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.


Introduction
The ras genes was first isolated as the oncogene [1][2][3], and it was shown that its product Ras possesses GTPase activity [4]. Ras and structurally related proteins form the small GTPase family, a superfamily of monomeric GTP binding proteins (G-protein), which exists in various organisms [5,6]. Numbers of studies of small GTPase have been reported in relation to their characteristics and distribution to variety of organisms [structures [7][8][9], functions [10,11] and oncogenesis [12][13][14][15] of mammals [16][17][18], plants [19] and fungi [20][21][22][23][24]]. Commonly, the small GTPase plays central roles in signal transduction across membranes via the interaction with GDP/GTP exchange factors (GEF), GTPase activation proteins (GAP) and other regulators [10,[25][26][27]. Among small GTPases, Ras subfamily members work with certain membrane receptors such as receptor tyrosine kinases (RTK) [28][29][30]. When these receptors accept extracellular signaling factors such as growth factors and hormones, signaling cascades start via the phosphorylation of proteins. Ras acts as a switch in the middle of such signaling cascades to regulate the downstream MAPK (mitogen-activated protein kinase) signaling pathway [28,31,32]. It is well known that the Ras/MAPK pathway exists in various signaling cassettes, and is crucially involved in the proliferation of a cell and the control of cell differentiation [33][34][35].
Invertebrates also possess Ras proteins [36][37][38][39][40][41][42]. Among invertebrates, a nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster have been used concentrically in various studies to elucidate the function of Ras, since they are appropriate for research using mutants. In C. elegans, various defective phenotypes during the development, which were casused by Ras pathway mutations, led the discovery of basic components and the framework of the RTK/Ras/MAPK signaling pathway [43]. Also in D. melanogaster, mutants related to Ras were studied and provided basic information on the function of Ras [44,45], after the identification of three Ras homologues [40][41][42][46][47][48]. Such studies revealed the importance of epithelial growth factor receptors (EGFR: one goup of RTK) and their downstream Ras pathways for various developmental events of Drosophila ranging from cell fate determination during embryogenesis to the control of cell apoptosis [45]. Imaginal disc proliferation and appendage differentiation during larvae/pupae stages [44,[49][50][51], cell fate determination during embryogenesis [52], polarization of body axes during oogensis [53] and the control of cell apoptosis [54] were intensively studied for Ras. Thus, we can acquire a lot of information on D. melanogaster Ras. On the other hand, in relation to Ras of other insect species, there are few reports of the characteristics and the mode of action. Furthermore, ras cDNA sequences of insects except for D. melanogaster have not been reported, although several cDNA sequences have been predicted from the genome data of some insect species.
During the growth of insects, the timing of developmental events, such as molting and metamorphosis, is strictly regulated by two peripheral hormones, juvenile hormone (JH) and 20hydroxyecdysone (20E) [55]. Recently, in relation to the body size determination of D. melanogaster, it was indicated that the secretion of ecdysone, the precursor of 20E, was regulated by the Ras signaling pathway [56]. Similarly, functional links between ecdysone and small GTPases during the development of Drosohila have begun to be reported [57][58][59]. These reports suggest that the modes of action of 20E, and probably JH, are closely associated with the Ras function during insect development. However, such relations shown so far are indirect, and the precise mode of such interactions is ambiguous. It is well known that the effects of peripheral hormones, especially the roles of 20E in the metamorphosis and function of JH, are not clear in Drosophila. Furthermore, precise roles of Ras of D. melanogaster and other insects are expected to be considerably different, since the patterns of the development and body structures are highly diverse among insect species. Therefore, Ras studies using various insect species are thought to provide more beneficial information on the direct link between the hormonal regulation of insect growth and the function of Ras in the molecular interaction level.
The silkworm Bombyx mori is an insect which is frequently used for endocrinological research, and is also suitable for genetic research. In addition, the effect of JH is very clear in B. mori compared with D. melanogaster. Thus, B. mori is advantageous species to study the interaction between the hormonal regulation of insect growth and the function of Ras. Moreover, the genome database, KAIKO-BASE (http://spg.dna.affrc.go.jp/KAIKObase/), which have been established recently, will promote such studies efficiently. In this study, to elucidate the existence of the Ras/MAPK pathway in B. mori and its contribution to growth, we identified three ras genes of B. mori (Bmras: Bmras1, Bmras2 and Bmras3) and determined their cDNA sequences. Furthermore, we precisely investigated the developmental expression profiles of Bmras by quantitative real-time PCR in various tissues. The results of this study suggest different roles of the three Bombyx Ras (BmRas: BmRas1, BmRas2 and BmRas3) during development.
mRNA Expression Analysis of Bmras during Development mRNA expression levels of three Bmras in various tissues of various developmental stages were measured using the qRT-PCR technique (Fig. 4). In whole body samples, Bmras2 (Fig. 4C) transcripts tended to be more abundant than Bmras1 (Fig. 4A) and BmRas3 (Fig. 4E) through the development. All ras transcripts were low level until the 4th instar, and gradually increased from day 6 of the 5th instar. They then showed a high expression in the pupal stage. However, when expression patterns of Bmras were compared precisely, there were differences among their expression patterns. Bmras1 and Bmras3 initially had an intensive expression, a moderate expression in the middle and a high expression again at the end of the pupal stage. In addition, they have small peaks of expression at the end of each larval stadium. On the other hand, Bmras2 were not highly expressed until the end of the pupal stage, with no peaks at the end of each larval stadium except for the one of the 5th stadium. Bmras3 was also expressed intensely in the early days of the embryonic stage. All Bmras were ubiquitously expressed in all tissues used in this study (Fig. 4B, D and F). They did not show obvious patterns of changes in their expression levels except for several peaks. In the Malpighian tube on day 9 of the 5th instar, all Bmras showed a clear peak of expression. Bmras1 and Bmras2 also showed a peak in the muscle on day 7 and day 3 of the 5th instar, respectively.

Localization of BmRas-GFP Fusion Proteins in Sf-9 Cells
Genes coding the constitutively active-type of mutant of three BmRas, which were fused with GFP, were transiently introduced into Sf-9 cells and the cellular localization of their products was observed. Non-fused GFP prepared as a control was widely distributed throughout the cell, although relatively high luminescence was observed in the nuclei (Fig. 5A). In contrast, three BmRas-GFP proteins were specifically localized on the cell membrane ( Fig. 5B, C and D), as in the case of mammalian Ras [60]. The weak GFP fluorescence observed in the cytoplasm is thought to be the result of products being transported to the membrane.

Sequence Analysis of Ras Family Members
Ras small GTPase is one of the most intensively studied molecules in signal transduction. However, its functions have not been investigated in insects except for D. melanogaster. Here, we try to clarify the function of Ras in B. mori, a model insect of endocrinological studies, to understand its roles in the hormonal regulation of insect development. From the sequence homology of Ras proteins, we cloned ras cDNAs from B. mori and determined their sequences. As in the case of D. melanogaster [40][41][42], three ras genes were identified (Fig. 1). Phylogenetic analysis revealed that three Ras of B. mori corresponded to those of Drosophila (Fig. 3). There were some differences in amino acid residues at effector interaction sites, GEF interaction sites and the C terminal CaaX motif among the subfamily of Ras. Such differences classified three BmRas into separate subgroups. In phylogenetic analysis, only BmRas1 was grouped into the 'authentic' Ras subfamily with DmRas1. On the other hand, BmRas2 and BmRas3 were classified into mammalian R-Ras and Rap subfamily with DmRas2 and DmRas3, respectively, and it was consistent with previous reports [8,61]. In some insect species, sequences which are highly homologous to BmRas1 have been predicted from genome data as Ras, and BmRas2 and BmRas3 as 'Ras-like' proteins in the database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Those 'Ras' proteins were also clustered with 'authentic' Ras ( Fig. 3), and 'Ras-like' proteins were with R-Ras or Rap. Therefore, it is likely that many insects possess counterparts of mammalian 'authentic' Ras, R-Ras and Rap. Both DmRas2 and mammalian R-Ras were reported to be involved in the formation of the neuronal network [62,63], suggesting that BmRas2 have similar roles. Recent studies including the genome database of D. melanogaster revealed that DmRas3 is identical to Drosophila Rap1 (roughened) [48,64]. Drosophila Rap1 was shown to relate to the regulation of cell shapes by forming adherens junctions [65,66], as in the mammalian Rap subgroup [67]. Therefore, BmRas3 (Rap) might also act in adherens junction formation. In comparison with the Ras subgroup, functions of R-Ras and Rap subgroups have not been elucidated adequately. Further study of insect Ras2 and Ras3 might bring the basis of functions of these subgroups.

Gene Expression Analysis of Bmras
In qRT-PCR analysis, transcripts of three B. mori Ras showed ubiquitous expressions, although their expression patterns were different each other. Therefore, it is predicted that three BmRas have different roles, and all are important for the development of B. mori. Their ubiquitous expression is consistent with D. melanogaster [68,69]. However, ras2, which was the most abundant in B. mori (Fig. 4), was less abundant than ras1 and ras3 in D. melanogaster. Thus, functions of these Ras proteins might be different among insect species. In this study, mRNA expression patterns of BmRas1 and BmRas3 were similar, and it was consistent with previous reports which indicated the related functions of Ras and Rap of D. melanogaster [70,71]. The spatial expression patterns of BmRas were also ubiquitous, indicating their widespread roles in B. mori. In Drosohila, Ras transcripts were markedly expressed in imaginal discs [69]. It is consistent with the intensive expression of BmRas transcripts after the late stage of 5th instar, the period of adult tissue development. However, Bmras were also expressed in the silk gland (a larval tissue) in this study. Thus, it is probable that BmRas exists generally in various tissues, but acts via different machineries in each organ.
The peak of expression in the Malpighian tube in 5th instar larvae on day 9, which was observed for all three BmRas, was interesting. In Drosophila, it was shown that Ras controls the  development of the Malpighian tube via EGFR in the embryonic period [72]. However, there have been no reports of changes occurring in Malpighian tube in larval stages. It might be related to imaginal disc growth in the puapl stage or signal regulation to excrete endogenous contents unnecessary for pupal metamorphosis. Further investigation is required to elucidate the meaning of Ras signaling activation at the Malpighian tube.
In this study, the expressions of three Bmras in the larval stage except for the late 5th stadium were low in comparison with embryonic and pupal stages. This period overlaps with that during which JH exists in a larval body, suggesting involvement of JH in suppression of ras expression. Therefore, the signaling of the control of cell differentiation by Ras proteins might be restricted by JH during growth period with limited cell differentiation in the larval stage, and then released in pupal stage with rapid reformation of body structures which is undergone through determination of the fate of various cells such as apoptosis. Elucidation of the function of JH for Bmras expression in specific tissues or cells is predicted to reveal the molecular action of JH and factors related to the action.

Membrane Localization of BmRas-GFP Fusion Proteins
Mammalian H-, K-and N-Ras have separate tissue-specific mRNA expression patterns [25]. Furthermore, these three Ras are targets of different modification of C terminal, namely isoprenyla-tion and palmitoylation [73]. Recent studies indicated that such tissue specificities and differences of the localization, which is controlled by C terminal modification, cause the differences in functions of mammalian three Ras [60] suggesting the C terminal modification in insects also plays an important role. We showed the membrane localization of three BmRas using GFP-fusion proteins (Fig. 5), which indicated that these proteins had modified C terminals to act in membrane signal transduction as generally seen with Ras proteins. It is predicted that further study of C terminal modification will reveal the molecular mechanism of Ras function in insects.
A recent study of D. melanogaster mutants, which have abnormal Ras/MAPK and other signaling pathways, showed that Ras and its downstream components, Raf and PI3K (phosphoinositide-3kinase), controls the body size of the fly, by regulating the timing of secretion of ecdysone from the prothoracic gland (PG) [56]. Ecdysone is synthesized in PG by the stimulation of the prothoracicotropic hormone (PTTH). Although the molecular mode of action of PTTH is not very clear, complex interactions of kinase pathways, for example protein kinase A, protein kinase C (PKC) and tyrosine kinase pathways, were suggested to be involved in the stimulation of PG by PTTH in Manduca sexta, via Ca 2+ and/or cAMP as second messengers [74]. Therefore, it is predicted that the Ras/MAPK signaling pathway strongly interacts with those signaling pathways to control the ecdysteroidogenesis. Further study for understanding the entire network of  In conclusion, we isolated and identified three genes of Ras family members of B. mori, BmRas1, BmRas2 and BmRas3, which corresponded to DmRas1, Ras2 and Ras3, respectively, and showed high sequence homology with their counterparts. mRNA expression analysis revealed that three BmRas are ubiquitously expressed, although they have remarkable expression in late 5th instar and pupal periods with different patterns each other. In addition, all three Bmras have a peak of expression in the Malpighian tube in the 5th instar on day 9. Furthermore, we showed the membrane localization of BmRas using a transient expression technique in insect cultured cells as GFP-fusion proteins. These data suggest that three BmRas have characteristics which are common among Ras family members, and thus play central roles in the cell differentiation and proliferation of B. mori. Further study will elucidate not only the relationship between the Ras/MAPK pathway and the development of B. mori, but also new components and the precise framework of the Ras-related signaling pathway.

RNA Isolation
B. mori, C145xN140 were routinely reared on an artificial diet at 26uC under a 12 hr light/12 hr dark photoperiod. Under these condition, durations of embryo, 1st, 2nd, 3rd, 4th and 5th instars and the pupal stage were 9, 4, 4, 5, 6, 12 and 11 days, respectively. For whole body samples, larvae were dissected and gut contents were removed, and then the whole insect bodies were used as samples. For tissue samples, insects were dissected and the epidermis, fat body, gut, muscle, silk gland and Malpighian tubule were separated. Total RNA was extracted from whole insect bodies from each day (24 hr period) in every stage and from tissues on all days of 4th and 5th instar larvae (24 hr period) using ISOGEN (NIPPON GENE Co., Tokyo, Japan), and purified by SV Total RNA Isolation System (Promega Co., Madison, WI, USA), which includes DNase treatment in its protocol. Total RNA was also extracted from whole insect bodies on the first day of the adult stage and pupal follicle cells. The numbers of insects used for RNA isolation were 40, 10, 5, 2-3, 1, 1, 1 and 1/day for the embryo stage, the larval stadium (1st, 2nd, 3rd, 4th, and 5th), the pupal stage and adult stage, respectively. Concentrations of RNA samples were determined using an UV spectrophotometer NanoDrop ND1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA).

cDNA Cloning
All primers used in this study were purchased from Tsukuba Oligo Service Co. (Ibaraki, Japan). First-strand cDNAs were synthesized from a total RNA sample of fat bodies of 5th instar day 3 larvae using Ready-To-Go RT-PCR Beads (GE Healthcare, Chalfont St Giles, UK) with oligo(dT) primers. The total RNA sample was treated with DNase (Promega), and 2 mg of the sample was used for cDNA synthesis. From amino acid sequence homologies among Ras proteins, degenerate primers were designed (Table 1). Reverse transcription PCR was conducted with primers ras1-F1 and -R1 for Bmras1, ras2-F1 and -R1 for Bmras2 and ras3-F1 and -R1 for Bmras3, respectively. The PCR program was as follows: initial incubation (95uC; 2 min) was followed by 35 repetitions of PCR cycle (95uC; 30 sec, 48uC; 30 sec, 72uC; 2 min). PCR products were then purified and their sequences were determined by ABI PRISM 3100 Genetic Analyzer (Applied Biosystems Co., Foster City, CA, USA) using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). For RACE, 2 mg of total RNA was used for first-strand cDNAs synthesis using SMART RACE cDNA Amplification Kit (TAKARA BIO Inc., Shiga Japan) according to the manufacture's instruction. The primary amplifications were performed by touchdown PCR using proof-reading KOD-PLUS (TOYOBO Co., Osaka, Japan) with primers ras1-rF1 and -rR1 for Bmras1, ras2-rF1 and -rR1 for Bmras2 and ras3-rF1 and -3rR1 for Bmras3, respectively (Table 1). PCR products were then further amplified using KOD-PLUS and nested primers, ras1-rF2 and -rR2 for Bmras1, ras2-rF2 and -rR2 for Bmras2 and ras3-rF2 and -rR2 for  Bmras3, respectively (Table 1). DNA sequences of amplified fragments were determined as mentioned above.

Quantitative Real-Time PCR (qRT-PCR)
Isolated total RNA was reverse transcribed using ReverTra -Plus-(TOYOBO) following the manufacturer's instruction. Briefly, 200 ng total RNAs was converted to cDNA with Oligo(dT) 20 by Moloney murine leukemia virus reverse transcriptase in 20 ml reaction volume. The reaction was diluted to 1/4 concentration with water and used as a template of qRT-PCR. cDNAs encoding full-length open-reading frames (ORFs) of BmRas1, BmRas2 and BmRas3 were amplified by PCR using primer pairs ras1-NdeIF and -BamHIR, ras2-NdeIF and -BamHIR and ras3-NdeIF and -BamHIR, respectively (Table 1), and ligated into pET-16b plasmid vector (Merck KGaA, Darmstadt, Germany). Serial dilutions of these constructs, namely pET16/BmRas1, pET16/ BmRas2 and pET16/BmRas3, were used as standards. As a reference gene, B. mori ribosomal protein 49 (Bmrp49) was chosen. The qRT-PCR standards and primers for Bmrp49 were prepared as described previously [75]. qRT-PCR primer pairs for Bmras1, Bmras2 and Bmras3 are listed in Table 1. The transcripts of Bmras, the same as Bmrp49, in prepared cDNA templates were quantified on a real-time thermal cycler, LightCycler 480 Real-Time PCR System (Roche Diagnostics, Indianapolis, IN, USA). qRT-PCR was carried out in 20 ml reaction volume containing 2 ml template cDNA (equivalent to 5 ng total RNA) or standard cDNA, 1 x SYBR Premix EX Taq (TaKaRa) and each primer. We used a PCR protocol of 1 cycle of denature at 95uC for 10 seconds; 20uC/second followed by 50 cycles of PCR at 95uC for 5 seconds; 20uC/second, 60uC for 20 second; 20uC/second. After the PCR stage, the absence of unwanted by-products was confirmed by automated melting curve analysis. The molar amounts of transcripts of each gene were calculated based on crossing point analysis, with standard curves generated from standard cDNA. The molar amounts of Bmras were normalized with Bmrp49 transcript levels in the same cDNA templates and used as indicators of the expression levels of each gene.

Expression of BmRas-GFP Fusion Proteins in Sf-9 Cultured Cells
Protein expression constructs of constitutively active mutants of three Bombyx Ras as GFP fusion proteins, namely BmRas1-Val 12 GFP, BmRas2Val 22 GFP and BmRas3Val 12 GFP, were prepared as follows, with refeence to a previous report [76,77]. A DNA fragment which codes GFP is introduced into the cloning site of pIB/V5-His-DEST plasmid vector (Invitrogen Co., Carlsbad, CA, USA). Inverse PCR was conducted to amplify mutated Bmras, in which a glycine residue at the corresponding position is substituted by a valine residue, using primers ras1-iF and -iR for BmRas1Val 12 GFP, ras2-iF and -iR for BmRas2-Val 22 GFP and ras3-iF and -iR for BmRas3Val 12 GFP ( Table 1). Templates of inverse PCR were pET16/BmRas1, pET16/ BmRas2 and pET16/BmRas3, respectively. Then amplified fragments were inserted into the C-terminal position of the GFP coding site to compose DNA sequences which code Ras-GFP chimeric proteins. These mutant constructs were used for transient transfection to Sf-9 cultured cells, a cell line from lepidopteran Spodoptera frugiperda. Sf-9 cells were routinely maintained in Sf-900 II (Invitrogen) at 27uC. For analysis by fluorescence microscopy, 8610 5 Sf-9 cells were seeded on the 18-mm micro cover glass (Matsunami Glass Ind., Osaka Japan) in 6-well plates (BD, Franklin Lakes, NJ, USA) and transfected with 1 mg plasmids using Cellfectin (Invitrogen) following the manufacturer's instruction. After 24 hr culture at 27uC, cells were washed three times with 1xPBS (10 mM sodium phosphate, 150 mM sodium chloride, pH = 7.4), fixed with 4% paraformaldehyde for 17 min at room temperature, and washed three times with 1xPBS again. The fixed cells were mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA), and observed using an FV1000 confocal microscope (Olympus, Tokyo, Japan). The images were processed using Photoshop 5.0 (Adobe, San Jose, CA, Japan).