Base sequences of highly repetitive components in nuclear DNAs from rat liver and rat-ascites hepatoma

Cancer Letters, 55 (1990) 201-208 Elsevier Scientific Publishers Ireland

201 Ltd.

Base sequences of highly repetitive components from rat liver and rat-ascites hepatoma Y. Ikeda, K. Nakamura,

Cell Biology Division, Toyama

930-01

Faculty

N. Iwakami,

of Pharmaceutical

in nuclear DNAs

Y. Hibino and N. Sugano

Sciences,

Toyama

Medical and Pharamceutical

Uniuersity,

Sugitani,

(Japan)

(Received

15 August

1990)

(Accepted

4 October

1990)

Summary

Introduction

A 370-bp highly repetitive component in each of the nuclear DIVASfrom rat liuer (RL) and ratascites hepatoma (AH) was isolated by Hindlll digestion and cloned in pUC9. Ten of the resulting clones were arbitrarily selected and sequenced. Heterogeneity size was found in 7 of the RL clones (366-369 bp), but in only 2 of the AH clones (369 bp). The sequence homology was 64.6% among the RL clones; 80.3% among the AH clones. The base compositions were AT-rich, ranging from 61.1 W to 64.7%. Many A and/or T runs consisting of 2-5 bases were interspersed throughout each The restriction sites reported sequence. preoiously [IZ], EcoRi, Haefll, Hindlfl, HinjZ and HphI sites, were confirmed in almost all of the clones. In the present experiment, 12 kinds of the sites were further found in both RL and AH clones.

Highly repetitive DNA sequences have been found in many eukaryotic species by restriction endonuclease digestion studies [17]. Of them, a 370-bp EcoRI repetitive component consisting of alternating 92- and 93-bp units was demonstrated to be present in the nuclear DNA from rat liver and was sequenced to be AT-rich (61.9%) [12]. Moreover, the 93-bp EcoRI repeat units in the nuclear DNA from rat liver were cloned and also sequenced to be AT-rich (58-64%) [16]. In contrast, a 372-bp Hind111 repetitive component containing 4 EcoRI sites at 93-bp intervals was reported to be present in and GC-rich (68%) in the DNA from Novikoff rat-ascites hepatoma [3,4]. Thus, to elucidate whether or not such a GC-rich sequence is a common feature of ascites-hepatoma and/or carcinoma cells, the present studies are concerned with the base sequences of Hind111 repetitive components in the nuclear DNAs from rat liver and rat-ascites hepatoma.

Keywords: ascites hepatoma; repetitive DNA; sequence

Materials and Methods

of

rat;

highly

Correspondence to N. Sugano.

0 1990 Elsevier Scientific Publishers 0304-3835/90/$03.50 Published and Printed in Ireland

Materials The ascites hepatoma AH414 has been established from a rat hepatocarcinoma induced by an azo dye [ 191. The hepatoma cells proIreland Ltd.

202

liferated actively until the 8-9th day after intraperitoneal transplantation to a male Donryu rat (120- 150 g) . To collect the ascites cells, the hepatoma-bearing rat was laparotomized on the 8th day. To free from the peritoneal exudate cells, the collected cells (105/ml) were cultured in a Dulbecco’s modified Eagle medium (Flow Lab.)/20% fetal bovine serum/kanamycin (100 pg/ml)/streptomycin (50 pg/ml)/NaHCO, (3.7 mg/ml) , with a plastic bottle (Falcon), under 5% CO, at 37°C for 4 days. The culture was repeated once in the same way. Liver was removed from a normal rat at the same age as the laparotomized rat. Isolation of Hind111fragments Nuclei were prepared from each of the liver and the ascites-hepatoma, as described previously [6,7]. Highly polymerized DNA was prepared from the nuclei according to the method of Marmur [9] and incubated with HindII1 (200 units/50 pg DNA, Takara) in 60 mM NaCI/lO mM Tris (pH 7.5)/7 mM MgCI, at 37°C for 3 h. The incubated material was extracted with equal vols. of phenol and then chloroform/isoamyl alcohol (24: 1, v/v). The final aqueous phase was dialyzed against distilled water and lyophilized. The lyophilized material was electrophoresed on a slab gel of 6% polyacrylamide at 30 mA for 3 h [ll]. To recover the Hind111 fragments, the band corresponding to 370 bp (base pairs) was cut out and electrophoresed with an ISCO sample concentrator (Model 1750). Extraction and purification of plasmid DNA DHl or JM109 cells (Escherichia co/i) carrying pUC9 plasmids were cultured in a LB medium/ampicillin (100 pg/ml) at 37°C for 15 h and subjected to lysis by alkali [ 141. The lysate was centrifuged in a Hitachi RP40 rotor at 17 500 rev. /min for 20 min. The supernatant was mixed with 0.6 vol. of isopropanol for 20 min and centrifuged at 2400 x g for 10 min. The pellet was rinsed with ethanol and dried in vacua. The dried material was dissolved in 10 mM Tris (pH 80)/l mM EDTA (TE buffer) and mixed with CsCl (1 g/ml)/ethidium bromide (540 pg/ml) . The mixture was centrifuged in a

Hitachi RP67VF- 134 rotor at 56 400 rev. /min at 15°C for 15 h. The band corresponding to the superhelical form was removed and washed with an equal vol. of n-butanol. The washed material was dialyzed against TE buffer and subjected to ethanol precipitation. The precipitate was dried in vacua and taken as the plasmid DNA. Cloning and sequencing of Hind111fragments The plasmid DNA (2-5 pg) was incubated with Hind111 (6 units as described above and heated at 65°C for 10 min. The heated material was dialyzed against 100 mM glycine (pH 9.5)/ 1 mM MgCI,/O. 1 mM ZnCl, and incubated with an alkaline phosphatase (5 units, Takara) at 37°C for 1 h. The incubated material was extracted with phenol and then chloroform/ isoamyl alcohol, as described above. The final aqueous phase was dialyzed against TE buffer and subjected to ethanol precipitation. The dried precipitate (0.1 pg DNA) was incubated with a T4 DNA ligase (350 units, Takara) in 66 mM Tris (pH 7.6)/10 mM dithiothreitol/6.6 mM MgCI,/O. 1 mM ATP/HindIII fragments (0.2-0.5 pg) at 14°C overnight. The incubated material was mixed with the competent DHl or JM109 cells (2.5 x 108) in 0.1 M CaCI, on ice for 30 min. The mixture was kept at 37°C for 2 min and cooled on ice for 5 min. Then, the cells were cultured at 37 “C for 60 min as described above. An appropriate vol. of the culture was transferred onto an agar plate of LB medium/ampicillin (100 pg/ml) spread with 100 mM IPTG (50 pl)/2% X-gal (20 ~1) and incubated at 37°C overnight. Each of the resulting white colonies was cultured again in the liquid medium/ampicillin (100 pg/ml) . The plasmid DNA was extracted and purified from the cultured cells, as described above. The purified DNA was incubated in 0.2 N NaOH at 37°C for 5 min and recovered by addition of ethanol/5 M ammonium acetate (25:2, v/v) at -80°C. The recovered DNA was rinsed with 70% ethanol and dried in vacua. The dried material was subjected to the sequence analysis according to a dideoxy method [ 151. The complementary sequence was determined with a reverse primer (Primer RV, Takara).

20

HinfI

30

Hinfl 40

EcoRI Hphl 50 60

HinfI 70

80 90

100 110

EcoRI 120

EcoRI 130

-TTcgcctag AAATTTTgaT TcAATTcgtg cAAATTTTTc tatatcacgA AcagtccacT TaTTactact gcggcctaTT gggAActAAc cAAATTcacc AAgTTa-tga gat-gggctc acAAAATTTT

gTTcgcctag AAATTTTgaT TccaTTcgtg AAAATTaTTc tatatcccgA Acagtccacg taTTactact gcggcctatc gggAActAAA tgAATTTacc gaTTTactca gatacggctc aggAAgTTTT

370 369

catAATTcTT TTAAgg-aca cataTTacAA gagcctgcta ctgggAActA ActgAATTca cAAAgAAAca gtgTTTccgT TcgTTAAtac gTTgctctgt ctcgAAAAAc gatAAAtcTT TAAAAgtaca tataTTTcAA gagtctgctc atgggAActA ActgaTTTca cAAAgAAAga gtgTTTcagT TcgTTAAAAc gTTgctctat ct-gAAtAAc

TatAAAtcTT TAAAAgtaca cataTTacAA gagcaggcta ctaggAActA ActgAATTca cAAAgAAAca gtgTTTcagt gcgTTAAAAc gTTgctctat cTTgAAtAAc

gatAAAtcTT TAAAAgtaca tataTTTcAA gagtctgctc atgggAActA ActgaTTTca cAAAgAAAga gtgTTTcagT TcgTTAAAAc gTTgctctat ct-gAAtAAc

gatAAAtcTT TAAAAgtaca cataTTacAA gagcctgcta ctgggAAcgA ActgAATTca cAAAgAAAca gtgTTTcagT TcgtgAAAAc gTTgctctat cTTgAAcAAc

gatAAAtcTT TAfJAAgcaca-AAATTacAA gagcctgcta cTTggAActA ActgAATTca cAAAggAAca gtgTTTcagT TcgTTAAAAc gTTgctctat cTTcAAtgac

TatAAAtcTT TAAAAgtaca cataTTacAA gagcaggcta ctaggAActA ActgAATTca cAPAgAAAca gtgTTTcagt gcgTTAAAAc gTTgctctat cTTgAAtAAc l l * l ** l l l l ** *** l * l* l l l ** *t *** l l l ** l *

c4

C6 c7

CB

c9

Cl0

ty is found among the clones.

Sequences of the clones (Cl-ClO) the position lacked single base. The restriction

Fig. 1.

C5

gatAAAtcTT TcAATTTaca cataTTacAA gagccggcta ctgggatctA ActgcaTTca catagAAAca gtgTTTcagT TcgTTAAAAc gTTgctctat cTTgAAtAAc

c3

62.7

64.7

62.6

64.0

63.2

64.0

62.8

62.4

64.0

62.9

AT%

of the HindlII-fragments from rat liver. A and T runs are emphasized by the capitals. Hyphen indicates sites reported previously [12] are underlined. Asterisk indicates the position at which the base heterogenei-

370

368

369

366 369

370

369

gatAAAtcTT TAAAAgtaca cacaTTacAA gagcctgcta ctgggAActA ActgAATTca cAAAgAAtcc gtgTTTcacT TcTTTAAAAc gTTgctctat cTTgAAtAAc

c2

369

BASES

Cl

EcoRI

280 290 300 310 320 330 340 350 360 370 TatAAAtcTT TAAAAgtaca cataTTacAA gagcaggcta ctaggAActA ActgAATTca cAAAgAAAca gtgTTTcagt gcgTTAAAAc gTTgctctat cTTg-atAAc

Hinf!

ATTcgcctag AAATTTTgaT TTcaTTcgTg AAAATTTTTc tatatcccgA AcagtccacT TaTTactact gag-cctaTT gggAAgAAAc tgAATTcacc AAgtatctga gaTTctgatc accAAATTTT -ATTcgcctag AAAATTTgaT TccgTTcgTg AAAATTTTcc tatatcccgA AcagtccacT TaTTactagt gcggcctaTT gggAActAAc cgAATTcacc atgTTactca gTTTcggctc accAAATTTT * l l* l *** l ** * * l * l * l *** t * **** * *t * *** l ****** l * * *** t l ** l

CB

c9

270

ATTcgcctag AAATTTTgaT TgcaTTcgtg AAAATTTTTc tatatcccgA AcactccacT TaTTactact gcggcctaTT gggAActAAc cgAATTccac AATTTTctga gaTTcggctc -ccAAATTTT

c7

Cl0

ATTcgcctag AAATTTTgaT TccgTTcgtg AAAATTTTcc tatatcccgA AcagtccacT TaTTactagT TcggcctaTT gggAActAAc cgAATTcacc atgTTactca gaTTcggctc accAAATTTT -gTTcgcctag AAATTTTgaT TccaTTcgtg AAAATTaTTc tatatcccgA Acagtccacg taTTactact gc=tatc gggAActAAA tgAATTTacc gaTTTactca gatacggctc aggAAgTTTT

c3

c2

c4

HinfI

c5 C6

140

Cl

HphI

***;_

tacAAgcatg tcccaTTggg AATTccctF

tacAAgcatg tcccaTTggg AActcactgA

tacAAgcatg tcccaTTggg AActcactgA cacAAgcgtg tcccaTTggg AAAtcactga

AAgcTTaTTa catgcgAAtc ctaTTgggAA cctactcAAT TcAAcatgat acTTagaTTc ccTTccTTAA AAtgTTgctc gataTTgAAA AgcAAActca tgcAAgcatg tcccaTTgg=actF * l * *** l * l l ** l l l * l * ** * ** * *** * l * l *** l ** l *

-AAgcTTaTTa catgcgAAtc ctaTTgggAA cctactcAAT TcAAcatgat acTTagaTTc cgTTccTTAA AAtgTTgctc gataTTgAAA AgcAAActca MgcTTaTTa cAAgtgAAtc ctaTTgggAA tctactgAAT Tcaccatgat acTTagaTTa cgTTccTTTT AAtgTTgctc tataTTgAAA AgcAAActca AAgcTTaTTa catgcgAAtc ctaTTgggAA cctactgAAT Tcaccatgat acTTagAAtc cgTTccTTAA AAtgtcgcta tataTTgAAA AgcAAActca AAgcTTaTTa catgcgAAtc ctaTTgggAA cctaTTgAAc TTaccAAgat acTTagaTTc cgTTccTTTa tatgTTgcta tataTTgAAA Agcacactca

-PAgcTTaTTa catgcgcatc ctaTTgggAA ccAAAtgAAT TcaccAAgat acTTTgaTTc tgTTccTTAA AAtgTTgcTT TataTTgAAA Agcacactca tacAAgcatg tcccaTTggg AActcactF AAgcTTaTTa catgtgAAtc ctaTTgggAA cctagtgAAT Tcaccatgat acTTagTTTc cgTTcctgAA AAtgTTgcta tatacggAAA Agcacactca tacAAgcatg tcccaTTggg AActcAAAK AAgcTTaTTa cAAgtgAAtc ctaTTgggAA tctactgAAT Tcaccatgat acTTagaTTa cgTTccTTTT AAtgTTgctc tataTTgAAA AgcAAActca cacAAgcgtg tcccaTTggg AAAtcactga

-AAgcTTaTTa catgcgAAtc ctaTTgggAA cctactcAAT TcAAcatgat acTTagaTTc cgTTccTTAA AAtgTTgctc gataTTgAAA AgcAAActca tacAAgcatg tcccaTTggg AActcactgA AAgcTTAAta cctgcAAAtc ctaTTTggAA cctatcgAAT Tcacgatgat acTTagaTTc ccTTcaTTAA AAtgTTgcta tataTTTAAA Agcacactca tacAAgcatg tcccaTTggg AActcactF

10

HinfI HaeIII EcoRI HphI Hphl 150 160 170 180 210 240 190 200 220 230 250 260 ATTcgcctag AAATTTTgaT TccgTTcgtg AAAATTTTcc tatatcccgA Acagtccacc taTTactact gcggcctaTT aggAActAAc cgAATTcacc atgTTactca gaTTcggctc accAAATTTT -ATTcacctag AAATTTTgaT TccaTTcgtg AAAATTTTTc tatatcccgA Aca-tcctcc taTTagtact gcggcctaTT aggAAcctac cgAATTcacc AAgTTactga gaTTcggctc accAAATTTT -ATTcgcctag AAAtaTTgat accaTTcgtg AAAATTTTTc tatatcccgA AcagtccacT TaTTactagt gc=taTT gggacctAAt cgacTTcacc AAgctactgt gaTTcggctc agcAAATTTT

Cl0

CB c9

C6 c7

C5

C3 c4

Cl c2

Hind111

E

Cl ’

HinfI

30

EcoRI HphI

50

HinfI

C8'

AAtgTTgctc tataTTgAAA Agcagactca tacAAgcatg tcccaTTggg AActcactF AAtgTTgctc AAtaTTgAAA ggcAAActca tacAAgcatg tcccaTTggg AActcactx

160

240

HinfI

HphI 250

cAAAtgcacc atgTTactca -gaTTcggctc accAAATTTT cgAATTcacc atgTTactca -gaTTcggctc accAAATTTT cgAATTcacc atgTTactca -gaTTcggctc acccAATTTT

gggAActAAc cgAATTcacc atgTTactca -gaTTcggctc accAAATTTT gggAActAAc cgAATTcacc atgTTacgca -gaTTcggctc accAAATTTT gggAActAAc cgAATTcacc atgTTactca -gaTTcggctc accAAATTTT

gggAActAAc cgAATTcacc atgTTactca gaTTcggctg accAAATTTT

230

EcoRI HphI

260 gggAActAAc cgAATTcacc atgTTactca gaTTTagcac accAAATTTT

220

ATTcgcctag AAATTTTgaT TccaTTcctg AAAATTTTTc tatatcccgA AcagtccacT TaTTactact gcggcctact gggAActAAc ATTcgcctag AAATTTTgaT TccaTTagtg AAAATTTTTc tatatcccgA AcagtccacT TaTTactact gcggcctact gggAActAAc ATTcgcctag AAATTTTgaT TccaTTcgtg AAAATTTTTc tatatcccgA Acagtccact caTTactact gcggcctact gggAActAAc ATTcgcctag AAATTTTgcT TccaTTcgtg AAAATTTT-c tatatcccgA AcagtccacT TafTactact gtggcctaTT gggAActAAc

150

Fig.2. underline

and

Truns

are emphasized

ClO' TAAAAAtctc tAAAAgtaca cataTTacAA gagcaggcta ctaggAActA ActgAATTca cAAAgataca gtgTTTcagT TcgTTAAAAc gTTgctctat cTTgAAtAAc l * l l * * * l ** l* l ** * l ** * l l * * l *

A

369

TAAAAAtctc tAAAAgtaca cataTTacAA gagcaggcta ctaggAActA ActgAATTca cAAAgataca gtgTTTcagT TcgTTAAAAc gTTgctctat cTTgAAtAAc

hepatoma.

369

gatAAAtcTT TAAAAgtaca catcgtacAA gtgcaggcta ctgggAActA ActgAATTca cagagAAAca gtgTTTcagT TcgTTAAAAc gTTgctctat cTTgAAtAAc

03' C9'

of the clones (Cl'-ClO') of HindIlI-fragments from rat-as&es and asterisk are the same as described in the legend to Fig. 1.

370 370

gatAAAtcTT TAAAAgtaca catagtacA4 gagcaggcta ctgggAActA ActgAATTca cAAAgAAAca gtgTTTcagT TcgTTAAAAc gTTgctctct cTTgAAtAAc

gatAAAtcTT TAAAAgtaca catagtAAAA gagcaggcta ctgggAActA AcggAATTca cagagAAAca gtgTTTcagT TcgTTAAAAc gTTgctctat ctagAAtAAc

C6' C7'

Sequences

370

TatAAAtcTT TAAAAgtaca cataTTacAA gagcaggcta ctaggAActA ActgAATTca cAAggAAAca gtgTTTcagT TcgTTAAAAc gTTgctctat cTTgAAtAAc gatAAAtcta tAAAAgtaca catcgtacAA gagcaggcta ctgggAActA ActgAATTca cagagAAAca gtgTTTcagT TcgTTAAAAc gTTgctctat cTTgAAtAAc

C4'

C5'

TatAAAtcTT gAAAggtaca cataTTacAA gagcaggcta ctaggAActA ActgAATTct cAAAgAAAca gtgTTTcagT TcgTTAAAAc gTTgctctat cTTgAAtAAc

63.1

63.1

61.1

62.4

62.4

62.7 61.1

62.7 62.7

62.4

AT%

by the capitals. Hyphen,

370 370

370 370

C3'

370

BASES

TatAAAtcTT TAAAAgtaca cagaTTacta gagcaggcta ctaggAActA ActgAATTca cAAAgAAAca gtgTTTcagT TcgTTTAAAc gTTgctctat cTTgAAtAAc

EcoRI

C2'

290

Cl 0

280

300 310 370 350 360 330 340 320 gatAAAtcTT TagAAgtacg cataTTacAA gagcctgcta ctgggAActA ActgAATTca cagagAAAca gtgTTTccgT TccTTAAAAc gTTgctctat cTTgAAtAAc

270

cgAATTcacc atgTTactca gaTTcggctA AAcAAAcTTT ClO' ATTcgcctag AAATTTTgcT TccaTTcgtg AAAATTTT-c tatatcccgA AcagtccacT TaTTactact gtggcctaTT gggAActAAc cgAATTcacc atgTTactca gaTTcggctA AAcAAAcTTT l l * ** ** l l l l ** l* * l l* ** l * t * *

C9'

CB'

C6' C7'

C5'

C3' C4'

C2'

Cl'

140

HaeIII 180 170 190 200 210 ATTcgcctag AAATTTTgaT TccaTTcgtg AAAATTTTTc tatatcccgA AcagtccacT Tagtactact gcggcctaTT - - -ATTcgcctag AAATTTTgaT TccAAtcgtg AAAATTTTTc tatatcccgA Acagtccacl laliactact gcggcctaTT -ATTcgcctag AAATTTTgaT TccaTTcgtg AAAATTTTTc tatatccAAA Acagtccacc talractact gccgcctaTT - - -_ AAtcgcctag AAATTTTgaT TccaTTcgtg AAAATTTTTc gatatcccgA Acagaccacl IarTactact gcggcctaTT ATTcgcctag AAATTTTgaT TccaTTcgtg AAAATTTTTc tatatcccgA Acagtccact caTTactact gcggcctact

HinfI

ClO' AAgcTTaTTa catgcgAAtc ctaTTgggAA cctactcAAT TcgcAAtgat acTTggaTTc cgTTccTTAA AAtgTTgctc AAtaTTgAAA ggcAAActca tacAAgcatg tcccaTTggg AActcactF * l * ** l ** l * ** * * *** l ** l

C9'

AAtgTTgctc tataTTgAAA AggAAActca tacAAgcatg tcccaTTagg AActcactz AAtgTTgctc tataTTgAAA AgcAAActca tacAAgcatg tcccaTTggg AActcactF

AAgcTTaTTa catgcgAAtc ctaTTgggAA cctactgAAT Tcaccatgat acTTagaTTc cgTTccacAA AAtgTTgctc tataTTgAAA Agcagactcc tacAAgcatg tcccaTTggg AActcactgA

AAgcTTaTTa catgcgAAtc ctaTTgggAA cctactgAAT Tcaccatgat acTTagaTTc cgTTccacAA AAgcTTaTTa catgcgAAtc ctaTTgggAA cctactgAAT Tcaccatgat acTTagaTTc cgTTccacAA AAgcTTaTTa catgcgAAtc ctaTTgggAA cctactgAAT Tcaccatgat acTTagaTTc cgTTccacAA AAgcTTaTTa catgcgAAtc ctaTTgggAA cctactcAAT TcgcAAtgat acTTggaTTc cgTTccTTAA

C6'

C7'

AAgcTTaTTa catgcgAAta ctaTTgggAA cctactcAAT Tcaccatgat acTTggaTTc cgTTccTTAA AAtgTTgctc gataTTgAAA AgcAAActca tacAAgcatg tcccaTTggg AActcactz

40

C4'

20

C5'

C3'

C2'

10

EcoRI 70 80 90 100 110 120 60 130 AAgcTTTTTa catacgAAtc ctaTTgggAA cctactgAAT TcaccatgTT acTTagagtc cgTTccTTAA AAtgTTgTTa cataTTgAAA Agcacactca tacAAgcatg tcccAAtggg AActcactgA AAgcTTaTTa catgcgAAtc ctaTTgggAA cctactcAAT Tcgccatgat acTTggaTTc cgTTccTTAA AAtgTTgctc gataTTgAAA AgcAAActca tacAAgcatg tcccaTTggg AActcactF AAgcTTaTTa catgcgAAcc ctaTTgggAA cctactcAAT TcgcAAtgat acTTggaTTc cgTTccTTAA AAtgTTgctc gataTTgAAA AgcAAActca tacAAgcatg tcccaTTggg AActcactr

Hind111

205

Resaltm and Discussion A 370-bp highly repetitive component in each of the nuclear DNAs from rat liver (RL) and ratascites hepatoma (AH) was isolated by Hind111 digestion and cloned in pUC9. Ten of the resulting clones were arbitrarily selected and sequenced (Figs. 1 and 2). The size heterogeneity was found in 7 of the RL clones (366-369 bp), but in only 2 of the AH clones (369 bp). The sequence longer than 370 bp was not found in any clones. Asterisk indicates the position at which the base heterogeneity is found among the clones. The frequency of the heterogeneity was higher in the RL clones than in the AH clones, in particular, at positions 231-237 in the RL clones (Fig. 1). In consequence, the sequence homology was 64.6% among the RL clones; 80.3% among the AH clones (Table I). However, the average of base compositions was about the same between both RL and AH clones: A, 32.2%, T, 31.18, G, 15.4% and C, 21.3% among the RL clones; A, 32.4%, T, 30.046, G, 15.4% and C, 22.2% among the AH clones. Many A and/or T runs consisting of 2-5 bases were interspersed throughout each sequence. The average number of A or T runs was about the same except 3T between both RL

Table 1. Base homology at each position in the sequences of Hind111fragments from rat liver (RL) and ratascites hepatoma (AH). Homology (%)

% Of total

No. of positions RL

AH

100 90 80 70 60 50 40

239 72 30 21 6 2 0

297 47 11 4 6 4 1

Total

370

370

RL 64.6 19.5 8.1 5.7 1.6 0.5 0.0 100

AH 80.3 12.7 3.0 1.1 1.6 1.1 0.2 100

and AH clones (Table II). A run adjacent to T run longer than 5 bp and/or the reverse were located at positions 67-72, 141-147, 161- 169, 254-260, 269-275 and 344-349. In consequence, highly AT-rich sequence was located at positions 254-275. Cruciform sequences consisting of 10 bases or more were found at positions 1 ll121, 160-170, 263-272 and 325-337. The restriction sites reported previously [ 121, EcoRI, HaeIII, HindIII, HinfI and HphI sites, are underlined. At alternating 92- and 93-bp intervals, 4 EcoRI sites were conserved in C2 and C8 of the RL clones; in Cl ’ , C5 ’ C7 ’ and C8 ’ of the AH clones. Another EcoRI site was irregularly located at positions 120-125 in C9 of the RL clones. In the present experiment, 28 kinds of the sites were further found. Of them, 12 sites, i.e., BsrI, DraI, FinI, HinflII, MaeI, MaeII, MseI, NlaIII, Nsp[7524]1, TaqI, TspEI and TthlllII sites, were present in both AH and RL clones. The others were found in only RL clones (the data are not shown here). The sequence of the most frequent base at each position is shown in Fig. 3. A 372-bp Hind111 repetitive component containing 4 EcoRI sites at 93-bp intervals was reported to be present in a highly GC-rich (68%) in the nuclear and the nucleolar DNAs from Novikoff rat-ascites hepatoma. In the subsequent report the 93-bp EcoRI repeat unit was sequenced to have a GC content of 36.6% 13-51. This complementary sequence is located at positions 319-41 in C5’ and C8’ of the AH clones (Fig. 2). At that time, a 370-bp EcoRI repetitive component consisting of 92- and 93-bp units in the nuclear DNA from rat liver was isolated and sequenced to have a GC content of 38.1% by Pech et al., [ 121. The cloned 93-bp EcoRI units were also sequenced to have a GC contents of 36-428 [16]. Of them, the complementary sequences of 2 clones (2B and 15B) are located at positions 135-227 in C5 ’ and C8 ’ of the AH clones. Moreover, 2B and 15B were shown to have identical sequences, suggesting that there was not an exceedingly large number of different sequence varieties within a given group. We also found that the sequences were iden-

206

Table II.

Clone

Number of A and T runs in the clones of Hind111fragments from rat liver and rat-ascites hepatoma. Run 2A

3A

4A

5A

% Of total A’

2T

3T

4T

5T

% Of total T’

c2 c3 c4 c5

17 18 15 18 16

6 5 5 5 7

5 5 4 3 4

0 0 0

59.5 59.2

2 0

53.5 61.3 58.5

24 21 24 22 19

2 5 5 2 6

2 2 1 3 3

1 1 1 1 0

60.4 60.3 61.5 60.4 57.6

C6 c7 C8 c9 Cl0

18 16 19 18 18

6 7 6 6 5

5 4 5 4 6

0 0 0 0 0

61.2 58.5 63.3 58.3 62.5

25 19 22 26 24

2 6 2 4 4

2 3 3 2 1

1 0 1 1 1

61.1 57.6 60.4 65.3 61.6

Mean S.D.

17.3 1.3

5.8 0.8

4.5 0.8

-

59.6

22.6 2.4

3.8 1.7

2.2 0. 8

0.8 0.4

60.6 2.2

Cl’ C2’ C3’ C4’ C5’

19 17 17 17 17

4 7 7 6 4

4 4 5 5 5

0 0 0 0 0

56.4 60.2 62.5 59.0 55.9

22 24 26 25 22

3 3 1 2 1

2 1 1 1 2

2 2 2 2 1

62.3 62.3 61.6 63.6 55.6

C6’ C7’ C8’ C9’ ClO’

16 16 18 19 19

7 5 3 6 6

5 6 5 4 4

0 0 0 1 1

60.3 57.7 56.0 63.6 63.6

22 22 22 26 26

2 2 2 1 1

2 2 2 3 3

1 1 1 0 0

57.3 58.3 57.3 59.8 59.8

Mean SD.

17.5 1.2

5.5 1.4

4.7 0.7

-

59.5 3.0

23.7 1.9

1.8 0.8

1.9 0.7

1.2 0.8

59.8 2.6

Cl

2.8

*(No. of bases in runs/total no. of A or T) x 100. Cl-ClO, hepatoma. SD., standard deviation.

tical between C9 ’ and Cl0 ’ of the AH clones (Fig. 2). Such a GC-rich sequence as in the DNA from Novikoff ascites hepatoma was not found in any clones. The average of GC contents was 36.7% among the RL clones; 37.6% among the AH clones. All the resulting sequences were in good agreement with that reported by Pech et al. Binding of non-histone protein(s) to a highly

clones from rat liver; Cl ‘X10’,

clones from rat-ascites

repetitive DNA sequence has been evidenced by many investigations. In particular, o-protein from African green monkey cells was shown to bind with about equal affinity to any run of 6 or more AT tracks in duplex DNA [18]. A microtubule-associated protein from porcine brain, MAP,, was elucidated to bind preferentially to an AT-rich sequence in mouse satellite DNA (Sau96.1 fragment) [2]. We have reported

207

at positions 254-275

predicts a bent structure

AAGCTTATE ** CATGCGAAE ******** CTATTGGGZ * * *** * TCACCATG:~ ***** * * CCPAdA:~ [8,20]. Such a structure has been visualized elecG 60 70 60 N 90 100 ACTTAGATTC CGTTCCTTAA AATGTTGCTC TATATTGAAA AGCAAACTCA ***** * * * * * *** * * * * * ** * ** 110 130 150 120 140 TACAAGCATG TCCCATTGGG AACTCACTGA ATTCGCCTAG AAATTTTGAT * * * **** ** * ** * l * * 160 170 160 200 190 TCCATTCGTG AAAATTTTTC TATATCCCGA ACAGTCCACT TATTACTACT ***** ** * * * l *** ** * l * * * *

tron microscopically to be present in mouse, rat and o-green monkey satellite DNAs [lo]. Thus, the highly repetitive and AT-rich DNA associated with non-histone protein(s) might play an important role in the construction of a higher-order structure essential for mitotic division.

210 220 230 T GCGGCCTATT GGGAACTAAC CGAATTCACC AAGTTACT;A 'GATTCGG::: l* * **** * *t** ********** l *** *** **** 260 T 270 260 290 AG 300 ACCAAATTTT GATAAATCTT TAAAAGTACA CATATTACAA GAGCCTGCTA * * * * *** *** * * *** *** l *** l **** ** 310 320 330 350 A 340 CTGGGAACTA ACTGAATTCA CAAAGAAACA GTGT'TTCAGT TCGTTAAAAC ** * * ** * * * * * **$* ** *****

1

2

360 370 GTTGCTCTAT CTTGAATAAC * *** **

Fig. 3.

Sequence of the most frequent base at each posi-

tion in a 370-bp

Hind111 repetive component.

frequent base is represented homology of 60%

or more

The most

3

as the base which shows the

(Z 6/10 bases) at each posi-

4

tion in both RL and AH clones. The bases described on the sequence are available among the AH position 81 represents a base not determined the high degree of base heterogeneity. underlined. Asterisk indicates the position heterogeneity

is found

homology

is determined

among

clones. N at because of the base

associated protein MAP, preferentially binds to a dA/dT sequence present in mouse satellite DNA. EMBO J., 2, 1229- 1234. Fuke, M. and Busch, H. (1979) Hindlll-sensitive sites present once in every four repeats of EcoRI-sensitive sites in Novikoff rat hepatoma DNA. FEBS Lett., 99, 136-140. Fuke, M., Davis, F.M. and Busch, H. (1979) Localization of highly repeated Hindlll fragments of Novikoff rat DNA

6

7

structure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease. B&hem. Biophys. Res. Commun., 52, 504-510. Hibino, Y. and Sugano, N. (1985) An endodeoxy-

the clones. The sequence

to be 52.2%.

that the 370-bp EcoRI repetitive component in the nuclear DNA from rat liver has a selective affinity for the nuclear proteins of 107- and 115-kDa [l]. On the other hand, Radic et al. showed that the cloned mouse satellite DNA (AvaII fragment) is located with the centromeric region and contains a stable curvature which can be alleviated in the presence of distamycin A. This binds preferentially to AT-rich DNA [13]. Accordingly, the AT-rich region of the bend has been inferred to be recognized by a non-histone protein that might be involved in the condensation of centromeric heterochromatin. The 93-bp EcoRI repeat units in rat liver were also shown to be located at centromeres and telomeres of metaphase chromosomes [ 161. In the present studies, the highly AT-rich sequence

for nuclear proteins from rat liver. Biochem. lnt., 19, 871-880. Avila, J., de Garcini, EM., Wandosell, F., Villasante, A., Sogo. J.M. and Villanueva. N. (1983) Microtubule-

to the nucleolus. FEBS Lett., 102, 46-50. Fuke. M. and Busch, H. (1979) A highly repeated fragment in rat DNA. J. Cell Biol., 83, 188. Hewish, D.R. and Burgoyne, L.A. (1973) Chromatin sub-

5

EcoRI sites are at which

Asano, S., Hibino, Y., Ikeda, Y., lwakami, N. and Sugano, N. (1989) Affinity of a DNA with highly repetitive sequence

10

ribonuclease activity in nuclei from rat-ascites hepatoma. Cancer Lett., 29, 245-254. Koo, H-S., Wu, H-M. and Crothers, D.M. (1986) DNA binding at adenine thymine tracts. Nature, 320,501-506. Marmur, J. (1961) A procedure for the isolation of deoxyrtbo-nucleic acid from microorganisms. J. Mol. Biol., 3. 208-218. Martinez-Balbas, A., Rodriguez-Campos, A., Garcia-

11

Ramirez, M., Sainz, J., Carrera, P., Aymami, J. and Azorin, F. (1990) Satellite DNAs contain sequences that induce curvature. Biochemistry, 29, 2342-2348. Peacock, A.C. and Dingman, C.W. (1967) Resolution of

8 9

12

13

multiple rtbonucleic acid species by polyacrylamide gel electrophoresis. Biochemistry, 6, 1818-1827. Pech, M., Igo-Kemenes, T. and Zachau, H.G. (1979) Nucleotide sequence of a highly repetitive component of rat DNA. Nucleic Acids Res., 7, 417-432. Radii, M.Z., Lundgren, K. and Hamkalo,

B.A. (1987) Cur-

vature

condensation

of

mouse

heterochromatin.

14

Sambrook,

satellite

DNA

and

of

Cell, 50, 1101-1108.

J., Fritsch, E.F. and Maniatis, T. (1989) Small-

scale preparations

of plasmid

DNA.

Lysis by alkali.

In:

208

16

Molecular Cloning. A Laboratory Manual, 2nd ed. pp. 1.25-1.28. Editor: C. Nolan. Cold Spring Harbor Laboratory Press, USA. Sager, F. (1981) Determination of nucleotide sequences in DNA. Science, 214, 1205-1210. Sealy, L., Hartley, J., Donelson, J., Chalkley, R., Hut-

17

chison, N. and Hamkalo, B. (1981) Characterization of a highly repetitive sequence DNA family in rat. J. Mol. Biol., 145, 291-318. Singer, M.F. (1982) Highly repeated sequences in mam-

15

malian genomes. In: Int. Rev. Cyto., Vol. 76, pp. 67-112. Editors: G.H. Bourne and J.F. Danielli. Academic Press, New York.

18

19

20

Solomon,

M.J., Strauss,

F. and Varshavsky,

A. (1986) A

mammalian htgh mobility group protein recognizes any strech of six A T base pairs in duplex DNA. Proc. Natl. Acad. Sci. USA, 83, 1276-1280. Sugano, N., Hibino, Y., Choji, Y. and Maeda, H. (1982) Anticarcinogenic actions of water-soluble and alcoholinsoluble fractions from culture medium of Lentinus edodes mycelia. Cancer Lett., 17, 109-114. Ulanovsky, L.E. and Trifonov, E.N. (1987) Estimation of wedge components

in curved DNA. Nature, 326,720-722.