Current Genetics

Current Genetics (1984) 8 : 379-385

© Springer-Verlag 1984

Transfer RNAs and tRNA genes of

Viciafaba chloroplasts

M. Mubumbila, E. J. Crouse, and J. H. Weft

Institut de Biologic Mol6culaire et Cellukaire du CNRS, Universit6 Louis Pasteur, 15 rue Descartes, F-67084 Strasbourg Cedex, France

Summary. Isolated chloroplasts from broad bean and common bean were found to contain a minimum of 31 and 32 tRNA species, respectively. These individual chloroplast tRNAs were 32p-labeled in vitro and hybridized to DNA fragments obtained upon digestion of broad bean and common bean chloroplast DNAs with various restriction endonucleases. At least 30 tRNA genes were localized on the physical maps of the two chloroplast genomes. Comparison of the broad bean tRNA gene map to that of common bean revealed DNA sequence rearrangements, such as inversions, insertions/deletions and duplications, within these two members of the Leguminosae family. Key words: Chloroplast tRNAs - tRNA/DNA hybridizations - tRNA gene mapping - DNA sequence rearrangements and duplications

Introduction

Chloroplasts from higher plants have been shown to contain multiple copies of a circular DNA molecule which varies in size from 120 to 182 kilobase pairs (kbp) depending on the plant species (for recent reviews, see Bohnert et al. 1982; Buetow 1982; Whitfeld and Bottomley 1983). Analysis of more than 20 higher plant chloroplast DNAs (cpDNAs) by electron microscopy and/or restriction endonuclease cleavage revealed that most of these plastid genomes contain a large region, ranging from 14 to 28.5 kbp, which is present twice, in an inverted orientation. Each repeated region contains a set of rRNA genes. Exceptions to this general organization of chloroplast genomes have been reported for three of

Offprint requests to: M. Mubumbila

the six analyzed members of the leguminous family. In contrast to larger cpDNAs (ca. 150 kbp) of mung bean (Vigna radiata), soybean (Glycine max) and common bean (Phaseolus vulgaris), the smaller cpDNAs (ca. 120 kbp) of broad bean (Vicia faba), pea (Pisum sativum) and chickpea (Cicer arietinum) do not contain any detectable repeated regions and have only one set of rRNA genes (Koller and Delius 1980; Chu et al. 1981 ; Palmer and Thompson 1981, 1982; Chu and Tewari 1982; Ko et al. 1983; Mubumbila et al. 1983; Palmer et al. 1983; Spielmann et al. 1983). Because of these differences in the size of the genomes and the number of rRNA genes between the Phaseolae and Viciae tribes of the legumes, a more detailed comparison of the chloroplast genomes and their gene products is particularly interesting. At the present time, it is not known if the smaller chloroplast genomes encode less genetic information than the larger ones. Physical maps, showing the location of the tRNA genes, have been reported for the higher plant cpDNAs of spinach (Bohnert et al. 1979; Driesel et al. 1979)Spirodela (Groot and van Harten-Loosbroek 1981), common bean (Mubumbila et al. 1983), maize (Selden et al. 1983) and tobacco (Bergmann et al. 1984). In this report, the number of broad bean chloroplast tRNAs and the location of the tRNA genes on the physical map of broad bean cpDNA are presented and compared to the corresponding data of other higher plants, especially to those of common bean.

Materials and methods Chloroplast tRNAs from broad bean (Vicia faba cv. dreifachweisse) and common bean (Phaseolus vulgaris var. Saxa) were isolated and fractionated by two-dimensional gel electrophoresis, visualized by methylene blue staining, recovered from the gel and

380

M. Mubumbila et al. : Chloroplast genes for Vicia faba tRNAs

PheOThr+Pr° ( ~ O Trp AlaV (:~}B

GIyf"~L" '1 Lys,,,.~ ~ /van

His

O Leul 0~2

OLeu2 (~Leu3

oTYr

-"~

U ( ~ M e t 1 .... C Val2 /'~Asn !'" Met2

(-J~. D " ' O i'..!

E ~

F p ~ A s p l +Argl

..'"'G a

1

ld,m

:...".)Leu1

Fig. la, b. Fractionation of total tRNAs from chloroplasts of a Vicia faba and b Phaseolus vulgaris by two-dimensional polyacrylamide gel electrophoresis. Diagrammatic representation of methylene blue stained gels after electrophoretic separation of chloroplast tRNAs in a 10% polyacrylamide (4 M urea) gel for 40 h at 450 V in the first dimension and 20% polyacrylamide (4 M urea) gel for 140 h at 350 V in the second dimension. The diagrams indicate the amino acid accepted by each identified tRNA, as revealed by aminoacylation of the tRNA after extraction from the gel. Weak spots are indicated by dotted circles. Capital letters correspond to unidentified "RNAs. Note that in several cases two or more broad bean tRNAs comigrate whereas the corresponding common bean tRNAs are much better separated

OSerl

',....

G ThrP~) ~,~{") Leu2 PhelpdY--~Phe2/'% Met.~Trd 1 P.'>H ~ [ Argl Q HisOV~keu3 Gtylf~ ~_Asnl U Ala ~..,¢ "" .j ".... Gly2 J Va 1 Asn2

Arg2 O K

O

(i.:'~

Ser2 ~

O

":~:

0 Met2

O llel

o0

Val2

0 Lys

..-"...C

g

Ser 3

O

.

lle2

b

identified by aminoacylation using E. coli aminoacyl-tRNA synthetases and 3H-amino acids as described by Burkard et al. (1982). Both total chloroplast tRNAs and individual, identified, tRNA species were treated with snake venom phosphodiesterase (Worthington, Freehold N J) to remove the terminal nucleotide(s) of the -CCA end, reextracted with phenol-chluroform, and enzymaticaUy labeled at the 3' end (Silberklang et al. 1977) in the presence of [a-32p]ATP, CTP and yeast tRNA nucleotidyl transferase (Rether et al. 1974). Broad bean and common bean chloroplast DNAs were isolated according to the methods described either by Bohnert and Crouse (1981) or by Koller and Delius (1980) and cleaved by the restriction endonucleases SalI, PstI, KpnI, Sinai or XhoI. DNA fragments were separated by horizontal agarose gel electroph0resis, stained with ethidium bromide, photographed under ultraviolet light (Polaroid 665 film), transferred from tlle gels to nitrocellulose filters, and hybridized with individual, labeled, tRNAs as described by Driesel et al. (1979) and by Mubumbila et al. (1983).

Results Fractionation and identification o f broad bean chloroplast t R N A s

T o t a l t R N A s were e x t r a c t e d f r o m i s o l a t e d c h l o r o p l a s t s o f b r o a d bean, labeled in v i t r o b y t h e y e a s t t R N A n u c l e o t i d y l transferase r e a c t i o n , w h i c h is specific f o r tRNAs, and fractionated by two-dimensional polyacryla m i d e gel e l e c t r o p h o r e s i s . T h e m e t h y l e n e blue s t a i n e d gel revealed t h e p r e s e n c e o f a t least 26 spots, o f w h i c h all b u t t h e slowest, s p o t A in Fig. la, were s h o w n b y autoradiography to contain tRNAs. After fractionation of unlabeled chloroplast tRNAs a n d e x t r a c t i o n o f t h e R N A species f r o m t h e gel, a t o t a l

381

M. Mubumbila et al. : Chloroplast genes for Viciafaba tRNAs Table 1. Identified chloroplast tRNAs and tRNA genes of broad bean and common bean tRNA specific for

Ala Arg Asn Asp Cys Glu Gin Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

No. of isoacceptors identified

No. of isoacceptors hybridized to cpDNA

No. of genes mapped

Broad bean

C o m m o n bean

Broad bean

C o m m o n bean

Broad bean

C o m m o n bean

1 2 1 2 . . . 1 1 2 3 1 2 1 1 2 1 1 1 2

1 2 2 -

1 2 1 2

1 1 2 -

1 2 to 4 1 1 to 3

2 2 to 3 2 to 4 -

2 to 4 1 2 5 1 2 to 3 1 2 2 to3 2 1 2 2 to 3

1 or 2 1 4 4 1 1 to 2 2 2 3 2 1 1 2 to 3

30 to 38

31 to 37

25

. . .

. . .

. . .

. . .

. . .

2 1 2 3 1 2 2 1 3 1 1 1 2

2 1 2 3 1 1 1 1 2 1 1 1 1

2 1 2 3 1 1 2 1 3 1 1 1 1

27

24

24

of 25 tRNA species, corresponding to 17 amino acids, were identified by aminoacylation using E. coli aminoacyl-tRNA synthetases and 3H-amino acids (Table 1). These 25 tRNA species are located in 19 regions of the gel (see Fig. 1a). The only amino acids for which tRNAs have not been identified are cysteine, glutamine and glutamic acid. Single tRNA species have been identified for alanine, asparagine, glycine, histidine, lysine, phenylalanine, proline, threonine, tryptophan and tyrosine, and isoaccepting species have been revealed for seven other amino acids: two each for arginine, aspartic acid, isoleucine, methionine, serine and valine, and three for leucine. Six tRNAs (B to G in Fig. la), which are tRNAs because they were labeled by yeast tRNA nucleotidyl transferase, could not be aminoacylated. These RNAs might consist of precursor tRNA molecules, partially degraded tRNA molecules, and/or the unidentified tRNAs specific for cysteine, glutamine and glutamic acid. Not all broad bean chloroplast tRNAs were fractionated to homogeneity by two-dimensional polyacrylamide gel electrophoresis. Several RNA spots accepted more than one amino acid, showing that two or more tRNAs have comigrated and could not be separated by this technique. In particular, tRNA~.° comigrated with tRNAThr; tRNA1Asp with tRNA1Arg, tRNA2Asp with tRNA2ne; and tRNAxta with tRNAGly,tRNAxMet ;tRNAYal and tRNA~(al (see Fig. la).

The two-dimensional gel electrophoretic fractionation of common bean chloroplast tRNAs gave a better resolution and revealed 27 tRNA species, specific for 16 amino acids (Table 1), and 5 unidentified tRNAs (G to K in Fig. lb) which can be labeled by the yeast tRNA nucleotidyl transferase reaction. These unidentified tRNAs probably include tRNA species specific for aspartic acid, cysteine, glutamine and glutamic acid, which were not identified by aminoacylation.

Hybridization of tRNAs to cpDNA fragments To determine which DNA fragments contain tRNA genes, individual broad bean chloroplast tRNA species were labeled in vitro and hybridized to filter-immobilized broad bean and common bean cpDNA fragments obtained by restriction endonuclease cleavage. Furthermore, in order to map the genes for the above-mentioned comigrating tRNAs from broad bean, heterologous hybridizations between broad bean cpDNA fragments and individual, identified, chloroplast tRNAs from common bean, maize or spinach were performed. Table 2 summarizes the homologous and the heterologous hybridization results. In addition to the identified tRNAs, two unidentified tRNAs (tRNA xB and tRNA xE) from broad bean chloroplasts also hybridized to broad bean cpDNA.

382

M. Mubumbila et al. : Chloroplast genes for Vicia faba tRNAs

Table 2. Summary of hybridization results of individual chloroplast tRNAs to broad bean and common bean chloroplast DNA fragments obtained by restriction endonuelease cleavage Broad bean chloroplast tRNA specific for

Gene symbol

Alab Argl Axg2b Asn Aspl Asp2 Glyl b Gly2b His Ilel b Ile2b Leul b Leu2b Leu3b Lysb Met2 Phe Prob Set1b Set2b Set3b Thrb Trp Tyrb Val2b XBc XE Xd

trnA

a b c d e f g

trnR1 trnR2 trnN trnD1

trnD2 trnG1

trnG2 trn H trnI1 trnI2 trnL1 e trnL2f trnL3

trnK trnM2 trnF f

trnP trnS1 trnS2 trnS3 trnT g trnW trnY g

trnV2 trnX1 trnX2 trnX3

Broad bean cpDNA fragmentsa

Common bean cpDNA fragmentsa

Sail

Kpnl

PstI

XhoI

S3b Sla, S3a, $4 S3b S3b Sla, S3a, $4 Sla, S3a Sla, S3b Sla, S3b S1b S3b S3b Sla, S3a Sla, S3a S3a Slb Slb Slb Sla, Slb Slb $2 Slb S/a, Slb $2 Sla, Slb S3b Sla, S3b $4 Slb, $2, S3b

K2 K4, K6, K7a K2 K3 K4, K6, K7a K4, K7a K2, K3 K2, K3 K5 K2 K2 K7a, K10, K11 K7a, K10 K7a K5 K5 K7a K4, K5 K7a K8 K7a K4, K7a K1 K4, K5 K2 K2, K7a K10 K2, K3, K6, K9

P2, P5 P2, P5 P2, P5 P10 P10 P9 P3, P4 P2, P5 P7 P3, P5 P8 P9 P7 P7 P1, P6 P10 P1 P7 P1, P6 P10 P6 P2, P5 -

X9 X3, X5 X3, X5 X2 X2 X4 X6 X9 X2 X4 X5 X4 X4 X2 X1, X2 X2 X1 X2 X1, X2 X2 X2 X4 -

Fragment nomenclature is based on relative size as observed by migration in agarose gels Results confirmed by hybridization using common bean, maize or spinach tRNA isoacceptor Probably contains at least two tRNAs which comigrate in the polyacrylamide gel Contains a mixture of tRNAs including tRNAAla, tRNA1Met, tRNAy al, tRNA2Val and tRNA Gly Confirmed by sequencing data (Bonnard et al. 1984) Confirmed by sequencing data (Bonnard, Weil and Steinmetz, pers. comm.) Confirmed by sequencing data (Kuntz, Weil and Steinmetz, pets. comm.)

The location of the tRNA genes on the physical map of broad bean cpDNA is shown in Fig. 2a. The tRNA genes are distributed around the circular DNA molecule; however, in some cases, several genes are located on the same cpDNA fragment. A m i n i m u m of 9 tRNA genes (trnA, trnI1, trnI2, trnG1, trnG2, t r n V 2 , t r n N , trnR2 and trnX1) are encoded in the Sal fragment S3b which is k n o w n to contain the rRNA genes. Within another DNA fragment, Kpn fragment K7a, at least 9 tRNA genes are also present (namely, trnF, trnS1, trnS3, trnT, trnL1, trnL2, trnL3, trnD1 and/or trnR1, and trnX1). A minim u m of 4 tRNA genes (trnD1 and/or trnR1, trnP, trnT and trnY) are locatedin Kpn fragment K4. Kpn fragment K5 contains trnH, trnK, trnM2, trnY and trnP, whereas Kpn fragment K10 carries trnL2 and trnX2. Because t R N A Leu hybridized to Kpn fragments K10 and K l l

(Table 2), Kpn fragment K l l , which had not been clearly mapped (Ko et al. 1983), must be between Kpn fragments K4 and K10, as predicted by Bohnert (unpublished mapping data). Two comigrating tRNAs of broad bean, tRNA1Asp and tRNA Arg (Fig. la), which were shown to hybridize to Kpn fragments K4 and K7a (see above) also hybridized to Kpn fragment K6 (Table 2), so that the corresponding gene(s), trnD1 and/or trnR1, may be present three times in the broad bean chloroplast genome. Another comigrating group of tRNAs, which contains tRNA Ala, tRNA Gly, tRNA1Met, tRNAy al and tRNA~( al (Fig. la) and is called tRNA x (Table 2), hybridized to four Kpn fragments (K2, K3, K6 and K9), indicating that trnM1 and t r n V 1 are possibly located on Kpn fragments K6 and/or K9 because trnG, trnV2 and trnA are mapped on the adjacent Kpn fragments K2

M. Mubumbila et al. : Chloroplast genes for Viciafaba tRNAs (trnM1) (trnV1)

383 and K3. The remainder of the tRNA genes, trnS2 and trnW, are located on Kpn fragments K8 and K1, respectively.

Discussion

Comparison o f broad bean and common bean chloroplast tRNAs Although individual chloroplast tRNAs, specific for all 20 amino acids, have not yet been isolated and identified from broad bean or common bean, the chloroplasts of another legume, pea, are known to contain at least one tRNA for each of the 20 amino acids (Meeker and Tewari 1980). In the present study, at least 31 tRNA species have been fractionated from broad bean chloroplasts. Of these tRNAs, 25 were identified by aminoacylation and found to be specific for 17 amino acids. The tRNAs for the remaining three amino acids (cysteine, glutamic acid and glutamine) are probably among the 6 unidentified tRNAs. The failure to identify some tRNA species may be due either to the inactivation of the tRNAs during gel electrophoresis (performed in the presence of 4 M urea) so that they can no longer be aminoacylated, or to the inactivation of the corresponding aminoacyl-tRNA synthetases. Similar problems have been encountered in a study on tRNAs and aminoacyl-tRNA synthetases of pea chloroplasts (Meeker and Tewari 1980). From c o m m o n bean chloroplasts, 27 tRNAs, specific for 16 amino acids, and 5 unidentified tRNAs were reported previously (Mubumbila et al. 1980, 1983). The total number of tRNA species found in the chloroplasts of broad bean and common bean is about the same (at least 31 and 32, respectively) and is approximately the minimum number expected, according to the wobble hypothesis (Crick 1966), to read all 61 sense codons. The actual number of chloroplast tRNA genes probably exceeds this number o f t R N A species. Meeker and Tewari (1980, 1982) reported from tRNA/DNA saturationhybridization studies that both pea and spinach cpDNAs contain about 40 tRNA genes. In our preparations of broad bean and common bean tRNAs, additional isoaccepting tRNA species for some amino acids may be present in the polyacrylamide gel of the fractionated

Fig. 2a, b. Location of tRNA genes on the restriction endonuclease cleavage site map of a Vicia faba and b Phaseolus vulgaris cpDNAs. The smallest DNA segment to which hybridization was observed is indicated for each chloroplast tRNA tested, o, Sail cleavage site; v, Psti cleavage site; v, KpnI cleavage site; e, Sinai cleavage site; *, Xhol cleavage site. For tRNA gene nomeclature, see Table 2, second column. The positions of the cleavage sites on the broad bean chloroplast genome (a) are based on data from Koller and Delins (1980), Palmer and Thompson (1982),

Ko et al. (1983) and Bohnert (personal communication). Only one of the two populations of common bean cpDNA (b) is shown (see Mubumbila et al. 1983; Palmer 1983). The two populations differ in the orientation of the small single-copy region relative to the large single-copy region. The blackened segments in the common bean cpDNA map corresponds to the inverted repeat regions, each of which contains a rDNA unit (rrnA and rrnB, respectively)

384 tRNAs, either comigrating with another isoacceptor or present in an amount below the level of detection. Comparison of the sequenced protein genes which are encoded in the chloroplast genome (reviewed by Bohnert et al. 1982; Whitfeld and Bottomley 1983; Crouse et al. 1984) revealed a bias in codon usage. The various isoacceptors specific for one given amino acid are present in different amounts, as observed in the intensity of the various spots in the polyacrylamide gels. The electrophoretic migration pattern of broad bean chloroplast tRNAs in a two-dimensional polyacrylamide gel shows many similarities to that of common bean chloroplast tRNAs. Although more RNA spots were observed in the gel of common bean chloroplast tRNAs (Fig. lb), as compared to broad bean chloroplast tRNAs (Fig. la), the total number of identified and unidentified tRNAs in each case is approximately the same (Table 1 and cf. Fig. la to lb). This is due to the fact that several broad bean tRNAs (e.g., tRNA Ala, tRNA Gly, tRNA Met, tRNA val and tRNA~(al) comigrate in the polyacrylamide gel, whereas in common bean a better, but not always a complete, separation was obtained.

Comparison of the chloroplast tRNA gene maps of broad bean and common bean The broad bean chloroplast genome is known to contain one gene for each of the 16S, 23S, 4.5S and 5S species of rRNAs. The 4.5S and 5S rRNAs have been sequenced (Bowman and Dyer 1979; Dyer and Bowman 1979). All four rRNA genes are located within one region (less than 9 kbp) of the cpDNA molecule (Delius and Koller 1980; Sugiura 1980; Sun et al. 1982, Ko et al. 1983). In the present study, a minimum of 30 tRNA genes have been located on the map of broad bean cpDNA (a figure similar to the number of tRNA genes found for common bean cpDNA, which is somewhat larger). This was accomplished by combining the results of homologous and heterologous hybridizations (using individual broad bean and common bean chloroplast tRNAs) to broad bean cpDNA fragments. Common bean chloroplast tRNAs were used because 11 broad bean chloroplast tRNAs (specific for Thr, Pro, Ala, Gly, Vall, Val2, Metl, Aspl, Argl, Asp2 and Ile2) couldnot be purified to homogeneity by two-dimensional polyacrylamide gel electrophoresis. The use of common bean tRNAs in heterologous hybridization experiments to broad bean cpDNAs was justified, as both broad bean and common bean chloroplast tRNAs hybridized to the same fragments of common bean cpDNA. Comparison of the broad bean and common bean chloroplast tRNA gene maps (cf. Fig. 2a to 2b) reveals that several groups of tRNA genes are common to both cpDNAs: e.g., trnA and trnI2, trnP, trnT and trnY;

M. Mubumbila et al. : Chloroplast genes for Viciafaba tRNAs trnH and trnK; trnL1, trnF, trnS3 and trnM2. However, there are some differences between the. two maps: For instance, in common bean trnL1, trnL2 and trnL3 are located in the large signle-copy region, the repeated regions and the small single-copy region, respectively, whereas in broad bean these three genes are located on the small Kpn fragment K7a (Table 2;cf. Fig. 2a to 2b). Furthermore, broad bean contains another DNA region (the adjacent Kpn fragments K11 and K10) which also encodes trnL1 and trnL2 (respectively). There are other sets of tRNA genes which appear to be present twice on the broad bean chloroplast genome: trnG1 and trnG2 are found on Kpn fragments K2 and K3 (whereas in common bean these two genes are both found only on Pst fragment P10) and trnY is found close to trnP and trnT on Kpn fragments K4 and K5 (whereas in common bean, trnY is found close to only one of the two sets of trnP and trnT). The serial order of corresponding DNA segments in chloroplast genomes of different plants has been compared by heterologous DNA-DNA hybridization. Whereas this order was similar among species of the Solanaceae family (Fluhr and Edelman 1981), between tobacco and spinach (Fluhr and Edelman 1981)and amongAtriplex, cucumber and spinach (Palmer 1982), extensive rearrangements have been reported in some, but not all, members of the Leguminosae family. Rearrangements have been observed when either pea or broad bean cpDNAs was compared to mung bean cpDNA and when pea cpDNA was compared directly to broad bean cpDNA (Palmer and Thompson 1981; 1982). On the other hand, the chloroplast genomes of mung bean, soybean and common bean, each of which has the rRNA-encoding inverted repeat regions, are essentially identical in linear sequence organization, except for some small insertions/deletions (Palmer et al. 1983). In our study of broad bean and common bean cpDNAs, chloroplast tRNAs from both species were used as probes to compare the organization of tRNA genes. The use of tRNAs as probes revealed finer details of the serial order of homologous sequences. The overall organization of these genomes is consistant with data on DNA sequence rearrangements presented by Palmer and Thompson (1981, 1982), but duplication of some broad bean cpDNA sequences encoding tRNA genes, in addition to rearrangements of tRNA genes, has been observed (Fig. 2a). Insertions/deletions within chloroplast genomes is not unique to the legumes. For example, in the genus Oenothera, the physical maps of the five genetically distinct cpDNA types have been shown to be colinear, although variations in size of some homologous cpDNA fragments were observed (Gordon et al. 1982). It is interesting to note that insertions/deletions are frequently observed in cpDNAs near the junction of the large singlecopy region and the inverted repeat regions. These junc-

385

M. Mubumbila et al. : Chloroplast genes for Viciafaba tRNAs tions appear to be more subject to changes than the remainder of the genome. In fact, comparison of the junctions of maize (Selden et al. 1983) and wheat (Mubumbila, unpublished results) to those of spinach (Dfiesel et al. 1979), tobacco (Bergmann et al. 1984) and soybean (Spielmann et al. 1983) reveals the loss of one of the two trnH genes in the three latter cpDNAs. In both broad bean and common bean, there is only one trnH present in a fragment which also carries the trnK. These two tRNA genes are also present on the same DNA fragment which hes at the junction in tobacco, wheat and maize. This suggests that the event, which caused the disappearance (or the appearance) of the second copy of the inverted repeat region, took place in the vicinity of this trnH/trnK-containing fragment. This region seems to have undergone other rearrangements, since in common bean trnF1 and trnM2 are close to trnH and trnK, while in broad bean trnT, trnP and trnY seem to have been inserted in this region. It should be pointed out that in broad bean the rDNA unit is flanked by a trnV2 gene next to the 16S rRNA gene and by a trnN gene next to the 23S rRNA gene, and that this organization is found in the two rDNA units present in the cpDNAs which have inverted repeats, namely comm o n bean, maize and tobacco (Mubumbila et al. 1983; Selden et al. 1983; Bergmann et al. 1984). Therefore the rearrangements have occurred outside a "maintained" segment containing trnV2, 16S rDNA, trnl2, trnA, 23S rDNA, 4.5S rDNA, 5S rDNA and trnN.

Acknowledgements. The authors thank Dr. H. J. Bohnert (University of Arizona, Tucson) for supplying us with his unpublished physical map of broad bean cpDNA; Drs. J. D. Palmer (Duke University, Durham), A. Steinmetz (I.B.M.C., Strasbourg) and B. Koller (EMBL, Heidelberg) for stimulating discussions and helpful suggestions; Dr. R. Gi6g~ and P. Romby (I.B.M.C., Strasbourg) for the gift of yeast tRNA nucleotidyl transferase and Ms. C. Schaeffer for help in the preparation of this manuscript.

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Communicated by H. K6ssel Received March 15, 1984