Kinetic complexity of RNA molecules

J. MOE. Biol.

(1972) 63, 21-39

Kinetic MAX

L.

Complexity

BIRNSTIEL, BRUCE

of RNA Molecules H.

SE&s?

AND

IAN

F.

PURDOM

Medical Research Council Epigenetics Research Group. Department of Genetics, University of Edinburgh, West Mains Edinburgh EH9 3JN, Scotland (Received 7 June 1971 and in revised form 20 September

Road,

1971)

A procedure has been developed for calculating the base sequence complexity of polyribonucleotides using RNA-DNA hybridization on membrane filters. Optimal rate temperature ( TOPt) in hybridization media containing 6 x SSC (SSC is 0.15 M-Nacl, 0.015 M-sodium citrate) (pH 7.2), 50% formamide or 1.15 M-sodium acetate (pH 5), 60% formamide were determined for a variety of RNA species. were found to be unexpectedly high for all RNA species The To,, in formamide investigated. It is suggested that the high T,,, are caused by increased stability of RNA-DNA hybrids in these incubation mixtures. It is demonstrated that RNA-DNA reactions with homogeneous RNA species present in excess are most easily characterized by C,t+ where C, is the molar concentration of RNA nucleotides in solution and t& is the time taken to reach the 50yo saturation value. Within certain limits C,t+ is independent of the amount of DNA on filter disks and the degree of fragmentation of the RNA. C,tk increases linearly with the base sequence complexity of RNA molecules. Using complementary RNA derived from a balanced synthetic RNA-@X DNA hybrid wo show that the slope of the straight linear standard curve which links C,4& to base sequence complexity is 9.3 x 10e9 moles.sec/l. dalton for 6 x SSC, 50% forms,mide. In 1.15 M-sodium acetate (pH 5), 60% formamide, the slope is 2.3 x 10-a moles.sec/l. daltons. Applications of the kinetic standard curve for evaluation of purity and concentration of isolated messenger RNA’s are discussed. We show that transfer RNA of both Escherichia coli and Xenopw laevis is made up from 30 to 40 basic nucleotide sequences distinguishable by RNA-DNA hybridization. In Xenopus there are some 6500 cistrons for tRNA and so each tRNA sequence is, on average, 160-fold reiterated. 5 s RNA of Xenopus, coded by some 9000 cistrons, and ribosomal RNA of Bacillus subtilis, rabbit and Xenopus coded by 8, 250 and 610 cistrons, respectively, each contain a single family of nucleotide sequences and are therefore highly conserved in these species.

1. Introduction The object of the experiments described in this paper is to develop a simple procedure for determining the base sequence complexity of polyribonucleotides using RNA-DNA hybridization techniques and by this method to establish the complexity of a few selected RNA species by comparison to standards of known genetic complexity. Previous workers have established a relationship between the rate of renaturation and base sequence complexity of DNA (Britten & Kohne, 1968; Wetmur & Davidson, 1968). DNA renaturation results from the collision of complementary strands and t On leave

from

St. Jude

Children’s

Research

Hospital, 21

Memphis,

Tennessee,

U.S.A.

22

M.

L.

BIRNSTIEL,

B.

II.

SELLS

AND

I.

F.

PURDOM

follows second-order kinetics. Thus, the half period of reassociation is inversely proportional to the molar concentrations of the DNA complements. Cot?, the product of the initial molar concentration C, (expressed as moles deoxynucleotide/l.) and half time of reassociation t, (see) is a measure of the base sequence complexity of the renaturing DNA (Britten & Kohne, 1968). In RNA-DNA hybridization experiments, where the RNA is well in excess of the complementary DNA, the hybridization reaction can conveniently be presented by the straight linear curve of a double reciprocal plot, l/hybridization versus l/time (Bishop, 1969). We provide further examples in this paper that this method of presenting the hybridization reactions, although thermodynamically not understood, is very useful for the evaluation of kinetic data and we further show that it yields saturation values comparable to those obtained by standard saturation experiments. At a given R;“U’A input the rate of hybridization is directly proportional to concentration of the RNA and inversely proportional, roughly, to the genetic complexity of the RNA (Bishop, 1969), thus as in the case of DNA renaturation, the reaction is governed by the molar concentration of nucleotide sequence in solution. From our experiments we have determined conditions of optimal rate for RNA-Dh‘A hybridization by the Gillespie & Spiegelman (1965) procedure for which we have adapted the low-temperature formamide technique of McConaughy, Laird & McCarthy (1969). First we show that an RNA of given base sequence is most simply characterized by C& the product of molar concentration of RNA sequences and the time necessary to reach half saturation; second, we developed a standard curve which correlates, with some precision, the rate of hybridization with base sequence complexity of RNA; third, we discuss possible applications of the kinetic analysis of RNA-DNA hybridization in determining the purity of messenger RNA, of messenger concentrations in RNA mixtures and the degree of base sequence divergence within RNA species derived from repetitive DNA sequences.

2. Materials

and Methods

(a) Materials n-[2-3-3H]Valine (29 Ci/m-mole), L-[3H]leucine (34 Ci/m-mole), [5-3H]uridinc ( > 24 Ci/m-mole) and 3H-labelled nucleoside triphosphates were obtained from R’adiochcmical Centre, Amersham, England. E. coli tRNA used in charging experiments was purchased from General Biochemicals (Chagrin Falls, Ohio, U.S.A.). Frozen E. coli MRE 600 were kindly provided by the Microbiological Research Establishment, Porton, England. @X 174 virus particles and E. co.% RNApolymerase were purchased from Miles-Seravac, Maidenhead, England. A kidney (Rafferty) cell line of Xenopus Zaewti was a gift from Dr J. Gall (New Haven, Corm.). Rabbit kidney cells RK13 were purchased from Flow Laboratories, Irvine, Scotland and Falcon tissue culture flasks from Falcon Plastics, Oxnard, Calif., U.S.A. (b) Preparation

of

DNA

DNA was prepared from nucleated blood cells. Toads were decapitated and the blood collected in 0.15 M-N&I, 0.1 M-EDTA, lye /I-mercaptoethanol, pH 8.0 (100 to 200 ml./toad), and lysed with 1% SDS? (final concn). The DNA was prepared according to a modified Marmur procedure (1961) using repeatedly RNase (O.O1o!o) and pronaso (O.OS’$h). For the preparation of human placental or rabbit liver DNA, the tissue was first homogenized in an Omnimix blender with 3 vol. of a. solution containing 0.4 M-sucrose, 0.001 ar&Cl,, 0.05 M-Tris, pH 7.2. The homogenate was filtered through cheese cloth, the filtrate 1961) lysed by addition of 1 y. SDS and treated with 10 vol. saline-EDTA (Marmur, deproteinized as above. Xenopus

t Abbreviation

used:

SDS,

sodium

dodecyl

sulphate.

KINETICS

OF RNA-DNA

23

HYBRIDIZATION

E. co& cells were lysed in saline-EDTA, 1% SDS (Marmur, 1961). B. subtilis cells were washed in 0.1 M-NaCl, 0.01 M-EDTA, 0.01 M-Tris, pH 8, and resuspended in 20 vol. of the same medium. Lysozyme to a concentration of 0.5 mg/ml. was added and left at 0°C for 5 min. SDS was then added to a final concentration of 1%. The cell suspension was heated to 37°C and 5 min later, pronase (0.5 mg/ml.) was added. After 15 min the lysate was cooled to room temperature. Both E. coli and B. subtilis DNA were deproteinized as above..

(c) Preparation of 3H-labelled ribosomal RNA Pormanent tissue culture lines from Xen0pu.s Zaevis and rabbit kidney were grown in. Falcon 75-cm2 bottles in Eagles MEM, supplemented with O.ll”,& NaHCO,, 10% foetal. bovine strum (Flow Laboratory) and antibiotics (streptomycin 130 i.u./ml.; penicillin 100 i.u./ml.). For Xenopus cells the medium was diluted by addition of 15% (vol.) water.. The cells were labelled by incubation for 36 to 72 hr with 50 to 100 &i/ml. [3H]uridino (2 24 Ci/m-mole) and chased for 36 hr with 4 changes of medium containing a loo-fold. excess of unlabelled uridinc. The incubation medium was removed, the cells detached by addition of the RNA extraction medium (containing 0.15 M-NaCl, 0.01 M-Tris (pH 7.5)., 0.05% sodium dodecyl sulphate (Sigma, London) ) and by scraping the flask with a rubber t,ipped spatula. The lysate was homogenized gently in a glass-Teflon homogenizer and dcproteinized by shaking with an ecpral vol. of water-saturated phenol added at room temperature. After centrifugation, the supernatant was re-extracted with phenol. This procedure yields RNA free from DNA. To the supernatant, 0.1 vol. of a 3 M-NaCl solution was added and the RNA precipitated by addition of 2.5 vol. of ethanol. After storage: overnight at -2O”C, the RNA was collected, washed 3 times in ethanol, dissolved in 0.02 M-sodium acetate pH 5, and fractionated on a 5 to 4096 sucrose gradient (0.1 M-NaCl, 0.02 M-sodium acetate, 0.001 M-EDTA, pH 5.0) in a Spinco SW 25.2 rotor at 15’C, 24,000 rev./min for 18 hr. To the 28 s and 18 s peak fractions, 0.1 vol. of 3 M-NaCl was added and the RNA recovered by precipitation with 2.5 vol. of ethanol. The specific activities of rRNA preparations ranged from 4 x lo5 to 3.1 x lo6 disint.,l min/pg RNA. The rRNA was shown to be free from other hybridizable material by hybridization with a complete range of DNA fractions taken from a CsCl density gradient (Birnstiol, Speirs, Purdom, Jones & Loening, 1968). In all cases hybridization was confined to the high density rDNA, leaving the main band DNA u&belled (Fig. l(a) and (c) ). 1,abelled B. subtilis and Proteus mirabilis rRNA had a specific activity of 3.2 x lo* and 3.8 ;< lo5 disint./min/pg, and were the gift of Dr J. Bishop (Edinburgh). The bacterial rRNA was shown to hybridize exclusively to high density bacterial DNA (cf. Purdom, Bishop 8; Birnstiel, 1970); (Fig. l(d)) and hence was free from contaminating messenger RNA. (d) Preparation

unlabelled 5 s RNA amZ Xenopus laevis Tissue culture cells were labelled for 3 days with 100 &i [3H]uridine/ml. RNA from labclled cells or livers from Xenopus toads were extracted as above. The RNA was passed through a 200-cm Sephadex CT100 column (bed vol, 630 ml.) which had been equilibrated with 0.01 M-sodium acetate, pH 5; 0.0001 M-EDTA according to Brown & Littna (1966). ‘rho flow rate was 12 ml./hr. The relevant peak fractions were collected, concentrated, if necessary, by lyophilization and used for hybridization. The 3H-labelled 5 s and 3Hlnbelled tRNA’s had a specific activity in excess of 2.8 x lo6 disint./min/pg. When annealed t,o a complete range of CsCl density gradient fractions, hybridization of 3H-labolled 5 s RNA was found to be exclusively with the lighter fractions of the gradient (Fig. l(a) ). 3Hlabclled tRNA, in the presence of an excess of unlaballed 5 S, 18 s and 28 s rRNA, annealed to DNA of slightly heavier than average density (Fig. l(b) ). Both these findings are in accordance with the observation of Brown & Weber (1968) and attest to the hybridiz&ion specificity of the preparation. transfer

of labelled and RNA from

(e) Preparation of sH-labelled complementary Approximately 6 to 15 x 1013 @X particlesweresuspended 0.035 nl-borate buffer (pH 9.1), 0.5% SDS and incubated 3 hr. The solution was made 1% Sarcosyl and the DNA CsCl density gradient (25”C, 42,000 rev./min pav = 1.720

RNA

from

@X

174

in 0.1 M-NaCl, 0.025 M-EDTA , with 0.05% pronase at 37°C for purified by banding in a 5.5ml. g/cm3) in c2 Spinco no. 50 angle

24

M.

L.

BIRNSTIEL,

B.

H.

SELLS

(a)L

Rotor

centre

AND

I.

F.

PURDOM

Mfufeu.5

lb) -I

I

I

!

!

u!’

\

‘/RNA

/” -

----+

FIG. 1. Hybridization of Xenopus 4 s, 5 s and rRNA rabbit and B. subtdis rRNA to C&l during gradient fractions of homologous DNA. 50 pg DNA in 4 ml. of 0.1 x SSC, together with M. Euteus DNA as a density marker, were added to 5.2 g CsCl and centrifuged at 42,000 rev./min at 25°C in an MSE 10 x 10 ml. rotor for 36 hr. Ten-drop fractions were collected and challenged at T,,, for 10 half-lives (tt) with (a) Xenpous 5 s RNA (0.014 pg/ml.) or 28 s + 18 s rRNA (3 pgglml.); (b) Xenopus tRNA (0.42 pg/ml.); (c) rabbit 28 s + 18 s rRNA (3 ,ug/ml.) and (d) B. subtilis 23 s + 16 s rRNA (2 pg/ml.).

rotor (Flamm, Bond & Burr, 1966), the fraction containing the O.D. peak dilutod 4-fold by addition of water and the DNA pelleted overnight in a Spinco no. 50 rotor at 45,000 rev./min and 15°C. The DNA pellet was dissolved in a small vol. of 0.01 M-Tris, pH 7.5. The @X RNA was transcribed in vitro according to the protocol of Chamberlin & Berg (1964). The incubation mixture contained in 1 ml., 40 mM-Tris buffer, pH 8,1 mM-ATP, CTP, GTP and UTP, each at a specific activity of N 50 mCi/m-mole, 4 mM-MgCl,, 1 mM-MnCl,, 12 m&r-%mercaptoethanol, 50 units of E. coli polymerase and 25 pg @X DNA. After incubation for 50 min at 37”C, the solution was chilled, made up to 2.5% Sarcosyl and deproteinized (twice) by shaking with an equal vol. of phenol. The supernatant was passed through a Sephadox G75 column (bed vol., 100 ml.) which had been equilibrated with 0.5 x SSC and the excluded fraction collected. The RNA : DNA ratio of the product was 1.06 by radioactivity. When examined in the Model E at 44,000 and 56,000 rev./min the product banded in C&SO, at 1.503 g/cm3 (Fig. 2) with only traces of U.V. absorbing material at 1.64 g/cm3 (where RNA is expected to band). Both these findings show that the RNA-@X DNA is a balanced hybrid (cf. Chamberlin & Berg, 1964). The RNA was dissociated from the DNA by a brief exposure (45 set) of the hybrid to 0.1 M-NaOH at O”C, quickly neutralized by addition of Tris buffer and 0.1 M-HCl and freed from DNA by centrifugation in a preparative C&SO1 gradient in a Spinco SW39 rotor (Chamberlin & Berg, 1964). The RNA was recovered, dialysed against 0.1 x SSC and lyophilized. The specific activity of the RNS was 8.5 x lo5 disint./min/pg. (f)

Preparation

of aminoacyl

symthetases

The synthotases were prepared from frozen E. coli MRE 600. The ground with 60 g of Al,O, and extracted with 150 ml. of Tris, 0.05 M, pH was fnst centrifuged at 3000 g for 10 min and then at 6000 g for 10 min

cells (30 g) were 7.5. The mixture to remove Al,O,

KINETICS

OF

RNA-DNA

--Rotor

FIG. g/em-3)

2. Isopycnic was added

centrifugation as a density

and cell debris. The supernatant 27,500 rev./min in the no. 30 The supernatant was removed cellulose column (50 ml. bed 7,5) to remove tRNA (Wong, pooled in small fractions and frozen state.

25

HYBRIDIZATION

centre

of @X RNA-DNA marker (see Text).

hybrid

in CSZSOI.

Xenopua

DNA

(1.422

was adjusted to 0.01 M-MgCl, and centrifuged for 4 hr at of a Spinco ulbracentrifuge to remove ribosome particles. and adjusted to 0.33 ~-Kc1 and passed through a DEAE vol. pre-equilibrated with 0.05 M-Tris. HCl in 0.33 M-KCl, pH Mustard & Herbert, 1969). The peak fractions were collected, frozen. The enzymes were stable for at least one month in the

rotor

(g) Preparation

of

charged transfer

RNA

The hybridization studies with tRNA were performed by using tRNA charged with a labelled amino acid. The charged tRNA was prepared at 37°C in a l-ml. reaction mixture (Waters & Novelli, 1968) containing the following components: 100 pmole /3-mercaptoethanol, 1.2 mg tRNA and [3H]leucine (2 pg and 0.5 mCi) or [3H]valine (2.4 mg and 1 mCi). The incubation time was 15 min and was initiated by the addition of 0.3 mg of crude synthetase preparation. Following charging, the reaction mixture was chilled and an equal vol. of 0.1 x SSC-saturated phenol was added. The mixture was shaken at room temperature for 10 to 15 min, centrifuged, the water layer removed, and passed through a coarse G25 Sephadex column to remove small molecular weight components. The fractions containing the bulk of the radioactivity were combined and stored for use in hybridization studies. (h)

Preliminary

procedure

before charging

To ensure that the tRNA’s specific for valine were saturated with amino acid, the subsequent steps were followed: a series of incubations were performed in the charging medium with a standard amount of tRNA and varying concentrations of the amino acid under consideration but a constant amount of radioactivity. The number of counts obtained in the charged tRNA were determined at the different amino-acid concentrations. The lowest concentration of amino acid necessary to saturate the standard amount of RNA was obtained by measuring the specific activity (cts/min/mg RNA) at the various concentrations of amino acid, then determining the concentration which produced a drop in specific activity proportional to the amount of ammo acid added. The amount of enzyme used was sufficient to complete the charging reaction within 10 to 15 min at 37°C. The rate of charging was determined by removing samples at intervals after enzyme addition and measuring the number of 10% trichloroacetic acid-precipitable counts.

26

&!I. L.

BIRNSTIEL,

(i) Preparation

B.

II.

of 3H-labelled

SELLS

AND

transfer

I.

RNA

F.

PURDOJI

from

E. coli

To prepare high specific activity tRNA, E. coli (pyr-, Zm-, and vit B-) was used. A suspension of exponentially growing cells with an absorbance of 0.08 (at 660 nm) wns incubated in a minimal medium containing 5 mCi E3H]uridine (6 mg/ml.) until tho supply of uridino was exhausted (usually at an absorbance of 0.35 to 0.5). After a further 30 min, unlebellod uridine was added to a final concentration of 300 pg/ml. and the incubation continued until the absorbance reached 0.6 to 0.7. To isolate the 3H-labelled tRNA the following procedure (Morell, Smith, Dubn:rkl & Marmur, 1967) was used. The cells were harvested by centrifugation and disrupted by grinding with Al,O,. The mixture was then extracted at 0 to 4°C with buffer (0.005 al-Tris and 0.01 M-MgCl,, pH 7.5). The extract was centrifuged at 5000 g for 10 min t,o rcmovo A1,03 and then at 24,000 g for 20 min to remove debris. To the supcrnat.ant DNtLsc W~LS added a final concentration of 5 pg/ml. and dextran sulphate 500 (60 pg/ml.) t#o inhibit. RNase. The preparation was then centrifuged at 38,000 rev./min for 3 hr in a Spinco no. 40 rotor to sediment ribosome particles. Following centrifugation the upper 3/4 of the supcrnatant was removed and added to an equal vol. of phenol (saturated with 0.1 ): SSC). The RNA was extracted by shaking t#he mixture for 15 min. The water layer containing the RNA was separated by centrifugation and the RNA precipitated by the addition of 0.1 vol. of 30yb potassium acetate and 2.5 vol. of absolute ethyl alcohol. The RNA was dissolved in 0.1 x SSC re-precipitated and washed with absolute ethyl alcohol and finally dissolved in 0.1 x ssc. (j) Hybridization

Focedure

13 mm HAWP Millipore filters were washed overnight in 2 x SSC. They were pl~~ced in stainless steel Swinney filtration apparatus (Millipore, U.K. Ltd.) fittod with lo-ml. Perspex tubes and washed with 5 ml. of 2 x SSC. DNA, usually in 0.5 ml. of 0.1 >: SSC, was denatured by addition of an equal vol. of 1 N-NaOH, which was neutralized 15 to 20 min later, by the addition of 4 vol of a medium containing 3 RI-N&I, 1 N-HCl and 1 M-Tris (pH 8.0) in a ratio of 2 : 1 : 1. The solution was filtered at a rate of 0.2 ml./min through t,he prepared HAWP disks, which were then washed with 5 ml. of 6 x SSC. The filters were heated at 80°C for 2 hr in a vacuum oven, marked with a pencil and stored at --20°C (Gillespie & Spiegelman, 1965). Before use, the DNA containing filters were soaked in 6 x SSC, 50: formnmidr. Fol kinetic studies the hybridization medium was brought to optimum temperature (soo below) and wetted-filters rapidly immersed and agitated at the appropriate temperature. Thz volume of incubation medium was sufficient to allow the filters to move around freely in tho solution. For sampling, the filters were removed at various time intervals, and placed in a large vol. of ice cold 6 x SSC. The filters were washed by the batch method (Birnstiel et al., 1968) by placing them in a 2-1. beaker with 11. of 2 x SSC at room temperature and keeping them under continuous agitation for 30 min for each exchange (3 times). The filters then were incubat,ed with 10 pg RNase/ml. for 20 min in 2 x SSC at room temperature, rinsed with 2 x SSC and vacuum dried before counting in the scintillation counter. Tho background controls using heterologous bacterial DNA instead of eukaryotic DNlZ were less than 0.5% of the homologous saturation values. 3H-labelled aminoacyl-tRNA’s were incubated in 600,; formamide, 1.15 ,Ir-sotlium (acetate), pH 5 at 46°C. Incubation for 2 hr in this medium led to only a minor discharge ( < 10%) of the [3H]valine-tRNA [3H]leucine-tRNA (Fig. 3). The washing procedure was carried out ss above except that there was no RN&se treatment. When aminoacyl-t RNA from E. co& was annealed to Xenopus DNA, the background was < 7 y0 of the homologous reaction. Both background noise and hybridization levels were unaffected by the add&ion of 10 or 100 units of T,-RNase/ml. in the final wash, nor was the hybridization : noise ratio improved (Table 1). (k)

Dissociation

of RNA-DNA

hybrids

rRNA-DNA hybrids from Xenopus and B. subtilis and cRNA-@X DNA formed at optimal temperatures as described above. The filters were washed method and treated with RNase 8s described above. The filters were then

hybrids wcrc: by the batch incubated in

KINETICS

OF RNA-DNA

HYBRIDIZATION

27

FIG. 3. Stability of [3H]leucine-tRNA and [3H]valine-tRNA during hybridization. E. coli tRNA charged with [3H]leucine or [3H]valine were incubated in 1.15 M-sodium acet,atc (pH 5), 60% formamide at 46°C. At times indicated a sample was withdrawn and injected intu 2 ml. of water, 100 pg yeast RNA (Sigma, London) was added and the RNA precipitated with a,n equal filters,

vol. of 5% trichloroacetic washed 3 times with 5%

acid in the trichloroacetic

cold. The precipitate was acid, dried, and counted.

TABLE

recovered

on hfillipore

1

Effects of T, RNase on the stability of [3H]leucine-transfer RNA-DNA hybrids Counts hybridized No T1 RNase treatment Xenopus

DNA

E. cobiDNA

T, RNase treatment

10 units/ml.

73 1237

83 1180

100 units/ml. 63 1133

0.1 x SSC, 0.1% diethylpyrocarbonate at 20°C for 30 mm to remove the nuclease activity, rinsed exhaustively in 0.1 x SSC and dried at room temperature. To determine the temperature at which 50% of the RNA was released from the filter (T,), the filters were placed in 1 ml. of 0.1 x SSC or 6 x SSC; 50% formamide and heated in temperature increments of approximately 5°C. After 6 mm at each temperature, the released RNA was recovered by trichloroacetic acid precipitation and counted.

3. Results In all experiments to be described here, the concentration of RNA was well in excess of the complementary DNA, usually by a factor of 50 to 100. The time-course of each reaction is presented conveniently as a double reciprocal plot, since this allows simple determination of both reaction half time and saturation value (cf. Bishop, 1969). (a) Effects of temperature

on rate of RNA-DNA

hybridization

We have established the optimal temperatures for maximal initial rates of reaction in 6 x SSC (pH 7*2), 50% formamide or in 1.15 M-sodium acetate (pH EGO), GOT/, formamide for a variety of RNA components. Up to 25 pg DNA bound to 13 mm millipore HAWP filters were challenged with O-1 to 3 pg homologous RNAjml. A time-interval of incubation was selected appropriate for each RNA concentration to allow hybridization to approximately 20% of the saturation value. The counts hybridized were taken as a measure of initial rate reaction. Figure 4 gives a selection of temperature-dependent rate curves. The temperature at which the reaction rates are maximal are well defined. From Table 2 it may be seen

28

M.

L.

BIRNSTIEL,

B.

H.

SELLS

AND

(a)

I.

F.

PURDOM

(b)

200 3oc 2oc

100

IOC I

C

0 Cd)

IOC

200

5C

100

2 -5 5 .2 .s :: .e 2 a

0

0

100

100

-c \c g :$ $7 4 B

50

0 (hl

0 100

400

50

200

Temperature

FIG. 4. Initial rate of RNA-DNA hybridization as a function of temperature. Filters contained approximately 25 pg DNA. The RNA concentration and time of incubation were chosen in such a way that about 20% of the complementary DNA reacted. (a) to (f) 6 x SSC, 50% formamide; (g) and (h) 1.15 sodium acetate (pH 5), 60% formamide; (a) Xenopw 5 s RNA, (b) Xenopus 28 s + 18 s (upper) and 18 s rRNA (lower), (c) HeLa rRNA, (d) rabbit 18 s rRNA, (e) E. coli 16 s RNA, (f) B. subtilis rRNA, (g) Xenopus 28 s RNA, (h) E. coli tRNA. Unlabelled rRNA and 5 s RNA were added in 20 and 2 times excess.

that the temperature optima (T,,,) are high, especially The T, in 0.1 x SSC and 6 x SSC; 50% formamide of RNA-DNA hybrids (discussed below). (b) Effects of fragment

size of RNA

for the ribosomal RNA species. are also listed for a selection

molecules

We have determined the effect of fragment size of ribosomal RNA molecules on the rate of hybridization reaction as follows: 28 s rRNA of Xenopus Eaevis in 0.1 x SSC was exposed to 0.1 N-NaoH, at 20°C for 0, 3, 10 and 30 minutes to yield RNA with

x

SSC, 50% formamide

50 53 63

53 53 53 42 60 57 54 59 62 56 62

1 y0 G + C arc taken

+ 60% formamide

4 s E. coli tRNA 23 + 16 s B. subtilis 28 s Xenopw rRNA

In 1.15 M-N&

16 8 E. cdi rRNA 23 + 16 s B. subtiZis 23 + 16 s P. mdrabiZis (PX cRNA 4 s Xenopus tRNA 6 s Xenopua rRNA 18 s Xenopwr rRNA 28 + 18 s Xenopus rRNA 28 + 18 s HeLa 18 s Rabbit 28 + 18 s Rabbit

In 6

RNA

GSCt content (%I

from

F. Amaldi

46 47 52

60 58 55 45 54 61 62 64 65 61 62

2

(1969);

Birnsticl

78

66

75

17, (hybrid) 6 x SSC, 50% formamide

et al. (1968);

69 69 69 64 73 71 70 73 75 71 75

Brown

hybrids

& Weber

Brown

81

77

85 Littna

20

70

(1966)

16

18

(hybrid) To,, (hybrid) 6 x SSC, 500/6 formamide

T,

76

80

(1968);

Tm (DNA) 0.1 x ssc

80

63

74

T, (hybrid) o-1 x ssc

for RNA-DNA

Tm (DNA) 6 x SSC, 50% formamide

rate and melting temperatures

T opt 6 x SSC, 50% formamide

Optimal

TABLE

9 11 14 19 19 10 8 9 10 10 13

T, (DNA)To,, (hybrid) 6 x SSC, 50% formamide

30

M.

L.

BIRNSTIEL,

B.

H.

SELLS

TABLE

Effect of fragment Sample

no.

S-values of 28, 9, 5 and hybridized to Xenopus preparations, irrespective means, in practice, that cules do not have to be

I.

F.

PURDOM

3

size on hybridization Cts/min hybridized in 20 min

S

1 2 3 4

AND

28 8 5 3.5

415 408 376 401

3.5 s RNA. When samples of these RNA preparations were DNA on disks the rate of hybridization was similar for all of initial molecular weight before incubation (Table 3). This for the purpose of kinetic comparison the various RNA molebrought to similar size. (c) Effects of DNA

concentrations

Gillespie & Spiegelman (1965) noted that the hybridization reaction is strongly influenced by RNA concentration, but that the concentration of DNA was less important. To study the effects of the amount of DNA bound to membrane filters more quantitatively, we prepared disks containing heterologous bacterial DNA (10 pg) with the addition of 0.01 to 1.0 pug Xenopus rDNA (Birnstiel et al., 1968). When these filters were challenged with radioactive Xenopus rRNA the absolute initial rate of hybridization (at 20 min reaction time) was proportional to the rDNA input (Table 4). When the initial rate is divided by the hybridization saturation value it is evident that the relative rates (i.e. percentages of the rDNA hybridized at a given time) are similar for the different concentrations, at least at low rDNA input. At low input of complementary DNA, therefore, the relative rate of hybridizat)ion is unaffected by t’he amount of DNA on the disks. The important practical consequence of this finding is that when studying specific DNA sequences under condition of RNA excess, the degree of reiteration of these sequences (i.e. the amount of complementary DNA) has little effect on the relative rate of hybridization. Consequently, when investigating the rate of hybridization of cellular RNA’s which are encoded by DNA segments of disparate multiplicities, a comparison of the rate for a given RNA concentration can be obtained simply by measuring the time required to reach 50% saturation of those sequences (see below). TABLE

Hybridization

of ribosomal

Amount of rDNAt bLg/dW

RNA

RNA hybridized

at 20 min 65 730 2031 3496

0.01 0.10 0.33 1.0 t Disks

also contained

4

to Xenopus

ribowmal

(cts/min) at

t

ratio

= co

cts/min, cts/min,

t = 20 t = co 0.32 0.36 0.28 0.17

200 2020 6940 20100

10 pg of Proteus

DNA

mirabilis

DNA.

KINETICS

OF

RNA-DNA

HYBRIDIZATION

31

(d) Effects of RNA concentration The initial rate of hybrid formation free in solution is proportional to the initiad concentration of RNA molecules (Nygaard & Hall, 1964). For the Gillespie & Spiegelman procedure the rate of hybrid formation throughout the entire reaction is a simple function of the initial RNA concentrations (Bishop, 1969). In accordance with this observation it may be shown (Fig. 5) that when B. subtilis rRNA is annealed to homologous DNA on membrane filters, tt, i.e. the time taken to reach 50% satura.tion value, is inversely proportional to the rRNA concentration. By multiplying the concentration of RNA (C,; moles/nucleotides/l.) and tt (set) we arrive at a product which is a constant. We shall show below that under standard conditions of incubation the constant C,t, is determined by the base sequence complexity of the RNA.

006

I/Time(hr)

FIG.

5. Effect

of RNA

concentration

on rate

of DNA. The filters contained 25 pg of B. subtilis DNA. increased to keep the RNA:DNA ratio constant data are expressed as double reciprocal plot (Bishop, saturation value has been reached. It may be concentration (cf. Bishop, 1969).

hybridization

of B. subtilis

The for

volume of incubation all RNA concentrations. 1969). At l/ye saturation seen that t+ is inversely

(e) Xequencecomplexity

rRNA

to homologous

at low RNA input was The hybridization =0.02, 50% of the proportional to RNA

qf RNA molecules

Since the (molar) concentration of the nucleotide sequences in solution determines the rate of hybridization, a given amount of RNA made up from a few, or a simple base sequence, hybridizes more quickly than the same amount of RNA containing a large diversity of base-sequences. Thus, when cRNA is synthesized in vitro on A phage, T4 or E. coli DNA and is hybridized to the homologous DNA, h phage cRN.4 reacts fastest, and T4 cRNA reacts faster than the E. coli transcript (Bishop, 1969). In order to determine the slope of the curve which correlates base sequence complexity and rate of hybridization, we synthesized a balanced RNA-DNA hybrid on single-stranded @X 174 DNB using the protocol of Chamberlin & Berg (1964). The RNA : DNA ratio was calculated both from the incorporation of labelled nucleotides and from the buoyant density of the hybrid in C&30, and was found to be close to one (see Materials and Methods). RNase resistant duplexes on incubation of t.he cRNA were less than 5% hence the cRNA contained few self complementary sequencej. The complexity of the RNA transcript is close to that of template DNA, i.e. 1.6 x 106

M.

32

L.

BIRNSTIEL,

I

B.

H.

'

-)

SELLS

AND

I.

F.

PURDOM

I

(b)

Ii-L---33

I

2

I 4

I 6

I 8

I IO

I/hour

6. Double reciprocal plots of RNA-DNA hybridization (a) for 6 x SSC, 50% formamide; (b) 1.15 M-sodium aoetate, 60% formamide. Filters contained 0.01 pg @X DNA with 10 pg Xenopus DNA added as a carrier. For hybridization of bacterial RNA, the filters contained 10 to 20 pg DNA, for hybridization of eukaryotic DNA, 25 pg filters were used. 3H-labelled tRNA preparations were supplemented by addition of S-fold excess of m&belled homologous rRNA and an equal amount of unlabelled 5 s RNA, separated by G 100 gel filtration (see Materials and Methods). The amounts of RNA shown in Fig. 6 are pg/ml. FIG.

x

SW;

50%

formamide

; 60:/,

formamide

*t The

cC,ncentration

of 3H-labellod

4 s E. coli valyl-tR,NAt 4 s E. coli tRNA (total, charged wit,h valine) 4 s h’. cold tRS-1 (total, charged with leucine) 4 s E. eoli 3H-labelled tRNA 4 s Xenopus tRNA 23 + 16 s B. subtilis rRNA 28 s Xenopus rRNA

In 1.15 .\r-Na+

174 cRNA E. coli tRNA x 16 s B. subtilis rRNA x 16 s P. wvirabilis rRNA Xenopus tRNA Xenopus I-RNA 18 s Xenopus rRNA 28 s Xenopus rRNA 28 + 18 s Xenopus rKNA 28 + 18 s Rabbit rRNA

OX 4 s 23 23 4s 5s

In 6

RNA

valyl-tENA

from

the level

of incorporation

of [3H]vsline

39 42 66 74

60 6.5 x lo3 6 6.1 x lo2

0.06 0.009 0.64 0.051

0.025 0.025 1.6 1.5 was calculated

25

-

-

0.7* 29

24-27 9-14 19-24 24 12-17 04-0.8 7-12 16-20 24-30 24-34

t, (at 3 PLg) (min) @we)

molecules

0.025

1 60 8 6 6.5 x lo3 9x103 6.1 x 1Oa 6.1 x lo2 6.1 x 1Oa 2.5~10~

Genomic redundancy

of RNA

3 -

complexity

0.0032 -

95 0.06 0.64 0.41 0.009 0.020 0.024 0.051 0.075 0.03

Complementary DNA (o/o)

and kinetic

5

0.025 0.025

1.6 0.025 1.6 1.6 0.025 0.038 0.7 1.5 2.2 2.5

Analytical complexity (daltons x 10-s)

Analytical

TABLE

into

tRNS.

18-24 24 37 42

17

0.4 17

15 6.7 12 13 7.8 0.37 5.3 11 16 17

c&t* x 103 (moles.sec/l.) (average)

0.95 1.05 Kinetic standard l-7

0.45

0.018 0.71

standard 0.78 1.4 1.5 0.90 iz 0.043 0.61 1.3 1.9 2.0

Kinetic

Kinetic complexity (daltons x 10T6) (average)

34

MM. L.

BIRNSTIEL,

B.

H.

SELLS

AND

I.

F.

PURDOM

daltons (Sinsheimer, 1959). We have determined the C,t, for this RNA species at optimal temperature. From Figure 6(a), it may be seen that at 0.94 pg @X cRNA ml. saturates half of the DNA complements in 80 minutes. A variety of cellular RNA’s have also been investigated at T,,,. Since 5 s RNA hybridizes very fast, the concentrations of this RNA waskept between0.1 to 0.5 pg/ml. For the other RNA speciesa range of 0.5 to 6 pg was employed. The exact composition of the reaction mixture is given in Figure 6(a) and (b). TheseFigures show the double reciprocal plots for the various RNA speciesin 6 x SSC, 50% formamide (pH 7.2), or 1.15 M-sodium acetate (pH 5), 60% formamide. The reaction half times (tJ were determined for all RNA species.Using the inverse relationships between t+ and concentration (Bishop, 1969; Fig. 5) we have calculated t+for an RNA concentration of 3 pg/ml. for each speciesand tabulated the observed rangesof values, together with the corrcsponding average Crt+ in Table 5.

4. Discussion (a) Optimal

rate temperatures for hybridization in the presence of formamide

of RNA

to DNA

Nygaard & Hall (1964) demonstrated that in O-3M-Nacl, 0.05 M-!lh’iS, pH 7.5, the initial rate of hybridization of T4 RNA to homologousDNA was optimal at 67”C, a temperature some16°Clower than the T, of the T4 RNA-DNA hybrid. This temperature differential is noticeably smaller than that for DNA renaturation where the rat’e of reassociationhas beenshown to be maximal about 25°Cbelow the meandenaturation temperature (Wetmur & Davidson, 1968). McCarthy & Church (1970) have recently reviewed the effect of reaction temperatures on the fidelity of basepairing. Generally, little is known about the temperature dependence of the kinetics of RNA-DNA reactions. Hybridization of rRNA is routinely performed at 67°C in media containing 2 or 6 x SSC.In initial experiments we observed that under these conditions, t’emperatures in excessof 80°Cwere required to permit maximal rate of hybridization of eukaryotic rRNA. Since these temperatures were deleterious to the stability of the RNA we have consequently supplemented these media with high concenhrations of formamide. Addition of this compound allows maximal rate of annealing at relatively low temperatures (McConaughy et al., 1969). We find that a solution composedof 6 x SSC and 50% formamide provides conditions allowing a reasonablerate of hybridizat,ion while eliminating largely the problems of RNA instability. Table 2 lists the T,,, for various RNA species.The values are compared t’o the T, of RNA-DNA hybrids determined by dissociation of RNase treated hybrids in either 6 x SSC, 50% formamide or O-1 x SSC (Fig. 7). We also list the T, of the DNA complementsas predicted from the G + C content of the RNA. For this purpose we determined the optical melting curves for Xenopus (40% G -k C), E. coli (50% G + C) and 01. Zuteus (71% G + C) DNA in 6 x SSC, 50yo formamide and in 0.1 x SSC (not shown) and calculated the T, according to the predicted G + C content of the DNA complements by linear interpolation. It should be noted that the T, of RNA-DNA hybrids are those obtained for undegraded RNA. Reduction of rRNA to 3 to 4 s for instance, reduces the T, by approximately 5°C. An unexpected finding is that RNA-DNA hybrids appear to be more stable in 6 x SSC; 50% formamide, by some2 to 6”C, than native DNA of comparable G+C

KINETICS

OF

RNA-DNA

35

HYBRIDIZATION

80

60

20

40

60

80

100

Temperature

40

60

80

100

(deg. C)

FIG. 7. Melting temperature of RNA-DNA hybrids. DNA was challenged with saturating amounts of homologous RNA and dissociated in the Materials and Methods section. (a) Dissociation in 6 x SSC, 50% formamide; (b) dissociation in 0.1 x SSC; @X RNA-DNA hybrid; -O-O-, B. subtilis rRNA-DNA hybrid; -A--A--, rRNA-DNA hybrid.

as described -O--O--, Xenopzcs

content (cf. Table 2), and by up to 8°C when compared to reassociated DNA duplexes (Grunstein, unpublished observations). By contrast the T, of the same RNA-DNA hybrids dissociated in O-1 x SSC are equal to or somewhat lower than, those of DNA complements and in this they conform to the generally accepted rule that RNA-DNA hybrids are less stable than corresponding native DNA segments (cf. Walker, 1969). For RNA of high molecular weight T,,, for RNA hybridization in 6 x SSC; 507/o formamide is approximately 10 deg. C below the T, of corresponding DNA complements. The differential for low molecular weight RNA is larger, but here the T,,, is likely to be lower because of the reduced stability of short RNA-DNA hybrids. When the T, of the hybrid and T,,, are both determined at high formamide concentrations, a temperature differential as high as 20°C is obtained (see Table 2) and in this the reaction resembles the hybridization of mRNA to T4 DNA (Nygaard & Hall, 1964). Our combined data suggest that the high concentration of formamide imparts high stability to RNA-DNA hybrids and consequently produces high optimal rate temperatures and high T,. We have also measured T,,, in an incubation mixture similar to that of Weiss, Hsu, Foft & Scherberg (1968) and of Daniel, Sarid & Littauer (1970) which allows the measurement of the kinetics of 3H-labelled aminoacyl-tRNA hybridization with little hydrolysis of the aminoacyl ester bond (see Materials and Methods; Fig. 3). In this medium, the formamide concentration is increased to 60% and the pH lowered to pi. T,,, are listed in Table 2. There are probably at least four characteristics of the RNA which influence T,,,. These are: fragment size, fidelity of base pairing, G $ C content and secondary structure of the RNA. Amongst the RNA species the To,, of rRNA is highest of all, and T, (DNA)-To,, (hybrid) smallest. In rRNA a large proportion of the nucleotide sequences are present in double-stranded pin-loop form. This could lead to a severe sequestration of nucleotide sequences and thus slow down the reaction by lowering of the effective nucleotide concentration in solution. It is possible that in this case a large

36

31. L.

BIRNSTIEL,

B.

H.

SELLS

AND

I.

F.

PURDOM

portion of the base sequence must first be made available by melting of the doublestranded RNA region. Consistent with this idea it is observed that B. subtilis and Xenopus rRNA when heated to optimal rate temperatures in 6 x SSC, 50% formamide appear to be largely melted but still maintain 15% of their hyperchromicity (Speirs, unpublished observations). The optimal rate temperatures observed probably represent a compromise between the melting of RNA molecules and the stability of the initial RNA-DNA hybrids around the nucleation sites. From the high optimal rate temperatures it would appear that previous annealing experiments with formamide, especially those utilizing rRNA, mere carried out ~\ell below the optimal discriminating temperatures of annealing. It’is noteworthy that, the saturation values for RNA’s such as Xenopus and B. subtilis rRNA are identical at optimal and sub-optimal temperatures, while at higher temperatures the value is reduced. Saturation levels were determined at t = co from all double reciprocal plots and these are listed in Table 5. They fall well within the ranges published by other laboratories. The hybridization saturation value of @X cRNA represents an interest)ing case. Here, apparenbly most of the @X DNL4 bound to filters is available for hybridization and no large “hybridization factor” (Bishop & Robertson, 1969) exists for this RNA species. (b) Choiceof standardsfor the determination of rates of hybridization Because of the relatively well-defined temperature optima it is essential to standardize the reaction condition rigorously. Once this has been achieved, RNA-DNA hybridization may be used to analyse the base sequence complexity of RNA molecules. The procedure is relatively simple because within the limits stated above, the degree of fragmentation of t,he RNA and t,he mass of the complement’ary DNS on membrane filters have little influence on the relative rate of RXA-DNA hybridization. d small molecule such as Xenopus 5 s R,NA encoded for by a multiplicity of some 9000 DNS segments (cf. Table 5) can be compared without difficulty to the relatively large bacterial ribosomal RNA, which is complementary to only a few DNA segments per genome. In choosing kinetic standards we have avoided self-complement’ary cRNA since here neither t’he true complexity nor the concentration of freely react,ive RKA is adequately known because of self annealing of the RNA during the hybridization reaction. Standards provided by in vivo t’ranscripts from repetitive sequences could always be open to the objection that minor heterogeneity amongst redundant genes might measurably influence the rate of hybridization. Other problems with cellular RNA’s might arise from poor locu s specificity of hybridization reactions (McCa~rthy & Church, 1970). For example, the base sequences in E. coli rRPLTA are similar enough to cross-hybridize between 23 s and 16 s cistrons (Attardi, Huang & Kabat, 1965; Mangiarotti, Apirion, Schlessinger & Silengo, 1968) so that the kinetic complexity of this RNA is not clearly predictable. In our hands, E. coli rRNA has so far provided the only exception to the hybridization series of Table 5, in that it appears to react more slowly by a factor of 1.7 than B. subtilis rRNA. But we have not been able to test this RNA for its hybridization purity and so this observation must be further investigated. For these reasons we have used cRNA from a balanced (PX RNA-DNA hybrid for the prime kinetic standard, while we consider RNA species such as 5 s RNA of Xenopus laevis and 23 s and 16 s rRNA of B. subtilis (which do not cross-hybridize) as

KINETICS

OF

RNA-DNA

HYBRIDIZATION

3’7

secondary kinetic standards. When the kinetic complexity of these secondary standards (and eukaryotic rRNA’s) are calculated by comparison to the 0X cRNA (Table 5), it is clear that in all cases the kinetic complexity closely approximates the molecular weight of the RNA’s. For most cellular RNA’s the complexity is in fact slightly less than anticipated and this may be caused by the faster reaction of G + C rich RNA molecules compared to the low G + C standard. For 6 x SSC, 50% formamide the slope which links C& and complexity is 9,3 x 10es moles.sec/l. daltons. From Table 5 it may also be deduced that the slope of the standard curve for 1.15 M-sodium acetate (pH 5); 60% formamide is 2.3 x lows’ moles.sec/l. daltons. (c) Kinetic complexity of RNA molecules In this discussion the analytical complexity of an RNA is defined as its molecular weight, while the kinetic complexity, also measured in daltons is defined as the rate at which RNA-DNA hybrids form and is determined by comparison to the above mentioned kinetic standards. These concepts have already been usefully employed when dealing with DNA renaturation experiments (Wetmur & Davidson, 1968). At its simplest the relationship between C,t, and base-sequence complexity underlines the long-recognized fact that a given amount of RNA made up from simple sequences supports rapid hybridization while an equal amount of more complex RNA molecules anneals more slowly. Under our condition of incubation, saturation is closely approximated in 6.5 minutes for 3 pg 5 s RNA/ml. Longer incubation only ma,gnifies the contribution of contaminants in the RNA preparation. Conversely, the reaction of bacterial ribosomal RNA requires a period of 240 minutes to achieve the same degree of saturation. Where a messenger RNA fraction has been isolated which is suspected to code for a single protein, the purity of this RNA can be quickly established. From the mole.. cular weight of this RNA (analytical complexity) a C,t+ can be calculated. By comparison to the experimentally obtained Crtt it may be established whether analytical and kinetic complexity coincide, i.e. whether the mRNA in question is a single species or a mixture of different RNA species. In hybridization experiments, using messenger RNA coding for globin (Williamson, Morrison & Paul, 1970) and, for histone (Kedes & Birnstiel, 1971) it was noted that hybridization was rapid, but no quantitative analyses of the kinetics have yet been made. As pointed out by Britten (1968), the kinetics of hybridization should be helpful in the determination of the concentration of messenger RNA’s Wherever the base-sequence complexity of the messenger is known, measurement of t; will yield C,. Finally, RNA species such as tRNA, 5 s and rRNA are encoded by highly redundant genes, and kinetic analysis can be used to determine whether the gene transcripts form one, two or more families of base sequences. Five examples are given below. (1) There are 5 to 8 ribosomal cistrons in the genome of B. subtilis (Oishi & Sueoka,, 1965, cf. Table 5). In agreement with the renaturation kinetics of isolated bacterial ribosomal DNA complements (Kohne, 1969) we show that the 23 s and the 16 s bacterial rRNA possesses a kinetic complexity, which is that of a single family of base sequence of w 1.4 x lo6 daltons. (2) Hybridization experiments indicate that of the order of 0.02% of Xenopus DNA are complementary to 5 s RNA (Brown & Weber, 1968, cf. Table 5). From this it may be calculated that 5 s cistrons are some 9000 times repetitive in the haploid genome of

38

M.

L.

BIRNSTIEL,

B.

H.

SELLS

AND

I.

F.

PURDOkT

laevis. Fingerprint analysis of this RNA species (Williamson, personal communication) has shown this speciest’o be largely homogeneous,Consistent with this we find that 5 s RNA anneals some 40 times faster than @X cRNA. 5 s RNA, although encoded by highly repetitive cistrons, has a kinetic complexity closet,o that of a single family of RNA molecules. (3) The ribosomal cistrons from Xenopus laevis and rabbit are approximately 450 to 800 and 250 to 700-fold redundant (cf. Table 5, Birnstiel et al., 1966; Brown & Weber, 1968; Moore & McCarthy, 1968). The analytical complexity of amphibian and mammalian 28 s and 18 s rRNA molecules combined are 2.2 and 2.5 x 106 daltons, respectively (Loening, 1968). The Crts values at optimal rate temperatures are listed in Table 5. By comparison to the kinetic standards it may be seenthat the kinetic complexities of these eukaryotic rRNA’s are closely similar to the analytical complexity. It follows that most, if not all, rRNA moleculesbelong to a single family of nucleotide sequences.In both Xenopus and rabbit the ribosomal sequencesare highly conserved. (4) In our hands, O*O58o/o of the E. coli genome is complementary to tRNA. This comparesto values of 0.04 to O.O6o/ofound by Morel1 et al. (1967) and Zehavi-Willner & Comb (1966) for E. coli and 0*07% for B. s&ilk DNA by Oishi & Sueoka (1965). For all 60 tRNA cistrons combined we anticipate an analytical complexity of approximately 1.5 x 106. As may be seen from Table 5 [3H]uridine labelled E. coli tRNA exhibits a complexity of only O-7 to 0.8 x lo6 daltons. tRNA labelled by acylation with [3H]leucine or [3H]valine exhibits a composite complexity of 0.60 and 0.71 x 10E daltons. The kinetic complexity of unfractionated E. coli tRNA, uniformly labelletl with [3H]uridine or acylated with [3H]valine or [3H]leucine is about half the value of the predicted analytical complexity. This can be explained in two ways. 1. The over-all reaction may be accelerated by the presenceof a few tRNA speciesin disproport,ionately high concentrations. The rapid annealing rate of tRNA labelled with [3H]leucine may reflect this aspect of the hybridization. 2. Some sequencesamongst the tRNA speciesmay be similar enough that they are not resolved from one another by hybridization experiments. This is perhapsdocumented by the finding that [3H]valinetRNA annealsto a multiplicity of DNA segmentsbut behaves kinetically asif it were homogeneous. (5) The results presented in Table 4 indicate that Xenopus tRNA is encoded by approximately 6500 cistrons. Its kinetic complexity, however, is similar to that of E. coli tRNA which is coded for by 60 cistrons. In spite of this great difference in redundancy between the bacterial and amphibian speciesthe base-sequence complexity is similar. Thus, the Xenopus tRNA genome contains N 40 families of tRNA base sequenceseach of which is reiterated about 160-fold. These data are interesting in terms of what is known about the heterogeneity of tRNA’s examined by various column procedures. Novelli and his colleagues (Novelli, 1969) using reverse-phase chromatography have demonstrated a number of subspeciesof tRNA for each amino acid in both animal and bacterial cells. Yang & Novelli (1971) have demonstrated in rat, mouseand man that the iso-accepting speciesof tRNA for the 20 amino acids are separable into some 60 peaks. Our kinetic studies are in agreement with these studies in that they suggest limited base sequenceheterogeneity despite the high degree of genetic redundancy of the tRNA cistrons. Xenopus

The technical assistance of Mrs Joan O’Neil J. 0. Bishop for supplying us with labelled studies were supported in part by the Damon

is greatly appreciated. Wo wish to thank Dr B. subtilis and P. mirabilis rRNA. These Runyon Memorial Fund.

KINETICS

OF

RNA-DNA

HYBRIDIZATION

39

REFERENCES Amaldi, F. (1969). Nature, 221, 95. Attardi, G., Huang, P. C. & Kabat, S. (1965). Proc. Nat. Acud. Sci., Wash. 53, 1490. Birnstiel, M. L., Speirs, J., Purdom, I., Jones, K. & Loening, U. E. (1968). Nature, 219, 454. Birnstiel, M. L., Wallace, H., Sirlin, J. L. & Fischberg, M. (1966). Nut. Cancer Inst. Monogr.. 23, 431. Bishop, J. 0. (1969). Biochem. ,T. 113, 805. Bishop, J. 0. & Robertson, F. W. (1969). Biochem. J. 115, 253. Britten, R. J. (1968). Carnegie Inst. Yrb. 67, 335. Britten, R. J. & Kohne, D. E. (1968). Science, 161, 529. Brown, D. D. & Littna, E. (1966). J. ~%foZ. Biol. 20, 95. Brown, D. D. & Weber, C. S. (1968). J. Mol. Biol. 34, 661. Chnmberlin, M. & Berg, P. (1964). J. Mol. BioZ. 8, 297. Daniel, V., Sarid, S. & Littauer, U. Z. (1970). Science, 167, 1682. Flamm, W. G., Bond, H. E. & Burr, H. E. (1966). Biochim. biophys. Acta, 129, 310. Gillespie, D.& Spiegelman, S. (1965). J. Mol. BioZ. 12, 829. Kedes, L. & Birnstiel, M. L. (1971). Nature, 230, 165. Kohne, D. E. (1969). Biophys. J. 8, 1104. Loening, U. E. (1968). J. Mol. BioZ. 38, 355. McCarthy, B. J. & Church, R. B. (1970). Ann. Rev. Biochem. 39, 131. McConaughy, B. L., Laird, C. D. & McCarthy, B. J. (1969). Biochemistry, 8, 3289. Mangiarotti, G., Apirion, D., Schlessinger, D. & Silengo, L. (1968). Biochemistry, 7, 456. Marmur, J. (1961). J. iqToZ. BioZ. 3, 208. Moore, R. L. & McCarthy, B. J. (1968). Biochem. Genetics, 2, 75. Morell, P., Smith, I., Dubnau, D. & Marmur, J. (1967). Biochemistry, 6, 258. No\relli, C;. D. (1969). J. Cell. Physiol. Suppl. 1, 74. Nygaard, A. P. & Hall, B. D. (1964). J. Mol. BioZ. 9, 125. Oishi, M. & Sueoka, N. (1965). Proc. Nat. Acad. Sci., Wash. 54, 483. Purdom, I., Bishop, J. 0. & Birnstiel, M. (1970). Nature, 227, 239. Sinsheimor, R. L. (1959). J. Mol. BioZ. 1, 37. Walker, P. M. B. (1969). Progress in Nucleic Acid Research, 9, 301. Waters, L. C. & Novelli, G. D. (1968). Biochem. Biophys. Res. Comm. 32, 971. U’eiss, S. B., Hsu, W., Foft, J. W. & Scherberg, N. H. (1968). Proc. Nat. Acad. Sci., Wash. 61, 114. Wotmur, J. G. & Davidson, N. (1968). J. Mol. BioZ. 31, 349. Williamson, R., Morrison, M. & Paul, J. (1970). Biochem. Biophys. Res. Comm. 40, 740. Wong, J. T., Mustard, M. & Herbert, E. (1969). Biochim. biophys. Acta, 174, 531. Yang, W. K. & Novelli, G. D. (1971). Methods in Embryology XII, Part C. Methods in Nucleic Acids, ed. by Grossman & Moldavo. Zehavi-Willner, T. & Comb, D. G. (1966). J. Mol. Biol. 16, 250.

Note added i-n proof: Yaniv & Barr&l (1971, Nature New shown that valine-tRNA from E. coli is separable into three valine,, and valinez,-tRNA were shown to be identical but tRNA shared 55 out of 77 bases with valine,-tRNA. This is tion data (Table 5) which reveal the presence of three closely in the E. coli genome.

Biol., 223, 113) have recently subspecies. The sequences of for six residues, while valine,consistent with our hybridizasimilar genes for valine-tRNA