Abstract
Normally, many granules containing uric acid accumulate in the larval integument of the silkworm, Bombyx mori. These uric acid granules cause the wild-type larval integument to be white or opaque, and the absence of these granules results in a translucent integument. Although about 30 B. mori loci governing larval translucency have been mapped, most have not been molecularly identified yet. Here, based on a structural analysis of a deletion of chromosome 14 that included the oa (aojyuku translucent) locus, we concluded that the BmHPS5 encoding a Bombyx homolog of the HPS5 subunit of biogenesis of lysosome-related organelles complex-2 is the candidate for the oa locus. Nucleotide sequence analyses of cDNAs and genomic DNAs in three mutant strains, each of which were homozygous for the respective allele of the oa locus (oa, oa 2, and oa v), revealed that each mutant strain has a frame shift or a premature stop codon (caused by deletion or nonsense mutation, respectively) in the BmHPS5 gene. Our findings indicate that some genes that cause the translucent phenotype in Bombyx, some HPS-associated genes in humans, and some genes that cause mutant eye color phenotypes in Drosophila are homologous and participate in an evolutionarily conserved mechanism that leads to biogenesis of lysosome-related organelles.
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Acknowledgments
This work was supported by the KAKENHI grants (Nos. 22128004 and 24658048), and the Professional Program for Agricultural Bioinformatics (MEXT), Japan. The silkworm strains were provided by the National Bioresource Project (NBRP, MEXT), Japan. We are grateful to M. Kawamoto for his technical assistance. We thank the members of the Institute for Sustainable Agro-ecosystem Services, University of Tokyo, for their support in mulberry cultivation. We also thank Lisa Onaga (Nanyang Technological University) for critical review. T. Fujii was a recipient of the JSPS Fellowship for Young Scientists.
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10709_2012_9694_MOESM1_ESM.pdf
Supplementary material Fig 1 Differences between the sequences of the molecular marker, 38-01. The black bar denotes the DraI site (TTTAAA). g60, Nl/+ . Sakado, B. mandarina with +/+ (PDF 35 kb)
10709_2012_9694_MOESM2_ESM.pdf
Supplementary material Fig 2 Sequence alignments of HPS5 orthologs from four species. Human = Homo sapiens HPS5 (NP852608), Mouse = Mus musculus HPS5 (AAH82542), Fly = Drosophila melanogaster HPS5 (CG9770). Multiple sequences were aligned using CLASTALW, which is available at http://www.genome.jp/tools/clustalw/. Underlined residues of the BmHPS5 (aa 64-aa 164) shows homology to the WD40 repeat domain. (PDF 181 kb)
10709_2012_9694_MOESM3_ESM.pdf
Supplementary material Fig 3 Molecular characterization of the Df(14)Nl chromosome that lacks the oa locus. a, B. mandarina with +/+ genotype. b, Nl/+. c and d are +/+ and Nl/+ larvae obtained from the cross between B. mandarina (a) and Nl/+ (b). M, molecular size marker (100 bp ladder). Arrowheads indicate 500 bp. (A) Amplification pattern of PCR markers that were polymorphic between the g60 strain (Nl/+) and B. mandarina (+/+). To detect the polymorphisms in the g60 strain (Nl/+) and B. mandarina (+/+), the PCR product from marker 38-02 was digested with Dra I. The Dra I site is indicated in Fig S1. See Fig. 2 for the location of the molecular markers. When 155-01, 40-01, 81-01, and 53-01 were used, band intensity differed between two alleles in the F1 individual with +/+ genotype (lane c). These differences were presumably caused by the difference of the amplification efficiency of the PCR between two alleles. When 81-01 and 53-01 were used, band intensity of the two allele in the F1 individual with Nl/+ genotype (lane d) were different between two alleles. These differences were presumably caused by the same mechanism as lane c. B, Direct sequencing of the 40-02 marker indicated that the Df(14)Nl chromosome lacked this marker. These results indicated that Df(14)Nl chromosome lacked three markers, 155-01, 40-01, and 40-02, while this chromosome retained four other markers, 38-01, 38-02, 81-01, and 53-01. (PDF 92 kb)
10709_2012_9694_MOESM4_ESM.pdf
Supplementary material Fig 4 Sequence alignments of the predicted BmHPS5 proteins from four oa alleles. p50T, +/+. o56, oa/oa. o57, oa 2/oa 2. l32, oa v/oa v. (PDF 112 kb)
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Fujii, T., Banno, Y., Abe, H. et al. A homolog of the human Hermansky–Pudluck syndrome-5 (HPS5) gene is responsible for the oa larval translucent mutants in the silkworm, Bombyx mori . Genetica 140, 463–468 (2012). https://doi.org/10.1007/s10709-012-9694-1
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DOI: https://doi.org/10.1007/s10709-012-9694-1