Comp. Biochem. Physiol. Vol. 72B,No. 4. pp. 531 to 535, 1982 0305-0491/82/080531-05503.00/0 Printed in Great Britain © 1982PergamonPress Ltd HIGH MOBILITY GROUP NON-HISTONE CHROMOSOMAL PROTEINS FROM THE FRUIT FLY CERATITIS CAPITATA GABRIEL MARQUEZ,FRANCISCO MONTEROand LulS FRANCO Departamento de Bioquimica, Facultad de Ciencias Quimicas, Universidad Complutense, Madrid-3, Spain (Received 4 December 1981) Abstract--l. Electrophoresis in acid-urea polyacrylamide gels of HMG proteins extracted from pharate adults of the fruit fly Ceratitis capitata by 5% perchloric acid and 0.35 M NaCI yields twelve main bands. 2. The electrophoretic pattern of these proteins largely depends on the developmental stage of the insect. 3. Some of the proteins so obtained behave like canonical HMG proteins from vertebrates when fractionated by CM-Sephadex chromatography or by the ethanol-HCl procedure. INTRODUCTION High Mobility Group (HMG) non-histone chromo- somal proteins have been reported to occur in all the eukaryotes examined to date: animals (Goodwin et al., 1978a), plants (Spiker et al., 1978) and yeast (Spiker et al., 1978; Weber & Isenberg, 1980). These proteins can be extracted from chromatin or nuclei by either 5% perchloric acid or 0.35 M NaC1 and several methods of fractionation have been described (Good- win et al., 1978a). Based upon their physical and chemical properties, HMGs can be classified into two different subsets. The first one contains those HMG proteins that have a molecular weight of about 26,000. HMG1 and HMG2 are the prototypes of this subset, to which trout HMG-T also belongs (Good- win et al., 1978a). The second subset contains proteins of lower molecular weights (8000-10,000). HMG14 and HMG17 fall into this subset, and so does the trout-specific H6 (Goodwin et al., 1978a) and, per- haps, some other recently described minor HMGs (Goodwin et al., 1980). Proteolysis often occurs during the isolation of HMG proteins and this results in the appearance of a number of polypeptides in the extracts, which causes complex electrophoretic patterns (Goodwin et al., 1978b). The degradation can be prevented by extract- ing the tissue directly with 5% perchloric acid (Good- win et al., 1978a), but this procedure is only useful with tissues such as thymus; in other instances, this method does not prevent the contamination by non- chromosomal proteins. We have previously reported the isolation of an HMG-like protein fraction from Ceratitis capitata, which was termed C1 (Mfirquez et al., 1981) on account of its similarities with the Drosophila melano- gaster protein D1, isolated by Rodriguez Alfageme et al. (1976). These proteins are soluble in 5% perchloric acid and their amino acid composition resembles those of calf thymus HMG1 and HMG2, although they differ in several physical properties, namely, their capacity to interact with DNA and their conforma- txon (Marquez et al., 1981). Wooley et al. (1981) have recently reported the presence of some HMG-like polypeptides in Drosophila melanogaster and they have found that antisera against calf thymus HMG14 and HMG17 bind to Drosophila melanooaster poly- tene chromosomes, preferentially at the heat-shock puffs; however, a definite identification of HMGs similar to those present in mammals has not been reported in insects. In the present paper we show that HMG proteins 1, 2, 14 and t7 may also occur in the fruit fly Ceratitis capitata. MATERIALS AND METHODS Crude chromatin from Ceratitis capitata pharate adults or larvae was obtained as described elsewhere (Franco et al., 1974). Total HMG proteins were isolated by extracting the chromatin with either 5% perchloric acid (Sanders & Johns, 1974) or 0.35 M NaC1 (Goodwin & Johns, 1973; Goodwin et al., 1975). In order to minimize proteolysis during the preparation of chromatin, the saline washings (Franco et al., 1974) were omitted in some instances. Fractionation of crude HMG proteins was carried out by ethanol-HC1 precipitation (Goodwin et al., 1977) and CM-Sephadex chromatography (Goodwin et al., 1975). Electrophoresis was carried out in 15% polyacrylamide gels in the presence of 2.5 M urea (Panyim & Chalkley, 1969). Double-gel electrophoresis was run according to the method of Johns and Forrester (1971). Hydrolysis of pro- tein samples was carried out at 105°C with 5.7 M HCI con- taining 0.1% (w/v) phenol, in evacuated sealed tubes for 24 hr. Amino acid analyses were performed with a Durrum amino acid analyzer, model D-500. RESULTS AND DISCUSSION Figure lb shows the electrophoretic pattern of the proteins extracted from chromatin of pharate adults by 5% perchloric acid, and precipitated with 3 volumes of acetone. The three main top bands corre- spond, from top to bottom, to C1 proteins (M~,rquez et al., 1981), histone H1 (Franco et al., 1977) and a puparium-specific protein (Franco et al., 1974). Although C1 proteins are HMG-like (M~irquez et al., 1981), this fractionation procedure shows a distinctive 531