Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a rate-limiting photosynthetic enzyme that catalyzes carbon fixation in the Calvin cycle. Much interest has been devoted to engineering this ubiquitous enzyme with the goal of increasing plant growth. However, experiments that have successfully produced improved Rubisco variants, via directed evolution in Escherichia coli, are limited to bacterial Rubisco because the eukaryotic holoenzyme cannot be produced in E. coli. The present study attempts to determine the specific differences between bacterial and eukaryotic Rubisco large subunit primary structure that are responsible for preventing heterologous eukaryotic holoenzyme formation in E. coli. A series of chimeric Synechococcus Rubiscos were created in which different sections of the large subunit were swapped with those of the homologous Chlamydomonas Rubisco. Chimeric holoenzymes that can form in vivo would indicate that differences within the swapped sections do not disrupt holoenzyme formation. Large subunit residues 1–97, 198–247 and 448–472 were successfully swapped without inhibiting holoenzyme formation. In all ten chimeras, protein expression was observed for the separate subunits at a detectable level. As a first approximation, the regions that can tolerate swapping may be targets for future engineering.

In Saccharomyces cerevisiae, HMR/HML, telomeres and ribosomal DNA are heterochromatin-like regions in which gene transcription is prevented by the silent information regulator (Sir) complex. The Sir complex (Sir2, Sir3 and Sir4) can spread through chromatin from the silencer. Boundaries prevent Sir complex spreading, and we previously identified 55 boundary genes among all ~6,000 yeast genes. These boundary proteins can be distinguished into two types: those that activate transcription to prevent spreading of silencing, and those that prevent gene silencing by forming a boundary. We selected 44 transcription-independent boundary proteins from the 55 boundary genes by performing a one-hybrid assay and focused on GIC1 (GTPase interaction component 1). Gic1 is an effector of Cdc42, which belongs to the Rho family of small GTPases, and has not been reported to function in heterochromatin boundaries in vivo. We detected a novel boundary-forming activity of Gic1 at HMR-left and telomeric regions by conducting a chromatin immunoprecipitation assay with an anti-Sir3 antibody. We also found that Gic1 bound weakly to histones in two-hybrid analysis. Moreover, we performed domain analysis to identify domain(s) of Gic1 that are important for its boundary activity, and identified two minimum domains, which are located outside its Cdc42-binding domain.

The aim of this study was to obtain primary information about the global diversity of garlic (Allium sativum L.) by evaluating morphological, physiological and isozyme variation. A total of 107 garlic accessions collected worldwide were grown in Yamaguchi, Japan. Five morphological traits (bulb weight, bulb diameter, number of cloves per bulb, number of bulbils and scape length) and one physiological trait (bolting period) of the collected garlic showed wide variation. Meanwhile, a total of 140 garlic accessions, including the 107 mentioned above, were characterized by leucine aminopeptidase (LAP) and phosphoglucoisomerase (PGI) isozyme analyses; they clearly showed polymorphisms in putative isozyme loci (Lap-1, Lap-2 and Pgi-1). Allelic frequencies were estimated in each group of accessions categorized by their geographical origin, and the observed (H_o) and expected (H_e) heterozygosities were calculated. The allelic frequencies differed between groups. A principal component analysis based on morpho-physiological data indicated a grouping of the garlic accessions into Central Asian and Northern Mediterranean groups as well as others. We discuss the roles of artificial and natural selection that may have caused differentiation in these traits, on the assumption that ancestral domesticated garlic populations have adapted in various regions using standing variation or mutations that accumulated during expansion, and have evolved along with human-preferred traits over a long history of cultivation.

Ovarian cancer is the fourth most common cancer in women worldwide. It is very heterogeneous at the clinical, histopathological and molecular levels and is caused by the accumulation of genetic and epigenetic changes in regulatory genes. More than 90% of ovarian cancers are epithelial in origin. Ovarian cancer is typically asymptomatic in its early stages, and, due to difficulties in early detection, most ovarian cancers are diagnosed at an advanced stage. The positive predictive value of CA-125, a routinely used serum protein marker, is < 30%; therefore, for effective screening, there is a need to develop a marker with high sensitivity for early detection. Development of blood-based biomarkers that detect DNA methylation in cell-free tumor-specific DNA is now being considered as a potential approach for the early diagnosis of cancer. Our objective in this study was to develop an absolute quantitative method, the MethyLight assay, to detect the promoter methylation status of two tumor suppressor genes. We analyzed the methylation level of the promoter regions of these genes in 42 tumor samples using the MethyLight assay. SOX1 promoter methylation was significantly higher in cancer samples than in normal samples (P = 0.011), whereas this difference between cancer and normal samples was not significant for DAPK1 promoter methylation (P = 0.18), when analyzed separately in a singleplex assay, whereas the detection frequency and significance level increased several-fold when these genes were analyzed together in a multiplex assay (P = 0.0004). The sensitivity was found to be 62% and 83% for DAPK1 and SOX1, respectively, when analyzed separately in the singleplex assay, but increased to 90% in the multiplex assay when either or both of the SOX1 and the DAPK1 gene promoters showed methylation.

Reduced-genome Escherichia coli strains lacking up to 38.9% of the parental chromosome have been constructed by combining large-scale chromosome deletion mutations. Functionally redundant genes involved in essential processes can be systematically identified using these reduced-genome strains. One large-scale chromosome deletion mutation could be introduced into the wild-type strain but not into the largest reduced-genome strain, suggesting a synthetic lethal interaction between genes removed by the deletion and those already absent in the reduced-genome strain. Thus, introduction of the deletion mutation into a series of reduced-genome mutants could allow the identification of other chromosome deletion mutations responsible for the synthetic lethal phenotype. We identified a synthetic lethality caused by disruption of nfo and xthA, two genes encoding apurinic/apyrimidinic (AP) endonucleases involved in the DNA base excision repair pathway, and two other large-scale chromosome deletions. We constructed temperature-sensitive mutants harboring quadruple-deletion mutations in the affected genes/chromosome regions. Using these mutants, we identified two multi-copy suppressors: holC, encoding the chi subunit of DNA polymerase III, and yoaA, encoding a putative DNA helicase. Addition of the yoaA disruption increased the methyl methanesulfonate (MMS) sensitivity of xthA single-deletion or xthA nfo double-deletion mutants. This increased MMS sensitivity was not suppressed by the presence of multi-copy holC. These results indicate that yoaA is involved in MMS sensitivity and suggest that YoaA functions together with HolC.

Siniperca chuatsi (Basilewsky), a demersal piscivore, is an endemic freshwater fish species in China. For the purpose of genomics research, we have constructed the first bacterial artificial chromosome (BAC) library for S. chuatsi. The BAC library comprised a total of 84,480 clones with an average insert size of 124.6 kb and less than 2.5% empty clones, corresponding to a 10.5-fold coverage of the S. chuatsi genome. The probability of isolating genes of interest was more than 99%. To validate the library, we screened 220 superpools and found that 1–19 were positive for six SSR markers, while none was positive for two mitochondrial gene markers. Therefore, the S. chuatsi BAC library will provide useful genomics resources and tools for cloning, functional genomics research and identification of economically important genes in this species.