Functional homology of mammalian and yeast RAS genes

Yeast spores lacking endogenous RAS genes will not germinate. If such spores contain chimeric mammalian/yeast RAS genes or even the mammalian H-ras gene under the control of the galactose-inducible GAL10 promoter, they will germinate in the presence of galactose and produce viable haploid progeny dependent on galactose for continued growth and viability. These results indicate that the biochemical function of RAS proteins is essential for vegetative haploid yeast and that this function has been conserved in evolution since the progenitors of yeast and mammals diverged.     

哺乳动物转录因子DNA结合谱

基因差异表达是生物发育和对刺激作出应答的分子基础,转录因子在这种基因差异表达中发挥着重要的调控作用。因此,要弄清楚转录因子调控基因差异表达的机理,就必须鉴定出它们全部的靶基因并构建其操纵的转录调控网络。对基因组DNA的序列特异性结合是转录因子调控基因转录的关键环节,因此,要鉴定转录因子的靶基因,就必须从它们与DNA相互作用的分子水平,鉴定它们能够识别并结合的全部DNA序列,即转录因子DNA结合谱。近年来随着DNA微阵列芯片和高通量DNA测序技术的产生和快速发展,出现了建立转录因子体内及体外DNA结合谱的一系列革命性的新技术,对该领域的研究带来重大影响。这些新技术主要包括建立转录因子体内DNA结合谱的染色质免疫沉淀-芯片技术(ChIP-chip)和染色质免疫沉淀-测序技术(ChIP-Seq),以及建立转录因子体外DNA结合谱的双链DNA微阵列芯片技术(dsDNA microarray)、指数富集配体系统进化-系列分析基因表达技术(SELEX-SAGE)、结合-n-测序技术(Bind-n-Seq)、多重大规模并行SELEX技术(MMP-SELEX)、凝胶迁移实验-测序技术(EMSA-Seq)和高通量测序-荧光配体互作图谱分析技术(HiTS-FLIP)。文章将对这些新技术做一综述。     

Striking similarities between the regulatory mechanisms governing yeast mating-type genes and mammalian major histocompatibility complex genes.

Expression of a mammalian major histocompatibility complex (MHC) class I gene is in part regulated by a silencer DNA sequence element which binds a complex of silencer factors. This negative regulatory system is shown to be strikingly similar to the yeast alpha 2 mating-type repression system. A moderate DNA sequence homology exists between the MHC class I silencer DNA element and the yeast alpha 2 operator. Mammalian silencer factors specifically bind to the yeast alpha 2 operator DNA and also specifically interact with a yeast alpha 2-binding protein. Furthermore, the alpha 2 operator functions as a silencer element in mammalian cells when placed upstream of a MHC class I promoter.     

A shuttle system for transfer of YACs between yeast and mammalian cells.

The development of a system for shuttling DNA cloned as yeast artificial chromosomes (YACs) between yeast and mammalian cells requires that the DNA is maintained as extrachromosomal elements in both cell types. We have recently shown that circular YACs carrying the Epstein-Barr virus origin of plasmid replication (oriP) are maintained as stable, episomal elements in a human kidney cell line constitutively expressing the viral transactivator protein EBNA-1. Here, we demonstrate that a 90-kb episomal YAC can be isolated intact from human cells by a simple alkaline lysis procedure and shuttled back into Saccharomyces cerevisiae by spheroplast transformation. In addition, we demonstrate that the 90-kb YAC can be isolated intact from yeast cells. The ability to shuttle large, intact fragments of DNA between yeast and human cells should provide a powerful tool in the manipulation and analysis of functional regions of mammalian DNA.     

Structural characterization of mammalian bHLH-PAS transcription factors

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