Expression systems for use in actinomycetes and related organisms

There have been significant advances in genetic and molecular approaches to understanding the physiology of organisms belonging to the genera Mycobacterium, Corynebacterium, Nocardia and Streptomyces. This review discusses recent advances in heterologous protein expression in members of the actinomycete group, including codon usage, post-translational modification and inducible gene expression.     

Divergence in gene expression related to variation in host specificity of an ectomycorrhizal fungus

Ectomycorrhizae are formed by mutualistic interactions between fungi and the roots of woody plants. During symbiosis the two organisms exchange carbon and nutrients in a specific tissue that is formed at the contact between a compatible fungus and plant. There is considerable variation in the degree of host specificity among species and strains of ectomycorrhizal fungi. In this study, we have for the first time shown that this variation is associated with quantitative differences in gene expression, and with divergence in nucleotide sequences of symbiosis-regulated genes. Gene expression and sequence evolution were compared in different strains of the ectomycorrhizal fungus Paxillus involutus; the strains included Nau, which is not compatible with birch and poplar, and the two compatible strains Maj and ATCC200175. On a genomic level, Nau and Maj were very similar. The sequence identity was 98.9% in the 16 loci analysed, and only three out of 1075 genes analysed by microarray-based hybridizations had signals indicating differences in gene copy numbers. In contrast, 66 out of the 1075 genes were differentially expressed in Maj compared to Nau after contact with birch roots. Thirty-seven of these symbiosis-regulated genes were also differentially expressed in the ATCC strain. Comparative analysis of DNA sequences of the symbiosis-regulated genes in different strains showed that two of them have evolved at an enhanced rate in Nau. The sequence divergence can be explained by a decreased selection pressure, which in turn is determined by lower functional constraints on these proteins in Nau as compared to the compatible strains.     

A comprehensive functional analysis of tissue specificity of human gene expression

Background

Periodic patterning of iterative structures is a fundamental process during embryonic organization and development. Studies have shown how gene networks are employed to pattern butterfly eyespots, fly bristles and vertebrate epithelial appendages such as teeth, feathers, hair and mammary glands. Despite knowledge of how these features are organized, little is known about how diversity in periodic patterning is generated in nature. We address this problem through the molecular analysis of oral jaw dental diversity in Lake Malawi cichlids, where closely related species exhibit from 1 to 20 rows of teeth, with total teeth counts ranging from around 10 to 700.

Results

We investigate the expression of conserved gene networks (involving bmp2, bmp4, eda, edar, fgf8, pax9, pitx2, runx2, shh and wnt7b) known to pattern iterative structures and teeth in other vertebrates. We show that spatiotemporal variation in expression pattern reflects adult morphological diversity among three closely related Malawi cichlid species. Combinatorial epithelial expression of pitx2 and shh appears to govern the competence both of initial tooth sites and future tooth rows. Epithelial wnt7b and mesenchymal eda are expressed in the inter-germ and inter-row regions, and likely regulate the spacing of these shh-positive units. Finally, we used chemical knockdown to demonstrate the fundamental role of hedgehog signalling and initial placode formation in the organization of the periodically patterned cichlid dental programme.

Conclusion

Coordinated patterns of gene expression differ among Malawi species and prefigure the future-ordered distribution of functional teeth of specific size and spacing. This variation in gene expression among species occurs early in the developmental programme for dental patterning. These data show how a complex multi-rowed vertebrate dentition is organized and how developmental tinkering of conserved gene networks during iterative pattern formation can impact upon the evolution of trophic novelty.     

Improving the sensitivity and specificity of protein homology search by incorporating predicted secondary structures

In this paper, we improve the homology search performance by the combination of the predicted protein secondary structures and protein sequences. Previous research suggested that the straightforward combination of predicted secondary structures did not improve the homology search performance, mostly because of the errors in the structure prediction. We solved this problem by taking into account the confidence scores output by the prediction programs.     

Improving bgl1 gene expression in Saccharomyces cerevisiae through meiosis in an isogenic triploid

Introducing large numbers of target genes into the chromosome of Saccharomyces cerevisiae via δ-sequence-mediated integration is a good strategy for exploring the effects of gene dosage on expression and secretion of heterologous proteins. The expression of exogenous genes might be further improved through meiosis in an isogenic triploid. Here, a stable strain A-8 was screened from 35 sexual spore colonies obtained from an isogenic triploid integratively expressing bgl1 from Aspergillus aculeatus. The corresponding β-glucosidase activity in this strain was increased by ~120 % compared with the parent strain BGL-a. Measurement of doubling time, flow cytometry, and mating experiments further confirmed that A-8 was a spore-forming strain obtained from a triploid parent. Thus, combining δ-integration and meiosis in an isogenic triploid is a promising approach for improving the expression of exogenous proteins in S. cerevisiae.     

Allelic variation in gene expression identified through computational analysis of the dbEST database

Differential expression between the two alleles of an individual and between people with different genotypes has been commonly observed. Quantitative differences in gene expression between people may provide the genetic basis for the phenotypic difference between individuals and may be the primary cause of complex diseases. In this paper, we developed a computational method to identify genes that displayed allelic variation in gene expression in human EST libraries. To model allele-specific gene expression, we first identified EST libraries in which both A and B alleles were expressed and then identified allelic variation in gene expression based on the EST counts for each allele using a binomial test. Among 1107 SNPs that had a sufficient number of ESTs for the analysis, 524 (47%) displayed allelic variation in at least one cDNA library. We verified experimentally the allelic variation in gene expression for 6 of these SNPs. The frequency of allelic variation observed in EST libraries was similar to the previous studies using the SNP chip and primer extension method. We found that genes that displayed allelic variation were distributed throughout the human genome and were enriched in certain chromosome regions. The SNPs and genes identified in this study will provide a rich source for evaluating the effects of those SNPs and associated haplotypes in human health and diseases.     

糜子抗旱节水相关基因PmMYB的克隆及表达分析

根据在糜子抗旱节水分子基础研究中获得的一个糜子MYB基因的EST序列, 以其序列及水稻MYB18基因的序列为基础设计引物, 扩增得到1 739 bp的全长基因组序列。序列分析表明, 其包含121 bp(347~467 bp)和93 bp(599~691 bp)的两个内含子, 3个外显子; 全长cDNA序列为1 525 bp, 其中3′非翻译区为212 bp, 5′非翻译区为41 bp, 编码区为1 272 bp, 共编码424个氨基酸, C-端存在一个丝氨酸(Ser, S)丰富区。该基因具有两个典型的MYB类转录因子基因的DNA结合区(DNA-binding domain), 分别为13~63、66~114位氨基酸, 属于典型的R2R3-MYB转录因子。对其与水稻、玉米、火炬松、拟南芥、辣椒、陆地棉、大麦及茄子等9种植物的MYB基因的R2、R3重复区的氨基酸序列多重比较, 表明R2R3重复序列在植物中具有较高的保守性; 基于氨基酸序列的编码区系统进化树分析表明, 不同植物的MYB基因遗传分化很大, 序列相似性为32%~84%, 其中糜子MYB基因与水稻的MYB18相似程度最高(84%), 与大麦和玉米的相似性分别为46%和41%。通过半定量RT-PCR对其表达模式分析表明, 该基因在水分胁迫和干旱后复水条件下上调表达, 与糜子抗旱节水紧密相关。该基因的克隆为进一步探讨利用该基因改良其他植物的抗旱节水性奠定了良好的基础。     

Species-specificity of DNA trimer densities in chromosomes and their use in the classification of closely related organisms

16S rDNA sequences are conventionally used for classification of organisms. However, the use of these sequences is sometimes not successful, especially for closely related species. For better classification of these organisms, several methods that are genome sequence-based have been developed. Sequence alignment-based methods are tedious and time-consuming, as they need conserved coding sequences to be identified and deduced prior to sequence alignment. Likewise, method that relies on gene function needs genes to be assessed for function similarity. Other alignment-free methods, which are based on particular genome sequence properties, so far have been complex and not species-specific enough for classification of organisms below genus level. The present study found that the ratios of DNA trimer frequencies to chromosomal length were species-specific. Density of a trimer in a chromosomal sequence was defined as the average frequency of the trimer per 1 kbp. The species-specificity of trimer densities in chromosomes of many closely related bacteria was compared in parallel with 16S rDNA sequences in these same bacteria. The results of these comparisons indicate that trimer densities in chromosomes can be used to simply and efficiently classify the organisms below genus level.     

Ranking differentially expressed genes from Affymetrix gene expression data: methods with reproducibility, sensitivity, and specificity

Background  

To identify differentially expressed genes (DEGs) from microarray data, users of the Affymetrix GeneChip system need to select both a preprocessing algorithm to obtain expression-level measurements and a way of ranking genes to obtain the most plausible candidates. We recently recommended suitable combinations of a preprocessing algorithm and gene ranking method that can be used to identify DEGs with a higher level of sensitivity and specificity. However, in addition to these recommendations, researchers also want to know which combinations enhance reproducibility.     

Anomeric specificity of the monosaccharide carrier in yeasts and yeast-like organisms

The anomeric specificity of monosaccharide uptake was investigated in 42 species of yeasts and related mycelium-forming fungi. Differences in the uptake of anomers were determined by the following methods. (1) Shift of anomeric equilibrium in the outer medium caused by preferential uptake of one of the anomeric forms was monitored polarimetrically as induced mutarotation. (2) The uptake of 1¹⁴C-D-glu-cose by cells was examined after addition of freshly prepared solutions of α-or β-D-glucose. Most of the organisms examined display theSaccharomyces-type preference for the α-anomers of glucose and xylose which is caused by the higher affinity of the monosaccharide carrier for the α-pyranose configuration. The following genera show this type of preference (the number of species is given in parentheses):Saccharomyces (5),Schizosaccharomyces (1),Endomycopsis (2),Eremascus (1),Endomyces (1),Pichia (1),Hansenula (1),Debaryomyces (2),Lipomyces (1),Willia (1),Nematospora (1),Kluyveromyces (2),Candida (6),Torulopsis (5),Cryptococcus (1). No anomeric specificity was shown by the following genera:Nadsonia (1),Dipodascus (2),Rhodotorula (5),Sporobolomyces (2),Bullera (1),Rhodosporidium (1). A parallel investigation of the concentration dependence of glucose uptake indicates that most yeasts possess a constitutive monosaccharide carrier characterized by the following features: a high maximum rate of uptake, a relatively low affinity, and preference for α-anomers. Besides this carrier the majority of these microorganisms possess a glucosetransporting carrier with a higher affinity and a lower capacity.