Ribosomal RNAs Synthesized by Isolated Squid Nerves and Ganglia Differ from Native Ribosomal RNAs *²Carla Perrone-Capano, *Marianna Crispino, *Enrico Menichini, ‡Barry B. Kaplan, and *Antonio Giuditta *Dipartimento di Fisiologia Generale e Ambientale, Universita ` di Napoli “Federico II,” and ² Istituto Internazionale di Genetica e Biofisica, CNR, Naples, Italy; and ‡Division of Intramural Research Programs, NIMH, NIH, Bethesda, Maryland, U.S.A. Abstract: The large rRNA of the squid comprises two chains that may be dissociated by heating at 65°C. A single chain constitutes the small rRNA. Surprisingly, the RNAs synthesized by dissected squid fin nerves and stellate nerves and ganglia differed in size from native rRNAs and did not manifest thermal instability. Nonethe- less, they resembled native rRNAs in relative abundance, subcellular distribution, lack of poly(A), and metabolic stability. In addition, newly synthesized RNA was local- ized in nerve and glial cells, as shown by autoradio- graphic analysis, and was assembled into 80S ribo- somes, which supported the synthesis of neuron-specific neurofilament proteins. Following incubation of nerves and ganglia for 10 h, native rRNAs started to disappear, while two major newly synthesized RNAs progressively accumulated. As a result, after 20 h, native rRNAs were substituted by the two novel RNAs. With use of 32 P- cDNA synthesized from the latter RNAs as a probe, the novel RNAs demonstrated a considerable degree of ho- mology with native rRNA in northern analysis. Taken to- gether, the data suggest that in dissected squid nerves and ganglia, the synthesis of native rRNAs is gradually terminated while two novel rRNAs are being synthesized, presumably as a correlate of reactive gliosis and/or neu- ronal degeneration/regeneration. Key Words: Ribosomal RNA—RNA synthesis—Squid—Nerve lesion—Ganglia. J. Neurochem. 72, 910 –918 (1999). In cephalopod mollusks, the large subunit of rRNA contains two nucleotide chains of unequal molecular mass (960 and 880 kDa, respectively) joined together by regions of hydrogen bonds with low thermal sta- bility. In contrast, the small rRNA subunit is a single chain with molecular mass identical to that of the larger moiety of the large rRNA (Belmonte et al., 1979; Cammarano et al., 1980; Giuditta et al., 1980; Rapallino et al., 1988). During perfusion studies of the isolated squid giant axon incubated with [ 3 H]uridine (Perrone Capano et al., 1993), the two major newly synthesized RNAs appearing in the axon perfusate were tentatively identified as rRNAs in view of their number, abundance, and gross electrophoretic mobilities in nondenaturing gels. How- ever, upon further analyses, their electrophoretic mobil- ities in denaturing and nondenaturing gels were found to differ significantly from those of native rRNAs. In addi- tion, the larger RNA species did not display signs of thermal instability. To confirm these unexpected obser- vations, the properties of the two major RNAs synthe- sized in vitro by squid nerves and ganglia were examined in greater detail. Our present data confirm that the RNAs synthesized in vitro differ in size and thermal stability from native rRNAs but establish their resemblance to native rRNAs in a number of other key features. MATERIALS AND METHODS Dissection of nerves and ganglia Adult squid (Loligo pealii) were kept in tanks with circulat- ing sea water at the Marine Biological Laboratory (Woods Hole, MA, U.S.A.) and used within 2 days of their capture. Fin nerves were readily visualized and dissected from decapitated animals, following partial removal of the fins. Stellate nerves and ganglia were dissected from eviscerated mantles kept in filtered sea water at 8 –10°C, according to a routine procedure (Rapallino et al., 1988). In vitro incubation Nerves and ganglia were incubated at room temperature (18 –20°C) in artificial sea water (ASW; 460 mM NaCl, 10 mM KCl, 55 mM MgCl 2 , 11 mM CaCl 2 , 0.6 mM KHCO 3 , 10 mM Tris-Cl, pH 7.8; 0.5 ml/nerve or ganglion). To minimize bac- terial contamination, ASW was routinely filtered through 0.2- m-pore cellulose nitrate filters (sterile ASW). The medium contained 100 Ci/ml [5,6- 3 H]uridine (30 Ci/mmol; ICN) to radiolabel newly synthesized RNA. [ 35 S]Methionine (100 Ci/ ml, 1,104 Ci/mmol; ICN) was used to label newly synthesized proteins and [methyl- 3 H]thymidine (100 mCi/ml, 40 – 60 Ci/ Resubmitted manuscript received September 7, 1998; revised manu- script received October 26, 1998; accepted October 26, 1998. Address correspondence and reprint requests to Dr. C. Perrone- Capano at Dipartimento di Fisiologia Generale e Ambientale, Via Mezzocannone 8, Naples 80134, Italy. Abbreviations used: ASW, artificial sea water; DOC, sodium deoxy- cholate; NF, neurofilament; nt, nucleotides; rRNA*, newly synthesized rRNA; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid. 910 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry