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K. P. Gopinathan - One of the best experts on this subject based on the ideXlab platform.
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comparative analysis of the development of the mandibular salivary Glands and the labial Silk Glands in the mulberry Silkworm bombyx mori
Gene Expression Patterns, 2005Co-Authors: R Parthasarathy, K. P. GopinathanAbstract:The mulberry Silkworm, Bombyx mori has a pair of salivary Glands arising from the mandibular segment, in addition to the labial Silk Glands which are generally considered as modified salivary Glands. Here we report the characterization of salivary Glands and the comparative gene expression profiling of the Silk and salivary Glands. The two independent salivary Glands made up by 330 cells, grow about 1000 fold during larval development. These individual Glands extend up to the $T_1$ thoracic segment unlike Silk Glands with fused anterior ends and extending up to the caudal region. The salivary Glands also undergo endomitosis resembling the Silk Glands. The B. mori homologue of the homeotic gene Deformed (BmDfd) was expressed in the mandibular and maxillary segments in stage 17 embryo and got localized to the centre of the mandibular segment at stage 18 to form the salivary gland placodes. The expression was also seen in the distal ends of the leg appendages after blastokinesis (stage 22). Only low variations in BmDfd expression ranging from 1.6 to 2.1 fold were apparent during embryonic development. BmDfd expression was observed in the salivary Glands all through the larval instars but not in the Silk Glands. The transcription factor, Forkhead and the segment polarity gene, Wingless were expressed throughout the salivary Glands, the latter confirming the absence of physiological compartmentation within these Glands unlike the Silk Glands. The expression of Amylase and Fibrohexamerin was restricted to the salivary and Silk Glands, respectively and therefore, served as molecular markers for these tissues.
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dna polymerase alpha primase complex from the Silk Glands of the non mulberry Silkworm philosamia ricini
Biochemical Journal, 1994Co-Authors: Somashekarappa Niranjanakumari, K. P. GopinathanAbstract:The DNA content in the Silk Glands of the non-mulberry Silkworm Philosamia ricini increases continuously during the fourth and fifth instars of larval development indicating high levels of DNA replication in this terminally differentiated tissue. Concomitantly, the DNA polymerase alpha activity also increases in the middle and the posterior Silk Glands during development, reaching maximal levels in the middle of the fifth larval instar. A comparable level of DNA polymerase delta/epsilon was also observed in this highly replicative tissue. The DNA polymerase alpha-primase complex from the Silk Glands of P. ricini has been purified to homogeneity by conventional column chromatography as well as by immunoaffinity techniques. The molecular mass of the native enzyme is 560 kDa and the enzyme comprises six non-identical subunits. The identity of the enzyme as DNA polymerase alpha has been established by its sensitivity to inhibitors such as aphidicolin, N-ethylmaleimide, butylphenyl-dGTP, butylanilino-dATP and antibodies to polymerase alpha. The enzyme possesses primase activity capable of initiating DNA synthesis on single-stranded DNA templates. The tight association of polymerase and primase activities at a constant ratio of 6:1 is observed through all the purification steps. The 180 kDa subunit harbours the polymerase activity, while the primase activity is associated with the 45 kDa subunit.
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isolation and characterization of dna polymerase epsilon from the Silk Glands of bombyx mori
Journal of Biological Chemistry, 1993Co-Authors: Somashekarappa Niranjanakumari, K. P. GopinathanAbstract:Abstract The Silk gland of Bombyx mori, an endomitotically replicative tissue shows high levels of DNA polymerases alpha, delta, and epsilon activities. The ratio of polymerase alpha to that of delta plus epsilon is maintained at 1.1 to 1.3 in both the posterior and middle Silk Glands for the entire duration of late larval development. The three activities copurify in the initial stages of fractionation through phosphocellulose and DE52 but polymerase alpha gets resolved from the others on hydroxylapatite column. Separation between polymerase delta and epsilon is achieved by chromatography on QAE-Sephadex. DNA polymerase epsilon is a heterodimer comprising of 215- and 42-kDa subunits. The activity is maximum at pH 6.5 and the Km values for dNTPs vary between 3-9 microM. The enzyme possesses an intrinsically associated exonuclease activity which functions in the mismatch repair during DNA synthesis. Both polymerase and 3'-->5' exonuclease activities are associated with the 215-kDa subunit. By itself, DNA polymerase epsilon is processive and the catalytic activity is not enhanced by externally added bPCNA (Bombyx-proliferating cell nuclear antigen, an auxiliary protein for DNA polymerase delta). The enzyme resembles polymerase delta in having the exonuclease activity and in its response to aphidicolin or substrate analogs, but could be distinguished from the latter by its lack of response to the bPCNA and sensitivity to dimethyl sulfoxide. The two enzymes show partial immunological cross-reactivity with each other but no immunological relatedness to polymerase alpha. The absence of the repair enzyme DNA polymerase beta and the presence of substantial levels of polymerase epsilon in the Silk Glands suggest a possible role for the latter in DNA repair in that tissue.
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dna polymerase delta from the Silk Glands of bombyx mori
Journal of Biological Chemistry, 1992Co-Authors: Somashekarappa Niranjanakumari, K. P. GopinathanAbstract:The Silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle Silk Glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori Silk Glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3' --> 5' exonuclease activity which participates in proofreading by mismatch match repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori Silk Glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.
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characterisation of the dna polymerase α primase complex from the Silk Glands of bombyx mori
FEBS Journal, 1991Co-Authors: Somashekarappa Niranjanakumari, K. P. GopinathanAbstract:Silk gland cells of Bombyx mori undergo chromosomal endoduplication throughout larval development. The DNA content of both posterior and middle Silk gland nuclei increased by 300,000 times the haploid genomic content, amounting to 18 rounds of replication. The DNA doubling time is approximately 48 h and 24 h during the fourth and fifth instars of larval development. However, DNA content does not change during the interim moult. Concomitant with DNA content, DNA polymerase activity also increases as development progressed. Enzyme activity is predominantly due to DNA polymerase alpha with no detectable level of polymerase beta. DNA polymerase alpha from Silk gland extracts was purified to homogeneity (using a series of columns involving ion-exchange, gel-filtration and affinity chromatography), resulting in a 4000-fold increase in specific activity. The enzyme is a heterogeneous multimer of high molecular mass, and the catalytic (polymerase) activity is resident in the 180-kDa subunit. The enzyme shows a pI of 6.2 and the Km values for the dNTP vary over 5-16 microM. The polymerase is tightly associated with primase activity and initiates primer synthesis in the presence of ribonucleoside triphosphates on a single-stranded DNA template. The primase activity is resident in the 45-kDa subunit. The enzyme is devoid of any detectable exonuclease activity. The abundance of DNA polymerase alpha in Silk Glands and its strong association with the nuclear matrix suggest a role in the DNA endoduplication process.
Osman Parlak - One of the best experts on this subject based on the ideXlab platform.
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fenoxycarb modulates ecdysone receptor b1 and programmed cell death of the anterior Silk gland of Silkworm bombyx mori
Entomological Science, 2011Co-Authors: Ebru Goncu, Osman ParlakAbstract:Fenoxycarb, O-ethyl N-(2-(4-phenoxyphenoxy)-ethyl) carbamate has been shown to be one of the most potent juvenile hormone analogues against a variety of insect species. In the present study, topical application of fenoxycarb to fifth-instar larvae of the Silkworm Bombyx mori (Lepidoptera: Bombycidae) was performed immediately after the fourth ecdysis (on day 0), day 3 and day 6 of the instar and then its effects on the anterior Silk Glands (ASG) and ecdysone receptor B1 (EcR-B1) protein were investigated during larval pupal development. Fenoxycarb application increased the instar length and prevented metamorphic events, depending on the application time. The ASGs of B. mori undergo programmed cell death during the larval–pupal metamorphosis and an insect steroid, 20-hydroxyecdysone (20E), triggers this cell death. The exact mechanism by which 20E and juvenile hormone regulates programmed cell death in insect tissues is poorly understood. To gain insights into how juvenile hormone regulates metamorphic events like programmed cell death in the anterior Silk Glands, we analyzed the progression of programmed cell death with morphological observations and biochemical experiments like acid phosphatase activity and DNA electrophoresis. Then we examined the EcR-B1 protein levels and their relationships with programmed cell death. Our results indicated that fenoxycarb modulates programmed cell death of the anterior Silk Glands and EcR-B1 protein level, depending on the application time. Fenoxycarb may exhibit its effects in at least two different ways: (i) acting on prothoracic gland secretory activity; and/or (ii) regulation of EcR-B1 expression in the anterior Silk Glands for programmed cell death process.
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morphological changes and patterns of ecdysone receptor b1 immunolocalization in the anterior Silk gland undergoing programmed cell death in the Silkworm bombyx mori
Acta Histochemica, 2009Co-Authors: Ebru Goncu, Osman ParlakAbstract:The Silk gland is a specific larval tissue of Lepidopteran insects and begins to degenerate shortly before pupation. The steroid hormone ecdysone triggers the stage specific programmed cell death of the anterior Silk Glands during metamorphosis in the Silkworm, Bombyx mori. The anterior Silk gland expresses ecdysone receptors, which are involved in regulation processes in response to ecdysone. In this study, the morphological changes, immunohistochemical localization and protein levels of ecdysone receptor B1 (EcR-B1) in the anterior Silk gland of B. mori were investigated during programmed cell death. Morphological changes observed during the degeneration process involve the appearance of large vacuoles, probably autophagic vacuoles, which increase in number in pupal anterior Silk Glands. No macrophages were found in the Silk gland during the prepupal and pupal stage unlike in apoptosis, which strongly suggests that programmed cell death of the anterior Silk gland is carried out by autophagy. Morphological changes of the Silk Glands were accompanied by changes in the immunolocalization and protein levels of EcR-B1. The differences in tissue distribution and protein levels of EcR-B1 during the programmed cell death indicate that the receptor plays a major role in the modulation and function of ecdysone activity in Bombyx anterior Silk Glands. Our results indicate that EcR-B1 expression may be important for the process of programmed cell death in the anterior Silk Glands.
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some autophagic and apoptotic features of programmed cell death in the anterior Silk Glands of the Silkworm bombyx mori
Autophagy, 2008Co-Authors: Ebru Goncu, Osman ParlakAbstract:Programmed cell death has been subdivided into two major groups: apoptosis and autophagic cell death. The anterior Silk gland of Bombyx mori degenerates during larval-pupal metamorphosis. Our findings indicate that two types of programmed cell death features are observed during this physiological process. During the prepupal period, pyknosis of the nucleus, cell detachment,and membrane blebbing occur and they are the first signs of programmed cell death in the anterior Silk Glands. According to previous studies, all of these morphological appearances are common for both cell-death types. Autophagy features are also exhibited during the prepupal period. Levels of one of the lysosomal marker enzymes-acid phosphatase-are high during this period then decrease gradually. Vacuole formation begins to appear first at the basal surface of the cell, then expands to the apical surface just before the larval pupal ecdysis. After larval-pupal ecdysis, DNA fragmentation, which is the obvious biochemical marker of apoptosis, is detected in agarose gel electrophoresis, which also shows that caspase-like enzyme activities occur during the programmed cell death process of the anterior Silk Glands. Apoptosis and autophagic cell death interact with each other during the degeneration process of the anterior Silk gland in Bombyx mori and this interaction occurs at a late phase of cell death. We suggest that apoptotic cell death only is not enough for whole gland degeneration and that more effective degeneration occurs with this cooperation.
Chengliang Gong - One of the best experts on this subject based on the ideXlab platform.
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lowering the blood glucose of diabetes mellitus mice by oral administration with transgenic human insulin like growth factor i Silkworms
Journal of Agricultural and Food Chemistry, 2012Co-Authors: Renyu Xue, Guangli Cao, Wenlin Zhou, Xiaojian Zheng, Yang Wang, Zhonghua Pan, Chengliang GongAbstract:To evaluate the biological activity of the posterior Silk Glands of transgenic Silkworms expressing human insulin- like growth factor I (hIGF-I), we bred hIGF-I-transgenic Silkworms through eight generations by continuously selecting with green fluorescence and G418. The G8 transgenic Silkworms were confirmed by polymerase chain reaction and dot blotting, and their posterior Silk Glands were removed from the fifth instar larvae to make freeze-dried powders. Enzyme-linked immunosorbent assay results showed that the expression level of hIGF-I in the posterior Silk Glands of G8 transgenic Silkworm is approximately 493 ng/g of freeze-dried powder. When the freeze-dried powder was administrated by gavage to diabetes mellitus (DM) mice, the blood glucose in DM mice significantly decreased (P < 0.05) in a time- and dose-dependent manner compared with that of DM mice orally administrated with distilled water and normal freeze-dried powders made of untreated Silk Glands. These results demonstrated that hIGF-I expressed in posterior Silk Glands of transgenic Silkworms could reduce blood glucose by oral administration.
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expression of hgm csf in Silk Glands of transgenic Silkworms using gene targeting vector
Transgenic Research, 2012Co-Authors: Renyu Xue, Huimei Chen, Linlin Cui, Guangli Cao, Wenlin Zhou, Xiaojian Zheng, Chengliang GongAbstract:The Silk gland of the Silkworm is a highly specialized organ that has the wonderful ability to synthesize and secrete Silk protein. To express human granucyto-macrophage colony-stimulating factor (hGM-CSF) in the posterior Silk Glands of gene-targeted Silkworms, a targeting vector pSK-FibL-L-A3GFP-PH-GMCSF-LPA-FibL-R was constructed, harboring a 1.2 kb portion of the left homogenous arm (FibL-L), a 0.5 kb portion of the right homogenous arm (FibL-R), fibroin H-chain-promoter-driven hGM-CSF and Silkworm actin 3-promoter-driven gfp. The targeting vector was then introduced into the eggs of Silkworm, and the transgenic Silkworms were verified by PCR and DNA hybridization after being screened for the gfp gene. Western blotting analysis using an antibody against hGM-CSF demonstrated a specific band with a molecular weight of 22 kD in the Silk Glands of the G3 generation transgenic Silkworms. The level of expression of hGM-CSF in the posterior Silk Glands of the G3 generation transgenic Silkworms was approximately 2.70 ng/g of freeze-dried powdered posterior Silk gland. These results showed that the heterologous gene could be introduced into the Silkworm genome and expressed successfully. Further more, the exogenous genes existing in the G5 transgenic Silkworm identified by PCR confirmed its integration stability. In addition, the Silk Glands containing expressed hGM-CSF performed the function of significantly increasing leukocyte count of CY-treated mice in a time-and-dose-dependent manner.
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reducing blood glucose level in tidm mice by orally administering the Silk Glands of transgenic higf i Silkworms
Biochemical and Biophysical Research Communications, 2011Co-Authors: Liang Cong, Guangli Cao, Wenlin Zhou, Xue Renyu, Pan Zhonghua, Zheng Xiaojian, Chengliang GongAbstract:Abstract To realize the secretory expression of human insulin-like growth factor-I (hIGF-I) in the posterior Silk Glands (PSGs) of transgenic Silkworms, the piggyBac transposon vector pigA3GFP-fibHS-hIGF-i.e.-neo containing a neomycin-resistance gene ( neo ), green fluorescent protein gene ( gfp ) and human insulin-like growth factor I ( hIGF -I) gene controlled by the Bombyx mori fibroin heavy chain gene ( fib -H) promoter with its downstream signal peptide sequence, and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 gene ( A 3) promoter were transferred into Silkworm eggs by sperm-mediated gene transfer. Transformed Silkworms were obtained after being screened for green fluorescence and by the antibiotic G418. In the PSGs of the transformed Silkworms, a specific band representing hIGF-I could be detected by Western blotting, and the content of the hIGF-I estimated by ELISA was approximately 1.84 μg/gram of cocoon and 19.18 μg/gram of freeze-dried PSG powder. To further estimate the biological activity of the expressed hIGF-I, streptozotocin-induced TIDM mice were orally administered with the PSG powder of the transgenic Silkworms, the results showed the blood glucose levels of mice were significantly reduced, suggesting that the the PSGs powder of transgenic hIGF -I Silkworms could possibly be used as a perorally administered medicine.
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expression of the higf i gene driven by the fhx p25 promoter in the Silk Glands of germline Silkworm and transformed bmn cells
Biotechnology Letters, 2011Co-Authors: Guangli Cao, Renyu Xue, Wenlin Zhou, Yang Wang, Chengliang GongAbstract:The expression of the human insulin-like growth factor (hIGF-I) gene driven by the Fhx/P25 promoter in the Silk Glands of transgenic Silkworms (Bombyx mori) and in transformed Silkworm cells, was achieved using BmN cells transfected with a piggyBac vector, pigA3GFP-Fhx/P25-hIGF-ie-neo containing a neomycin-resistance gene (neo), a green fluorescent protein gene (gfp), an hIGF-I gene, and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 (A3) promoter. We selected stably transformed BmN cells expressing hIGF-I using the antibiotic G418. The expression level of hIGF-I was about 450 pg in 3 × 106 cells, determined by ELISA. The piggyBac vector was transferred into the Silkworm eggs using sperm-mediated gene transfer. The expression level of hIGF-I per gram fresh posterior Silk Glands of G4 transgenic Silkworms was approx. 150 ng.
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expression of the hgm csf in the Silk Glands of germline of gene targeted Silkworm
Biochemical and Biophysical Research Communications, 2010Co-Authors: Guangli Cao, Renyu Xue, Huimei Chen, Haifang Jia, Chengliang GongAbstract:Abstract To express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene in the Silk Glands of transformation Silkworm ( Bombyx mori ) based on gene-targeting, two fragments from fibroin heavy chain gene ( fib -H) of Silkworm were cloned and sequenced. One fragment contains the 1st exon and its downstream 1st intron’s partial sequence; and the other fragment contains the 1st intron’s partial sequence and the 2nd exon’s partial sequence. Then the two fragments, as homologous arm, were inserted into pSK to generate a gene-targeted vector, pSK-HL-A3GFP-FLP-GM-CSF-FLPA-HR in which a gfp gene driven by A3 promoter and an hGM - CSF gene under the control of fibroin light chain ( fib -L) promoter were included. The vector was transferred into the Silkworm eggs using sperm-mediated gene transfer. After being screened for green fluorescent, the transformation Silkworm was obtained, whose genome was verified by PCR and dot hybridization to confirm whether the target genes had been integrated into the Silkworm genome. Furthermore, in the posterior Silk Glands of the G4 generation transformation Silkworms, a specific band with the molecular weight of 22 kDa could be detected by Western blotting with an antibody against hGM-CSF, and the expression level of the hGM-CSF estimated by ELISA was approximately 1.26 ng per gram fresh posterior Silk gland.
Janice S. Edgerly - One of the best experts on this subject based on the ideXlab platform.
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The spinning apparatus of webspinners – functional-morphology, morphometrics and spinning behaviour
Scientific Reports, 2015Co-Authors: Sebastian Büsse, David Mcmillan, Kyle Hohu, Thomas Hornschemeyer, Janice S. EdgerlyAbstract:Webspinners (Insecta: Embioptera) have a distinctly unique behaviour with related morphological characteristics. Producing Silk with the basitarsomeres of their forelegs plays a crucial role in the lives of these insects – providing shelter and protection. The correlation between body size, morphology and morphometrics of the spinning apparatus and the spinning behaviour of Embioptera was investigated for seven species using state-of-the-art methodology for behavioural as well as for morphological approaches. Independent contrast analysis revealed correlations between morphometric characters and body size. Larger webspinners in this study have Glands with greater reservoir volume, but in proportionally smaller tarsi relative to body size than in the smaller species. Furthermore, we present a detailed description and review of the spinning apparatus in Embioptera in comparison to other arthropods and substantiate the possible homology of the embiopteran Silk Glands to class III dermal Silk Glands of insects.
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the spinning apparatus of webspinners functional morphology morphometrics and spinning behaviour
Scientific Reports, 2015Co-Authors: Sebastian Büsse, David Mcmillan, Kyle Hohu, Thomas Hornschemeyer, Janice S. EdgerlyAbstract:Webspinners (Insecta: Embioptera) have a distinctly unique behaviour with related morphological characteristics. Producing Silk with the basitarsomeres of their forelegs plays a crucial role in the lives of these insects – providing shelter and protection. The correlation between body size, morphology and morphometrics of the spinning apparatus and the spinning behaviour of Embioptera was investigated for seven species using state-of-the-art methodology for behavioural as well as for morphological approaches. Independent contrast analysis revealed correlations between morphometric characters and body size. Larger webspinners in this study have Glands with greater reservoir volume, but in proportionally smaller tarsi relative to body size than in the smaller species. Furthermore, we present a detailed description and review of the spinning apparatus in Embioptera in comparison to other arthropods and substantiate the possible homology of the embiopteran Silk Glands to class III dermal Silk Glands of insects.
Cheryl Y. Hayashi - One of the best experts on this subject based on the ideXlab platform.
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golden orb weaving spider trichonephila clavipes Silk genes with sex biased expression and atypical architectures
G3: Genes Genomes Genetics, 2021Co-Authors: Sandra M Correagarhwal, Paul L Babb, Benjamin F Voight, Cheryl Y. HayashiAbstract:Spider Silks are renowned for their high-performance mechanical properties. Contributing to these properties are proteins encoded by the spidroin (spider fibroin) gene family. Spidroins have been discovered mostly through cDNA studies of females based on the presence of conserved terminal regions and a repetitive central region. Recently, genome sequencing of the golden orb-web weaver, Trichonephila clavipes, provided a complete picture of spidroin diversity. Here, we refine the annotation of T. clavipes spidroin genes including the reclassification of some as non-spidroins. We rename these non-spidroins as spidroin-like (SpL) genes because they have repetitive sequences and amino acid compositions like spidroins, but entirely lack the archetypal terminal domains of spidroins. Insight into the function of these spidroin and SpL genes was then examined through tissue- and sex-specific gene expression studies. Using qPCR, we show that some Silk genes are upregulated in male Silk Glands compared to females, despite males producing less Silk in general. We also find that an enigmatic spidroin that lacks a spidroin C-terminal domain is highly expressed in Silk Glands, suggesting that spidroins could assemble into fibers without a canonical terminal region. Further, we show that two SpL genes are expressed in Silk Glands, with one gene highly evolutionarily conserved across species, providing evidence that particular SpL genes are important to Silk production. Together, these findings challenge long-standing paradigms regarding the evolutionary and functional significance of the proteins and conserved motifs essential for producing spider Silks.
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dissection of Silk Glands in the western black widow latrodectus hesperus
Journal of Arachnology, 2018Co-Authors: Crystal R Chaw, Cheryl Y. HayashiAbstract:The Silk Glands of the Western black widow Latrodectus hesperus Chamberlin & Ivie, 1935 are morphologically and functionally distinct. Studies of spider Silk Glands often show only high magnification images of sections or drawings of Glands, making differentiation of dissected Glands difficult. We dissect all of the gland types from L. hesperus females and show their gross morphology with light microscopy. Our micrographs portray the distinct morphologies and relative sizes of each Silk gland type, consistent with prior descriptions of the Silk apparatus of Latrodectus spiders. Notably, we verify the presence of two differentiated pairs of aggregate Silk Glands and spigots, thus resolving a discrepancy in the literature.
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Evolutionary shifts in gene expression decoupled from gene duplication across functionally distinct spider Silk Glands
Scientific Reports, 2017Co-Authors: Thomas H Clarke, Cheryl Y. Hayashi, Jessica E. Garb, Robert A. Haney, R. Crystal Chaw, Nadia A AyoubAbstract:Spider Silk synthesis is an emerging model for the evolution of tissue-specific gene expression and the role of gene duplication in functional novelty, but its potential has not been fully realized. Accordingly, we quantified transcript (mRNA) abundance in seven Silk gland types and three non-Silk gland tissues for three cobweb-weaving spider species. Evolutionary analyses based on expression levels of thousands of homologous transcripts and phylogenetic reconstruction of 605 gene families demonstrated conservation of expression for each gland type among species. Despite serial homology of all Silk Glands, the expression profiles of the glue-forming aggregate Glands were divergent from fiber-forming Glands. Also surprising was our finding that shifts in gene expression among Silk gland types were not necessarily coupled with gene duplication, even though Silk-specific genes belong to multi-paralog gene families. Our results challenge widely accepted models of tissue specialization and significantly advance efforts to replicate Silk-based high-performance biomaterials.
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proteomic evidence for components of spider Silk synthesis from black widow Silk Glands and fibers
Journal of Proteome Research, 2015Co-Authors: R C Chaw, Sandra M Correagarhwal, Thomas H Clarke, Nadia A Ayoub, Cheryl Y. HayashiAbstract:Spider Silk research has largely focused on spidroins, proteins that are the primary components of spider Silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with Silk synthesis are virtually unknown. Previous analyses of tissue-specific RNA-seq libraries identified 647 predicted genes that were differentially expressed in Silk Glands of the Western black widow, Latrodectus hesperus. Only ∼5% of these Silk-gland specific transcripts (SSTs) encode spidroins; although the remaining predicted genes presumably encode other proteins associated with Silk production, this is mostly unverified. Here, we used proteomic analysis of multiple Silk Glands and dragline Silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform Silk Glands and detect 17 SST encoded proteins in major ampullate Silk fibers. The observed proteins include known Silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel Silk-associated proteins. Major and minor ampullate Glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major and minor ampullate Silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider Silk gland transcriptomes.
