Comp. Biochem. Physiol. Vol. 92B, No. 4, pp. 605~08, 1989 0305-1M91/89 $3.00 + 0.00 Printed in Great Britain Pergamon Press plc

ISOLATION A N D CHARACTERIZATION OF HISTONES FROM ANOPHELES ALBIMANUS WEIDEMANN

STEVE MILLER and SONJA THORNTON

Control Technology Branch, Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, Atlanta,

GA 30333, USA

(Received 17 May 1988)

Abstract--1. Histones from Anopheles albimanus adults were prepared by a combination of techniques including chromatin isolation and selective extractions.

2. The anopheline histones were identified on acid urea gels by comparing their electrophoretic profile with that of calf thymus histones and histones isolated from other tissue.

3. Excellent separation of histones was obtained after the extractions by a single electrophoretic run. 4. In addition to the five major classes of histones found in eukaryotes, a sixth class was detected and

tentatively identified as histone H5. 5. This is the first report of histone H5 and its function in insects.

INTRODUCTION

The nucleosome is the fundamental structural unit o f eukaryotic chromosomes. It consists of a "core re- g ion" containing the histone octamer (H2A, H2B, H3, and H4) surrounded by 140-145 base pairs of helical D N A in approximately 1.75 turns. The fifth histone class, HI is located at the outside of the junction of successive nudeosomes interacting with the core particles and the D N A molecule (Finch et al., 1975; Kornberg, 1974; McGhec and Felsenfield, 1980). Substantial heterogeneity exists in nucleosome components, and it may affect expression of different chromatin segments (Urban et al., 1979). Histone modification and histone-nonhistone interaction pro- duce a change in their affinity for D N A thus facili- tating structural changes that enable selective gene expression. To define the molecular organization of the chromosome, it is essential to determine the complexity and variability of histones.

Five main histone classes o f Drosophila have been identified (Cohen and Gotchel, 1971; Pa l io ta and Berlowitz, 1970; Oliver and Chalkley, 1972a, b; AI- fageme et al., 1974). The major five histones were also found in the fruit fly Ceratitis capitata (Franco et al., 1974). The core histone variants of the meal moth Plodia interpunctella were identified in three devel- opmental stages by Patarya et al. (1984). The histone classes of mosquitoes have not been described. This study reports on the preparation of chromatin from pharate adult anopheline mosquitoes and on the selective extraction o f histones from chromatin. The electrophoretic mobilites of the histones were com- pared with calf thymus histones.

MATERIALS AND METHODS Insects

Mosquitoes from a laboratory colony of Anopheles albi- manus originally obtained from Panama were used in all experiments. Larvae were reared on essential larvae nutri-

*Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or the U.S. Department of Health and Human Services.

ents at a temperature of 27°C and relative humidity of 60%-70%. Pupae were placed in distilled water for emergence. Pharate adults were selected for the study (Hinton, 1968).

Isolation of nuclei and chromatin

Anopheles albimanus pharate adults (5.0g) were sus- pended in ice cold Buffer A (0.25 M sucrose, 20raM Tris-HCl, pH 8.2, 3.0 mM CaCI2, 50 mM NaHSO 3, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM 2-mercaptoethanol) and homogenized by five strokes with a tight-fitting motor driven homoge- nizer. The suspension was filtered through cheesecloth, and the pellet was homogenized and filtered twice more. The combined filtrate was centrifuged at 800g for 10 min. The nuclear pellet was further purified by three washing cycles in Buffer B (20raM Tris-HCl, pHS.2, 0.15M NaCI, 1.0 mM PMSF, and 10 mM 2-mercaptoethanol). The final pellet was suspended in Buffer B containing 0.25 M sucrose and layered onto a cushion of 1.7 M sucrose/Buffer B and centrifuged 25,000 rev/min for 3 hr in the SW 41 rotor of a Beckman L2-65 B ultracentrifuge. The resulting pellet was suspended in Buffer B and dialyzed against the same buffer. Chromatin that had separated out during dialysis was dispersed by mild shearing in an ultrasonic disintegrator.

Extraction of histones from chromatin

Chromatin was suspended in 0.25M HC1/10mM 2-mercaptoethanol/5mM dithiothreitol/1 mM PMSF. The suspension was stirred at 5°C and centrifuged at 12,100 g for 20 min. The solubilized proteins were precipitated from the supernatant by the addition often volumes of acetone. After standing overnight at -20°C, the precipitate was collected by centrifugation at 12,100 g for 20 min, washed three times with cold acetone, and suspended in 2% NaCI-75% eth- anol. After 10 minutes of stirring, the mixture was centri- fuged at 12,100g for 20min. The extraction was repeated and all supernatants were saved. To ensure complete re- moval of histories, the resulting precipitate was extracted twice with 2.0 M NaC1, each time with stirring followed by centrifugation. The combined 2% NaC1-75% ethanol and 2.0 M NaC1 supernatants were dialyzed against 20 mM HCI and evaporated to dryness under vacuum. The histones were dissolved in 20 mM HCI and stored at -20°C.

Polyacrylamide gel electrophoretic separation of histories

Histories were analysed electrophoretically by using a GE-2/4 LS Pharmacia* gel electrophoretic apparatus. Gels

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606 STEVE M1LLtR and S()NJA ]'HORNI'ON

were cast containing 15% acrylamide, 0.4% N,N'- bisacrylamide, 0.9 N acetic acid, and 2.5 M urea. The elec- trode chamber contained 0.9 N acetic acid and 2.5 M urea. Gels were prerun for 300 volt hours before the addition of samples. In general, 50/~g histones (30 #~I) in 4 M urea/0.9 N acetic acid/0.5 mM mercaptoethanol/0.02% pyronine Y/20% sucrose were added to the sample slots. A potential of 200 V was applied for 900 V hr. After electrophoresis, the gels were fixed in 11.5% trichloroacetic acid and were stained overnight with 0.1% amido black 10 B in ethanol water-acetic acid (4:4:1 by vol.). Excess stain was removed by washing in the stain solvent. Calf thymus histones were obtained from Boehringer Mannheim Biochemicals and used as standards.

Analytical methods

Protein concentrations were determined after extraction of histones by the method of Lowry et al. (1951). Bovine serum was used as a standard. The concentration of DNA in chromatin was estimated by the diphenylamine method of Burton (1968) with calf thymus DNA as standard. RNA was determined by the orcinol method of Schneider (1957) with a purified RNA standard.

RESULTS

There are limitations in available methods for preparing pure histones. Extraction of histones from whole tissue or even nuclei yields components that include basic proteins of ribosomes, Dilute HC1 commonly used for extracting histones also extracts nonhistone chromosomal proteins. These obser- vations were confirmed in our early studies and therefore required developing a method for isolation of anopheline histones. A marked improvement oc- curred in the preparation of histones when Triton X-100, 2-mercaptoethanol and phenylmethylsulfonyl fluoride (PMSF) were used in the homogenizing mixture, and the nuclei were repeatedly washed and centrifuged at low speed. Triton X-100 removed adhering cytoplasmic contaminants, 2-mercapto- ethanol prevented oxidation of histones, and PMSF prevented complications due to degradation. It was also essential to use chromatin as a starting material and to selectively extract histories from chromatin.

Anopheles albimanus chromatin was prepared as described under Methods and had a 1.00/0.91 DNA histone ratio when the DNA content is taken as 1.00. This ratio agrees with values obtained from other insects (Elgin and Hood, 1973; Pitel and Durzan, 1978). The capacity ofchromatin for transcription by an exogeneous RNA polymerase depends on the DNA:histone ratio (Wormhoudt and Sellos, 1981). To ensure complete removal of high purity histones from chromatin, chromatin was first extracted with 0.25 N HCI followed by selective removal of histones from the acid extract with 2.0 M NaCI-75% ethanol and 2.0 M NaCI.

Anopheline histones extracted from chromatin were compared electrophoretically with available his- tones from calf thymus. Figure 1 shows the results of polyacrylamide gel electrophoresis in an acidic sys- tem containing 2.5 M urea. Electrophoresis was car- ried out at a pH 2.5 to minimize aggregation effects which are known to occur above pH 3.0. Anopheles albimanus histones were resolved into six major classes, and the histones appear to be free of con-

tamination. Anopheline histoncs arc unique because of the presence of a histone whose mobility is between histone HI and H3 (Fig. 1). This histone was desig- nated histone H5. During the final stages of erythro- poiesis in birds, a novel histone H5 between histone HI and H3 is associated with chromatin condens- ation in the red blood cells (Grimmond and Holmes, 1981). Panyim and Chalkley (1969b) tentatively identified a lysine rich histone in nonreplicating tissue similar in mobility to histone H5 and designated it as histonc I . Anopheline histone HI showed I sub- fraction and is considered the most variable of the histones. This histone class differs from other his- tones in amino acid composition and secondary structure (Zalenskaya and Zalensky, 1980).

The anopheline core histones (H2A, H2B, H3, and H4) separated as distinct bands and the electro- phoretic mobilities were similar to calf thymus his- tones except histone H4. The distinct separation of anopheline histories is an interesting feature since most of the core histones from other tissues were not clearly resolved (Vanflelercn, 1982" Simpson, 1978: Dick and Johns, 1969: Bagi and Hidvegi, 1983: Zalenskaya and Zalensky, 1980). The presence of a single band for each core histone indicates that modification of these histones had not occurred during the pharate adult stage. Extraction with 2% NaC1 75% ethanol essentially removed all the his- tones from the acid extract since identical bands of less intensity were observed from the 2.0 M NaCI extract.

Calf thymus histones have been separated into five major classes designated H 1, H2A, H2B, H3, and H4 (Johns, 1967, Panyim and Chalkley, 1969a). Histone HI was not present in the natural mixture of calf thymus histones (Fig. 1). In addition, the classes of calf thymus histones were not as clearly resolved as anopheline histones. This is probably due to differences in the method of isolation and extraction of histones and demonstrates the effectiveness of the presented methodology for the preparation of histones.

DISCUSSION

In eukaryotes, most of the transcription restriction of DNA is undoubtedly due to histones (Kostraba and Wang, 1975). DNA is compacted with histones in the nucleosomal core in a way that is accessible for polymerase and effector molecules. The nucleosomal "spacer region" is associated with variable amounts of histones HI and/or H5 and differs between cells of the same species and different species.

To find conditions for the preparation and electro- phoretic analysis of nucleosomal core and spacer region histones in Anopheles albimanus, the isolation and extraction of histones were examined under various conditions. A method was developed that allows the preparation of all major histone com- ponents. It was essential to use chromatin as the starting material and to selectively extract histones from chromatin. All conditions other than the one presented, resulted in large amounts of nonhistone chromosomal proteins and histones as shown by multiplicity of electrophoretic bands (unpublished results).

Anopheles albimanus histones 607

H1

H3 H2B H2A

H3 H2B

H4

A B C D Fig, I. Polyacrylamide gel electrophoresis of calf thymus and Anopheles albimanus histones. (A) Mixture of histones from calf thymus obtained from Boehringer Mannheim as reference. (B) Histone fractions H2A and H4 obtained from Boehringer Mannheim as reference. (C) 2% NaCI-75% ethanol extract from Anopheles albimanus adults after 0.25 M HC1 extraction. (D) 2.0 M NaC1 extract from Anopheles albimanus adults after 0.25 M HCI and 2% NaCI-75% ethanol extractions. The conditions for

electrophoresis are described in Materials and Methods.

A striking fact, in this study, is the presence of a histone class in Anopheles albimanus adults ten- tatively identified as histone HS. The condensation of chromatin is correlated with and possibly caused by the appearance of a tissue specific histone termed histone H5 (Appels et al., 1972; Simpson, 1978; Grimmond and Holmes, 1981). Cerebellar and pig electrophoretic patterns reveal a band whose mobility is similar to histone H5 and termed histone 1 ° (Shaw and Huang, 1970). It has not been established whether histone H5 is identical to histone 1 °. Further identification of histone H5 in anopheline adults requires amino acid analysis since the electrophoretic approach for histones is limited because mobility measurements depend on molecular weight and the presence of charged amino acids. This is the first report of histone H5 in insects.

Electrophoretic analysis of Anopheles albimanus histones also revealed the presence of histone H1 and H1 subfraction. This class of histones is fairly well conserved although the number of subfractions vary in species and differ in the amount of amino acids. Data favor the assumption that the principal struc- tural rearrangement of chromatin related to repres- sion and derepression of genes occurs at the supra- nucleosomal level known to be stabilized by histone H1 (Worcell et aL, 1977). Histones H5 and HI may be considered variants of the same class, although differences are extensive as shown by electrophoretic

mobilities in this study and in other investigations (Isenberg, 1979).

The Anopheles albimanus core histones (H2A, H2B, H3, and H4) are similar in electrophoretic mobility to calf thymus histones, except histone H4. Histones H2A and H2B have greater variability as compared to histone H3 in many organisms (Von Holt et al., 1979). H2A and H2B are not only structural elements of the nucleosome but also may interact with molecules outside the nucleosome core and thus have additional species and tissue function (Kadura et aL, 1983). Histories H3 and H4 occur in the globular region of the molecule in a sequence that is rich in hydrophobic residues. These histones are conserved and arginine rich, however, recent findings show that Euglena and Tetrahymena H3 and H4 are not arginine rich (Jardine and Leaver, 1978). In addition, the gel mobilities of both yeast histone H3 and Tetrahymena H3 are distinctly different from that of calf thymus (Mardian and Isenberg, 1978).

In summary, the method presented for chromatin isolation and the subsequent specific extractions of histones make it possible to clearly resolve these proteins by polyacrylamide gel electrophoresis. The method will be useful in determining the composition and electrophoretic patterns of histones in devel- opmental stages of strains and species of mosquitoes. It is also reasonable to assume that changes in gene activity (gene amplification) as a result of exposure to

608 STEVE MILLER and SONJA THORNTON

insecticides, may be accompanied by changes in his tone patterns. These pa t te rns can be determined by the me thod presented in this communica t ion .

Acknowledgements--The authors thank Dr Robert Novak for his criticisms and suggestions and Loraine Novak for critical reading of the manuscript.

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