Sequencing of FAT c2 interacting protein ( ip 45) coding gene.
Department of Science, Kumamoto University073- s4106
Yuuya Uehara

Abstract
DNA sequencing is based on dideoxy method which was originals developed by Dr. Frederic
Sanger in 1977, and still improving. In modern biology, DNA sequencing is a fundamental
method to analyze genetic information of variety biological systems.
In this experiment, we analyzed a plasmid DNA by using fluoresceinated ddNTP and
automated sequencer, and the cDNA involved in the plasmid was identified by homology searchusing internet.

Materials and Methods
The concentration of plasmid, named as pEGFP-007 (for nip), was determined by UV260nm
absorbance using Eppendorf spectrophotometer (Biophotometer).
To analyze the DNA sequence, 16µg of DNA solution (mixture of 0.21µl of plasmid with
13.9µl of H O) was mixed with 2µl of sequence mix containing fluoresceinated ddNTP and 1µlof sequencing primer (primer sequence is “ 5’CGAGAAGCGCGATCACAT3’ “) and 1µg of
buffer. The mixture was incubated as follows using thermal cycler : 20 seconds of denature at
96℃, 20 seconds of annealing at 50℃ and 3 minutes of extension at 60℃ (these 3 steps were
performed 40 cycles) .
60µl of 99% ethanol and 5µg of stop solution (containing NaOAc, EDTA and glycogen) was
added to the reaction and the DNA fragments were precipitated by centrifugation at 14000 rpm
and 4℃ for 2 minutes using centrifuge. The precipitate was rinsed twice with 70% ethanol.
After 15 minutes of desiccation, the precipitate dissolved in Sample Loading Solution (SLS).
DNA sequencing was carried out using Beckman sequencer.

Results
The result of DNA sequencing and amino acid sequence which is translated based on the
DNA sequence is shown in Figure 1.
From a result of homology search using Blast program, this plasmid DNA was identified as
NFAT c2 interacting protein (Nip45). cRNA consists of 3388 nucleotides and cording region

consists of 1239 nucleotides (from 23 to 1261 nucleotides) . So it is deduced that Nip 45 protein
consists of 413 amino acids.

Discussion
First, I want to discuss about content of stop solution which contains NaOAc, EDTA andglycogen. NaOAc contains sodium acetate, and plays in a role to form salt precipitate(DNA - Na ). EDTA is a chelating agent, and it can keep enzyme inactivate state (Mg isessential for activation of DNA polymerase). Glycogen is coprecipitated with DNA molecules.
Second, I want to discuss about Nip45. Definition of Nip45 is “Mus musculus nuclear factor of
activated T-cells, cytoplasmic, calcineurin-dependent 2 interacting protein”. The function of
Nip45 is protein binding: interacting selectively with any protein or protein complex (a complex
of two or more proteins that may include other non protein molecules). The process which
Nip45 takes part in is cytokine production, protein modification and regulation of DNA
dependent transcription. For example in T lymphocytes, covalent modification of Nip45 byarginine methylation is important mechanism of regulating the expression of nuclear factor of
activated T cells (NFAT)-dependent cytokine genes. Nip45 substantially augments interleukin(IL)-4 gene transcription, whereas repression of IL-4 gene transcription is mediated throughtumor necrosis factor receptor-associated factor (TRAF) family of proteins interaction with
Nip45. TRAF is generally known to function as adapter proteins that transducer signals from thetumor necrosis factor receptor superfamily.

Acknowledgments
We are grateful to Teaching Assistants for doing this experiment. This experiment was
supported by laboratory of Dr. Saito.

References
1. Novatchkova M, Bachmair A, Eisenhaber B and Eisenhaber F. Proteins with two SUMO-likedomains in chromatin-associated complexes: the RENi (Rad60-Esc2-NIP45) family.BMC
Bioinformatics. 2005; 6: 22
2. Mowen KA, Schurter BT, Fathman JW, David M and Glimcher LH (2004) Arginine
methylation of NIP45 modulates cytokine gene expression in effector T lymphocytes. Mol.
Cell. 2004; 15(4): 559-571
3. Lieberson R, Mowen KA, McBride KD, Leautaud V, Zhang X, Suh WK, Wu L and Glimcher
LH. Tumor necrosis factor receptor-associated factor (TRAF)2 represses the T helper cell
type 2 response through interaction with NFAT-interacting protein (NIP45). J Exp Med. 2001;
194(1):89-98

1 ......................ATGGCGGAACCACTGAGGGGACGTGGTCCGAGGTCCCG
1.......................-M--A--E--P--L--R--G--R--G--P--R--S--R
39 CGGTGGCCGAGGCGCTCGGAGAGCCCGAGGCGCCCGTGGCCGGTGTCCTCGCGCCCGGCA
13 --G--G--R--G--A--R--R--A--R--G--A--R--G--R--C--P--R--A--R--Q
99 GTCTCCGGCTAGGCTCATTCCAGACACCGTGCTTGTGGACTTGGTCAGTGACAGCGACGA
33 --S--P--A--R--L--I--P--D--T--V--L--V--D--L--V--S--D--S--D--E
159 AGAGGTCTTGGAAGTCGCAGACCCAGTAGAGGTGCCGGTCGCCCGCCTCCCCGCGCCGGC
53 --E--V--L--E--V--A--D--P--V--E--V--P--V--A--R--L--P--A--P--A
219 TAAACCTGAGCAGGACAGCGACAGTGACAGTGAAGGGGCGGCCGAGGGGCCTGCGGGAGC
73 --K--P--E--Q--D--S--D--S--D--S--E--G--A--A--E--G--P--A--G--A
279 CCCGCGTACATTGGTGCGACGGCGGCGGCGGCGGCTGCTGGATCCCGGAGAGGCGCCGGT
93 --P--R--T--L--V--R--R--R--R--R--R--L--L--D--P--G--E--A--P--V
339 GGTCCCAGTGTACTCCGGGAAGGTACAGAGCAGCCTCAACCTCATTCCAGATAATTCATC
113 --V--P--V--Y--S--G--K--V--Q--S--S--L--N--L--I--P--D--N--S--S
399 CCTCTTGAAACTGTGCCCTTCAGAGCCTGAAGATGAGGCAGATCTGACAAATTCTGGCAG
133 --L--L--K--L--C--P--S--E--P--E--D--E--A--D--L--T--N--S--G--S
459 TTCTCCCTCTGAGGATGATGCCCTGCCTTCAGGTTCTCCCTGGAGAAAGAAGCTCAGAAA
153 --S--P--S--E--D--D--A--L--P--S--G--S--P--W--R--K--K--L--R--K
519 GAAGTGTGAGAAAGAAGAAAAGAAAATGGAAGAGTTTCCGGACCAGGACATCTCTCCTTT
173 --K--C--E--K--E--E--K--K--M--E--E--F--P--D--Q--D--I--S--P--L
579 GCCCCAACCTTCGTCAAGGAACAAAAGCAGAAAGCATACGGAGGCGCTCCAGAAGCTAAG
193 --P--Q--P--S--S--R--N--K--S--R--K--H--T--E--A--L--Q--K--L--R
639 GGAAGTGAACAAGCGTCTCCAAGATCTCCGCTCCTGCCTGAGCCCCAAGCAGCACCAGAG
213 --E--V--N--K--R--L--Q--D--L--R--S--C--L--S--P--K--Q--H--Q--S
699 TCCAGCCCTTCAGAGCACAGATGATGAGGTGGTCCTAGTGGAAGGGCCTGTCTTGCCACA
233 --P--A--L--Q--S--T--D--D--E--V--V--L--V--E--G--P--V--L--P--Q
759 GAGCTCTCGACTCTTTACACTCAAGATCCGGTGCCGGGCTGACCTAGTGAGACTGCCTGT
253 --S--S--R--L--F--T--L--K--I--R--C--R--A--D--L--V--R--L--P--V
819 CAGGATGTCGGAGCCCCTTCAGAATGTGGTGGATCACATGGCCAATCATCTTGGGGTGTC
273 --R--M--S--E--P--L--Q--N--V--V--D--H--M--A--N--H--L--G--V--S
879 TCCAAACAGGATTCTTTTGCTTTTTGGAGAGAGTGAACTGTCTCCTACTGCCACCCCTAG
293 --P--N--R--I--L--L--L--F--G--E--S--E--L--S--P--T--A--T--P--S
939 TACCCTAAAGCTTGGAGTGGCTGACATCATTGATTGTGTGGTGCTAGCAAGCTCTTCAGA
313 --T--L--K--L--G--V--A--D--I--I--D--C--V--V--L--A--S--S--S--E
999 GGCCACAGAGACATCCCAGGAGCTCCGGCTCCGGGTGCAGGGGAAGGAGAAACACCAGAT
333 --A--T--E--T--S--Q--E--L--R--L--R--V--Q--G--K--E--K--H--Q--M
1059 GTTGGAGATCTCACTGTCTCCTGATTCTCCTCTTAAGGTTCTCATGTCACACTATGAGGA
353 --L--E--I--S--L--S--P--D--S--P--L--K--V--L--M--S--H--Y--E--E
1119 AGCCATGGGACTCTCTGGACACAAGCTCTCCTTCTTCTTTGATGGGACAAAGCTTTCAGG
373 --A--M--G--L--S--G--H--K--L--S--F--F--F--D--G--T--K--L--S--G
1179 CAAGGAGCTGCCAGCTGATCTGGGCCTGGAATCCGGAGATCTCATCGAAGTCTGGGGCTG
393 --K--E--L--P--A--D--L--G--L--E--S--G--D--L--I--E--V--W--G--*1239 A...........................................................
-...........................................................

Figure1. Coding region sequence and amino acid sequence of ip45. Aminoacids are showed one-letter abbreviations. Bases are shaded blue, and amino acids
are shaded red. mRNA sequence (3388 base pair) isn’t showed here.

Identification of Small Ubiquitin-related Modifier 1 (SUMO1) in theplasmid named as pEGFP-hs
Kazuyo Hamada
Department of Biological Science, Faculty of Science, Kumamoto University
Discussion

Material & Methods
The concentration of plasmid named as

Small Ubiquitin-related Modifer (SUMO)pEGFP-hs, was determined by UV260nm

is a typical ubiquitin-like protein. SUMO

absorbance

protein looks like the Ubiquitin well. In the

using

the

spectrophotometer

mammal, it is reported that four isoforms(Eppendorf).
To analyze the DNA sequence, 0.35µg ofexist, SUMO-1, SUMO-2, SUMO-3, andplasmid DNA was mixed with 4µl of the

SUMO-4, and form a protein structure

sequence mix containing ddNTP, dNTP,

similar to the ubiquitin very well. On the

DNA polymerase, and 2µl of sequencing

other hand, SUMO-1 shows the sequence

primer(5’-CGAGAAGCGCGATCACAT-3’).

similarity with the ubiquitin only by 18%,

The mixture was incubated as follows using

and the character on the surface of the protein

thermalis greatly different.

cycler(Applied

Biosystems):

denature (96˚C for 20 seconds), annealing

Ubiquitin is consisted of 76 amino acids(50˚C for 20 seconds), and extension (60˚C

and used to modify other proteins. The

for 3 minutes).

enzyme that takes part in making to the

99% ethanol was added to the reaction and

SUMO

modification

processisalso

the DNA fragments were precipitated byanalogous to that of the ubiquitin. While thecentrifugation at 14000rpm for 2minutes

ubiquitin becomes the tag of the degradation

using centrifugal analysis. The precipitateof proteins, such a function has not been

was rinsed twice with 70% ethanol andrepeated in SUMO. In the degradation

dissolved in SLS. DNA sequence was carriedprocess,out using Dye-terminator.

antagonist of ubiquitin.

SUMO

may

function

asan

SUMO is consisted of about 100 aminoacids. The length and weight are different
Results

according to the kind of the proteins and the

The analysis gave the following DNA

originating in organisms. For example,

sequence, which consisted of about 680 basehuman of SUMO-1 is composed of 101

pairs (Fig.1). We carried out a homology

amino acids. SUMO has many functions, andsearch, and this sequence was identified asplays a role in a number of cellular processes

SUMO1.

such as nuclear transport, DNA replication
and repair, mitosis and signal transduction.

SUMO is added to the target protein, lysine,

361 GGAGGAAGAAGATGTGATTGAAGTTTATCAGGAACAAACGGGGGGTCATTCAACAGTTTA

though the covalent bound by a chain of

246 GGAGGAAGAAGATGTGATTGAAGTTTATCAGGAACAAACGGGGGGTCATTCAACAGTTTA

enzyme reactions that look like making to82 --E--E--E--D--V--I--E--V--Y--Q--E--Q--T--G--G--H--S--T--V--*ubiquitin well. In other worlds, SUMO is421 GATATTCTTTTTATTTTTTTTCTTTTCCCTCAATCCTTTTTTATTTTTAAAAATAGTTCT

one of the modification factors after the

306 Gtranslation of the protein.

481 TTTGTAATGTGGTGTTCAAAACGGAATTGAAAACTGGCACCCCATCTCTTTGAAACATCT541 GGTAATTTGAATTCTAGTGCTCATTATTCATTATTGTTTGTTTTCATTGTGCTGATTTTT

1 GCAGCCGCGGTGTTGTGCTGTGGGGAAGGGAGAAGGATTTGTAAACCCCGGAGCGAGGTT

601 GGTGATCAAGCCTCAGTCCCCTTCATATTACCCTCTCCTTTTTAAAAATTACGTGTGCAC............................................................

661 AGAGAGGTCACCTTTTTCAGGACATTGCATTTTCAGGCTTGTGGTGATAAATAAGATCGA............................................................

721 CCAATGCAAGTGTTCATAATGACTTTCCAATTGGCCCTGATGTTCTAGCATGTGATTACT

61 CTGCTTACCCGAGGCCGCTGCTGTGCGGAGACCCCCGGGTGAAGCCACCGTCATCATGTC

781 TCACTCCTGGACTGTGACTTTCAGTGGGAGATGGAAGTTTTTCAGAGAACTGAACTGTGG.......................................................ATGTC

841 AAAAATGACCTTTCCTTAACTTGAAGCTACTTTTAAAATTTGAGGGTCTGGACCAAAAGA.......................................................-M—S

901 AGAGGAATATCAGGTTGAAGTCAAGATGACAGATAAGGTGAGAGTAATGACTAACTCCAA

121 TGACCAGGAGGCAAAACCTTCAACTGAGGACTTGGGGGATAAGAAGGAAGGTGAATATAT

961 AGATGGCTTCACTGAAGAAAAGGCATTTTAAGATTTTTTAAAAATCTTGTCAGAAGATCC

6 TGACCAGGAGGCAAAACCTTCAACTGAGGACTTGGGGGATAAGAAGGAAGGTGAATATAT

1021 CAGAAAAGTTCTAATTTTCATTAGCAATTAATAAAGCTATACATGCAGAAATGAATACAA

2 --D--Q--E--A--K--P--S--T--E--D--L--G--D--K--K--E--G--E--Y—I

1081 C

181 TAAACTCAAAGTCATTGGACAGGATAGCAGTGAGATTCACTTCAAAGTGAAAATGACAAC.

66 TAAACTCAAAGTCATTGGACAGGATAGCAGTGAGATTCACTTCAAAGTGAAAATGACAAC

Fig.1. The nucleic acid sequence and amino acid22 --K--L--K--V--I--G--Q--D--S--S--E--I--H--F--K--V--K--M--T--T

sequence of human SUMO1 encoding gene. Top line

241 ACATCTCAAGAAACTCAAAGAATCATACTGTCAAAGACAGGGTGTTCCAATGAATTCACT

is the nucleic acid sequence, which contained no

126 ACATCTCAAGAAACTCAAAGAATCATACTGTCAAAGACAGGGTGTTCCAATGAATTCACT

coding and coding region of mRNA. Middle line is the

42 --H--L--K--K--L--K--E--S--Y--C--Q--R--Q--G--V--P--M--N--S--L

nucleic acid sequence, which contained coding region

301 CAGGTTTCTCTTTGAGGGTCAGAGAATTGCTGATAATCATACTCCAAAAGAACTGGGAAT

of m RNA. Bottom line is amino acid sequence.

186 CAGGTTTCTCTTTGAGGGTCAGAGAATTGCTGATAATCATACTCCAAAAGAACTGGGAAT
62 --R--F--L--F--E--G--Q--R--I--A--D--N--H--T--P--K--E--L--G—M

Acknowledgement
I would like to express my deepest
appreciation to Mr. Saito and teachingassistants.

Reference
http://www.ncbi.nlm.nih.gov/
http://au.expasy.org/
http://www.ensembl.org/index.html

Sequencing of the human SUMO1 encoding gene
Momoyo Nomaguchi
Faculty of Science, Kumamoto Univercity.

Materials & Methods[1] Determination of the plasmid DNA
We prepared the dilute solution of plasmidDNA, and then determined the concentration of thesolution by absorptiometry. The name of thisplasmid was ‘pEGFP - hs SUMO1.’[6] Homology search
Homology search was carried out with NCBI’s
websites: http://www.ncbi.nlm.nih.gov/. We got
the corresponding amino acids sequence. The
function of analysis gene was estimated withliterature.

[2] Sequencing reaction
Results
After adjustment of 13µl of the DNA solution, we
added to this solution the following reagents: 2µl of
the primerDNA, 4µl of the sequence mix containing
fluoresceinated ddNTP, dNTP, and DNA
polymerase, and 1µl of Buffer. The sequence ofthe primer is 5’-CGAGAAGCGCGATCACAT-3’,
which correspond to the part of hs SUMO1 gene
specifically. Amplification reaction was carriedout with the thermal cycler (Applied Biosystems).
[3] Ethanol precipitation
To stop the sequencing reaction, we added 5µl of
the STOP solution containing NaOAc, EDTA, andGlycogen, and 60µl of 99% ethanol to the reaction
solution. After centrifugation at 14,000rpm for
15min, the supernatant was removed and the cDNA
was precipitated. 200µl of 70% ethanol was added.
After centrifugation at 14,000rpm for 2min, thesupernatant was removed.
When thecentrifugation was repeated again, the cDNA pellet
was collected. For the sequencing, we added 30µl
of SLS solution, and scrambled.

The analysis gene was found to be human SUMO1
encoding gene (Fig.1). The gene has 1081 bases intotal. Base 116-421 of cDNA is coding region, but
the others are non-coding region.

Discussion‘Small ubiquitin-like modifier’ is formal name of
SUMO. The SUMO protein’s 3D structure issimilar to ubiquitin. So, in a phase of
post-translational modification, Sumoylation andubiquitination are believe to function in similar but
distinct biological processes.

Acknowledgement
We thank T. Sasano and Y. Yasutake of graduate
students of Kumamoto University for helpful
technical guidance.

[4] Sequencing
DNA sequencing was carried out with a DNA
sequencer(Beckman). About 2 hours later, sequence
was terminated.

References[5] Input the sequence

Hong-Lin Su and Steven S. -L. Li.
(2002) Molecular features of humanubiquitin-like SUMO genes and their encoded
proteins. Gene 296:1-2, 21.

For homology search, we input the DNA sequence
to Sequence assistant (Vector) according to the
manufacturer’s protocol.

Fig.1
Nucleic acid sequence andcorresponding amino acidsequenceof
SUMO1
encoding gene.
Top lineshows non-coding and coding
region of SUMO1 encoding
cDNA. Middle line shows
coding region of SUMO1
encoding cDNA.
Bottomline shows corresponding
amino acids sequence to
SUMO1 encoding cDNA.

1 GCAGCCGCGGTGTTGTGCTGTGGGGAAGGGAGAAGGATTTGTAAACCCCGGAGCGAGGTT............................................................
............................................................
61 CTGCTTACCCGAGGCCGCTGCTGTGCGGAGACCCCCGGGTGAAGCCACCGTCATCATGTC.......................................................ATGTC.......................................................-M--S
121 TGACCAGGAGGCAAAACCTTCAACTGAGGACTTGGGGGATAAGAAGGAAGGTGAATATAT
6 TGACCAGGAGGCAAAACCTTCAACTGAGGACTTGGGGGATAAGAAGGAAGGTGAATATAT
2 --D--Q--E--A--K--P--S--T--E--D--L--G--D--K--K--E--G--E--Y--I
181 TAAACTCAAAGTCATTGGACAGGATAGCAGTGAGATTCACTTCAAAGTGAAAATGACAAC
66 TAAACTCAAAGTCATTGGACAGGATAGCAGTGAGATTCACTTCAAAGTGAAAATGACAAC
22 --K--L--K--V--I--G--Q--D--S--S--E--I--H--F--K--V--K--M--T--T
241 ACATCTCAAGAAACTCAAAGAATCATACTGTCAAAGACAGGGTGTTCCAATGAATTCACT
126 ACATCTCAAGAAACTCAAAGAATCATACTGTCAAAGACAGGGTGTTCCAATGAATTCACT
42 --H--L--K--K--L--K--E--S--Y--C--Q--R--Q--G--V--P--M--N--S--L
301 CAGGTTTCTCTTTGAGGGTCAGAGAATTGCTGATAATCATACTCCAAAAGAACTGGGAAT
186 CAGGTTTCTCTTTGAGGGTCAGAGAATTGCTGATAATCATACTCCAAAAGAACTGGGAAT
62 --R--F--L--F--E--G--Q--R--I--A--D--N--H--T--P--K--E--L--G--M
361 GGAGGAAGAAGATGTGATTGAAGTTTATCAGGAACAAACGGGGGGTCATTCAACAGTTTA
246 GGAGGAAGAAGATGTGATTGAAGTTTATCAGGAACAAACGGGGGGTCATTCAACAGTTTA
82 --E--E--E--D--V--I--E--V--Y--Q--E--Q--T--G--G--H--S--T--V--*
421 GATATTCTTTTTATTTTTTTTCTTTTCCCTCAATCCTTTTTTATTTTTAAAAATAGTTCT
306 G...........................................................
-...........................................................
481 TTTGTAATGTGGTGTTCAAAACGGAATTGAAAACTGGCACCCCATCTCTTTGAAACATCT............................................................
............................................................
541 GGTAATTTGAATTCTAGTGCTCATTATTCATTATTGTTTGTTTTCATTGTGCTGATTTTT............................................................
............................................................
601 GGTGATCAAGCCTCAGTCCCCTTCATATTACCCTCTCCTTTTTAAAAATTACGTGTGCAC............................................................
............................................................
661 AGAGAGGTCACCTTTTTCAGGACATTGCATTTTCAGGCTTGTGGTGATAAATAAGATCGA............................................................
............................................................
721 CCAATGCAAGTGTTCATAATGACTTTCCAATTGGCCCTGATGTTCTAGCATGTGATTACT............................................................
............................................................
781 TCACTCCTGGACTGTGACTTTCAGTGGGAGATGGAAGTTTTTCAGAGAACTGAACTGTGG............................................................
............................................................
841 AAAAATGACCTTTCCTTAACTTGAAGCTACTTTTAAAATTTGAGGGTCTGGACCAAAAGA............................................................
............................................................
901 AGAGGAATATCAGGTTGAAGTCAAGATGACAGATAAGGTGAGAGTAATGACTAACTCCAA............................................................
............................................................
961 AGATGGCTTCACTGAAGAAAAGGCATTTTAAGATTTTTTAAAAATCTTGTCAGAAGATCC............................................................
............................................................
1021 CAGAAAAGTTCTAATTTTCATTAGCAATTAATAAAGCTATACATGCAGAAATGAATACAA............................................................
............................................................
1081 C..
..

Analysis of DNA sequence and gene function
Department of Science, Biology Selection Kumamoto University
Yamato Kojimoto
Materials and Methods
The concentration of plasmid, named as pEGFP-009, was determined by UV260nm
absorbance using Bio photometer spectrophotometer (Eppendorf).
To analyze the DNA sequence, 0.4μl of plasmid DNA was mixed with 2μl of thesequence mix (containing dye-terminator ddNTP and dNTP and DNA polymerase), 1μl ofsequencing primer, 1μl of primer DNA and 1μl of reaction buffer. The mixture wasincubated conditions using thermal cycler. 60μl of 99% ethanol and 5μl of STOP
solution was added to the reaction and the DNA fragments were precipitated bycentrifugation at 4℃, 14000rpm using MX-300 (Tomy). The precipitate was rinsed
twice with 200μl of 70% ethanol and dissolved in 30μl of SLS. DNA sequencing
was carried out using CEQ8000 (BECKMAN). A given sequence was searched byhomology search with NCBI of web site.
Results
We got the DNA sequence of pEGFP-009 and found that it contains Homez cDNA sequence(Figure1). Homez cDNA consists of 5633 bases and the protein-coding region is from 96 to
1652 nucleotide. We found that Homez protein contains structure of homeodomain leucine
zipper.
Discussion
Homez means homeodomain leucine zipper-encoding gene.
There are several regions within Homez in vertebrate homeobox gene including three atypicalhomeodomains, two leucine zipper-like motifs, and an acidic domain. The gene is ubiquitously
expressed in human and murine tissues, although the expression pattern is more restricted
during mouse development. Genomic analysis revealed that human and mouse genes are locatedat 14q11.2 and 14C, respectively, and are composed of two exons.
Acknowledgments
We are grateful to Ms.Muramatsu and Ms.Kawasaki for doing this experiment. This experiment
was supported by laboratory of Dr.Saitoh.
References
・Proc Natl Acad Sci U S A. 2003 September 2; 100(18): 10358–10363.

GCTGTATGTAGCTTATTGTTAAAATGCTTGGCCACCGACTCCTGCCCAGTTTAGACTTCCCAGCTGTCTCTGAAGGGTACAAACCAGAGCACGACATGAGTCCTAATAAAGATGCCAGCAGTCTCAACAGCTCTGCAGCAGGGCTTGTCTGTCTCCCGCCAGTCTCAGAGGAGCTACAGCT-M—-S—-P—-N—-K—-D—-A—-S—-S—-L—-N—-S—-S—-A—-A—-G—-L—-V—-C—-L—-P—-P—-V—-S—-E—-E—-L—-Q—-L
TGTGTGGACTCAAGCCATCCAAACCAGTGAGCTGGATGGAAATGAGCACCTGCTACAAGCCTTCAGCTACTTCCCCTATCCAAGCCTGGCAGATATCGCTCTCCTCTGCCTGCGGCATGGGCTGCAGATGGAGAAGGTAAAGACCTGGTTCATGGCCCAACGTCTCCGCTG--V—-W—-T—-Q—-A—-I—-Q—-T—-S—-E—-L—-D—-G—-N—-E—-H—-L—-L—-Q—-A—-F—-S—-Y—-F—-P—-Y—-P—-S—-L—-A—-D—-I—-A—-L—-L—-C—-L—-R—-H—-G—-L—-Q—-M—-E—-K—-V—-K—-T—-W—-F—-M—-A—-Q—-R—-L—-R—-C
CGGCATTAGCTGGTCATCTGAAGAGATAGAGGAGACTCGTGCCAGAGTGGTGTACCACCGAGATCAGCTCCTTTTCAAGTCTCTTCTCTCTTTCACACAGCAGTCAGTGAGGCCCCCACAGGAGAGGCCTCCAGTGCTCCGTCCGGAGCAGGTTGCTCTTGGACTGAGTCC--G—-I—-S—-W—-S—-S—-E—-E—-I—-E—-E—-T—-R—-A—-R—-V—-V—-Y—-H—-R—-D—-Q—-L—-L—-F—-K—-S—-L—-L—-S—-F—-T—-Q—-Q—-S—-V—-R—-P—-P—-Q—-E—-R—-P—-P—-V—-L—-R—-P—-E—-Q—-V—-A—-L—-G—-L—-S—-P
TCTGGCGCCTAGTGAGCAGCCCACTCATATGAAAGGATTGAAGGTCGAGCCTGAGGAGCCCTCTCAGGTATCACAGCTGCCACTGAATCATCAGAACGCTAAGGAG
CCTCTGATGATGGGCAGCAGAACATTCTCCCACCAATCAGACTGTCAGGATCTTCAGATCAGTGG--L—-A—-P—-S—-E—-Q—-P—-T—-H—-M—-K—-G—-L—-K—-V—-E—-P—-E—-E—-P—-S—-Q—-V—-S—-Q—-L—-P—-L—-N—-H—-Q—-N—-A—-K—-E—-P—-L—-M—-M—-G—-S—-R—-T—-F—-S—-H—-Q—-S—-D—-C—-Q—-D—-L—-Q—-I—-S—-G
CCTCTCTAAGGAGCAGGCAGGCAGAGGTCCTGACCAGTCATGTGGAAAGACTGCTTCCTGGAATCACTTCACAGCTGTCCATCAGCCAGATAAGCCCGCATCGGTC
TCATTACTTGATAATAGTTGTAAGGAGGAATCCGAACCTAGTGGAATACCTCCATCTTCCTCTAC--L—-S—-K—-A—-E—-Q—-A—-G—-R—-G—-P—-D—-Q—-S—-C—-G—-K—-T—-S—-W—-N—-H—-F—-T—-A—-V—-H—-Q—-P—-D—-K—-P—-A—-S—-V—-S—-L—-L—-D—-N—-S—-C—-K—-E—-E—-S—-E—-P—-S—-G—-I—-P—-P—-S—-S—-S—-T
CTCTTCTCCCTCTTTCCAGGCATTGGCTAATGGAACCACTGCCACCCCTAAGCCCCTCCAGCCTTTGGGTTGTATCTCACAGTCACTCTCACCTAGTAAGAAGGCA
CTGTCTCCACAAGTAGAACCACTCTGGCCCCAAAGGCTATGGAATAACTCAGAACCAAATTCAGC--S—-S—-P—-S—-F—-Q—-A—-L—-A—-N—-G—-T—-T—-A—-T—-P—-K—-P—-L—-Q—-P—-L—-G—-C—-I—-S—-Q—-S—-L—-S—-P—-S—-K—-K—-A—-L—-S—-P—-Q—-V—-E—-P—-L—-W—-P—-Q—-R—-L—-W—-N—-N—-S—-E—-P—-N—-S—-A
TGGCCCCACAGAATACCTTTCCCCAGATATGCAACACCAGCGAAAGACTAAGCGGAAAACCAAAGAACAGTTGGCCATCCTCAAATCCTTTTTCCTACAGTGCCAATGGGCACGACGAGAAGATTACCATAAATTAGAACAGATCACTGGTTTACCTCGCCCGGAGATCAT--G—-P—-T—-E—-Y—-L—-S—-P—-D—-M—-Q—-H—-Q—-R—-K—-T—-K—-R—-K—-T—-K—-E—-Q—-L—-A—-I—-L—-K—-S—-F—-F—-L—-Q—-C—-Q—-W—-A—-R—-R—-E—-D—-Y—-H—-K—-L—-E—-Q—-I—-T—-G—-L—-P—-R—-P—-E—-I—-I
TCAATGGTTTGGTGACACACGATATGCCTTGAAGCATGGACAGCTGAAATGGTTTCGGGACAATGCAGTACTTGGTACCCCTAGTTTTCAAGATCCAGCCATTCCTACACCTTCAACTCGTTCCTTGAAAGAATGGGCCAAGACACCACCTCTACCAGCCCCACCGCCCCC--Q—-W—-F—-G—-D—-T—-R—-Y—-A—-L—-K—-H—-G—-Q—-L—-K—-W—-F—-R—-D—-N—-A—-V—-L—-G—-T—-P—-S—-F—-Q—-D—-P—-A—-I—-P—-T—-P—-S—-T—-R—-S—-L—-K—-E—-W—-A—-K—-T—-P—-P—-L—-P—-A—-P—-P—-P—-P
ACCAGATATACGACCTTTGGAGAAGTACTGGGCAGCCCACCAGCAGCTGCAGGAAGCTGATATCCTTAAACTGAGTCAGGCATCACGACTAAGCACTCAGCAAGTG
TTGGACTGGTTTGACTCTCGATTGCCTAAGCCAGCAGAAGTGGTAGTTTGTCTAGATGAAGAAGA--P—-D—-I—-R—-P—-L—-E—-K—-Y—-W—-A—-A—-H—-Q—-Q—-L—-Q—-E—-A—-D—-I—-L—-K—-L—-S—-Q—-A—-S—-R—-L—-S—-T—-Q—-Q—-V—-L—-D—-W—-F—-D—-S—-R—-L—-P—-K—-P—-A—-E—-V—-V—-V—-C—-L—-D—-E—-E—-D
CGAGGAGGATGAGGAGGATGAACTGCCAGAAGATGGTGAGGAAGAGGAGGAGGAGGAGGAAGATGATGATGATGGTGATGATGATGTCATCATATGGGACTGAGGGAGGCTGAGGCAAAGGGGAGTCTTTACTGAAATATTAACCAACTTAGTGACTGTTCAAACCTTTTTGAAATTG--E—-E—-D—-E—-E—-D—-E—-L—-P—-E—-D—-G—-E—-E—-E—-E—-E—-E—-E—-E—-D—-D—-D—-D—-G—-D—-D—-D—-V—-I—-I—-W—-D—-*—
CTCTTGTAAAAAGTTTTGTGTTCCTGTGTAGGCAAAGGGGGTAATCTGCAGTAAACTGGAGAGGGTGACCAGGACAAACTGAAGTATTGGATCAACACAAATCCAATTCTCCAGCCTCCGTGTAGAGAGAGTTTGGGATCTGGGCAGATACTGGCTGAAAAGAGCTCTTGCTTTGCTCTTGCTCCTTAGGACTCTTTAAGTCGTGTCTCAGC
CCCACATCTACAGGGAACCTGAATGGAGAGAAGTATTGTGATCCAATTGATCCCAGTGCACCCCAGCATTGTCAGAGACTCCTTGTGCCTGGCCTGTCTTTCTGAG
CACATGTTCAGGCACACTGCAGCTAGTGTTTGCAGGGTCTTGGCTTTATAACAGGCACATCGTCAGACCCCTGATAATGTCTGACTCGATTTGTCACTAATTCACA
GCAGCCTGTTCTTGCTTTTGACACTGGGGCCAATCTGCTCTCTACCATTTTATTTCTATGAAATCCCCTCTTTGCTTACTATTGTCTCAGGGTAAATCTTTACATAGAGAGTTTGTGAAACTTTTTAATAATCAGAGTGTGAATATTAGCCTGTGTATTTATTGGAGGCGGGGATGAGCCGGCATAGTGACTGTGACTACTATACTTGGCAGGTAGAGGCAGGAGGATCAAGGGGTTCAAGGTCAGCCTTGACTACATATGAAGTTAAAGGCCAACCTGGACTATGTGAGACTCTGTTTTGAAACATAACAGTGAGTCAGAGGAGAAGGGCGGTGGGGTAGTTACAGGTTTGGATTTACAAACCTGACACTATTTTCTGCCCAGATTGATATAGATTGTTTGGTTGGTTTTTTAATTATTTTTT
CCTTTTTTAACTAGTGCTTCAATTTGTATCTAACAAGAGATGTTTCCCTCTTTATAGGATCTGTCTTGTGGACAGAAGGACCATGGTCTTCTAACTCAATGATGTTCTAGAACTGGGAATTACTGGTAACAATTGGTTAGTCATCTGAAAATAGAGTGCAAGACCCAAATTGCTTAATAATAAAAAAAGGCCAAAAATAAAATTGTTTCTTT
TCTCCACTTTGCCACTTCTGTGTTTCAAGATTTATCTCCGTCTATCTATCCATCCACTTTCCTTTAGGTATATGAGTACACTGTTGCTGTCTTCAGACACACCGGAAGGGGGCATAAGATCCCACTACAGATGGTTGTGAGCCACCATGGGGTTGATGGGAATTGCACTCAGGACCTCTGGAAGAGCAGTGTTCTTAACTGCTGAGCCATCTCTCTAGCCCCATCTTCACTTTCTTAAATGCTTGTCTTGAACTCACTGTGAAGCCATGGCTGCTGCTGTTCTTATCTTCCTGTTTCCATCTCCTAAACGCTGATTAGAAGTATGTGCCATTATGCCCAGCTTATGCAGTACTGGGGATCAAACCAGGCCTTTATGTATGGTAGGCAGGTACTCTGCCAACTGGGCTACATCTGTACCTAGCCTGAGTCTAGGAATTTAATGCTGGCATGTAGCCCAGTGGTAGACCTCTTACCTAGCATGTGTGACACCCTAGTTTCAAGCCCAGCATCACTTTAAAATCATTTCTGGT
TTAGTTTATTTTTTTAAAGAGTTAGTCAGGACACGCTGCAGTAGATAAAAATTGAAGACAGTAATTCAGCATCTATTGCTACATGCATGGTTTGCAGCCATCAGATGACAAGGTTTATTGGCCCTCCAGCAGTGACAACAGGTTGTAACTCCACTTGCTCATCCTACCATTTGAGAAACTATTTTAGCAAGAGTGGCAGAAGCCATAAAAGGGTATGTTAATGATTTTTTTCCTCTTTATTAGTCTATATACCTTACTAAGGTGGAAATCTACCTGTTGTCAAAGCTGGAAATGTATTCTACCTATTGTTTGGATAAAGGAAAATCAGAACATTTCATAGCAGCTGGGTGTAGAACAATAGGTAAAAAGTATAATACATGAGGGTGGGGAACAGTTGTTTATTAACATTGGCCACAGGATAGCAACGTCCAGAGCTAAGCAGTGAAATCCAGAAATGAACCAGGATAAATATATTATCTAGTCTTCCTATGCCAAAGCATGAGGCTAAGGATTTTTGCAACCTTAGTCTCAAACTAGGAAATGAAAGGCCGCACATATGGGAGAGATGAAGATCTTCCTGAAGCCCTGGGGAAGACAGCCACTGAGATGGCACTTGCCACCAAGTCTAGAGACTTG
AGTTGGATCCCCAGGACTAACTTTGTTGGAAAGAACTGACTCCTGCAAGTTGTCCTCAGACCCCCCACAAGTACGTACACAAAGTAAACAAAACCCAGGGGGCTGG
AGACGTGGCTCAGCAGTTTAGTATGCTTTGTGCCGGGCTCCCCAGCACCCATGTCAGTCAGCTCACACGCTGCCTATAACCCTGGCTTTAGGGGATCATACACCCTCTTCTGGTCTCTGAGGGCACCTGCATCACAGGCACCCATATGCAAGTAGTATACCCTCATACAACACACACAGTTTTTAAAAATTTTTTTAATGTTTGGAGAGAAAGACATGTAAGATAACGTTCAAGAAAACAAGCTGCAGACTGAAACCAGGAACCAGCAAGTGCCCATAGCCCCTGAGAGAACTGGACACTGCAGGCCAACCTTGAGCATTATGCACAGAGCAGACCTGGAAGAGGGATGTCTATCTGGAAATGTTCCATCCGTCTTTCCTGGCTAGTCCCATCTGCCTCTGTTGGTAGAACGTCTGCAGCAGAGAACAGATTTGCATGTTATGACAATACAGACTACTAAAGGTGTCCTACCAATAAAGTTTTATTGAACAAAATCAACTGCTACTGCTTCTCCATGGTTTTTCCTCATTGTATGATGACTAACTTACAAACAAAAAGAAATTTTGTTTGAGACAGGGTCTCATTATTGCTATTGCTATGTAGACCATGGCTGGCCTTGAATTCACAGCAATCTGC
CTGCTTCTGTTTTCCAAAAGGGCAGGGATTAAAGGTGCCACCATGCCCAACCTAGATTAAGAGCTGATGCGTCCTGTGAGACAAGGCCTTTGTTGTTACTGAAGAA
ACTGTACGCTTCAGTGCTTGATGATGATGATGTAAAATATCTGAGGAACTTTGGGAAGATTTGGAACATCTGACTACTGTATCAAAAGACAATTACCAGGGAGGCA
AAGATCTTACACCTCTAGTGATGTGGTACTTTTCAGTAACTATTTTTAACTTTATAATGTTTTATTTTGAAGTTTTCATATTTATGGATGTTTTGTTGAATGTATT
TCTGTGCTCAGAGACAACTCTGGAGATCCCCTGGGCTGGAGTTACAGAGGATTGTGATCCACCATGTGGGTGCTGGAATCGAACCCAGGTACCCTGGAAGAGCAGCCACTATTCTTAACTGCTGAATCATCTCTCCAGTCCCAATAGGTTTTGTTTTAAAACAGTAGTTCTTAACCTGTTGGTCTCCACCCCTTTCGGGTCAAATGACCCTTTCACAGGAGTCACATATCAGATATCCTGCATATCAGATATTTATATGATTCATAACAATAGCAGAGTTACAGTTATGAAGTAGTAATAATTCTATGGTTGGGGTCACCACACATGAGGGACTGTATTAAAGGGTTACAGCAGTAGGAAGGCTAGGAACTACTGTTTTAAAGGATAGACCAGAATAATGAACCTGTCATCCAATATCAACAAA
ACTCGTGGCTAATATTTATTGTTCTTTCTATTGCTTCCTTTTAGTAAATTATACTAAAGCAAAATCTAGATACCATTTAACTTCATAAAATTTTTATTTACATATATATGGATATGTACTTGTTTGTGTTTGTGTATTTGTGAGTAAATACAAAGTCATCAGACCTGAAAAAAAAAAAAAAAAAAAAAAAAAAA

Figure1. cDNA sequence and amino acid sequence of Homez.There are 5633 nucleotides in Homez cDNA,
gray line indicates the coding region of Homez (96 – 1652 nucleotide). Blue line shows the deduced aminoacid sequence.