Ribosomal RNA homologies in flowering plants

339

Biochimica et Biophysica Acta, 349 (1974) 339--350 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98011

RIBOSOMAL RNA HOMOLOGIES IN FLOWERING PLANTS: COMPARISON OF THE NUCLEOTIDE SEQUENCES IN 5.8-S rRNA FROM BROAD BEAN, DWARF BEAN, TOMATO, SUNFLOWER AND RYE

J A N E WOLEDGE*, M.J. C O R R Y and P.I. PAYNE

Agricultural Research Council Unit of Developmental Botany, 181A Huntingdon Road, Cambridge, CB3 ODY, (U.K.) (Received January 11th, 1974)

Summary Broad bean 5.8-S rRNA was digested separately with T~ and pancreatic A ribonucleases and the resulting oligonucleotides (27 and 19, respectively) were fractionated by two-dimensional electrophoresis. The oligonucleotides were further analysed and the nucleotide sequences of most of them determined. They were compared with those from 5.8~ rRNA of dwarf bean, tomato, sunflower and rye. The results suggest that the sequence of the molecule has been highly conserved during the evolution of the flowering plants. Introduction Homologies of the ribosomal RNAs between species of Angiosperms have so far been studied by molecular hybridisation [1,2]. Different applications of the technique have produced conflicting results. Trewavas and Gibson [1] tested the ability of rRNA of various plant species to displace 32 P-labelled pea rRNA from a homologous DNA--RNA hybrid. They concluded that the nucleotide sequences of the rRNAs from species of monocotyledonous and dicotyledonous plants differed considerably. Vodkin and Katterman [2] annealed the rRNAs of various plant species to the DNA of a single species and measured the thermal stabilities of the hybrids. While they agree with Trewavas and Gibson [1] that there is considerable variation of rRNA sequences within the monocotyledons, they disagree by suggesting the nucleotide sequences of rRNAs of dicotyledons are highly conserved.

Abbreviation: CMCT, N-cyelohexyl-N~-2-(4-morPholinyl)-ethylcarbodiimide p-toluene sulphonate.

* Permanent address:- Grassland Research Institute, Hurley, Maidenhead, Berkshire, SL6 5LR, Great Britain.

340 The degree of homology between rRNAs from different species of flowering plants can be determined with certainty only from nucleotide sequences. In plants and animals, the primary gene product of rDNA is the ribosomal precursor RNA which is processed to 25-S, 18~S and 5.8-S RNAs (refs 3--6). The 25-S and 18-S rRNAs contain about 4000 and 2000 nucleotide residues, respectively, and a detailed sequence analysis of them would be impracticable. The 5.8-S rRNA in contrast, contains only about 150 residues [7]. We have compared nucleotide sequences in 5.8-S rRNAs from five flowering plant species and from the results we suggest that the molecule has been highly conserved, both amongst dicotyledons and between dicotyledons and the monotyledon studied. Materials and Methods Seeds of tomato (L y copersicum esculentum ), sunflower (Helianthus anuus ) and dwarf bean (Phaseolus vulgaris) were obtained from Thompson and Morgan Ltd, Ipswich, Suffolk, U.K., and broad bean (Vicia faba) from Carters Seeds Limited, Llangollen, Wales, U.K. Embryos isolated from rye grain (Secale cereale, obtained from Nunns Corn and Coal Company Ltd, Woodbridge, Suffolk, U.K.) by the method of Johnston and Stern [8] were provided by Miss S. Sen. Pancreatic A ribonuclease, snake venom phosphodiesterase and Escherichia coli alkaline phosphatase (Worthington Biochemical Corp.) were obtained from Cambrian Chemicals Ltd, Croydon, Surrey, U.K. and T~ ribonuclease (Sankyo Co.) from Calbiochem Ltd, London, U.K. Cellulose acetate strips (Schleicher and Schull) measuring 75 cm by 3 cm were obtained from Anderman and Company Ltd, London, U.K., Kodirex X-ray film for autoradiography was from Kodak Ltd, London, U.K. and N-cyclohexyl-N'-2-(4-morpholinyl)-ethylcarbodiimide p-toluene sulphonate (CMCT) (Fluka AG) from Fluorochem Ltd, Glossop, Derbyshire, U.K. Ribonuclease U~ was kindly provided by Dr G.G. Brownlee.

Incorporation of 32p and extraction of R N A Root ribosomal R N A (rRNA) was labelled with ortho[ 32p] phosphate by growing 8--10~lay-old seedlings for 24--48 h with their roots immersed in 200 ml of distilled water which contained 80 ~g chloramphenicol/ml and 50/~Ci carrier free ortho[32P]phosphate/ml. The medium was continually aerated during the incubation. For incorporation of radioactivity into rye nucleic acids, ten 0.1-ml aliquots of a 10 Ci/l solution of ortho[32P] phosphate were dried onto different areas of a petri dish. Approx. 130 rye embryos were imbibed on one of these areas in I ml 2 % (w/v) sucrose and 50 #g/ml chloramphenicol. At about 5-h intervals, the embryos and solution were transferred to other areas of 32 p and R N A was extracted after 48 h of germination. R N A was extracted by the method of Hastings and Kirby [9 ] as described previously [10]. High molecular weight R N A and associated 5.8~S were separated from the bulk of the 4~S and 5~S R N A s by high speed cent~ifugation at 3 • 1 0 7 X gay min (usually 2 . 7 3 • 1 0 s × gay for 1 1 0 rain at 5°C in a B e c k m a n - S p i n c o 60 Ti fixed-angle rotor ray = 6.3 cm). The pellet was dissolved in 0.5%

341 (w/v) sodium dodecylsulphate, 0.15 M sodium acetate buffer, pH 6.0, to a final concentration of 0.1 mg RNA/ml. To dissociate the 5.8~S rRNA from 25-S rRNA the solution was heated at 70°C for 15 min, rapidly cooled and RNA precipitated from it by adding two volumes of ethanol and storing at --10°C overnight.

Polyacrylam ide gel electrophoresis The precipitated RNA was dissolved in 0.3--0.5 ml of 0.5% (w/v) sodium dodecylsulphate, 10% (w/v) sucrose, 0.15 M sodium acetate buffer, pH 6.0, and subjected to electrophoresis [11] for 4.5 h at 70 V in a 10 cm × 0.3 cm × 7.5 cm deep slab of 10% polyacrylamide gel with a 0.5-cm deep 2.6% spacer. After electrophoresis the spacer gel and top 1 cm of the slab were cut away and the remainder autoradiographed for 30 min to locate the 5.8~S rRNA band. The 5.8-S rRNA was recovered from the gel by homogenising the band in 0.2 M NaC1, 0.02 M potassium acetate buffer, pH 5.6, and concentrated by DEAEcellulose column chromatography [12]. The RNA content of the eluate was made up to 40 pg with purified, unlabelled E. coli high molecular weight RNA. Finally RNA was precipitated by adding two volumes of ethanol and storing overnight at --10 ° C. Enzymic digestion of RNA and fractionation of the products RNA was digested to completion with T1 or pancreatic A ribonucleases as previously described [12]. The digestion products were fractionated by twodimensional electrophoresis [13] and their positions were located by autoradiography. Analysis of oligonucleotides The oligonucleotide loci were cut out, placed in scintillation vials containing 10 ml of 0.7% (w/v) 5-(4-biphenylyl)-2-(4-t-butylphenyl)-l-oxa-3, 4-diazole in toluene, and radioactivity was measured in a Packard scintillation counter. The DEAE-cellulose paper discs were washed in toluene, then in ethanol and dried. Oligonucleotides were eluted with 30% (w/v} triethylamine carbonate, pH 10, containing 20 mg hydrolysed yeast RNA/ml [13]. Pancreatic A, T1 and U2 ribonuclease digestions of the oligonucleotides were carried out as previously described [12]. The 3'-terminal nucleotide of 5.8-S rRNA was determined by comparing the results of: (1) digestion of the 3'-oligonucleotide with 10 pl of 0.2 mg snake venom phosphodiesterase per ml of 0.01 M MgC12 and 0.05 M Tris--C1buffer, pH 8.9, for 2 h at 37°C with (2) hydrolysis of the 3'-oligonucleotide with 0.2 M NaOH [13]. Additional sequence information was obtained by reacting some of the TI ribonuclease oligonucleotides with CMCT before pancreatic A ribonuclease digestion [13]. Results

Analysis of sequences in broad bean 5. 8-8 rRNA An autoradiograph of a two-dimensional fractionation of the oligonucleotides produced after TI ribonuclease digestion of root 5.8~S rRNA is shown in

342

~1$

O ~

P~lO

Fig. 1. A u t o r a d i o g r a p h o f f r a c t i o n a t e d p r o d u c t s f r o m b r o a d b e a n 5.8-S r R N A a f t e r d i g e s t i o n w i t h (a) r i b o n u c l e a s e T1 a n d (b) p a n c r e a t i c A r i b o n u c l e a s e . F r a c t i o n a t i o n w a s b y e l e c t r o p h o r e s i s in c e l l u l o s e a c e t a t e ( d i r e c t i o n 1) a n d in D E A E - c e l l u l o s e p a p e r w i t h 7% (v/v) f o r m i c a c i d ( d i r e c t i o n 2). S e q u e n c e a n a l y s e s o f t h e o l i g o n u c l e o t i d e s a r e given in T a b l e s I a n d II. O l i g o n u c l e o t i d e T 4 3 w a s p r e s e n t in s u b m o l a r a m o u n t s b u t w a s c o n s i d e r e d t o b e a n a u t h e n t i c p r o d u c t o f 5.8-S r R N A as P 2 8 w a s p r e s e n t i n m o l a r q u a n t i t i e s . All o t h e r f r a g m e n t s p r e s e n t in s u b m o l a r (less t h a n 0 . 5 M) a m o u n t s w e r e a s s u m e d t o b e contaminants.

Fig. la. Identical fingerprints were obtained with 5.8-S rRNA from the leaf and stem of the same plant species [14]. The sequences of most of the oligonucleotides were deduced after separate digestion with pancreatic A ribonuclease, U2 ribonuclease and pancreatic A ribonuclease after modification with CMCT (Table I). Oligonucleotides T2 4, T2 ~ and T30 gave inconclusive results after CMCT modification and their sequences remain unknown. Pancreatic A ribonuclease digestion of oligonucleotide T43 produced Gp and a product which migrated slightly faster than A--Gp at pH 3.5. The prod-

TABLE I A N A L Y S I S O F T H E C O M P L E T E T 1 R I B O N U C L E A S E D I G E S T I O N P R O D U C T S O F B R O A D B E A N 5.8-S r R N A Oligonucleotide

Pancreatic A ribonuclease digestion p r o d u c t s

Ribonuclease U2 digestion products

P r o d u c t s of p a n c r e a t i c A ribonuclease digestion after CMCT modification

Sequence deduced

Experimental m o l a r yield* *

T1 T2 T3 T5 T6 T7 T8 T9 TI1 TI2 TI3 TI4 T15 TI6 TI8 TI9 T20 T21 T22

Gp Cp, G p A--Gp A--A--Gp CP3, G p Cp, A - - C p , Gp CP2, A - - C ~ , U p , u n k n o w n * Cp, A - - A - - G p CP, A - - A - ' C p , Gp Up, G p A--Up, Gp Up, A--Gp Cp2, Up, ~ CP2, A - - U p , G p A--A--Up, Gp A - - A - - A - - U p , Gp A - - A - - U p , Cp3, G p A - - A - - C p , CP2, A - - U p , G p UP2, Gp UP2, Cp, G p A - - C p , Up20 Cp 2, G p A - - U p , Cp 2, Up, A - - G p A - - U p , A - - C p . UP2, G p UP3 , CP2, G p ( A - - U P ) 2 , U p , Cp, A - - G p A - - A - - U p , X p * , Up° A - - G p UP4, Cp° Gp unknown*, Gp

-----C - - A p , C---Gp C - - C - - A p , (C, U')Ap -C--Ap, Ap, C--Gp -Ap, U--Gp --C--Ap, (U,C)Gp A--Ap, Ap, U--Gp -A--Ap, Ap, (U,C3)GP AP2, C---C--Ap, ( U , C ) G p --Ap, (C3,U2)GP A p , C - - C - - A p , U - - U - - A p , Gp Ap, U--Ap, (C,U2)GP -(U2,C) Ap, U--Ap, Ap, Gp AP3° U - - U - - X p * , Gp ---

------------U--Gp ----A--U---Cp, G.p -U--Gp, (C,U)Gp. U--Cp -(A--U,U)A--Gp --A--G_p ---

Gp C--Gp A--Gp A--A--Gp C--C--C--Gp C--A--C--Gp ~--C--A, C--U--A)NoH** C--A--A--Gp C--A--A---C'--Gp U--Gp A--U--Gp U--A---Gp C--C--U--Gp C--A--U--C--Gp A--A--U--Gp A--A--A--U--Gp A--A--U--C--C---C--Gp A--A---C---C--A--U--C--Gp U--U--Gp U--C--U---Gp A--C(U2C2)GP C--C--A--U--U--A--Gp A--U--A---C--U--U--Gp (U3.C2)GP A--U--A--U--U--~--A--Gp A--A--U--U--X*--A---Gp (U4,C)Gp

6 4.1 2.5 1.0 1.0 0.95 0.96 0.93 0.72 4.2 2.0 0.87 0.92 0.92 0.57

T23 T24 T25 T26 T27 T28 T29 T30 T43

-

-

U n d e r l i n e d bases h a v e b e e n m o d i f i e d b y C M C T t r e a t m e n t . * See t h e t e x t . * * N = a n u n i d e n t i f i e d n u c l e o t i d e ( i d e n t i f i e d as U O H in t h e t e x t ) . ** * E x p r e s s e d r e l a t i v e to t h e m e a n ~adioactivity f o u n d in t h e f o u r t r i n u c l e o t i d e s p o t s A - - A - - G p , A - - u - - G p , U - - A - - G p , U--U---Gp. 2 2

0.86 0.81 0.83 2.1 0.85 0.92 0.79 0.93 0.80 0.86 1.0 0.89 0.5

o~

344 uct was hydrolysed by alkali to two unidentified nucleotides (designated Yp and Zp) in the ratio of 2:1. These had R B values on 3 MM paper at pH 3.5 of 0.92 and 0.70, respectively (B = the reference dye xylene cyanol FF). When the alkaline hydrolysis was preceeded by an alkaline phosphatase digestion, Yp and inorganic phosphate were the only products detected. Therefore the sequence of this product is Y--Y--Zp and that of T43 is Y--Y--Z--Gp. An unusual product was also produced when oligonucleotide T2 9 was digested with pancreatic A ribonuclease. It migrated just faster than A--Cp and was resistant to snake venom phosphodiesterase and alkaline hydrolysis and therefore assumed to be another minor base (designated Xp). Oligonucleotide T8 occurred on the G graticule of the fingerprint although it contained Up. This and the absence of Gp suggests that T8 contains the 3'-end of 5.8-S rRNA Complete snake venom phosphodiesterase digestion of Ts produced pU, pC and pA in equal amounts whilst alkaline hydrolysis produced Up, Cp and Ap in the ratio 1:3:2. Therefore UOH is the 3'-terminal nucleoside of Ts and Cp is its 5'-terminal nucleotide. The sequence of T8 is either C--C--A-C--U A - - U o H or C - - U - - A - - C - C - A - - U o H (Table I). N o oligonucleotide with a 5'-phosphate was detected. An autoradiograph of a two-dimensional fractionation of the oligonucleoTABLE II ANALYSIS OF THE COMPLETE PANCREATIC A RIBONUCLEASE DIGESTION PRODUCTS OF BROAD BEAN 5.8-S rRNA Oligonucleotide

T 1 ribonuelease digestion p r o d u c t s

Sequence deduced

Experimental molar yieldt

PI

Up Cp A--Cp A--A--Cp G--Cp A--Up G--A--Cp A--G---Cp G--A--A---Cp G--Up Cr--G-~p G--A~Up (G,A--G)Cp A--A--Cr--Up G--A--A--Up G--A--A--Up A--Cr--A--A--Up ~ U p G---G--A--Up (G,A--G)Up **

13

PI7 PI8 PI9 P20 P22 P23 P24 P28

Up Cp A--Cp A--A---Cp Gp, Cp A--Up Gp, A---Cp A--Gp, Cp Gp, A--A--Cp Gp, Up Gp 2 , Cp Gp, A--Up Gp, A--Gp, Cp A--A--Gp, Up Gp, A--A--Up Gp, A--A--A--Up A--C~, A--A--Up Gp 2, Up Gp 2 , A--Up Gp, A--Gp, Up A--A--Gp, Gp, u n k n o w n *

P30

Xp

Xp

P2 P3 P4 P5 P6 P7 P8 P9 PI3

PI4 PI5 PI6

12 2.3 1.0 4.8 2.8 1.0 1.0 1.7 4.0 0.85 3.3

0.80 0.91 0.91 1.0 1.0 0.72 0.72 0.72 0.98* * * 0.96

* Same u n k n o w n as present in digests o f T43 and i d e n t i f i e d in the t e x t as Y--Y--Zp. ** See t h e t e x t . *** Calculated assuming P28 is a s e p t a n u e l e o t i d e , see the t e x t . t E x p r e s s e d relative t o the m e a n radioactivities f o u n d in the five trinucleotide s p o t s A - - A - - C p . Cr--A---Cp, A--G---Cp, ~ p , G--A--Up and ~ U p . 3

345 tide products after complete pancreatic A ribonuclease digestion of 5.8~S rRNA is shown in Fig. lb. Unique sequences of all but three of the products could be deduced after a T~ ribonuclease digestion (Table II). T~ ribonuclease cleaved oligonucleotide P28 to Gp, A--A--Gp and a product with an RB value of 0.65 which was identified as Y--Y--Zp in the analysis of T43 • Since A--A--Gp occurred on the T~ ribonuclease fingerprint there must be a pancreatic A ribonuclease oligonucleotide containing the sequence -G--A--A--G--. The complete sequence of P2 s is therefore G--A--A--G--Y-Y--Zp. The complete sequence of oligonucleotides P16 and P24 remain unknown.

Sequence analysis of 5.8-S rRNA from dwarf bean, tomato, sunflower and rye The 5.8-S rRNA from dwarf bean, tomato, sunflower and rye were digested to completion with T1 ribonuclease and the products were separated by two-dimensional electrophoresis (Fig. 2). The fingerprints were similar both to each other and to the TI ribonuclease fingerprint of broad bean 5.8~q rRNA. Pancreatic A ribonuclease fingerprints were prepared for each species of 5.8-S rRNA and were also similar in all five plant species (not shown). Sequences of oligonucleotides T 1 - 6 , Tg, T10, T 1 2 - 1 4 , TI 8, T~ 9, T22, T32, and T39 (Table III) were deduced after their digestion with pancreatic A ribonuclease. Similarly oligonucleotides P 1 - 9 , PI 3-1 s, P 1 7 - 2 0 , P22, P23, P2 s, P26 and P29 (Table IV) were sequenced after a T1 ribonuclease digestion. Two sunflower 5.85 rRNA oligonucleotides were sequenced by comparing T~ and pancreatic A ribonuclease fingerprints. The presence A--A,A--A--Up (P26 ) (Table IV) indicates that the sequence of T37 (which contains A--A-A--A--Up, Cp and Gp) is C--A--A--A--A--U--Gp (Table III, Fig. 2). Similarly the presence of a second molecule of A--A--Gp. (Ts) implies that P2 ~ has the sequence G--A--A--G--Cp (Table IV). Sequences were assigned to most of the other oligonucleotides by comparison with the sequence data obtained from broad bean. If an oligonucleotide occurred at the same position on the fingerprint as a broad bean oligonucleotide and yielded the same secondary digestion products, it was assumed to have the same sequence. Oligonucleotides TT, T1 ~, T1 s, T16, T2 o, T21, T23, T24--T26, T29, and T4 3 (Table III) and P2 s (Table IV) were sequenced in this way. Oligonueleotide T27 of dwarf bean and tomato and T30 of dwarf bean, tomato and rye were assumed to be identical to T27 and T30 of broad bean although unique sequences of the latter two are not known (Table III). There are some oligonueleotides which are common to several fingerprints but not present in broad bean. Because these had identical fingerprint positions and secondary digestion products their sequences were assumed to be the same. Oligonucleotides TI 7 and T34 --T36 were of this kind. Since T36 occurs in dwarf bean, tomato, sunflower and rye it seems likely that there is an homologous sequence in broad bean 5.85 rRNA. This would be the case if the sequence of T36 was A--U--A--U--U--C--C--Gp which differs from T2 s (A--U--A--U--U--C--A--Gp) by only one base. Similarly, oligonucleotide T40 of rye was assigned the sequence A--U--A--C--C--U--Gp to be homologous to T26 (A-U--A--C--U--U---Gp) of broad bean, dwarf bean, tomato and sunflower.

346 {a) =

°:.

It

T~

~o

,4

0T.

Tr¢

O~

~Tm

-O

~4g

oQ -0

,B

Fig. 2. A u t o r a d i o g r a p h of t r a c t i o n a t e d T1 r i b o n u c l e a s e d i g e s t i o n p r o d u c t s of 5.8-S r R N A f r o m (a) d w a r f b e a n , (b) t o m a t o , (c) s u n f l o w e r a n d (d) r y e . T h e s e q u e n c e s o r c o m p o s i t i o n s of all o l i g o n u c l e o t i d e s axe listed a n d c o m p a r e d in T a b l e III.

Oligonucleotide T3 s only occurs in rye 5.8-S rRNA, T3 i in t o m a t o , T3 3 in sunflower and T44 in dwarf bean; sequences can neither be deduced for nor assigned to t h e m (Table III). Discussion An examination of Tables III and IV suggests the nucleotide sequences of 5.8 S rRNA from broad bean, dwarf bean, t o m a t o , sunflower and rye have clear resemblances, b u t are n o t identical. The degree of similarity can be determined exactly only by comparing complete sequences, but our results give a reliable estimate if two assumptions are made: (1) when two oligonucleotides migrate to the same position on a figerprint and are cleaved to the same secondary digestion products, t h e y have the same sequence; (2) when identical oligonucleotides containing four or more bases occur in 5.8-S rRNA f r o m two plant species their positions in the intact molecule are the same. Initially the TI ribonuclease oligonucleotides of 5.8-S rRNA from different pairs of plant

347

TM

(c)

(d)

species were compared. Mono-, di- and trinucleotides and the 5'- and 3'-ends were excluded. The values below are the numbers of bases in the c o m m o n oligonucleotides expressed as a percentage of the total number of bases in the oligonucleotides of four or more residues. These values, however, are underestimates of the true homologies as only identical oligonucleotides were included. Some of the nucleotides m a y have changed in the excluded oligonucleotides but the others remain homologous. In some instances the relationships between changed oligonucleotides is uncertain and remaining homologies cannot be determined. However, there are four cases where a single base change has ocPlant

Broad bean

Dwarf bean

Tomato

Sunflower

Dwarf bean

80% 81% 67% 64%

81% 66% 74%

80% 77%

75%

Tomato

Sunflower Rye

348

TABLE

III

DISTRIBUTION OF T 1 RIBONUCLEASE PLANT SPECIES

DIGESTION

Oligonucleotide

T1

Broad bean

PRODUCTS

OF 5.8-S rRNA

AMONG

THE FIVE

Dwarf bean

Tomato

Sunflower

Rye

T2 T3 T4 T5

Gp C--Gp A--Gp C--C--Gp A--A--Gp

-* Q -~ • --> • -~ •

• • • • •

• • • • • •

• • • • •

• • • • •

T6 T7 T8 T9 T 10

C---C---C--Gp C--A---C--Gp (C---C--A,C--U--A)UoH C--A--A--Gp A--A--C--Gp

• --~ • • -* • -

• • •

• • _ • -

• •

• •

•

• •

TI 1 TI2 T13 TI4 T1s

C--A--A--C--Gp U--Gp A--U--Gp U--A--Gp C--C--U--Gp

-'~ • "~ • --~ O • • --~ •

• • • •

• • • • •

• • • • •

• • • • •

T16 TIT TI8 TI9 T20

C--A--U----C--Gp (A--C,C,U)Gp A--A--U--Gp A--A--A--U--Gp A--A--U--C---C--.C--Gp

~

• • • ---> •

•

• •

• •

• •

• • • •

•

• •

T21 T22

A--A--C--C--A--U---C--Gp U--U--Gp

-+ • --> 0 0

• •

• •

• 00

• •

T23 T24 T25

U----C--U--Gp A--C--U--U---C---C--Gp C----C--A--U--U~A--Gp

• -'~ • •

• •

• •

• •

•

T26 T27 T28 T29 T30

A--U--A--C--U--U--Gp (U3,C2)GP A--U--A--U--U--C--A--Gp A--A--U--U--X--A--Gp (U4,C)Gp

• •

• •

•

• •

• •

•

T31 T32 T33 T34 T35

(U2,C3)GP U--U--U~U--Gp (A--U,C 3,U)Gp (A---C,C 3 , U ) G p (C,A--U)Gp

T36 T37 T38 T39 T40

A--U--A--U--U--C--C--Gp C--A--A--A--A--U--Gp (U2,C4)GP C-"C--A--A--A--Gp A--U--A---C----C--U---Gp

"1"43 T44 3r 5t

Y--Y--Z--Gp (C,U)Gp C--C--A--C--U~No pA--U--Gp

~

• • • • • -

• • • •

• •

•

-

• •

• • •

• •

• "-> •

H

• •

• • • @

• -

•

• -

• oligonucleotide present in m o l a r quantities; • • oligonueleotide present in bimolar quantities; - , oligo. nucleotide absent; ~ oligonueleotide present in all five species; NOH, an unidentified nueleoside.

349 TABLE

IV

DISTRIBUTION OF PANCREATIC A R I B O N U C L E A S E PRODUCTS OF 5.8-S r R N A A M O N G F I V E P L A N T SPECIES •

oligonucleotide present; - , oHgonueleotide absent; ~

Oligonucleotide

Broad bean

THE

oligonucleotide present in all five species. Dwarf bean

Tomato

Sunflower

Rye

• • • • •

• • • • •

• • • • •

P1

Up

P2

Cp

'~ • --~ •

P3 P4

A---Cp A--A---Cp

-~ ~

P5

G--Cp

--~ •

• • • • •

P6 P7 P8 P9

A--Up G--A--Cp A--Cr---Cp Cr--A--A---Cp

"~ -'~ "~ "~

• • • •

• • • •

• • • •

• • • •

• • • •

P13 P14

Cr--Up G--Cr----Cp

"~ • "-~ •

• •

• •

• •

• •

P15

Cr--A--Up

"-~ •

•

•

•

•

P16

(G,A--G)Cp

P17

P18

A--A--G---Up Cr--A--A--Up

P 19

Cr--A--A--A--Up

• •

•

•

-

-

-

"> • -'> •

• •

• •

• •

• •

•

•

•

-

•

P20

A--G--A~A--Up

-'~ •

•

•

•

•

P22 P23

G--G--Up G---G---A--Up

~ • -'~ •

• •

• •

• •

• •

P24 P25

(G,A--G) Up A--A--A--Cr--Cp

~

• -

• •

• -

• -

• -

P26 P27

A--A--A--A--Up Cr--A--A--Cr--Cp

-

-

-

• •

-

P28

Cr--A--A--G--Y--Y--Zp

P29

A--Cr--A--G---Cp

P30

Xp

P31

(G2, A - - G ) C p

--~ •

•

•

•

•

-

-

•

-

-

•

•

•

"~ • .

• .

.

.

•

cuffed but the homologies remain clear; A--U--A--U--U--C--C-Gp (T3 6 ) iS replaced by A--U--A--U--U--C--A--Gp (T28) in broad bean 5.8-S rRNA, A--U--A--C- U--U--Gp (T26 ) by A--U--A--C--C--U--Gp in rye, C--Gp (T2) and A--A--A--U--Gp (TI 9 ) by C--A--A--A--A--U--Gp (T37 ) in sunflower and C--U--U--U--U--Gp (T30 ) by G--U--U--U--U--Gp (T32 ) again in sunflower. If the homologous bases in these oligonucleotides are added to those used to calculate the above values, the following percentages are obtained:

Plant

Broad bean

Dwarf bean

Tomato

Sunflower

Dwarfbean Tomato Sunflower Rye

87% 88% 83% 75%

81% 75% 79%

87% 81%

81%

350

We therefore conclude that the 5.8-S rRNA molecule has been highly conserved both amongst dicotyledons and between dicotyledons and the one monocotyledon studied. In a parallel study of the sequence of 5-S rRNA from the same five species of flowering plants [ 1 2 ] , we found a greater conservation of sequence (95--98%). The 5.8-S rRNA comprises only about 3% of the total rRNA but is processed from the same ribosomal precursor molecule as the 25-S and 18-S rRNAs [3--6]. If 25-S and 18-S rRNAs have been conserved to the same extent as the 5.8-S rRNA during the evolution of the flowering plants, then our results suggest a much greater degree of homology than do the results obtained by molecular hybridisation. References 1 2 3 4 5 6 7 8 9 10 11 12 13

Trewavas, A.J. and Gibson, I. (1968) Plant Physiol. 43, 4 45--447 Vodkin, M. and K a t t e r m a n , F.R.H. (1971) Genetics 6 9 , 4 3 5 - - 4 5 1 Leaver, C.J. and Key, J.L. (1970) J. Mol, Biol. 4 9 , 6 7 1 - 6 8 0 Rogers, M.E., Loening, U.E. and Fraser, R.S.S. (1970) J. MoL Biol. 49, 6 8 1 - 6 9 2 Udem, S.A. and Warner, J.R. (1972) J. Mol. Biol. 65, 2 2 7 - - 2 4 2 Rubin, G.M. and Sulston, J.E. (1973) J. Mol. Biol. 7 9 , 5 2 1 - - 5 3 5 Payne, P.I. and Dyer, T.A. (1972) Nat. New Biol. 235, 145--147 J o h n s t o n , F.B. and Stern, H. (1957) Nature 1 7 9 , 1 6 0 - - 1 6 1 Hastings, J.R.B. and Kirby, K.S. (1966) Blochem. J. 100, 532--539 Payne, P.I. and Dyer, T.A. (1971) Biochim. Biophys. Acta 228, 167--172 Loening, U.E. (1969) Biochem. J. 113, 131--138 Payne, P.I., Corry, M.J. and Dyer, T.A. (1973) Biochem. J. 135, 845--851 Brownlee, G.G. (1972) Determination of sequences in RNA, North-Holland publishing Co., Amsterdam 14 Payne, P.I., Woledge, J. and Corry, M.J. (1973) FEBS Lett. 35, 327--330