Jerome M. Eisenstadt

The protein-synthesizing systems from the cytoplasm and the chloroplasts of Euglena gracilis*

Chloroplasts from Euglena gracilis , free of contamination by cytoplasmic ribo-somes, promote the incorporation of amino acids into acid-insoluble material. The activity is highly dependent on a Euglena supernatant fraction, an ATP-generating system and transfer RNA. It is inhibited by ribonuclease, puromycin and chloramphenicol. Light produces a marked inhibition of the incorporation by chloroplasts. In the absence of the ATP-generating system, however, the incorporation is stimulated by light. The activity of the chloroplasts is somewhat stimulated by the ribonucleoside triphosphates and inhibited by actinomycin. This is tentatively considered as evidence for DNA-dependent RNA polymerase activity. Ribosomes derived from the chloroplasts are considerably less active than the intact chloroplasts. Their activity is greatly enhanced by the addition of template RNA. The intact chloroplasts are not stimulated by this material. The chloroplast ribosomal unit active in protein synthesis shows a sedimentation value of 60 s. The corresponding value for the cytoplasmic ribosomes is 70 s. The two types of ribosomes also differ markedly with respect to nucleotide composition of the RNA. The cytoplasmic ribosomes, obtained by a procedure similar to that used for the preparation of the chloroplast particles, are not stimulated by template RNA. They are also insensitive to chloramphenicol. A possible correlation between the properties of the two types of ribosomes and the characteristics of chloroplast and cytoplasmic protein synthesis in vivo is suggested.

Deoxyribonucleic acid from the chloroplasts of Euglena gracilis

Abstract 1. 1. DNA preparations from Euglena chloroplasts were found to contain a major component with a density of 1.684 in addition to the nuclear component with a density of 1.708. Partial lysis of the chloroplasts resulted in the removal of the nuclear component. A component with a density of approx. 1.692 was also associated with the chloroplast preparations. 2. 2. The temperature of melting of the chloroplast DNA was 78–80°, as compared to 89–91° for the nuclear DNA. 3. 3. Nucleotide analysis of the chloroplast DNA indicates an overall G+C content of about 25 mole%. The 5-methylcytosine of Euglena DNA is concentrated in the nuclear material. 4. 4. The amount of DNA per chloroplast appears to be about the same as that in an Escherichia coli cell. 5. 5. The nucleotide composition of the ribosomal RNA of Euglena chloroplasts seems to be influenced by that of the chloroplast DNA.

TEMPLATE AND RIBOSOMAL RIBONUCLEIC ACIDS ASSOCIATED WITH THE CHLOROPLASTS AND THE CYTOPLASM OF EUGLENA GRACILIS.

Zone centrifugation of the RNA derived from the cytoplasm of Euglena gracilis produces one broad peak (19 s). The chloroplast RNA yields two components (14 s and 19 s). The two chloroplast components appear to have the same nucleotide composition, which is sharply different from that of the cytoplasmic 19 s material. Template RNA, studied through its ability to stimulate preincubated Escherichia coli ribosomes, appears as two well-defined sedimentation peaks (11 to 13 s and 22 s). Similar sedimentation values for template RNA were obtained with chloroplast and cytoplasmic RNA, but the proportion of the heavy and light components was markedly different in the two preparations. The chloroplasts contain a considerably greater amount of template RNA than does the cytoplasm. It is also higher than that of Esch. coli. Lysis of the chloroplasts results in a separation of part of this material from the ribosomes. This template RNA can be recovered in the 100,000 g supernatant fraction of the lysed chloroplasts. The partially depleted chloroplast ribosomes are dependent on added template RNA for their amino-acid-incorporating activity. The cytoplasmic ribosomes, which are considerably more active than the chloroplast particles, contain much less template RNA. Furthermore,a preincubation of the cytoplasmic ribosomes which produces a partial dependency on added RNA templates fails to reduce their template RNA content. The results indicate that ribosomes from various sources differ with respect to their interaction with endogenous template RNA. A possible regulatory role for the ribosomes in the process of protein synthesis is suggested.

Ribosome Formation from Subunits: Dependence on Formylmethionyl-transfer RNA in Extracts from E. coli

RIBOSOMES from bacterial, plant and animal cells consist of two subunits: a larger one (in E. coli the 50S subunit) and a smaller one (in E. coli the 30S subunit)1. Apparently a 30S and a 50S subunit associate to form a 70S ribosome in E. coli when the synthesis of a protein molecule is initiated; the ribosome persists during the synthesis and dissociates into subunits when the synthesis is completed2–7. There is suggestive evidence for the occurrence of the following events during the process of peptide chain initiation. First, a 30S subunit forms a complex with messenger RNA and formylmethionyl-tRNA (F-Met-tRNA) in the presence of GTP and initiation factors; subsequently, a 50S subunit is attached to this complex5–13.

Chloramphenicol-resistant mutants of human HeLa cells.

Chloramphenicol is one of a number of antibiotics which specifically inhibit mitochondrial protein synthesis in eukaryotic cells, while not affecting the cytoplasmic system [ 1,2]. Some mutations conferring resistance to chloramphenicol could directly affect the mitochondrial protein synthesizing system. Studies of respiratory-deficient mutants of yeast and of Neurospora have shown that mitochondrial proteins are products of both cytoplasmic extrachromosomal determinants and of chromosomal genes [3]. The Neuro spora poky mutation, which affects mitochondrial ribosomes, exhibits nonMendelian inheritance [4]. The existence of erythromycin-, mikamycin-, and chloramphenicol-resistant yeast mutants which likewise show cytoplasmic inheritance [5-S] suggest that, in mammalian cells also, resistance to chloramphenicol may be mitochondrially coded. Extrachromosomally inherited mitochondrial mutations in mammalian cells are essential to determine gene locations of mitochondrial proteins and to study nuclear-mitochondrial interactions in mitochondrial biogenesis. This paper describes the isolation and general properties of mutants of human HeLa cells resistant to chloramphenicol.

Retention of Both Parental Mitochondrial DNA Species in Mouse-Chinese Hamster Somatic Cell Hybrids

Interspecific somatic cell hybrids were isolated following the fusion of an oligomy-cin-resistant derivative of LM(TK−) mouse cells to a chloramphenicol-resistant derivative of AK412 Chinese hamster cells. Hybrids were selected in either HAT medium, HAT plus chloramphenicol (CAP), HAT plus oligomycin (OLI), or HAT plus chloramphenicol and oligomycin. Cytogenetic analysis of the hybrids indicated that their karyotype reflected the sum of the parents. Hybrids selected in HAT medium alone or HAT plus OLI retained primarily mouse mitochondrial DNA while those selected in HAT plus CAP, or HAT plus CAP plus OLI retained both species of mitochondrial DNA. There was no evidence for mitochondrial DNA recombination, despite the continued growth of these hybrids in CAP plus OLI. Hybrids that were removed from dual antibiotic selection for over three months retained both species of mitochondrial DNA in approximately equal amounts with no detectable loss or rearrangement.

Adenosine 3',5' cyclic monophosphate in Euglena gracilis.

Abstract Euglena gracilis contains in high concentration the enzymes for the synthesis and degradation of cyclic AMP. The synthetic enzyme, adenyl cyclase is mainly associated with a particulate fraction which sediments at 7,000–30,000xg whereas the degradative enzyme, 3′5′ nucleotide phosphodiesterase, is soluble (does not sediment at 78,000xg). The adenyl cyclase activity is stimulated somewhat by prostaglandins and by catecholamines, agents which markedly stimulate cyclase in appropriate mammalian tissues. There is no detectable activity of guanyl cyclase, the enzyme which synthesizes cyclic GMP. Euglena also contains a cyclic AMP stimulated protein kinase which is associated with a particulate fraction sedimenting at 30,000xg.

Supermelanotic hybrids derived from mouse melanomas and normal mouse cells

Hybrids formed between HPRT− Cloudman mouse melanoma and normal cells were isolated. The parental origin of the hybrids was verified by isozyme and karyotype analyses. These hybrid cells differed in two major characteristics from hybrids of melanoma and established fibroblastic cells. (1) They grew as tumors when injected into mice, and (2) they expressed differentiated melanocytic functions. At least one of the differentiated functions was overexpressed. The specific activity of tyrosinase was 3–20 times higher in the hybrid cells than in the parental mouse melanoma. The overexpression of tyrosinase in these hybrid cells has been stable for more than a year, has been transmitted to subclones of the original hybrid cell lines, and has been expressed in tumors that grew after injections of hybrid cells into animals.

Sedimentation characteristics of template RNA from rabbit reticulocytes and rat liver

Abstract After zone centrifugation of RNA from rabbit reticulocytes and rat liver, the material active in stimulating amino acid incorporation by Escherichia coli ribosomes is concentrated in the 18-S region. This result is obtained with RNA extracted with phenol at 4°. In RNA extracted at 30°, most of the activity is present in the 28-S region. Recentrifugation of the 18-S fraction from reticulocyte RNA leads to a sharp 15–16-S activity peak. The relative leucine, isoleucine and valine content of the material synthesized as a result of stimulation by reticulocyte RNA fractions approaches that of rabbit globin.

Ribonucleic acids from the mitochondria of bleached Euglena gracilis z: I. Isolation of mitochondria and extraction of nucleic acids

Abstract Intact mitochondria can be sedimented at 5000 × g from cell-free homogenates prepared from exponential phase cultures of bleached Euglena gracilis z. Mitochondria purified in Renografin gradients consume 1.35 μmoles O2 per mg protein per h and contain only DNA with a density of 1.688 g/cm3. The spectrum of acid-hydrolyzed RNA from purified, intact, biochemically-active mitochondria is different from that of either E. gracilis chloroplast or cytoplasmic RNA hydrolyzates.