Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis

We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly(dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently directionally amplified by PCR, and then recombined. The amplified products (approximately 100 ng) show superior representation and reproducibility of original gene expression, especially for genes expressed in more than 20 copies per cell, compared with those obtained by a conventional PCR protocol, and can effectively be used for quantitative PCR and EST analyses. The cDNAs are then subjected to another PCR amplification with primers bearing the T7 promoter sequence. The resultant cDNA products are gel purified, amplified by one final cycle and used for isothermal linear amplification by T7 RNA polymerase to synthesize cRNAs for microarray hybridization. This protocol yields cDNA templates sufficient for more than 80 microarray hybridizations from a single cell, and can be completed in 5-6 days.     

Generation of a novel isogenic trisomy panel in human embryonic stem cells via microcell-mediated chromosome transfer

Aneuploidy is the gain or loss of a chromosome. Down syndrome or trisomy (Ts) 21 is the most frequent live-born aneuploidy syndrome in humans and extensively studied using model mice. However, there is no available model mouse for other congenital Ts syndromes, possibly because of the lethality of Ts in vivo, resulting in the lack of studies to identify the responsible gene(s) for aneuploid syndromes. Although induced pluripotent stem cells derived from patients are useful to analyse aneuploidy syndromes, there are concerns about differences in the genetic background for comparative studies and clonal variations. Therefore, a model cell line panel with the same genetic background has been strongly desired for sophisticated comparative analyses. In this study, we established isogenic human embryonic stem (hES) cells of Ts8, Ts13, and Ts18 in addition to previously established Ts21 by transferring each single chromosome into parental hES cells via microcell-mediated chromosome transfer. Genes on each trisomic chromosome were globally overexpressed in each established cell line, and all Ts cell lines differentiated into all three embryonic germ layers. This cell line panel is expected to be a useful resource to elucidate molecular and epigenetic mechanisms of genetic imbalance and determine how aneuploidy is involved in various abnormal phenotypes including tumourigenesis and impaired neurogenesis.     

Curvature-dependent recognition of ethanolamine phospholipids by duramycin and cinnamycin

Duramycin is a 19-amino-acid tetracyclic lantibiotic closely related to cinnamycin (Ro09-0198), which is known to bind phosphatidylethanolamine (PE). The lipid specificity of duramycin was not established. The present study indicates that both duramycin and cinnamycin exclusively bind to ethanolamine phospholipids (PE and ethanolamine plasmalogen). Model membrane study indicates that the binding of duramycin and cinnamycin to PE-containing liposomes is dependent on membrane curvature, i.e., the lantibiotics bind small vesicles more efficiently than large liposomes. The binding of the lantibiotics to multilamellar liposomes induces tubulation of membranes, as revealed by electron microscopy and small-angle x-ray scattering. These results suggest that both duramycin and cinnamycin promote their binding to the PE-containing membrane by deforming membrane curvature.     

Two X family DNA polymerases, λ and μ, in meiotic tissues of the basidiomycete, Coprinus cinereus

The X family DNA polymerases λ (CcPolλ) and μ (CcPolμ) were shown to be expressed during meiotic prophase in the basidiomycete, Coprinus cinereus. These two polymerases are the only members of the X family in the C. cinereus genome. The open reading frame of CcPolλ encoded a predicted product of 800 amino acid residues and that of CcPolμ of 621 amino acid residues. Both CcPolλ and CcPolμ required Mn²⁺ ions for activity, and both were strongly inhibited by dideoxythymidine triphosphate. Unlike their mammalian counterparts, CcPolλ and CcPolμ had no terminal deoxynucleotidyl transferase activity. Immunostaining analysis revealed that CcPolλ was present at meiotic prophase nuclei in zygotene and pachytene cells, which is the period when homologous chromosomes pair and recombine. CcPolμ was present in a slightly wider range of cell stages, zygotene to diplotene. In analyses using D-loop recombination intermediate substrates, we found that both CcPolλ and CcPolμ could promote primer extension of an invading strand in a D-loop structure. Moreover, both polymerases could fully extend the primer in the D-loop substrate, suggesting that D-loop extension is an activity intrinsic to CcPolλ and CcPolμ. Based on these data, we discuss the possible roles of these polymerases in meiosis.     

Long-term prevalence of antimicrobial-resistant Salmonella enterica subspecies enterica Serovar infantis in the broiler chicken industry in Japan

Eleven broiler isolates of Salmonella Infantis obtained between 1989 and 1998 were examined for antimicrobial susceptibility and pulse field gel electrophoresis (PFGE) profiles. Seven strains of S. Infantis isolated after 1993 harbored similar antimicrobial susceptibilities to the recent isolates between 2001 and 2003. In comparison of PFGE profile with 22 isolates obtained from 22 apparently healthy broiler chickens between 2001 and 2003, the predominant cluster included the seven strains isolated after 1993. We could not clarify the reasons why the serovar has been prevalent in the broiler industry for a long time, but current antimicrobial usage is not always linked to its prevalence.     

Rhizopus delemar is the proper name for Rhizopus oryzae fumaric-malic acid producers

The zygomycete Rhizopus oryzae currently is identified by sporangiophore morphology and growth temperature, but heterogeneity of the species has been reported. We examined the suitability of organic acid production as an effective taxonomic character for reclassification of the species. Strains were divided into two groups, LA (lactic acid producer) and FMA (fumaric-malic acid producers) according to organic acid production. These grouping were confirmed as phylogenetically distinct because analyses of rDNA ITS, lactate dehydrogenase B, actin, translation elongation factor-1alpha and genomewide AFLP resolved the same two exclusive clusters, corresponding with the organic acid grouping. Reclassification of strains in the FMA group as R. delemar was proposed.