Genes & Genetic Systems
Online ISSN : 1880-5779
Print ISSN : 1341-7568
ISSN-L : 1341-7568
Volume 88, Issue 2
Displaying 1-7 of 7 articles from this issue
Review
  • Manabu Yoshikawa
    Article type: Review
    2013 Volume 88 Issue 2 Pages 77-84
    Published: 2013
    Released on J-STAGE: July 06, 2013
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    Trans-acting small interfering RNAs (tasiRNAs) are plant-specific endogenous siRNAs that control non-identical mRNAs via cleavage. The production of tasiRNAs is triggered by cleavage of capped and polyadenylated primary TAS transcripts (pri-TASs) by specific miRNAs. Following miRNA-directed cleavage, either 5′ or 3′ cleavage fragments are converted into double-stranded RNAs (dsRNAs) by RNA-DEPENDENT RNA POLYMERASE 6. The dsRNAs are processed to tasiRNAs by DICER-LIKE 4 in a phasing manner. There are two forms of pri-TASs; One has a single miRNA target site that is targeted by 22-nucleotide microRNAs, and the other has two miR390 target sites. Secondary siRNAs that are important for the amplification of RNA silencing are defined as siRNAs whose production is initiated by the cleavage of primary small RNA-containing RNA-induced silencing complexes. Thus, tasiRNA production is a model system of secondary siRNA production in plants. This review focuses on the production of tasiRNAs that are endogenous secondary siRNAs.
Full papers
  • Amaliawati Ahmad Latiffi, Abu Bakar Salleh, Raja Noor Zaliha Raja Abd. ...
    Article type: Full paper
    2013 Volume 88 Issue 2 Pages 85-91
    Published: 2013
    Released on J-STAGE: July 06, 2013
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    The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme’s capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZαB and pPICZαB) and transformed into P. pastoris strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The α-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in GS115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa.
  • Naohiko T. Miyashita, Hiroko Iwanaga, Suliana Charles, Bibian Diway, J ...
    Article type: Full paper
    2013 Volume 88 Issue 2 Pages 93-103
    Published: 2013
    Released on J-STAGE: July 06, 2013
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    Supplementary material
    Bacterial community structure was investigated in five tropical rainforests in Sarawak, Malaysia and one temperate forest in Kyoto, Japan. A hierarchical sampling approach was employed, in which soil samples were collected from five sampling-sites within each forest. Pyrosequencing was performed to analyze a total of 493,790 16S rRNA amplicons. Despite differences in aboveground conditions, the composition of bacterial groups was similar across all sampling-sites and forests, with Acidobacteria, Proteobacteria, Verrucomicrobia, Planctomycetes and Bacteroidetes accounting for 90% of all Phyla detected. At higher taxonomic levels, the same taxa were predominant, although there was significant heterogeneity in relative abundance of specific taxa across sampling-sites within one forest or across different forests. In all forests, the level of bacterial diversity, estimated using the Chao1 index, was on the order of 1,000, suggesting that tropical rainforests did not necessarily have a large soil bacterial diversity. The average number of reads per species (OTUs) per sampling-site was 8.0, and more than 40–50% of species were singletons, indicating that most bacterial species occurred infrequently and that few bacterial species achieved high predominance. Approximately 30% of species were specific to one sampling-site within a forest, and 40–60% of species were uniquely detected in one of the six forests studied here. Only 0.2% of species were detected in all forests, while on average 32.1% of species were detected in all sampling-sites within a forest. The results suggested that bacterial communities adapted to specific micro- and macro-environments, but macro-environmental diversity made a larger contribution to total bacterial diversity in forest soil.
  • Misuzu Nosaka, Akemi Ono, Aiko Ishiwata, Sae Shimizu-Sato, Kiyoe Ishim ...
    Article type: Full paper
    2013 Volume 88 Issue 2 Pages 105-112
    Published: 2013
    Released on J-STAGE: July 06, 2013
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    Small RNAs, such as small interfering RNAs (siRNAs) or microRNAs (miRNAs), regulate gene expression at transcriptional and posttranscriptional levels in eukaryotes. miRNAs are processed from duplexes formed on single-stranded RNA. They regulate expression of their target gene either by cleaving mRNA or supressing translation. In general, the primary miRNA transcripts are synthesized by RNA polymerase II and processed similarly to mRNAs. MIRNA genes are usually located in transcriptionally active euchromatic regions. In contrast, siRNAs are processed from duplexes made of two RNA molecules. One of them is often derived from a transposable element (TE) or from repetitive sequences that reside in heterochromatic regions. The other strand is synthesized by the RNA-dependent RNA polymerase on the first strand as a template. siRNAs establish epigenetic marks in parasitic DNA such as TEs, thus they usually act in cis. The rice miRNA miR820, encoded by CACTA TEs (five copies, located on different chromosomes), reduces the expression of the de novo DNA methyltransferase gene OsDRM2. Because miR820 is derived from silent TEs, in which the heterochromatic histone modifications are enriched, the mechanism of MIR820 transcription could be expected to differ from typical miRNAs. Here we show that the primary transcript of MIR820 is mainly derived from the CACTA TE copy on chromosome 7 (MIR820b). Histone modification and DNA methylation status around MIR820b differed from that of the other four loci. These unique epigenetic modifications in MIR820b were only found around the miR820 coding region. We conclude that MIR820b transcription may depend on the unique epigenetic modifications, which in turn may be established by the action of miR820 in cis. This suggests a dual function of miR820 in cis and in trans.
  • Yusaku Yasuno, Yoshihiro H. Inoue, Masa-Toshi Yamamoto
    Article type: Full paper
    2013 Volume 88 Issue 2 Pages 113-126
    Published: 2013
    Released on J-STAGE: July 06, 2013
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    Sex ratio distortion, which is commonly abbreviated as sex-ratio, has been studied in many Drosophila species, but the mechanism remains largely unknown. Here, we report on the sex-ratio mutant of D. simulans named excess of females (exf). The third chromosomal recessive mutation results in a sex ratio of approximately 0.2 or less (males/total). Cytological observation demonstrated that meiosis appeared to be completed normally, but that most Y chromosome-bearing nuclei failed to elongate during spermiogenesis, as revealed by fluorescence in situ hybridization using sex chromosome-specific probes. These aberrant nuclei contained membranous inclusions as revealed by electron microscopic analysis. Most of the aberrant exf spermatids failed to individualize and mature, suggesting that a later stage of spermiogenesis is involved in prevention of production of sperm with abnormal morphology. On the one hand, in exf seminal vesicles, sperm nuclei with a length of 5–8.5 μm were occasionally observed, in addition to those with wild-type sperm dimensions, that is, a length of approximately 10 μm. Thus, spermatids with less severe nuclear defects can escape elimination and be released into the seminal vesicles as mature sperm. Furthermore, we constructed His2AvD-GFP and ProtamineB-eGFP transgenic lines in D. simulans, and examined the processes involved in replacement of chromatin proteins over a time course, according to nuclear morphology. We found that both normal and abnormal sperm heads demonstrated equal chromatin replacement during late spermiogenesis. Our results suggest that exf belongs to a unique class of meiotic drive systems in that (1) intranuclear membranous inclusions cause failure of nuclear shaping of Y-bearing spermatids without affecting the histone-protamine transition, and (2) a portion of the aberrant spermatids differentiate into mature sperm; these are transferred to and stored by females.
  • Tie-Bo Zeng, Hong-Juan He, Feng-Wei Zhang, Zheng-Bin Han, Zhi-Jun Huan ...
    Article type: Full paper
    2013 Volume 88 Issue 2 Pages 127-133
    Published: 2013
    Released on J-STAGE: July 06, 2013
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    The Dlk1-Dio3 imprinted domain on mouse chromosome 12qF1 contains three paternally expressed protein-coding genes and multiple maternally expressed long or short noncoding RNA genes. All these imprinted genes are regulated by IG-DMR located between Dlk1 and Meg3/Gtl2. Recently, several novel imprinted noncoding RNAs were identified in the intergenic region of this domain, although the exact number of imprinted genes within the region is unclear. Here, we report that a novel noncoding RNA, AK003491, located between Meg3/Gtl2 and Meg8, is maternally expressed in E15.5 brain, tongue, heart, lung, liver and kidney tissues. In situ hybridization analysis at E15.5 shows AK003491 is predominantly expressed in forebrain, tongue, thymus, somites, lung and liver. Quantitative real-time RT-PCR analysis confirms this expression pattern and detects highest expression in tongue. While the AK003491 expression pattern at postnatal day 1 is similar to E15.5, AK003491 expression at postnatal day 30 is mainly restricted to the brain. These results expand the number of known imprinted long noncoding RNAs in this domain, and contribute to further investigation of their regulatory mechanism and function.
  • Yi-Deun Jung, Ja-Rang Lee, Yun-Ji Kim, Hong-Seok Ha, Keon-Bong Oh, Gi- ...
    Article type: Full paper
    2013 Volume 88 Issue 2 Pages 135-142
    Published: 2013
    Released on J-STAGE: July 06, 2013
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    The potential risk of porcine endogenous retrovirus (PERV) transmission is an important issue in xenotransplantation (pig-to-human transplantation). Long terminal repeats (LTRs) in PERV elements show promoter activity that could affect neighboring functional genes. The methylation status and promoter activities of 3 LTR structures (PERV-LTR1, LTR2, and LTR3 elements) belonging to the PERV-A family were examined using luciferase reporter genes in human liver cell lines (HepG2 and Hep3B). The PERV LTR3 element exhibited hypomethylation and stronger promoter activity than the other LTR elements in human liver cells. We also performed comparative sequences analysis of the PERV LTR elements by using bioinformatics tools. Our findings showed that several transcription factors such as Nkx2-2 and Elk-1 positively influenced the high transcriptional activity of the PERV LTR3 element.
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