筛选抑制酿酒酵母生长的人类基因

【目的】基于人类基因文库,构建一个筛选抑制酿酒酵母生长的人类基因的方法,并运用此方法筛选含有生长抑制性人源蛋白质的酿酒酵母,用于分析人类基因的生理功能及其抑制剂的寻找。【方法】利用Gateway~(TM)重组技术将人类蛋白质编码基因构建到酿酒酵母表达质粒中。得到的质粒分别转化酿酒酵母细胞中,分析哪些基因的表达会抑制酿酒酵母的生长,并用绿色荧光蛋白标签对典型候选基因在酿酒酵母中的定位进行观察。【结果与结论】本研究建立了抑制酿酒酵母生长的人类基因的筛选方法,并运用此方法成功地从2991个人类蛋白质编码基因中筛选到29个显著抑制酿酒酵母生长的基因。其中一些是引起人类疾病的致病基因。例如,PDLIM4参与到骨质疏松症和前列腺癌的形成和发展,但其生理功能尚不清楚。我们的研究可能为揭示这些候选基因的功能和调节机制提供新的途径。     

Expression of human plasma protein genes in ageing transgenic mice

Introduction of human plasma protein genes into the mouse genome to produce transgenic mice furnishes an in vivo model for correlating chromosomal DNA sequences with developmental and tissue-specific expression. The liver produces an array of plasma proteins that circulate throughout the body contributing to homeostasis. Non-hepatic tissue sites of synthesis have been identified where a local provision of plasma proteins in needed. Analysis of expression of human plasma protein genes in ageing transgenic mice appears especialy promising in identifying DNA sequences that respond to environmental adversities such as inflammatory factors, hormonal changes and metal toxicity. The results indicate that human genes encoding and controlling liver plasma proteins serve as useful models for studying genetic regulation in the background of development and ageing.     

Confirmation of the chromosomal localization of human lamp genes and their exclusion as candidate genes for Salla disease

Summary Salla disease is an inherited lysosomal storage disorder caused by accumulation of free sialic acid in the lysosomes. Lamp genes, lamp A and lamp B (lysosome associated membrane proteins), are the first known genes encoding for human lysosomal membrane proteins. Absence of linkage in a large group of families shows that lamp genes are not involved in Salla disease. The lamp genes were localized, using Southern hybridization in hamster — human hybrid cell panels, to chromosomes 13 (lamp A) and X (lamp B).     

Computational identification of human mitochondrial proteins based on homology to yeast mitochondrially targeted proteins

MOTIVATION: Patients with defects of the mitochondrial respiratory chain due to mutations in nuclear genes are often undiagnosable due to the lack of information about the role of these genes. We therefore sought to produce a novel dataset of human nuclear-encoded mitochondrial proteins. RESULTS: We have used the web-based computer program Mitoprot to predict which proteins in the Saccharomyces cerevisiae genome are targeted to mitochondria. We then used this protein dataset to identify the homologous human proteins in the Unigene database using TBLASTN from NCBI. Human proteins with an Expectation value <10(-5) and an Identity >30% were accepted as true homologues of the yeast proteins. These human proteins were then reanalyzed with Mitoprot. The final set of proteins comprises a dataset of 361 human mitochondrially targeted proteins with homology to all S.cerevisiae mitochondrially targeted proteins. One hundred twenty eight of these proteins are novel and are of unknown function. SUPPLEMENTARY INFORMATION: Supplementary tables will be available from http://www.sickkids.ca/Robinsonlab/     

Close physical linkage of the genes encoding the pNR-2/pS2 protein and human spasmolytic protein (hSP)

Trefoil proteins contain a conserved domain of distinctive structure. Three human trefoil proteins have been described to date of which the human spasmolytic polypeptide (hSP) and pNR-2/pS2 proteins have a similar pattern of expression in normal tissues. The genes encoding these two proteins were isolated from a human DNA library. Preliminary experiments suggested that some recombinants contained both genes. Southern hybridisation showed that all the recombinants were derived from a single stretch of DNA spanning 45 kb and suggested that the hSP gene was located downstream of the pNR-2/pS2 gene. Further experiments demonstrated that the two genes are transcribed in the same direction and that the distance between the 3′ end of the pNR-2/pS2 gene and the 5′ end of the hSP gene is 12.5 kb. The close linkage of these two genes is evidence that they have evolved by gene duplication and that their similar pattern of expression in normal tissues could result from the retention of common regulatory elements. Received: 3 June 1996 / Revised: 13 September 1996     

Insights into the Evolutionary Features of Human Neurodegenerative Diseases

Comparative analyses between human disease and non-disease genes are of great interest in understanding human disease gene evolution. However, the progression of neurodegenerative diseases (NDD) involving amyloid formation in specific brain regions is still unknown. Therefore, in this study, we mainly focused our analysis on the evolutionary features of human NDD genes with respect to non-disease genes. Here, we observed that human NDD genes are evolutionarily conserved relative to non-disease genes. To elucidate the conserved nature of NDD genes, we incorporated the evolutionary attributes like gene expression level, number of regulatory miRNAs, protein connectivity, intrinsic disorder content and relative aggregation propensity in our analysis. Our studies demonstrate that NDD genes have higher gene expression levels in favor of their lower evolutionary rates. Additionally, we observed that NDD genes have higher number of different regulatory miRNAs target sites and also have higher interaction partners than the non-disease genes. Moreover, miRNA targeted genes are known to have higher disorder content. In contrast, our analysis exclusively established that NDD genes have lower disorder content. In favor of our analysis, we found that NDD gene encoded proteins are enriched with multi interface hubs (party hubs) with lower disorder contents. Since, proteins with higher disorder content need to adapt special structure to reduce their aggregation propensity, NDD proteins found to have elevated relative aggregation propensity (RAP) in support of their lower disorder content. Finally, our categorical regression analysis confirmed the underlined relative dominance of protein connectivity, 3′UTR length, RAP, nature of hubs (singlish/multi interface) and disorder content for such evolutionary rates variation between human NDD genes and non-disease genes.     

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks

Background  

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure.     

Expression profiles of switch-like genes accurately classify tissue and infectious disease phenotypes in model-based classification

Background  

Large-scale compilation of gene expression microarray datasets across diverse biological phenotypes provided a means of gathering a priori knowledge in the form of identification and annotation of bimodal genes in the human and mouse genomes. These switch-like genes consist of 15% of known human genes, and are enriched with genes coding for extracellular and membrane proteins. It is of interest to determine the prediction potential of bimodal genes for class discovery in large-scale datasets.     

Detection of Mutations in Tumor Suppressor Genes

Defects in vital genes occur in a high percentage of human diseases, including cancer. Defects could be due to the accumulation of mutations in the genes leading to the production of faulty proteins. Although the biological significance of such mutant proteins still remains in question, recent experiments have demonstrated that genes overproducing faulty proteins are often associated with tumor cell growth. Thep53tumor suppressor gene is the most frequently mutated gene yet identified in human cancer. It is mutated in wide variety of human cancers. Missense mutations are common for thep53gene and are essential for the transforming ability of the oncogene. The wild-typep53gene may directly suppress cell growth or indirectly activate genes that are involved in growth suppression. Thus inactivation of wild-typep53by point mutation may contribute to transformation. Therefore, identification of such mutations have potential clinical implications. Recently, polymerase chain reaction-based advanced molecular techniques had a profound impact on the detection and identification of such mutations. These techniques are sensitive and quantitative tools for the study of the pathogenesis of neoplastic diseases at the single-cell level.     

Emergence,Retention and Selection: A Trilogy of Origination for Functional De Novo Proteins from Ancestral LncRNAs in Primates

While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.     

Synthesis of frataxin genes by direct assembly of serial deoxyoligonucleotide primers and its expression in Escherichia coli

Frataxin, a small nuclear-encoded protein targeted to mitochondria, is known to play an important role in both the mitochondrial respiratory chain and iron homeostasis. The protein is highly conserved in most eukaryotic organisms with no major structural changes, suggesting that it serves a crucial function in all organisms. Recently, purified frataxin was used as a therapeutic treatment of Friedreich’s ataxia, a common degenerative disorder that results from a frataxin protein deficiency, by directly applying the protein to the diseased cells. In this report, we describe a novel and rapid method of synthesizing genes encoding frataxin proteins for the purpose of efficient protein production. The artificial yeast and human frataxin genes were synthesized by direct assembly of serial deoxyoligonucleotide primers designed based on the optimal nucleotide sequences. When we tested the expression of these synthetic genes in two E. coli host strains, the yeast frataxin gene was expressed 20 folds higher in Rosetta (DE3) cells than in BL21 (DE3) cells, whereas the expression levels of human frataxin were similar in both E. coli strains. Attenuation of the Fenton reactions by the purified yeast and human frataxin proteins was observed under the defined conditions, which suggests that the recombinant frataxin proteins are active and functional. The procedure described here could be applied to many known genes or to generate novel synthetic genes that can be redesigned by arranging functional domains from previously identified genes and to study the structure and function of synthetic recombinant proteins and potential usage.     

Duplication and extensive remodeling shaped POTE family genes encoding proteins containing ankyrin repeat and coiled coil domains

The POTE family genes encode a highly homologous group of primate-specific proteins that contain ankyrin repeats and coiled coil domains. At least 13 paralogous POTE family genes are found on 8 human chromosomes (2, 8, 13, 14, 15, 18, 21 and 22), which can be sorted into 3 groups based on sequence similarity. We identified by a database search a group of additional human ankyrin repeat domain proteins, of which ANKRD26 and ANKRD30A are the best characterized; these are more distant homologs of POTE family proteins. A comprehensive comparison of the genomic organization indicates that ANKRD26 has the genomic structure of the possible ancestor of ANKRD30A and all POTE family genes. Extensive remodeling involving segmental loss and internal duplication appears to have reshaped the ANKRD30A and POTE family genes after the primal duplication of the ancestor gene. We also identified a mouse homolog of human ANKRD26, but failed to find a mouse homolog that bears the structural characteristics of any of the POTE family of proteins. The mouse Ankrd26 may serve as a useful model for the study of the function of human ANKRD26, ANKRD30A and POTE family proteins.     

Derivation of an interaction/regulation network describing pluripotency in human

Identification of the key genes/proteins of pluripotency and their interrelationships is an important step in understanding the induction and maintenance of pluripotency. Experimental approaches have accumulated large amounts of interaction/regulation data in mouse. We investigate how far such information can be transferred to human, the species of maximum interest, for which experimental data are much more limited. To address this issue, we mapped an existing mouse pluripotency network (the PluriNetWork) to human. We transferred interaction and regulation links between genes/proteins from mouse to human on the basis of orthologous relationship of the genes/proteins (called interolog mapping). To reduce the number of false positives, we used four different methods: phylogenetic profiling, Gene Ontology semantic similarity, gene co-expression, and RNA interference (RNAi) data. The methods and the resulting networks were evaluated by a novel approach using the information about the genes known to be involved in pluripotency from the literature. The RNAi method proved best for filtering out unlikely interactions, so it was used to construct the final human pluripotency network. The RNAi data are based on human embryonic stem cells (hESCs) that are generally considered to be in a (primed) epiblast stem cell state. Therefore, we assume that the final human network may reflect the (primed) epiblast stem cell state more closely, while the mouse network reflects the (unprimed/naïve) embryonic stem cell state more closely.