However, little is known about that the genetic structure of the canine LINE-1 gene, the molecular relationship between the canine LINE-1 element and the LINE-1 element TVT-LINE inserted
Trang 1J O U R N A L O F
Veterinary Science
J Vet Sci (2002), 3(4), 285-292
Abstract6)
LINEs (lo n g in te rsp e rse d n u c le a r e le m e n ts or lon g
interspersed repeated DNA e lem ents) contains tw o o pe n
re a din g fra m e s (ORFs ), ORF 1 an d ORF 2 We a n aly se d
th e ORF 2 lo ca te d in th e 5' re g ion to th e first e xon of
oncogene c-myc in canine transmissible ve n e re al tumor
(TVT) cell We also show e d the transcription a ctiv atio n
w a s in du c e d by th is TVT-LINE se qu e n c e u sin g CAT
as sa y To id e n tify th e m u tatio n of tu m or su p pre ss or
ge n e , s e qu e n c e an a ly sis of p53 fro m TVT c e ll w a s
pe rfo rm e d We id e n tifie d th e po in t m u tatio n of 964
n u cle otid e (T→C) re s u ltin g in th e ch a n ge of am in o
ac id (P h e →Se r) of p53 tu m or s u pp re s so r p ro te in
Introduction
Mammalian genomic DNA contains several families of
frequently repeated sequences and some have been shown to
be mobile elements, such as SINE and LINE [9,21] They
are known to be mostly flanked by direct repeats of 7-20
base pairs (bp) that are believed to result in the duplication
of target site LINEs, long interspersed nuclear elements or
long interspersed repeated DNA elements, are considered to
be a nonviral retroposon found in all mammals The LINE-1
elements, which are mostly truncated at the 5‘ end existed
on the genomic DNA with 10,000 to 50,000 copies [5] and
they encode two open reading frames(ORFs), ORF1 and ORF2
From the results of sequence analysis and purified components,
the ORF2 of the LINE-1 encodes such biological functions as
reverse transcriptase, endonuclease and possibly RNase H
[8,15,17] In contrast, the ORF1 has no activity showing a
known enzymatic function [13] The LINE-1 also can transpose
near to oncogenes or other structural genes, and occasionally
＊Corresponding author: Chul-Joong Kim
Laboratory of Infectious Diseases, College of Veterinary Medicine,
Chungnam National University, Daejeon 305-764, Korea
Tel : +82-42-821-6783, Fax : +82-42-823-9382
E-mail : cjkim@cnu.ac.kr
causes malignant diseases, such as transmissible venereal tumor (TVT) in dog [11,12] and human breast carcinoma [19] The p53 tumor suppressor protein plays a central role in the maintenance of genomic integrity Inactivation of the p53 tumor suppressor gene by point mutation and translocation events has been associated with a large number of human neoplasms [6] Recently similar mutations within the canine cancer types including thyroid carcinoma [3], mammary tumors [22], osteosarcoma [10], circumanal gland adenoma [18] and lymphoma [23] The vast clinical knowledge concerning the identification and treatment of canine cancer and the apparent similarity of p53 inactivation in the tumors of some cancer patients identifies canine p53 as a potential target for anti-cancer therapy in the dog [24] The canine TVT is a naturally occurring neoplastic disease that affects the external genitalia of both sexes It is transmitted during coitus In addition to the natural mode
of transmission, the TVT can be transferred from one dog to another by experimental transplantation of living tumor cells [2] Canine TVT cells have truncated LINE-1 sequences from the 5‘region to the first exon of oncogene c-myc [1, 11, 12] However, little is known about that the genetic structure of the canine LINE-1 gene, the molecular relationship between the canine LINE-1 element and the LINE-1 element (TVT-LINE) inserted in rearranged c-myc (rc-myc) of TVT cell and the functional relationship between the TVT-LINE element and TVT disease
To estimate the molecular structures of the canine LINE-1 element and the TVT-LINE integrated in TVT, we determined their DNA sequences In addition, we have examined the transcription activation property of TVT-LINE element and investigated the point mutation of p53 gene in TVT cells Here we report the complete ORF2 DNA sequences of the canine LINE-1 element and the TVT-LINE element isolated from the TVT lines in Korea and the results of experiments that examine the transcription activity property of TVT-LINE element using the CAT assay
Sequence Analysis of Canine LINE-1 Elements and p53 Gene in Canine Transmissible Venereal Tumor
Young-Ki Choi and Chul-Joong Kim*
Department of Clinical and Population Sciences College of Veterinary Medicine University of Minnesota,
1988 Fitch Avenue St.Paul, MN 55108
Laboratory of Infectious Diseases, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea
Received J une 7, 2002 / Accept ed November 15, 2002
Trang 2286 Young-Ki Choi and Chul-Joong Kim
Materials and Methods
Sa m p le s an d D NA P re p ara tion
TVT tissues were removed surgically from the TVT dogs
and were frozen and kept at -80℃ until used Genomic DNA
was extracted as described by Okumura et al [20] The
canine fibroma cell line A72 was cultured in Dulbeccos
modified Eagles medium(DMEM) supplemented with 10%
fetal bovine serum and antibiotics Baby hamster kidney
(BHK) cell line was cultured in MEM medium contained
10% tryptose phosphate, 20mM HEPES and 2mM
gulutamine with 5% fetal bovine serum and antibiotics The
cell line A72 was used as a source to isolate total RNA and
BHK-21 cell line was used as a target cell for CAT assay
and production of the virus like particles of SFV
Co n stru c tion of plam ids
5′truncated LINE-1 element of TVT (TVT-LINE) was
subcloned into pBLCAT2 vector {PBL cat (F); TVT-LINE
inserted into Xba I and Bam H I sites upstream of CAT
gene, PBL cat (P) ; TVT-LINE inserted into S m a I and Kpn
I sites of downstream of CAT gene, PBL cat (5D); 5′deleted
TVT-LINE which was digested with Hind III restriction
enzyme was inserted into Hind III and Bam H I sites
upstream of CAT gene, PBL cat (3D); 3′deleted TVT-LINE
which was amplified with primers T8 (5′-GCAGAAAGATC
ATTTTGAAAGAAGG-3′) and T9 (5-CTACCACCCCTAGTT
CGTTT-3) was inserted into Xba I and BamHI sites
upstream of CAT gene (Fig 1)
P CR, DNA m an ip u latio n an d D NA s e qu e n cin g
The PCR was performed with synthetic primers in 50㎕
reaction volume for 30 cycles Long and Accurated(LA) PCR
was performed in 50㎕ reaction volume for 10 sec at 98℃
and for 15 min at 68℃ for 30 cycles The total RNAs were
extracted from a canine fibroma cell line A72 and primary
TVT tissue with TRIZOL reagent (GIBCOl, USA), and
mRNAs were purified with Oligotex-dT 30 (Takara, J apan)
The cDNA templates were synthesized by reverse
trans-cription (RT)-PCR from 5㎕ of mRNA using a first strand
synthesis kit (Pharmacia, Sweden) The amplified PCR
products and pBluescript SK(+) cloned DNA fragments were
sequenced by the dideoxy chain-termination method using a
373S sequencer (Perkin Elmer Applied Biosystems Division,
USA) with a Taq Dye Deoxy Terminator Cycle Sequencing
FS Kit Synthetic DNA primers were used for the PCR,
RT-PCR, LA-PCR and DNA sequencing The DNA sequence
data were analyzed by GENETYX-MAX software (Software
Development Co Ltd, Japan) for multiple sequence alignments
and homology search
Ch lo ra m p h e n ic ol a ce ty ltra n sfe ra se (CAT) as sa y
TVT-LINE cloned pBLCAT plasmids were transformed
into BHK and A72 cell lines by electroporation using
Bio-Rad Gene Pulser 10㎍ of plasmid DNAs was used for
each 1×106 BHK and A72 cells After 48 hrs of transfection, the cells were harvested at 250×g for 10min 4℃ After 3 times washing with 5ml of pre-cooled PBS (4℃), the cell pellet was mixed with 1ml of lysis buffer (MOPS-buffered saline containing Triton X-100, pH 6.5) and stand for 30min
at room temperature The cell lysates were spinned in a microcentrifuge at 15,000×g for 10min at 4℃ Cell extracts were used for ELISA test using CAT ELISA kit (Boehringer Mannheim GmbH, Germany) or stored at -70℃ until used Protein standard curve was made with Bio-Rad protein assay (Bio-Rad, CA) 100㎍ of cell extract protein was used per well of CAT ELISA plate The absorbance of the samples at 450 nm using a THEROMO max microtiter plate (ELISA) reader
Results
D NA s e qu e n ce o f Can in e LINE-1 ORF2
To obtain the functional canine LINE-1 sequence, RT-PCR was performed with mRNA from cell line A72 The ORF2 was constructed from six overlapping PCR fragments The six PCR products generated contiguous 4,251-bp cDNA sequences encoding 1,275 amino acids (hatched region of LINE-1 in Fig 2) A comparison with the human ORF2 revealed that canine ORF2 sequences had a 63% homology with those of human ORF2 at the amino acid level
D NA s e qu e n ce s of TVT-LINE in rc-m yc o f TVT
To estimate the relationship between the TVT-LINE and canine LINE-1, the TVT-LINE region was amplified with several primers and was determined for its sequences Four TVT-specific primers (T1 to T4) reported by Ameriglio et al [1] were used for the PCR but some non-specific DNA fragments were observed in normal dog samples Therefore three genomic DNA primers (T6, T7 and T8) flanking the TVT-LINE elements were newly synthesized, and the TVT-LINE was amplified from three TVT samples When the primer pairs of T6-T7 or T6-T8 were used in PCR, normal samples yielded 267bp or 158bp bands, which were different from those from the TVT samples, 1,645bp and 1,536bp, respectively These results suggested that the primer pairs of T6-T7 or T6-T8 were specific primer pairs for diagnosis of TVT using PCR method These PCR products were extracted from the gel, were determined for DNA sequences directly, and the DNA sequences of the TVT-LINE were aligned with those of the canine ORF2 region (Fig 2) The DNA sequences of the inserts from three TVT lines were identical with the 1,378bp in size and were flanked by 10bp direct repeats (ATTCTCTGGC) The sequence homology analysis with canine ORF2 suggested that the TVT-LINE contained a 416bp length homologous to the complementary strand of canine LINE-1 and was followed
by a 5bp deletion; DNA sequences of the TVT-LINE showed
a 98 % and 63 % homology to canine LINE-1 and human LINE-1 sequences, respectively
Trang 3Sequence Analysis of Canine LINE-1 Elements and p53 Gene in Canine Transmissible Venereal Tumor 287
F ig 1 TVT-LINE element as a whole or truncated from was subcloned into pBLCAT2 vector.
C A AG G C T C TC A T T C AA A A T G GA A G G A GA G A T A AA G A G C TT C C A A GA C A G G CA G G A A CT G A A A GA A T A T GT G A C C TC C A A A 8 0
C C AG C T C T GC A A G A AA T T T T AA G G G G GA C T C T TA A A A T TC C C C T TT A A G A AG A A G T TC A G T G GA A C A G TC C A C A AA A A C A 1 6 0
A G GA C T G A AT A G A T AT C A T G AT G A C A CT A A A C TC A T A T CT C T C A AT A G T A AC T C T G AA T G T G AA C G G G CT T A A T GA C C C C 2 4 0
A T CA A A A G GC G C A G GG T T T C AG A C T G GA T A A A AA A G C A GG A C C C AT C T A T TT G C T G TC T A C A AG A G A C TC A T T T TA G A C A 3 2 0
G A AG G A C A CC T A C A GC C T G A AA A T A A AA G G T T GG A G A A CC A T T T AC C A T T CG A A T G GT C C T C AA A A G A AA G C A G GG G T A G 4 0 0
C C AT C C T T AT A T C A GA T A A A CT A A A A TT T A C C CC A A A G AC T G T A GT G A G A GA T G A A GA G G G A CA C T A T AT C A T A CT T A A A 4 8 0
G G AT C T A T CC A A C A AG A G G A CT T A A C AA T C C T CA A T A T AT A T G C CC C G A A TG T G G G AG C T G C CA A A T A TA T A A A TC A A T T 5 6 0
A T TA A C C A AA G T G A AG A A A T AC T T A G AT A A T A AT A C A C TT A T A C TT G G T G AC T T C A AT C T A G CT C T T T CT A T A C TC G A T A 6 4 0
G G TC T T C T AA G C A C AA C A T C TC C A A A GA A A A G AG A G C T TT A A A T GA T A C A CT G G A C CA G A T G GA T T T C AC A G A T AT C T A C 7 2 0
Trang 4288 Young-Ki Choi and Chul-Joong Kim
A G AA C T T T AC A T C C AA A C T C AA C T G A AT A C A C AT T C T T CT C A A G TG C A C A TG G A A C TT T C T C CA G A A T AG A C C A CA T A T T 8 0 0
G G GT C A C C AA T C G G GT C T G A AC C G A T AC C A A A AG A T T G GG A T C G TC C C C T GC A T A T TC T C A G AC C A T A AT G C C T TG A A A T 8 8 0
T A GA A C T A AA T C A C AA C A A G AA G T T T GG A A G G AC C T C A AA C A C G TG G A G G TT A A G G AC C A T C CT G C T A AA A G A T GA A A G G 9 6 0
G T CA A C C A GG A A A T TA A G G A AG A A T T AA A A A G AT T C A T GG A A A C TA A T G A GA A T G A AG A T A C AA C C G T TC A A A A TC T T T G 1 0 4 0
G G AT G C A G CA A A A G CA G T C C TG A G G G GG A A A T AC A T C G CA A T A C AA G C A T CC A T T C AA A A A C TG G A A A GA A C T C AA A T A C 1 1 2 0
A A AA G C T A AC C T T A CA C A T A AA G G A G CT A G A G AA A A A A CA G C A A AT G G A T CC T A C A CC C A G G AG A A G A AG G G A G TT A A T A 1 2 0 0
A A GA T T C G AG C A G A AC T C A A CG A A A T CG A A A C CA G A A G AA C T G T GG A A C A GA T C A A CA G A A C CA G G A G TT G G T T CT T T G A 1 2 8 0
A A GA A T T A AT A A G A TA G A T A AA C C A T TA G C C A GC C T T C TT A A A A AG A A G A GA G A G A AG A C T C AA A T T A AT A A A A TC A T G A 1 3 6 0
A T GA G A A A GG A G A G AT C A C T AC C A A C AC C A A G GA A A T A CA A A C G AT T T T A AA A A C A TA T T A T GA A C A G GT A T A C GC C A A T 1 4 4 0
A A AT T A G G CA A T C T AG A A G A AA T G G A CG C A T T CC T G G A AA G C C A CA A A C T AC C A A A AC T G G A AC A G G A AG A A A T AG A A A A 1 5 2 0
C C TG C A C A GG C C A A TA A C C A GG G A G G AA A T T G AA G C A G TC A T C A AA A A C C TC C C A A GA C A C A AG A G T C CA G G G C CA G A T G 1 6 0 0
G C TT C C C A GG G G A A TT T T A T CA A A C G TT T A A A GA A G A A AT C A T A CC T A T T CT C C T A AA G C T G TT T G G A AA G A T A GA A A G A 1 6 8 0
G A TG G A G T AC T T C C AA A T T C GT T T T A TG A A G C CA G C A T CA C C T T AA T T C C AA A A C C AG A C A A AG A C C C CA C C A A AA A G G A 1 7 6 0
G A AT T A C A GA C C A A TA T C C C TG A T G A AC A T G G AT G C A A AA A T T C TC A A C A AG A T A C TG G C C A AT A G G A TC C A A C AG T A C A 1 8 4 0
T T AA G A A A AT T A T T CA C C A T GA C C A A GT A G G A TT T A T C CC C G G G AC A C A A GG C T G G TT C A A C AC C C G T AA A A C A AT C A A T 1 9 2 0
G T GA T T C A TC A T A T CA G C A A GA G A A A AA C C A A GA A C C A TA T G A T CC T C T C AT T A G A TG C A G A GA A A G C AT T T G A CA A A A T 2 0 0 0
A C AG C A T C CA T T C C TG A T C A AA A C T C TT C A G A GT G T A G GG A T A G AG G G A A CA T T C C TC G A C A TC T T A A AA G C C A TC T A C G 2 0 8 0
A A AA G C C C AC A G C A AA T A T C AT T C T C AA T G G G GA A G C A CT G G G A GC C T T T CC C C T A AG A T C A GG A A C A AG A C A G GG A T G T 2 1 6 0
C C AC T C T C AC C A C T GC T A T T CA A C A T AG T G G T GG A A G T CC T A G C CT C A G C AA T C A G AC A A C A AA A A G A CT T T A G GG G C A T 2 2 4 0
T C AA T T T G GC A A A G AA G A A G TC A A A C TC T C C C TC T T C G CC G A T G AG A T G A TC C T C T AC A T A G AA A A C C CA A A A G TC T C C A 2 3 2 0
C C CC A A G A TT G C T A CA A C T C AT G C A G CA T T G T GG T A G C GT G G C A GG A T A C AT C A T C AA T G C C CA G A A A TC A G T G GC A T T T 2 4 0 0
C T AT A C A C TA A C A A TG A G A C TG A A G A AA G A G A AA T T A A GG A G T C AA T C C C AT T T A C AA T T G C AC C C A A AA G C A T AA G A T A 2 4 8 0
C C TA G G A A TA A A C C TA A C C A GG G A G G TA A A G G AT C T A T AC C C T C AA A A C T AT A G A A CA C T T C TG A A A G AA A T T G AG G A A G 2 5 6 0
A C AC A A A G AG A T G G AA A A A T AT T C C A TG C T C A TG G A T T GG C A G A AT T A A T AT T G T G AA A A T G TC A A T G TT G C C C AG G G C A 2 6 4 0
A T TT A C A C GT T T A A TG C A A T CC C T A T CA A A A T AC C A T G GA C T T T CT T C A A AG A G T T AG A A C A AA T T A T TT T A A A AT T T G T 2 7 2 0
G T GG A A T C AG A A A A AA C C C C GA A T G G CC G G G G GA A T T T TA G G A A AA A A A A CC A T G T CT G G G G GC A T C T CA A T G C CA G A T T 2 8 0 0
T C AG G T T G TA C T A C AA A G C T GT G G C T AT C A A G AC A G T G TG G T A C TG G C A C AA A A A C AG A C A C AT A G A T CA G T G G AA C A G A 2 8 8 0
* * * * ** * * * * ** * * * * ** * * * * **
A T AG A G A A CC C A G A AG T G G A CC C T G A AC T T T A TG G G C A AC T A A T CT T C G A TA A A G G AG G A A A GA C T A T CC A T T G GA A G A A 2 9 6 0
* * ** * * * * T* * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * A* * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * *
Trang 5Sequence Analysis of Canine LINE-1 Elements and p53 Gene in Canine Transmissible Venereal Tumor 289
A G AC A G T C TC T T C A AT A A A T GG T G C T GG G A A A AT T G G A CA T C C A CC T G C A GG A G G A TG G A A C TA G A C C CC T C T C TT T C A C 3 0 4 0
* * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * *A * * * * *A * * A * ** A * * * ** * * * * A* * * * * ** * * * *
C C TA C A C A AA G A T G AA C T C C AA A T G G AT G A A A GA T C T A AA T G T C AG A C C A GA T T C C AT C C A A AT C C T A GA G G A G AA C A C A 3 1 2 0
* A ** * * * * ** * * * A ** * * * A ** * * * * ** * * * * ** * * * * ** * * * * ** * * A * ** * * * * ** * A * * ** * * * * ** * A * * ** * * * *
G G CC A C A C AA T T T T TG A A A T CG G C C A CC G T A A CT T C T T GC A A G A TA C A T C CA C G A A GG C C A A AG A A A C AA A A G C AA A A A T 3 2 0 0
* * *A * * * * ** * * * * ** * * C * ** * * * * TA * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * A * * ** * * * * ** * * * * ** * * * *
G A AC T A T T GG G A C T TC A T C A AG A T A A GA A G C T TT T G A C AC G C A A AG G A T A CA G T C A AC A A A A CT A A A A GA C A A C CT A C A G 3 2 8 0
* * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** C * * * ** * * G * *A G A G A
A A TG G G A G AA G A T A TT T G C A AA T G A C GT A T C A GA T A A A GG G C T A GT T T C C AA G A T C TA T A A A GA A C T T AT T A A A CT C A A C 3 3 6 0
* T - - - * ** * * * * ** * * * * ** * * * * A* * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * *
A C CA A A G A AA C A A A CA A T C C AA T C A T GA A A T G GG C C A A AG A C A T GA A C A G AA A T T T CA C A G A GG G A G A CA T A G A CA T G G C 3 4 4 0
* * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * A * * ** * * * * ** * * * * ** * * C * ** * * * * A* A * * * ** * * * * ** * * * *
C A AC A T G C AC A T G A GG A A A T GC T T T G CA T C A A TT G C C A TC A G G G AA A T A C AA A T C A AA A C C A CA A T G A GA T A C C AC C T C A 3 5 2 0
* * ** * * * * ** * * * * *A * * * * ** * C * * ** * * * C ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * *
C A CC A G T G AG A A T G GG G A A A AT T A A C AA G G C A GG A A A C CA C A A A TG T T G G AG A G G A TG C G G A GA A A A G GG A A C C CT T T T A 3 6 0 0
* * ** * * * * ** * * * * ** A * * * ** * * * * ** * * * * ** * * * * A* * * * * ** * * * * ** * * * * ** T * * * ** * * * * ** * * * * ** C * * *
C A CT G T T G GT G G G A AT G T G A AC T G G T GC A G C C AC T C T G GA A A A C TG T G T G GA G G T T CC T C A A AC A G T T AA A A A T AT A C C T 3 6 8 0
* * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * G * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * *
G C CC T A T G AC C C A G CA A T T G CA C T G T TG G G G A TT T A C C CC A A A G AT A C A A AT G C A A TG A A A C GC C G G G AC A C C T GC A C C C 3 7 6 0
* * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** T * * * ** * * * * ** * * * *
C G AT G T T T AT A G C A GC A A T G GC C A C G AT A G C C AA A C T G TG G A A G GA G C C T CG G T G T CC A A C G AA A G A T GA A T G G AT A A A G 3 8 4 0
* * ** * * * * C* * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * *
A A GA T G T G GT T T A T GT A T A C AA T G G A AT A T T A CT C A G C TA T T A G CA A T G A CA G A T A CC C A C C AT T T G C TT C A A C GT G G A T 3 9 2 0
* * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * A* * * * * ** A * * * ** * * * * ** * * * * ** * * * * ** * * * *
G G AA C T G G AG G G T A TT A T G C TG A G T G AA G T A A GT C A G T CA G A G A AG G A C A AA C A T T AT A T G T TC T C A T TC A T T T GG G G A A 4 0 0 0
* * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * *
T A TA A A T A AT A G T G AA A G G G AA T A T A AG G G A A GG G A G A AG A A A T GT G T G G GA A A T A TC A G A A AG G G A G AC A G A A CG T A A A 4 0 8 0
* * ** * * * * ** * * * * ** * * * * ** A * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * *A * * * *
G A CT G C T A AC T C T G GG A A A C GA A C T A GG G G T G GT A G A A GG G G A G GA G G G C GG G G G G TG G G A G TG A A T G GG T G A C GG G C A C 4 1 6 0
* * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * *
T G GG G G T T AT T C T G TA T G T T AG T A A A TT G A A C AC C A A T AA A A A A TA A A T T AA A A A A AA A A G A TG A A A C CA C A G A AA A A A A 4 2 4 0
* * ** T * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * * ** * * * T A* * * * A ** * T * * ** T * A * AT * * * A T* A * A T ** * * * *
* * ** * T * * C* T T C T CT G G C C
Fig 2 NA sequences of the ORF2 canine LINE-1 and the TVT-LINE The ORF2 of caine LINE-1 has one open reading
frame from 178 to 4002 nucleotide The DNA sequences of the TVT-LINE in TVT lines are compared with those of canine LINE-1 The underlined below the sequences shows the reverse complementary 416-bp sequences of TVT-LINE The ten direct repeats found in the TVT-LINE are marked by a box
Trang 6290 Young-Ki Choi and Chul-Joong Kim
Activation property te st of TVT-LINE elem ent in vitro
To investigate the activation property of the truncated
LINE-1 element inserted in the upstream of the c-m yc gene
(TVT-LINE), we performed CAT assay in the BHK and A72
cell lines Four plasmids were constructed with pBLCAT2
vector [TLPBL cat (F); TVT-LINE element was inserted
upstream of the CAT gene, TLPBL cat (P); TVT-LINE
element was inserted downstream of the CAT gene, TLPBL
cat; 5′D) and 3′(D) truncated TVT-LINE element was
inserted upstream of the CAT gene] Each of these 10㎕
DNA was transformed into 1×106 - 2×106 BHK and A72
cells at the electroporation condition of the 25μF/0.83KV
After 48 hours of transfection infected cells were harvested
and then determined the CAT concentration of each infected
cell lines (Fig 3) TLPBL cat (F) contained BHK cell lines
and A72 cell lines were shown 0.29 - 0.89 ng/ml and 0.21
-0.63 ng/ml of CAT enzyme activity, respectively However,
TLPBL cat (P) and TLPBL cat (D) showed similar CAT
activity with control cell lines These results suggested that
TVT-LINE element in the upstream of c-m yc oncogene could
activate the c-m yc gene in the TVT cell line.
Fig 3 Activation property test of TVT-LINE elements in
vitro using CAT assay Control; pBLCAT2 plamid, BHK(F)
and A72(F); pBLCAT-TVT(F) plasmid, BHK(P) and A72 (P)
; pBLCAT-TVT(P) plasmid, BHK(D) and
A72(D);pBLCAT-TVT(5D) and (3D)
Id e n tifica tion o f p oin t m u ta tion o f p 53 g e n e in TVT
The p53 gene is frequently mutated in diverse human
cancer [7, 14] The isolation of canine p53 cDNA was carried
out using a RT-PCR method and canine p53 gene specific
primers (caFOR: 5′-ATGGAGGAGTCGCAGTCAGAC -3′,
caREV: 5′-TTTT ATGGCGAGAGGTAGATTGC-3') from the
primary TVT tissues This fragment was purified at the 0.7
% agrose gel and cloned into TA cloning vector (Novagen,
USA) and then nucleotide sequence was determined These
sequences were compared with normal canine p53 gene
mRNA from Genbank (Fig 4) We found a single point
mutation at 964 nucleotide position from T → C From the
this point mutation amino acid was changed from Phe to Ser at codon 316
To confirmed the point mutation at the TVT genomic DNA, we designed the primers (ca p53 931: 5′-CGCAAAA GAAGAAGCC ACTA-3′and caREV) for the exon 9 and 10 The PCR products were purified at the 0.7% agarose gel and cloned into TA cloning vector and then sequenced We confirmed the point mutation at the TVT genomic DNA samples These results suggested that codon 316 might be the one of the causes of oncogenesis in the TVT
Discussion
In this study, we determined the DNA sequences of the ORF2 for canine LINE-1 element and those of the LINE-1 integrated in c-myc gene of TVT However, we failed to isolate the full sequence of ORF1 for canine LINE-1 element
At an initial investigation, we tried to isolate canine LINE-1 element form the genomic libraries, but no functional LINE-1 gene encoding for ORF1 and ORF2 was isolated A comparison
of the DNA sequence of a genomic clone 16 with those of the functional LINE-1 from cDNA clone reveled that clone 16 has nucleotide mutations or deletions, although the sequence identity between both clones was over 93% These results indicated that even if genomic LINE-1 genes integrate with high copies in a canine genome and they would be transcripted
by arbitrary promotes, the functional LINE-1 protein would not be produced as much The functional ORF2 of LINE-1 was constructed from six RT-PCR products, but a long full-length ORF2 was not amplified in this study This evidence suggests that the transcripts of LINE-1 have a small quantity of total RNA from canine cell line A72 In fact, we could not detect and distinct signals for LINE-1 gene by Northern blot analysis with total RNA from A72 (data not shown) From molecular analysis of canine LINE-1 and TVT-LINE integrated in TVT lines, the TVT specific insert in
front of c-m yc gene appears to originate from the 3 region
of canine LINE-1 Katzir et al.[11, 12] reported that the 1,314-bp LINE-1-like element flanked by 10-bp direct repeats integrate into the c-myc region of TVT, which is 64-bp smaller than the TVT-LINE of our study with different nucleotides However, the 10-bp repeat sequence and the molecular basic structure, including the inversion and deletion, commonly occur in inserts of TVT lines from Israel,
J apan and Korea This result indicates that the insertion of
a truncated LINE-1 element upstream of the c-myc gene may be the genetic event specific for the tumorignesis of TVT disease
An interesting question remains whether the TVT is caused by insertion of the truncated LINE-1 element
upstream of the c-m yc gene Katzir et al.[12] pointed out the
possibility that the inserted repetitive sequence has and
effect on the transcriptional activity of the c-m yc gene To
investigate this question, we performed CAT assay of TVT-LINE element When TVT-LINE element was inserted
highest lowest mean
Trang 7Sequence Analysis of Canine LINE-1 Elements and p53 Gene in Canine Transmissible Venereal Tumor 291
in the upstream of CAT gene, CAT enzyme concentration
was raised but the concentrations of CAT enzyme were not
consistent as 0.284 - 0.89 ng/ml This result suggests that
the insertion of a truncated LINE-1 element upstream of
c-m yc gene can activate the c-m yc gene but maybe the other
factors are more needed
Loss of p53 function is a common event in many types of
human [6,14] and animal [24] Although there is evidence
that p53 functions through several mechanism, it is clear that
the ability of p53 to act as a sequence specific transcriptional activator can play an important role in mediating both cell cycle arrest and apoptosis [3,18]
Mutant p53 can regulate the expression of the endogenous
c-m yc gene and is a potent activator of the c-m yc promoter
[6] We showed here that mutation of p53 gene at codon 316
in the TVT tissues although it wasn't existed from exon 3
to 8 where were commonly occurred mutation in the human but it maybe the one of the etiological reasons of TVT
AGCCAAGTCTGTTACTTGGACGTACTCCCCTCTCCTCAACAAGTTGTTTTGCCAGCTGGC 6 0
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
AGCCAAGTCTGTTACTTGGACGTACTCCCCTCTCCTCAACAAGTTGTTTTGCCAGCTGGC 3 9 0
GAAGACCTGCCCCGTGCAGCTGTGGGTCAGCTCCCCACCCCCACCCAATACCTGCGTCCG 1 2 0
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
GAAGACCTGCCCCGTGCAGCTGTGGGTCAGCTCCCCACCCCCACCCAATACCTGCGTCCG 4 5 0
CGCTATGGCCATCTATAAGAAGTCGGAGTTCGTGACCGAGGTTGTGCGGCGCTGCCCCCA 1 8 0
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
CGCTATGGCCATCTATAAGAAGTCGGAGTTCGTGACCGAGGTTGTGCGGCGCTGCCCCCA 5 1 0
CCATGAACGCTGCTCTGACAGTAGTGACGGTCTTGCCCCTCCTCAGCATCTCATCCGAGT 2 4 0
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
CCATGAACGCTGCTCTGACAGTAGTGACGGTCTTGCCCCTCCTCAGCATCTCATCCGAGT 5 7 0
GGAAGGAAATTTGCGGGCCAAGTACCTGGACGACAGAAACACTTTTCGACACAGTGTGGT 3 0 0
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
GGAAGGAAATTTGCGGGCCAAGTACCTGGACGACAGAAACACTTTTCGACACAGTGTGGT 4 3 0
GGTGCCTTATGAGCCACCCGAGGTTGGCTCTGACTATACCACCATCCACTACAACTACAT 3 6 0
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
GGTGCCTTATGAGCCACCCGAGGTTGGCTCTGACTATACCACCATCCACTACAACTACAT 6 9 0
GTGTAACAGTTCCTGCATGGGAGGCATGAACCGGCGGCCCATCCTCACTATCATCACCCT 4 1 9
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
GTGTAACAGTTCCTGCATGGGAGGCATGAACCGGCGGCCCATCCTCACTATCATCACCCT 7 5 0
GGAAGACTCCAGTGGAAACGTGCTGGGACGCAACAGCTTTGAGGTACGCGTTTGTGCCTG 4 7 9
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
GGAAGACTCCAGTGGAAACGTGCTGGGACGCAACAGCTTTGAGGTACGCGTTTGTGCCTG 8 1 0
TCCCGGGAGAGACCGCCGGACTGAGGAGGAGAATTTCCACAAGAAGGGGGAGCCTTGTCC 5 3 9
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
TCCCGGGAGAGACCGCCGGACTGAGGAGGAGAATTTCCACAAGAAGGGGGAGCCTTGTCC 8 7 0
TGAGCCACCCCCCGGGAGTACCAAGCGAGCACTGCCTCCCAGCACCAGCTCCTCTCCCCC 5 9 9
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
TGAGCCACCCCCCGGGAGTACCAAGCGAGCACTGCCTCCCAGCACCAGCTCCTCTCCCCC 9 3 0
★ GCAAAAGAAGAAGCCACTAGATGGAGAATATTCCACCTTTCAGATCCGTGGGCGTGAACG 6 5 9
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
GCAAAAGAAGAAGCCACTAGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAACG 9 9 0
CTATGAGATGTTCAGGAATCTGAATGAAGCCTTGGAGCTGAAGGATGCCCAGAGTGGAAA 7 1 9
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
CTATGAGATGTTCAGGAATCTGAATGAAGCCTTGGAGCTGAAGGATGCCCAGAGTGGAAA 1 0 5 0
GGAGCCAGGGGGAAGCAGG 7 3 8
| | | | | | | | | | | | | | | | | | |
GGAGCCAGGGGGAAGCAGG 1 0 6 9
Fig 4 Sequence comparison of p53 DNA binding region from TVT cell and normal cell Point mutation (T→C) at No 964
is indicated with asterisk (Phe→Ser in amino acid)
Trang 8292 Young-Ki Choi and Chul-Joong Kim
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