©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Increased DNA-bending Activity and Higher Affinity DNA Binding of High Mobility Group Protein HMG-1 Prepared without Acids (*)

(Received for publication, November 22, 1994; and in revised form, February 3, 1995)

James P. Wagner Deanne M. Quill David E. Pettijohn (§)

From the Department of Biochemistry, Biophysics, and Genetics, University of Colorado Cancer Center and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recently, DNA ring closure assays showed that high mobility group protein HMG-1 and its close homolog HMG-2 mediate sequence-independent DNA flexion. This DNA-bending activity appears to be central to at least some of the recently elucidated functions of HMG-1/2, such as the enhancement of progesterone receptor DNA binding. Here we show that standard purification procedures utilizing perchloric and trichloroacetic acid can produce HMG-1 significantly deficient in its abilities to bind and bend double-stranded DNA, while acid-independent methods purify HMG-1 that is superior in these respects. Significant losses of DNA ring closure activity were seen upon limited 2-5-h exposures of non-acid-purified HMG-1/2 to perchloric acid and/or trichloroacetic acid. Measurements of the apparent DNA dissociation binding constant (K) of acid-extracted preparations of HMG-1 gave a wide range of values, and only those preparations demonstrating little DNA ring closure activity had K values near the previously published value (10M). The highest ring closure activities and lowest K (<3 times 10M) were obtained for HMG-1 purified without acids. These combined results support the use of alternative, non-acid purification procedures for preserving the DNA-bending activity of HMG-1/2 and suggest that past procedures utilizing acids have led to an underestimation of the affinity of HMG-1 for DNA.


INTRODUCTION

The high mobility group protein HMG(^1)-1 was first identified over two decades ago (1) and has been indirectly linked to a number of eukaryotic cellular processes, such as transcription(2, 3, 4) , recombination(5) , and cellular dedifferentiation (6) , yet its precise role in these processes is only just beginning to be elucidated. In this regard, several laboratories recently described a hitherto unknown activity of HMG-1, the ability to induce sequence-independent DNA flexion, or bending(7, 8, 9) . More significantly, there is evidence that this ability to alter DNA structure by bending may be important to at least some of the cellular functions of the protein.

Paull et al.(7) , for example, have demonstrated that HMG-1 (or the closely related HMG-2) can functionally substitute for the bacterial DNA-bending protein HU in vitro in a recombinational DNA inversion reaction requiring a sharp bending and looping of the DNA, and it is speculated that HMG-1 may function in analogous recombination reactions in eukaryotic systems. Oñate and colleagues (9) have recently demonstrated that HMG-1 serves as an accessory factor in facilitating the binding of the human progesterone receptor to its target DNA sequences in vitro, with evidence for a mechanism based on DNA bending. Studies in our laboratory (^2)also showed a similar stimulatory effect of HMG-1 on transcription factor NF-kappaB, with additional evidence for an effect mediated through DNA bending. The combined results suggest that HMG-1, by altering DNA structure, may be functioning to cooperatively assist the bindings and interactions of transcription factors and other proteins that require energetically unfavorable DNA distortions, such as loops or bends.

During our recent experiments using HMG-1 purified by standard denaturing/oxidizing methods involving perchloric/trichloroacetic acid extractions, we assayed many preparations of the protein that possessed suboptimal DNA-associated activities, i.e. higher dissociation constants, lower DNA-bending activities, and reductions in ability to stimulate binding of transcription factors. Here, we demonstrate that these activities of HMG-1 are improved when HMG-1 is prepared by alternative methods that avoid acids. Our results also indicate that past purification techniques have led to an underestimation of the affinity of HMG-1 for double-stranded DNA. These combined results support the use of alternative non-acid procedures in purifying the protein.


MATERIALS AND METHODS

DNA Ring Closure Assays

T4 ligase-dependent ring closure assays and the origin of the linear DNA fragments used in these assays have been previously described(9) .

Purification of HMG-1 Using Acids

Several preparations of acid extracted calf thymus HMG-1 were purified in the laboratory of Dr. Raymond Reeves, Washington State University, by a modification of the acid-dependent method of Adachi et al.(10) . These procedures included exposures of up to several hours to 5% perchloric acid (PCA) and 25% trichloroacetic acid with final purification on a polybuffer exchange column, PBE 94 (Pharmacia Biotech Inc.)(10) .

Acid-independent Purification of HMG-1 and HMG-2 from Rat Liver

Rat liver nuclei were prepared from fresh rat livers following the method of Blobel and Potter(11) , but including 5 mM DTT and 2 mMpara-chloromercuribenzenesulfonate (pCMBS), a protease inhibitor, in the liver homogenate and TKM buffer (50 mM Tris, pH 7.5, 25 mM KCl, 5 mM MgCl(2)). Phenylsulfonyl fluoride was not used because of a report that it stimulates proteolysis of HMG-1 and HMG-2(12) . Nuclei were centrifuged at low speed and resuspended immediately at 4 °C for 30 min in a severalfold excess volume of hypotonic lysis buffer: 0.1 mM PIPES, pH 6.8, 5 mM CaCl(2), 2 mM pCMBS, 5 mM DTT, 0.5% Triton X-100, 0.5% Nonidet P-40 (or Triton X-114). Nuclei were again centrifuged, and the supernatant was removed. After washing several times in hypotonic buffer minus detergents, nuclei were resuspended at 4 °C for 30 min in a severalfold excess volume of 0.35 M protein extraction buffer: 0.35 M NaCl, 20 mM Tris, pH 7.2, 12 mM MgCl(2), 5 mM EGTA, 5 mM DTT, 2 mM pCMBS. Nuclei were again centrifuged and the supernatant removed as the ``0.35 M extract.'' Nuclei were re-extracted in 0.35 M buffer, and the wash fraction was pooled with the bulk of the 0.35 M extract. The extract was immediately heated for 3 min in boiling water and cooled on ice for 10 min, the denatured proteins were removed by centrifugation, and the supernatant (containing HMG-1/2 and other heat-resistant proteins) was removed as the ``0.35 M boiled supernatant.'' (It was observed that rapid heating of the extract inhibited proteolysis of the 28-kDa form of HMG-1 to its 23-kDa form (p23), which was seen in the absence of heating and which was reported by others to form quickly in rat liver extracts(5) . 3 mM additional fresh DTT was added to the boiled extract, and the extract was stored on ice.

Following essentially the ``non-acid'' method of Adachi et al.(10) , but without prior dialysis of the extract, the 0.35 M boiled supernatant was chromatographed on a PBE 94 column equilibrated with 20 mM Tris, pH 7.5, 0.35 M NaCl, 5 mM DTT and using approximately 1.5 ml of PBE 94 resin per starting rat liver. HMG-2 and HMG-1 eluted at about the 0.6 M and 0.75 M points, respectively, in a 0.35-1 M NaCl gradient. These steps purified HMG-1 and HMG-2 to >90% homogeneity with 33% yield of starting ring closure activity (see Table 1). Purified HMG-1 and HMG-2 were desalted on Centriprep 10 devices (Amicon) versus 5 mM Hepes, pH 7.6, 5% glycerol, 0.1% Tween 20, and concentrated to 100 ng/µl by volume reduction on the Centripreps. Fresh DTT was added to 5 mM, and the proteins were frozen in 10-µl aliquots at -70 °C. Occasionally, a significant contaminant of about 40 kDa was retained with HMG-1 after the PBE column. Carboxymethyl cellulose was found to not bind this contaminant, but to avidly retain HMG-1, indicating that this resin may be useful for achieving further purity of HMG-1, if needed.



Perchloric and/or Trichloroacetic Acid Treatment of Non-acid-extracted HMG-1/2

Several different preparations of non-acid-extracted HMG-1 and HMG-2 (HMG-1/2) of varying degrees of purity were used to test the effects of PCA and trichloroacetic acid on the ring closure activity. Aliquots of the HMG-1/2 proteins, at protein concentrations between 45 ng/µl and 3 µg/ul total protein, were made 5% in PCA, and placed at 4 °C for different times as specified, and the acid was then neutralized by the addition of a 21-fold volume excess of 0.1 M Tris, pH 8. The Tris buffer also served to resuspend any precipitated protein. The solutions were reconcentrated by volume reduction on Amicon Microcon or Centricon microconcentrators (10,000-kDa molecular weight cutoff). Control aliquots not exposed to PCA were taken through the same manipulations as the acid treated aliquots, and equivalent amounts of each sample were assayed in the ring closure assay. The procedure for measuring trichloroacetic acid-induced inactivation was similar, except that the HMG-1/2 aliquots were made 25% in trichloroacetic acid and untreated control tubes received an equal volume of T.E. (10 mM Tris, 0.1 mM EDTA, pH 8). Although some adsorptive losses were observed during reconcentration on the Amicon devices in these inactivation experiments, the losses were found to be reproducible. Moreover, a PCA inactivation experiment utilizing a neutralizing buffer of higher pH (eliminating the need to reconcentrate the control and treated solutions) demonstrated an activity loss for the PCA-treated aliquot that was nearly identical to that measured using the microconcentrators.

Electrophoresis and Staining of Protein Gels

Electrophoresis of proteins was on 10% polyacrylamide gels with 4% stacking gels(13) . Gels were silver-stained as described elsewhere(14) . Tween 20 at 0.05% in gel buffers helped reduce silver stain backgrounds.

Quantitative Determination of Protein Concentrations

Pierce's colorimetric bicinchoninic acid assay was used to determine protein concentrations(15) . DTT was removed to use the standard protocol. Histone H1 (Life Technologies, Inc.) was used as a standard.

Amino Acid Sequencing

N-terminal Edman degradation protein sequencing was performed on non-acid-purified HMG-1 on an Applied Biosystems model 477A protein sequencer in the Microsequencing Facility of the University of Colorado Health Sciences Center. The identity of the protein as HMG-1 was verified by an exact match of the first 8 residues to the published sequence, G-K-G-D-P-K-K-P(16) .

HMG-1:DNA Binding Assayed via Gel Mobility Shift

Standard binding buffer contained 10 mM Tris-acetate, pH 7.5, 0.1 mM EDTA, 50 mM potassium glutamate, 2 mM MgCl(2), 10 mM DTT, 10% glycerol, 100 µg/ml bovine serum albumin. 0.1 ng of a P-labeled restriction fragment was incubated 15 min at 25 °C with specified amounts of HMG-1 protein (in molar excess of DNA) in 10 µl of binding buffer, and the mixture was layered onto an 8% polyacrylamide gel made up in 5 mM Hepes pH 7.6, 5 mM potassium glutamate, 5 mM DTT, 5% glycerol. Gels were loaded running at 10 V/cm to reduce the electrophoretic dead time and after the samples entered the gel matrix, electrophoresis was continued at 4 V/cm for 2 h at 25 °C. Recirculation of the cathodic and anodic buffers was necessary to maintain a neutral pH within the gel. After electrophoresis, gels were dried, visualized by phosphorimaging on a Molecular Dynamics Series 400 PhosphorImager, and quantitated using the ImageQuant software. The K(d) was estimated as the protein concentration at the point in the titration where half of the input DNA had been complexed with protein, with protein in molar excess(17) . Some gel shift experiments with linear DNA followed essentially the procedure of Pil and Lippard (18) which utilizes different parameters, including an alternate DNA binding buffer (designated here as buffer B): 10 mM Hepes pH 7.9, 1 mM EDTA, 10 mM MgCl(2), 10 mM KCl, 0.05% Nonidet P-40, 200 µg/ml bovine serum albumin, 0.1 mM DTT and 20% glycerol.


RESULTS

Purification of HMG-1 and HMG-2 without Using Acids

Studies using ligase-catalyzed DNA ring closure assays indicate that HMG-1 and HMG-2 are the major sequence-independent non-histone DNA-bending proteins in mammalian nuclei(7, 8, 9, 19) . During the course of our recent experiments, significant variations were observed in the DNA-bending activities of different preparations of HMG-1 made by standard acid extraction methods. To avoid the possible detrimental effects of acid, a non-acid procedure for the purification of HMG-1 and HMG-2 from rat liver nuclei, a modification of the non-acid method of Adachi et al.(10) , was developed. The procedure (see ``Materials and Methods'' and Table 1) isolates HMG-1 and HMG-2 in high purity, yield, and activity without the use of harsh acid treatments, exposure to strong oxidizing agents (perchloric acid) (20) , acetone treatments, or multiple rounds of freeze/thawing, and the proteins are maintained under reducing conditions (in 5 mM DTT or more) throughout the isolation.

The heating step in the procedure, although providing a manyfold purification of HMG-1/2 and inhibiting proteolysis (see ``Materials and Methods''), may also be somewhat harmful to the proteins, as about 25% of the starting ring closure activity is lost on boiling the 0.35 M NaCl extract 3 min (Table 1). Some of this loss is probably due to trapping of HMG-1/2 protein within the heat-sensitive proteins as they are precipitated, but some may also be due to inactivation. Ideally, a future method might substitute a milder procedure for this heating step. Adachi et al.(10) reported that HMG-1 and 2 can be purified directly from 0.35 M NaCl extracts of porcine thymus by chromatography on PBE 94. Our experience indicates that this is probably insufficient in purifying HMG-1/2 from rat liver.

Exposure of Active HMG-1 to Perchloric Acid and/or Trichloroacetic Acid Significantly Diminishes Its DNA Ring Closure Activity

Purified or partially pure preparations of non-acid-extracted HMG-1/2 were exposed to 5% PCA and/or 25% trichloroacetic acid for differing times to verify the suspicion that acids and oxidizing agents inactivate the proteins. A 4-h PCA treatment caused a significant drop in the amount of 88 bp of monomer circle formed during ring closure compared to that formed using untreated HMG-1/2 (Fig. 1, lanes 1 and 2). Densitometric quantitation shows this to be a 77% reduction in the ratio of monomer circle reaction product:total DNA for the treated sample compared to the unexposed control (data not shown). As described previously(21) , short DNA fragments significantly less than 150 bp do not form the monomer circle reaction product in the absence of active DNA-flexing proteins as demonstrated in control reactions (lane 3). Other experiments not shown here demonstrated that exposure to PCA for times as short as 3 h gave significant losses (>35%) of ring closure activity, and the activities generally showed further decreases with increasing times of exposure. It should be noted that it is not uncommon for the HMG proteins to be exposed to PCA for several hours or longer in large scale acid-dependent purification schemes. Trichloroacetic acid was also found to be detrimental to the DNA-bending activity of HMG-1/2 (Fig. 1, lanes 4-6). After a 2-h incubation in 25% trichloroacetic acid, the neutralized HMG-1/2 (lane 5) showed a substantial loss (45%) in ring closure activity relative to the untreated control (lane 4). Another aliquot (lane 6), after sequential exposure to 5% PCA for 5 h and 25% trichloroacetic acid for 2 h, lost nearly 70% of the original ring closure activity.


Figure 1: Effects of PCA and trichloroacetic acid on the ring closure activity of the HMG-1/2 proteins. Standard ring closure incubations contained, in addition to 10 ng of cohesively ended DNA and 40 units of T4 DNA ligase, the following: lane 1, an aliquot of partially pure HMG-1/2 prepared without acids; lane 2, an aliquot identical to the lane 1 material but exposed to PCA for 4 h on ice; lane 3, no additional proteins; lane 4, an untreated aliquot (approximately 5 ng) of non-acid-purified HMG-1/2 proteins; lane 5, an aliquot identical to the lane 4 material but exposed to trichloroacetic acid for 2 h on ice; lane 6, an aliquot identical to the lane 4 material but exposed to PCA for 5 h then trichloroacetic acid for 2 h on ice. It was previously demonstrated that the DNA bands migrating at the indicated positions MC and ML are the monomer circle and monomer linear DNA, respectively(21) . Lanes 1 and 2 utilized an 88-bp DNA fragment, lanes 3-6 a 94-bp fragment.



The above ring closure assays used amounts of HMG-1/2 less than required to convert all the DNA to monomer circles in the reactions. Under these conditions, there is a linear relationship between the amount of DNA cyclized and the amount of active HMG-1 in standard ring closure reactions (data not shown), suggesting that an observed fractional decrease in ring closure activity after acid treatment directly reflects the losses incurred.

Active HMG-1 Binds DNA with Higher Affinity than Previously Estimated

Gel mobility shift methods were developed to study the binding of HMG-1 to double-stranded DNA fragments of different sequences (see ``Materials and Methods''). A typical binding titration done at high protein:DNA molar ratios shows that roughly 50% of the DNA was bound at an HMG-1 concentration of 6 nM (Fig. 2, lanes 1-4). The retarded band is assumed to represent the complex of equimolar amounts of HMG-1 and the DNA fragment, because higher concentrations of HMG-1 result in a ladder of increasingly retarded bands (data not shown). Repeats of this experiment have indicated that the K(d) (the apparent K(d) for the equimolar protein:DNA interaction) of active HMG-1 is in the range 2-8 nM under standard conditions (see ``Materials and Methods''). When HMG-1 preparations demonstrating a K(d) in the 2-8 nM range were assayed in binding buffer B (see ``Materials and Methods''), K(d) values slightly higher (3-20 nM), but overlapping the former range, were found (Fig. 2, lanes 5-10).


Figure 2: Gel mobility shift assays titrating the binding of HMG-1 to linear DNA under different conditions. Lanes 1-4, incubations and electrophoresis on 8% polyacrylamide performed under standard conditions. Mixtures containing constant concentrations (0.06 nM) of P-labeled 207-bp linear DNA were incubated 15 min at 25 °C with the varied nanomolar concentrations of HMG-1 protein shown above the lanes. Lanes 5-10, incubations and electrophoresis performed under the conditions of Pil and Lippard (18) (see also ``Materials and Methods''). Mixtures containing constant concentrations (0.07 nM) of P-labeled 207-bp linear DNA were incubated with the varied nanomolar concentrations of HMG-1 shown above the lanes, before electrophoresis on a 10% polyacrylamide gel. Bands corresponding to the free and complexed DNA are indicated by Df and Dc, respectively.



Acid-extracted HMG-1 preparations showed much variability in their K(d) values, sometimes giving a K(d) of 10M or greater. The DNA binding data for several different preparations of HMG-1, including the corresponding K(d) of each preparation is given in Fig. 3. Also indicated (Fig. 3B) is the ring closure activity of each preparation, estimated by titrations of each in ring closure assays (data not shown). There was a consistent relationship between the K(d) of an HMG-1 preparation and its ring closure activity: those with the lowest K(d) values correspondingly had the highest specific ring closure activity (units/µg of HMG-1 protein). It is noteworthy that the preparation with the highest affinity for DNA (and also the most ring closure activity) was the preparation prepared by the non-acid procedure. The discrepancy between the low K(d) values determined here for active HMG-1 and earlier reports that the K(d) for HMG-1 was about 10M in buffer/salt mixtures similar to buffer B (18) (see also (22) ), may be attributable to purification using acids, non-ideal storage conditions, and/or use of recombinant HMG-1. Indeed, the acid-extracted preparation designated as prep 2 shows a K(d) approximating the previously reported values (Fig. 3).


Figure 3: DNA-associated activities of several different HMG-1 preparations. Panel A, binding titrations of three representative HMG-1 preparations to linear DNA. The approximate K values obtained from the data for these different preparations are as follows: non-acid-purified HMG-1, 2.5 nM; acid-extracted HMG-1 (prep 1), 32 nM; acid-extracted HMG-1 (prep 2), 650 nM. The sizes of the linear DNAs used in these titrations are 94, 144, and 207 bp, respectively, for the non-acid-extracted preparation, acid-extracted prep 1, and acid-extracted prep 2. Panel B, the corresponding specific DNA ring closure activity (units/µg of HMG-1 protein) of the three HMG-1 preparations described in Panel A. Units of ring closure activity are as described for Table 1.




DISCUSSION

Through its ability to bind and bend double stranded DNA in a sequence-independent manner, protein HMG-1 appears to be serving, at least in one of its roles, as a general accessory factor that facilitates the binding and interactions of other DNA-binding proteins (7, 9, 19, 23) .^2 In this regard, the results of the present report are of significance in that they demonstrated that the most common purification protocols utilizing perchloric and trichloroacetic acid extraction can lead to inactivation of HMG-1 as measured by two important parameters, its ability to bind DNA with high affinity and its ability to flex double stranded DNA as measured by DNA ring closure assays.

Short exposures to both PCA and trichloroacetic acid were shown to have significant detrimental effects on the ring closure activity of different non-acid-extracted preparations of HMG-1/2 (Fig. 1). These results are in agreement with several previous studies, undertaken before the DNA-bending activity of HMG-1/2 was known, which showed that these acids could affect other properties of HMG-1/2. Cockerill et al.(24) , for example, saw a significant difference in the circular dichroism spectrum of perchloric acid-extracted HMG-1 versus HMG-1 prepared under nondenaturing conditions, which was interpreted as a decrease of 24% in the relative alpha-helical content of the acid extracted HMG-1. Their results are particularly interesting in light of the recent NMR structural determination of one of the two conserved DNA-binding domains (or boxes) of HMG-1(25, 26) , which show it to be composed chiefly of three alpha-helices arranged in a V-shape, possibly providing the contacts that are responsible for constraining the DNA in a bent form. It seems possible that some of the losses in DNA-bending (and DNA-binding) activities observed in the present report for acid-extracted HMG-1 could be due to perturbations in the alpha-helices of these DNA binding domains.

Oxidation of cysteine sulfhydryl groups, to form intramolecular disulfide bridges, has been reported to occur rapidly during purification of the HMG-1/2 proteins in the absence of reducing agents or EDTA (27, 28) and to adversely affect the interaction of HMG-1/2 with histone H1(29) . Although not specifically examined in the present report, oxidation of cysteines, 3 of which lie within the two DNA binding domains of mammalian HMG-1/2(30, 31) , could be contributing to the adverse effects of the oxidizing PCA (20) and/or nonreducing environments seen here. The non-acid purification procedure outlined under ``Materials and Methods'' included 5 mM DTT in all buffers to minimize these effects.

The studies of HMG-1 binding to double-stranded DNA indicated a K(d) in the range of 2-8 nM when the interaction utilized protein prepared in the absence of acids and/or demonstrating high levels of ring closure activity (>200 units/µg HMG-1). It was noted that this K(d) differed by several orders of magnitude from published values(18, 22) . Experiments using different DNA fragments of unrelated sequence yielded similar K(d) values for the same HMG-1 preparations (results not shown), indicating that DNA sequence was not responsible for the discrepancy. The finding that the lowest K(d) values were obtained for non-acid-extracted HMG-1, while only acid-extracted preparations demonstrated values in the published range, suggests that past purification procedures utilizing acid extractions and other harsh treatments have led to an underestimation of the affinity of HMG-1 for DNA. Variations in the time of exposure to the acids or variations in the effects of the acids on different HMG-1 preparations may explain the wide range of binding affinities determined here for different acid extracted HMG-1 preparations and may be responsible for some of the conflicting reports in the literature about the properties of HMG-1/2.

There was a consistent correspondence between the ring closure activity and K(d) for individual preparations of HMG-1. Such a correlation is expected since the HMG-1/2 protein must first bind the DNA before it can bend it. Previous studies suggest that the DNA binding domains themselves may be primarily responsible for the DNA-bending activities of HMG-1/2 since each domain alone mediates DNA cyclization(9) . Titrations show, also as expected, that maximal ring closure activities occur at concentrations of HMG-1 above its K(d) and in molar excess of the DNA fragment in the ring closure incubation (data not shown).

The alternative procedure described here for purifying HMG-1/2 avoids acids and is as convenient to use as the conventional methods based on acid precipitations. In addition to producing HMG-1 and HMG-2 in high yields, purity, and activity, the early heating step in this procedure seems to minimize the proteolytic cleavage of HMG-1/2 that commonly occurs at early stages in rat liver extracts (5) using conventional protocols. It is anticipated that this approach will facilitate future studies of the HMG-1/2 proteins.


FOOTNOTES

*
This research was supported by United States National Institutes of Health Grant R01 GM 18243. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 303-270-8837; Fax: 303-270-3304.

(^1)
The abbreviations used are: HMG, high mobility group; PCA, perchloric acid; pCMBS, para-chloromercuribenzenesulfonate; DTT, dithiothreitol; PIPES, 1,4-piperazinediethanesulfonic acid; PBE, polybuffer exchanger; bp, base pair(s).

(^2)
J. P. Wagner, C. Kunsch, and D. E. Pettijohn, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. James McManaman (University of Colorado Cancer Center Microsequencing Core Laboratory, Denver) for performing the amino acid sequencing, Dr. Raymond Reeves (Washington State University, Pullman) for supplying acid-extracted HMG-1, and Drs. Yvonne Hodges-Garcia (University of Colorado Health Sciences Center, Denver) and Joan Betz (Regis University, Denver) for gifts of certain DNA fragments.


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