs were totally screened out, accounting for 15.8% of the non-redundant ESTs. It was discovered that the average distance of EST-SSSR was 1/4 096 bp in EST-SSRs distribution of C. bassiana. Trinucleotide repeats were the most abundant types with 419 repeated sequences. Regarding to C. militaris, totally 1 363 non-redundant ESTs were acquired, from which 1 117 EST-SSRs were screened, and rate of SSR sites in ESTs was 81.95%. The leading motif of SSR was nucleotide A. The 50 pairs of EST-SSR primers were designed according to the ESTs of C. bassiana, and preliminary test showed the 34 pairs of primers amplified clear fragments,accounting for 68% of all primers. Furthermore, the 39 of the 40 pairs of primers from the ESTs of C. militaris were found to be amplified as the clear fragments, accounting for 97.5%. The phylogenetic analysis revealed that different anamorph of Cordyceps spieces were divided into four branches.</p><p><b>CONCLUSION</b>The EST-SSR of Cordyceps had comparably higher utility value. The EST-SSR markers developed from ESTs of C. bassiana and C. militaris had well transferability in Cordyceps. And it was suggested that the EST-SSR markers should be an easy and effective way to assay molecular genetic structure of Cordyceps.</p>

Progress in lignocellulose deconstruction by fungi / 生物工程学报

Inefficient degradation of lignocellulose is one of the main barriers for the utilization of renewable plant biomass for biofuel production. The bottleneck of the biorefinery process is the generation of fermentable sugars from complicated biomass polymers. In nature, the main microbes of lignocelluloses deconstruction are fungi. Therefore, elucidating the mechanism of lignocelluloses degradation by fungi is of critical importance for the commercialization of lignocellulosic biofuels. This review focuses on the progress in lignocelluloses degradation pathways in fungi, especially on the advances made by functional genomics studies.

Aspergillus niger as a potential cellular factory: prior knowledge and key technology / 生物工程学报

Aspergillus niger is an important industrial workhorse with extensive application in the sectors of industrial enzymes, heterogeneous proteins, organic acids and etc. The disclosure of its genomic sequence to the public brought the study of A. niger into the post-genomic era. Diverse omic data are being produced massively and rapidly, which largely upgrades our understanding to the hyperproduction mechanism of A. niger to a systems and molecular level. At meanwhile, its genetic operating system is becoming mature, which enables genome-scale genetic perturbation within A. niger. In conclusion, we are on the right way to redesign and engineer A. niger to an omnipotent cellular factory.

Neurospora crassa fmf-1 encodes the homologue of the Schizosaccharomyces pombe Ste11p regulator of sexual development.

The Neurospora crassa fmf-1 mutation exerts an unusual 'perithecium-dominant' developmental arrest; fmf-1 x fmf-1+ cross becomes arrested in perithecial development regardless of whether the mutant participates in the cross as the male or female parent. We localized fmf-1 to the LG IL genome segment between the centromere-proximal breakpoint of the chromosome segment duplication Dp(IL)39311 and the centromere. By mapping crossovers with respect to RFLP markers in this region we further localized fmf-1 to an approximately 34-kb-genome segment. Partial sequencing of this segment revealed a point mutation in the gene NCU 09387.1, a homologue of the Schizosaccharomyces pombe ste11+ regulator of sexual development. The fmf-1 mutation did not complement a NCU 09387.1 deletion mutation, and transformation with wild-type NCU 09387.1 complemented fmf-1. S. pombe Ste11 protein (Ste11p) is a transcription factor required for sexual differentiation and for the expression of genes required for mating pheromone signalling in matP and matM cells. If FMF-1 also plays a corresponding role in mating pheromone signalling in Neurospora, then protoperithecia in an fmf-1 x fmf-1+ cross would be unable to either send or receive sexual differentiation signals and thus become arrested in development.

Production of amorpha-4,11-diene in engineered yeasts / 药学学报

Plasmid-carrying Saccharomyces cerevisia (W303-1B[pYeDP60/G/ADS]) and genome-transformed S. cerevisia (W303-1B[rDNA:ADS]), both harboring amorpha-4,11-diene synthase (ADS) gene were constructed to investigate the production of amorpha-4,11-diene. The recombinant plasmid pYeDP60/G/ADS that harbors the ADS gene was transformed into S. cerevisiae W303-1B, resulting in the engineered yeast W303-1B[pYeDP60/G/ADS], which contains multi-copies of the plasmid. The ADS gene expression cassette was obtained by PCR amplification of the pYeDP60/G/ADS template, and then introduced into S. cerevisiae W303-1B to obtain the engineered yeast W303-1B[rDNA:ADS], in which the ADS gene was integrated into the rDNA locus of the yeast genome through the homologous recombination. GC-MS analysis confirmed that both of the engineered yeasts could produce amorpha-4,11-diene. Moreover, the amorpha-4,11-diene yield of W303-1B[pYeDP60/G/ADS] was higher than that of W303-1B[rDNA:ADS]. Southern blot analysis showed that there is only one copy of ADS gene in the genome of W303-1B[rDNA:ADS]. It implied that the amorpha-4,11-diene yield can be improved by increasing the ADS gene copies.

Molecular cloning, expression and characterization of a serine proteinase from Japanese edible mushroom, Grifola frondosa: solving the structure - function anomaly of a reported aminopeptidase

The N-terminal amino acid sequence of an aminopeptidase from Japanese edible mushroom, Grifola frondosa, was reported to have high similarity with that of a serine proteinase from basidiomycete, Agaricus bisporous (Nishiwaki and Hayashi, 2001). The full-length cDNA and the corresponding genomic DNA of the enzyme were cloned, based on the reported N-terminal amino acid sequence. The predicted open reading frame (ORF) of the cloned cDNA, encoding a product of 379 amino acids, was expressed in E. coli using pET expression vector. The expressed pro-enzyme (40 kDa) underwent autolysis to produce the mature protein (30 kDa) and a pro-peptide (10 kDa). The mature protein and the pro-peptide remained tightly bound to each other and could not be separated by Ni-NTA metal affinity chromatography or Q-Sepharose ion-exchange chromatography. The enzyme was inactive in the bound form. Upon treatment with subtilisin, the bound pro-peptide was further hydrolyzed and a high serine proteinase activity was recovered. No aminopeptidase activity was detected at any stage of the protein processing. These results clearly indicated that the N-terminal amino acid sequence and the function of the reported aminopeptidase were not derived from the same protein entity and hence caused the structure-function anomaly.

SCGPred: a score-based method for gene structure prediction by combining multiple sources of evidence / 基因组蛋白质组与生物信息学报·英文版

Predicting protein-coding genes still remains a significant challenge. Although a variety of computational programs that use commonly machine learning methods have emerged, the accuracy of predictions remains a low level when implementing in large genomic sequences. Moreover, computational gene finding in newly sequenced genomes is especially a difficult task due to the absence of a training set of abundant validated genes. Here we present a new gene-finding program, SCGPred, to improve the accuracy of prediction by combining multiple sources of evidence. SCGPred can perform both supervised method in previously well-studied genomes and unsupervised one in novel genomes. By testing with datasets composed of large DNA sequences from human and a novel genome of Ustilago maydi, SCG-Pred gains a significant improvement in comparison to the popular ab initio gene predictors. We also demonstrate that SCGPred can significantly improve prediction in novel genomes by combining several foreign gene finders with similarity alignments, which is superior to other unsupervised methods. Therefore, SCG-Pred can serve as an alternative gene-finding tool for newly sequenced eukaryotic genomes. The program is freely available at http://bio.scu.edu.cn/SCGPred/.

Functional genomics in the rice blast fungus to unravel the fungal pathogenicity / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.

Application of semi-nested polymerase chain reaction targeting internal transcribed spacer region for rapid detection of panfungal genome directly from ocular specimens.

BACKGROUND: The incidence of fungal endophthalmitis has dramatically increased in recent years and rapid detection of fungi using nucleic acid-based amplification techniques is helpful in management. AIM: To evaluate semi-nested polymerase chain reaction (PCR) targeting internal transcribed spacer (ITS) region for detection of panfungal genome in ocular specimens. STATISTICAL ANALYSIS USED: Z test for two proportion. MATERIALS AND METHODS: Standardization of PCR targeting ITS primers was carried out by determining analytical sensitivity and specificity. The sensitivity and specificity of PCR was determined by serial tenfold dilutions of C. albicans (ATCC 24433) DNA and DNA extracts of laboratory isolates of Aspergillus fumigatus, Fusarium lichenicola (4), other fungal and closely related bacterial strains and also human DNA. Semi-nested PCR was applied onto a total of 168 ocular specimens with clinically suspected fungal etiology during 2003-2005. RESULTS AND CONCLUSIONS: PCR was specific and sensitive to detect 1fg of fungal DNA with ITS primers. PCR detected fungal genome in 90 (53.57%) in comparison with the conventional technique, positive in 34 (20.23%) by smear examination and in 42 (25%) by culture. The increase in clinical sensitivity by 28.57% using PCR was found to be statistically significant { P < 0.001 using Z test for two proportion}. The accuracy of the test was found to be 70.85%. PCR proved to be a rapid diagnostic technique for detection of panfungal genome directly from clinical specimens.

Minimization of transcriptional temporal noise and scale invariance in the yeast genome

The analysis of transcriptional temporal noise could be an interesting means to study gene expression dynamics and stochasticity in eukaryotes. To study the statistical distributions of temporal noise in the eukaryotic model system Saccharomyces cerevisiae, we analyzed microarray data corresponding to one cell cycle for 6200 genes. We found that the temporal noise follows a lognormal distribution with scale invariance at the genome, chromosomal and sub-chromosomal levels. Correlation of temporal noise with the codon adaptation index suggests that at least 70% of all protein-coding genes are a noise minimization core of the genome. Accordingly, a mathematical model of individual gene expression dynamics was proposed, using an operator theoretical approach, which reveals strict conditions for noise variability and a possible global noise minimization/optimization strategy at the genome level. Our model and data show that minimal noise does not correspond to genes obeying a strictly deterministic dynamics. The natural strategy of minimization consists in equating the mean of the absolute value of the relative variation of the expression level (alpha) with noise (eta). We hypothesize that the temporal noise pattern is an emergent property of the genome and shows how the dynamics of gene expression could be related to chromosomal organization.

Protoplast transformation of Mortierella isabellina with hygromycin B resistance plasmid PD4 / 生物工程学报

A strain Mortierella isabellina M6-22-4, which was sensitive to hygromycin B, was selected by treating parental spores with N-methyl-N' -Nitro-N-nitrosoguanidine (MNNG). Protoplasts of the strain Mortierella isabellina M6-22-4 were transformed successfully to hygromycin B resistance using the PD4 plasmid, which contains the Escherichia coli hph gene under the control of Mortierella alpina his H4.1 promoter. The PD4 plasmid was introduced by PEG/CaCl2 treatment. Transformation frequencies of 1.6 - 2.8 transformants/microg of DNA were achieved. Then they were successively incubated to non-selected PDA plates for 10 generations. About 31.6% transformants only from digested plasmid were mitotically stable and showed different hygromycin B resistance when they were incubated back to selection plates. The results of PCR and Southern analysis in three transformants indicated that the plasmid PD4 had been integrated into the fungal genome with 1 - 2 copies. This is the first report of Mortierella isabellina transformation system and supplies an important tool for further research into genetic manipulation of this filamentous fungus.

Rapid improvement of lipase production in Penicillium expansum by genome shuffling / 生物工程学报

In the present study, the genome shuffling was used to improve lipase production of Penicillium expansum. A lipase producing mutant strain-Penicillium expansum FS8486 and a wild type of Aspergillus Tamarii FS-132 isolated from soil of a volcano in Xinjiang were used as the parental strains. After two rounds of genome shuffling, several elite daughter strains were screened. The lipase activity in one of the daughter strains was increased 317% over the starting strain FS8486. Comparisons of the morphology, RAPD (Random Amplification of Polymorphic DNA) polymorphism and the fatty acid compositions between the daughter and the parental strains suggested that the filial generation were generated by genome shuffling. In this study, the genome shuffling used successfully first time in eukaryotic microorganism and increases the production of the desired metabolite in short time, the study will be useful to spread the genome shuffling in eukaryotic microbial breeding.

Genome-wide expression profiling of the response to terbinafine in Candida albicans using a cDNA microarray analysis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Candida albicans is the most frequently seen opportunistic human fungal pathogen. Terbinafine is an allylamine antifungal agent that has been proven to have high clinical efficacy in the therapy of fungal infections, the mechanism of action of terbinafine involves the specific inhibition of fungal squalene epoxidase, resulting in ergosterol deficiency and accumulation of intracellular squalene. We used cDNA microarray analysis technology to monitor global expression profile changes of Candida albicans genes in response to terbinafine treatment, and we anticipated a panoramic view of the responses of Candida albicans cells to the representatives of allylamine antifungal agents at the molecular level in an effort to identify drug class-specific and mechanism-independent changes in gene expression.</p><p><b>METHODS</b>Candida albicans strain ATCC 90028 was exposed to either medium alone or terbinafine at a concentration equivalent to the 1/2 minimal inhibitory concentrations (MICs, 4 mg/L) for 90 minutes. RNA was isolated and gene expression profiles were compared to identify the changes in the gene expression profile using a cDNA microarray analysis. Differential expression of 10 select genes detected by cDNA microarray analysis was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>A total of 222 genes were found to be responsive to terbinafine, including 121 up-regulated genes and 101 down-regulated genes. These included genes encoding membrane transport proteins belonging to the members of the ATP-binding cassette (ABC) or major facilitator superfamily (MFS; CDR1, AGP2, GAP6, PHO84, HOL3, FCY23, VCX1), genes involved in stress response and detoxification (CDR1, AGP2, HOL3), and gene involved in the ergosterol biosynthesis pathway (ERG12). The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis.</p><p><b>CONCLUSIONS</b>The up-regulation of the gene encoding the multidrug resistance efflux pump CDR1 may contribute to the terbinafine resistance in Candida albicans. However, the precise roles of other affected genes remain unclear, further studies of these genes and their respective products that play roles in the context of antifungal resistance are warranted.</p>

Development and application of multiplex polymerase chain reaction for the etiological diagnosis of infectious endophthalmitis.

BACKGROUND: Uniplex polymerase chain reaction (PCR) for detection of bacterial and panfungal genome has been applied onto a large number of intraocular fluids facilitating management of infective endophthalmitis. AIM: To develop and apply a novel, rapid multiplex polymerase chain reaction (mPCR) to detect the presence of eubacterial, Propionibacterium acnes and panfungal genomes in intraocular fluids from patients clinically diagnosed to have infective endophthalmitis. SETTINGS AND DESIGN: Prospective study. MATERIALS AND METHODS: Conventional methods of direct microscopy by KOH/calcofluor mount, Gram's staining and culture were done on 30 (19 Aqueous humor-AH and 11 Vitreous fluid-VF) intraocular specimens and mPCR done for simultaneous detection of eubacterial, P. acnes and panfungal genomes. RESULTS: mPCR detected an infectious etiology in 18 (60%) of 30 intraocular specimens. Eubacterial genome was detected in 12 (40%) specimens, P. acnes genome in 4 (13.3%) specimens and panfungal genome in 2 (6.6%) specimens. mPCR results correlated with those of uniplex PCR. mPCR results were available within 5-6 hours after receipt of specimen, as against 8 hours required for each uniplex PCR with three separate thermalcyclers for their completion. Consumption of Taq polymerase was reduced considerably for mPCR. CONCLUSION: mPCR is a cost effective, single tube method for the simultaneous detection of eubacterial, P. acnes and panfungal genomes in intraocular specimens from patients with infective endophthalmitis. It is a more rapid procedure than uniplex PCRs and requires only a single thermalcycler.