Current Genetics

Current Genetics (1983)7:265-272

© Springer-Verlag 1983

In vivo Transcriptional Products of the Chloroplast DNA of Euglena gracilis Kim P. Dix 1 and James R. Y. Rawson 2 1 Department of Molecular and Population Genetics, 2 Department of Botany, University of Georgia, Athens, GA 30602, USA

Abstract. Cloned chloroplast restriction endonuclease DNA fragments were used as hybridization probes to identify the in vivo transcriptional products of the chloroplast genome from the alga Euglena gracilis. Total cellular RNA was size fractionated by electrophoresis in denaturing gels and transferred to nitrocellulose paper. Individual plasmids containing specific chloroplast DNA fragments were radioactively labeled in vitro and hybridized to the immobilized RNA. The stable RNAs in the chloroplast were identified on the basis of their size and their origin on the chloroplast genome. Several transcripts were shown to be developmentally expressed. Some transcripts showed a possible precursor-product relationship. The rDNA was shown to be transcribed as a large transcript and then processed to t h e mature rRNAs. Key words: Euglena - Chloroplast transcripts

certain regions of the chloroplast DNA is developmentaily regulated. The purpose of the following experiments was to determine the size of the large stable RNAs which were transcribed from specific restriction endonuclease DNA fragments of the Euglena chloroplast genome and to determine whether there might be a precursor-product relationship between any of the these RNAs. The design of these experiments was to use radioactively labeled recombinant plasmids containing chloroplast restriction endonuclease DNA fragments as hybridization probes to identify complementary RNAs that had been separated on the basis of their molecular weights in denaturing gels.

Materials and Methods Euglena Cell Growth. Euglena graeilis Klebz (Z strain, The Cul-

Introduction The complexity and the abundance of RNA transcribed from the chloroplast DNA of Euglena gracilis has been measured (Rawson and Boerma 1976; Chelm and Hallick 1976; Chelm et al. 1978 and 1979; Rawson et al. 1981). These studies showed that (1) a large fraction of the transcripts derived from chloroplast DNA are present in dark grown Euglena cells, (2) the majority of these transcripts are present throughout chloroplast development, and (3) the abundance of the RNA derived from

Offprint requests to: J. R. Y. Rawson Abbreviations used: kbp - kilobase pairs; kb - kilobases; SDS sodium dodecylsulfate; SSC - 0.15 M NaC1 and 0.015 M sodium acetate; rRNA - ribosomal RNA; rDNA - ribosomal DNA; RuBPCase - ribulose-l,5-bisphosphate carboxylase

ture Collection of Algae at the University of Texas at Austin, no. 753) cells were grown in a heterotrophic medium (Rawson and Boerma 1976). Chloroplast development proceeded in cells as described by Rawson and Boerma (1976) and Rawson et al. (1981).

RNA Isolation. Total cell RNA was isolated from cells that had been grown continuously in the dark (0-h RNA) and from cells which had been grown in the dark and then illuminated for 48 h. Five to ten gm of ceils (wet weight) were suspended in 250 ml of Extraction Buffer (0.1 M NaC1, 1 mM MgC12, 50 mM TIis-HC1 pH 9.0, 100 mM 2-mercaptoethanol and 100 #g/ml ethidium bromide) and adjusted to 2% (w/v) SDS with a 20% stock solution. 400 ml of a phenol-chloroform mixture (1 : 1) were added and the entire mixture was homogenized in a Waxingblender for 5 min. The aqueous phase was separated from the organic phase by centrifugation at 7,000 rpm for 30 min. The organic phase was extracted with 100 ml of Extraction Buffer and the two aqueous phases were combined. The nucleic acids were precipitated with one tenth volume of 3 M sodium acetate and 2 volumes of ethanol and the precipitate collected by centrifugation at

266

K.P. Dix and J. R. Y. Rawson: Transcripts of Chloroplast DNA

Table 1. Properties of recombinant molecules containing chloroplast DNA Recombinant plasmid or tambda phage

Chloroplast restriction endonuclease DNA fragment

pECR1 pECR2 pECR4 pECR5 pECR7 pECR11 pECR13 pECR16 pECR17 pECR18 Ch9.EC(EcoRI-E) pECP1 pVK51 pVK52 pECS1 pECBS1 pECPvR1 pECPvR2 pECSH 1 pECRB1 pECRB2

EcoRI-I EcoRI-F EcoRI-J' EcoRI-C EcoRI-S EcoRI-O EcoRI-M EcoRI-G EcoRI-D EcoRI-H EcoRI-E PstI-E BamHI-D BamHI-E SalI-C BamHI-SalI-1 PvuI-EcoRI-1 PvuI-EcoRI-2 SalI-HindlII-1 EcoRI-BamHI-1 EcoRI-BamHI-2

Size of chloroplast DNA fragment (kbp) 4.7 7.3 3.6 10.3 1.2 2.7 3.1 7.0 8.8 5.5 7.3 7.0 6.7 6.2 4.3 2.8 2.8 3.2 2.1 0.9 1.9

5,000 rpm for 10 rain. The precipitate was then suspended in 35 ml of Extraction Buffer, extracted with an equal volume of phenol and again precipitated with 0.3 M sodium acetate and 2 volumes of ethanol. The precipitate was collected by centrifugation (5,000 rpm, 10 min), suspended in 10 ml of TE (10 mM Tris-HC1 pH 8.0 and 1 mM EDTA) and adjusted to 2 M LiCI with a 4 M stock solution. The RNA was precipitated over night at 4 °C, collected by centrifugation at 5,000 rpm for 10 rain, suspended in 10 ml of TE and precipitated twice more with sodium acetate and ethanol. The final precipitate was suspended in water at concentrations of approximately 1 - 5 mg/ml.

Construction of Recombinant Plasmids. Recombinant plasmids containing chloroplast DNA from Euglena gracilis were constructed in vitro by ligation of chloroplast restriction endonuclease DNA fragments to plasmid vectors containing selectable genetic markers as described by Rawson et al. (1981), Rawson and Andrews (1982) and Andrews and Rawson (1982). Table 1 lists the recombinant plasmids and the chloroplast DNA fragments contained in each plasmid that were used in these experiments. Experiments involving recombinant DNA molecules were carried out under P1-EK1 containment conditions as specified by the National Institutes of Health (U.S.) Guidelines for Research Involving Recombinant DNA Molecules. Construction of Lambda Recombinants. A recombinant phage containing the EcoRI-E chloroplast DNA fragment was plaque purified from a library consisting of lambda phage Charon 9 and EcoRI DNA fragments of Euglena DNA (Curtis and Rawson 1981). The library was screened using the recombinant plasmid pECRS1 as a hybridization probe (Andrews 1981). The plasmid pECRS1 consisted of a 0.6 kbp EcoRI-SalI DNA fragment that

was completely contained in the EcoRI-E fragment. The resulting lambda recombinant phage was Ch9.EC(EcoRI-E) (see Table 1).

Propagation and Isolation of PlasmMs and Lambda Recombinant DiVAs. Plasmid DNAs were isolated as described by Andrews and Rawson (1982). Lambda recombinant phage were propagated and the phage DNA isolated as described by Curtis and Rawson (1981). Nick-translation of Plasmid DNA. Recombinant DNAs were labeled in vitro with [a32p]dCTP by nick-translation (Rigby et al. 1977) as modified by Curtis and Rawson (1981). The specific activity of the DNA was greater than 5 x 107 cpm/tzg. Gel Electrophoresis of RNA. Total cell RNA (0-h or 48-h) from Euglena was precipitated with 1/10 volume of 3 M sodium acetate and 2 volumes of ethanol. The RNA precipitate was collected by centrifugation in a microfuge, suspended in 2 0 - 4 0 #1 of 50% (v/v) formamide, 6% (v/v) formaldehyde, 18 mM Na2HPO 4 and 2 mM NaH2PO4, incubated at 60 °C for 10 min and cooled to 4 °C. One fifth volume of the same buffer containing Bromophenol Blue and 25% glycerol was added and the RNA immediately placed on either an agarose (1.5-2.0%, w/v)or polyacrylamide (15%, w/v) gel containing 6% (v/v) formaldehyde, 18 mM Na2HPO 4 and 2 mM Na2HPO 4. The gel buffer, consisting of 3% (v/v) formaldehyde, 18 mM Na2HPO 4 and 2 mM NaH2PO4, was circulated during electrophoresis and a voltage potential of 2 0 - 3 0 volts was applied for a period of time sufficient to cause the Bromophenol Blue to migrate approximately 80% of the length of the gel. The wells containing RNA markers were stained with acridine orange and photographed using longwave U.V. light. E. eoli rRNA (1.65 and 3.3 kb), tRNA (0.08 kb) and TMV-RNA (6.3 kb) were used as molecular weight markers. Preparation of Northern Imprints and Hybridization of DNA. RNA was eluted from either agarose gels or polyacrylamide gels onto strips of MiUipore filter paper (HA, 0.45 taM) according to Mangiarotti et al. (1981). These Northern imprints were preincubated in 6 x SSC, 0.5% SDS and 5 x Denhardt's solution (Denhardt 1966) for 2 h at 60 °C. 32p-labeled DNA ( 1 - 1 0 x 106 cpm) was hybridized in 6 x SSC, 0.5% SDS and Denhardt's solution to preincubated Northern imprints for 1 8 - 2 4 h at 60 ° C. Following hybridization, the Northern imprints were washed extensively at 60 °C in an excess of 2 x SSC and 0.5% SDS. Autoradiographs were prepared of the Northern imprints by exposing them to film (Kodak XR-1) in the presence of intensifier screens at - 8 0 °C (Swanstrom and Swank 1978).

Results Characterization o f R e c o m b i n a n t DATA Molecules Carrying Chloroplast D N A T h e r e c o m b i n a n t D N A m o l e c u l e s listed in T a b l e 1 contain restriction endonuclease DNA fragments representing 73 k b p o f u n i q u e n u c l e o t i d e s e q u e n c e s o f t h e 138 k b p c h l o r o p l a s t g e n o m e o f Euglena. Figure 1 s h o w s a restrict i o n e n d o n u c l e a s e m a p o f t h e Euglena c h l o r o p l a s t D N A ( G r a y a n d Hallick 1 9 7 7 a n d 1 9 7 8 ; Hallick, p e r s o n a l comm u n i c a t i o n ) i n d i c a t i n g w h e r e t h e d i f f e r e n t D N A fragm e n t s are l o c a t e d .

K. P. Dix and J. R. Y. Rawson: Transcripts of Chloroplast DNA

267 Table 2. Size of transcripts to which chloroplast restriction endonuelease DNA fragments hybridize a

I. 2, 3. 4, 5,

REGION A

EcoRI BAMHI PSTI PvuI SALI

3 kbp

Region of chloroplast DNAb

Cloned chloroplast DNA fragment

Size (kb) of RNA hybridizing to chloroplast DNA fragment

A

SalI-C EcoRI-S SalI-HincllII-1 EcoRI-E EcoRI-I EcoRI-J' EcoRI-C PvuI-EcoRI-1

6.0 5.0*, 3.8, 3.1, 2.5*, 0.8 6.0, 5.0*, 3.8, 3.1, 2.5 5.0, 3.8, 3.1, 2.5* 0.8 c 3.2, 2.5, 2.0, 1.4" 6.8, 5.9, 3.8*, 0.6* 6.8, 5.9, 4.4, 3.8*, 1.4" 6.8, 5.9, 4.4, 3.8*

B

PvuI-EcoRI-2 EcoRI-O EcoRI-D

BamHI-SalI-1 EcoRI-G

3.8, 3.1, 2.4, 1.4" 2.5, 0.5 6.2, 5.7, 5.0, 4.3, 3.7, 2.8, 2.4, 1.8", 1.3, 1.0, 0.7,0.5", 0.4 4.3, 3.7", 1.8, 1.3 4.3, 4.1, 3.7*, 1.8", 1.3, 0.8, 0.6 3.7", 1.4, 0.9* 7.2, 5.8, 5.2, 3.7*, 1.8, 0.9*

C

BamHI-D BamHI-E EcoRI-BamHI-1 EcoRI-BamHI-2

6.0, 4.9, 3.3", 1.65",0.7,0.4 6.0, 4.9, 3.3", 1.65", 0.7, 0.4 6.0, 0.14" 0.14

Unmapped

EcoRI-M

1.6

~'; 1 I

II

I~I

I II I I I

I I I

ii I II

I I I

I I I

~E~oRI-XI

L I I I J EcoRI-E

I I I

I I I

E#RI-C

1

PstI-E EcoRI-H

EcoRI-J' l PvuI-EcoRI-1

E dR1~S

SA-~LI-HINDIII-1 REGION B I

I I I

II II II

i II I

2 kbp

II

PvuI--~coRI~0 EcoRI-2

I r I

] ] I I J

i I I I I

i J I I

Ec(~RI t I -D jEcoRI' IH I i EcoRI-G I EcoRI-F , Ps,I-E l l~_J BAMHI-SALI-'I

REGION C

5S 23S v 16S ! B R

i I I I

~

5S

,! R

23S ~ 16S

B

5S , q

i 165

16S

I I

I I

i

IEcoRI_ II

II

EcoRI-

23S

I kbp

i

BAMHI-I~

I I

!BAMHI-2]

|

BAMHI-E

I

BAMHI-D

iI

Fig. 1. Restriction endonuclease map of the chloroplast DNA. The circular map shows the sites on the chloroplast DNA that are recognized by five restriction endonucleases. These restriction endonucleases are from the outside to the inside of the circle: (1) EcoRI, (2) BamHI, (3) PstI, (4) PvuI, and (5) SaIL The abbreviations for the restriction endonuclease sites shown in the linearized maps of regions A, B and C are R = EcoRI, H = HindIII, S =SalI, P = PstI and PV = PvuI. Below the linearized maps of regions A, B and C, the solid lines between restriction endonuelease sites represent those regions of the chloroplast DNA that were cloned into plasmid vectors (see Table 1). The chloroplast DNA fragment EcoRI-M has not yet been located on the restriction endonuclease map of the chloroplast DNA

Characterization o f I n vivo Transcriptional P r o d u c t s

Total cellular R N A s were size fractionated b y electrophoresis in denaturing gels and a N o r t h e r n imprint prepared by transfering the R N A to nitrocellulose filter paper. The size o f the stable in vivo transcriptional prod-

a Total cell RNA was size fractionated by electrophoresis in denaturing agarose and/or polyacrylamide gels and transferred to nitrocellulose paper to form a Northern imprint. Radioactively labeled plasmids containing restriction endonuclease DNA fragments from the chloroplast genome of Euglena were individually hybridized to these Northerns imprints to locate complementary RNA sequences. The size of the RNAs to which the DNAs hybridized was estimated as described in the materials and methods. The RNA species which show the highest concentration of complementary nucleotide sequences (greatest intensity on autogradiograph) upon hybridization of a specific DNA fragment are indicated by an asterisk (*). Those sets of RNAs in which there is no one transcript which is more abundant than another have no RNAs marked with an asterisk b See Fig. 1 for location of region A, B and C c The gel used to prepare the Northern imprint for monitoring this RNA was 2% agarose and the electrophoresis of the RNA was such that the large RNAs did not move appreciably into the gel. Thus, t h e larger transcripts to which the EcoRI-E fragment hybridized appeared very near the origin of the gel unresolved from one another

ucts c o m p l e m e n t a r y to each chloroplast D N A f r a g m e n t were d e t e r m i n e d by individually hybridizing the plasrnids listed in Table 1 to these N o r t h e r n imprints. The size o f all the transcripts and the m o s t a b u n d a n t transcripts d e t e c t e d in this fashion are summarized in Table 2. F o r ease o f discussion, the chloroplast g e n o m e has been sub-

268

K.P. Dix and J. R. Y. Rawson: Transcripts of Chloroplast DNA that transcript was transcribed from a DNA sequence contained in the DNA fragment in question. The possibility of the RNA originating from a nucleotide sequence similar to a part of a given hybridization probe, but actually located elsewhere on the chloroplast DNA was ruled out o n the basis of the fact that (other than the DNA sequences coding for the rRNA) none of the DNA fragments used in these studies showed cross hybridization with another DNA fragment (Rawson and Boerma, unpublished results). Where possible, plasmids that contalne d chloroplast DNA fragments which mapped adjacent to one another were used as hybridization probes to identical RNA samples run in adjacent wells on the same gel. Similar sized RNAs that were probed by two such DNA fragments were assumed to represent a single identical transcript originating from adjacent nucleotide sequences contained in these two DNA fragments. Finally, some of the DNA fragments used in these experiments were either partially overlapping with or completely contained in another DNA fragment. Such DNA fragments afforded an opportunity to localize more precisely the region of the chloroplast genome from which certain transcripts originated.

Fig. 2. Hybridization of the EcoRI-C, PvuI-EeoRI-1 and EeoRI-J' fragments to in vivo transcriptional products. 0-h and 48-h total cell RNA (10 jag/well) was separated by electrophoresis in a formaldehyde denaturing gel and transferred to nitrocellulose paper. 32p-labeled plasmids containing the chloroplast DNA fragments EcoRI-C, PvuI-EeoRI-1 and EeoRI-J' were hybridized to 0 h and 48 h total cell RNAs that were run in wells immediately adjacent to one another. RNAs complementary to the radioactively labeled DNAs were detected by autoradiography. The locations of the molecular weight standards (in kb) are indicated to the left in the figure

divided into three regions (Andrews and Rawson 1982); A, B and C (see Fig. 1). The transcriptional products of each of these regions will be considered separately. First, since complete recombinant DNA molecules (vector and chloroplast DNA fragment) were used as hybridization probes, it was important to determine whether the vectors used to construct these DNA molecules hybridized to any specific Euglena transcripts. The plasmids pACYC184, pBR322, PBR325 and pBR313 have already been shown not to hybridize to Euglena RNA (Rawson et al. 1981). Curtis and Rawson (1981) showed that the lambda phage Charon 9 did not hybridize to EugIena RNA. Therefore, we can expect that any RNAs to which these recombinant DNA molecules hybridize must be transcripts that originate from the chloroplast DNA of Euglena. The following logic was used in evaluating the data generated from these experiments. When a chloroplast DNA fragment hybridized to a given RNA, it was assumed that either all of that transcript or a portion of

Transcriptional Products o f Region A The SalI-C DNA fragment hybridized to six different RNAs ranging in size from 0.8 to 6.0 kb. The EcoRI-S and SalI-HindIII-1 fragments are completely contained in the SalI-C fragment. The EcoRI-S fragment hybridized to the same transcripts, except for the 0.8 kb RNA, as did SalI-C. The SalI-HindIII-1 fragment hybridized to the same transcripts, except for the 0.8 and 6.0 kb RNA, as did SalI-C. This suggests the following: one, the 0.8 kb transcript must originate from DNA sequences to the right of EcoRI-S; and two, the 6.0 kb RNA must be transcribed from DNA sequences to the right of the HindIII site in SalI-C. The major RNA species detected by hybridization of the EcoRI-E fragment was 0.8 kb. This same RNA transcript was also detected by hybridization of the SalI-C fragment and, therefore, must originate from DNA sequences common to both SalI-C and EcoRI-E. The EcoRI-I fragment hybridized to all or parts of four transcripts ranging in size from 1.4 to 3.2 kb. The 1.4 kb transcript showed the most intense hybridization signal indicating that it was either present in relatively high concentration or that the other RNAs contained only short nucleotide stretches complementary to EcoRM. The hybridization of the EcoRI-C and -J' and the PvuI-EcoRI-1 fragments to their complementary transcripts in 0-h and 48-h RNAs is shown in Fig. 2. The EcoRI-J' fragment hybridized to four different RNAs ranging in size from 0.6 to 6.8 kbp. The EcoRI-C frag-

K. P. Dix and J. R. Y. Rawson: Transcripts of Chloroplast DNA

269 Transcriptional Products o f Region B

Fig. 3. Hybridization of the EcoRI-H, PstI-E and EcoRI-D fragments to in vivo transcriptional products. 48 h total cell RNA (10 #g/well) was separated by electrophoresisin a formaldehyde denaturing gel and transferred to nitrocellulose paper. 32p. labeled plasmids containing the chloroplast DNA fragments EcoRI-H, PstI-E and EcoRI-D were hybridized to these RNAs. RNAs complementary to the radioactively labeled DNAs were detected by autoradiography. The locations of the molecular weight standards (in kb) are indicated to the left in the figure

ment is located immediately adjacent to EcoRI-J' and hybridized to five differently sized RNAs (1.4 to 6.8 kb). Three of these RNAs (6.8, 5.9 and 3.8 kb) were detected by hybridization of both the EcoRI-J' and -C fragments. The 4.4 and the 1.4 kb RNA transcripts hybridized only to the EcoRI-C fragment. The PvuI-EcoRI-1 fragment, which consists of approximately 27% of the EcoRI-C fragment, hybridized to four RNA species similar in size to those RNAs detected by hybridization of theEcoRI-C and -J' fragments. Therefore, the 0.6 kb RNA species complementary to the EcoRI-J' fragment must originate from a region of the chloroplast DNA entirely to the left of the EcoRI site that separates the EcoRI-C and -J' fragments. Similarly, the 1.4 kb RNA species is complementary only to EcoRI-C and, therefore, must originate from chloroplast DNA sequences to the right of the PvuI site in this same DNA fragment. The expression of several of these RNAs appears to be developmentally regulated (Fig. 2.). The 1.4 kb RNA transcribed from the EcoRI-C fragment is present in very low quantities in 0-h RNA and increases substantially in concentration during chloroplast development (48-h). Similarly, the 3.8 kb RNA, to which the EcoRI-C and -J' fragments hybridized, increases in quantity when dark grown Euglena cells are placed in the light for 48 h. The 0.6 kb RNA, to which the EcoRI-J' fragment hybridized, also increases in concentration during this same period of time.

The PvuI-EcoRI-2 fragment contains the gene for large subunit of RuBPCase (Stiegler et al. 1982). It hybridizes to four RNAs ranging in size from 1.4 to 3.8 kb. The 1.4 kb transcript is that RNA with the highest concentration of complementary nucleotide sequences and probably represents the mature mRNA coding for the large subunit of RuBPCase. The EcoRI-O fragment hybridizes to two RNAs (0.5 and 2.5 kb). A combination of the high specific activity of the radioactive DNA used to hybridize to these transcripts and the relatively long period of time required to visualize by autoradiography these RNAs suggested that these transcripts are probably present in very low concentrations in the cell. The hybridization of the EcoRI-D and -H and the PstI-E fragments to their complementary RNAs is shown in Fig. 3. The EcoRI.D fragment hybridized to thirteen different RNAs ranging in size from 0.4 to 6.2 kb. The PstI-E fragment, which overlaps part of the EcoRI-D fragment, hybridizes to four RNAs. These four RNAs are similar in size to four of the RNA species to which the EcoRI-D fragment is complementary. The EcoRI-H fragment hybridized to seven different sized RNAs (0.6 to 4.3 kb). Four of these RNAs were also detected by the hybridization of the PstI-E and the EcoRI-D fragments. Since the EcoRI-D and -H fragments are immediately adjacent to one another and the PstI-E fragment overlaps 3.8 kbp and 3.2 kbp of the EcoRI-D and -H fragments, respectively, these four transcripts must contain some common nucleotide sequences. The hybridization of the BamHI-SalI-1 and the EcoRI-G fragments to their complementary transcripts in 0-h and 48-h RNA is shown in Fig. 4. The BamHISalI-1 DNA fragment hybridized to three RNAs ranging in size from 0.9 to 3.7 kb. The EcoRI-G fragment hybridized to six RNAs species whose sizes varied from 0.9 to 7.2 kb. The BamHI-SalI-1 fragment, which overlaps with part of both the EcoRI-H and -G fragments, hybridized to a single transcript similar in size (3.7 kb) to a transcript which the EcoRI-H fragment hybridized and to a transcript similar in size (0.9 kb) to which the EcoRI-G fragment hybridized. Thus, the 3.7 kb transcript must originate from DNA sequences which begin in the PstI-E fragment and end prior to the left of the EcoRI-G fragment. The 0.9 kb transcript must originate from DNA sequences to the left of the Sag site in EcoRI-G and to the right of the EcoRI site ending the EcoRI-H fragment. At least two of the RNAs (3.7 and 0.9 kb) which hybridized to both the BamHI-SalI-1 and EcoRI-G fragments appear to increase substantially in concentrations during greening of Euglena cells. The 1.8 kb RNA complementary to the EcoRI-G fragment and the 1.4 kb

270

K. P. Dix and J. R. Y. Rawson: Transcripts of Chloroplast DNA Fig. 4. Hybridization of the BamHI-SalI-1 and EcoRI-G fragments to in vivo transcriptional products. 0-h and 48-h total cell RNAs (10 #g/well) were separated by electrophoresis in a formaldehyde denaturing gel and transferred to nitrocellulose paper. 32p-labeled plasmids containing the chloroplast DNA fragments BamHI-SalI-1 and EcoRI-G were hybridized to 0 and 48 h. RNAs that were complementary to these radioactively labeled DNAs were detected by autoradiography. The locations of the molecular weight standards (in kb) are indicated to the left in the figure

the possible detection of other less abundant transcripts (Rawson et al. 1981).

Transcriptional Products o f Region C Region C contains the genes for the chloroplast rRNA (Rawson et al. 1978) and several tRNAs (Orozco et al. 1980). When the BamHI-D and -E fragments were hybridized to RNA on Northern imprints, the predominant RNA species detected were the 23S (1.65 kb) and 16S (3.3 kb) chloroplast rRNAs (Fig. 5). If the autoradiographs were overexposed, two larger RNAs (6.0 and 4.9 kb) were seen as well as two smaller transcripts (0.7 and 0.4 kp) (not shown). The larger RNAs probably represent precursors molecules to the mature rRNAs. The largest of these transcripts could represent the transcription o f a single rDNA repeat. The significance of the two smaller transcripts is as yet unclear. The EcoRI-BamHI-1 fragment hybridized to at least one of the larger RNA transcripts (6.0) and to a 0.14 kb species of RNA. The BamHI-EcoRI-2 fragment also hybridized to a 0.14 kb RNA. 5S RNA is approximately this size and part o f this 5S gene is located on the EcoRI-BamHI-1 fragment, but there are no 5S RNA sequences on the BamHI-EcoRI-2 fragment (Orozco et al. 1980). Thus, it is unlikely that the 0.14 kb transcript to which the BamHI-EcoRI-2 fragment was 5S RNA. The EcoRI-M fragment, which has not yet been located on the EcoRI restriction endonuclease map o f the chloroplast genome, hybridizes to a single 1.6 kb transcript.

Discussion Fig. 5. Hybridization of the BamHI-D fragments to in vivo transcriptional products. 48-h total cell RNA (10 #g/well) was separated by electrophoresis in a formaldehyde denaturing gel and transferred to nitrocellulose paper. 32p-labeled plasmid containing the chloroplast DNA fragment BamHI-D was hybridized to this RNA. RNAs complementary to this DNA was detected by autoradiography. Lane A and B represent a short exposure and a much longer exposure, respectively, of the same nitrocellulose filter to the film. The arrows indicated the hybridization of this DNA fragment to a 4.9 and a 6.0 kb transcript. The locations of the molecular weight standards (in kb) are indicated to the left in the figure

RNA complementary to the BamHI-SalI-1 fragment are also light regulated but are not present in the cell at concentrations similar to that of the 3.7 and 0.9 kb transcripts. When the EcoRI-F fragment was hybridized to RNA on Northern imprints, it hybridized to the rRNA transcripts, which because of their concentration, obscured

The number of kilobases of RNA to which many o f the chloroplast restriction endonuclease DNA fragments of Euglena hybridize was usually greater than the size of DNA fragment used to probe the RNAs. In some cases this can be explained by the fact a DNA fragment need only hybridize to a fraction of a RNA molecule in order for that RNA molecule to be detected. This also explains the observation that some of the RNAs detected by one DNA fragment also hybridize to an adjacent DNA fragment. In other cases, the sum of the sizes o f the RNAs detected by hybridization to a given DNA fragment was significantly greater than would be expected from the size of the DNA fragments in question. The best example of this latter phenomona is seen when one compares the size of the PruI-EcoRI-1 fragment (2.8 kbp) and the total number of kilobases of RNA (20.9 kb) to which this DNA fragment hybridized. It is not possible for a DNA fragment o f this size to be complementary to this many unique RNA nucleotide sequences. Therefore, some re-

K. P. Dix and J. R. Y. Rawson: Transcripts of Chloroplast DNA

271

gions of the different sized transcripts to which the

species detected in this fashion are limited to relatively stable transcripts. The argument that some of the larger transcripts detect in these studies may be precursor RNAs is dependent upon the fact that the total size of the various RNAs detected by hybridization of a DNA fragment and its surrounding DNA fragments is significantly greater than the size of the DNA used to probe these RNA sequences. The data presented here suggests that there may be precursor RNAs arising from DNA sequences in

PvuI-EcoRI-1 fragment hybridize must be identical. This same phenomona is also seen in the hybridization of the SalI-C, the PstI-E, the BamHI-D and -E fragments to RNA on Northern imprints. The presence of identical nucleotide sequences in two or more transcripts can be explained in several ways. One, the largest transcript may be a precursor to one or more of the smaller RNAs. Two, there may be multiple sites of initiation and/or termination of transcription of the same gene. Three, the smaller transcripts could represent relatively stable in vivo or in vitro degradation products o f the larger RNAs. Four, some of the transcripts could be the result of symmetrical transcription. The possibility of symmetrical transcripts arising from certain regions of the chloroplast genome of Euglena has been suggested earlier (Rawson et al. 1981). The SalI-C and EcoRI-H and -J' fragments were shown to hybridize to more RNA than could be accounted for by asymmetrical transcription. In the studies reported here, these three DNA fragments all hybridize to more RNA (kb) than can be accounted for by the size of these individual DNA fragments. The vast majority of the transcripts detected by the procedures outlined above are present in dark grown Euglena cells. Some of these transcripts appear to be constitutively expressed while others increase in concentration during the greening of the alga cells. There does not appear to be any case where a transcript is present only in cells undergoing chloroplast development. The 0.9 kb RNA identified by the hybridization of overlapping regions of the EcoRI-G and BamHI,SalI-1 DNA fragments probably represents the transcript from the 273 nucleotide open reading frame recently sequenced by Orozco and Hallick (1982). Keller et al. (1982) have shown that a 32 kilodalton photogene is located on the EcoRI-I fragment. A gene coding for a polypeptide of this size would code for a transcript of at least 0.75 kb in size. In the experiments reported here, no such transcript was observed. The smallest stable transcript detected by hybridization of the EcoRI-I fragment was a 1.4 kb RNA. The majority of the experiments reported here were done using a gel system which was designed to detect RNA molecules greater than 400 bp in size. This would preclude the detection of small RNAs such as tRNAs. The sizes of the transcripts reported here are probably accurate to within 10% of the number of kilobases reported. The lower limit of the concentration of the transcripts detected by these procedures can be estimated from previous work (Rawson et al. 1981). The transcripts from the EcoRI-O fragment are present at the level of 10-20 copies per cell or represent approximately 10-4% of the total RNA in the cell. Another limitation of the design of these experiments is the fact that the RNA

SalI-C, SalI-HindlII-1, EcoRI-S, EcoRI-J', EcoRI-C, EcoRI-G, BamHI-SalI-l, EcoRI-D and BamHI-D and -E. The BamHI-D and -E fragments, which both contain a complete nucleotide sequence for the chloroplast rDNA, hybridize to the same transcripts, but this does not necessarily imply that both these DNA sequences are transcribed in vivo. The suggestion that genes on the chloroplast genome are transcribed as precursors and/or polycistronic mRNAs is not without precedence. Zurawski et al. (1982) have shown that genes for the/3 and e subunits of the ATPase coupling factor in spinach are transcribed as polycistronic mRNAs which exist in the chloroplast as several different sized mRNAs. They also suggested that these different sized RNAs may be due to either multiple transcriptional initiation sites or post-transcriptional processing.Krebbers et al. (1982) have suggested that the/3 and e subunits of the same enzyme complex in the chloroplast of maize also be translated from a polycistronic mRNA. Palmer et al. (1982) have shown that in several plants there are at least two different sized mRNAs containing complementary nucleotide sequences to the gene for the large subunit of RuBPCase. If, in fact, many of these large transcripts found in Euglena are precursors to mature RNAs, then they are relatively stable and are not immediately processed to mature transcripts upon transcription.

Acknowledgements. This work was supported by research grants PCM 8141949 and PCM 8200949 from the National Science Foundation.

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Communicated by C. S. Levings, III Received March 7, 1983