Paper 7



Nature of Steady – State and Newly Synthesized Mitochondrial Messenger Ribonucleic Acids in Mouse Liver and Ehrlich Ascites ** Tumor Cells+


Kolari S. Bhat*, Gouder R, Kantharaj** and Narayan G. Avadhani*


Dept. of Biology, University of Pennsylvania, Philadelphia , USA; ** National

College, Bangalore 56004, India.



ABSTRACT:  The steady-state mitochondrial mRNAs in Ehrlich ascites tumor cells and mouse liver were identified by the Northern blot analysis using nick translated mt DNA and 32P labeled cDNA and were compared with poly (A)-containing RNA synthesized in vitro in isolated mitoplasts containing RNAs in different mouse tissues are identical. The results of Northern blot analyses suggest that there may be at least two different modes of m RNA maturation depending upon if the reading frames are interrupted by tRNA cistrons or not.  m RNAs for reading frames with adjacent tRNA cistrons downstream appear to be processed from very short-lived precursors. In contrast mRNAs coded by adjacently located reading frames with no interrupting tRNA genes such as URF3 – Cyt ox III an URF5 – Cyt b are processed from relatively long-lived precursors. The in vitro pulse – labeling studies also show that almost all the poly (A)-containing mRNAs are transcribed at nearly identical rates, suggesting that the major regulation of mt gene expression may occur at the level of translation or mRNA decay. The present experiments have also identified a 1.8 poly (A)-containing RNA as the putative URF5 mRNA.


Mitochondria from different mammalian cells contain a circular genome of about 16-kb 1ong DNA (Borst, 1972; Dawid et al., 1976). Recent DNA sequence analyses have shown that mt genomes from human (Anderson et al., 1981), mouse (Bibb et al., 1981) and bovine (Anderson et al., 1982) cells contain information for coding 2 rRNA, 22 tRNA and 13 potential mRNAs.   Of the 13 mRNA reading frames, 5 have been identified as genes coding for 3 mitochondrially synthesized subunit of cytochrome C oxidase, designated as Cyt ox I, Cyt ox II and Cyt ox III, subunit of 6 of ATPase, and the 42KD Cyt b protein (Anderson et al, 1981), 1982; Bibb et al, 1981).  The products of the remaining eight reading frames designated as URFs have not been identified.  By use of partial nucleotide sequencing and physical mapping methods, putative mRNA coded by 12 different reading frames located on the H strand of the mt genome have been identified in both human (Gelfand & Attardi, 1981; Montoya et al, 1981; Ojala et al,!9810 and mouse (Battey, & Clayton,1978; Van Etten et al, 1982) systems.  The precise mode of transcription and the maturation pathway for a number of mRNA coded by me mammalian cells, however, remain to be elucidated. Similarly, it is unknown if the mt genome is expressed uniformly irrespective of tissue types or if there are differential rates of transcription and turnover of mRNAs is different tissues.


Recent studies in our laboratory showed that digitonin treated mitoplasts can actively synthesize proteins resembling in vivo mt translation products (Bleat et at., 1981, 1982) and also accurately transcribe and process mt- specific rRNAs, in vitro (Kantharaj et al, 1983).  In the present paper, we have used this in vitro mitoplasts system to study the mode of synthesis of poly (A) containing RNA, putative mt RNAs and compared them with the steady-state mt mRNAs from mouse liver and Ehrlich ascites tumor cells. Our results show that the in vitro mitoplast system can synthesize almost all of the Poly (A)-containing mRNAs detected in the steady state mtRNA.  Furthermore, the mRNA patterns in Ehrlich ascites mt and mouse liver mt are identical, suggesting no detectable tissue-specific variations in mt gene expression.


Experimental Procedures.

Materials: Ultra pure grade guanidinium thiocyanate, formamide, and formaldehyde were purchased from Fluka Chemical Crop. Electrophoresis grade Agarose, restriction endonucleases, and the nick-translation kit were purchased from Bethesda Research Laboratories.  AMV reverse transcriptase was obtained from Life Sciences Inc. (dT) 8-12 was purchased from Collaborative Research Laboratories. Sequenal grade NaDodSO4, optical grade CsCl, and ultrapure sucrose were from Pierce Chemical Co., Poly (U)-Sepharose 4B was purchased from Pharmacia. Methyl mercury hydroxide was from Alfa Ventron Corp. Other bio chemicals were purchased from Sigma Chemical Co. [32P]-UTP (>600 Ci /mmol), [32P]- CTP (> 600 Ci / mmol), and (> 600 Ci / mmol), were purchased from New England Nuclear Corp. Nitrocellulose membrane sheets for RNA blot transfer and Na 56 DEAE paper were purchased from Schleicher & Schuell.


Isolation of Mitochondria: Mitochondria were isolated from Ehrilich ascites tumor cells and also from mouse liver by the differential centrifugation method using sucrose-mannitol buffer (4 mM Hepes, pH 7,4, 70 mM sucrose, 220 mM mannitol, and 2 mM EDTA) essentially as described before (Niranjan & Avadhani 1980; Bhat et al., 1982; Kantharaj et al., 1983) were washed twice with mitochondrial isolation buffer containing 10 mM EDTA and used for preparing mitoplasts by the digitonin fractionation method ( 50 mg of digitonin/mg of mitochondrial protein) as described before (Bhat et al, 1982).


In Vitro labeling of mitochondrial RNA:  Mitochondrial RNA was labeled with 32 UTP and (32p) CTP by using the in vitro mitoplast system described before (Kantharaj et al., 1983). Freshly isolated mitoplasts were incubated in a buffer system containing 5m M Hepes (pH 7, 4), 60 mM KCl, 6mM Mg (CH3COO) 2, 5mM 2-mercaptoethanol, 3mM KH2PO4 (pH 7.4), and 0.14 M sucrose at a final concentration of 10 mg of protein/mL. The incubation mixture was supplemented with 2 mM ATP, 1mM GTP, 5 mM Creatine phosphate, 4 mM pyruvate, 0.2 mg/mL creatine phosphokinase and 100 uM each of 20 L- amino acids. The suspension was shaken at 35^C with added (32P)-UTP (600 Ci/mmol) and (32p)-CTP (600Ci/mmol), 150 Ci/mL each, for the required length of time. The mitoplasts were pelleted, washed twice with sucrose- mannitol buffer, and used for isolating the RNA.


Isolation RNA: Mitochondria (10-50 mg) were dissociated in 5 mL of guanidinium thiocyanate buffer (25 mM sodium citrate, PH 7.0, 5.0 M guanidinium thiocyanate, 0.1 M 2-mercaptoethanol, and 0.5% sodium laurylsarcocinate), and RNA was isolated by repeated extraction with phenol CHCI3 essentially as described before (Kantharaj et al, 1983). Finally, RNA was pelleted through CsCl as described by Chirgwin et al (1979). Poly (A) - containing RNA was isolated by the poly (U)-Sepharose adsorption method (Ricca et al., 1981).


Preparation of Mitochondrial DNA Probes: Escherichia coli C600 transformed with pAM1 DNA was obtained from Dr. David Clayton.  pAM1 DNA consists of the entire mouse mitochondrial genome cloned in pACYC 177 plasmid at the unique Hae II site (Martens & Clayton,1979). Cells were grown in L both (Martens & Clayton, 1979) and plasmid DNA was isolated by the clear lysis method of Clewell & Helinski (1972). The Plasmid DNA was digested with restriction enzymes Eco RI, Bgl II, Hpa l, HinD III and Apa l as required and resolved on 0.8% Agarose slab gels. The DNA from gel slices were electro blotted to DEAE Paper, eluted by extraction with 1 M NaCl, and precipitated with 2.5 volumes of ethanol. DNA restriction fragments were nick translated with [32p]-d CTP (>3000 Ci/mmol) by using a kit supplied by Bethesda Research Laboratories.


Preparation of cDNA:  cDNA to mitochondrial pol (A)-containing RNA was prepared as described before (Avadhani, 1979).  The incubation mixture (50 ul contained50mM Tris-HCl (pH 8.3), 40mM KCl, 10mMMgCl2, 10 mM dithiothretol, 5ug of Actinomycin D, 5ul of (dT) 12-118, 40 ug of total mitochondrial RNA, 200uM each of dATP, dGTP, and dTTP, 50 uCi of [32P] CTP >3000 Ci/mmol), and 40 units of AMV reverse transcriptase.  The incubation was carried out at 42^C for 1 hour.  The unincorporated 32P-labeled nucleotides were removed by passing the reaction mixture through Sephadex G-100 following alkaline hydrolysis (Venetiener & Leader, 1974).


Electrophoresis of RNA:  Mitochondrial RNA was electrophoresed on denaturing methylmercury-agarose gels (Baily & Davidson, 1976).  In some experiments, mtRNA was also electrophoresed on formaldehyde-Agarose gels (Lehrach et al, 1977).  The gels were stained with EtBr, and RNA bands were visualized under UV light.  In Northern blot experiments (Alwine et al, 1977), RNA from the gel was blotted onto nitrocellulose sheets (Yomas, 1980) and probed with 32P-labeled nick- translated DNA or 32P-labeled cDNA as described before 9kantharaaj et al, 1983).



Characteristics of Mitochondrial DNA probes. The entire mouse mt genome cloned in pACYC177 Plasmid (Martens & Clayton, 1979) was used to prepare the DNA probes specific for various mRNA reading frames. The restriction maps for HindIII, Hpa1, and Apal are shown in Figure 1A. The positioning of 12S. 165, rRNAs, tRNAs, and presumptive mRNA genes on the mt genome as indicated in Figure 1A was based on the DNA sequence data reported by Bibb et, al. (1981). The 3.7-kb pACYC plasmid is inserted through a unique Hae ll site on the mt genome located about 70 nucleotides upstream from the 3' end of the 16S rRNA coding sequence as shown in Figure 1B. A complete digestion of pAMI DNA with HindIII, Hpal, and Apal  yields 10 fragments of 4.14, 3.77, 2.59, 2.41, 1.94, 1.46, 1.25, 0.98, 0.88, and 0.53 kb as shown in Figure 1B. These restriction fragments will be referred to as fragments 1-10 as indicated in Figure 1B. It should be noted that under the electrophoretic condition used, fragments 7 and 8 migrate as a doublet. DNA from the top eight bands (including the doublet containing fragments 7 and 8) were eluted and used for preparing the nick-translated probes. Fragment 10 was not used to prepare the probe because of insufficient recovery.


Identification of Transcripts: The transcripts corresponding to different reading frames were identified by the Northern blot analysis. As shown in Figure 2, total RNA from Ehrlich ascites mt was electrophoresed on methyl mercury gels, blotted onto a nitrocellulose membrane (Thomas, 1980), and probed with 32 P-labeled nick -translated restriction fragment 1-9. As shown in Figure 2 and Table I, the results of Northern blot analysis indicate the presence of some long-lived precursors in addition to at least 10 transcripts, which map precisely at the regions of 12 different mRNA reading frames. For example, fragment I, which contains the 3’–end region (740 nucleotides) of the 16S rRNA gene and over 90% of the Plasmid vector, hybridizes to the 1.6 – kb 16S rRNA.




FIGURE: 1 Genetic and restriction endonuclease maps pAMI DNA (A). The positioning of tRNA (I) 12S and 16S rRNAs, and genes to Bibb et al (1981). The tRNA Projecting outside the circles are coded by the H strand, and those projecting inside the circles are coded by the L strand. The dashed areas denote the position of various reading frames. The dashed areas positioned inside the circle represent the only L strand open reading frame, designated URF6 (Bibb et al, 1981).  OH and the arrow (with solid line) indicate the origin and the direction of H-strand synthesis, respectively. The arrow with a dotted line indicates the direction of H strand transcription. The restriction sites for HindIII, HpaI, and ApaI are indicated by arrows inside the circular. (B) The pAMI DNA was digested to completion with HindIII, HpaI and ApaI and electrophoresed n 0.8% Agarose gels, and the DNA bands were visualized with EtBr as described under experimental procedure. The Eco RI fragments of  l and fX174 DNA were run as standard markers.



FIGURE 2: Northern blot analysis of Ehrlich ascites mitochondrial RNA using 32P – labeled nick-translated DNA mtRNA (8-10 mg) was electrophoresed on 1.7% Agarose gels containing 10mM methyl mercury hydroxide, trans-blotted onto nitrocellulose paper, and probed with nick – translated DNA as described under Experimental Procedures. The number on top of each lane indicates the DNA fragment numbers as shown in Figure 1 and Table I, 16S, 12S, 18S, and 5S RNAs were used as molecular weight markers.


In addition to two smaller transcripts of 0.7 and 0.4 kb of unknown function detected by this probe, two minor transcripts of about 2.1 and 1.3 kb are also detected in the total mtRNA from Ehrlich ascites cells. Preliminary results indicate that these two minor transcripts are the H-stand transcription products. Furthermore, fragment 4 containing the remaining portion of 16S rRNA gene, the 12S rRNA gene, and the D loop area hybridizes to 16S (1.6kb) and 12S (0.96Kb) rRNA, 0.4-kb transcripts of unknown function.

Similar low molecular weight rRNA-related transcripts were also detected in mtRNA from mouse LA9 cells by Van Etten et al (1982).


Table 1. A segment of reading to transcripts detected by Northern Blot Analysis:

Probe used

(fragment used)

Estimated transcript size (KB)

Gene assignments

Predicted values





16S rRNA









Precursor of URF5


Cyt b








Cyt ox I

Cyt ox II








16s rRNA

12s rRNA














Prι Cyt ox III-URFALATPase6

Cyt ox III











Pre Cyt ox III-URFA6L-ATPase6

ATPase6-URFA6Lcyt ox III
















Pre URF5








The molecular weights of RNAs as shown n Fig.2 were calculated on the basis of migration in comparison with standard RNA markers (16s, 12s, 18s and 5s).  The predicted values presented in the parentheses were calculated n the basis of DNA sequence analysis by Bibb et al 1981 and further corrected to account for average 50-60 poly (A) residues.


Fragment 2 contains the most part of URF5 gene, genes coding for URF6 and Cyt b and a small portion of D loop area (See Figure 1). Use of nick – translated fragment 2 as a probe identifies three transcripts of 2.4, 1.8, and 1.2 kb and also some minor low molecular weight RNA. We have recently obtained evidence that the low molecular weight species are the L strand transcripts of unknown function (K.S. Bhat and N.G Avadhani, unpublished results). The 2.4 and 1.8 transcripts appear to be related the URF5 reading frame since both of them are also identified by fragment 9 which contains the 5’   end of this reading frame (Bibb et all 1981).  In addition, fragment 9 also identifies smaller transcripts of about 1.72 kb which appears to correspond to URF4L-URF4 reading frame which contains overlapping sequences [see Anderson et al (1981, 1982) and Bibb et al (1981)].  Fragment 5 contains the remaining 5’ end of URF4L – URF4 reading frames, the entire length of URF3 reading frame and the 3’ end of Cyt ox III gene. This probe identifies three relatively more abundant transcripts of 1.72, 0.85, and 0.4 kb and a less abundant transcript of 1.67 kb.  On the basis of the previous observation in HeLa and mouse mt systems (Montoya et al., 1981; Van Etten et al., 1982) and also the hybridization pattern obtained with fragment 9 above, the 1.72 – kb transcript appears to be mRNA coding for URF4L – URF 4, and the 0.85 and 0.4 – kb transcripts are putative mRNAs coding for Cyt-ox III and URF3 gene products. The low abundant 1.67 – kb RNA not detected in previous experiments (Montoya et al, 1982).





Figure: 3 Northern blot analysis of mouse liver mtRNA using 32P – labeled nick – translated DNA probes. The details are as described in Figure 2 except that mouse liver mtRNA was used. Various rRNAs were used as molecular weight markers as shown in Figure 2.


Attardi al., 1982., Van Etten et al., 1982) appears to be the common

precursor of Cyt ox III – URFA6L – AT Pase 6 reading frames. This assumption is supported by the fact that fragment 6 which contained the 5’ end of the Cyt ox III gene and URFA6L – ATPase 6 reading frames also identifies a relatively less abundant 1.67 – kb transcript in addition to a major transcript about 0.85 kb. The latter is a doublet (Figure 2, lane 6) and contains 0.82 – and 0.89 – kb transcripts representing matured Cyt ox III and URFA6L – ATPase 6 mRNAs. Fragments 7 and 8 together hybridize to 1.6 kb and 16S rRNA and two transcripts of about 0.9 and 1.05kb. In a separate experiments (not reported here) using EcoR1 fragment containing a portion of 16S rRNA gene and the entire URF1 coding region (Kantharaj et al, 1983), we have observed that the 0.97 kb RNA is the product of the URF1 gene. The 1.05 kb RNA, therefore, appears to be transcribed from URF2 reading frame.


Qualitative and Quantitative comparison of Transcription Pattern in Different Tissues. In order to determine variations in the relative levels of expression of mt genes or any tissues specific differential expression of the in genome, we have compared the transcription patterns of mouse liver and Ehrlich ascites cell mt using two different procedures. First, the steady-state mRNA is total mtRNA from mouse liver was studied by the Northern blot experiments using the specific DNA restriction fragments (see Figures 1 and 2 and Table I) as hybridization probes. These results, presented in Figure 3, show that the steady-state transcripts detected in mouse liver mtRNA are nearly identical with those detected in total mtRNA but LES cells with respect to size. The only exceptions are the mouse species of 2.1- and 1.3-kb RNA, which are identified by, probe 1 in the Ehrlich ascites mtRNA but not in the mouse liver mtRNA (Figure 3). Although not shown here, mtRNA from other tissues such as kidney and brain yield essentiality similar patterns. Second, qualitative and quantitative comparisons were made with a cDNA probe.

In this experiment, total mtRNA from mouse liver and Ehrilich ascites cells was probed with 32P-labeled cDNA prepared to poly(A)-containing RNA from Ehrlich ascites cells was probed with transcripts of about seven different size classes in total mtRNA from Ehrlich ascites cells (Figure 4, lane 1 ). RNA populations of identical sizes and relative intensities are also detected in the total RNA from mouse liver mt (Figure 4, lane 2), suggesting that the overall transcription patterns in different cell types are identical. In both cases, the poly(A)-containing RNA species resolve as major complex bands in the size range of about 1.8, 1.5-1.6, 1.1-1.2, 0.9-1.0 and 0.7- 0.8kb.


There are also two less abundant species in the size range of 2.3 – 2.4 and 3.5 – 3.6 kb. A 2.4 – kb RNAs having the properties of a URF 5 precursor was also detected with specific DNA probes (Figures 2 and Table I). Although not shown here, the 3.5 3.6 kb species detected by the cDNA probe hybridizes to fragment 2, suggesting that it may be a common precursor of URF5 – Cyt b mRNAs.  Use of 32P – labeled cDNA prepared to mouse liver mt poly (A) – containing RNA yields essentially an identical pattern of transcripts and the relative levels of expression different cell types are nearly identical.


Analysis of Newly Synthesized Mitochondrial mRNA: In an attempt to determine the rates and extent of transcription of various mRNA reading frames, we have studied the nature of poly (A) – containing RNA pulse labeled in vitro in isolated mitoplasts. As shown in Figure 5, the in vitro system transcribes the entire mt genome as shown by in vivo pulse labeling HeLa cell mtRNA (Murphy et al, 175) and S1 nuclease mapping of DNA-RNA heteroduplexes in mouse LA 9 cells (Battey & Clayton, 1978). The in vitro pulse – labeled RNA hybridizes to all three large fragments of 11.8 – 5.8 and 2.2 – kb DN fragments obtained by Bgl II and Eco RI digestion.  A small fragment obtained by 0.2-kb DNA electrophoreses out of the gel under these conditions. Further digestion of the 11.8- kb DNA of Cyt b; see Kantharaj et al. (1983) to completion with HpaI and HindIII yields 6 fragments all of which hybridize to in vitro pulse-labeled poly(A) – containing RNA (see Figure 5, lane 2), suggesting that the entire genome is transcribed under these conditions. Although not shown here, the in vitro labeled RNA contains both the H strand L strand complements.


Fig.5:                                         Fig 6:

Figure 5:  Extent of transcription of mt genome under in vitro conditions.  Ehrlich ascites mitoplasts were pulse labeled for 30 min with [32P] UTP and [32P] CTP as described under experimental procedures, and the labeled RNA was hybridized to mt DNA restriction fragments by Southern procedures.  Lane 1, pAM1 DNA digested to completion with E.coR1 and Bgl! lane 2, the 11.8-kb DNA from lane 1, further digested to completion with Hapa1and HindIII.  In both cases, DN fragments were electrophoresed on 8% Agarose gels.  About 15ug of total RNA (3.2 x 10^4 cpm/ug) was used as the probe. The E.coR1 fragments of Lambda and pi X 174 DNA were run as standard markers.                                            


Figure 6: Comparison of newly synthesized and steady State poly (A) containing RNA.  In vitro pulse labeled RNA as described in Figure 5 was fractionated into Poly (A)-containing RNA and poly (A)-lacking RNA species by poly (U)-Sepharose chromatography.  About 2x10^3 cpm each of 32P-labeled total RNA (lane 1), poly(A)-lacking RNA (lane 2), and poly(A)-containing RNA (lane3) as well as 10 ug of RNA from unlabeled mitoplasts was electrophoresed on 1.7% Agarose –methyl mercury gels as described under experimental procedures.  Lanes 1-3 was processed directly for autoradiography.  Lane 4 was transblotted on Nitrocellulose paper and probed with 32P-labeled cDNA to Ehrlich ascites mt poly (A)-containing RNAs as detected in steady state mtRNA.  16S, 12S, and 5S RNAs were used as molecular weight markers.




 The electrophoretic patterns of in vitro 32P labeled RNA [total RNA, poly (A) – lacking RNA, and also poly (A) – containing RNA] are shown in the Fig 6 (lanes 1-3).  Both the total RNA and poly (A) lacking RNA contain 12S and 16S rRNA and 5s RNA presumed to be tRNAs as major components, in addition to RNAs of heterogeneous size distribution.  The poly (A)-containing RNA contains at least 14 different species, 9 of which appear to be relatively more abundant.  A comparison of the in vitro labeled RNA with steady state poly (A)-containing RNA detected by the cDNA probe (figure. 6, lane 4) shows a striking similarity in that all of the transcripts detected in the steady state RNA present in vivo pulse labeled species.  These results suggest that the in vitro system accurately transcribes and processes all of the mRNAs expressed under the in vivo conditions.  The minor species of in vitro labeled RNA not detected in the steady state RNA may represent short-lived precursors.  Further more, the major populations of poly (A)-containing RNA indicated by the arrows in Figure 6 (lane 3) corresponding in size with various mRNA reading frames as shown in Figure 2 and Table 1 are detected in nearly equimolar ratios (cpm in the band/ molecular weight), suggesting that almost all of the mRNAs are transcribed at nearly the same rates.  Finally, although results are not shown, the newly synthesized poly (A)-containing RNAs in mouse liver mitoplasts are exactly similar to those of Ehrlich ascites mitoplasts as shown in the Figure 6(lane 3).



A recent study from our lab showed that digitonin treated mitoplasts from Ehrlich ascites cells can accurately transcribe and process mt 16S and 12S rRNAs (Kantharaj et al, 1983). Also two groups have independently shown that yeast mt particles can synthesize precursors of 21S and 15S rRNAs under in vitro conditions (Boerner et al, 1981; Groot et al, 1981).  This in vitro system were also shown to convert the pre 21S rRNA to mature 21S rRNA by splicing a 1.1 kb long sequence (Boerner et al, 1981; Groot et al, 1981).  Furthermore, several investigators have shown that isolated mt particles from Ehrlich ascites cells as well as; from rat liver can synthesize poly (A)-containing RNA (Aujame & Freeman, 1973; Rose et al, 1975; Cantatore et al, 1976).  The accurate synthesis and processing of poly (A) - containing RNAs having the properties of matured mtRNA have not yet been reported in any of the mt systems.  The results presented in this paper show that the entire mt genome is transcribed under these in vitro conditions.  Furthermore, all of the putative mt RNAs detected in the steady-state mt RNAs are transcribed and processed accurately in this vitro system.  Our inability in the past to demonstrate the synthesis of poly (A) containing RNA by isolated mt particles, therefore, appears to be largely due to suboptimal labeling conditions employed (Avadhani et al, 1974, 1975).


With a view to understand the mode of transcription and maturation of mouse mt mRNA, we have studied the nature of steady state mRNA coded by the mt genome and compared them with the newly synthesized mRNA species.  The results of Northern blot analysis using specific nick translated mt DNA probes show the presence of 12 discrete transcripts of 0.4-2.4kb in addition to 12S and 16S rRNA in the total mt RNA (steady state RNA).  The results also show that the patterns of steady state transcripts are identical in both Ehrlich ascites tumor cell and mouse liver mtRNA.  The general pattern of steady state RNA observed in this study is similar to those previously reported from HeLa (Gelfand & Attardi, 1981; Montoya et al, 1981: Ojala et al, 1981) and mouse liver mt (Van Etten et al, 1982) systems.  A relatively abundant 7s poly (A)-containing RNA species believed to be the L strand product of the D-loop area in the HeLa cell mt system (Attardi et al, 1982) was not detectable either in the steady state RNA or in the newly synthesized species of mouse mt system.  In agreement with previous studies in HeLa (Attardi et al, 1981) and mouse (Van Etten et al, 1983) mt systems, the nick translated DNA probes used in the present study also failed to detect significant levels of steady state transcripts corresponding to the only L strand open reading frame, URF6.  It is likely that these L strand products in the mouse mt system are rapidly degraded without significant accumulation.


Nucleotide sequence analysis studies have shown that in both HeLa and mouse mt systems, reading frames for URFS and Cyt b are localized on the H strand and separated by a 600nuclootide stretch which dose not contribute any specific information (Anderson et al. 1981; Bibb et al.. 1981). The opposite strand (L strand) of this 600-nucteotide stretch, however, contains an open reading frame designated URF6. On the basis of the 5'-end sequencing of RNA it was reported that a 2.4-kb poly (A)-containing RNA mapping between the start of the URF5 reading frame and the beginning of the Cyt b gene may be putative mRNA for the URF5 gene (Anderson et al., l981; Montoya et al.. 1981; Ojala ct al., 1981). On the basis of hybridization with DNA restriction fragments, a transcript of similar size was thought to be putative URF5 mRNA in the mouse mt system (Van Etten et al., 1982). In the present study, in addition to this 2.4-kb RNA, we have identified a transcript of 1.85 kb coded by the URF5 reading frame (Figures 2 and 3 and Table I).  In steady-state RNA the 2.4-kb RNA appear to be the less abundant of the two species as determined by both nick-translated DNA and cDNA probe. In the newly synthesized RNA, on the other hand, the 2.4-kb species is a relative’s more abundant species, suggesting that it may be an immediate precursor of the mature 1.85-kb mRNA. In addition to the 1.2-kb Cyt 6 mRNA, 1.85-kb URF5 mRNA, and a 2.4-kb putative precursor, a low abundant transcript of 3.5 kb coded by the Cyt b-URF5 area is detectable in the steady-state RNA (using the cDNA probes). This 3.5-kb RNA appears to be a common precursor from the Cyt b-URI'·'5 region. The Northern blot analysis using specific nick-translated probes in the present study has detected a relatively less abundant 1.67kb species having the properties of a common precursor of the Cyt ox III-URFA6L-ATPase 6 region.


The comparative analysis of steady state and newly synthesized poly (A)-containing RNA (Figures 2-4 and 6) suggests the presence of two different types of precursor RNAs. A number of very low abundant species not detected in the steady state RNA and the three species of relatively more abundant precursors of 1.67, 2.4 and 3.5KB corresponding to the Cyt. ox III-URFA6L-ATPase 6 coding regions and the URF5-cyt-b region.  It should be noted that both 12S and 16S rRNA genes and most of the mRNA reading frames, other than the Cyt ox III-URFA6L-ATPase 6 and URF5-cyt b frames are separated by tRNA cistrons [see Figures1 and Bibb et al (1981)]. Thus as indicated in our recent study on mt rRNA processing (Kantharaj et al, 1983), and also as predicted by others (Van Etten et al, 1980; Attardi et al, 1982), mRNAs for reading frames which contain juxtaposed tRNA cistrons may be processed rapidly from the nascent transcripts.  In addition, the results of the present study also suggest that mRNAs for reading frames that are continually arranged without interrupting tRNAs are deleted from relatively longer-lived precursors.  It is therefore likely that there are at least two modes of mRNA maturation; one involving the endonucleolytic cleavage of the nascent transcripts using the secondary structure of tRNA as signals (Van Etten et al, 1980) and the secondary mechanism for processing relatively long lived precursors.  The molecular signals for the second type of processing remain unknown.


Results showing nearly identical specific activities for all of the putative mt RNAs and the newly synthesized poly (A)-containing RNA population (Figure 6) suggest that they may all be transcribed at similar rates. These results also support the view that the mRNAs or their immediate precursors are derived form a common transcript (or growing chain) as proposed by Attardi et al. (1982). That differential expression of mt genome as indicated in vastly different levels of individual mt translation products (Bhat et al., 198I, I982; Ching & Attardi, 1982) may be determined either by differential turnover of mt mRNAs as previously indicated (Avadhani, 1979) or by the translation efficiencies of individual mRNAs. In conclusion, the mode of transcription and maturation of mt mRNA in different mouse cells appears to be identical. In this respect, a recent observation in our laboratory on the qualitative and quantitative difference between the translation products of Ehrlich ascites and mouse liver mt, (Bhat et al.; 1981, 1982) may be related to posttranscriptional control mechanisms. The in vitro mitoplast system capable of transcribing almost all of the poly (A)-containing RNAs resembling authentic mt RNAs may prove useful for further elucidation of the mt mRNA maturation process and in the identification of processing signals.


Acknowledgements: We are thankful to Dr. D. A. Clayton for providing the DNA clones used in this study. We also thank Nina Leinwand for helping with the preparation of the manuscripts.

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