Multiple transcripts detected by Northern blot analysis may represent in vivo transcript size heterogeneity or breakdown products of a single transcript. Similar.
Journal of Biochemical and Biophysical Methods, 18 (1989) 183-194 Elsevier
183
BBM 00730
A convenient procedure for the isolation of intact translatable mRNA by potassium iodide gradient centrifugation P. Mfinzner * and J.
Voigt
Institut [~r Allgemeine Botanik und Botanischer Garten, Universitit't Hamburg. OhnhorststroJ3e 18, D-2000 Hamburg 52, F.R.G.
(Received 10 August 1988) (Accepted 20 December 1988)
Summary High concentrations of KI were found to efficiently protect R N A against degradation by RNases. When a sufficient amount of solid KI was added to cell lysates or subcellular fractions (9 g per 10 ml), the solutions could be stored at room temperature for several days without measurable degradation of mRNA. Ribonucleic acids were selectively sedimented when these KI-containing solutions were centrifuged at 72000 × g for 24 h. The RNA pellets were found to be readily dissolved in bidistflled water and the redissolved RNA could be immediately submitted to oligo(dT)-cellulose chromatography to isolate the poly(A)~containing RNA. However, extraction with phenol/chloroform was found to be necessary, if total RNA or poly(A)-minus R N A was to be analysed. This procedure was found to be superior to other methods currently in use - especially with respect to the isolation of intact, translatable high-molecularweight mRNA. Key words: mRNA; RNA isolation; KI-gradient centrifugation; In vitro translation; Northern blot analysis
Introduction Analysis of gene expression requires the isolation of intact RNA molecules. Multiple transcripts detected by Northern blot analysis may represent in vivo transcript size heterogeneity or breakdown products of a single transcript. Similar Correspondence address: J. Voigt, Botanisches Institut der Technischen Universitiit Braunschweig, MendelssohnstraBe 4, D-3300 Braunschweig, F.R.G. * Present address: Botanisches Institut der Technischen Universit~it Brannschweig, MendelssohnstraBe 4, D-3300 Brannschweig, F.R.G. 0165-022X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
184 problems arise when differential gene expression is studied during developmental processes by in vitro translation and subsequent immunochemical analysis of the translation products. We investigated several genes which are differentially expressed during the vegetative celt cycle of the unicellular green alga Chlamydornonas reinhardtii some of which encode mRNA species larger than 4 kb. During such cell cycle experiments, aliqaots have to be taken from synchronized cultures at different times throughout the day of experiment and were processed to isolate the RNA. Since the nuclei from all the samples were isolated in order to determine the S-phase by an in vitro replication assay [1], we were not able to apply the guanidine thiocyanate procedure of Chirgwin et al. [2]. Other methods commonly in use [3,4] were found to be unsatisfactory with respect to simplicity, processing time, yield, intactness and biological activity of the resultant RNA. Better results were obtained with the recently published procedure of Herrin and Michaets [5], which includes digestion of RNases by proteinase K E6] and inhibition of RNases by ribonucleoside-vanadyl complexes [71. However, even the method of Herrin and Michaels [5] proved to be too time consuming for our cell cycle experiments and was furthermore found to be unsatisfactory for the isolation of mRNA species encoding polypeptides larger than 80 kDa. Therefore, we have developed a more convenient procedure for the isolation of intact mRNA which we described here. The method is based on our observation that high concentrations of K1 efficiently protect RNA against degradation by RNases and the well-known separation of RNA, DNA and proteins by KI-gradient centrifugation [8,9].
Materials and Methods [3H]Poly(U) (spec. act. 20 Ci/mmol) and the mixture of 14C-methylated standard proteins (spec. act. 35 ~Ci/mg) were purchased from Amersham-Buchter (Braunschweig, F.R.G.). [c~-32p]Deoxycytidine-5'-triphosphate (spec. act. 3000 Ci/mmol), L-[35S]metlfionine (spec. act. 800 Ci/mmol) and Gene Screen nylon membrane were obtained from New England Nuclear Chemicals (Dreieich, F.R.G.). Oligo(dT)-cellulose, rabbit reticulocyte lysate in vitro translation system, nick-translation system, AMV reverse transcriptase, DNA-polymerase I and RNase H were purchased from GIBCO-BRL (Eggenstein, F.R.G.). The recombinant plasmid pVM 43 was prepared by molecular cloning the HindIII-digested chloroplast DNA from Chlamydomonas reinhardtii into the HindIII site of pUC 8 (Miinzner, Voigt and W/Sstemeyer, Unpubl.). The cloned chloroplast-DNA fragment was found to hybridize with EcoRI fragment R17 and BarnHI fragment Ba3 of the chloroplast genome [10].
Cells The Chlamydomonas reinhardtii wild-type strain 137C(+) and the cell wall-deficient mutant CW-15 were grown in a high-salt medium containing 0.2% (w/v)
185 sodium acetate as recently described [1]. Volvox globator, Spirotaenia erythrocephala and Chlorella ellipsoidea were grown under fight (2000 lx) in a WEES medium [11]. These cultures were constantly bubbled with filtered air. The cells were collected by centrifugation at 6000 X g for 10 rain (2°C). The cells were washed and resuspended in ice-cold homogenization buffer (375 mM sorbitol, 20 m M KC1, 5 m M MgC12, 1 mM MnC12, 2 mM EDTA, 14 mM 2-mercaptoethanol, 40 m M Tris-HC!, p H 7.5) to a cell density of 1 X 10 s cells/ml.
Isolation of RNA A. Isolation ofRNA fromprotoplasts
Protoplasts were lysed by addition of 1 / 2 0 vol. of 10% (v/v) Nonidet P-40. More than 95% of the cells were found to be lysed after 5-8 rain at 0 ° C as judged by light microscopy. The lysate was centrifuged at 1500 x g for 5 min and the supernatant adjusted to 10 m M E D T A and 50 m M 2-mercaptoethanol. Unless otherwise stated, 9 g KI were added per 10 ml and the solution stored at room temperature. The KI-containing solution was centrifuged for 24 h at 72 000 × g and 15 ° C in a Kontron TST 28 rotor (Kontron, Ztirich, Switzerland). Under these conditions, the R N A was found to be quantitatively sedimented. The supernatants were discarded and the walls of the centrifuge tubes were thoroughly cleaned. The pelleted R N A could be readily dissolved in 0.5 mM E D T A / 1 0 mM Tris-HCl, p H 7.5. The same procedure could be used to isolate RNA from subcellular fractions.
B. Isolation of RNA from walled cells Procedure 1 Suspensions of walled algae were adjusted to 10 m M E D T A and 10 m M ribonucleoside-vanadyl complexes [7]. After addition of proteinase K (final concentration 0.5 m g / m l ) and N-lauroyl-sarcosine (final concentration 2%, w/v), the suspensions were incubated for 1 h at 37 o C. After centrifugation for 10 rain at 20000 x g, 9 g KI were added per 10 ml of the supernatants and the solutions stored at room temperature. Isolation of R N A was performed by KI-gradient centrifugation as described above (procedure A). This procedure was found to be efficient for the isolation of intact R N A from Chlamydomonas reinhardtii wild-type cells or VoIvox globator colonies. Chlorella ellipsoidea or Spirotaenia erythrocephala cells, however, could not be effectively lysed by treatment with proteinase K and detergents. Breakage of these walled algae was achieved by sonication (procedure B2) or by passing the cell suspension through a prechilled French press [5]. Procedure 2 Cell suspensions were adjusted to 10 m M E D T A and 10 m M ribonucleoside-vanadyl complexes and submitted to sonication at 0 o C. Cell breakage waS checked by light microscopy. The homogenates were centrifuged for 10 min at 2 0 0 0 0 × g and 2°C. The supernatants were then adjusted to 2% ( v / v ) Nonidet P-40. After 5 min at 0 ° C, 9 g KI were added per 10 ml and the solutions stored at room temperature. RNA was isolated by KI-gradient centrifugation as described in procedure A.
Fraetionation of RNA into poly(A)-eontaining and poly(A)-minus species R N A pellets obtained by KI-gradient centrifugation could be readily dissolved in 0.5 m M E D T A / 1 0 mM Tris-HC1, p H 7.5, at 0 ° C. The solutions were heated for 5
186 min at 80 ° C and chilled in ice. After addition of NaC1 (final concentration: 500 mM) the RNA solutions were subjected to chromatography on oligo(dT)-cellulose columns [12], which were pretreated overnight with 30% (v/v) H202, washed with bidistilled water and equilibrated with 500 mM NaC1/0.2 mM E D T A / 1 0 m M Tris-HC1, p H 7.5. The RNA solutions were passed through the oligo(dT)-cellalose colunms twice. Thereafter, the columns were washed successively with the equilibration buffer (3 column vol.) and 100 mM NaC1/0.2 m M E D T A / 1 0 m M Tris-HC1, p H 7.5 (10 column vol.). The poly(A)-containing R N A was then eluted with 0.2 m M E D T A / 1 0 mM Tris-HC1, p H 7.5, preheated to 80°C. The eluate fractions still contained considerable amounts of protein as judged by comparing the extinctions observed at 260 and 280 nm. These contaminating proteins could be removed by re-binding and elution from the affinity matrix. Eluate fractions of the second oligo(dT)-cellulose column were combined and adjusted to 100 m M NaCk After addition of 2.5 vol. ethanol, the poly(A)-containing R N A was precipitated overnight at - 2 0 ° C and pelleted by ultracentrifugation at 1 1 2 0 0 0 × g and 2 ° C, using the Kontron TST 28 rotor. The pellet was washed with 70% (v/v) ethanol, dried and redissolved in 0.2 mM E D T A / 1 0 m M Tris-HC1, p H 7.5. The poly(A)-containing R N A was stored a t - 70 ° C. If the second round of oligo(dT)-cellulose chromatography had to be omitted, the poly(A)-containing RNA eluted from the affinity matrix was extracted twice with phenol/chloroform/isoamylalcohol (49 : 49 : 2) at room temperature prior to ethanol precipitation.
A garose-gel electrophoresis Since RNA obtained by KI-gradient centrifugation still contains contaminating proteins, pretreatment with p h e n o l / c h l o r o f o r m is required prior to fractionation of crude RNA by agarose-gel electrophoresis. The RNA pellets were dissolved at 0 ° C in 0.1 mM E D T A / 1 0 m M Tris-HC1, p H 7.5 (TE) and the solutions were extracted 4-times at room temperature with p h e n o l / c h l o r o f o r m / i s o a m y l a l c o h o l (49 : 49 : 2). After addition of NaC1 (final concentration: 0.2 M) and 2.5 vol. ethanol, the R N A was precipitated overnight at - 20 ° C. The precipitates were collected by centrifugation at 112000 × g and 2 ° C using the Kontron TST 28 rotor. After washing with 70% (v/v) ethanol, the pellets were dried and redissolved in a sma11 volume of T E buffer. Electrophoresis of RNA samples was performed on 1.2% agarose gels. 20/xg of total RNA were applied per lane. The gels were stained with ethidium bromide [13].
Northern blot analysis Total cellular RNA was denaturated with glyoxal [14], fractionated electrophoretically in 1.2% agarose gels, and blotted onto Gene Screen filter membranes (New England Nuclear Chemicals). D N A fragments for use as probes were labelled with [a-32p]dCTP by nick-translation [15]. The filter Nots were hybridized with labelled D N A probes at 4 2 ° C in the presence of 50% (v/v) formamide and subsequently washed as described in the Gene Screen manual (New England Nuclear Chemicals).
187
Synthesis of 32P-labelled DNA-RNA hybrids and 32p-labelled DNA double strands 32p-labelled DNA-RNA hybrids were prepared by reverse transcription of poly(A)-containing RNA in the presence of [a-32p]dCTP using oligo (dT) as primer [16]. Labelled DNA double strands were obtained by incubation of cDNA-RNA hybrids with RNase H, DNA polymerase I and deoxyribonucleoside triphosphates [171. The radioactively labelled nucleic acids were separated from non-incorporated [~-32p]dCTP by chromatography on Sephadex G-50 columns. Cell-free translation Poly(A)-containing RNA was translated in a rabbit reticulocyte lysate translation system (GIBCO-BRL) according to the manufactorer's recommandations using L-[35S]methionine as radioactive label. The translation products were analyzed by SDS/polyacrylamide-gel electrophoresis according to Laemmli [18] and subsequent fluorography [19] using Kodak X-AR5 X-ray film. R e s u l t s and D i s c u s s i o n
As shown in Fig. 1, degradation of [3H]poly(U) by RNase A (Fig. 1A) or RNases present in lysates of C. reinhardtii CW-15 cells (Fig. 1B) is inhibited by high
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Fig. 1. Effect of KI on the degradation of [3Hlpoly(U) by RNase A (A) and endogenous RNases of C. reinhardtii (B). 2.5/~Ci of [3Hlpoly(U ) were incubated at 20 ° C with either 1 ml RNase A (5 /,g per ml 0.1 m M E D T A / 1 0 m M Tris-HC1, pH 7.5) or 1 ml of C. reinhardtii homogenates in the presence or
absence of KI (0.9 g dissolved in 1 ml). Aliquots (100 /~1) were taken at the indicated times and plated onto Whatman 3MM filter discs. The filters were subsequently washed with 10% (w/v) trichloroacetic acid (TCA), 5% (w/v) TCA (4-times), and methanol (twice), dried and measured for radioactivity after addition of 5 ml Lipoluma (Baker Chemicals) using a liquid-scintillation counter (Tri Carb 300, Packard).
188
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Fig. 2. Purification of RNA by KI-gradient centrifugation. Solid KI was added to the 1500×g supernatant of C. reinhardtii lysates. After addition of either radioactively labelled DNA-RNA hybrids (e 0) or labelled double-stranded DNA ([]. . . . . . N), the solutions were subjected to ultracentrifugation using the Beckman SW 28 rotor. After 24 h, the gradients were fractionated and 0.1 ml aliquots measured for radioactivity (A, C). 2 vol. of bidistilled water and 5 vol. of ethanol were added to the fractions and the nucleic acids precipitated overnight at -20 °C. Precipitated nucleic acids were collected by centrifugation, dried, redissolved iv_ 500 /zl bidistilled water and desalted by ethanol precipitation. The precipitates were collected by centnfugation, washed with 70% (v/v) ethanol, dried and redissolved in 20/~1 bidistilled water. Aliquots were then analyzed for mRNA by in vitro translation using the rabbit reticulocyte lysate system (B, D). Conditions for KI-gradient centrifugation (A, B) 11 g KI added per 10 ml lysate and centrifugation at 112000× g; (C, D) = 9 g KI added per 10 ml lysate and centrifugation at 72 000 × g.
c o n c e n t r a t i o n s of KI. Tiffs f i n d i n g led us to develop a p r o c e d u r e for the isolation of intact, translatable m R N A based o n the w e l l - k n o w n s e p a r a t i o n of R N A , D N A a n d p r o t e i n b y K I - g r a d i e n t centrifugation [8,9]. U s i n g s t a n d a r d c o n d i t i o n s for K I - g r a d i ent centrifugation [9], m R N A was detected b y i n vitro t r a n s l a t i o n i n the lower third of the gradient (Fig 2B), whereas D N A - R N A h y b r i d s were f o u n d i n the m i d d l e a n d D N A d o u b l e strands in the u p p e r third of the gradients (Fig. 2A). Since fractionation of the gradients a n d s u b s e q u e n t detection of m R N A b y a n i n vitro t r a n s l a t i o n assay were f o u n d to be very i n c o n v e n i e n t for the isolation of R N A , we m o d i f i e d the c o n d i t i o n s for K I - g r a d i e n t c e n t r i f u g a t i o n b y decreasing the K I c o n c e n t r a t i o n a n d b y lowering the speed of centrifugation. U n d e r these m o d i f i e d c o n d i t i o n s for K I - g r a d i e n t centrifugation, m R N A was f o u n d to be q u a n t i t a t i v e l y s e d i m e n t e d (Fig.
189
Fig. 3. Ethidium bromide-stained agarose gel of total R N A from C. reinhardtii prepared by KI-gradient centrifugation. The different RNA samples (20 /~g) analyzed in lanes a - c were isolated from homogenates which were stored in the presence of KI for I h (a), 1 day (b) or 4 days (c) at room temperature.
2D), whereas DNA double strands and DNA-RNA hybrids were detected in the upper and lower part of the gradients (Fig. 2C), respectively. RNA pellets obtained by this modified KI-gradient centrifugation could be readily dissolved, in contrast to RNA pelleted through CsC1 cushions [2,4]. The redissolved RNA could be directly subjected to oligo(dT)-cellulose chromatography to isolate the poly(A)-containing RNA. Analysis of total RNA or poly(A)-minus RNA, however, requires a pretreatment with phenol/chloroform to remove residual proteins. Since RNase activities are effectively inhibited by KI (Fig. 1), crude homogenates or subcellular fractions adjusted to high KI concentrations could be stored at room temperature for several days without detectable degradation of RNA (Figs. 3-5): analysis of RNA samples by agarose-gel electrophoresis revealed that the integrity of the RNA was not affected when the KI-containing homogenates were stored for several days at room temperature prior to isolation of RNA by KI-gradient centrifugation (Fig. 3). The 25 S, 18 S and 16 S ribosomal RNAs were intact. The 23 S rRNA species was found to be absent in all the RNA samples and apparently contained an internal nick. RNA samples isolated by the procedure of Herrin and Michaels [5] or by the guanidine thiocyanate procedure of Chirgwin et al. [2] also lacked this rRNA species. In order t o ascertain whether mRNA is also preserved during storage of homogenates in the presence of KI, the RNAs samples were also subjected to Northern blot analysis. Using a cloned chloroplast DNA fragment as hybridization probe, which is localized within a single copy region of the chloroplast genome of C. reinhardtii (EcoRI fragment R17; [10]), definite transcripts of 2.7 and 3.2 kb were detected in all the RNA samples (Fig. 4a-c). Therefore, no degradation of the corresponding transcripts could be detected even after storage of the homogenates at room temperature for 4 days. Since the cloned DNA fragment used as probe does not contain rRNA or tRNA genes [10,20], this finding clearly
190
Fig. 4. Northern blot analysisof the RNA samples shownin Fig. 3 using pVM43 as hybridizationprobe.
indicates that mRNA is also preserved during storage of crude homogenates in the presence of KI. RNA pelleted by KI-gradient centrifugation was redissolved and immediately submitted to oligo(dT)-cellulose chromatography. The poly(A)-containing RNA was translated in the rabbit reticulocyte cell free system, and the radioactively labelled translation products were anMyzed by SDS-polyacrylamide gel electrophoresis and fluorography. As shown in Fig. 5A, the pattern of in vitro translation products was not changed, when the homogenates were stored in the presence of K1 for different time periods prior to KI-gradient centrifugation and subsequent isolation of the poly(A)-containing RNA° Furthermore, storage of the homogenates in the presence of KI did not affect either the yield or the translation efficiency of the poly(A)-containing RNA (Table 1). RNA pellets obtained by K[-gradient centrifugation can be stored at - 70 ° C for several weeks without detectable degradation of m R N A (data not shown). Translation efficiency was found to be considerably increased by re-binding and elution of the poly(A)-containing RNA from the affinity matrix (Table 1). However, definite radioactively labelled polypeptides larger than 97 kDa were missing in the translation products of these m R N A preparations (Fig. 5A). These large in vitro translation product were obtained, when the second round of oligo(dT)-cellulose chromatography was omitted (Fig. 5B). Both Northern blot analysis and cell free protein synthesis have shown that high concentrations of KI efficiently protect m R N A in crude homogenates against degradation by RNases. This finding is important because R N A can be easily isolated from crude homogenates by KI-gradient centrifugation. The procedure described has proved to be superior to published methods for the isolation of intact, translatable mRNAo As compared to the phenol/chloroform extraction with or
191
Fig. 5. Fluorogram of SDS-polyacrylamide gels comparing the in vitro translation products of poly(A)containing RNA isolated from C. reinhardtii homogenates which have been stored for different time periods at room temperature in the presence of KI. (A) Poly(A)-containing RNA which was purified by two cycles of binding to and elution from oligo(dT)-cellulose chromatography. The RNA samples were prepared from homogenates which were stored in the presence of KI for 1 h (a), 1 day (b), 2 days (c) and 4 days (d), respectively. (B) Poly(A)-containing RNA which was purified by only one round of oligo(dT)-cellulose chromatography followed by phenol/chloroform treatment. The RNA samples were prepared from homogenates which were stored in the presence of KI for 1 h (a) and 4 days (b), respectively:
without pretreatment with proteinase K [3,6], higher yields of large mRNA species were obtained. Contaminations of the RNA preparations with DNA or reduced yields of RNA due to trapping effects have never been observed in contrast to the guanidinium thiocyanate procedure [2], which includes the separation of RNA from DNA by ultracentrifugation through a CsC1 cushion [2,4]. Furthermore, RNA pellets obtained after KI-gradient centrifugation can be more readily dissolved than RNA pelleted through CsC1 cushions. The most striking advantage of the KI procedure, however, is due to the fact that it is considerably less laborious and time consuming than all the published methods for the isolation of mRNA including the
TABLE 1 STORAGE OF CRUDE HOMOGENATES IN THE PRESENCE OF POTASSIUM IODIDE DOES NOT AFFECT YIELD AND TRANSLATION EFFICIENCY OF POLY(A)-CONTAINING RNA Time of storage a
0 1 2 4
First round of oligo(dT)-cellulose chromatography
Second round of ofigo(dT)-cellulose chromatography
Yield (%)
Translation efficiency (103 cpm//~g RNA) b
Yield (%)
Translation efficiency (103 cpm//~g RNA) b
3.5 3.9 3.2 3.4
2.900 3.300 2.500 3.100
1.9 2.1 1.7 2.0
10.200 9.800 8.700 9.400
a Storage at room temperature. b Incorporation of [35S]methionine.
192 recently published procedure of Herrin and Michaels [5]. Therefore, the K I procedure was found to be better suited to our investigations of the gene expression during the vegetative cell cycle of Chlamydomonas reinhardtii. During these experiments, aliquots have to be taken from synchronized cultures at different times throughout a cell cycle period of 24 h. To determine the S-phase, intact nuclei have to be isolated and analyzed for D N A poIymerase activities [1]. The 1500 × g supernatants are processed for the isolation of RNA. Therefore, our observation that addition of K1 to crude homogenates or subcellular fractions results in a preservation of m R N A against degradation by endogenous RNases proved to be very useful because this finding enabled us to interrupt the preparation at an early stage without loss of intact RNA. Preliminary experiments revealed that the above described procedure is also suitable for animal tissues (brain and liver) and could also be adapted for the isolation of intact, translataNe m R N A from walied algae. As already shown by Herrin and Michaels [5], intact R N A could be extracted from C. reinhardtii wild-type cells by incubation with N-lauroyIsarcosine (2%, w / v ) and proteinase K (0.5 m g / m l ) in the presence of ribonucleoside-vanadyl complexes. This procedure was also found to be effective for the extraction of R N A from Volvox globator. In case of other walled green algae Ilke CMorella and Spirotaenia, the cells had to be broken by sonication because treatmem with protease and detergents did not effectively lyse these ceils. When solid K[ was added to the 1500 × g supernatants of these homogenates (0.9 g KI added per ml), intact R N A could be isolated by KI-gradient centrifugation. Therefore, with prudent choices of tissue preparation and cell breakage, the KI procedure should prove to be generally useful in isolating intact, translatable m R N A .
Simplified description of the method and its applications We have found that KI is a potent inhibitor of RNases. After addition of KI, cell lysates or subcellular fractions can be stored at room temperature for several days without measurable degradation of mRNA. RNA can be isolated from KI-containingcell lysates or subceliular fractions by KI-gradient centrifugation, an established, but time consuming procedure for the fractionation of DNA, RNA and protein. Therefore, we have modified the KI-gradient centrifugation in order to selectively sediment RNA. RNA pelleted by this modified KI-gradient centrifugation can be readily dissolved and subsequently fractionated by oligo(T)-cellulosechromatographywithout pretreatment with phenol/chloroform to obtain the poly(A)-containingRNA. This method for the isolation of intact, translatable mRNA was found to be superior to other procedures currently in use - especially with respect to experiments concerning the regulation of genes which are differentially expressed during short time periods.
AcknoMedgement This work has kindly been supported by a grant (Vo 327/1) from the Deutsche Forschungsgemeinschaft.
193
References 1 Voigt, J. and Mtinzner, P. (1987) The Chlamydomonas cell cycle is regulated by a light/dark-responsive cell-cycle switch. Planta 172, 463-472. 2 Chirgwin, J., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases. Biochemistry 18, 5294-5299. 3 Kirby, K.S. (1968) Isolation of nucleic acids with phenolic solvents. Methods Enzymol. 12, 87-92. 4 Gli~in, V., Crkvenjakov, R. and Buys, C. (1974) Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13, 2633-2637. 5 Herrin, D. and Michaels, A. (1984) Isolation, fractionation and analysis of intact, translatable mRNA from walled algal cells. Plant Mol. Biol. Reporter 2, 24-28. 6 Wiegers, U. and Hilz, H. (1971) A new method using 'proteinase-K' to prevent mRNA degradation during isolation from HeLa ceils. Biochem. Biophys. Res. Commun. 44, 513-519. 7 Berger, S.L. and Birkenmeier, C.S. (1979) Inhibition of intractable nucleases with ribonucleosidevanadyl complexes: isolation of messenger ribonucleic acid from resting lymphocytes. Biochemistry 18, 5143-5159. 8 Wolf, H. (1975) A procedure for simultaneous preparation of large amount of DNA and RNA by the use of potassium iodide gradients. Anal. Biochem. 68, 505-511. 9 Cooper, J.A. and Moss, B. (1979) Translation of specific vaccinia virus RNAs purified as RNA-DNA hybrids on potassium iodide gradients. Nucleic Acids Res. 6, 3599-3612. 10 Rochaix, J.D. (1978) Restriction endonuclease map of the chloroplast DNA of Chlamydomonas reinhardtii. J. Mol. Biol. 126, 597-617: 11 Kies, L. (1967) Uber Zellteilung und Zygotenbildung bei Roya obtusa (Br~b.) West et West. Mitt. Staatsinst. Allg. Bot. Hamburg 12, 35-42. 12 Aviv, H. and Leder, P. (1972) Purification of biologically active globin messenger RNA by chromatography on oligo-tliymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69, 1408-1412. 13 Maniatis, T., Fritsch, E.F. and Sambrock, J. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 14 McMaster, G.K. and Carmichael, G.G. (1977) Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74, 4835-4838. 15 Rigby, P.W.J., Dieckman, M., Rhodes, C. and Berg, P. (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237-251. 16 Verma, I.M., Temple, G.F., Fan, H. and Baltimore, D. (1972) In vitro synthesis of DNA complementary to rabbit reticulocyte 10 S RNA. Nature New Biol. 235, 163-167. 17 Gubler, U. and Hoffman, B.J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. 18 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. 19 Laskey, R.A. and Mills~ A.D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341. 20 Bergmann, P., Schneider, M., Burkhard, G., Weil, J.H. and Rochaix, J.D. (1985) Transfer RNA gene mapping studies on chloroplast DNA from Chlamydomonas reinhardtii. Plant Sci. 39, 133-140.
