DNA Sequencing of the Mutated Ubiquitin Encoding Gene
Department of Science, Biological Science Selection, Kumamoto University
MICHITO NAKASHIMAof 70 % ethanol was added to the precipitation and theIntroduction
1Ubiquitin is a protein composed of 76 amino acids .

sample was centrifuged at 4 ºC, 14000 rpm, 2 min. This

This protein is involved in various vital phenomenons

washing operation was performed 2 times. The

2(e.g. protein decomposition, and DNA repair ) . The

precipitation removed the supernatant was dried up andPlasmid D is a plasmid that has cDNA encoding the30 μl of SLS solution was added. The sample was putted

ubiquttin protein. The plasmid appears to contain aon ice 30 min.

mutation, but the site of the mutation is unclear. In order(5) DNA sequencing was carried out using Genetic

to identify the mutation, I investigate the DNAAnalyzer.

sequences by dideoxy chain termination method .
Results
Materials & Methods

DNA sequencing was determined and found a(1) DNA concentration was determined by visualizing

nucleotide sequence of the mutated ubiquitin encoded inethidium bromide stained agarose gel electrophoresis.

the plasmid D. By comparing sequences of amino acids(2) 1.5 μl of buffer, 2 μl of primer DNA M4 and 2 μl ofof Wild type ubiquitin to that of Plasmid D, I found thatsequence mix contains fluoresceinated ddNTP, dNTP

C-terminal amino acid Glycine is mutated to Threonine.

and DNA polymerase were mixed into 14.5 μl of(Fig. 1)

prepared DNA solution contains 0.2 μg of plasmidDNA.

Discussion(3) PCR reaction was performed using Thermal Cycler.

C-terminal of ubiquitin participates in some reactions

The reaction was progressed under the condition as(e.g. connecting with ubiquitin-activating enzyme,follows; 20 sec of denature at 96 ºC, 20 sec of annealing

forming of poly-ubiquitin, binding to target protein) 3 .

at 50 ºC and 3 min of extension at 60 ºC. The reaction

The mutation of C-terminal might influence these

was repeated 25 cycles.

reactions.

(4) 5 μl of STOP solution containing NaOAC, Glycogenand EDTA and 60 μl of 99 % ethanol were added to theReferences

sample and the DNA fragments were precipitated by1.Ciechanover A.Cell Death Differ. 2005 Sep;12(9):1167-77.

15 min centrifugation at 4 ºC, 14000 rpm. To wash the2.Hershko A.Cell Death Differ. 2005 Sep;12(9):1158-61.

precipitation, after the supernatant was removed, 200 μl

3.Rose I.Cell Death Differ. 2005 Sep;12(9):1162-6.

Wild type ubiquitin : MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGG
Mutated ubiquitin : ***************************************************************************T
Fig. 1 Comparison of sequence of amino acids between the wild type ubiquitin and the ubiquitin protein encoded bythe plasmid D. Top lane is sequence of amino acids of the wild type ubiquitin. Bottom lane is that of the mutatedubiquitin. The “*” marks the idential amino acids. The pink box indicate the site of mutation.

Sequencing of Arabidopsis thaliana ubiquitin encording gene
Shota Matsuyama
Faculty of Science, Program for Biological Environment, Kumamoto University, Japan
Introduction

carboxyl-terminal glycine residue. This residue attach

Ubiquitin is a protein which made of 76 amino acids. A

cysteine residue of E1 and ubiquitin is transferred fromplasmid has genes encoded ubiquitin. It is suggested that

E1 to E2 (2). Then ubiquitin binds lysine residue ofthe plasmid contains a mutation, but I don’t know thetarget protein by E3 and the function of protein ismutated region. To clear the mutation, I decide tocontrolled (3). In this experiment, glycine residue

determine the nucleotide sequence and estimate thechanged isoleucine, so I think that ubiquitination can not

mutated amino acids.

occur.
A) …GGTGGTTGAB) …RGG

1) A plasmid sample called as “R” was given by Mr.

…GGTATCTGA…RGIObata. DNA concentration was determined by ethidium…***…**Materials and Methods***bromide stained gel.
2) 0.2g of the sample was mixed with 12l H2O, 2lFig.1

primer DNA, 1.5l Buffer, 2l sequence mix and theThe nucleic acid sequence and amino acid sequence

mixture was incubated as follows using thermal cyclerof Arabidopsis thaliana ubiquitin encording gene(Applied Biosystems):96℃for 20 seconds denature,50℃A) The nucleic acid sequence of wild type ubiquitinfor 20 seconds annealing,60℃ for 3 minutes extension.

(upper line) compare to the sequence of mutation

This cycle repeated 25 times.

ubiquitin (lower line). * means the identical nucleotide3) 99% ethanol was added to the reaction and DNA wassequence.

precipitated by centrifugation at 14000 rpm for 15B) The amino acid sequence of wild type ubiquitin

minutes. The precipitate was washed twice with 70%(upper line) compare to the sequence of mutation

ethanol and dissolved in SLS.

ubiquitin (lower line). * means the identical amino acid4) DNA sequence was carried out using dye-terminator.

residues.

Results

References

The analysis gave me DNA sequence and amino acids(1) The ubiquitin-proteasome system regulates plant hormone signaling.

sequence of ubiquitin(Fig.1). I carried out a homology
search. The last amino acid of wild type is glycine(G),Santner A, Estelle M.
Plant J. 2010 Mar;61(6):1029-40.

but glycine changed isoleucine(I) in sample.

(2) Ubiquitin Enzymes, Ubiquitin and Proteasome Activity in Blood

Discussion

Mononuclear Cells of MCI, Alzheimer and Parkinson Patients.

Ubiquitin is used modification of other protein andUllrich C, Mlekusch R, Kuschnig A, Marksteiner J, Humpel C.

concerns several vital phenomenon. All of eucaryotes

Curr Alzheimer Res. 2010 May 11. [Epub ahead of print]

have almost same amino acids sequence, but eubacteria
don’t have them. Ubiquitin conjugation needs three(3) The E3 ubiquitin ligase RNF5 targets virus-induced signaling adaptor forenzymes, the ubiquitin-activating enzyme E1, theubiquitination and degradation.

ubiquitin-conjugating

Zhong B, Zhang Y, Tan B, Liu TT, Wang YY, Shu HB.

enzymeE2andthe

ubiquitin-protein ligase E3 (1). Ubiquitin has theJ Immunol. 2010 Jun 1;184(11):6249-55. Epub 2010 May 5.