Hiroko Hagiwara

Analysis of the nucleotide sequence of chromosome VI from Saccharomyces cerevisiae.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VI (270 kb) has revealed that it contains 129 predicted or known genes (300 bp or longer). Thirty–seven (28%) of which have been identified previously. Among the 92 novel genes, 39 are highly homologous to previously identified genes. Local sequence motifs were compared to active ARS regions and inactive loci with perfect ARS core sequences to examine the relationship between these motifs and ARS activity. Additional ARS sequences were predominantly observed in 3′ flanking sequences of active ARS loci.

Genome Sequence of the Basidiomycetous Yeast Pseudozyma antarctica T-34, a Producer of the Glycolipid Biosurfactants Mannosylerythritol Lipids

ABSTRACT The basidiomycetous yeast Pseudozyma antarctica T-34 is an excellent producer of mannosylerythritol lipids (MELs), members of the multifunctional extracellular glycolipids, from various feedstocks. Here, the genome sequence of P. antarctica T-34 was determined and annotated. Analysis of the sequence might provide insights into the properties of this yeast that make it superior for use in the production of functional glycolipids, leading to the further development of P. antarctica for industrial applications.

Expression Profiles of Transcripts from 126 Open Reading Frames in the Entire Chromosome VI of Saccharomyces cerevisiae by Systematic Northern Analyses

Chromosome VI of Saccharomyces cerevisiae contains 126 open reading frames (ORFs), and the functions of proteins encoded by 80 ORFs are still unknown. In this report, we have systematically examined the expression profiles of all 126 ORFs on chromosome VI under five kinds of growth conditions by quantitative Northern hybridization. A series of Northern analyses and reverse transcription polymerase chain reactions have revealed that more than 64 novel ORFs are transcribed. Two ORFs (YFL059w and YFR011c) are specifically expressed in the presence of galactose. Two ORFs (YFL012w and YFR032c) are specifically transcribed in sporulation. Six ORFs (YFL049w, YFL035c, YFL010c, YFR006w, YFR010w and YFR017c) are abundantly expressed in many growth conditions.

Genome and Transcriptome Analysis of the Basidiomycetous Yeast Pseudozyma antarctica Producing Extracellular Glycolipids, Mannosylerythritol Lipids

Pseudozyma antarctica is a non-pathogenic phyllosphere yeast known as an excellent producer of mannosylerythritol lipids (MELs), multi-functional extracellular glycolipids, from vegetable oils. To clarify the genetic characteristics of P. antarctica, we analyzed the 18 Mb genome of P. antarctica T-34. On the basis of KOG analysis, the number of genes (219 genes) categorized into lipid transport and metabolism classification in P. antarctica was one and a half times larger than that of yeast Saccharomyces cerevisiae (140 genes). The gene encoding an ATP/citrate lyase (ACL) related to acetyl-CoA synthesis conserved in oleaginous strains was found in P. antarctica genome: the single ACL gene possesses the four domains identical to that of the human gene, whereas the other oleaginous ascomycetous species have the two genes covering the four domains. P. antarctica genome exhibited a remarkable degree of synteny to U. maydis genome, however, the comparison of the gene expression profiles under the culture on the two carbon sources, glucose and soybean oil, by the DNA microarray method revealed that transcriptomes between the two species were significantly different. In P. antarctica, expression of the gene sets relating fatty acid metabolism were markedly up-regulated under the oily conditions compared with glucose. Additionally, MEL biosynthesis cluster of P. antarctica was highly expressed regardless of the carbon source as compared to U. maydis. These results strongly indicate that P. antarctica has an oleaginous nature which is relevant to its non-pathogenic and MEL-overproducing characteristics. The analysis and dataset contribute to stimulate the development of improved strains with customized properties for high yield production of functional bio-based materials.

Fine De Novo Sequencing of a Fungal Genome Using only SOLiD Short Read Data: Verification on Aspergillus oryzae RIB40

The development of next-generation sequencing (NGS) technologies has dramatically increased the throughput, speed, and efficiency of genome sequencing. The short read data generated from NGS platforms, such as SOLiD and Illumina, are quite useful for mapping analysis. However, the SOLiD read data with lengths of <60 bp have been considered to be too short for de novo genome sequencing. Here, to investigate whether de novo sequencing of fungal genomes is possible using only SOLiD short read sequence data, we performed de novo assembly of the Aspergillus oryzae RIB40 genome using only SOLiD read data of 50 bp generated from mate-paired libraries with 2.8- or 1.9-kb insert sizes. The assembled scaffolds showed an N50 value of 1.6 Mb, a 22-fold increase than those obtained using only SOLiD short read in other published reports. In addition, almost 99% of the reference genome was accurately aligned by the assembled scaffold fragments in long lengths. The sequences of secondary metabolite biosynthetic genes and clusters, whose products are of considerable interest in fungal studies due to their potential medicinal, agricultural, and cosmetic properties, were also highly reconstructed in the assembled scaffolds. Based on these findings, we concluded that de novo genome sequencing using only SOLiD short reads is feasible and practical for molecular biological study of fungi. We also investigated the effect of filtering low quality data, library insert size, and k-mer size on the assembly performance, and recommend for the assembly use of mild filtered read data where the N50 was not so degraded and the library has an insert size of ∼2.0 kb, and k-mer size 33.

A novel TATA-binding protein-binding protein, ABT1, activates basal transcription and has a yeast homolog that is essential for growth.

ABSTRACT Identification of a novel mouse nuclear protein termed activator of basal transcription 1 (mABT1) that associates with the TATA-binding protein (TBP) and enhances basal transcription activity of class II promoters is described. We also identify mABT1 homologous counterparts in Caenorhabditis elegans and Saccharomyces cerevisiae and show the homologous yeast gene to be essential for growth. The mABT1 associated with TBP in HeLa nuclear extracts and with purified mouse TBP in vitro. In addition, ectopically expressed mABT1 was coimmunoprecipitated with endogenous TBP in transfected cells. More importantly, mABT1 significantly enhanced transcription from an adenovirus major late promoter in a reconstituted cell-free system. We furthermore demonstrate that mABT1 consistently enhanced transcription from a reporter gene with a minimal core promoter as well as from reporter genes with various enhancer elements in a cotransfection assay. Taken together, these results suggest that mABT1 is a novel TBP-binding protein which can function as a basal transcription activator.

ANALYSIS OF A 36.2 KB DNA SEQUENCE INCLUDING THE RIGHT TELOMERE OF CHROMOSOME VI FROM SACCHAROMYCES CEREVISIAE

The nucleotide sequence of a 36·2‐kb distal region containing the right telomere of chromosome VI was determined. Both strands of DNA cloned into cosmid clone 9965 and plasmid clone pEL174P2 were sequenced with an average redundancy of 7·9 per base pair, by both dye primer and dye terminator cycle sequencing methods. The G + C content of the sequence was found to be 37·9%. Eighteen open reading frames (ORFs) longer than 100 amino acids were detected. Four of these ORFs (9965orfR017, 9965orfF016, 9965orfR009 and 9965orfF003) were found to encode previously identified genes (YMR31, PRE4, NIN1 and HXK1, respectively). Six ORFs (9965orfR013, 9965orfF018, 9965orfF006, 9965orfR014, 9965orfF013 and 9965orfR020) were found to be homologous to hypothetical 121·4‐kDa protein in the BCK 5′ region, Bacillus subtilis DnaJ protein, hypothetical Trp‐Asp repeats containing protein in DBP3‐MRPL27, putative mitochondrial carrier YBR291C protein, Salmonella typhimurium nicotinate‐nucleotide pyrophosphorylase, and Escherichia coli cystathionine β‐lyase, respectively. The putative proteins encoded by 9965orfF018, 9965orfR014 and 9965orfR020 were found to be, respectively, a new member of the family of DnaJ‐like proteins, the mitochondrial carrier protein and cystathionine lyase. The nucleotide sequence reported here has been deposited in the DDBJ/GenBank/EMBL data library under Accession Number D44597.

Fifteen open reading frames in a 30.8 kb region of the right arm of chromosome VI from Saccharomyces cerevisiae.

The nucleotide sequence of cosmid clone 9765, which contains 30·8 kb of the right arm of chromosome VI, was determined. Both strands were sequenced, with an average redundancy of 8·17 per base pair by both dye primer and dye terminator cycle sequencing methods. The G+C content of the sequence was found to be 40·3%. Fifteen open reading frames (ORFs) greater than 100 amino acids and one tRNA‐Tyr gene (SUP6) were detected. Seven of the ORFs were found to encode previously identified genes (HIS2, CDC14, MET10, SMC2, QCR6, PH04 and CDC26). One ORF, 9765orfF010, was found to encode a new member of the Snf2/Rad54 helicase family. Three ORFs (9765orfR002, 9765orfR011 and 9765orfR013) were found to be homologous with Schizosaccharomyces pombe polyadenylate binding protein, Escherichia coli hypothetical 38·1‐kDa protein in the BCR 5′ region, and transcription regulatory protein Swi3, respectively. The sequence may be found in the DDBJ, EMBL and GenBank nucleotide sequence databases under Accession Number D44602.

Hybrid De Novo Genome Assembly Using MiSeq and SOLiD Short Read Data.

A hybrid de novo assembly pipeline was constructed to utilize both MiSeq and SOLiD short read data in combination in the assembly. The short read data were converted to a standard format of the pipeline, and were supplied to the pipeline components such as ABySS and SOAPdenovo. The assembly pipeline proceeded through several stages, and either MiSeq paired-end data, SOLiD mate-paired data, or both of them could be specified as input data at each stage separately. The pipeline was examined on the filamentous fungus Aspergillus oryzae RIB40, by aligning the assembly results against the reference sequences. Using both the MiSeq and the SOLiD data in the hybrid assembly, the alignment length was improved by a factor of 3 to 8, compared with the assemblies using either one of the data types. The number of the reproduced gene cluster regions encoding secondary metabolite biosyntheses (SMB) was also improved by the hybrid assemblies. These results imply that the MiSeq data with long read length are essential to construct accurate nucleotide sequences, while the SOLiD mate-paired reads with long insertion length enhance long-range arrangements of the sequences. The pipeline was also tested on the actinomycete Streptomyces avermitilis MA-4680, whose gene is known to have high-GC content. Although the quality of the SOLiD reads was too low to perform any meaningful assemblies by themselves, the alignment length to the reference was improved by a factor of 2, compared with the assembly using only the MiSeq data.

Construction of a positive selection marker by a lethal gene with the amber stop codon(s) regulator

A novel positive selection marker for Escherichia coli transformation was developed. The marker consisted of a DNA fragment encoding the C-terminal ribonuclease domain (CRD) of colicin E3 (colE3) and one or more amber stop codons between the initiation codon and the E3-CRD coding sequence. The toxicity of the marker was controlled by the suppressor activity the host cells possessed. This allowed both effective selection and propagation of the vector possessing the maker by selecting appropriate hosts from among those widely distributed: sup + strains for selection and sup 0 strains for propagation respectively. The insert DNA fragment was introduced onto the vector by replacing the marker DNA. The transformants harboring the vector with an insert grew, but those without an insert were effectively removed by the killing activity of E3-CRD encoded on the marker DNA. The marker was also successfully applied to λ phage display vector.