Synthetic Gene DataBase
 

Synthetic Gene 96


 
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Field NameNatural GeneSynthetic Gene
SGDB Gene ID8396
GenBank AccessionNC_001522
GenBank GI9627056
Gene NameBPV-1 L2BPV-1 HBL2
Gene Length (bp)14101410
Speciesbovine papillomavirus type 1 (synthetic)Aethiops sabeus (African Green Monkey)
StrainsCos-1: kidney fibroblast cells
CDSatgagtgcacgaaaaagagtaaaacgtgccagtgcctatgacctgtacaggacctgcaag
caagcgggcacatgtccaccagatgtgatacgaaaggtagaaggagatactatagcagat
aaaattttgaaatttgggggtcttgcaatctacttaggagggctaggaataggaacatgg
tctactggaagggtggccgcaggtggatcaccaaggtacacaccactccgaacagcaggg
tccacatcatcgcttgcatcaataggatccagagctgtaacagcagggacccgccccagt
ataggtgcgggcattcctttagacacccttgaaactcttggggccttgcgtccaggggtg
tatgaggacactgtgctaccagaggcccctgcaatagtcactcctgatgctgttcctgca
gattcagggcttgatgccctgtccataggtacagactcgtccacggagaccctcattact
ctgctagagcctgagggtcccgaggacatagcggttcttgagctgcaacccctggaccgt
ccaacttggcaagtaagcaatgctgttcatcagtcctctgcataccacgcccctctgcag
ctgcaatcgtccattgcagaaacatctggtttagaaaatatttttgtaggaggctcgggt
ttaggggatacaggaggagaaaacattgaactgacatacttcgggtccccacgaacaagc
acgccccgcagtattgcctctaaatcacgtggcattttaaactggttcagtaaacggtac
tacacacaggtgcccacggaagatcctgaagtgttttcatcccaaacatttgcaaaccca
ctgtatgaagcagaaccagctgtgcttaagggacctagtggacgtgttggactcagtcag
gtttataaacctgatacacttacaacacgtagcgggacagaggtgggaccacagctacat
gtcaggtactcattgagtactatacatgaagatgtagaagcaatcccctacacagttgat
gaaaatacacagggacttgcattcgtacccttgcatgaagagcaagcaggttttgaggag
atagaattagatgattttagtgagacacatagactgctacctcagaacacctcttctaca
cctgttggtagtggtgtacgaagaagcctcattccaactcgagaatttagtgcaacacgg
cctacaggtgttgtaacctatggctcacctgacacttactctgctagcccagttactgac
cctgattctacctctcctagtctagttatcgatgacactactactacaccaatcattata
attgatgggcacacagttgatttgtacagcagtaactacaccttgcatccctccttgttg
aggaaacgaaaaaaacggaaacatgcctaa
atgagcgcccgcaagagagtgaagcgcgccagcgcctacgacctgtacaggacctgcaag
caggccggcacatgtccaccagatgtgatccgaaaggtggagggcgacaccatcgccgac
aagatcctgaagttcggcggcctggccatctacctgggcggcctgggcatcggaacatgg
tctactggaagggtggcggccggcggctcaccaaggtacaccccactgcgcaccgccggc
tccacctcctccctggcctccatcggatccagagccgtgaccgccgggacccgcccctcc
atcggcgcgggcatccctctggacaccctggaaactcttggggccctgcgccctggcgtg
tacgaggacaccgtgctgcccgaagcccctgccatcgtgacccctgacgccgtgcctgca
gactccggcctggacgccctgtccatcggcacagactcctccaccgagaccctgatcacc
ctgctggagcctgagggccccgaagacatagccgtgctggaactccagcccctggaccgc
ccaacctggcaggtgagcaatgctgtgcaccagtcctctgcctaccacgcccctctccag
ctgcaatcctccatcgccgagacatctggtttagaaaatatttttgtaggaggctcgggt
ttaggggataccggcggcgagaacatcgagctgacctacttcggctccccccgcaccagc
acgccccgcagtattgcctctaaatcacgtggcattttaaactggttcagcaagcggtac
tacacccaggtgcccaccgaagatcccgaagtgttctcctcccagaccttcgccaacccc
ctgtacgaggccgagcccgccgtgctgaagggccctagcggccgcgtgggcctgtcccag
gtgtacaagcctgataccctgaccacacgtagcggcacagaggtgggcccccagctgcat
gtcaggtactccctgtccaccatccatgaggatgtggaggctatcccctacaccgtggat
gagaacacccagggcctggccttcgtgcccctgcatgaggagcaggccggcttcgaggag
atcgagctcgacgatttcagcgagacccatcgcctgctgccccagaacacctcctccacc
cccgtgggcagcggcgtgcgcagaagcctgatccctacccgagagttcagcgccacccgg
cctaccggcgtggtgacctacggctcccccgacacctactccgctagccccgtgaccgac
cctgattctacctctcctagcctggtgatcgacgacaccaccaccacccccatcatcatc
atcgacggccacacagtggatctgtacagcaggaactacaccctgcatccctccctgctg
aggaagcgcaagaagcgcaagcatgcctaa
5' End
3' End
NotesRecoded nucleotide sequence edited manually. Expect human error. See original paper.
Expression VectorpCDNA3pCDNA3
Assay MethodsImmunofluorescence, Western Blot, Northern BlotImmunofluorescence, Western Blot, Northern Blot
ResultsProtein level undetectable by immunofluoresence and western blot. mRNA level normal (relative to GAPDH internal standard).Protein level much higher in immunofluoresence and western blot assays compared to wt. Densitometric analysis indicates at least 1000x increase in protein level. mRNA level same as wt.
Protein Function
Recoding PurposeTo improve expression
Synthesized ByAuthors
Recoding MethodALL codons with BPV:Mammalian usage ratios >2 were replaced with more suitable codons and MOST
codons with ratios >1.5 were replaced. (303/1410 bps substituted)
Publication Author(s)Zhou, J.; Liu, W. J.; Peng, S. W.; Sun, X. Y.; Frazer, I.
Corresponding AuthorIan Frazer
Corresponding AddressCentre for Immunology & Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland 4102, Australia.
Publication Year1999
Publication TitlePapillomavirus capsid protein expression level depends on the match between codon usage and tRNA availability
AbstractTranslation of mRNA encoding the L1 and L2 capsid proteins of papillomavirus (PV) is restricted in vivo to differentiated epithelial cells, although transcription of the L1 and L2 late genes occurs more widely. The codon composition of PV late genes is quite different from that of most mammalian genes. To test the possibility that PV late gene codon composition determines the efficiency of PV late gene expression in some cell types, synthetic bovine papillomavirus type 1 (BPV1) late genes were constructed with codon composition modified to resemble the typical mammalian gene. Expression of these genes from a strong promoter in Cos-1 cells was compared with expression of wild-type BPV1 late genes from the same promoter. Both unmodified and modified PV late genes were transcribed in Cos-1 cells, but only the codon-modified genes were translated. In vitro translation of wild-type but not synthetic BPV1 L1 mRNA was markedly enhanced by addition of aminoacyl-tRNAs. Codon composition thus limits BPV1 late gene translation in Cos-1 cells, and this limitation can be overcome by modification of the codon composition of the genes or by provision of excess tRNA. Replacement of codons in the green fluorescent protein (gfp) gene with those frequently used in PV late genes did not alter gfp transcription in Cos-1 cells but almost abolished translation, supporting the hypothesis that the observed differences in efficiency of translation of modified and unmodified PV capsid genes were related to codon usage rather than mRNA structure. As tRNA populations vary within and between tissues in the same eukaryotic organism, we speculate that matching of tRNA availability to codon usage may be one determinant of the restriction of expression of PV late genes to differentiated epithelium.
JournalJ Virol. 73(6): 4972-82.
SummaryAuthors worked with synthetic bovine papillomavirus type 1 (BPV-1). The L1 and L2 capsid proteins are only translated in differentiated epithelial cells. They hypothesized that codon composition of the host cell was one factor that determines expression efficiency. All codons with BPV:Mammalian usage ratios greater than 2.0 were replaced with more suitable codons and most codons with ratios greater than 1.5 were replaced (See Table 1 in paper for details). The authors modified the codon composition to resemble a typical mammalian gene. The genes were then expressed in Cos-1 cells the authors found that the wild-type gene was not translated but the recoded gene was. The increase in expression was at least 1000x for both the L1 and L2 capsid proteins. The authors also found that by adding aminoacyl-tRNAs to a mammalian in vitro environment (rabbit reticulocyte lysate), they could enhance the translation of wild-type L1 mRNA. The recoded gene did not have higher translation efficiency as codon composition was not a (severely) limiting factor. Conversely, recoding a humanized (synthetic) GFP gene to represent the viral genome significantly inhibited its expression in Cos-1 cells, further supporting that codon composition can limit expression. In general, transcription of heterologous genes was neither hindered by an alien environment nor enhanced by recoding, indicating that increased protein expression is a result of change in translation efficiency and not change in mRNA levels.
CommentsExcellent paper with multiple related experiments and a lot of material to consider.
Discussion
PubMed ID10233959
Submitter NameZheng, Yuanpu
Submitter AddressUMBC
Entry ConfirmationNo
 
 

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