Identification and Expression Analysis of Ras Gene in Silkworm, Bombyx mori

Ras proteins play important roles in development especially for cell proliferation and differentiation in various organisms. However, their functions in the most insect species are still not clear. We identified three ras cDNAs from the silk worm, Bombyx mori. These sequences corresponded to three Ras of Drosophila melanogaster, but not to three mammalian Ras (H-Ras, K-Ras, N-Ras). Subsequently, the expression profiles of ras were investigated by quantitative real-time PCR using whole body of individuals from the embryonic to adult stages, and various tissues of 4th and 5th instar larvae. Each of three Bombyx ras showed different expression patterns. We also showed membrane localization of their products. These results indicate that the three Bombyx Ras are functional and have different roles

Insect Adenine Nucleotide Translocases

Mitochondrial adenine nucleotide translocase (ANT) specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4) and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4) is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1) and the testis-specific paralogue (BmANTI2). The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran Plutella xylostella (PxANTI1), but not those of other insect species (or PxANTI2), restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4

Primer sequences.

<p>(A) Degenerate primers; (B) Primers for RACE; (C) Primers for full length cDNA constructs; (D) Primers for inverse PCR; (E) Primers for quantitative RT-PCR.</p

Localization of Ras-GFP fusion proteins in Sf-9 cells.

<p>GFP (A), Ras1-GFP (B), Ras2-GFP (C) and Ras3-GFP (D) proteins were translated in Sf-9 cultured cells, and the localization of these proteins was observed with their GFP signals by confocal microscopy (left pannels). Right panels show Nomarsky microscope images of left panels. GFP luminescence is localized in nuclei in the case of non-fused GFP (A), but on the plasma membrane, and weakly in the cytoplasm, in cases of fusion proteins (B, C and D).</p

Alignment of amino acid sequences of Ras superfamily members.

<p>Bm, Ce, Dm and Mm means <i>B. mori</i>, <i>C. elegans</i>, <i>D. melanogaster</i> and <i>M. musculus</i>, respectively. The accession number of each sequence is described in Results. Identical and homologous amino acid residues are highlighted and shaded, respectively. Amino acids which are important for GTP/Mg²⁺ binding are boxed. Amino acids of the GEF interaction site and the effector binding site are shown by arrowheads and asterisks, respectively. The C terminal isoprenylation site is indicated with dots.</p

Phylogenic tree constructed using primary sequences of Ras superfamily members.

<p>An unrooted UPGMA tree was prepared using CLC FREE WORKBENCH VER. 4.01 (CLC Bio A/S, Aarhus, Denmark). References for sequences are shown in Results. A bootstrap value is attached to each node, and the value is a measure of the confidence in the branch. The number of replicates in bootstrap analysis is adjusted to 100.</p

Changes in the mRNA expression levels of <i>Bmras1</i>, <i>Bmras2</i> and <i>Bmras3</i> during development.

<p>mRNA samples were harvested from various organs of various developmental stages from the embryo to adult of <i>B. mori</i> at 24-hours interval. Transcripts of <i>Bmras</i> in these samples were quantified by qRT-PCR. Relative expression levels in whole body (A) and tissues (B) of <i>Bmras1</i>, whole body (C) and tissues (D) of <i>Bmras2</i>, whole body (E) and tissues (F) of <i>Bmras3</i> against <i>Bmrp49</i> are shown. Expression levels in whole body samples are indicated by solid squares. Changes in organs, namely, the epidermis, fat body, silk gland, muscle, Malpighian tubles and gut are shown by solid circles, solid triangles, open circles, solid diamonds, open squares and open triangles, respectively.</p

Scalable Generation of High-Titer Recombinant Adeno-Associated Virus Type 5 in Insect Cells

We established a method for production of recombinant adeno-associated virus type 5 (rAAV5) in insect cells by use of baculovirus expression vectors. One baculovirus harbors a transgene between the inverted terminal repeat sequences of type 5, and the second expresses Rep78 and Rep52. Interestingly, the replacement of type 5 Rep52 with type 1 Rep52 generated four times more rAAV5 particles. We replaced the N-terminal portion of type 5 VP1 with the equivalent portion of type 2 to generate infectious AAV5 particles. The rAAV5 with the modified VP1 required α2-3 sialic acid for transduction, as revealed by a competition experiment with an analog of α2-3 sialic acid. rAAV5-GFP/Neo with a 4.4-kb vector genome produced in HEK293 cells or Sf9 cells transduced COS cells with similar efficiencies. Surprisingly, Sf9-produced humanized Renilla green fluorescent protein (hGFP) vector with a 2.4-kb vector genome induced stronger GFP expression than the 293-produced one. Transduction of murine skeletal muscles with Sf9-generated rAAV5 with a 3.4-kb vector genome carrying a human secreted alkaline phosphatase (SEAP) expression cassette induced levels of SEAP more than 30 times higher than those for 293-produced vector 1 week after injection. Analysis of virion DNA revealed that in addition to a 2.4- or 3.4-kb single-stranded vector genome, Sf9-rAAV5 had more-abundant forms of approximately 4.7 kb, which appeared to correspond to the monomer duplex form of hGFP vector or truncated monomer duplex SEAP vector DNA. These results indicated that rAAV5 can be generated in insect cells, although the difference in incorporated virion DNA may induce different expression patterns of the transgene

Two Adenine Nucleotide Translocase Paralogues Involved in Cell Proliferation and Spermatogenesis in the Silkworm <i>Bombyx mori</i>

<div><p>Mitochondrial adenine nucleotide translocase (ANT) specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous <i>ANT</i> genes (<i>ANT1-4</i>) and utilize these paralogues in different types of cells. The fourth paralogue of <i>ANT</i> (<i>ANT4</i>) is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1) and the testis-specific paralogue (BmANTI2). The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran <i>Plutella xylostella</i> (PxANTI1), but not those of other insect species (or PxANTI2), restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4.</p></div