Eur. J . Biochem. 123, 165-170 (1982) ( \ FEBS 1982

C1 Proteins: a Class of High-Mobility-Group Non-histone Chromosomal Proteins from the Fruit Fly Ceratitis capitata

Gabriel MARQUEZ, Federico MORAN, Luis FRANCO, and Francisco MONTERO

Departamento de Bioquimica, Facultad de Ciencias, Universidad Complutcnse, Madrid

(Received July 22/November 17, 1981)

1. CM-cellulose chromatography of a fraction soluble in 5 o/, perchloric acid from Cerutitis cupitutu chromatin yields three proteins, C l a l , Cla2 and Clb, which have been purified to electrophoretical homogeneity.

2. C l a l , Cla2 and C l b analyse like high mobility group (HMG) non-histone chromosomal proteins, although they do not exactly correspond with those from vertebrates. It is proposed that C1 proteins, as well as Drosophilu D1 [Rodriguez Alfageme et al. (1980) Chromosomu, 78, 1 - 311 are representative of a class of insect-specific HMG proteins. Tryptic fingerprints show that C l a l and Cla2 are very similar, but C l b is a somewhat distinct protein. Circular dichroism studies have shown that these preparations do not appreciably fold on increasing ionic strength.

3. The interactions between DNA and C1 proteins have been studied. These proteins precipitate DNA in 0.1 5 M NaCI, 0.015 M sodium citrate and the precipitation curves are cooperative. Soluble complexes between C1 proteins and DNA could be prepared in low ionic strength media and their thermal denaturation profiles obtained. C1 proteins do not destabilize DNA under the conditions used to prepare the complexes but the three proteins stabilize DNA to a different degree. From these studies it has been concluded that the association constant of C l b to DNA is smaller than that of C l a l and Cla2.

High mobility group (HMG) non-histone chromosomal proteins can be extracted from chromatin or nuclei by either 5 % perchloric acid or 0.35 M NaCI. Their amino acid com- position is unusual as they have a large number of both basic and acidic amino acid residues (for a review, see [l]). They have been reported to occur in all the eukaryotes examined to date: animals [l], plants [2] and yeast [2,3]. Most of the HMG proteins described to date fall into two families. The proteins of the first family have a molecular weight of about 26000 and they share some other physical and chemical properties. HMGl and HMG2 are the most representative proteins of this class, to which trout HMG T also belongs [l]. The HMG proteins of the second family have a small molecular weight (8000- 10000) and they also have some common structural characteristics. HMG14 and HMG17, which are, together with HMGI and HMG2, the major HMG proteins in mammals, fall into this family, and so do the trout-specific H6 [I] and, perhaps, some other minor HMG proteins, such as the recently-described HMGl8, HMG19A and HMGI 9B [4].

Early studies on the primary structure of HMG proteins revealed a unique feature in HMGI and HMG2 sequences, namely the presence of long acidic peptides. The C-terminal region of HMGI contains a peptide with a continuous amino acid sequence of 41 glutamic and aspartic acid residues. A similar, but not identical, peptidc is present in HMG2 [5].

Some details of the higher order structure of HMG proteins are also known. It is now well established that calf thymus HMGI and HMG2 can fold in salt solutions and the folded conformation of both proteins is 40-50% helical [6,7]. In

Ahhrc~i~iutions. HMG proteins, high mobility group non-histonc chro- mosomal proteins; NaCl/Cil. 0.15 M NaCI, 0.015 M sodium citrate; 0.1 x NaCl/Cit, NaCI/Cit diluted 10-fold; CD, circular dichroism; I , , melting

temperature.

~ ~~

contrast, HMG14 and HMG17 are not able to fold and they remain in solution as random-coiled polypeptides [8,9].

Despite the large number of studies that have been carried out (see, for instance, [lo- 15]), the functions of the HMG proteins are not yet known. The characterization of new HMG proteins, and the subsequent comparative studies, may help to understand their functions. In the present paper we describe a study of a class of putative HMG proteins of the fruit fly Ceratitis cupitatu. We have previously reported the isolation of an HMG-like protein fraction from this insect [16], and a similar protein was also isolated from Drosophilu melunoguster by preparative electrophoresis by Rodriguez Alfageme et al. [17], who termed it D1. The amino acid composition of D1 [18] is very similar to that of the Ceratitis fraction [16] and both preparations also share some other properties, as will be further discussed. These similarities prompted us to name our HMG- like fraction C l (after Ceratitis) [19].

The protein D1 is present in many chromomeres of the salivary gland chromosomes, but it seems to be preferentially present in chromatin containing sequences d(A-A-T-A-T) and d(A-A-T-A-T-A-T) [IS]. It is not known whether this pref- erential binding may play a role in the genetic activity of chromatin, and the answer to such a question must surely await a better molecular knowledge of this class of proteins. The present study is aimed at the fractionation and characterization of Ceratitis C1 proteins, as an attempt to improve the molecular knowledge that we have just referred to.

MATERIALS AND METHODS

C1 proteins were isolated from crude histone H1 by a modification of the previouly described method [I 61. Crude H1 was dissolved in borate buffer (7.5mM boric acid, 3mM

166

NaOH, pH 8.8) to a concentration of 10 mg/ml and 20 ml were loaded onto a CM-cellulose column (35 x 2.5 cm). The sample was eluted with a linear gradient of NaCl formed by mixing 350 ml of borate buffer and 350 ml of 1 .O M NaCl in the same buffer (method I). The resolution of C1 proteins was improved by eluting the sample with 180ml of borate buffer before starting the gradient of NaCl (method 11). The flow rate was 20 ml/h and fractions of 4 ml were collected in both methods. When necessary, C1 proteins were further purified by isoelec- tric precipitation, as described under Results.

Peptide maps of C1 proteins were obtained after tryptic digestion. Proteins, 0.4 mg, were dissolved in 60 p1 of bidistilled water and 40pl of 0.2M N-ethylmorpholine at pH8.1 were added. The digestion was started by adding 10 p1 of a solution of trypsin (1 mg/ml, treated with I.-I -tosylamido-2-phenylethyl chloromethyl ketone) and the solutions were incubated at 37 'C for 4 h and then dried under vacuum. The residues were dissolved in 40 pl of bidistilled water and aliquots ( z 10 pl) were taken for peptide fingerprinting, which was carried out by the two-dimensional technique of Bitar and Wittmann-Liebold [20] on cellulose thin-layer plates (Cel-300, Macherey-Nagel). The dried plates were sprayed with a solution of ninhydrin (1 g/l) in ethanol/acetic acid (50 : 450, v/v). In some instances, peptides were recovered for amino acid analysis [20] and then the ninhydrin reagent was threefold diluted with ethanol.

The interactions between DNA and C1 proteins were studied, as a first approach, by precipitation. DNA was prepared from calf thymus by the method of Kay et al. [21] and it was dissolved in NaCl/Cit to a concentration of 0.55 mg/ml. Protein solutions (1.3mg/ml in NaCl/Cit) were added in varying amounts to 0.3-ml aliquots of DNA solution and the final volume was adjusted to 1 ml with NaCl/Cit. The mixtures were shaken and centrifuged at 12000 x g for 30 min and the absorbance of the supernatants were measured at 260 nm to determine the percentage of DNA precipitated. The complexes of DNA and C1 proteins are soluble in low-ionic-strength media, such as 0.1 x NaCl/Cit, 0.25 mM EDTA, 25 mM NaCI, 1 mM Tris/HCl, pH8. Soluble complexes were prepared by mixing DNA and protein solutions in the above-mentioned media ; in all the experiments the final mixtures contained 0.08 mg DNA and varying amounts of protein in a volume of 3 ml.

Thermal denaturation studies were carried out in a Beckman DU-8 spectrophotometer. Temperature was in- creased at 0.33' C/min and the absorbance (average of five values) was recorded every 0.5 C. Hyperchromicity, H ( t ) , was calculated according to the following equation: H ( t ) = 100 [A2hO(t)-A2h0(25 T)]/A260(250C). Data were analysed by means of a desk computer to obtain the derivative melting curves. From every set of five consecutive values of H ( t ) , the best-fitting line was adjusted by linear regression. The resulting slope was taken as A H ( t ) / d t at the middle temperature.

Circular dichroism of proteins was recorded in a Mark 111 dichrograph (Jobin-Yvon) and the percentage of a-helix was determined as described elsewhere 1221. The spectra of un- diluted DNA-protein soluble complexes were recorded in 1 -cm path-length cuvettes between 330 and 250 nm.

Total HMG proteins from calf thymus were prepared by the method of Sanders and Johns [23].

Electrophoresis was carried out in 15 '%; polyacrylamide gels in the presence of 2.5 M urea [24]. Proteins and peptides were hydrolysed at 105 C in constant-boiling HC1, containing 0.1 y, (w/v) phenol, in evacuated sealed tubes during 24 h. Amino acid analyses were pet-formed with a Durrum amino acid analyser model D-500. Tryptophan was determined spectro-

photometrically [25]. Protein concentration was determined as previously described [22]. Molecular weights were estimated by electrophoresis [26] using calf thymus histones as standards.

RESULTS

Fractioiiatioii and Properties of' C1 Proteins

The fractionation of C1 proteins largely depends on the conditions of CM-cellulose chromatography. A preliminar report of the results using chromatographic method 1 has been given elsewhere [I 91. Briefly, the use of a gradient of NaCl from the beginning of the elution results in the presence of C1 in two fractions. The first one elutes as a symmetrical peak with the void volume of the column, whereas the second one elutes only when the concentration of NaCl is 0.3 M. The presence of C1 in two different and well-resolved chromatographic fractions was considered as an indication that two forms of C1 may exist, and they were referred to as C l a and Clb. These proteins were not recovered in a pure form, but it was found that both C l a and C l b were only slightly soluble at pH5.4 and acidifying the corresponding chromatographic fractions to pH 5.4 resulted in the precipitation of C l a and C l b as electrophoretically homo- geneous proteins [I 9).

Method I1 provided a better resolution than method I. Fig. 1 shows the chromatographic profile of crude HI. The most important difference between both methods is that the proteins that elute as a single peak with the void volume are now resolved into three peaks (1, 2 and 3 in Fig. 1).

The electrophoretic analysis of the more relevant chro- matographic fractions is shown in Fig. 2. Fraction 3 gives a single band in urea/acetic acid polyacrylamide gels, but bands with the mobility of C1 are also present in chromatographic fractions 1, 2 and 4. Taking advantage of the low solubility of C1 proteins at pH 5.4 it has been possible to purify them from chromatographic fractions I and 4, but, although a C1 protein is the major component of the chromatographic fraction 2 (see Fig. 2), the protein content of this fraction was so small that we did not succeed in purifying it. An example of the purification procedure is given in Fig. 3, which shows that it was possible to recover the C1 component from the chromatographic frac- tion 1 almost quantitatively and as an electrophoretically homogeneous protein.

The two purified C1 proteins, namely the one obtained from chromatographic fraction 1 (Fig. 3c) and that present in fraction3 are further referred to as C l a l and Cla2, because they were found in the previously-described unresolved C1 a fraction.

F r a c t i o n number

Fig. 1. Elutiorz profile of ' clir.ornulox,.ruphj' of crude H I on CM-rc~llulo.rc~. (- ~ -) Absorbance at 230nm. (-- -) NaCl concentration. C1 proteins are prcscnt in li-actions 1 -4 and pure HI is recovered in fraction 5

I67

Table 1. Amino uc.itl c~ompositioii o f C'lal, CluZ untl C l h / J Y J I ? I Ceriititis capitata conipurc~el i v d h thur of Drosophila mehogas te r D l [ I H ]

Amino acid Amount i n c1 H I C l a l C l a 2 C l b D1 [I81

mo1/100mol

Cysteine n.d." n.d." Aspartic acid 16.4 17.5 Threonine 5.3 5.3 Serinc 7.8 8.6 Glutamic acid 12.0 11.9 Proline 5.7 4.4 Glycinc 10.0 11.8 Alanine 8.9 8.5 Valine 3.9 2.8 a b c d e

,iyl ~ . l ~ ~ c t r o l ~ l z o l e s i , ~ putterti.s of crude HI ( a ) und qf ~ <>, ru.siwctivelj) ohtuitwd h j CM-c~ellulosc clzronitr-

Migration is from top to bottom

Methionine lsolcucine Leucine Tyrosine Phenylalanine Lysinc Histidine Arginine Tryptophan

0.0 2.9 3.9 1.8 1.9

' 12.5 0.0 6.9 0.4b

0.0 2.4 2.6 1.1 0.8

14.2 0.0 8.1 O.0b

The C1 protein present in chromatographic fraction 4, previously referred to as C l b [19], appears as a minor component (Fig. 2e) but it was also purified to electrophoretic homogeneity by acidifying fraction 4 to pH 5.4.

Typical yields of pure C l a l , Cla2 and C l b were, re- spectively, 1.2,2.9 and 0.5 mg/kg insect. Due to the low yield of C1 proteins, method I1 was used only when the resolution of C la into C l a l and Cla2 was necessary. Otherwise, method I was preferred because it allows one to isolate a large amount of C l a free from other contaminant proteins.

Although C l a l , Cla2 and C l b run together in acid/urea gels their mobility in sodium dodecylsulfate gels are slighty different, revealing some minor differences in molecular weights. The estimated molecular weights are as follow: 25700 for C la l and Clb , and 28200 for Cla2.

Table 1 shows the amino acid composition of C l a l , Cla2 and C1 b. Though related, the composition of these proteins shows some distinctive features. The content of hydrophobic amino acids is smaller in C1 b, which also is more basic than C l a proteins. C l a l and Cla2 are far more similar to each other,

11.d."

16.3 5.3 8.2

12.8 7.6

12.1 8.6 2.7 0.7 1.7 1.4 0.0 0.0

12.2 1.9 8.6 0.0b

0.2 16.0 3.1

10.6 10.7 8.0

13.2 9.6 4.8 0.2 1.7 1.5 0.7

< 0.1 11.5 1.3 7.4

< O . l

Basiciacidic 0.68 0.76 0.78 0.83 Hydrophobic 14.8 9.7 6.5 8.9

a Not detected. Spectrophotomctrically determincd [25].

although C l a l has more hydrophobic residues and is less basic than Cla2. These features may account for the different chromatographic behaviour of the three proteins. Some other distinctive characteristics are seen in the composition of these proteins: for instance, C l b lacks aromatic residues and it has methionine and histidine.

A number of similarities are also evident when the tryptic maps are compared (Fig. 4). Again, these similarities are more evident between C l a l and Cla2 than between C1 b and either C l a l or Cla2. The regions11 and 111 in Fig. 4 are more interesting for comparative purpouses, because the spots in region1 are mainly due to free lysine and dipeptides. It is interesting to note that the fingerprints of C1 a l , C1 a2 and C1 b show the presence of some negatively-charged peptides and the pattern of these acidic peptides in C l a l and Cla2 is very similar. The acidic peptides of C l a l are now being studied and preliminary evidence shows that they possess 8 - 10 amino acid residues and that aspartic and glutamic acids account for at least two-thirds of them. Large peptides such as those occurring in HMGl and HMG2 from calf thymus, have not been detected.

CD spectra of C l a and C l b (Figs) show that these preparations are not able to fold to a significant level on increasing ionic strength. Even in the presence of 1 .O M NaCI these preparations are only 2 helical and no other ordered structure could be detected. C l a proteins did not show dichroism in the region 250- 300 nm although they have aromatic residues (Table 1).

Interactions bt'tiveeri D N A crnd CI Proteins

Fig. 6 shows the precipitation curves of DNA with C1 a and C l b in NaCl/Cit. The sigmoidal shape of both curves may

168

100

8 0 -

- - e - -

a L

O 60

z 4 0 -

@J 20 &

- c 0 ._ + .- a " ._

-

o r

b 0

L m L-2

C oo i:>

Fig. 4. Tr~ /~ t i c , f b zgcr~r in t s of ( u ) C l u l , ( h i CIu2, ( c ) Clh. Electrophoresis (horizontal) and chromatography (vertical) was carried out on ccllulosc thin- plates by the technique of Bitar and Wittmann-Licbold [20]. Solid lines indicate intense spots and dashcd contours indicate weak spots

200 220 240 200 2 2 0 240 Wavelength ( n r n )

Fig. 5 . CD spectra of 7.4 p M .solutioi7.s o f CI ptwteiiis in the,firr-iiltra~~iolet region. (A) C l a i n water, pH 3.5; (B) Cla in 1 .OM NaCI, pH 7.0; (C) C l b i n water, pH 3.5; (D) C1 b in 1.0 M NaCI, pH 7.0. The spectra were recorded in a 0.05-cm path-length cell. The curves give the expcrimental spectra and the points correspond to the best-fitting theorctical spectra calculated as described elsewhere [22]. No significant ionic-strength-induced changes were observed

suggest that the precipitation is a cooperative process. C l b precipitates DNA more readily than C1 a as evidenced by : (a) 50 precipitation occurs at an input protein/DNA ratio of about 1.2 instead of about 1.7; (b) total precipitation of DNA occurs at an input ratio Clb/DNA of about 2.0, whereas the precipitation curve of DNA by C l a shows a slow increase from 85

The stoichiometry of the insoluble complexes between C1 b and DNA has been studied by determining the content of protein and DNA in the sedimented complexes. DNA content was obtained by subtracting the amount of DNA in the supernatant from the input amount of DNA. The content of protein in the complexes was measured as described in Materials and Methods after dissolving the protein in 0.25 M HCI. It has been found that even when all DNA has been precipitated, i.e. at input ratios Clb /DNA = 2, a large amount of protein (.=70"/,) remains free in solution. The ratio C1 b/DNA in the precipitated complex incrcases linearly from the beginning of the precipitation when the input ratio C1 b/DNA increases. The ratio protein/DNA in the insoluble complex reaches a value of 1.6 for an input ratio of 5. These results suggest that the value of the association constant of C1 b to DNA is small, although this protein is able to precipitate DNA at undersaturating levels.

at Cla /DNA = 2 to 90 "/, at C1 a/DNA = 5.

I I

The C D spectra of soluble complexes between DNA and either C l a l or Cla2 (see Materials and Methods) and that of free DNA show no significant differences. This finding shows that the conformation of DNA is little or not at all different from the B form upon interaction with these proteins.

The interaction of DNA with C l a l , Cla2 and C l b in low- ionic-strength media is characterized, however, by a definite effect upon thermal denaturation. Fig. 7 shows the derivative melting profiles of the complexes prepared in 0.1 x NaCl/Cit. The complex between C l a l and DNA shows a transition at about 67' C, i.e. some 3 'C lower than that of naked DNA. Moreover, this transition is characterized by a flattened derivative curve that may indicate a loss of cooperativity in the denaturation of DNA upon interaction with C l a l . This low cooperativity is not noticeable in the complex C1 a2-DNA, which shows a transition at a t, similar to that of free DNA, but a considerable percentage of DNA melts at higher tempera- tures. Complexes were also prepared at a protein/DNA ratio of 0.2. Their derivative melting profiles (not shown in Fig. 7 ) retain the characteristics of the curves depicted in this figure, i.e. one broad transition in the Cla l -DNA complex and two transitions ( t , z 70 'C and tk zz 85'C) in the Cla2-DNA complex. There are, however, quantitative differences attribut- able to the varying protein/DNA ratio. For instance, the area under the peak of thc second transition of the Cla2-DNA complex diminishes when lowering the protein/DNA ratio to 0.2 and the transition of the Cla l -DNA ( r = 0.2) complex, although broader than that of DNA, gave a peak sharper than that of Fig. 7.

The interaction with C l b causes the DNA to melt entirely at a temperature higher than that of naked DNA (Fig. 7). This effect was also noticeable when the complex was prepared at a

T e m p e r a t u r e , t ( " C )

Fig. 7. Deriwtiw rriclring p r o f i l m of (- -) cu1jtkyniu.s D N A , ( . . . . . . .) c~inpki~.u hctti.ecn D N A arid C l u l , (-- ) conzple.\- bet iwen D N A uiz Clu-7 und (-.-. - i coriiplr~x hefwecv? D N A und C l h . Complexes were prepared in 0.1 x NaCIICit at prolein/DNA ratio of 0.5 (w/w)

protein/DNA ratio of 2.5, although the t , was, in this instance, 82 C. The possible significance of this effect will be discussed later.

When the complexes were prepared in 0.25 mM EDTA, the differences between C l a l and C la2 were not so noticeable (Fig. 8). In both complexes DNA shows a biphasic melting curve and, probably, no significance can be attached to the minor differences between both curves. The curves obtained at a ratio protein/DNA of 0.2 (not shown) exhibits the obvious decrease of the area under the second peak and the concomitant increase of the peak corresponding to free DNA.

Complexes between C l a l or Cla2 and DNA were also prepared in 0.25 mM EDTA, 25 mM NaCI, 1 mM Tris/HCl, pH 8.0. As in Fig. 8, there appear two well-resolved transitions, although, as a result of increasing ionic strength, their melting temperatures are 73 C and S3'C.

DISCUSSION

The functions of the HMG proteins are not yet well understood and any attempt to define them has to be based on circumstantial criteria, such as those given by Goodwin et al. [l]. These criteria are fulfilled by C1 proteins. In a previous report, we suggested that C1 appeared to be an HMG non- histone chromosomal protein, mainly based on the amino acid composition (nearly equal amounts of basic and acidic res- idues) and their solubility in both 0.25 M HC1 and 5 % perchloric acid [16]. In the present paper we give an additional piece of evidence, namely the DNA-binding capacity. On the other hand, C1 proteins are closely related to the protein D l isolated from Drosophila melanogaster. Apart from the similar- ities in amino acid composition. C1 is also stained blue- greenish with amido black and its electrophoretic mobility is smaller than that of calf thymus HMGI (Fig. 3). Some of the similarities between D1 and C1 have been pointed out by Rodriguez Alfageme et al., who also discussed the similarities between these insect proteins and the HMG proteins of vertebrates [18]. We are entirely in agreement on these two points.

The classification of C1 and D1 proteins into one of the two above-mentioned HMG families is not an easy task. C1 proteins share some properties with HMGl and HMG2 as

I \ I

30 40 50 60 70 80 90 100 Tempera ture , t ("C)

Fig. 8. D r r i w t i w i i icl i i t igI~r(~/; le~s of i--) c.ci1ftlijrnu.s D N A , i . . . . . .)

c,onzpki,s h c t l t , r c , l l D N A urid C l a l urid (---- conip1r.u h o i w n D N A u t l d

CIrr2. Complexes were prepared in 0.25mM EDTA, pH 8.0, at a proleiniDNA ratio of 0.5 (w/w)

evidenced by some of the present results, especially the large molecular size. The presence of tyrosine and phenylalaninc, as well as the relative abundance of hydrophobic residues in Cla l and Cla2, also resembles some compositional features of H M G l and HMG2. On the other hand, the absence of methionine in C l a proteins is feature found in HMG14 and HMG17. C1 proteins, in common with HMG14and HMG17, do not fold on increasing ionic strength. The last statement is not, however, an absolute one, because perchloric acid, which has been used to isolate C1 proteins, might induce irreversible damage to their native conformation. This is not the case when 10% trichloroacetic acid is used to precipitate the HMG proteins by the method of Goodwin and Johns [27], because both HMGl and HMG2, prepared by this method, are able to fold with a high a-helical content [7], but there is no evidence as to the damage that perchloric acid may cause to C1 proteins. I n this connection we should mention that HMG-T, which is reputed to belong to the HMGl and HMG2 family, does not fold to a significant degree in saline solution and also shows chromatographic behaviour similar to that of C1 proteins [28]. Due to these considerations, we think that C1 proteins (and, presumably, DI) may be representative of a new class of insect- specific HMG proteins, although HMG-T seems to be their closest counterpart in vertebrates.

The present studies have shown that C1, formerly reported as a single protein [16], can be resolved at least into three components. C l a l and Cla2 are different in their tryptic fingerprints and in their chromatographic bebaviour. Nevertheless, they do not differ very markedly, and the possibility exists that the resolution of C l a in sever' 'L 1 com- ponents, such as C l a l and Cla2, reflects the existence of microheterogeneity in Cla. In contrast, C l b seems to be a protein distinct from C 1 a, although they are closely related. Preliminary evidence [29] shows that Drosophila D1 can also be fractionated into several components by CM-cellulose chro- matography. This heterogeneity might then be characteristic of this class of insect-specific HMG proteins.

Javaherian et al. [30] have claimed that calf thymus HMGl and HMG2 may destabilize DNA in the presence of NaCl in the concentration range 25 - 75 mM. We have not found any indication of destabilization of DNA in our complexes in 0.25 mM EDTA, 25 mM NaCI, 1 mM Tris/HCl, pH 8.0, one of the buffers used by these authors. When the complex C la l -

170

DNA was prepared in 0.1 xNaCl/Cit, some DNA melts between 50 and 60 'C (Fig. 7), but, as discussed above, this behaviour may be attributable to a loss of cooperativity rather than destabilization.

The interaction of C1 b with DNA seems to be characterized by a low affinity constant, as deduced from two findings. Firstly, large amounts of free protein are still in solution when all DNA has been precipitated as an undersaturated complex. Secondly, the thermal denaturation curve of the Clb-DNA complex in 0.1 x NaCl/Cit is monophasic (Fig. 7) and all the DNA in the complex melts some 5 ' C higher than free DNA. The absence o f a transition at 70' C (i.e. the t , of naked DNA) when DNA is not saturated may be interpreted in terms of a low affinity constant in the binding of C l b to DNA, according to the theoretical treatment of McGhec [31].

The interactions between C1 proteins and DNA have been studied in the present paper with calf DNA. However, the present results may be extrapolated to the interactions between C1 proteins and Cevatitis capitata DNA because the techniques used, namely precipitation and low-resolution melting of DNA, are not aimed at specific interactions. Further work is now being carried out in our laboratory to investigate any possible sequence-specific interaction between C1 proteins and Crraritis DNA.

At the present time we do not know whether the diversity of C1 proteins corresponds with diverse functions. As we have previously stated, D1 seems to bind to many chromomeres although it binds more specifically to well-defined chromo- somal loci [18]. These results have not been confirmed by Howard et al. [32] who have shown that monoclonal antibodies against D1 only bind to some chromosomal loci. We think that the diversity of D1 proteins [29] might account for the disagreement between both groups of workers, because Rodriguez Alfageme et al. did not use monoclonal antibodies [I 81. If this interpretation is correct, the diversity of C1 and D1 proteins might be connected with a variety of functions.

This work has been supported in part by grant 4239179 from the Contisicin A.wsoru (ke Itivestigucibn Cient(fica y Ti.ctiicu (Spain). We are indebted to Prof. A. M. Muiiicio for his valuable suggestions and to Mr G. Gonzikz de Buitrdgo for excellent technical assistance.

REFERENCES

1 . Goodwin, G. H., Walker, J . M. &Johns, E. W. (1978) in Cell Nuc1eii.s (Busch, H., ed.) volVI, pp. 131 -219, Academic Press, New York.

2. Spiker. S., Mardian, J . K. W. & Isenberg, 1. (2978) Biochem. Bi(Jph.l'S. Rcs. Commun. 82, 129- 135.

3. Wcbcr, S. & Isenberg, I. (1980) Biod~et?ii.slry, 19, 2236- 2240.

4. Goodwin, G. H., Brown. E., Walker, J. M. & Johns, E. W. (1980) Bioclzitn. Biop/iy.s. Ac/u, 623, 329 - 338.

5. Walker. J . M.. Goodcrhdm, K . &Johns, E. W. (1979) Biocl im. J . 170, 253-255.

6. Cary, P. D., CraneRobinson, C., Bradbury, E. M., Javaherian. K., Goodwin. G. H. &Johns, E. W. (1976) Eur. J . Bioclien?. 62, 583- 590.

7. Baker, C., Iscnberg, I . , Goodwin, G. H. & Johns, E. W. (1976)

8. Abcrcrombie, B. D., Kneale, G. G., Crane-Robinson, C., Bradbury, E. M., Goodwin, G. H., Walker, J . M. &Johns, E. W. (1978) Eur. J . Biochmi. 84, 173 - 177.

9. Javaherian, K. & Amini, S. (1978) Biochrr~i. Biopliys. Res. Cornniun. 85, 1385 - 1391.

BiCJC/l<~/Jli.T/,7', / j , 1645 - 1649.

10. Vidali, G., Boffa, L. C. & Allfrey, V. G. (1977) c'c4l. 12, 409-415. 11. Levy, W. B., Wong, N. C. W. & Dixon, G. H. (1977) Pruc. Nut1 Acad.

12. Weisbrod, S. & Weintraub, H. (1979) P t w . Nurl Acud. SL,i. US.4, 76.

13. Lcvy, W. B. & Dixon, G, H. (1978) Nucleic A d s Res. /Z, 4155-4163. 14. Goodwin, G. H. &Johns, E. W. (1978) Biochirn. Biophys. Actcl, 510,

15. Goodwin, C. H., Mathew, C. G. P.. Wright, C. A,, Venkov. C. D. &

16. Franco, L., Montcro, F. & Rodriguez-Molina. J . J. (1977) FEBS Lett.

17. Rodriguez Alfageme, C., Rudkin, G. T. & Cohen, L. H. (1976) Proc.

18. Rodriguez Alfageme, C., Rudkin, G. T. & Cohen, L. H. (1980)

19. Marquez, G., Morin, F., Barbero, J . L., Montero, F. & Franco, L.

20. Bitar, K . G. & Wittmann-Liebold, B. (1975) Hoppe-Sejlw's Z . Pltjsiol.

21. Kay, E. R.. Simmons, N . S. & Dounce, A. L. (1952) J . Am. Chem. Soc.

22. Barbcro, J . L., Franco, L., Montero, F. & Morin, F. (1980)

23. Sanders, G. &Johns, E. W. (1974) Biocheni. Soc. Trans. 2, 547-550. 24. Paiiyim, S. & Chalkley, R. (1969) Arch. Biochctii. B ioph~:~ . 130, 337-

346. 25. Donovan, J . w. (1969) in Phj:.Yicul Principles and Tecliniques oJ'Prorein

C/ienii.sf~y (Leach, S. J., ed.) pp. 101 - 170, Academic Press. New York.

Sci. U S A , 74, 2810-2814.

630 - 634.

279 - 284.

Johns, E. W. (1979) Nucleic Acids Res. 7 , 1815-1835.

78, 317-320.

Nut/ Acud. Sci. USA, 73, 2038-2042.

C~Jr(JtMOSOt7lU ( h ' / . ) 78, 1 - 31.

(1981) Ahstr. FEBS Meet. Mon-SI-15.

Chwn. 356, 1343-1352.

74, 1724-1728.

BiOc~hC'?Jii.S/ry, 10, 4080 - 4087.

26. Panyim, S. & Chalklcy. R. (1971) J . Bid. Cltem. 246, 7557-7560. 27. Goodwin, G. H. & Johns, E. W. (1973) Eur. J . Biuclieni. 40, 215-219. 28. Brown, E., Goodwin, G. H., Mayes, E. L. V., Hastings, J . R. B. &

29. Guerrero, I., Marquez, G., Montero, F. & Alonso, C. (1981) 2nd

30. Javaherian, K., Sadeghi, M. & Lin, L. F. (1979) Nucleic Acidr Res. 6 ,

31. McGhec, J . D. (1976) BiOpo/J'ntfw, 15. 1345-1375. 32. Howard, G. C.. Abmayr, S. M., Shinefeld, L. A., Sato, V. L. & Elgin, S.

Johns, E. W. (1980) Biochrm. J . 191, 661 -664.

FESBE Congress. abstr. 245.

3569-3580.

C. R. (1981) J . C d B i d . 88, 219-225.

G. Marquez, F. Morin, and F. Montero, Departamento de Bioquimica, Facultad dc Ciencias, Uiiiversidad Complutense, Madrid-3, Spain

L. Franco, Departamento de Bioquimica, Facultad dc Cicncias Biologicas, Burjasot, Valencia, Spain