C一代数与Table代数

综述了Ｃ—代数，ｔａｂｌｅ代数的基本概念、发展历史，并报告了若干新发展，新结果。本文源于作者在１９９３年１０月２５～２９日湖北大学群论研讨会上的几个系列报告。     

Restriction enzyme-generated siRNA (REGS) vectors and libraries

Small interfering RNA (siRNA) technology facilitates the study of loss of gene function in mammalian cells and animal models, but generating multiple siRNA vectors using oligonucleotides is slow, inefficient and costly. Here we describe a new, enzyme-mediated method for generating numerous functional siRNA constructs from any gene of interest or pool of genes. To test our restriction enzyme-generated siRNA (REGS) system, we silenced a transgene and two endogenous genes and obtained the predicted phenotypes. REGS generated on average 34 unique siRNAs per kilobase of sequence. REGS enabled us to create enzymatically a complex siRNA library (>4 x 10(5) clones) from double-stranded cDNA encompassing known and unknown genes with 96% of the clones containing inserts of the appropriate size.     

Localization of muscle gene products in nuclear domains

The localization of gene products is central to the development of cell polarity and pattern specification during embryogenesis. To monitor the distribution of gene products encoded by different nuclei in the same cell in tissue culture, we fused cells of different species to form multinucleated non-dividing heterokaryons. In previous fusion studies, cell-surface antigens and organelles contributed by disparate cell types intermixed within minutes. Using heterokaryons produced with differentiated muscle cells, we demonstrate here that a muscle membrane component, the Golgi apparatus mediating its transport, and a sarcomeric myosin heavy chain are localized in the vicinity of the nuclei responsible for their synthesis. These results provide direct evidence that products (organelle, membrane and structural proteins) derived from individual nuclei can remain localized in myotubes, a finding with implications both for neuromuscular synapse formation and for the carrier state of Duchenne muscular dystrophy.     

Following the signal sequence from ribosomal tunnel exit to signal recognition particle

Membrane and secretory proteins can be co-translationally inserted into or translocated across the membrane. This process is dependent on signal sequence recognition on the ribosome by the signal recognition particle (SRP), which results in targeting of the ribosome-nascent-chain complex to the protein-conducting channel at the membrane. Here we present an ensemble of structures at subnanometre resolution, revealing the signal sequence both at the ribosomal tunnel exit and in the bacterial and eukaryotic ribosome-SRP complexes. Molecular details of signal sequence interaction in both prokaryotic and eukaryotic complexes were obtained by fitting high-resolution molecular models. The signal sequence is presented at the ribosomal tunnel exit in an exposed position ready for accommodation in the hydrophobic groove of the rearranged SRP54 M domain. Upon ribosome binding, the SRP54 NG domain also undergoes a conformational rearrangement, priming it for the subsequent docking reaction with the NG domain of the SRP receptor. These findings provide the structural basis for improving our understanding of the early steps of co-translational protein sorting.     

Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia

Primary ciliary dyskinesia most often arises from loss of the dynein motors that power ciliary beating. Here we show that DNAAF3 (also known as PF22), a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes before their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in individuals from families with situs inversus and defects in the assembly of inner and outer dynein arms. Knockdown of dnaaf3 in zebrafish likewise disrupts dynein arm assembly and ciliary motility, causing primary ciliary dyskinesia phenotypes that include hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a PF22-null mutant cannot assemble any outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests that DNAAF3 (PF22) acts at a similar stage as other preassembly proteins, for example, DNAAF2 (also known as PF13 or KTU) and DNAAF1 (also known as ODA7 or LRRC50), in the dynein preassembly pathway. These results support the existence of a conserved, multistep pathway for the cytoplasmic formation of assembly competent ciliary dynein complexes.     

Structure of eEF3 and the mechanism of transfer RNA release from the E-site

Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA-eEF1A-GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.     

In vivo selection using a cell-growth switch

A major obstacle to stem-cell gene therapy rests in the inability to deliver a gene into a therapeutically relevant fraction of stem cells. One way to circumvent this obstacle is to use selection. Vectors containing two linked genes serve as the basis for selection, with one gene encoding a selectable product and the other, a therapeutic protein. Applying selection in vivo has the potential to bring a minor population of genetically corrected cells into the therapeutic range. But strategies for achieving in vivo selection have traditionally relied on genes that confer resistance to cytotoxic drugs and are encumbered by toxicity. Here we describe a new system for in vivo selection that uses a 'cell-growth switch', allowing a minor population of genetically corrected cells into the therapeutic range. But strategies for achieving in vivo selection have traditionally relied on genes that confer resistance to cytotoxic drugs and are encumbered by toxicity. Here we describe a new system for in vivo selection that uses a 'cell-growth switch', allowing a minor population of genetically modified cells to be inducibly amplified, thereby averting the risks associated with cytotoxic drugs. This system provides a general platform for conditionally expanding genetically modified cell populations in vivo, and may have widespread applications in gene and cell therapy.