TC003322
Fly Ortholog:
COP9 signalasome subunit 1b
Student Name:
Casey Horan and Sean Ogurek
School Name:
Miami University
Year of Study:
2018
Gene Information:
The CSN complex is important in regulating E3-cullin RING ubiquitin ligases, which have been shown to have several functions including DNA replication and most important to us, appendage development(1). Though the exact molecular function of our subunit, CSN1b, is not entirely understood, it has been suggested to play a role in enzymatic function, cell cycle control and proliferation, development, cell signaling and protein metabolism. It has also been shown to be important in protecting Capicua, a transcriptional regressor of epidermal growth factor genes from abnormal expression, suggesting that it has a role in tumor suppression (2). CSN1b’s Drosophila ortholog has been shown to be important for proper development of both the legs and antennae.
NCBI Link:
iBeetle Link:
Tribolium Transcript: 5' UTR:
ATTGTATTTGTAGTTCAAATGACTTGTGCAAATAGTCCGAACGCCGGGCCGGGAAATGTCGATGTGAAAGAAGGCAAAAATAAACCACAGACGGAGAAATATTAATA (KD)
Tribolium Transcript: CDS:
ATGCCGCTCCAAATGCAGACCATTGTGGCCCTGAACGCAGTGGAGCCCATGCAAGTGGACGCTCCCCCCGAAGACAACGACAACAATGAGGAGGAGAGCTACATTGTTGAGAATCCCTCCCTTGATCTGGAAGTCTACGCCAACAGCTACTCGGGCTTGGCCAAGCTCTACCGCCTCATTTTCATCATAGACCACTGTCCCTCTCTGCGTCTGGAAGCCCTAAAAATGGCCATTTCGTACGTAATGACCACCTACAACGTGAACTTGTACCAAGTGCTCCACCAGAAGCTGGCCGAAGTGACGACACAAATGAGCGTGCCAGACGTGGCCGCCACTTCCACCTCCCAAGACATCCCAGTTATAGACAACCTCTGGATGGAAACCCGCTCGAAAAAGGCCGCCCTTAAGCTGGAGAAACTCGACAATGACTTGAAGAGCTACAAGTCCAACTCGATCAAGGAGAGCATCAGGCGGGGTCACGACGACCTAGGGGAGCACTATCTAGACTGTGGCGATTTGTCCAACGCTTTGAAATGCTTTTCCCGGGCGAGAGACTACTGCACTAGCGGCAAACACGTGGTCAACATGTGCCTCAACGTTATCAAAGTTTCGGTTTATTTGCAAAACTGGTCGCACGTCCTGAGTTATGTTTCAAAGGCTGAGAGCACTCCCGACTTCACCGAAGCCCAGACGAAAGACTCGAACCAGGTCATTGTGACCAAGCTGAAATGCGCCGCAGGTTTGGCCGAACTTGCTACAAAAAAATACAAATCTGCAGCCAAACACTTCCTGCAAGCCAATCTAGACCACTGTGACTTTCCTGAGCTGCTTTCGCCAAATAATGTCGCTATGTATGGCGGGTTGTGCGCTTTGGCCACGTTTGATCGGCACGAGTTGCAAAAAAACGTGATTTTTAGTAGTTCGTTTAAGTTGTTTCTGGAGTTGGAACCGCAACTGCGCGACATTATTTTCAAATTCTACGAGTCAAAATACGCGTCTTGTTTGAAGTTGCTTGATGAGATTAAGGACAACTTGCTCTTGGACATGTACATCGCTCCGCATATCAATACTTTGTACACACAAATACGAAACCGCGCTTTGATCCAGTATTTCAGTCCATACTTGTCCGCGGATATGCATAAGATGGCCATTGCATTCAATCGAACCGTGCCAGCGCTGGAGGACGAGCTGATGCAGCTGATTCTGGATGGGCAGATCCAGGCCAGGATCGATTCGCATAATAAAATTCTGTTTGCGAAGGACGTTGACCAAAGAAGTACGACGTTTGAGAGGAGTTTGTTGATGGGGAAGGAGTACCAGAGGAGGACTAGGATGTTGATTTTGCGAGCGGCGGTTCTAAAAAGCAAGATACATGTGAAGAGCCCGCAAAGGGACACGTCTGGCCAGAGTGGGGAAGTGACAGTGGCGCCAGGCACTAGTAGCGTTTTGTCGGCGCGAAATTGA
Tribolium Transcript: 3' UTR:
ACTACTTGCTGATAGTTATTTTCTGTGAACTTGTATTAAATTTATTTTCCTTTTCCATGTA (KD)
Tribolium Protein:
MPLQMQTIVALNAVEPMQVDAPPEDNDNNEEESYIVENPSLDLEVYANSYSGLAKLYRLIFIIDHCPSLRLEALKMAISYVMTTYNVNLYQVLHQKLAEVTTQMSVPDVAATSTSQDIPVIDNLWMETRSKKAALKLEKLDNDLKSYKSNSIKESIRRGHDDLGEHYLDCGDLSNALKCFSRARDYCTSGKHVVNMCLNVIKVSVYLQNWSHVLSYVSKAESTPDFTEAQTKDSNQVIVTKLKCAAGLAELATKKYKSAAKHFLQANLDHCDFPELLSPNNVAMYGGLCALATFDRHELQKNVIFSSSFKLFLELEPQLRDIIFKFYESKYASCLKLLDEIKDNLLLDMYIAPHINTLYTQIRNRALIQYFSPYLSADMHKMAIAFNRTVPALEDELMQLILDGQIQARIDSHNKILFAKDVDQRSTTFERSLLMGKEYQRRTRMLILRAAVLKSKIHVKSPQRDTSGQSGEVTVAPGTSSVLSARN
Forward Primer 1:
CTGCGTCTGGAAGCCCTAAA
Reverse Primer 1:
TTCGAGTCTTTCGTCTGGGC
Forward Primer 2:
ACATGTACATCGCTCCGCAT
Reverse Primer 2:
TTGCTTTTTAGAACCGCCGC
Gel Picture:
Cloned Fragment:
CTGCGTCTGGAAGCCCTAAAAATGGCCATTTCGTACGTAATGACCACCTACAACGTGAACTTGTACCAAGTGCTCCACCAGAAGCTGGCCGAAGTGACGACACAAATGAGCGTGCCAGACGTGGCCGCCACTTCCACCTCCCAAGACATCCCAGTTATAGACAACCTCTGGATGGAAACCCGCTCGAAAAAGGCCGCCCTTAAGCTGGAGAAACTCGACAATGACTTGAAGAGCTACAAGTCCAACTCGATCAAGGAGAGCATCAGGCGGGGTCACGACGACCTAGGGGAGCACTATCTAGACTGTGGCGATTTGTCCAACGCTTTGAAATGCTTTTCCCGGGCGAGAGACTACTGCACTAGCGGCAAACACGTGGTCAACATGTGCCTCAACGTTATCAAAGTTTCGGTTTATTTGCAAAACTGGTCGCACGTCCTGAGTTATGTTTCAAAGGCTGAGAGCACTCCCGACTTCACCGAAGCCCAGACGAAAGACTCGAA
Injection concentration:
1ug/ul
Number Injected:
18
Number Survived to Adult:
2
Number w/ RNAi Phenotype:
14
Percent Penetrance:
7%
Assay Description:
N/A
RNAi Phenotype Description:
The RNAi phenotypes observed did not align with the original hypothesis. Unlike Drosophila, leg and antennae morphological development was unaffected when CSN1b was knocked down in Tribolium. Instead, the observed phenotype was abnormal elytra pigmentation. Elytra were stripped, with partially pigmented stripes and partially translucent stripes.
RNAi Phenotype/Assay Images:
Assay Data:
N/A
Discussion:
The observed result was partially translucent, striped elytra. The abnormal pigmentation, however, is most likely an indirect effect of CSN1b knockdown in Tribolium. The newly formulated, working hypothesis is that CSN1b is responsible for proper development and vascularization of the most anterior portion of elytra in Tribolium. In Drosophila, the observed LOF phenotype was a developmental transition of antennae to leg like structures. In this case, the antennae became sclerotized and lost aristae. The results of knockdown in both Tribolium and Drosophila indicated that gene function has diverged. While CSN1b controls proper development of legs and antennae in Drosophila, it is apparent from the study conducted that the Tribolium ortholog is responsible for proper elytra development.
Notebook:
Annotated Genbank File:
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