人膜补体调节蛋白MCP、CD59在稳定转染的细胞中的共表达及功能研究

采取内核糖体进入位点(IRES)策略构建含人的膜补体调节蛋白基因MCP和CD59的cDNA的双顺反子真核表达载体pcDNA3-MCPIRESCD59,以磷酸钙沉淀法转染NIH3T3细胞,用 G418筛选阳性克隆,并研究MCP和CD59双基因在稳定细胞系中的共表达及保护功能.PCR实验结果显示双基因稳定整合在异源细胞NIH3T3的染色体上,RT-PCR及Western印迹实验分别从 RNA水平和蛋白质水平证实了人补体调节蛋白分子MCP和CD59在细胞系中皆获得同步表达.检测连续传代30次的NIH3T3 pcDNA3-MCPIRESCD59,结果表明人MCP和CD59基因仍稳定整合在细胞基因组中,并未随着传代而丢失,为稳定的转双基因细胞系.补体依赖的细胞毒反应表明,pcDNA3-MCPIRESCD59转染细胞由于MCP和CD59的共表达获得了高于MCP或CD59单一表达时所提供的保护功效,能更好地抑制人补体依赖的细胞毒作用的发生,保护宿主细胞免受人补体的攻击.以上结果表明,所构建的双基因重组表达载体实现了不同人补体调节蛋白基因高效转移和高水平共表达,在克服超急性排斥反应的基因治疗中有潜在的应用价值.     

Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae

A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac⁺ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac⁺ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac⁺ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield. © 1998 John Wiley & Sons, Ltd.     

Three-dimensional architecture of ribosomal DNA within barley nucleoli revealed with electron microscopy

To elucidate the topological positioning of ribosomal RNA genes (rDNA) and nucleolar structure in three dimensions, we examined the localization of rDNA using in situ hybridization (ISH) analysis by scanning electron microscopy (SEM). The rDNA genes within the three-dimensional architecture of nucleoli were detected on chromatin fibers that connect a thick strand-like structure and a protrusion of rDNA into the inner nuclear hole where the nucleolus is formed. This novel use of ISH together with SEM is useful for the analysis of nucleolar structure in detail. Furthermore, rDNA was detected at the periphery of the fibrillar centers (FCs) of the nucleolus using immuno-gold labeling together with transmission electron microscopy (TEM). In situ hybridization with TEM confirmed that rDNA is naked and thus active in the FCs of nucleoli; ISH with SEM confirmed that rDNA is not covered with ribonucleo proteins at the protruding point and is thus inactive. We also show that the distribution pattern of FCs differs from sample to sample. These results indicate that rDNA is transcribed dynamically in a time- and region-specific manner over the course of the cell cycle.     

LEU2 gene homolog in Kluyveromyces lactis.

A DNA fragment that can complement the leu2 mutation of Saccharomyces cerevisiae was cloned from the genomic library of Kluyveromyces lactis. The nucleotide sequence revealed an open reading frame of 362 codons, 75% homologous to S. cerevisiae LEU2 gene. The upstream region contained a CCGGAACCGG sequence identical to the site of leucine-specific control of LEU2. Further upstream, there is a partial open reading frame homologous to rat ribosomal protein L7.     

How Nature Morphs Peptide Scaffolds into Antibiotics

The conventional notion that peptides are poor candidates for orally available drugs because of protease‐sensitive peptide bonds, intrinsic hydrophilicity, and ionic charges contrasts with the diversity of antibiotic natural products with peptide‐based frameworks that are synthesized and utilized by Nature. Several of these antibiotics, including penicillin and vancomycin, are employed to treat bacterial infections in humans and have been best‐selling therapeutics for decades. Others might provide new platforms for the design of novel therapeutics to combat emerging antibiotic‐resistant bacterial pathogens.     

II. Yeast sequencing reports. The two genes encoding yeast ribosomal protein S8 reside on different chromosomes,and are closely linked to the hsp70 stress protein genes SSA3 and SSA4

A 7·4 kb segment of chromosome II was sequenced and analysed. This segment is part of the 25 kb insert of cosmid clone α1004.10 which is located on the left arm of chromosome II. Sequence analysis revealed four open reading frames (ORFs), of which two had been characterized previously (SSA3, AAR2) and one was not identified. The other ORF was precisely 600 bp long and the deduced protein sequence predicted a very basic protein (pI=11·1; molecular weight=22·5 kDa). Evidence was found that the ORF is the S40 ribosomal protein gene (RPG) S8. Consensus splice signals were found in the 5′ leader sequence and also potential RPG-specific sequences. Chromoblot analysis revealed a second copy of the S8 RPG on chromosome IV or VIII. This copy is also closely linked to an hsp70 protein gene, SSA4. The sequence has been deposited in the EMBL data library under accession number Z26879.     

Localization of ribosomal and telomeric DNA sequences in intact plant nuclei by in-situ hybridization and three-dimensional optical microscopy

We have combined the use of three-dimensional (3-D) fluorescence microscopy and computer image processing of images with in-situ hybridization to analyse the 3-D organization of interphase nuclei in plants. In sections of root tips of Pisum sativum, using cDNA probes, we have shown that telomeres are arranged around the nuclear periphery and that the ribosomal genes in this species appear to exist in discrete, 3-D domains.     

Molecular taxonomy of the yeasts

The term ‘yeast’ is often taken as a synonym for Saccharomyces cerevisiae, but the phylogenetic diversity of yeasts is illustrated by their assignment to two taxonomic classes of fungi, the ascomycetes and the basidiomycetes. Subdivision of taxa within their respective classes is usually made from comparisons of morphological and physiological features whose genetic basis is often unknown. Application of molecular comparisons to questions in yeast classification offers an unprecedented opportunity to re-evaluate current taxonomic schemes from the perspective of quantitative genetic differences. This review examines the impact of molecular comparisons, notably rRNA/rDNA sequence divergence, on the current phenotypically defined classification of yeasts. Principal findings include: 1) budding ascomycetous yeasts are monophyletic and represent a sister group to the filamentous ascomycetes, 2) fission yeasts are ancestral to budding and filamentous ascomycetes, 3) the molecular phylogeny of basidiomycetous yeasts is generally congruent with type of hyphal septum, presence or absence of teliospores in the sexual state, and occurrence of cellular xylose.     

Sequence,map position and genome organization of the RPL17B gene,encoding ribosomal protein L17b in Saccharomyces cerevisiae

Sequence analysis of the newly defined SSU81 gene revealed an adjacent open reading frame (ORF) encoding a protein whose deduced amino acid sequence is identical to that of ribosomal protein L17. The DNA sequence of this region is different from that of the RPL17A gene and therefore represents a duplicate gene encoding L17. We have designated this gene RPL17B. The RPL17B coding region is split by an intron that occurs in the same position (codons 14/15) as the intron in RPL17A. The RPL17B promoter region includes two TATA boxes, a canonical UAS_RPG motif, and several pyrimidine-rich tracts. RPL17B was mapped by CHEF and lambda clone grid hybridization blots to the right arm of chromosome V, linked to the TRP2 and RAD51 genes. A partial ORF was identified adjacent to RPL17B and SSU81 that is homologous to an ORF (designated A509) physically linked to RPL17A. This observation, and the identical position of the introns within the RPL17 genes, suggest that one RPL17 locus arose by duplication and translocation of the other. The complete 3·8 kbp DNA sequence encompassing RPL17B has been entered in the GenBank data library under Accession Number U15653.