Characterisation of histones in the salivary glands of Rhynchosciara americana larvae

Cell Differentiation, 8 ( 1 9 7 9 ) 3 8 3 - - 3 9 4 © E l s e v i e r / N o r t h - H o l l a n d Scientific Publishers Ltd.

383

CHARACTERISATION OF HISTONES IN THE SALIVARY GLANDS OF R H Y N C H O S C I A R A A M E R I C A N A LARVAE*

MANUEL TROYANO PUEYO, CARLOS EDUARDO WINTER and F R A N C I S C O J.S. L A R A

Departamento de Bioqu[mica, Instituto de Qu[mica, Universidade de S~o Paulo, CP20780, SlT"oPaulo (Brasil) Accepted June 16th, 1979

T h e h i s t o n e s f r o m t h e salivary glands o f Rhynchosciara americana larvae were identified. T h e e l e c t r o p h o r e t i c p a t t e r n s o f t h e p r o t e i n s s t u d i e d r e s e m b l e t h a t o f calf t h y m u s h i s t o n e s , i n c l u d i n g t h e H 1 h i s t o n e , w h i c h in Rhynchosciara h a s a l o w e r e l e c t r o p h o r e t i c m o b i l i t y in u r e a / p o l y a c r y l a m i d e gels b u t s h o w s a m o l e c u l a r w e i g h t i d e n t i c a l t o t h e corr e s p o n d i n g h i s t o n e o f calf t h y m u s , as j u d g e d b y S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s .

Previous results (Pueyo et al., 1975} have shown that histone synthesis in polytene cells of salivary glands of R. americana larvae, occurs in the cytoplasm in small polyribosomes containing 3--4 ribosomes. These are active in histone synthesis by the time of maximal DNA synthesis and the opening of DNA puffs in the gland chromosomes. The observation that the last replication cycle in the gland cells is a highly synchronised phenomenon (Socker and Pavan, 1974, Machado-Santelli and Basile, 1975) which lasts throughout the last 5--6 days of the larval stage (Cordeiro and Meneghini, 1973) renders this system attractive for the study of the temporal relationship between DNA synthesis and that of the different histones. However, these proteins were not previously studied in Rhynchosciara. The aim of this paper is to describe the experiments that led to the characterisation of histones of Rhynchosciara americana salivary glands. MATERIALS AND METHODS

Animals Larvae of Rhynchosciara americana were raised under laboratory conditions as previously described (Lara et al., 1965). All the experiments were carried out with larvae from the 4th instar which comprises most of the larval life. For practical purposes the 4th instar was divided by Terra et al. (1975) * T h e h i s t o n e n o m e n c l a t u r e used in this p a p e r is t h e o n e p r o p o s e d in t h e Ciba F o u n d a t i o n S y m p o s i u m 28, " T h e S t r u c t u r e a n d F u n c t i o n o f C h r o m a t i n " , Elsevier, A m s t e r d a m , ( 1 9 7 5 ) . T h e c o r r e s p o n d e n c e b e t w e e n this n o m e n c l a t u r e a n d t h a t o f J o h n s a n d B u t l e r ( 1 9 6 2 ) is: H, = F~; H2B = F2b; H2A = F2a 2 ; H 3 = F 3 a n d H 4 = F~al.

384

into periods which account for important modifications in the physiology as well as in the morphology of the larvae. Among these we can make reference to the 3rd period when the animals start to spin the collective c o c o o n at approx. 49 days after egg hatching. This period usually goes on for 5 or 6 days toward the 4th period (2--3 days) after which the animals enter the 5th period {approx. 57 days after egg hatching) and a burst of DNA synthesis is detected in the salivary gland chromosomes, the maximum synthesis of DNA taking place coupled with the maximum opening of the 2B puff. Usually this period is 1--2 days long and after it the animals enter the 6th period which is characterised by the maximum opening of the 3C-puff in the salivary gland chromosomes.

Histone extraction from the salivary gland nuclei The nuclei of a b o u t 200 salivary glands of larvae from either 3rd or 5th periods of the 4th instar were prepared as described previously by Santelli et al. {1976). The excess of buffer was drained off and the sedimented nuclei were then suspended in 5 ml of a 0.4 N H2SO4 and 50 mM NaHSOa solution. The suspension was transferred to a Dounce homogeniser where the nuclei were vigorously homogenised during 1 h at 0--4°C. After the extraction of basic proteins, the suspension was centrifuged at 10 000 g for 20 min in a Sorvall refrigerated centrifuge. The supernatant was then precipitated with 4 vols. of 90% ethanol for 24 h at - 1 0 ° C . The sediment was washed once with the same ethanol solution and stored under vacuum until subsequent analysis.

Preparation of chromatin from salivary glands The m e t h o d employed for this purpose is essentially the one described b y Elgin and H o o d (1973) for the purification of Drosophila egg chromatin. It m a y be summarised as follows: 40 salivary glands from animals in the appropriate stage o f larval development were homogenised in a mini-Dounce (capacity for 2 ml) in 1 ml of buffer (10 mM Tris--HC1; 100 mM NaC1; 10 mM MgC12 and 250 mM sucrose, pH 7.6). After homogenisation the suspension was centrifuged in the cold at 3000 g for 5 min to recover crude chromatin. The sediment was washed twice with the same buffer, twice with 5 mM Tris--HCl (pH 8.0) and once with 1 mM Tris--HC1 (pH 8.0) by centrifugation at 10 000 g for 10 min in the cold. After the last washing, the chromatin was suspended in 5 mM Tris--HC1 (pH 8 . 0 ) c o n t a i n i n g 100 ug/ml crystalline a-amylase from B. subtilis a n d then incubated for 20--30 min at 37°C. This enzymatic treatment was found convenient in order to improve the purity of the chromatin preparation. After this treatment the chromatin was washed with 10 ml of 5 mM Tris--HC1 (pH 8.0) and resuspended in 1.5 ml of the above buffer which was layered over a cushion of 3.0 ml of 1.7 M sucrose made up in the same buffer. The chromatin preparation was then spun in a SW 65 K rotor of a Spinco L3-50 ultracentrifuge at 35 000 rev./min for 105 min at 4°C. Subsequently, the chromatin was recovered from the

385 b o t t o m of the tube, washed three times with 10 mM Tris--HCl (pH 8.0), and immediately used for the extraction of basic proteins.

Histone extraction from purified chromatin of salivary glands Chromatin preparations obtained by the above procedure were transferred to a mini-Dounce with the aid of 1.0 ml of 0.4 N H2SO4 plus 50 mM NaHSO3 solution. The volume was adjusted to 2 ml with the same solution and the suspension was vigorously homogenised for I h at 4°C in order to extract histones. After the extraction, the suspension was centrifuged at 10 000 g for 10 min in the cold and the sediment discarded. To the supernatant was added 100 pl o f a 1 mg/ml protamine sulphate solution made up in 0.4 N H2SO4 (in some instances 50 pl of 1 mg/ml solution of crystalline a-amylase were used instead of protamine sulphate). The aim of the addition of protamine sulphate to the histone solution is to improve the recovery of basic proteins after their precipitation with 0.2 vols. of 1 g/ml TCA for 20 min at 0--4°C (Franco et al., 1974). After precipitation with TCA, the proteins were sedim e n t e d by centrifugation at 10 000 g for 10 min in the cold, washed twice with acetone/HC1 (99 : 1), once with pure acetone (Cohen and Gotchel, 1971) and then dired and stored under vacuum at - 1 0 ° C prior to analysis in polyacrylamide gels.

Histone electrophoresis in urea/polyacrylamide gels Rhynchosciara histones prepared from either nuclei or purified chromatin were analysed in 15% acrylamide gels containing 6.5 M urea as previously described b y Pangina and Chalkley (1969).

Histone electrophoresis in SDS-polyacrylamide slab gels; molecular weight estimation Histones prepared from the purified chromatin, as well as calf thymus histones (Sigma Chemical Co.) and proteins of known molecular weight, were first treated as described by Winter et al. (1977). Briefly this comprises dissolution in a buffer (10 mM Tris; 0.5 mM EDTA; 2.5% SDS; 2.5% ~mercaptoethanol; 5% sucrose) and denaturation by heating up to 50°C for 1 h. This procedure ensures m o n o m e r analysis in SDS-polyacrylamide gels. After this, 20 pl of a 1 mg/ml Rhynchosciara histone solution together with the other proteins were analysed in t w o different electrophoretic systems, both based on the buffers described by Laemli (1970) and on the apparatus assembled according to Studier (1973). The functional differences between the t w o procedures will be described shortly. The electrophoresis of proteins was performed for 4 h at room temperature; after this the gels were fixed for 1 h with 10% TCA (w/v), at 4 ° C, stained overnight in a 0.1% Coomasie Blue R (Sigma Chemical Co.), 95% ethanol and 10% acetic acid solution, and finally destained for 8 h in a 25% ethanol plus 10% acetic acid solution.

Electrophoresis in gradient slab gels. Histones from Rhynchosciara were

386

run together with purified H~ t h y m u s histone (obtained from the commercial preparation according to Johns, 1964) and the other standard proteins in a linear SDS-polyacrylamide gel slab gradient formed b y using t w o communicating chambers connected to a peristaltic p u m p (LKB Producter AB) which are filled with equal volumes of t w o solutions made up in 8% and 15% acrylamide, according to Laemli (1970), and containing 1.0% and 5.0% glycerol respectively to stabilise the gradient during the polymerisation. The molecular weight of the separated histone bands was then estimated as described by Lambin et al. (1976) from a log T* versus log mol. wt plot.

Electrophoresis in gel slabs o f constant acrylamide concentration. Five slabs of this kind of gel were prepared, using in each one 8,10,12,14 and 16% acrylamide solutions respectively. These were prepared as described by Winter et al. (1977). The same proteins as before (except the H1 t h y m u s histone) were analysed in this system. Their migration allowed us to work o u t log R m * * versus %T plots for all the proteins. These plots gave us the K ' r ¢ (Ferguson, 1963) The molecular weight of Rhynchosciara histones was then estimated from t h e K' r versus mol. wt plots as r e c o m m e n d e d b y Hayashi et ak (1974). RESULTS AND DISCUSSION

The approach for the identification of histones is based on their isolation and on the determination of several characteristics of these proteins. Among these the determination of the amino acid composition of each separated band assumes special importance. However, in some instances, as in the case of Rhynchosciara salivary glands, this cannot be easily accomplished, since it would be necessary to dissect a great number of larvae to obtain enough mass of each histone to perform such analyses. The assumption that the histones of salivary glands are the same as those from the rest of the animal allows us to use the whole larva and consquently facilitates the amino acid determination. However, this could n o t be done with Rhynchosciara since some very active proteases, probably deriving from other organs of the larva (W. Terra, pets. comm., caused extensive protein degradation. Thus, the salivary gland histones were identified by other criteria which are widely used in the field. These criteria are: origin, electrophoretic behaviour, staining properties and molecular weight determination.

Origin In all experiments described here, the proteins were extracted with * T = (g o f a c r y l a m i d e + g o f b i s a c r y l a m i d e ) / v o l u m e × 1 0 0 ( H j 6 r t e n , 1 9 6 2 ) . ** R m is t h e r a t i o o f t h e d i s t a n c e m i g r a t e d b y t h e p r o t e i n s a n d t h e t r a c k i n g d y e ( b r o m o p h e n o l blue). ¢ K r is t h e r e t a r d a t i o n c o e f i c i e n t o f t h e p r o t e i n in t h e gel.

387

0.4 N H2SO4 either from nuclei (Santelli, 1976) or from purified chromatin (Elgin and Hood, 1973), Thus, one can expect that almost all the extracted proteins are histones. The purity of the preparation can be checked by comparing the electrophoretic mobility of the main bands with that of standard histones of calf thymus.

Electrophoretic behaviour The method employed for the analysis of the basic proteins extracted

r

R []

H3o x,..

-.,,1 %

H1,H3+H2B,. H2A,.-

Dw []

Fig. 1. Electrophoretograms of calf thymus histones and nuclear basic proteins of Rhynchosciara salivary glands. Both thymus and Rhynchosciara proteins were solubilised in 0.9 N acetic acid containing 20% sucrose, and 50 ul samples were loaded onto the top of 7.5 cm × 0.6 cm gels of 15% acrylamide which were prepared according to Panyim and Chalkley (1969). The electrophoresis solution was 0.9 N acetic acid. The other conditions were: 2.5 mA/gel during 5.0 h at 6--10°C. Migration was from the positive to negative pole. (T) electrophoretogram of calf thymus histones (purchased from Sigma Chem. Co.); (R) electrophoretogram of basic proteins extracted from nuclei of 200 salivary glands of 5th period larvae, as described in Materials and Methoda. After electrophoresis the gels were stained for 2 h in a solution made up of 0.1% amido black; 7% acetic acid and 20% ethanol at room temperature. Destaining was carried out for 30-40 h in a 10% acetic acid solution.

388 either from nuclei or purified chromatin was that of Panyim and Chalkley (1969), which allows in a simple and reproducible way, the separation of the histone complement from several organisms into 5 main bands (Panyim et al. 1971). Figure 1 illustrates the results obtained when thymus histones and Rhynchosciara basic proteins are separated by this technique. In the gel labelled T (Fig. 1) calf t h y m u s histones were analysed. The several bands correspond in increasing order of mobility to the oxidised H3 histone, followed b y the H1, H3, H2B, H2A and H4 histones. The band H3ox was observed to be c o m p o s e d (results n o t shown) of two sub-bands, each of which corresponds to a dimer arising from the oxidation of the H3 histone which in t h y m u s contains t w o cysteine residues (Fambrough and Bonnet, 1969). The electrophoretic mobility of such bands is roughly equivalent to half of that of the m o n o m e r (Panyim et al., 1970) as can be seen in Fig. 1. In this figure the gel labelled R, shows the results obtained in the analysis of nuclear basic proteins of R. americana. The bands marked 1 to 6 can be identified by comparing their mobilities with that of calf t h y m u s as demonstrated below.

Bands 1, 3 and 6. The electrophoretic mobilities of the H3 and H4 histones are independent from their source due to the higher degree of conservation of their amino acid sequences t h r o u g h o u t evolution (Fambrough and Bonner, 1968; De Lange et al., 1969). This criterium allows us to identify the bands 3 and 6 as being the histones H3 and H4 respectively of Rhynchosciara. With respect to band 1, we can see that its mobility is half that of band 3 identified as the H3 histone. This fact agrees with the observations of Oliver and Chalkley (1972) in Drosophila for the dimer of the H3 histone. In this case only a dimer is observed, instead of t w o as in the case of calf t h y m u s (results n o t shown). The unique dimer reflects the presence of only one cysteine residue in the m o n o m e r (Oliver and Chalkley, 1972). Thus, the band 1 in Rhynchosciara was identified as the dimer of the H3 histone. Band 2. This band is identified as the H1 histone, because except for the band corresponding to the H3 dimer, it is the band having lowest electrophoretic mobility. This identification is justified because the HI histone is that of lowest mobility in all organisms studied until now. The comparison b e t w e e n gels T and R in Fig. 1 shows that the mobility of the Rhynchosciara H~ histone (band 2) is lower than that of the similar calf t h y m u s histone, as expected from identical observations made in other insects (Cohen and Gotchel, 1971; Oliver and Chalkley, 1972; McMaster-Kaye and Kaye 1973; Franco et al., 1974; Alfageme et al., 1974). Besides this, it was observed that the previous t r e a t m e n t of the whole preparation with DTT does not change the position of band 2 (results not shown). This fact precludes the possibility that band 2 can be a dimer of the H3 histones. Bands 4 and 5. Due to the region where they migrate in the gel these bands are tentatively identified as the H2A and H2B histones respectively. This

389 TABLE

I

RELATIVE SOURCES

ELECTROPHORETIC

MOBILITIES

OF

HISTONES

FROM

VARIOUS

Histones from Rhynchosciara and calf thymus were analysed in polyacrylamide gels containing 6.5 M urea as described in the text. Their mobilitiesare compared with that of the histones from other sources previously described, and are given as percent of the mobility of the H 4 hlstone which is considered as being 100%. Our data were obtained from 12 determinations in the case of Rhynehosciara and from 4 determinations in the case of calf thymus.

Source

Histone

Reference

H,

H3

H2A

H2B

H4

Drosophila (sal. gland)

62

81

86

92

100

House cricket (Cecus)

62

78

83

88

100

C. capitata R. americana

67 61 +_2 66 -+ 2 65

82 76-+ 6 77-+ 2 82

89 80-+ 6 85+- 2 88

93 86+- 6 80-+ 2 85

100 100 100 100

Calf thymus Rat uterus

Oliver and Chalkley (1973) McMaster-Kaye and Kaye (1973) Franco et al. (1974) aOurdata b Our data Barker (1971)

a Measured in 4 gels b Measured in 12 gels i d e n t i f i c a t i o n c o r r e s p o n d s t o an inversion o f t h e i r position w h e n c o m p a r e d t o t h y m u s histones and it is based o n t h e o b s e r v a t i o n o f P a n y i m et al. ( 1 9 7 1 ) , a c c o r d i n g t o which t h e s e p a r a t i o n f r o m t h e p h y l l u m m a m m a l i a in the evolut i o n a r y scale leads t o an inversion o f t h e H2B, H2A o r d e r as observed in t h y m u s , so t h a t t h e increasing o r d e r o f these t w o p r o t e i n s in Drosophila is H2A, H2B ( C o h e n a n d G o t c h e l , 1 9 7 1 : Oliver and Chalkley, 1 9 7 2 ) . T h e c o m parison o f mobilities o f basic p r o t e i n s in Rhynchosciara with t h a t o f histones f r o m o t h e r insects, is a n o t h e r e l e m e n t o f i d e n t i f i c a t i o n (see Table I).

Stain properties A n o t h e r c r i t e r i u m o n which we can establish t h e n a t u r e o f Rhynchosciara basic n u c l e a r p r o t e i n s is based o n t h e stain p r o p e r t i e s o f histones with a m i d o black. T h e H1 a n d H2B histones are stained purple, while t h e o t h e r bands are stained blue. This p r o p e r t y can be studied b y o b t a i n i n g t h e a b o r b a n c e s o f t h e respective bands in t w o visible wavelengths, 4 5 0 a n d 700 nm, in t h e same m a n n e r as C o h e n and G o t c h e l ( 1 9 7 1 ) did in t h e case o f Drosophila histones. A c c o r d i n g t o these a u t h o r s , t h e bands c o r r e s p o n d i n g t o t h e HI and H2BhiSt o n e s m u s t e x h i b i t l o w e r a b s o r b a n c e in near red wavelengths ( 7 0 0 n m ) t h a n in n e a r blue wavelenths ( 4 5 0 n m ) . T h e r e c i p r o c a l m u s t b e t r u e f o r t h e H3 and H2A histories. Figure 2 shows t h a t t h e characteristic changes in absorb a n c e o c c u r as e x p e c t e d o n the bands previously identified as the HI, H2B and H3, H2A histones o f Rhynchosciara.

390

B

A

O


O

<~ 0.1

5

0

I

10

Cm

[]

i I

Fig. 2. Optical scanning at two different wavelengths of electrophoretically separated Rhynchosciara salivary gland histones. Basic proteins of 200 salivary glands of 3rd period larvae were extracted from nuclei and analysed in acrylamide gels as described in the caption of Fig. 1. Running time 3.5 h. The abcissa refers to dimensions of the recorder chart. (A) gel scanning at 450 nm; (B) gel scanning at 700 nm; (C) stained electrophoretogram of the preparation.

Molecular weight The molecular weights of Rhynchosciara histones were estimated by using t w o different methods. First, comparing in polyacrylamide gel-SDS electrophoresis the Dipteran basic proteins with histones of calf thymus and other proteins of well known molecular weight, where we changed the acrylamide concentration in each gel from 8% to 16%. This allowed us to work out a plot of K'r versus molecular weight (see Fig. 3A), so that it is possible, according to Hayashi et al. (1974), to obtain accurate values for the molecular weights of histones. In the second m e t h o d we compared Rhynehosciara

391

*S

A

I,..

10

~6

1.10

~0 I'08

1.06

~ O

1.04

i'M

eO 1.02

I

I.OO i

I

J

t

10 20 30

t

i

I

I

40 50 60 70 -3

MWxlO

0.08 4.0

I 4.2

A 4.4

I 4.6

i 4.8

log M W

Fig. 3. Plots used to estimate the histones molecular weights. (A) the log of acrylamide concentration at which the protein migrates after electrophoresis in a linear acrylamide gradient, is plotted versus the log of the tool. wt. The equation obtained by linear regression is: log T = (--01503) log tool. wt + 1.7224 (r = 0.9982). ( B ) t h e retardation coefficient is computed from Ferguson plots (Ferguson, 1964) and is plotted versus the tool. wt of the proteins. The equation obtained by linear regression is: --K'r × 100 =- (0.0838) mol. w t + 4.5242 (r= 0.9880). The proteins used as standards are: Myoglobin (M) = 17 200; Chymotrypsinogen ( Q ) = 25 700; Egg albumin ( O ) = 43 000; Muscle pyruvate kinase ( P ) = 57 000. Bovine serum albumin ( S ) = 68 000. These molecular weights were obtained from Weber et ai., (1972).

histones with that of calf t h y m u s and with standard proteins in a same gel plate made up of a linear polyacrylamide gradient from 8% to 15% acrylamide (see Fig. 3B). The four-band pattern obtained by using the first procedure (not shown here) agrees with that obtained for histones in similar electrophoretic systems (Panyim and Chalkley, 1971; Panyim et al., 1971; Hayashi et al., 1974} b u t it may be improved using a linear polyacrylamide-SDS gel gradient as we did in the second m e t h o d , with which it is possible to resolve these proteins into 5 very well-separated bands (Fig. 4). The results in terms of molecular weight are shown in Table II. We can see that: (a) the calculated molecular weight for calf thymus and Rhynchosciara H1 histone are the same, regardless of the m e t h o d employed for this determination although the value found in our case is higher than the tool. wt

392

T

R Y BP

H1Q

o

1

Qo 2 3 f 5

OM

[] Fig. 4. Electrophoretogram of proteins analysed in SDS-polyacrylamide slab gel gradient. Proteins of known molecular weight were treated separately in a buffer (62 mN Tris--HCl; 5 mM EDTA; 2.5% SDS; 2.5% beta-mercaptoethanol and 5% sucrose at pH 6.8 (Winter et al., 1977), during 1 h at 50°C until totally solubilised. The basic proteins of Rhynchosciara were extracted from the purified chromatin of 200 salivary glands of 5th period larvae as described in Materials and Methods, and solubilised as above for standard proteins. Samples containing 5 ug of each standard protein were loaded onto the top of SDS polyacrylamide slab gel gradients prepared as described in Materials and Methods. The same was done with 20 ul of 1 mg/ml solution of Rhynchosciara basic proteins. The electrophoresis was carried out employing the Laemli (1970) buffer at 6.6 V/cm during 4 h at room temperature. After electrophoresis, the slab was fixed in 10% TCA (w/v) for 1 h and stained overnight in a solution containing 0.1% Coomasie Blue R (Sigma Chemical Co), 10% acetic acid and 45% ethanol. For destaining, a solution of 8% acetic acid and 25% ethanol was used. The molecular weights of the standard proteins, labelled with capital letters on the figure, are based on data from Weber et al., (1972): S = 68 000 (serum albumin); P = 57 000 (pyruvate kinase);O = 43 000 (egg albumin); Q = 25 700 (chymotrypsinogen); M = 17 000 (myoglobin). In the labelled slots, were run the following samples: Slot T: here was run the H 1 histone obtained from a commercial preparation of calf thymus histones (Sigma Chemical Co.) using the method of Johns (1962). Slot R: were run the histones obtained from Rhynchosciara. These proteins are labelled 1, 2, 3, 4, and 5 in the order of increasing electrophoretic mobility. Migration was from negative to positive pole.

value of approx. 21 000 reported for the vertebrate histone (Delange and Smith, 1971; Panyim and Chalkley, 1971); (b) the molecular weights for the other Rhynchosciara histones are in good agreement with the values reported for the corresponding histones from other sources (Panyim and Chalkley, 1971). In view of the results shown in Table II we identify in Fig. 4 the bands 1, 2, 3, 4, and 5 as the histones H,, H3, H2A, H2B and H4 respectively.

393 TABLE II THE M O L E C U L A R WEIGHT OF R H Y N C H O S C I A R A S A L I V A R Y GLAND HISTONES AS CALCULATED BY TWO METHODS USING SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS The molecular weight of Rhynchosciara histones and that o f the H~ thymus histone were calculated by using slab gels made up in a polyacrylamide gradient (method A) and by using several slab gels of different polyacrylamide concentrations (method B). The results obtained are compared with the data found in the literature for Rhynchosciara histories and for the H 1 histone from thymus, the only vertebrate protein analysed in the present work. Histone molecular weights H1

H3

H2A

H2B

H4

Reference

Method A: acrylamide gradient in slab gels Rhynchosciara Calf thymus

32000 32 000

15 100 nd a

14200 nd

13000 nd

11 300 nd

this paper this paper

Method B" constant acrylamide concentration in slab gels R h y nchosciara Calf thymus

29 800 29 800

14 100 nd

10 200 nd

this paper this paper

21000

14000

14000

12 500

11 000

21000

15 324

15000

13 774

11 282

(Panyim and Chalkley, 1971 ) (cited in Huberman, 1975)

Values for the calf thymus histones found in the literature

12 300 nd

aNot determined.

Clearly, we failed to correct the molecular weight of the H1 histones b y using the K ' r v e r s u s mol. wt plots (Hayashi et al., 1974). Despite this we can remark that if the mobility differences observed in SDS-acrylamide gel electrophoresis reflect molecular weight differences, then it is possible to predict that the molecular weight of the H~ histone from both Rhynchosciara and t h y m u s are the same and correspond when corrected to approx. 21 000. This situation is n o t found in Drosophila, in which H1 histone has a molecular weight higher than the corresponding protein of thymus and other organisms {Cohen and Gotchel, 1971; Oliver and Chalkley, 1972; Alfageme et al., 1974). On the other hand, as the electrophoretic mobility of the Rhynchosciara H l histone in urea/polyacrylamide gels is lower than that of the same protein of thymus (see Fig. 1 and Table I) we can predict that similarly to what was observed in Drosophila, its basicity is lower than that of the vertebrate protein. Together, these facts also can indicate that the basicity of the Rhynchosciara H~ histone is lower than that of the Drosophila H~ histone.

394 O u r results i n d i c a t e t h a t w i t h i n the r e s o l u t i o n o f t h e t e c h n i q u e s e m p l o y e d , t h e c h a r a c t e r i s t i c s o f t h e HI h i s t o n e f r o m p o l y t e n e c h r o m o s o m e s o f d i f f e r e n t species (Drosophila a n d Rhynchosciara) are n o t e x a c t l y t h e s a m e . It is possible t h a t t h e d a t a o b t a i n e d in Drosophila m a y n o t be r e p r e s e n t a t i v e o f t h e insect H1 h i s t o n e a n d p r o b a b l y t h a t s o m e d i f f e r e n c e s o b s e r v e d in t h a t protein are n o t strictly r e l a t e d to t h e c h a r a c t e r i s t i c s t r u c t u r e o f t h e p o l y t e n i c chromosome. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y grants f r o m F u n d a c ~ o de A m p a r o ~ Pesquisa d o E s t a d o de S~o Paulo ( F A P E S P ) ( G r a n t s no. 7 4 / 1 6 6 a n d 1 6 / 0 4 0 7 ) a n d Cons¢lho N a c i o n a l de D e s e n v o l v i m e n t o Cietifico e T ~ c n o l h g i c o (CNPg) ( G r a n t no. SIP 0 4 / 0 0 3 ) . M.T.P. a n d C.E.W. were g r a d u a t e fellows f r o m FAPESP. REFERENCES Alfageme, C.R., A. Zweidler, A. Mahovald, and L.H. Cohen: J. Biol. Chem. 249, 3729-3736 (1974). Cohen, L.H. and B.V. Gotchel: J. Biol. Chem. 246, 1841--1848 (1971). Cordeiro, M. and R. Meneghini: J. Mol. Biol. 78,261--274 (1973). DeLange, R.J. and E.L. Smith: Annu. Rev. Biochem. 40, 279--314 (1971). Elgin, S.R.C. and L.E. Hood: Biochemistry, 12, 4984--4991 (1973). Fambrough, D.M. and J. Bonnet: J. Biol. Chem. 243, 4434--4439 (1969). Ferguson, K.A.: Clin. Exp. Metabolism 13,985--1002 (1964). Franco, L., F. Monteiro, J.M. Navlet, J. Perera and M.C. Rojo: Eur. J. Biochem. 48, 53-61 (1974). Hayashi, K. E. Matrutera and Y. Ohba: Biochim. Biophys. Acta 342, 185--194 (1974). Hj~rten, S.: Arch. Biochem. Biophys. suppl. 1,147--151 (1962). Huberman, J.A.: Annu. Rev. Biochem. 42, 355--378 (1973). Johns, E.W. and J.A.V. Butler: Biochem. J. 82, 15--18 (1962). Johns, E.W.: Biochem. J. 92, 55--59 (1964). Laemli, U.K.: Nature 227,680--685 (1970). Lambin, P., D. Rochu and J.M. Fine: Anal. Biochem. 74, 567--575 (1976). Lara, F.J.S.H. Tamaki and C. Pavan: Am. Nat. 99,189--191 (1965). Machado-Santelli, G.M. and R. Basile: Ciencia e Cultura 27,167--174 (1975). McMaster-Kaye, R. and J.S. Kaye: Arch. Biochem. Biophys. 156, 426--436 (1973). Oliver, D.R. and R. Chalkley: Exp. Cell Res. 73,295--302 (1972). Panyim, S. and R. Chalkley: Arch. Biochem. Biophys. 130,337--346 (1969). Panyim, S., R. Chalkley, S. Spider and D.R. Oliver: Biochim. Biophys. Acta 214, 216-221 (1970). Panyim, S., D. Bilek and R. Chalkley: J. Biol. Chem. 246, 4206--4215 (1971). Panyim, S. and R. Chalkley: J. Biol. Chem. 246, 7557--7560 (1971). Pueyo, M.T., M.F. Bonaldo and F.J.S. Lara: Cell Differentiation 4, 257--263 (1975). Santelli, R.V., G.M. Machado-SanteUi and F.J.S. Lara: Chromosoma 56, 237--248 (1976). Stocker, A.J. and C. Pavan: Chromosoma 45, 295--319 (1974). Studier, F.W.: J. Mol. Biol. 79, 237--248 (1973). Terra, W.R., A.G. de Bianchi, A.G. Gambarini and F.J.S. Lara: J. Insect. Physiol. 19, 2097--2106 (1973). Weber, K., J.R. Pringle and M. Osborn: Methods Enzymol. 26, 3--27 (1972). Winter, C.E., A.G. de Bianchi, W.R. Terra and F.J.S. Lara: Chromosoma 61, 193--206 (1977).