Mitochondrial nucleic acids as internal standards for blot hybridization analyses

Mitochondrial Nucleic Acids as Internal for Blot Hybridization Analyses Clifford

G. Tepper,*

Mary

M. Pater,?

Alan

Pater,t

Standards

Hui-min

Xu,*

and

*Department of Laboratory Medicine and Pathology, UMD-New Jersey Medical School, Newark, New Jersey 07103; and tFaculty of Medicine, Health Science Center, Memorial St. John’s, Newfoundland, Canada AlB3V6

Received

November

George

P. Studzinski*

185 South Uniuersit?i

Orange Auenw, of Newfoundland,

6,lSSl

A plasmid, designated ~72, constructed from human lung carcinoma DNA inserted into the promoterless herpes simplex virus thymidine kinase gene pML-TKBgl II vector, hybridizes strongly to human nucleic acids on Southern and Northern blots. The portion of the DNA insert responsible for the strong signal following hybridization to human DNA or RNA is a 167-bp 3 terminal portion of the mitochondrial 16s ribosomal RNA gene. The expression of this gene is constitutive in the several human cell lines that were tested and is unaffected by exposure to cytotoxie chemicals that alter the expression of nuclear genes. This plasmid offers an excellent tool for studies of perturbations of gene expression and for controlling for the variations in sample preparation, loading, and transfer in Southern or Northern analysis of nucleic acids. Q nxw ~cedemic PIG... loo.

Quantitative Northern blot analysis relies on analyzing equivalent amounts of RNA in different samples, on uniform transfer of the RNA, and on reproducible efficiency of hybridization between different experiments. This is frequently addressed by determining the steadystate mRNA levels of “housekeeping” genes such as l&S rRNA, P-a&in, or glyceraldehyde-3.phosphate dehydrogenase. However, while these genes appear to be stably expressed under some conditions, marked variation in their steady-state levels can be observed when growth rates vary, or following exposure of cells to antimetabolites or drugs (l-4). A recently reported specific example is the downregulation of the fi-actin gene in HL-60 cells exposed to 1,25-dihydroxyvitamin D, [1,25(OH),D,]’ observed by Solomon et al. (5). ‘Abbreviations used: 1,25(OH),D,, 1,25-dihydroxyvitamin D,; mt, mitochondrial: eMEM, modified Eagle’e medium; SDS, sodium do-

We have found that under most circumstances mitochondrial (mt) gene expression is subject to less perturbation than the commonly used housekeeping genes and can therefore be used in Northern blot analysis as a superior control for the variations in recovery during cell lysis and sample preparation and in loading, transfer, and hybridization of nucleic acid samples (6,7). In this report we describe the construction, specificity, and uses of a vector containing a fragment of mtDNA. MATERIALS

AND

METHODS

Subline Gl was subcloned from an early passage of HL-60 human promyelocytic leukemia cells (a generous gift of Dr. R. W&on). Subline 240 was cloned from HL60 cells purchased from the American Type Culture Collection (Rockville, MD). This subline shows delayed response to inducers of mono&c differentiation while Gl cells are highly sensitive to those agents. U937 human monocytic leukemia, K562 human myelogenous leukemia, and COLO 320 human colon carcinoma cell lines were also purchased from the ATCC. These cell lines were grown as suspension cultures in RPM1 1640 medium (Mediatech, Washington, DC) supplemented with 10% fetal calf serum (Hyclone Laboratories, Inc., Logan, UT), 2 rn~ glutamine (Mediatech), and 50 IU/ ml penicillin-50 pg/ml streptomycin (Mediatech), in a 37°C incubator with a humidified environment of 5% CO, in air. The A-549 human lung adenocarcinoma cell line was grown in modified Eagle’s medium (a-MEM). Mouse Lta TK- cells were maintained as monolayer cultures in a-MEM and 10% fetal calf serum as previously described (8). decylsulfate; TK, thymidinekinase; ND1,NADHdebrdrogenasesub. unit 1; TGFB, transforming growth factor 8; SSC, standard citrate.

saline

TEPPER

128 DNA

Isolation

Isolation

High-molecular-weight DNA was isolated from samples of 10’ cells by a slight modification of established methods (9,lO). Spectrophotometric readings at 260 and 280 nm were used to determine both the concentration and the quality of the DNA (11).

Isolation

of

Mitochondria

and

mtDNA

Mitochondria were isolated according to the “onestep” procedure described by Bogenhagen and Clayton (12). The entire isolation was performed at 4’C; 108cells were washed in TD buffer (134 rn~ NaCl, 5 rn~ KCl, 0.7 rn~ Na,HPO,, 2.5 rn~ Tris, pH 7.5) followed by homogenization in Mg reticulocyte standard buffer (10 rn~ N&l, 1.5 rn~ MgCl,, 10 rn~ Tris, pH 7.5). The mitochondria were stabilized against osmotic shock by the addition of one-sixth volume of 2.0 M sucrose, 35 rn~ EDTA, 50 rn~ Tris, pH 7.5. The nuclei were then pelleted and their DNA was isolated as previously described for whole cells. The supernatant was layered over 1.5 M sucrose, 5 rn~ EDTA, 10 rn~ Tris, pH 7.5, and centrifuged. The interface of this one-step sucrose gradient contained the mitochondria that were collected and then pelleted by high-speed centrifugation. The mtDNA was isolated by resuspension of the mt pellet in 10 rn~ N&l, 10 rn~ EDTA, 50 rn~ Tris, pH 7.5, and lysis was by the addition of SDS (final concentration of 0.5%). The DNA was then purified by phenol extraction and ethanol precipitation.

Southern

ET AL

Blot

Analysis

DNA samples (10 pg) were electrophoresed through 1.2% agarose gels and subsequently depurinated and denatured as described by Wahl et al. (13). The DNA was then transferred to Biotrans nylon membranes (ICN) in 20x SSC for 16-24 h according to the method of Southern (14). Immobilization of the DNA was accomplished by air-drying the membranes followed by baking in an 80°C oven for 2-5 h. The membranes were prehybridized for 8 h at 42°C in hybridization buffer containing 50% formamide, 5X SSC, 5~ Denhardt’s solution, 200 pg/ml denatured salmon sperm DNA, and 0.5% SDS. Probes were either nick-translated (15) or 5’.end-labeled (16) and then added to the buffer (1.2 X lo6 cpm/ml hybridization buffer) and hybridization was continued for 16-24 h (17). Membranes were successively washed in 2X, 0.5X, andO.lX SSC bufferscontainingO.l% SDS at room temperature for 15 min each, followed by a final wash in 0.1X SSC/O.l% SDS at 42°C for 30 min. While still damp, the membranes were wrapped in plastic wrap and exposed to X-ray film at -80°C.

of

Total

Cellular

RNA

RNA was isolated from cell cultures using the guanidinium/cesium chloride method (l&19). The RNA was dissolved in water and quantitated spectrophotometritally at 260 nm. In addition, its integrity was verified by the presence of 18s and 28s ribosomal RNA bands on 1.2% agarose minigels. Northern

Blot

Analysis

Twenty micrograms of each RNA sample was denatured and electrophoresed on 1.2% formaldehyde-denaturing agarose gels in 4.morpholinepropanesulfonic acid buffer at 5 V/cm for 4 h (20,21). The gels were rinsed with water several times and soaked in 10X SSC for 45 min. Transfer of RNA to nylon membranes, hybridization conditions, and washing were identical to those for Southern analysis. When it was necessary to increase the stringency of hybridization, a 0.1X SSC/ 0.1% SDS wash was performed at 65”C, instead of 42’C. The plasmid, pRyc 7.4 (Dr. G. Rovera), contained a 1.2. kbp cDNA insert composed of exons 2 and 3 of the cmyc protooncogene inserted at the P&I site of pBR322. The phagemid HHC189 was a construct containing a 1.1.kbp p-actin cDNA inserted into the EcoRI site of pBluescript SK- (ATCC). RESULTS Construction

of p72

Clone ‘72 (~72) containing 600 bp of mtDNA was constructed in the following manner. DNA from the human lung adenocarcinoma cell line A-549 was digested to completion with Mb01 and subsequently ligated into the BglII-digested and dephosphorylated pML-TK-BglII vector, which contains a promoterless thymidine kinase (TK) gene of the herpes simplex virus. Exogenous DNA inserts containing promoters were detectedby their ahility to induce TK expression. An average of one insert per plasmid was obtained in ampicillin-resistant bacterial colonies. Pools with 0.3 pg plasmid DNA from each of five minipreps of such were tested on Lta TK- cells. Two pools were found to produce colonies of cells growing in hypoxanthine-aminopterin-thymidine (HAT) selection medium. The p71-~75 pool produced approximately 50 colonies in two 60.mm plates of approximately 5 x lo6 TK- cells. p72 was identified as the expressing construct when resistant colonies with p72 alone were obtained in three of three such plates. Sequencing

of

the p72

Insert

The DNA sequences from vided the enhancer-promoter pression of the TK gene were TK sequence proximal end of

the p72 insert that proactivity to allow the exthen characterized. The the insert was sequenced

BLOT

HYBRIDIZATION

by the chemical cleavage method of Maxam and Gilbert (22). To this end, phosphatase-treated Sau3AI fragment (600 bp) was kinased with [y-‘*P]ATP. This was digested with HincII, yielding 532- and 68-bp fragments. The larger fragment was electroeluted and the first 264 bp were sequenced. This fragment contains a typical TATA box sequence 136-140 nucleotides from the Mb01 site of insertion upstream ofthe TK sequence. There is also a transcription start site, pyrimidine-adenine-pyrimidine (TAC) sequence, 24-26 hp downstream of the TATA box. No ATG sequence was found downstream of the putative mRNA start site before the Mb01 site used for the construction of the ~‘72 plasmid. Thus, the ATG from the TK gene is expected to be the initiator codon for this construct. Analysis

of the

Insert

Sequence

An analysis of the 264.bp sequence was performed with the PC/Gene system, data banks release 3.0 (updated in 1990). When the sequence was analyzed in the inverse complementary arrangement, 100% homology was found to human mitochondrial DNA sequences. A data bank search for splice junctions found one potential splice acceptor site 162 nucleotides upstream from the MboI insertion site. Two possible sites of binding to eukaryotic ribosomes were found at nucleotides 123 and 172. These data, and the consensus TATA and mRNA initiation site sequences, suggest that the ~72 insert acts as a promoter for the TK gene. The entire 16,569-bp human mitochondrial genome has been sequenced and all of its genes located, including those for the 12s and 16s rRNAs, 22 tRNAs, and 13 peptides (23,24). The 264.bp sequence aligns with nucleotides 3063-3326 on the &DNA map. This corresponds to the 3’.end of the 16s rRNA gene (positions 3063-3229), the entire tRNA’““‘oUR’ gene (positions 3230-3304), and the 5’-end of the gene for NADH dehydrogenase subunit 1 (positions 3307-3326). The remaining 336 bp of the SouSAI fragment extend more 3’ into the latter gene. Thus, the ~72 insert corresponds to positions 3063-3662 of the mitochondrial genome as shown in Fig. 1. Although the mitochondrial genome contains no introns, the exonlintron splice site mentioned above maps closely to the junction of the 16s rRNA and tRNAL”‘om’ genes (map position 3224). Nucleotide 123 (mtDNA map position 3185) has the highest potential of being a ribosome binding site since it is located in the 16s rRNA gene. Validation of the ~72 Insert DNA Probe

As a Mitockondrinl

Uncut DNA from K562 and COLO 320 cells was fractionated on a 1.2% agarose gel and transferred nylon membrane. The ~‘72 insert was excised from construct with BglII, nick-translated, and used

sizeto a the as a

ANALYSIS

OF DNA

129

probe for homologous sequences on the membrane. A DNA band of 16.6 kbp was detected (Figs. 2A and 2B). This is the mitochondrial genome (-16.6 kbp) since nuclear DNA migrates as a smear of much higher molecular weight (223 kbp), if it enters the gel at all. DNA was also obtained from COLO 320 cells exposed to ehemotherapeutic agents such as the topoisomerase II inhibitors, VP-16 and VM-26, and the DNA synthesis inhibitor, Am-C. The cellular mitochondrial DNA content was not affected by any of these agents (Fig. 2B). DNA isolated from HL-60 (clones Gl and 240), U937, K562, and COLO 320 cells was digested with BarnHI and subjected to a similar analysis. The BamHI digestion generated a single, approximately 16.6-kbp DNA fragment corresponding to the full-length, linearized mitochondrial genome (Fig. 3A). This is the expected result since mtDNA possesses only one restriction site (i.e., position 14262) for BamHI (25). The presence of a single BamHI recognition site was confirmed in another experiment with HL-60 (Gl) and U937 cells (Fig. 3B). EcoRI-digested DNA from these cell lines was also analyzed. As shown in Fig. 3B, a single, distinct band corresponding to a DNA fragment of approximately 8 kbp was visualized. This was also expected since EcoRI cuts mtDNA at nucleotide positions 4125 and 12,644 (25). generating two fragments, one of which consists of 8.05 khp encompassing 12 genes, including the entire 16s rRNA and tRNALe”‘“oR’ genes and most of the NADH dehydrogenase(1) gene, and this fragment hybridizes to the ~72 probe (Fig. 3B). In addition to the HL-60 (Gl) and U937 cell lines, Southern analysis of EcoRI digests of HL-60 (240), K562, and COLO 320 DNA was performed, yielding the same 8.0-kbp band (Fig. 4A). The membrane was then stripped of the ~72 probe and hybridized with an end-labeled synthetic oligonucleotide probe (20.mer) corresponding to the first 20 bp of the inverse complement of the 264.bp sequence and therefore specific for the 16s rRNA gene (Fig. 4B). Identical bands were visualized on this Southern blot with respect to fragment size and relative band intensities using either the ~72 probe or the 16s rRNA gene-specific oligamer, showing that the ~72 probe detects the mitochondrial 16s rRNA gene. However, a much longer autoradiogram exposure was required when the oligamer was used and this was therefore less useful as a probe for a loading control. To further validate the nature of the probe, DNA isolated from HL-60 (Gl) mitochondria was digested with EcoRI followed by Southern blot analysis. DNA from isolated nuclei and whole cells was analyzed for comparison. As seen in Fig. 5, the HL-60 (GI) mtDNApreparation (lane 1) yields a signal intensity much higher than that of the nuclear and whole cell DNA preparations (lanes 2 and 3, respectively) when the blot is probed with ~72. This was direct evidence that the ~72 probe is specific for mitochondrial DNA. The light 8-kbp band in

TEPPER

130

1’

ET AL

(subunit’,tI

(subunit 2)

16s rRNA Synthetic IO-mu

In ail the cell types tested, three RNA species of approximately 2.6,1.6, and 1.2 kb were detected on North-

em blots probed with ~72 (Fig. 6A), while only two were seen when the 16s rRNA 20.mer was used (Fig. 6B). The latter were superimposable on the lower two bands of the p72-probed blots. The rate of migration on the gel indicates that the 1.6.kb signal results from hybridization of either probe to the 16s rRNA gene transcript. The 1.2. kb and 2.Gkb (very faint) bands seen in these blots probably represent cross-hybridizations of the probes to nuclear-encoded messages rather than to the

FIG. 2. Hybridization of the p72 probe to the full~length mitocbon~ drial genome in DNA samples. Southern blot analysis was performed on undigested DNA from (A) I%62 and(B) COLO 320 cells. In addition to untreated cells, DNA samples from cell cultures erposed to “P-16 (5 &, “M-26 (5 PM), and Ax-C (10 w, for 12 b were analyzed. A HindIII digest of h bacteriophage DNA was used for molecular weight markers CM).

FIG. 3. Identification of mxtochandrial DNA by the ~72 probe: (At IO cg of BornHI-digested DNA from HL-60 (Gl and 240). U937, K562. and COLO 320 cells was subjected to Southern blot analysis as described under Materials and Methods. (B) In a confirmatory experiment. BomHl IB) and EcoRI (E) digests of HL-60 (Gl) and U937 DNA were analyzed with nick-translated ~72.

the nuclear DNA sample was most likely the result of contamination of the isolated nuclei with mitochondria since the procedures for isolation of both organelles share the same initial steps. Northern HL-60,

Blot Analysis U937, K562,

of and

Total Cellular RNA COLO 320 Cells

from

BLOT

HYBRIDIZATION

FIG. 4. Southern analysis ofEcoRI-digestedDNAwith oligonucleotide probe. Conditions and cell lines were BSin Fig. 3. Autodiagrams are of a membrane hybridized (A) with labeled ~72 insert, followed by stripping and (B) rehybridization with Wend-labeled mitochondrial 16s rRNA aligomer.

NADH dehydrogenase subunit 1 (NDl) gene transcript, or other mitochondrial gene transcripts, for the following reasons: (i) The ND1 message is encoded by 956 bp of mt genome, so the size of the expected mRNA does not coincide with these bands, (ii) no significant homology can be found between the ~72 insert sequence and the remainder of the mitochondrial genome, (iii) the 1.2.kb band on the Northern blot is also detected with a synthetic 20-me1 oligonucleotide probe for 16s rRNA, and (iv) when blots prohed with ~‘72 were subjected to a high-stringency wash (i.e., 0.1X SSCIO.l% SDS at 65°C) this nonspecific binding was eliminated. M

1

2

3

8.0 kbp

FIG. 5. Greater ~72 signal intensity in B mitochondrial DNA preparation than in nuclear or whole cell preparations: IO pg of EcoRI-digested DNA from “L-60 (Gl) mitochondria (lane I), nuclei (lane 21, and whole cells (lane 3) was subjected to Southern blot analysis using the ~72 probe.

ANALYSIS

OF DNA

FIG. 6. Northern blot analvsis with mitochondrial DNA orobes. (A) ~72 and (B) the mitochon&ial16S rRNA gene 20.me1 weie used as wobes: 20 ,zg of total cellular RNA from HL-60 (G1 and 2401, US&, K562, and COLO 320 cells was analyzed 88 described under Materials and Methods. &es of the RNA bands were determined using a 0.24 to 9.5.kb RNA ladder (BRL) as a molecular weight marker (M). The final wash ~88 high stringency (0.1X SSC/O.l% SDS at 65°C).

Stable

Expression

of the

16-S rRNA Gene in K562 Cells to Chemical Compounds We have rehybridized Northern blots to labeled p72 probe in a routine manner following visualization of transcripts whose abundance varies under different experimental conditions and did not detect any differences due to the treatment used. For instance, K562 cells are induced to differentiate with the chemotherapeutic drug Am-C (26). This process is inhibited by preexposure to 1,25(OH),D, (27). Activation of the differentiation pathway results in the alteration of the steady-state mRNA levels of many genes, including the globin genes and several protooncogenes such as c-myc (26,ZS). In contrast, levels of 16s rRNA remain constant when K562 cells are exposed to Ara-C, or a combination of the two agents 1,25(OHLD,, (Fig. 7). Steady-state 16s rRNA levels also remained unchanged when K562 cells were treated with cycloheximide or actinomycin D (Fig. SA). Nuclear transcript levels were compared simultaneously with those of mitochondrial 16s rRNA by allowing the ~72 probe to remain hybridized to the 2.6- and 1.2-kb transcripts, as well as to the 1.6-kb 16s rRNA, by omitting the final wash of the membrane at 65”C, resulting in intense signals being produced by the cross-hybridizing nuclear transcripts. Treatment with cycloheximide resulted in stabilization and therefore greater abundance of the 2.6. and 1.2.kb nuclear transcripts, while the abundance of 16s rRNA remained steady. When RNA polymerase activity was inhibited by actinomycin D, the cross-hybridizing 2.6-kb band was eliminated and the 1.2.kb

following Exposure

TEPPER

132

L FIG. 7. Steady-state levels of mitocbondrial 16-S rRNA in K562 cells treatedwith 24 rm 1,25(OHJ,D,, IO PM Ara-C, or 1,25(OH),D, + AK-C. Northern blot anahsis was mrformed on RNA isolated from cells after the following treatments:;ntreated controls, lanes I and 9: I,%(OH),D, for 4 h, lane 2; Ara-C for 15 min. 4 h, and 24 h, lanes $5, and 7, respectively; and 1,25(OH),D, pretreatment (4 h) + Ara-C for I5 min, 4 h, and 24 h. lanes $6, and 8, respectively. A-DNA (Hind111 digest) was denatured and used as the molecular weight marker (MJ. The wash was high stringency

band reduced, but the 16s rRNA level was not altered. In contrast, c-myc mRNA levels were greatly reduced by treatment with actinomycin D (Fig. SB), as is expected of nuclear-encoded transcripts. Equal loading of the lanes was verified by rehybridization to the P-actin probe (Fig. SC). This demonstrates that transcription of 16s rRNA in the mitochondria is not affected by those agents and illustrates its usefulness as an internal standard for monitoring of gene expression. DISCUSSION In Northern blot analysis, it is important to assess the uniformity of sample preparation and placement, the efficiency of gel penetration, the transfer to membrane, and the hybridization to the probe. This is most often achieved by comparing the band intensity, following hybridization of the membrane with a radioactive probe for the gene transcript of interest with the intensity of the band that identifies the mRNA of one of the constitutively expressed genes. Such “housekeeping” genes are assumed to be expressed at a constant rate, and while this is often the case, situations in which the expression of such genes fluctuates are being encountered. For instance, stimulation of AKR-2B mouse embryo fibroblasts by transforming growth factor fi (TGFP) causes a rapid increase in the steady-state level of actin mRNA due to upregulation of transcription (29). In contrast, @- and y-a&in gene transcripts decrease in abundance during the adipogenic conversion of 3T3-Ll mouse fibroblasts (30). The level of glyceraldehyde-3phosphate dehydrogenase mRNA increases dramatically during this differentiation (31), but is downregulated in other instances such as when human A-549 lung adenocarcinoma cells are treated with 12-O-tetradecanoylphorbol-13.acetate (TPA) (1) and when AKR-2B cells are exposed to TGFP (1). In the Chinese hamster lung fibroblast line K12, the cDNA clone p3AlO detects

ET AL

a gene constitutively expressed at both the permissive (35°C) and nonpermissive (40.5”C) temperatures (32). Although this is a suitable reference gene for this study, its expression increases when K12 cells are subjected to glucose deprivation (33). In contrast, no variation in the levels of transcripts detected by the ~72 mitochondrial probe has so far been observed (6,7). A method employing the use of an external standard for the normalization of multiple RNA samples during Northern blot or RNase protection analyses has been described by Toscani et al. (34). The external standard consisted of an in vitro synthesized cRNA that was added to the cell lysate, thus becoming integral with the cellular RNA content. The authors demonstrated that the external control content remains at a fixed concentration in the total RNA sample during its isolation and analysis, and it permitted normalization of the mRNA abundance under experimental conditions that result in changes of the concentration of &-microglobulin or i3actin mRNA, the conventional internal standards (35). The normalization using as internal standards transcripts of mitochondrial genes (i.e., 16s rRNA), presented here, provides an alternative means to achieve this objective that also circumvents possible variations in recovery during cell lysis. It should be noted that both of these procedures for normalization are suitable for the examination of total cellular RNA, but not transcripts selected by oligo-(dT)cellulose for polyadenylation. In addition to the advantages of the critically important constant levels of expression and transcript stability, the use of the ~7‘2 probe offers the advantage of sensitivity since there are 103-10’ copies of the mitochondrial genome in each cell (12) (which was likely instrumental in the isolation of this probe) and the mi-

FIG. 8. Stability and continued expressmn of 16% rRNA in IS62 cells emosed to cvcloheximide (CHXI and actinomycin D (Act D). Total c&Iar RNA was isolated from untreated K562 cells (Co) and cells exposed for 4 h to eycloheximide (20 pg/mIJ or actinomycin D (5 &ml). (A) Northern blot analysis performed using the ~72 probe, under conditmns of low stringency of hybridization. The 1.6.kb mitochondrial 16s rRNA and the 2.6 and 1.2.kb cross-hybridizing nuclear transcripts are indicated. Molecular weights were determined usmg B 0.24. to 9.5.kh RNA ladder. ,B) The same membrane was stripped and rehyhridized with a c-my probe. (0 The membrane was stripped again and rehybridized to a o-a&in probe.

BLOT

HYBRIDIZATION

tochondrial rihosomal RNA genes are constitutively expressed (24) and are under the influence of a powerful promoter. The mitochondtial rRNA transcripts are approximately 60.fold more abundant than the most abundant mitochondrial mRNA, because there is a bidirectional transcriptional termination sequence beginning eight bases downstream from the 16s rRNA gene, and mitochondrial rRNA has a half-life Z- to .&fold longer than that of mitochondrial mRNA (25,36). The high abundance of mitochondrial rRNA relative to mitochondrial mRNA also helps explain why the p72 insert detects 16s rRNA, but not the ND1 transcript. Further, it should be stressed that no instance in which treatment of the cells with metabolic inhibitors or cytotoxic drugs for periods of 24 h or less resulted in a change in the level of transcripts which hybridize to the p72 probe has yet been encountered (6,7,27). The p72 probe is also useful for the analysis of Southern blots. The cellular content of mitochondrial DNA is altered by only a few chemical compounds that are not frequently used in eukaryotic cell studies, such as chloramphenicol, oligomycin, and ethidium bromide (37-411, and their effect on mitochondrial DNA requires several days to develop. Thus, mitochondrial DNA may provide a reference probe of choice in many experimental situations.

The authors are grateful to Joe Menonna for preparing the mito~ chondriaII6S rRNA olieonucleotide and Dr. Giovanni Rovera for the gift of the c-myc probe.“We also thank Claudine C. Marshall for her assistance in the preparation of this manuscript. The work WBB supported by NIH Grant ROl CA 44722.04 from the National Cancer Institute (G.P.S.) and by the MRC and NC1 of Canada (A.P. and M.M.P.). REFERENCES 1. Pertovaara, L., Sistonen, L., Bois, T . J., Vogt, P., Keski-Oja, J., and Alit& K. (1989) Mol. Cell. Btol. 9, 1255-1262. 2. Lin, A. Y., and Lee, A. S. (1984) Proc. Natl. Acod. Sei. USA 81, 988-992. 3. Lehtola, L., Nister, M., Holtta, E., Westermark, B., and Alit&, K. (1991) Cell ReguL. 2,651-r+%. 4. Schwartz. E. L., and Nilson, L. (1988) J. Cell, Physiol. 136,526530. 5. Solomon, D. H., O’DriscoII, K., Sosne, G., Weinstein, I. B., and Cayre, Y. E. (1991) Cell Growth Diffemntiution 2, 187-194. 6. Brslvi, Z. S., and Studzinski, G. P. (1986) J. Cell. Biol. 102,22342243. 7. B&vi, Z. S., and Studzinski, G. P. (1986) J, Cell. PhysioI. 181, 43-49. 8. Pa&, M. M., Peter, A., dihlayorca, G., and Smiley, J. R. (1982) Virology 117,536X40. 9. Enrietta, P. J.. Payne, L. N., and Hayman, M. J. (1983) CeN 36, 319-379. 10. Gross-Bellard. M., Oudet, P., and Chambon, P. (1972) Eur. J Riochem. 36,32.

ANALYSIS

133

OF DNA

II. Maniatie, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, New York. 12. Bogenhagen, D., and Clayton, D. (1974) J. Biol. Chem. 249, 7991-7995. 13. Wahl, G. M., Stern, M., and Stark. G. R. (1979) Pm. N&l. Aead. Sci USA 76, 3683-3687. 14. Southern. E. M. 11975) J Mel Bml. 98. 503-517. 15. Righy, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P. (19771 J. Mol. Bid. 113, 237-251. 16. Maxam. A. M., and Gilbert, W. (1977) Proc N&l. Acod Sci. USA 74.560-564. 17. Meinkoth, J., and Wahl, G. (1984) Anal Bioehem. 138,267-284. 18. Glisin, V., Crkvenjakov, R., and Byus, C. (1974) B~ochemktq 13, 2633-2637. 19. Chirgwin, J. M.. Przybyle, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Emchemistry 1% 5294-5299. 20. Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H. (1977,

Biochemists

16,

4743-4751.

21. Goldberg, D. A., (1980) Proc. N&l. Acad. Ser. USA 77,5194-5798. 22. Maxam, A. M., and Gilbert, W. (1980) in Methods in Enzymology ,Grossman. L.. and Moldave. K.. Eds.). Vol. 65.~11.499-560. Aca.. demic Press, &n Diego, CA. 23. Anderson, S.. Bankier, A. T., Barrell, B. G., deBruijn, M. H. L., Coulson, A. R., Drouin, J., Eperan, I. C., Nierlich, D. P., Roe, B.A., Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, R., and Young, I. G. (1981) Nature 290, 457-465. 24. Clayton, D. A. (1984) Annu. Reu Biochem. 53,573-594. 25. O’Brien. S. J. (1990) Genetic Maps, Locus Maps of Complex Geneomes. Book 5. Human Maps, Cold Spring Harbor Laboratory Press, New York. 26. Luisi-DeLuca, C.. Mitchell, T., Spnggs, D., and Kufe, D. W. (1984) J. Clm Inuest. ,4,821-827. 27. Moore. D. C., Carter, D. L., Bhandal, A. K., and Studzinski, G. P. (1991) Blood 77, 1452-1461. 28. Tonini, G. P., Radzioch, D., Gronherg, A., Clayton, M., Blasi, E., Benetton, G., and Varesio, L. (1987) Cancer Res. 47,4544-4547. 29. Leaf, E. B., Proper, J. A., Getz. M. J.. end Moses, H. L. (1986) J CeM

Physior

127.

83-88.

30. Bernlohr. D. A., Angus, C. W.. Lane, M.D., Bolanowski, M. A., and Kelley, T . J. (1984) Proc. NotI Acod. Sci. USA 81, 54685472. 31. Benito. M., Porras. A., Nebreda, A. R., and Santos, R. (1991) Science 253,565-568. 32. Lee, A. S., Delegeane, A., and Scharff, D. (1981) Proe. Natl. Acod. Sei USA 78, 4922-4925. 33. Lin, A. Y. and Lee, A. S. (1984) Proc. Natl Acad. Sci. USA 81, 988-992.

34. Toscani. A., Soprano, D. R., Casenza, S. C., Owen, T . A., and Soprano, K. J. (1987) Anal Biochem. 186,309-319. 35. Kelly, K., Cochran, B.. Stiles, C., and Leder, P. (1983) Cell 35, 603-610. 36. Ojela, D., Montoya, J., and Attardi, G. (1981) Nature 290,470474. 37. King, M. E., Godman, G. C., and King, D. W. (1972) J. Cell Biol 53, 127-142. 38. Lardy, H. A., Johnson, D., and McMurray, W. C. (1958) Arch Biochem

Biophys.

713.587-597.

39. Soslau, G., and Nass, M. K. (1971) J Cell Biol. 51, 514-524. 40. Desjardms, P., Frost, E., and Morais, R. (1985) Mol. Cell. Bill. 1163-1169. 41. Nass, M. M. K. (1970) Proe N&l. Aead. Sci. USA 67.1926-1933.

5.