Characterization of Allomyces genome

377

Biochimica et Biophysica Acta, 478 (1977) 377--391 © Elsevier/North-Holland Biomedical Press

BBA 99013 C H A R A C T E R I Z A T I O N O F A L L O M Y C E S GENOME

MUKTI OJI-IA, HANS TURLER * and GILBERT TURIAN Laboratoire de Microbiologie, Ddpartement de Biologie v~gdtale, * Ddpartement de Biologie moldculaire, Universitd de Gen~ve, 1211 Gen~ve 4 (Switzerland)

(Received March 15th, 1977)

Summary A l l o m y c e s arbuscula DNA isolated from whole cells (bulk DNA) is composed of a major (~) and two minor components (~ & 7) with b u o y a n t densities in neutral CsCI corresponding to 1.721, 1.710 and 1.702 g/cm 3, respectively. The DNA obtained from purified nuclei contains ~ c o m p o n e n t only. The c o m p o n e n t corresponds to mitochondrial DNA. The 7 c o m p o n e n t is also extranuclear b u t has n o t been characterized. The reassociation kinetics of sheared, bulk and nuclear DNA show that (i) 25% bulk and 10% of nuclear DNA reanneal very rapidly and contain highly repeated sequences; (ii) moderately repeated sequences, accounting for 15% of b o t h bulk and nuclear DNA, have a sequence complexity of approximately 7.2 • 106 daltons and are repeated about 320 times; (iii) the slow reannealing fraction accounts for a b o u t 60% of the genome and has kinetic properties similar to single copy sequences. The sequence complexity of this fraction was determined in relation to that of Escherichia coli. After a correction for the size of the repeated sequences the genome size of A. arbuscula was calculated to be 1.7 • 10 ~° daltons.

Introduction Reassociation kinetics of the DNA from higher organisms have shown that the genome is composed of groups of sequences reassociating at very fast, fast and slow rates [1]. The very fast and fast reassociating components consist of simple sequences that are repeated many times [1,2]. The repetitive sequences form a significant portion (10--80%) o f the total eukaryotic genome [3]. These sequences are interspersed between unique sequences and considered to have a regulatory function in the expression of the genes [4]. From the kinetics of hybridization of these fractions with rapidly labeled RNAs it is concluded that repetitive fractions are poorly transcribed [5] and that the majority of the

378 transcribed sequences hybriduze with the slowly reassociating fraction and follow a true second order kinetics indicating that they are composed mainly of single c o p y sequences [6]. Earlier we have reported DNA mediated transfer of sexual polarity and nucleotide sequence h o m o l o g y between Allomyces strains [7,8]. In this paper we present our results on the satellite composition and reassociation kinetics of A. arbuscula DNA. Materials and Methods

Culture conditions The A. arbuscula strain Bali 1 (Noack 101.33) was used throughout in this study. The stock cultures were maintained as sporophytes on YpSs agar slants [9]. The inoculum for experiments consisted of meiospores which were grown as gametophytic cultures [7,8]. Isolation of DNA DNA was labeled with [32p]orthophosphoric acid. The details of the labeling and DNA extraction have been described earlier [7,8]. Enriched nuclear DNA was isolated from mycelia which were homogenized in a mitochondrial extraction medium (to be published later). After a pre-centrifugation in a Sorvall GSA rotor at 10 000 rev./min, the supernatant was used for the isolation of mitochondria, and the pellet for the extraction of nuclear DNA. Purer nuclear DNA preparations were obtained by a modification of the procedure of Dill and Stock [10] (see accompanying paper). The isolated nuclei were lyzed in the lysing mixture and the DNA was extracted by the hydroxyapatite m e t h o d [7]. 14C-Labeled Escherichia coli DNA E. coli cells (strains 100 thymine-less) labeled with [J4C]thymine were kindly supplied by Mme. G. Kellenberger of the Department of Molecular biology, University of Geneva. The following procedure was used for the DNA extraction: Labeled E. coli cells were washed three times with DNA buffer (Tris • HC1 0.01 (pH 8)/EDTA 0.05 M), resuspended in the same buffer to 1/10 of the original culture volume and incubated at 37°C for 30 min with 100 pg/ml lysozyme. Sodium lauryl sulfate (0.5%) and pronase (1 mg/ml) were added and the lysate was incubated for 4 h. The homogenate was deproteinized for 15 min with phenol saturated with DNA buffer and the aqueous phase was recovered after centrifugation in a Sorvall SS 36 rotor at 5000 rev./min. The interphase was re-extracted with a small a m o u n t of DNA buffer. Two volumes of ethanol were added to the combined aqueous layers and the DNA was spooled on a glass rod. The DNA was dissolved in 1/10 SSC, treated with 50 pg/ml of RNAase for 1 h at 37°C deproteinized again with phenol and reprecipitated. Final purification of the DNA was done by CsC1 density gradient equilibrium centrifugation. Preparative CsCl centrifugation Approximately 90 pg DNA in 2.5 ml SSC was mixed with 3.2 g solid CsC1 in

379 polyallomer tubes. The density was adjusted to 1.700 g/cm 3, and the solution was centrifuged in MSE 10 × 10 rotor at 37 000 rev./min for 60 h at 15--20 ° C. Fractions were collected from the b o t t o m of the gradient with a " p e r p e x " p u m p and recorded either directly on a Gilford s p e c t r o p h o t o m e t e r with a flow through cuvette or A260 of individual fractions was measured after addition of 0.2 ml water.

Analytical ultracentrifugation Native, bulk or nuclear DNA in 0.01 M Tris • HC1 buffer (pH 7.2) was used for detection of satellite components. Samples for the analysis of repetitive sequences were taken from partially reassociated nuclear DNA. The density of the DNA samples was brought to 1.700 g/cm 3 by the addition of solid CsCI. 2--5 pg of the above samples were mixed with 1 pg marker DNA (Micrococcus lysodeikticus or mouse DNA) and centrifuged in a Spinco model E ultracentrifuge equipped with a photoelectric scanner. The centrifugation was done at 40 000 rev./min at 20°C for 24 h. The b u o y a n t densities were calculated according to the formula of Schildkraut et al. [ 11].

Optical melting profiles The denaturation profiles of the DNA samples were obtained with a Gilford thermoprogrammer model 2527. The samples were sealed in a Gilford model 2527-7 cuvette which has a path length of 1 cm and uses a thermoelectric device for heating. The samples were heated at a rate of l°C/min. The changes in temperature and hyperchromicity were recorded with a Beckman WW 600 Tarkan recorder attached to the thermoprogrammer and spectrophotometer. The increase in absorbance for each 0.5°C interval was normalized with respect to maximum hyperchromicity and plotted against temperature. The G + C values were calculated according to Mandel et al. [12].

Fragmentation of the DNA To reduce the fragment length, DNA samples were sheared either b y pressure drop or sonication. Fragmentation by pressure drop consisted of passing the DNA solutions through a needle valve with a pressure drop of 48 000 lb/inch 2. Fragmentation b y sonication was done in a Branson homogenizer using a micro tip. The DNA solutions in polyallomer tubes were equilibrated with N: and sonicated at full power bursts (60 W) in alternate cycles of 10 s sonication and 10 s cooling in ice for a total of 4 min. The sheared DNAs were first passed through a Metricel GA-3 (pore diameter 0.2 pm) filter and then through a chelex resin (Bio-rad. chelex 100) to remove heavy metal ion contaminations. The average size of the DNA fragments was determined by sedimentation in alkaline sucrose gradients or by electron microscopy [13].

Reassociation analysis Appropriate m o u n t s of DNA in 0.03 M phosphate buffer pH 6.8 were sealed in reaction vials, denatured at 105--110°C in ethylene glycol for 3 min and quickly plunged in ice. The phosphate buffer molarity of the DNA solution was adjusted to 0.12 M or 0.48 M b y addition of 4.8 M phosphate buffer (pH 6.8}. While the vials were still in ice, the reaction mixture was brought to 2 mM

380

EDTA and 0.4% sodium lauryl sulfate. Appropriate quantities were sealed either in capillary tubes or reaction vials and incubated either at 70 or 72°C depending upon the PB molarity. After incubation to the desired Cot values the reactions were terminated by plunging the reaction vials or capillaries in ice, and the contents processed on hydroxyapatite columns [ 7,8 ].

Purification of repeated and non-repeated sequences 5 mg of ultrasonically fragmented bulk DNA in 0.03 M phosphate buffer, 2 mM EDTA and 0.4% sodium lauryl sulfate was denatured for 3 min at 105 ° C. The phosphate buffer molarity was raised to 0.12 M and the mixture was incubated to a Cot of 4 at 70 ° C. The reaction was stopped (by plunging the reaction vial in ice) and the phosphate buffer molarity of the reaction mixture was brought to 0.03 M with ice cooled 0.001 M phosphate buffer. The solution was then quickly passed over a hydroxyapatite column equilibrated with 0.03 M phosphate buffer at 70 ° C. The duplexes were separated from single-stranded DNA as described earlier [7,8]. The fractions in 0.14 M and 0.48 M PB were pooled separately and dialyzed against 0.001 M phosphate buffer containing 2 mM EDTA. After extensive dialysis, the repeated sequenced (0.48 M phosphate buffer fraction} and single copy sequences (0.14 M fraction} were concentrated b y flash evaporation at 30°C, filtered through Metricel 0.2 p m filters and stored at --20°C tintfl further use. Results

Analytical CsCl density gradient equilibrium centrifugation In the analytical ultracentrifuge, bulk DNA resolves into 3 components (Fig. 1). In addition to the major c o m p o n e n t a, banding at a density of 1.721 g/cm 3, t w o light satellites/3 and 7 appear at 1.710 (/3) and 1.702 (7) g/cm3. The mole per cent G + C calculated from the densities are 62, 51 and 45% respectively.

u ¢-

o

.10 i. O

f g

1.702 1.710

1.~21

1.731

Density ( g m / c m 3 ) Fig. 1. Analytical CsC| density gradient equilibrium centrifugation o f w h o l e c e l l D N A o f A l l o r n y c e s arbuscula. D N A w a s i s o l a t e d a n d c e n t r i f u g e d t o e q u i l i b r i u m as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . D N A f r o m M i c r o c o c c u s l y s o d e i k t i c u s w a s u s e d as d e n s i t y m a r k e r ( 1 . 7 3 1 g / c m 3).

381

O.b.

i

0.4'

@

E i

0 ,0

i i

0.2

10

20 Fraction number

Fig. 2, H e t e r o g e n e i t y o f w h o l e cell A. arbuscula D N A . 9 0 #g of w h o l e cell D N A w a s s u b j e c t e d t o preparative CsC1 d e n s i t y g r a d i e n t e q u i l i b r i u m c e n t r i f u g a t i o n u n d e r c o n d i t i o n s d e s c r i b e d in Methods. A 2 6 0 o f individual f r a c t i o n s was m e a s u r e d . I n d i v i d u a l f r a c t i o n s i n d i c a t e d b y a r r o w s a n d m a r k e d 1 t o 4 w e r e t h e n s u b j e c t e d t o a n a l y t i c a l CsCI c e n t r i f u g a t i o n using M. lysodeikticus D N A as d e n s i t y marker (marker D N A p e a k s i n d i c a t e d b y d o t t e d lines).

The presence of two satellite DNAs was further demonstrated with samples taken from different regions of a preparative gradient (Fig. 2). Samples taken from the heavy region (1) and from the main band (2) consist exclusively of component (a). On the other hand samples taken from fractions marked by arrows 3 & 4, contain all three components of the bulk DNA (a, ~ & 7). In sample 3, ~ and 7 components are present in equal amounts whereas in sample 4, 7 component is the major peak. Analytical centrifugation of the DNA preparations from purified nuclei showed one symmetric band (Fig. 3). The possible existence of cryptic satellites was examined in samples enriched in either heavy, medium or light components. These samples were prepared by 3 step preparative gradient centrifugation (Fig. 4). All three preparations showed a single symmetric band in the analytical ultracentrifuge and no satellite either heavy or light could be observed. Highly repetitive sequences in eukaryotic DNAs are mostly of nuclear origin. In some cases they differ in density from main nuclear DNA as in mouse DNA [14] or they have the same over all base composition as in rat [14]. Rapidly reassociating DNA can however be separated from the rest of the DNA by

382

9

.O

1.T31 Top

Bottom Density(g/cm3)

F i g . 3 . A n a l y t i c a l C s C I d e n s i t y gradient equilibrium eentrifugation o f nuclear D N A of A. arbuscula: ( a ) w i t h d e n s i t y m a r k e r , (b) w i t h o u t . C o n d i t i o n s for the centrifugation are the s a m e as in F i g . 1 .

denaturation followed by a short term incubation to allow the renaturation of repetitive sequences only. On centrifugation in CsC1 at neutral pH the denaturated DNA being heavier than the renatured fraction forms a separate band. Analytical ultracentrifugations of heat denatured nuclear A. arbuscula DNA, and denatured DNA renatured to Cot of 0.001, 0.1 are shown in Fig. 5 and Table I. The banding profiles of these preparations were asymmetric. As expected the major portion remained single stranded. The renatured repetitive sequences did not separate into a distinct peak but formed a tail in the lighter region o f the profiles.

Reassociation kinetics Reassociation of the denatured, unfractionated total cell DNA (450 nucleotides fragment-length) is shown in Fig. 6. Approximately 25% of the DNA reassociated very rapidly (Cot 10-2), 15% somewhat less, but faster than the rest of the DNA which represents the slow reassociating fraction. The latter accounted TABLE

I

BUOYANT DNA

DENSITIES

OF BULK AND NUCLEAR

Solvent

SPECIES

State o f D N A

O F A. A R B U S C U L A

DNA

Density Major

Minor I

Minor I I

Bulk

N e u t r a l CsC1

Native

1.721

1.710

1.702

Nuclear Nuclear Nuclear

Neutral CsCl Neutral CsC1 Neutral CsCI

Native Denatured Renatured (partially)

1.721 1.739 1.728

----

----

O

H

t

p

Bottom

®

| Bottom

BOttOm

Bottom

soMo~

ToP

Fig. 4. A b s e n c e o f d e t e c t a b l e satellite D N A s in n u c l e a r D N A o f A. a r b u s c u ~ . I n a p r e p a r a t i v e CsCI grad i e n t 1 . 1 4 m g o f n u c l e a r D N A w a s c e n t r i f u g e d t o e q u i l i b r i u m u n d e r c o n d i t i o n s d e s c r i b e d in Materials a n d M e t h o d s . T h e g r a d i e n t s w e r e f r a c t i o n a t e d w i t h a u t o m a t i c r e c o r d i n g o f t h e A 2 6 0 n m in G i l f o r d s p e e t r o photometer. The DNA peaks were divided into a "heavy" (fractions towards the bottom of the gradient) a n d a 'q_ight" ( f r a c t i o n s t o w a r d s t h e t o p ) c o m p o n e n t . I n a s e c o n d p r e p a r a t i v e g r a d i e n t , t h e h e a v y a n d light f r a c t i o n s r e s p e c t i v e l y w e r e c e n t r i f u g e d a n d f r a c t i o n a t e d as d e s c r i b e d i n t h e t e x t . T h e h e a v y c o m p o n e n t f r o m the second centrifugatinn was divided into " h e a v y " (H fraction towards the b o t t o m of the gradient) a n d ' ~ m e d i u m " (M f r a c t i o n t o w a r d s t h e t o p o f t h e g r a d i e n t ) c o m p o n e n t s . T h e l i g h t c o m p o n e n t w a s likewise d i v i d e d i n t o " m e d i u m " ( f r a c t i o n s t o w a r d s t h e b o t t o m o f t h e g r a d i e n t ) a n d '~light" (1 f r a c t i o n s towards the top of the gradients) c o m p o n e n t s . The t w o t ~ e d i u m " c o m p o n e n t s were pooled and labeled as " m e d i u m d e n s i t y f r a c t i o n " . T h e " h e a v y " , " m e d i u m " a n d " l i g h t " c o m p o n e n t s w e r e t h e n s u b j e c t e d t o

384

d

0

a

b

A :

i, i ~p

I

1

1.721

,,700 1.739

1.700 ,.737

Bottom

Density ( g/cm 3)

Fig. 5. A n a l y t i c a l CsCI d e n s / t y g r a d / e n t e q u i l i b r i u m c e n t r i f u g a t i o n o f n a t i v e , h e a t d e n a t u r e d a n d p a r t i a l l y r e n a t u r e d n u c l e a r D N A o f A. arbuscula. (a) N a t i v e n u c l e a r D N A (4 ~ g ) ; (b) d e n a t u r e d n u c l e a r D N A in 0 . 1 S S C h e a t e d f o r 5 r a i n a t l l 0 ° C . A f t e r r a p i d c o o l i n g in ice t h e s o l u t i o n w a s a d j u s t e d t o 4 S S C ; (c) d e n a t u r e d n u c l e a r D N A i n c u b a t e d a t 7 2 ° C t o C o t 0 . 0 0 1 a n d (d) C o t 0 . 1 . T h e r e a c t i o n s w e r e s t o p p e d b y r a p i d c o o l i n g a n d t h e s a m p l e s w e r e s u b j e c t e d t o a n a l y t i c a l CsCI c e n t r i f u g a t i o n . S a m p l e s b , c a n d d w e r e c e n t r i f u g e d w i t h o r w i t h o u t m o u s e D N A s e r v i n g as d e n s i t y m a r k e r ( m o u s e m a i n b a n d d e n s i t y = 1 . 7 0 0 g/cm 3 ).

0

•

10-

D~ 50.

coO

70.

90-

,o2

,0,

,~-0

,o,

Cot (moles sec/liter)

,02

lo3

,o,

Fig. 6. R e a s s o c i a t i o n k i n e t i c s o f w h o l e eeU D N A o f A. arbuscula. D N A w a s s h e a r e d ( a v e r a g e f r a g m e n t l e n g t h 4 5 0 b a s e p a i r s ) , d e n a t u r e d a n d r e a s s o c i a t e d as d e s c r i b e d in M e t h o d s . D N A c o n c e n t r a t i o n f o r Cot v a l u e s f r o m 1 0 -3 t o 1 w a s 6 5 Mg/ml a n d f r o m i t o 2 0 0 0 w a s 5 6 5 Mg/rrd. T h e p h o s p h a t e b u f f e r m o l a r i t y a n d t e m p e r a t u r e o f i n c u b a t i o n f o r r e a c t i o n m i x t u r e s f r o m C ot 10 -3 t o 1 w e r e 0 . 1 2 M , p H 6 . 8 a n d 7 0 ° C and from 10 to 2000 were 0.48 M and 72°C respectively. Experimental data are represented by open circles a n d t h e t h e o r e t i c a l s e c o n d o r d e r c u r v e s h o w n b y c l o s e d circles.

385 T A B L E II C O M P U T E R A N A L Y Z E D R E A S S O C I A T I O N K I N E T I C S O F A. A R B U S C U L A

DNA *

Component

Fraction

Complexity ( n u c l e o t i d e pairs)

k

k (pu~e)

1 2

40 70

1 . 4 • 104 3.5 • 1 0 ~

30 0.021

75 0.0306

• D a t a f r o m Fig. 6 w e r e u s e d f o r t h e analysis.

for 60% of the total DNA and its reassociation kinetics were of second order as can be expected for single copy sequences. The data from the reassociation experiment were analyzed by a computer programme established by Britten et al. [15] (Table II) which fits possible second order components to the experimental curve and determines the reassociation rate contant " k " for each component. It also corrects the rate constant for sequence dilution and G + C mol per cent in the total reaction mixture to give a corrected reassociation rate " k " (pure). This corresponds to the rate constant of sequences constituting 100% of the reassociating DNA fragments with a G + C content of 50 tool per cent (as DNA of Escherichia coli). The very fast reassociating c o m p o n e n t accounts for 40% of the DNA and has a corrected reassociation constant, " k " (pure) of 75 and a kinetic complexity of 9.45 • 106 daltons (1.4 • 104 nucleotide palm). The repetition frequency of this c o m p o n e n t is 250 with respect to single copy component. The slow reassociating c o m p o n e n t comprises the unique or single c o p y sequences and accounts for 60% of the total sequences. It has a corrected rate constant of 0.03 M -I • s -I and a complexity of 2.27 • 101° daltons (3.5 • 107 nucleotide pairs). Fig. 7 represents the reassociation kinetics o f nuclear DNA. The curve is 0 10

,g

~,0-

10-3

i 10-2

i i0-1

i ]00

Cot (moles sec/liter)

a 10z

~ 102

i 103

Fig, 7. R e a s s o e i a t i o n k i n e t i c s o f n u c l e a r D N A , 2 m g D N A w a s f r a g m e n t e d ( a v e r a g e size 1 0 0 0 base pairs), d e n a t u r e d a n d r e a s s o e i a t e d as d e s c r i b e d in t h e M e t h o d s . D N A concentrations, phosphate b u f f e r m o l a r i t i a s and temperatuzes of i n c u b a t i o n w e r e as follows: b e t w e e n C o t 1 0 -3 ~ 1, 5 0 / z g ] m l , p h o s p h a t e b u f f e r molao r i t y 0 . 1 2 M a n d p H 6 . 8 , 7 0 ° C ; b e t w e e n 1 - - 1 0 0 0 , 2 m z / m l p h o s p h a t e b u f f e r 0 . 4 8 M, p H 6.8 a n d 7 2 ° C . T h e r e a s s o c i a t i o n m i x t u r e c o n t a i n e d 2 m M E D T A a n d 0.4% s o d i u m l a u r y l s u l f a t e . T h e r e a s s o c i a t e d D N A s w e r e s e p a r a t e d f r o m d e n a t u r e d D N A as d e s c r i b e d in t e x t .

386 0-

"0

3o

.g ~ ~o

90

I

10-3

10-2

I

10-1

J

100

I

I01

!

|0 2

I

10 3

Cot (moles s e c / l i t e r ) Fig. 8. Reassociation kinetics o f moderately and non-repeated components o f A. arbuscula D N A . 5.95 mg ultrasonically sheared DNA in phosphate buffer 0.1 M were heat denatured and brought to 0.12 M phosp h a t e b u f f e r , 2 m M E D T A , 0 . 4 % s o d i u m l a u r y l s u l f a t e as d e s c r i b e d in t h e m e t h o d s a n d r e a s s o c i a t e d t o COt o f 4 a t 7 0 ° C ( r e a s s o c i a t i o n 4 6 % ) . T h e r e a s s o c i a t e d ( r e p e a t e d s e q u e n c e s ) a n d single s t r a n d e d ( n o n r e p e a t e d s e q u e n c e s ) f r a c t i o n s w e r e r e c o v e r e d as d e s c r i b e d in the t e x t . T h e k i n e t i c s o f r e a s s o c i a t i o n o f the i n d i v i d u a l f r a c t i o n s w e r e o b t a i n e d as in Figs. 6 a n d 7. C o n d i t i o n s o f r e a s s o c i a t i o n : r e p e a t e d s e q u e n c e s , Cot 1 0 - 3 - - C 0 t 0 . 1 , 3 7 . 5 / ~ g D N A / m l a n d f r o m Cot 0 . 1 - - C 0 t 1 0 , 3 7 5 / z g D N A / m [ . T h e p h o s p h a t e b u f f e r molarity and temperature of incubation were 0.12 M and 70°C respectively and a minimum of 50/~g D N A / r e a c t i o n vial w a s u s e d . N o n - r e p e a t e d s e q u e n c e s : f r o m Cot 1 0 -3 t o Cot 1 0 , 3 3 . 5 / ~ g o f D N A , 0 . 1 2 M p h o s p h a t e b u f f e r a t 7 0 ° C a n d f r o m Cot 1 - - C 0 t 2 0 0 0 , 3 3 5 / ~ g D N A , p h o s p h a t e b u f f e r m o l a r l t y a n d t e m p e r a t u r e o f i n c u b a t i o n f o r r e a c t i o n s f r o m Cot 1 t o Cot 2 0 a n d f r o m Cot 2 0 t o 2 0 0 0 w e r e 0 . 1 2 M a n d 70°C and 0.48 M and 72°C respectively, o o, r e p e a t e d s e q u e n c e s ; • e, n o n - r e p e a t e d s e q u e n c e s .

clearly biphasic. Approximately 10% of the sequences reassociate very fast and probably include highly repetitive and inverted repeats. 15% of the sequences represent moderately repetitive sequences including rRNA and tRNA cistrons. The assessments of the proportions of moderately repetitive and single copy sequences are based on the theoretical second order kinetic curve which was calculated with a reassociation rate constant of 0.033 (C0t~, 30i and represents the possible rate of reassociation of single copy sequences. The reassociation kinetics of repetitive and single copy components are shown in Fig. 8. The repetitive fraction does n o t appear to be kinetically homogeneous since approximately 30% reassociated at a Cot of 10 -3. This can be considered as the very fast reassociating component, the rest of the fraction as moderately repetition component. The rate constant calculated from the Cot~ value (0.1) of the latter was f o u n d to be 10 -1 M • s -~. This value compares favourably with the approximate rate constant of this fraction in the bulk and nuclear DNA. The kinetics of reassociation of the single copy c o m p o n e n t show that approximately 6% of the DNA reassociated at a Cot of 10 -2 and 4% remained dissociated at a Cot of 1000. The Cot½of this fraction was f o u n d to be 30. The theoretical curve obtained with the rate constant of 0.033 M -~- s -~ gave a best fit to the experimental curve. The unique sequence reassociation rate in this experiment coincided with that obtained from the rate constant of the unique sequence c o m p o n e n t of the bulk and nuclear DNA shown in Figs. 6 and 7.

387

0' 10'

3o .g IJ

Z 7(

90.

0"3

,;-2

100

10'

,02

,03

Cot (moles sec/liter) Fig. 9. C o r e a s s o c i a t i o n of A. arbuscula non-repeated fraction and E. coil DNA. Non-repeated s e q u e n c e s were o b t a i n e d as d e s c r i b e d a b o v e a n d m i x e d w i t h i n d e p e n d e n t l y sheared E. coli DNA, d e n a t u r e d a n d zeass o c i a t e d . C o n d i t i o n s o f z e a u o e i a t i o n : Cot 10 -3 to Cot 1, 100 pg A. arbuscula DNA and 80 t~g E. coli DNA, phosp hate buffer m o l a r i t y 0.12 M and temperature of i n c u b a t i o n 70°C; from Cot 1 to Cot 2000, 220 p g / m l A. arbuscula DNA and 150 I~g E. coU DNA, pho s pha t e buffer rnolarity and t e m p e r a t u r e of i n c u b a t i o n from Cot 1--20 and Cot 20--2000 as d e s c r i b e d for Fig. 8.

The relative rate of reassociation of unique sequences of Allomyces was compared to E. coli and the results are shown in Fig. 9. 14C-Labeled E. coli DNA (sheared separately) was added to the slow reassociating fraction of AUomyces and was reassociated after denaturation and adjustment of its PB molarity to either 0.12 or 0.48 M. The Cot~ of E. coli and A. arbuscula were found to be 3.2 and 34 respectively. This indicates that under identical conditions of reascociation E. coil DNA reassociates a b o u t 10 times faster than the single copy sequences of AUomyces. The results from reassociation experiments are summarized in Table II. They show clearly that AUomyces DNA like other eukaryotic DNAs contains highly and moderately repetitive sequences. The number of copies/genome of different components was calculated by the ratio of Cot~ of the repeated sequences to that of single copy sequences. The kinetic complexity of the sequences in each c o m p o n e n t was calculated from its proportion in the genome and its repeat frequency. The non-repeated sequences in Allomyces DNA represent 65--80% of the genome and have, based on their reassociation rate, a complexity of 1.7 • 10 l° daltons (see discussion for the calculation). The intermediate reannealing component reassociates 320 times faster and, therefore, contains sequences repeated as many times per genome. The complexity of this sequence is 7.2 • 106 daltons. Thermal denaturation profiles of reassociated DNA show the precision of base pairing. A reduction in t h e mean thermal denaturation as compared to native DNA is an indication for base pair mismatching or sequence divergence [16]. Fig. 10 shows the melting profiles o f the reassociated DNAs. The reduced Tm (84°C) of the repetitive fraction reveals a certain degree of mismatching; this indicates that the very fast reassociating fraction contains a large number of very similar b u t not identical sequences. This agrees with what is k n o w n

388

'°° 1

~ A0-t i

40

60 80 Temperature °C

Fig. 10. Thermal denaturation profiles of native (e c i a t e d t o Cot 1 0 0 0 (~ -~) a n d r e p e a t e d D N A s ( a d e s c r i b e d in M e t h o d s .

100

-'), sheared (o ©), s h e a r e d D N A r e a s s o A). T h e d e t a i l s o f t h e p r o c e d u r e have b e e n

a b o u t the properties of repetitive sequence elements in other eukaryotic DNAs [1]. Reassociated (Cot 1000) bulk DNA melts at the same temperature as sheared native DNA which is a b o u t 1.5°C lower than unsheared native DNA. The difference in the melting profiles of reassociated bulk DNA and sheared native DNA is probably due to some mismatching in the reassociated repetitive sequences, nevertheless the identical Tm indicates that the majority of the sequences reassociated to near perfection. Discussion

One or more satellite DNAs have been found in most fungi. T w o of these, mitochondrial DNA and DNA carrying the ribosomal RNA cistrons (rDNA) have been well characterized. In general mitochondrial DNA appears as light satellite in analytical density gradient centrifugation. The position of the r R N A cistrons, which have been shown to be conserved during evolution [17--18], depends on the base composition of nuclear DNA which varies considerably in fungi [19]. Our results obtained by analytical ultracentrifugation show that in A l l o myces arbuscula :

(i) DNA from total cell consists of 3 components, ~, ~ and 7 with b u o y a n t density in CsC1 of 1.721, 1.710 and 1.702, respectively. (ii) Nuclear DNA shows only one band corresponding to ~. The ribosomal DNA sequences can n o t be separated by CsC1 density gradient centrifugation (see accompanying paper). The light satellites ~ and 7 are therefore of cytoplasmic origin. The ~ c o m p o n e n t has a G + C content corresponding to that of DNA isolated from purified mitochondria [19]. (iii) The repetitive fraction from the nuclear DNA does n o t form a clearly defined band b u t shows a broad shoulder (Fig. 5). The analysis by reassociation has shown that approximately 25% of the total nuclear DNA consists of repeti-

389 rive sequences (10% "zero t i m e " + very fast reassociating DNA and 15% moderately repetitive DNA). Such an a m o u n t should form a visible peak on analytical centrifugation. Since repetitive sequences do n o t reassociate with highly matched base pairs, it is difficult to determine accurately their base composition from b u o y a n t density measurements. Further, since the DNA samples for denaturation and renaturation consisted of fairly high molecular weight {unsheared, native DNA), the duplexes could have been highly mismatched. The fact that repetitive DNA does n o t appear as satellite band could further be explained by interspersion of the repetitive sequences between single copy sequences; since native DNA was used in the reassociation a very large amount of inter and intra-strand base pairing could have occurred, thus further increasing the chances of mismatching of base pairs. Reassociation kinetics of eukaryotic DNAs have clearly demonstrated that they contain "zero time", highly repetitive, moderately repetitive and single copy sequences [20--22]. In A. arbuscula, the "zero t i m e " reassociating DNA constitutes a b o u t 10% of the total DNA and probably consists of mainly inverted repeats. During renaturation, they form intramolecular doublestranded regions extremely rapidly. The kinetics of reassociation of the highly repetitive sequences have n o t y e t been studied in fungi. In AUomyces, this fraction accounts for about 25%. The moderately repetitive sequences have been shown to vary from 8--30% in fungi [23--26]. In A. arbuscula, the amount of these sequences in bulk and nuclear DNA is 10--15%. The kinetic complexity of these sequences (7.2 • 106 daltons, observed value and 9.46 • 10 ~, computer simulated value, see Table III) is close to the published values for other fungi [26--27]. However, this could be an over-estimate since the sequence divergence can cause a significant reduction in the reassociation rate [28]. The single copy sequences comprise approximately 75% of the nuclear genome and have a kinetic complexity of about 1.7 • 10 ~° daltons. This value has been obtained by comparing the reassociation rates of single copy sequences o f A. arbuscula and E. coli (see Fig. 8). When co-reassociated, the A. arbuscula single copy sequences were found to reassociate 10 times faster than E. coli. In earlier work [29,30] a relation was found between the rate of renaturation and % G + C and a correction factor was proposed to compare the relative rate of renaturation of t w o DNAs differing in their % G + C. After correction for the difference in G + C mole percent between E. coli (51%) and A. arbusucla (62%) [29,30], the non repeated portion of A. arbuscula was found to be 8.5 times (34/3.2 × 0.802) more complex than the E. coli genome. However, recently Gillis and de Ley [31] have analyzed the most reliable literature data along with their own measurements of rate of renaturation and found negligible effect of % G + C on the rate of renaturation. They proposed to disregard this effect until more precise reference data are available. AssumingE. coli genome to be 2.8 • 109 daltons and that in Allomyces the single copy sequences represent approximately 60% of the genome, the genome size can be estimated to be a b o u t 1.7 • 10 ~° daltons (2.8 • 109 × 10 × 0.6) if we disregard the effect of % G+C. Estimates of the haploid genome size are available for several fungi. Thus, among lower fungi, that of the O o m y c e t e Achlya ambisexualis is reported to be 11 times larger than E. coli [32]. The Zygomycete Mucor has species with geno-

390 TABLE III NUCLEOTIDE SEQUENCE FREQUENCY DISTRIBUTION HYDROXYAPATITE CHROMATOGRAPHY

O F A. A R B U S C U L A

GENOME USING

T h e p a r t i t i o n i n g o f t h e g e n o m e a n d a m o u n t o f e a c h f r a c t i o n w e r e c a l c u l a t e d f r o m Figs. 5 a n d 7. T h e s e q u e n c e c o m p l e x i t y of e a c h f r a c t i o n w a s c a l c u l a t e d f r o m t h e g e n o m e size m e a s u r e d b y t h e r a t e of reassoc i a t i o n o f single c o p y s e q u e n c e s u n d e r o p t i m a l c o n d i t i o n s ( 2 . 3 8 • 1 0 1 0 d a l t o n s ) .

Component

Proportion of the genome Bulk

Nuclear

V e r y fast r e p e t i rive

25

10

I n t e r m e d i a t e repetitive

10

10

Non-repetitive copy

65

80

Non-repetitive purified Non repetitive purified coreassociated with

2

C Ot~l

0,1 ( 0 . 1 ) *

32

(29) *

Copies/ genome

Sequence complexity (daltons)

320

7.2 - 106

1

30

1

29

1

1.7 • 1010

E. coli * T h e v a l u e s in p a r e n t h e s i s c o r r e s p o n d t o t h e Cot ½ o f n u c l e a r D N A c o m p o n e n t s .

mic mass ranging f r om 0.5 • 10 l° to 3 • 101° daltons and Phycomyces blakesleeanus 1.9 • 10 l° daltons [33]. Christiansen et al. [20] showed that among H e m i a s c o m y c e t e yeasts the genome size can vary from 0.6 • 101° to 3 • 101° daltons. T h a t o f the E u a s c o m y c e t e Neurospora crassa and Basidiomycete Coprinus lagopus have been r epor t e d to be 2 . 2 . 1 0 1 ° and 3 . 1 0 1 ° respectively [25]. Taking into a c c o u n t the relative accuracy o f the genome size estimates (+-10%) and the large variations r e p o r t e d for species belonging t o the same group (0.5--3 • 10 l° daltons), it is difficult t o draw any conclusions on the evolution o f genome size in relation to the t a x o n o m i c evolution o f fungi.

Acknowledgements We t h a n k Mr. Ren6 Bach for his e x p e r t assistance with t he c o m p u t e r analysis o f the reassociation kinetic data and Miss Arlette Cattaneo for her excellent technical assistance.

References 1 2 3 4 5

B r i t t e n , R. a n d K o h n e , D.E. ( 1 9 6 6 ) S c i e n c e 1 6 1 , 5 2 9 - - 5 4 0 B o s t o e k , C. ( 1 9 7 1 ) A d v . Cell Biol. 2 , 1 5 3 - - 2 2 1 Nagl, W. ( 1 9 7 6 ) A n n . Rev. P l a n t Physiol. 2 7 , 3 9 - - 6 9 D a v i d s o n , E.H. a n d B r i t t e n , R. ( 1 9 7 3 ) Q. R e v . Biol. 4 6 , 5 6 5 - - 6 1 3 F l a m m , W , G . , Walker, P.M.B. a n d M c C a l l u m , M. ( 1 9 6 9 ) J. Mol. Biol. 4 0 , 4 2 3 - - 4 4 3

391

6 Gelderman, A,H., Rake, A.U. and Brltten, R.J. (1971) Proc. Natl. Acad. Sci. U.S. 68,172--176 7 0 j h a , M. and Turian, G. (1971) Mol. Gen. Genet. 112, 49--59 8 0 j h a , M., Dutta, S.K. and Tutdan, G. (1975) Mol. Gen. Genet. 136,151--165 9 Emerson, R. (1941) Lloydia, 4, 77--144 10 Dill, B. and Stock, J.L. (1974) Arch. Microbiol. 96,281--289 11 Schildkraut, C.L., Mannur, J. and Doty, P. (1962) J. Mol. Biol. 4,430--443 12 Mandel, M., Igambi, L., Bergendahl, J., Dodson, Jr., M.L. and Scheltgen, E. (1970) J. Bacteriol. 101, 333--338 13 Davis, R., Simon, M. and Davidson, N. (1971) in Methods in Enzymology (Grossman, L. and Moldave, K., eds.), (Acad. Press. New York) 21 (D), 413---428 14 Walker, P.M.B. Nature (1968) 219,228--232 15 Britten, R.J., Graham, D.E. and Neufeld, B.R. (1974) in Methods in Enzymology (Grossman, L. and Moldave, K., eds.), (Acad. Press, New York) 29 (E), 363--418 16 Kohne, D,E. (1970) Q. Rev. Biophys. 33,327--375 17 Sinclair, J.H., Brown, D.D. (1971) Biochemistry 10, 2761--2769 18 Jerbi, S. (1976) J. Mol. Biol. 106,791--816 19 Storck, R. and Alexopoulos, C.J. (1970) Bacteriol. Rev. 34,126--154 20 Davidson, E.H., Hough, B.R., Amenson, C.S. and Britten, R.J. (1973) J. Mol. Biol. 77, 1--23 21 Church, R.B. and Georgiev, G.P. (1973) Mol. Biol. Reports 1.21--25 22 Wilson, D.A. and Thomas, C.A. (1974) J. Mol. Biol. 84,115---144 23 Christiansen, C., Bak, A.L., Stenderup, A. and Christiansen, G. (1971) Nat. New Biol, 231,176--177 24 Dutta, S.K., Penn, S.R., Knight, A.R. and Ojha, M. (1972) Experientia 28, 582~584 25 Dutta, S.K. and Ojha, M, (1972) Mol. Gen. Genet. 114,232--240 26 Dutta, S.K. (1974) Nucleic Acid. Res. 1, 1411--1419 27 Ohja, M, and Dutta, S.K. (1976) in The Filamentous Fungi, Vol. Ill, Chapt. 2, Pp. 8--27 (Smith, J.E. and Berry, D.R., eds.), Edward Amolds (Lond.) 28 Bonnet, T.I., Brenner, D.J., Neufeld, B.R. and Britten, R.J. (1973) J. Mol. Biol. 81,123--136 29 Wetmur, J.G. and Davidson, N. (1968) J. MoL Biol. 31,349--370 30 Seidler, R.J. and Mandel, M. (1971) J. Bacteriol. 106,608--619 31 Gillis, M. and de Ley, J. (1975) J. Mol. Biol. 98,447--464 32 Green Dodd, J., Horgen, P.A. and Straus, N.A. (1975) Cytobios 13, 31--36 33 Dusenbery, R.L. (1975) Biochim. Biophys. Acta 378,363--377