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わたしの備忘録、生物学とプログラミングが多いかも

膜電位プローブ

研究 神経科学 科学

ジャンル分け

chemical

  • DiO-DPA

    GEVI

  • Ace-mNeon, 2015 link

    • FRET-opsin
    • mouse & fly in vivo

  • Mac-mOrange, Mac-mCitrine 2014 link

    • FRET-opsin
    • slice

  • QuasAr2-palette 2014 link

    • FRET-opsin (eFRET)
    • culture

  • ASAP1 2014 link

    • Ci-VSD/P & cpGFP
    • slice

  • ArcLight 2012, 2013 link link

    • Ci-VSD/P and pHluorin
    • fly in vivo

総説 2011.
仕組みとかに深く触れててとても良い, が, ジャンルの発展が爆速なせいで古い.
eFRET probes (QuasAr2-paletteやAce(?))の登場前だね.
http://www.columbia.edu/cu/biology/faculty/yuste/Publications/PeterkaNeuron2011.pdf

電位感受性ドメイン

  • QuasAr
  • Ace
  • Mac
  • Ci-VSD/P

eFRET

eFRETは

FRETで何を検知するか

Traditionally, FRET is used to measure the physical distance between a donor and acceptor. However, we and others have also used FRET to measure changes in the absorption spectrum of a protein via its influence on nonradiative quenching of an attached fluorescent moiety20,21,22.
従来、FRETはドナー・アクセプター間の距離測定に用いられてきた。
しかし、我々や他のグループは、タンパク質の吸収スペクトラム変化を測定するためにFRETを用いてきた.
これは蛍光ドメインのクエンチングがスペクトラム変化に影響されることを利用している.
※筆者注: moiety - 分子の一部、モチーフ的意味合い Moiety (chemistry) - Wikipedia

Ace

Acetabularia rhodopsin

  • AR1: 246 AA, 27 kDa
  • AR2: 247 AA,

AR

“H+ -pumping rhodopsin from the marine alga Acetabularia”

An opsin-encoding cDNA was cloned from the marine alga Acetabularia acetabulum.
H+ -pumping rhodopsin from the marine alga Acetabularia. - PubMed - NCBI

AR complete CDS, Genbank Acetabularia acetabulum rhodopsin mRNA, complete cds - Nucleotide - NCBI

AR1

We cloned an intronless, nuclear-encoded opsin gene from an EST library of Acetabularia acetabulum. Acetabularia rhodopsin I (ARI) encodes a protein of 246 amino acids with molecular weight of 27 kDa.
We here reported cloning of a novel Acetabulariarhodopsin gene with a correct termination codon
from “Acetabularia rhodopsin I is a light-stimulated proton pump”
Acetabularia rhodopsin I is a light-stimulated proton pump. - PubMed - NCBI

AR2

AR2を見つけてきた論文.

ARII consists of 247 amino acid residues…
with a profile similar to that of ARI (or AR).

The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII.
“Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga”
Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga. - PubMed - NCBI

AR1もAR2も釣ってきたよ
AR1 complete CDS, Genbank Acetabularia acetabulum rhodopsin I (aopI) gene, complete cds - Nucleotide - NCBI
AR2 complete CDS, Genbank Acetabularia acetabulum rhodopsin II (aopII) gene, complete cds - Nucleotide - NCBI

Ace-mNeon

Here, we present fast GEVIs (<1-ms response) that fuse the Acetabularia acetabulum rhodopsin (Ace) (11) …
11. Tsunoda SP, et al. Biophys. J. 2006;91:1471–1479.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4904846/

引用11番. AR初クローニング2006論文

私がアラインメントしたところ、AR2を利用した模様. (アラインメント結果は下記)

簡易年表

  1. . AR(現在のAR1?)の発見と解析. Tsunoda, et. al.. “H+ -pumping rhodopsin from the marine alga Acetabularia”
  2. . AR1(ARと末端が違う)の発見と解析. Lee, et. all.. “Acetabularia rhodopsin I is a light-stimulated proton pump”
  3. . AR2の発見と解析. Wada, et. al.. “Crystal Structure of the Eukaryotic Light-Driven Proton-Pumping Rhodopsin, Acetabularia Rhodopsin II, from Marine Alga”

alignment AR1, AR2 and Ace-mNeon

AR2をAce-mNeonに使ってるみたい.

 CLUSTAL 2.1 Multiple Sequence Alignments


Sequence type explicitly set to DNA
Sequence format is Pearson
Sequence 1: HM070407.1_AR1_aopI__complete_cds    741 bp
Sequence 2: HM070408.1_AR2_aopII__complete_cds   834 bp
Sequence 3: AR_sequence_of_Ace2N-mNeon_GEVI      681 bp
Start of Pairwise alignments
Aligning...

Sequences (1:2) Aligned. Score:  58
Sequences (1:3) Aligned. Score:  56
Sequences (2:3) Aligned. Score:  78
Guide tree file created:   [clustalw.dnd]

There are 2 groups
Start of Multiple Alignment

Aligning...
Group 1: Sequences:   2      Score:10972
Group 2: Sequences:   3      Score:8967
Alignment Score 8584

CLUSTAL-Alignment file created  [clustalw.aln]

clustalw.aln

CLUSTAL 2.1 multiple sequence alignment


HM070408.1_AR2_aopII__complete      ------ATGGCTGACG---TTGAGACCGAAACCGGTATGATTGCCCAATG
AR_sequence_of_Ace2N-mNeon_GEV      ------ATGGCTGACG---TGGAAACCGAGACCGGCATGATTGCACAGTG
HM070407.1_AR1_aopI__complete_      ATGTCAAACCCTAACCCTTTCCAAACCACCTTGGGAACTGATGCTCAATG
                                          *   ** **    *  * ***      ** *    *** ** **

HM070408.1_AR2_aopII__complete      GATTGTGTTCGCTATTATGGCCGCAGCAGCAATAGCGTTCGGTGTTGCTG
AR_sequence_of_Ace2N-mNeon_GEV      GATTGTCTTTGCTATTATGGCTGCTGCTGCTATTGCTTTTGGAGTGGCTG
HM070407.1_AR1_aopI__complete_      GGTAGTTTTCGCCGTCATGGCTTTGGCTGCCATTGTTTTTTCCATCGCTG
                                    * * ** ** **  * *****    ** ** ** *  **     * ****

HM070408.1_AR2_aopII__complete      TGCATTTCCGTCCCTCTGAACTTAAGAGTGCTTACTACATCAACATCGCA
AR_sequence_of_Ace2N-mNeon_GEV      TGCACTTTCGGCCTTCAGAGCTGAAGAGCGCATACTATATCAACATTGCC
HM070407.1_AR1_aopI__complete_      TTTAATTCCGTCCCCTTCCTCTAAGATTGACTTACTACGTCAACATAGCC
                                    *  * ** ** **       ** *      * *****  ******* ** 

HM070408.1_AR2_aopII__complete      ATCTGTACCATTGCTGCTACCGCATACTATGCTATGGCCGTTAACTACCA
AR_sequence_of_Ace2N-mNeon_GEV      ATCTGCACTATCGCCGCTACCGCTTACTATGCAATGGCCGTGAACTACCA
HM070407.1_AR1_aopI__complete_      ATCTGCACTATTGCAGCGACCGCTTATTATGCTATGGCTGTTAACGGAGG
                                    ***** ** ** ** ** ***** ** ***** ***** ** ***     

HM070408.1_AR2_aopII__complete      AGAT------CTTACCATGAAT---------GGGGAAAGATAGGTTGTGT
AR_sequence_of_Ace2N-mNeon_GEV      GGAC------CTGACAATGAAT---------GGTGAAAGGCAGGTGGTCT
HM070407.1_AR1_aopI__complete_      GGACAACAAGCCCACCGCCGGTACAGGAGCGGATGAGCGTCAAGTTATAT
                                     **       *  **      *         *  **  *  * **  * *

HM070408.1_AR2_aopII__complete      ATGCCCGATATATTGATTGGGTTTTGACCACTCCCTTGTTATTGTTGGAC
AR_sequence_of_Ace2N-mNeon_GEV      ACGCAAGATATATTAACTGGGTGCTGACCACACCACTGCTCCTGCTCGAT
HM070407.1_AR1_aopI__complete_      ACGCCAGATACATTGATTGGGTTTTCACCACACCCTTGCTGTTATTAGAC
                                    * **  **** *** * *****  * ***** **  ** *  *  * ** 

HM070408.1_AR2_aopII__complete      TTGATCGTAATGACCAAGATGGGCGGAGTGATGATCTCTTGGGTCATCGG
AR_sequence_of_Ace2N-mNeon_GEV      CTCATCGTCATGACCAAGATGGGCGGAGTGATGATTTCTTGGGTCATCGG
HM070407.1_AR1_aopI__complete_      CTTGTATTGCTAACCAACATGCCTGCCACCATGATCGCCTGGATCATGGG
                                     *  *  *  * ***** ***   *     *****  * *** **** **

HM070408.1_AR2_aopII__complete      TGCTGATATTTTCATGATTGTCTTCGGTATTTTGGGAGCTTTCGAAGACG
AR_sequence_of_Ace2N-mNeon_GEV      CGCAGACATTTTCATGATCGTGTTTGGTATTCTGGGCGCCTTCGAGGATG
HM070407.1_AR1_aopI__complete_      AGCAGACATTGCCATGATTGCATTCGGTATCATCGGCGCATTCACCGTGG
                                     ** ** ***  ****** *  ** *****  * ** ** ***   *  *

HM070408.1_AR2_aopII__complete      AACACAAGTTTAAATGGGTGTACTTTATTGCCGGCTGTGTCATGCAAGCT
AR_sequence_of_Ace2N-mNeon_GEV      AACACAAGTTCAAATGGGTGTACTTTATCGCTGGATGTGTGATGCAGGCA
HM070407.1_AR1_aopI__complete_      GAAGCTA---CAAGTGGTTCTACTTTGTTGTCGGCTGCATCATGCTTGCC
                                     *  * *    ** *** * ****** * *  ** **  * ****  ** 

HM070408.1_AR2_aopII__complete      GTCCTAACCTACGGTATGTACAATGCTACTTGGAAAGATGACTTGAAAAA
AR_sequence_of_Ace2N-mNeon_GEV      GTCCTGACATACGGGATGTATAACGCCACTTGGAAAGACGATCTGAAGAA
HM070407.1_AR1_aopI__complete_      GTGCTAGCCTGGGGAATGATCAACCCAATCTTCAAGGAAGAACTTCAAAA
                                    ** **  * *  ** ***   **  * *  *  ** ** **  *  * **

HM070408.1_AR2_aopII__complete      GAGCCCCGAGTACCATAGCAGCTATGTCTCATTGTTGGTGTTCCTATCCA
AR_sequence_of_Ace2N-mNeon_GEV      AAGCCCCGAGTACCATAGCTCCTATGTCAGTCTGCTCGTCTTCCTGTCAA
HM070407.1_AR1_aopI__complete_      GCACAAGGAATACACCGGAGCTTACACCACCCTTTTGATTTACTTGATCG
                                       *   ** ***    *    **   *    *  *  * * * *     

HM070408.1_AR2_aopII__complete      TTTTGTGGGTTTTCTACCCCGTTGTCTGGGCTTTCGGAAGCGGCTCCGGA
AR_sequence_of_Ace2N-mNeon_GEV      TCCTCTGGGTGTTTTATCCTGTCGTGTGGGCTTTCGGGTCTGGTAGTGGC
HM070407.1_AR1_aopI__complete_      TCTTGTGGGTTATCTACCCCATCGTTTGGGGATTGGGGGCTGGCGGTCAC
                                    *  * *****  * ** **  * ** ****  ** **    **       

HM070408.1_AR2_aopII__complete      GTTTTGTCTGTCGATAATGAAGCCATCTTGATGGGAATTTTGGATGTTCT
AR_sequence_of_Ace2N-mNeon_GEV      GTGCTGTCCGTCGACAATGAGGCCATTCTCATGGGAATCCTGGATGTGCT
HM070407.1_AR1_aopI__complete_      ATCATAGGGGTGGATGTTGAGATCATCGCGATGGGAATCTTGGACTTGTT
                                     *  *    ** **   ***   ***    ********  ****  *  *

HM070408.1_AR2_aopII__complete      TGCAAAGCCTTTGTTTGGTATGGGATGTTTAATTGCCCACGAGACTATCT
AR_sequence_of_Ace2N-mNeon_GEV      CGCTAAGCCACTGTTTGGAATGGGGTGCCTCATTGCCCATGAGACTATCT
HM070407.1_AR1_aopI__complete_      GGCTAAGCCTCTATATGCCATCGGTGTGTTGATTACTGTGGAGGTTGTTT
                                     ** *****  * * **  ** **     * *** *    ***  * * *

HM070408.1_AR2_aopII__complete      TCAAGAAGATGGAGAGCAAGGAAACTTTGATTGAGAAGGTTGTTGCAACT
AR_sequence_of_Ace2N-mNeon_GEV      TCAAG---------------------------------------------
HM070407.1_AR1_aopI__complete_      ACGGAAAGTTGGGTAAGGAGGAAGCTTAGCCTTTGACTGCTTGA------
                                     *                                                

HM070408.1_AR2_aopII__complete      GCTCAAGCTATTGCTTGATTAAGACTTTGATTGAGAGTACATATTTCCAT
AR_sequence_of_Ace2N-mNeon_GEV      --------------------------------------------------
HM070407.1_AR1_aopI__complete_      --------------------------------------------------
                                                                                      

HM070408.1_AR2_aopII__complete      ATTATTTGTTTTTGTGTGTTCAGTTTATGACTTTCCTTCATTATTATTTT
AR_sequence_of_Ace2N-mNeon_GEV      --------------------------------------------------
HM070407.1_AR1_aopI__complete_      --------------------------------------------------
                                                                                      

HM070408.1_AR2_aopII__complete      GCATATAA
AR_sequence_of_Ace2N-mNeon_GEV      --------
HM070407.1_AR1_aopI__complete_      --------
                                            
clustalw.dnd

(
HM070407.1_AR1_aopI__complete_cds:0.32151,
HM070408.1_AR2_aopII__complete_cds:0.09684,
AR_sequence_of_Ace2N-mNeon_GEVI:0.11461);