Molec. gen. Genet. 163, 1 - 6 (1978) © by Springer-Verlag i978

Molecular Cloning of the Gene Region Coding for the Chloroplast rRNA of Euglena gracilis * U l r i c h C. K n o p f a n d E r h a r d Stutz Laboratoire de Physiologic v6g6tale et Biochimie, Universit6 de Neuchg~tel, Chantemerle 20, CH-2000 Neuchfitel, Switzerland

Summary. E. gracilis c h l o r o p l a s t D N A

Barn fragm e n t s E a n d D, c o d i n g for r R N A were c l o n e d separately using the p l a s m i d p B R 322 as v e c t o r a n d E. coli as host. The n e w l y c o n s t r u c t e d r e c o m b i n a n t plasraids E g c K S 8 a n d E g c K S 11 ( c o n t a i n i n g the Barn H I f r a g m e n t s E a n d D respectively) were a n a l y s e d a n d c h a r a c t e r i z e d b y gel electrophoresis, e l e c t r o n m i croscopy and analytical ultracentrifugation.

The three genes are t a n d e m l y a r r a n g e d in three rep e a t i n g units o f 5,600 base pairs length each ( G r a y a n d H a l l i c k , 1978; Jenni a n d Stutz, 1978). These three genes are l o c a t e d on the l i n k e d D N A f r a g m e n t s Barn D a n d Barn E as shown in F i g u r e 1. In o r d e r to study the r D N A region in m o r e detail it seemed a d v a n t a g e o u s to clone this D N A region. In this p a p e r we r e p o r t the m o l e c u l a r c l o n i n g o f the f r a g m e n t s Barn D a n d Barn E f r o m E. gracilis c t D N A using the recently c o n s t r u c t e d p l a s m i d p B R 322 (Boliv a r et al., 1977).

Introduction The

chloroplast from

the

unicellular green a l g a with a m o l e c u l a r weight o f a b o u t 92 x 106 d a l t o n s ( a b o u t 139,000 base pairs) ( M a n n i n g a n d R i c h a r d s , 1972). E a c h c h l o r o p l a s t c o n t a i n s several c i r c u l a r D N A m o l e c u l e s which a c c o r d i n g to the results f r o m D N A r e n a t u r a t i o n kinetics studies m u s t be identical (Stutz, 1970). This D N A is t r a n s c r i b e d in vivo, e s t i m a t e s o f t r a n s c r i p t i o n r a n g i n g f r o m 12 to 26% d e p e n d i n g on the g r o w t h c o n d i t i o n s a n d d e v e l o p m e n t a l stages o f the c h l o r o plasts ( C h e l m a n d H a l l i c k , 1976; R a w s o n a n d B o e r m a , 1976). The m o s t a b u n d a n t c h l o r o p l a s t D N A t r a n s c r i p t s are the r R N A (16s, 23s, 5s) a n d it was s h o w n t h a t three r R N A genes o c c u r p e r 92 x 106 daltons (Scott, 1973; K o p e c k a , C r o u s e a n d Stutz, 1977).

Materials and Methods

E. gracilis c o n t a i n s circular D N A

* An abstract of this work was presented at the 10th annual meeting of the Union Schweizerischer Gesellschaften fur Experimentelle Biologic, Davos 19th and 20th Mai, 1978. The recommendations of the Schweizerische Akademie fiir medizinische Wissenschaften for work with recombinant DNA-molecules were respected throughout this work. For offprints contact." Prof. E. Stutz Abbreviations." Ap=Ampicillin; Tc=Tetracycline-hydrochloride; Bam HI=endonuclease isolated from Bacillus amyloliquefaciens; Eco RI =endonuclease isolated from E. coli RYI3; Bgl II =endonuclease isolated from Bacillus globiggi; EDTA=Ethylene-dia-

mine-tetra-acetic-acid; ctDNA =chloroplast DNA.

Biomalerials and Growth-Media E. coil containing the plasmid pBR 322 was a kind gift of Drs

Heineker and Boyer, Department of Biochemistry and Biophysics, University of California, San Francisco, 94143, Ca, USA to U.C.K. It was grown in medium L (containing per liter: Bacto-Difco Tryptone, 10 g; Bacto Difco Yeast Extract, 5 g; NaC15 g; 1 M Tris-HCl, pH 7.5, 10ml; 1 M MgSO4-solution, 1 ml) in the presence of Ap (Sigma Chemical Co., St. Louis, 63178, Mo, USA) and (or) Tc (Calbiochem, San Diego, 92112, Ca, USA). E. coli C600 was a kind gift of Dr. Bickel, Department of Microbiology, Biozentrum, 4000Basel, Switzerland. The Z strain (#753) of E. gracilis was obtained from the culture collection of algae, Indiana University, Department of Botany, Bloomington, Ind., USA. It was grown and harvested as published previously (Kopecka, Crouse and Stutz, 1977).

Enzymes and BufJers

Bgl II was a kind gift of Dr. T. Bickel, Department of Microbiology, Biozentrum, 4000-Basel, Switzerland. Eco RI and Bam HI were purchased from Boehriuger Biochemicals, Mannheim, B.R.D. DNA-ligase from T4 infected E. coli was purchased from Miles Laboratories Inc., Elkart, 46514, Ind., USA. Bam HI buffer (10 x concentrated): 0.2 M Tris-HC1, pH 7.9; 0.I M MgC12; 0.2 M NaCI. Eco RI buffer (10 xconcentrated): 1 M Tris-HC1, pH 7.2; 0.5 M NaC1; 0.05 M MgC12. Buffer K: 60 mM Tris-HC1, pH 8.0; 1 mM EDTA, pH 8.0.

0026-8925/78/0163/0001~01.20

U.C. K n o p f and E. Stutz: Cloning of the Gene Region Coding for the Chloroplast r R N A of Euglena gracilis

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Fig. 1. EcoRI- Bgl II cleavage map of the rDNA region of E. gracilis chloroplast D N A (Jenni and Stutz, 1978). A segment of the chloroplast genome, corresponding to the linked D N A fragments Barn -E-E-D- is serially ordered along with adjacent fragments according to Gray and HalIick (1978). Three heavy bars below the Bam HI cleavage map mark those D N A segments which carry 16s+23s r R N A cistrons. Letters and numbers refer to cleavage fragments obtained from digests with enzymes mentionned at the right side of the bars, as characterized and mapped previously (Jenni and Stutz, 1978). A repeating unit of 5600 base pairs corresponds in size to the fragment Bam E. The scale (bottom line) is in 1000 base pairs (kb)

Isolation and Purification of DNA Preparation of plasmid D N A by amplification in the presence of chloramphenicol was performed according to Clewell (1972). The D N A was purified by a slight modification of the cleared lysate technique of Guerry et al. (1973). C t D N A was isolated from purified chloroplasts (Kopecka, Crouse, and Stutz, 1977) following in a first step the D N A isolation procedure of Kolodner and Tewari (I975). In a second step the D N A preparation was dialysed against buffer K after extraction of the ethidium-bromide with 3-methyl-Ibutanol. The dialysed solution was adjusted to a density of 1.69 g/ cm 3 (at 25°C) with CsC1, and 1 gg/mI of ethidium-bromide was added. Subsequently the D N A was centrifuged to equilibrium in a Beckman type 50 rotor (Beckman model L5-65), at 39,000 rpm, 20°C for 44 h. Nuclear and c t D N A could be visualized under UV and were separated by fractioning. The ctDNA was extracted again with 3-methyl-l-butanol and dialyzed against buffer K.

Fragmentation of DNA with Restriction Enzymes and Gel-Electrophoresis

The molecules were transformed into E. coli C600 with a slightly modified method described by Cohen et al. (1972). The bacteria were subsequently plated on medium I containing ampicillin and incubated at 37°C for 18 h. Colonies growing on this medium were replica plated on Ap and Tc containing plates. Colonies which were showing A l l and Tcs were selected and analysed for the presence of recombinant plasmids. This was done by isolating the plasmids followed by agarose gel-electrophoresis.

Analytical Ultracentrifugation Analytical CsC1 density gradient experiments were done using a Beckman Model E analytical ultracentrifuge equipped with a photoelectric scanning device. The centrifugation conditions were : AnG Ti rotor, 44,000 rpm, 25°C, 18 20 h; starting density of CsC1 solution was between 1.6995 1.7000 g/cm 3.

Electronmicroscopy and Measurement of the DNA-Molecules

Digestion of D N A with Bam HI was done in Barn HI buffer, with Eco RI and Bgl II in Eco RI buffer. The reactions were done at 37°C for 1 to 5 h. The reactions were stopped as described previously (Knopf, 1977) or by a heat-treatment (see below). Electrophoresis was done as described previously (Knopf, 1977). Horizontal agarose gels (23 x 13 x0.7 cm) were used. Electrophoresis buffer was: Tris-acetate, 0.1 M, pH8.2, 12.5mM Na-acetate, 2.5 m M Na-EDTA.

This was done as described previously (Knopf, 1977) except for the measurements which were done as follows: The photographs of circular molecules were magnified 10 x with a Durst Laborator 138S enlarger, projected and drawn with a pencil on paper and subseqently measured with a curvimeter. Each molecule was measured at least 2 times. Following the measurements, the size of the recombinant-DNA molecules was compared to the size of the pBR 322 molecule which was taken as 2,6 x 106 daltons (Bolivar et al., 1977) corresponding to 3940 base pairs.

Construction of Bacterial Strains Carrying Chimeric Plasmids

Results

E. gracilis total ctDNA (7 gg) and E. coli pBR 322 plasmid D N A

Construction of chimeric Plasmids

(1 gg) were mixed and incubated with Bam HI as described above. After 4 h the restriction enzyme was inactivated by heating the mixture to 60°C for 5 min. Subsequently, the sample (100 gl) was placed at 12°C and made up to 70 m M Tris-HC1, pH 7.5, 10 m M MgC12, 70 m M ATP, 10 m M DTT. DNA-ligase (0.01 Units) was added and the reaction was continued at 12°C for about 12 h.

The plasmid pBR 322 (Bolivar et al., 1977) contains the replication element of the Col E like plasmid pMB 1 with the Ap resistance gene from pSF 2124 and the Tc resistance gene ofpSC 101. It has a single

U.C. Knopf and E. Stutz: Cloning of the Gene Region Coding for the Chloroplast rRNA of Euglenagracilis

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Fig. 2. Gel electropherogram of ctDNA, and DNA from plasmids pBR 322, pEgcKS 8 and pEgcKS 11. 2-5 ~tg DNA samples were loaded and electrophoresed for 16 h at 1 mV.cm-a, 25° C. a) pEgcKS 8; b) pEgcKS 11 ; c) Barn HI digested pEgcKS 8; d) Bam HI digested pEgcKS 11; e) pBR 322; J) Bam HI digested pBR 322; g) Bam HI digested ctDNA

Bam H I site, located within the Tca gene, thus giving the possibility for direct selection o f bacterial strains having a piece o f E. graciIis c t D N A integrated at this site, by selecting for AI~ and Tcs colonies. In this study, a b o u t 1100 A p ~ colonies were replica-plated on m e d i u m I containing A p or Tc. A m o n g these we f o u n d 32 colonies which were AI~ and Tcs and thus fullfilled our selection scheme. Isolation of the plasmids of these strains followed by gel-electrophoresis showed, that m o s t of these potential r e c o m b i n a n t plasmids did not migrate as far as the plasmid p B R 322 and thus must differ in size. In Figure 2 we display the results f r o m gel electrophoresis o f the chimeric plasmids p E g c K S 8, containing potentially the D N A f r a g m e n t Barn E, and p E g c K S 11 (panel b) containing potentially the D N A fragment Barn D before and after digestion with Barn HI. U p o n digestion the plasmid p E g c K S 8 (Fig. 2, panel a) yields two D N A fragments (panel c) which migrate equal to the c t D N A fragment Barn E (panel g) and the linear f o r m o f the Bam digested plasmid p B R 322 (panel f). Similarly, the Bam H I digested plasmid p E g c K S 11 (panel d) yields two

Fig. 3. Gel electropherogram of ctDNA and DNA from pBR 322 and pEgcKS 8 digested with various restriction enzymes. The conditions for gel-electrophoresis were given in legends to Figure 2. a) Bam HI-Eco RI digested ctDNA; b) Bam HI digested pBR 322; c) Bam HI-Eco RI digested pBR 322; d) Barn HI-Bgl II digested pBR 322; e) Barn HI-Eco RI digested pEgcKS 8; J) Bgl iI digested pEgcKS 8; g) Barn HI-Bgl II digested pEgcKS 8; h) Barn HI-Bgl II digested ctDNA. The original photograph of the gel was cut and arranged in order to have the two reference banding patterns from the digested ctDNA at the margins. All eight samples were run in the same gel. Numbers on the left margin refer to the fragments from ctDNA digests with Bam HI-Eco RI and numbers on the right side refer to fragments from ctDNA digested with Bam HI-Bgl II Eco RI (see also Fig. 1)

fragments which migrate like the c t D N A fragment Barn D (panel g) and the linear plasmid p B R 322 (panel f).

Analysis of the chimeric Plasmids with Restriction Endonucleases The vector plasmid p B R 322 carries one Eco R I cleavage site a b o u t 300 base pairs f r o m the Bam H I site (Bolivar et al., 1977). Bam H I - E c o R I digestion, therefore, yields two fragments as seen in Figure 3c. The c t D N A fragment Barn E is cleaved twice by Eco R I (see Fig. 1). As a consequence the digestion o f the chimeric plasmid p E g c K S 8 with Bam H I and Eco R I should give four bands, two bands c o r r e s p o n d i n g to the fragments B a m - E c o 15 and 24 (2400 and 800

4

U.C. Knopf and E. Stutz: Cloning of the Gene Region Coding for the Chloroplast rRNA of Euglenagracilis

base pairs in length, respectively), and two bands corresponding to the Bam-Eco fragments of pBR 322. This is indeed the case as shown in Figure 3 e (compare with banding patterns in panels a and c). The 300 base pairs piece of plasmid pBR 322 is well visible in panel c but only faintly in panel e. The vector plasmid pBR 322 has no Bgl II cleavage site. The c t D N A fragment Barn E is cleaved twice by Bgl II (Fig. 1). Thus double digestion of pEgcKS 8 with Barn HI and Bgl lI must yield four kinds of fragments (Fig. 3 g), three ctDNA fragments and the linear form of the plasmid pBR 322 (Fig. 3 d). The three c t D N A fragments are equivalent to the BamEco fragments 16, 17 and 20 of 2300, 2000 and 1300 base pairs in length, respectively (Fig. 3 h and consult Fig. 1). Further evidence that pEgcKS 8 contains the Barn E fragment was derived from the banding pattern obtained after digestion wth Bgl II, only. In this case two fragments are generated (Fig. 3 f) one corresponding to the c t D N A fragment Bam-Bgl 17, having 2000 base pairs, which is equivalent to Bgl Q and a long chimeric D N A molecule of about 7500 base pairs, composed o f p B R 322 and the c t D N A Bam-Eco 16 and 20 of 2100 and 1100 base p a i r s in length, respectively. A similar analysis with pEgcKS 11 gave the results shown in Figure 4. Upon double digestion with Bam HI and Bgl II four D N A fragments are generated (Fig. 4b). Three fragments correspond to the c t D N A fragments Bam-Bgl 14, 16 and 17 with 2700, 2300, 2000 base pairs, respectively, and the top band corresponds to the linear form of the plasmid pBR 322. If pEgcKS 11 is double digested using Barn HI and Eco RI three bands corresponding to the ctDNA fragments Bam-Eco 15 and 16 having 2400 and 2100 base pairs can be seen in the gel (Fig. 4 d) as predicted from the cleavage map (Fig. 1). The top band is the shortened D N A fragment of pBR 322. The cut off 300 base pair piece cannot be seen on the bottom of this gel. If finally, digestion is done with Bgl II only the c t D N A fragment Bam-Bgl 17 with 2000 base pairs is produced along with a long chimeric D N A piece of about 8900 base pairs (faintly visible) composed of the pBR 322 genome and the two attached c t D N A fragments Bam-Bgl 14 and 17 with 2700 and 2000 base pairs respectively (Fig. 4c).

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Fig. 4. Gel electropherogram from ctDNA and DNA from pBR 322 and pEgcKS I1 digested with various restriction enzymes. The conditions for gel electrophoresiswere as given in the legend to Figure 2. a) Bam HI-Bgl II digested ctDNA; b) Bam HI-Bgl II digested pEgcKS 11; c) Bgl II digested pEgcKS 11; cO Barn HI-Eco RI digested pEgcKS 11. Numbers on the left margin refer to ctDNA fragments from Bam HI- Bgl II digests and on the right margin to ctDNA fragments from Barn HI-Eco RI digests (the corresponding reference banding pattern is not shown). The bands 15 and 16 in panel a are not clearly resolved in this gel. As shown previously, using gels of lower agarose concentrations (Jenni and Stutz, 1978) the respective DNA fragments differ in length by about 500 base pairs

8 molecules was 6.2 x 10 6 daltons (+_ 15 %), equivalent to 9400 base pairs. The average size of 24 monomers ofpEgeKs 11 molecules was 7.1 x 10 6 'daltons (_+ 11%) or about 10,700 base pairs. From gel-electrophoretic analysis we estimated the respective sizes as 9500 and 10,800 base pairs. Analysis by electron-microscopy showed that both samples contained, in addition to the monomers, also oligomers.

Buoyant Density Determinations Analysis by Electron-Microscopy Further evidence for integration of the c t D N A fragments Barn D and E into the vector plasmid is provided by electron-microscopic studies. The average size of twelve circular relaxed monomers of pEgcKS

We showed previously (Stutz and Vandrey, 1971) that the chloroplast r R N A cistrons were located in a D N A region with a relatively high G + C content of 41%, being well above the average G + C content of 21% of total ctDNA. We expected, therefore, that the

U.C. Knopf and E. Stutz: Cloning of the Gene Region Coding for the Chloroplast rRNA of Euglena gracilis

5

Discussion

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Fig. 5. Tracings (absorbance at 265 nm) of analytical CsCI density gradients from DNA samples digested with Barn HI restriction endonuclease. A) ctDNA; B) pBR 322; C) pEgcKS 8; D) pEgcKS 1I. Micrococcus lysodeikticus DNA (density, 1.731 g/cm3) was used as density marker.

D N A fragments Barn D and E would have buoyant densities of about 1.700 g/cm 3 in neutral CcC1. This is the case as shown in the respective density profiles C and D in Figure 5. F o r comparative reasons we also included the density profiles fi'om Bam H I digested total c t D N A (profile A) and plasmid p B R 322 (profile B). In profile A the major peak ( d = 1.685 g/cm 3) corresponds to bulk ctDNA. While the minor peak (shoulder) correlates in terms of buoyant density with the density of the isolated D N A fragments Bam D and Barn E. Using expanded scannings of the density profiles C and D, we determined the areas below the peaks. The area ratios between the two peaks with densities of 1.700 and 1.713 g/cm 3 in profiles C and D are 1.3 and 1.9, respectively. The ratios of the respective molecular weights are 1.4 and 1.8. Such a good coincidence is further evidence that the two chimeric plasmids contain the c t D N A fragments Barn E and Barn D.

The plasmid p B R 322 is a highly suitable vector for the molecular cloning of fragments from E. gracilis c t D N A . The screening procedure for recombinants is straight-forward, the vector plasmid is a relatively small and an easely handled D N A molecule. The relaxed replication of the chimeric plasmids allows the production of relatively large amounts of D N A in small cell batches. The chimeric plasmid pEgcKS 8 contains a D N A fragment corresponding to an entire 5600 base pairs repeating unit containing one r R N A gene set (Jenni and Stutz, 1978; Gray and Hallick, 1978). F r o m restriction endonucleases cleavage sites mapping studies done in this and other laboratories (e.g.J.R.Y. Rawson, personal communications) it looks as if the two contiguous Bam E fragments (Fig. 1) have identical base sequences. However, only by complete sequence analysis and using cloned Bam E fragments from several chimeric plasmids we will get a definite answer to this problem. The plasmid pEgcKS 11 contains in addition to the 5600 base pairs of repetitious D N A , additional 1300 base pairs (see Fig. 1) which according to Jenni and Stutz (1978) do not hybridize with 16+23s r R N A . This segment has an average G + C content of about 27%. This value can be calculated considering that the entire Barn D fragment has a G + C content of about 41%, while the segment coding for the r R N A has a G + C content of about 47% (Crouse, Vandrey and Stutz, I974) and makes up 70% of the size of the fragment Bam D. By similar reasoning we estimate that the c t D N A fragment Bam-Eco 24 which is part of the fragment Bam E (Fig. 1) has a G + C content of only 8%. These D N A stretches adjacent to ribosomal r R N A cistrons are, therefore, AT-rich and might have important control functions. Acknowledgements. This research was supported by grants No. 75NG03 to U.C.K. and 3.687.76 to E.S. from the Swiss National Science Foundation and a special gift from the Sandoz Foundation for the Advancement of Medical and Biological Sciences. The analytical ultracentrifuge was kindly made available by the Institute of Molecular Biology, University of Geneva, Geneva, Switzerland.

References

Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W., Crosa, J.H., Falkow, S. : Construction and characterization of new cloning vehicles, II. A multiple cloning system. Gene 2, 95-113 (1977) Chelm, B.K., Hallick, R.B. : Changes in the expression of the chloroplast genome of E. gracilis during chloroplast development. Biochemistry (Wash.) 15, 593-599 (1976)

6

U.C. Knopf and E. Stutz: Cloning of the Gene Region Coding for the Chloroplast rRNA of Euglena gracilis

Clewell, D.B. : Nature of Col-E1 plasmid replication in Eschericia coli in the presence of chloramphenicol. J. Bact. 110, 667 (1972) Cohen, S.N., Chang, A.C.Y., Hsu, L.: Non-chromosomal antibiotic resistance in bacteria: genetic transformation of E. coli by R-factor DNA. Proc. nat. Acad. Sci. (Wash.) 69, 2110 2114 (1972) Crouse, E., Vandrey, J.P., Stutz, E. : Comparative analysis of chloroplast and mitochondrial DNAs from E. gracilis. In : Proceedings of the third International Congress on Photosynthesis, Israel (Avron, M., ed.), pp. 1775-1786. Amsterdam: Elsevier 1974 Gray, P.W., Hallick, R.B. : Restriction endonuclease map of E. gracilis chloroplast DNA. Biochemistry (Wash.) 16, 1665 1671 (1977) Gray, P.W., Hallick, R.B. : Physical mapping of the E. gracilis chloroplast DNA and ribosomal RNA gene region. Biochemistry (Wash.) 17, 284-289 (1978) Guerry, P., Le Blanc, D.J., Falkow, S.: General method for the isolation of plasmid deoxyribonucleic acid. J. Bact. 116, 1064 1066 (1973) Jenni, B., Stutz, E.: Physical mapping of the ribosomal DNA region of E. gracilis chloroplast DNA. Europ. J. Biochem. 1978 in press Knopf, U.C. : Studies on the bacteriophage PS8 of Agrobacterium tumefaciens (Smith and Townsend) Conn: physico-chemical properties of its DNA. Microbios 17, 231~37 (1976)

Kolodner, R., Tewari, K.K. : The molecular size and conformation of the chloroplast DNA from higher plants. Biochim. biophys. Acta (Amst.) 402, 372 390 (1975) Kopecka, H., Crouse, E.J., Stutz, E.: The E. gracilis chloroplast genome: Analysis by restriction enzymes. Europ. J. Biochem. 72, 525 535 (1977) Manning, J.E., Richards, O.C.: Isolation and molecular weight of circular chloroplast DNA from E. gracilis. Biochim. biophys. Acta (Amst.) 259, 285-296 (i972) Rawson, J.R.Y., Boerma, C.L.: A measurement of the fraction of chloroplast DNA transcribed during chloroplast development in E. gracilis. Biochemistry (Wash.) 15, 588-592 (1976) Scott, N.S. : Ribosomal RNA cistrons in E. gracilis. J. molec. Biol. 81, 327 336 (1973) Stutz, E. : The kinetic complexity of E. gracilis chloroplast DNA. FEBS Letters 8, 25-28 (1970) Stutz, E., Vandrey, J.P.: Ribosomal DNA satellite of E. gracilis chloroplast DNA. FEBS Letters 17, 277-280 (1971)

Communicated

by H. Boyer

Received February 28, 1978