TWO DNA-BINDING NON~ISTONE C~ROMOSO~AL PROTEINS FROM MOUSE MYELOMA TUMOR CELLS WORST Max-Planck-Institut

(Rewired

Abstract-l. MOPC173

Two DNA-binding by hydroxyapatite

BL~THMANN

fiir Virusforschung. 16 January

5 vol of 0.32 M sucrose,

5 mM MgCI, with a Teflon-glass homogenizer, rotating at 1500rev/min for 20 strokes and

Myeloma tumors are thought to originate monoclonally from a single immunoglobulin producing plasma cell (Potter, 1972). Hence homogeneous cell populations with well defined transcriptional activities. the mRNA of immunoglobulin. are available and can be obtained in large amounts by subcutaneous transplantation as solid tumors in highly inbred mice. The nonhistone chromosomai proteins of one of these tumors. MOPC173. have been analysed in this study by hydroxyapatite chromatography and polyacrylamide gel electrophoresis. Gene activity in eukaryotes is generally believed to be regulated at least in part by molecules which bind to specific DNA sequences or sites. There is evidence that nonhistone chromosomal proteins are involved in this process. This class of proteins have long resisted isolation and fractionation into individual proteins due to their heterogeneity and tendency to aggregate. Hydroxyapatite chromatography (MacGillivray et al., 1972; Bliithmann rt al., 1975) and affinity chromatography on DNA columns (Van den Broek er al.. 1973; Allfrey it al., 1973; Kleinsmith, 1973: Bearden & Chandra, 1976: Wakabayashi et (II., 1974: Bliithmann, 1976: Bliithmann. 1978a: Thomas & Pate], 1976: Herrick & Alberts, 1976) have been shown to be useful methods for the isolation of nonhtstone proteins in preparative amounts. Both chromatographic procedures have been used in sequence in this study to isolate two DNA-binding nonhistone chromosomal proteins from mouse myeloma tumor cells. Their binding properties to double-stranded and single-stranded DNA, to repetitive and unique DNA as well as to RNA are presented in this paper.

centrifuged at 1200 9 for IO min. The crude nuclear pellet was resuspended in 5 vol of 0.25 M sucrose. 3 mM M&II. 0.5”,;, Triton X-100. the suspension underlayed in the centrifugation tube with 0.25 vol of 0.32 M sucrose, 3 mM MgC12 and centrifuged at 1200 y for IO mm The nuclear pellet was further purified by sedimentation through 2.2 M sucrose, 1 mM MgClz as described by Chaveau L’I al. (1956). The nuclear pellet was suspended in 50 vol of 0.08 M NaCI, 0.024 M EDTA. pH 8.0. and centrifuged at %OOg for IOmin. The nuclei were Iysed in 5Ovol of 0.01 M Tris. pH 8.0. with a Dounce homogenizer and the chromatin sedimented at 50009 for 10 min. This step was repeated. The chromatin sediment was extracted with 0.73 M NaCI. as described by Johns & Forrester (1969) to remove cytoplasmic contamination. The chromatin was resuspended twice in 50 vol of 0.01 M Tris pH X.0 and the resulting gel sedimented at 15,000 y for 10 min. For further purification the chromatin was centrifuged through I .7 M sucrose. as described by Marushige & Bonner (I 966). Chromatography

and chrQ~ati~1

Nuclei were isolated by a modificatjon of the method of Teng 41 at. (1971). The cell pellet was homogenized in 469

on hydmxyapatitr

Chromosomal proteins were chromatographed at 4 C on hydroxyapatite columns into 2 histone and 4 nonhistone fractions as described previously (Bliithmann rt d.. 1975).

Single-stranded DNA-agarose was prepared by the method of Schaller P? al. (1972). A 20 ml column containing about 20mg mouse DNA was processed as described (Bl~tbmann, 1976). Isolation

of DNA

DNA was isolated from nuclei of myeloma MOPCl73 as described (Bliithmann. 1976). The molecular weight of the DNA was 3.5 x IOh. Lahrling

PROCEDURE

Myeloma MOPC173 was kindly provided by Dr B. Mach (University of Geneva, Switzerland) and maintained as a solid tumor by subcutaneous passage into G-8 weekold BALB/Ca mice (GL. Bomhottg~rd, Danmark). of‘ rtuciri

1978)

employing nitrocellulose filters show that these DNA with a slight preference for repetitive DNA.

INTRODUCTION

Pri~~a~~tio~

West Germany

nonhistone chromosomat proteins were isolated from mouse myeloma chromatography and single stranded DNA-agarose affinity chroma-

tography. 2. Equilibrium competition binding experiments Droteins exhibit stronger binding to single-stranded binding to RNA. however. is poor.

EXPERIMENTAL

Tiibingen,

oj’ DNA

Mouse Ehrlich ascites tumor ceils were maintained in Eagle’s medium supplimented with IO”,, calf serum at a concentration of 5 x fO’cel&ml. Some of the culture (1500ml) were labeled with 15O#Yi (2-“Cl Thymidine and harvested after 24 hr DNA was prepared as described above. The size of native DNA was estimated with a Spinco model E ultracentrifuge employing U.V. optics. Sedimentation coe%cients were converted to lengths using Studier’s equation (1965). The isolated mouse DNA had a molecular

HORST BL~~THMANN

470 wclght of 3 x 10”. Its specific min~‘~g~‘_

activity

was

3850counts

Mouse DNA at a concentration of I mg/ml in 0.06 M sodium phosphat. pH 6.8 was sonicated in a Branson sonificr cqulpped ulth a microtlp to a molecular weight of 470.000 and head denatured. The phosphate concentration was adjusted to 0.17 M and renaturation allowed to occur for IO hr at 60 C to reach a Cot-value of 100. Single strands were separated from double strands on a hydroxyapatite column at 60 C as described by Britten Ct ul. (1974). Rcpctitivc and nonrepetitive DNA fractions were pooled and dlal>scd.

Nonhrstone fractions NH 111 and NH IV were pooled and dtalysed against 0.01 M Trls pH 7.4 containing I”, sarkosyl. Samples were brought to 1.66 M Cs,SO, in 0.01 M Tris pH 7.4 containing I”,, sarkosyl and centrifuged to cqullibrlum at 30,CGOrev/min for 90 hr at 20 C in a SW4lTi rotor. The tubes were punctured and the RNA containing fractions collected and dialysed against 0.01 Trls pH 7.4. The RNA had a sedimentation coefficient of s LO,_ = x.9

15 min) and the RNA in the supernatant determined by the orcinol method of Dische & Schwarz (1937) with E. co/i transfer RNA (Boehringer) as standard. The precipitate was suspended in 5 ml 5”,, trlchloroacetic acid and the DNA hydrolysed at 90‘ for 20 min. After the sample was cooled on ice. the TCA concentration was brought to IO”, and the precipitate collected after 60mm by centrifugation (I 7.000 y I5 min). The DNA in the supernatant was determined by the method of Keck (1956) as modified by Hubbard et uI. (1970) with calf thymus DNA (Sigma) as standard. The nonhistone proteins in the precipitate were dissolved in 0.4ml I N NaOH at room temperature and the protein content determined by the method of Lowry ~‘1 (I/. (1951) which was calibrated for nonhistone proteins by nitrogen determination according to Kjehldahl (Pregel & Roth, 1947). Motrrials Hydroxyapatite was prepared according to the method of Tiselius et al. (1956). The urea used throughout this study was prepared as a IO M stock solution and deionized on a column of Ionen-austauscher V mixed bed resin (Merck). The prepared buffers were stored in the cold and used within two days of preparation.

.~itroc~c//~c/ose ,fi/ft,r us.sa.t The nitrocellulose filter assay. originally developed by Riggs & Bourgeots (1968) and Riggs rl al. (1968) to mcasurc DNA-protein interactions, was used as described previously (Bliithmann. 1976).

Disconttnuous gel electrophoresis was performed in 0.56 x IOcm tubes according to Laemmli (1970) using 3”” stacking gels and IO”,, separating gels. Samples (0.1-0.X ml) containing about 30 pg nonhistone proteins were dialyscd against 0.06 M Tris pH 6.8. 2”,, SDS. IO”,, Glycerol and 5”,, 2-mercaptoethanol and heated for 1.5 min m boiling water prior to electrophoresis. The gels were stained with 0.25”,, Coomassie brilliant blue in 50”

Two DNA-binding nonhistone chromosomal proteins from mouse myeloma tumor cells.

TWO DNA-BINDING NON~ISTONE C~ROMOSO~AL PROTEINS FROM MOUSE MYELOMA TUMOR CELLS WORST Max-Planck-Institut (Rewired Abstract-l. MOPC173 Two DNA-bindi...
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