The results of an investigation into the properties of ribosomes associated with the endoplasmic reticulum in HeLa cells are reported in PART II. These results suggest that the majority of ribosomes in the mitochondrial fraction of HeLa cells are extramitochondrial, i.e. Regarding the informative role of mitochondrial DNA, the idea was suggested at the end of the last century that mitochondria are capable of semi-autonomous growth and division (21, 22).
This pattern of sensitivity to bacterial protein synthesis inhibitors indicates the presence of bacterial-type ribosomes in the mitochondria. Regarding the source of information for the synthesis of components of the mitochondrial protein synthesis system and components of mitochondrial membranes, several types of observations have pointed to dual control, nuclear and mitochondrial. Indeed, one of several components of the wild-type structural protein in yeast mitochondria was reported to be missing in preparations from the cytoplasmic petite mutant.
Regarding mitochondrial phospholipids, there is evidence that the fundamental steps in their biosynthesis occur in the endoplasmic reticulum and that they subsequently. The informative role of mitochondrial DNA is to analyze the primary gene products of mitochondrial DNA. In Biochemical Aspects of the Biogenesis of Mitochondria, ed. 1953), La Formation des Enzymes Respiratoires chez La Levure.
CHAPTER 1
As for the fractionation of the cytoplasmic structures themselves, since the vast majority (85–90%) of ribosomes and polysomes in HeLa cells are non-membrane bound (2), differential centrifugation allows the membrane fraction (which, in addition - 17 • 18 In the middle part of the gradient, in Figures 1A and 2, one recognizes a band of polysomes, which presumably correspond to the free ones. After a 30-minute pulse with H3-uridine, about twice as much of the newly synthesized RNA appears in the region of the rapidly sedimenting material, as associated with free polysomes (Fig. 1A).
Differential centrifugation of the total cytoplasmic extract separates the membrane fraction, 'contaminated by a small amount of free polysomes (Fig. 1B), from the bulk of this (Fig. 1C). 34;apparent" base composition of the mRNA extracted from the membrane fraction and from free polysomes after a 30-minute P32 pulse. The base composition previously found to be 10 for "total" cytoplasmic mRNA (i.e., the RNA extracted after a 30-minute P32 -pulse).minus pulse, of free polysomes and that part of the membrane-associated polysomes that.
Prompt analysis of RNA extracted from the membrane fraction after various periods of exposure to the drug reveals a fairly rapid decline (estimated half-life less than 1 h) of the mRNA fraction, which is compensated by the appearance of rRNA labeled; To obtain some information about the site of synthesis of these two mRN A classes, an analysis of the kinetics of label presentation in both fractions was performed.
CHAPTER 2
The capacity of fractions from different regions of the gradient to hybridize to P32-bound membrane-bound RNA is also shown in Figure 1. The interpretation that the RNase-resistant fraction of heterogeneous membrane-bound RNA corresponds to intramitochondrial RNA and RNase - the mRNA-sensitive fraction of ER-proximal polysomes is supported by another type of observation. In these experiments, because of the nuclear origin of rRNA in membrane-bound ribosomes, the 1· 3• 16-labeled RNA would not contribute to hybrid formation.
Biochemical and El\I evidence showed that the vast majority (>973) of ribosomes 8100 X r;. HJ-uridine, in the presence or absence of 1 µg/ml ethidium bromide, by lysis. As shown in Figure 3, treatment with ethidium bromide results in a significant reduction in RNA labeling of membrane-bound polysomes (approximately 43% reduction in the polysomal region of the gradient from fractions 8-36).
The mitochondrial fraction (containing most elements of the gross E.R.) was isolated by differential centrifugation from HeLa cells labeled for 30 minutes with [5-3. Fractions corresponding to the portion of the gradient indicated by the arrows were pooled and dialyzed for 30 min.
Effect of ethidium bromide on the labeling of the RNA of the E.-bound polysmes. Both types of experiments suggest that some of the RNA synthesized on mit DNA is associated with polysomes, possibly those of crude E. Two types of problems currently exist that complicate analysis of the mRNA of E. .
In the present experiments, any intramitochondrial polysomes will fuse with the polysomes of the rough E. In light of these two problems, definitive evidence for the preseD:ce in the polysomes of the rough E. In order to obtain information about the properties. of the bulk species mit-RNA, HeLa cells were labeled for 48 h.
In order to determine the distribution of the mit-RNA components in the sedimentation pattern of the long-term labeled RNA from the EDTA-treated mitochondrial fraction, hybridization with purified mit-DNA was used as an analytical tool. Furthermore, in order to be able to measure the concentration of the different RNA species homologous to mit-DNA, a low RNA to DNA ratio (much lower than that required to achieve DNA saturation with total mit-RNA) was used in the incubation mixtures .
Sedimentation Properties of RNA Species homologous to Mitochondrial DNA in Hela Cells
CHAPTER 4
- orthophosphate~
Analysis of the sedimentation behavior of the 16 S and 12 S species under denaturing conditions has shown that they are. The intercalating dye ethidium bromide has recently been reported to inhibit selective labelling, during a 2 h exposure of the cells to [ 3 H]-uridine, of the 1121 S11 and 1112 S" electrophoretic RNA species (Zylber, Vesco & Penman, 1969) and largely also 4 S RNA from the mitochondrial fraction of HeLa cells For analysis of the sedimentation properties of 16 S and 12 S RNA under denaturing conditions, sa:.a.ples.
In a one:n experiment investigating the effect of ethidium bromide (1 µg/ml.) on the labeling of the RNA of the mitochondrial fraction during a 2 hour exposure to [5-3H)-uridine in the presence of 0.04 µg/ ml . The cocipponents corresponding to the portions of the pattern in Figure 4b, indicated by arrows, were collected by ethanol precipitation and centrifugation, and run again in the presence of 14 c. Labeling of the 28 SP.NA after a 4 hour pulse, as measured after a second sucrose gradient.
The data are normalized for variation in the recovery of the mitochondrial fraction on the basis of O.D.200 associated with 4S RNA. After 22 hours of exposure of the cells to 32P-orthophosphate, almost all of the label in the sedimentation profile of RNA from the EDTA-treated mitochondrial fraction (Fig. 8) was found to be alkali sensitive. It appears that the 16 Sand 12 S components have a base co:r.iposition corresponding to the heterogeneous RBA.
Rather, the 16S RNA component of the sedimentation pattern appeared to move through. In the present work, the base composition of 21 S and 23S components was found to be of the high GC type, rather similar to that of 28 S ffiIA. Likewise, their methylation degree was fairly close to that of the main rRHA species.
What proportion of the sequences transcribed from mit DNA are represented in the discrete RNA species described above is not known. From the relative labeling rates of the 12 S and ethidium bromide-sensitive 4S species (under the reasonable assumptions that these rates fairly closely reflect the actual rates of synthesis of these components and that all ethidium bromide-sensitive 4S RNA is mi t-DHA encoded), the roughly estimate that around 10 molecules of
PART II
The results of these biochemical investigations, as well as electron microscopic studies of membrane-bound ribosomes i n. Under these conditions, less than 1% of the total [3H]thymidine-labeled cellular DNA was found in the cytoplasmic fraction.). For analysis of membrane-bound polysomes, an 8100 g membrane fraction was treated with 1% NaDOCt and centrifuged through a 15 to 30% (w/w) sucrose gradient in TKM (25 ml, prepared above 3 ml.
It has previously been shown (Attardi & Attardi, 1968) that after a very short pulse (3 min) with [3HJuridine, the majority of the newly synthesized RNA in the membrane fraction, if not all, is intramitochondrial;. Of the ribosomes present in the 15,800 g supernatant fraction, approx. 70% in the form of polysomes. RNA was extracted by the sodium dodecyl sulfate method from mitochondrial endoplasmic reticulum bands of control (a) and of the EDTA-treated sample (b) and from the material released into the EDTA supernatant fraction (c) and run on a 15 empty 30% sucrose gradient in sodium dodecyl sulfate buffer, as detailed in materials and methods (e).
4(a)) is due to the asynchronous arrival into the cytoplasm from the nucleus of the two newly synthesized rRNA components (Girard et al., 1965). The RNA was extracted from the mitochondria-endopla.srnic reticulum components of the EDTA-treated membrane fraction (banded in sucrose gradient), from the material released by EDTA, and from free polysomes with sodium dodecyl sulfate, as described in detail in Materials and Methods (f. ). 4(b) and (c)), and if it is assumed that the specific activity of the heterogeneous RNA after this labeling time is approx. twice that of afrRNA (due to its turnover (Attardi & Attardi, 1967)), representing 9 to 10% of total membrane-associated RNA.
As in the case of the RNA extracted from the total membrane fraction after exposure to EDTA (Fig. RNA was released by sodium dodeoyl sulfate from the components of the Na.DOC lysate of the EDTA-treated membrane fraction shown in Fig. In the prosonant work, the most elements of rough endoplasmic reticulum were recovered, together with most of the mitochondria, in the membrane fraction of 8100 g.
Therefore, these results indicate the association with the endoplasmic reticulum of the majority of ribosomes of the membrane fraction. Therefore, the amount of polysomes recovered from the membrane fraction by NaDOC treatment can only provide a minimal estimate of the fraction of membrane-bound ribosomes that are associated with mRNA in the living cell. It is not known which factors are involved in the EDTA-resistant attachment of 50 s subunits to the membrane.
Initial labeling of membrane-bound 18 S rRNA ribosomes can be detected after 30 min. -RNAs from membrane-bound 50 S subunits were mixed and run on a 15 to 30% sucrose gradient in SDS buffer in SW 25. b} Top tube aliquots from sedimentation sample 14.
