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An affinity chromatographic procedure, using ribosomal or tRNA covalently coupled to agarose through an adipic acid dihydrazide spacer, has been developed to isolate and identify the Escherichia coli ribosomal proteins which specifically bind to these RNA molecules. Transfer RNA, 5-S, 16-S and 23-S ribosomal RNA were readily immobilized on agarose-dihydrazide: the maximum amount coupled decreasing with increasing molecular size. Specific binding of ribosomal proteins to RNA was observed in buffer containing 0.3 M KCI and 0.02 M MgC12. Bound proteins were eluted with a high-salt, 2 M KCI, buffer containing 0.005 M EDTA. Two ribosomal proteins, identified as L18 and L25 by two-dimensional gel electrophoresis, bound tightly to E. cob 5-S RNA;' small amounts of a third protein, L5, also were bound. These proteins did not bind to immobilized tRNA nor did 30-S ribosomal proteins bind to 5-S RNA. Denatured 5-S RNA bound less Ll8 and L25 than the native species. Several 30-S and 50-S ribosomal proteins bind to immobilized tRNA. The major 503 subunit proteins tightly bound to tRNA were identified as L3, L4, L5, L7 and L8/L9. Smaller amounts of proteins L1, L2, L11, L16 and L21 were also observed. Proteins L2 and L16 were retarded by agarose-bound tRNA. Protein S3 was the major 30-S subunit protein bound to tRNA. Lesser quantities of proteins S6, S9, S13 and S18 also interacted with tRNA.

Axotomy induces profound morphological changes in the neuronal cell body 2,a,1°. Neurons whose axons lie within the central nervous system of higher vertebrates (intrinsic neurons) show little or no functional regenerative capability 4,8. However, many mammalian neurons whose axons lie outside the CNS, e.g., sensory and motor neuronsS, 15 and some intrinsic CNS neurons of lower vertebrates, such as teleosts and amphibia, can regenerate functional axons and re-establish normal synaptic fieldsT,9,11. This recuperative response of the perikaryon to axotomy provides a model for the study of regeneration as well as synaptogenesis and other events thought to underlie neuronal growth, recognition and plasticity. Regeneration of functional retinotectal fibers following optic nerve crush in goldfish is associated with a number of quantifiable biochemical changes in the retina, including increased nucleoside uptake and phosphorylation 1. There is also an enhanced labeling of cytoplasmic poly(A)-containing RNA 1 and of the microtubule protein tubulin 6. The enhanced radioisotopic labeling of goldfish retinal tubulin following optic nerve crush could be attributed either to its increased synthesis or decreased degradation. Since the former explanation could reflect regulation at the transcriptional or translational level and axotomy had resulted in enhanced labeling of poly(A)containing RNA, we investigated whether retinal poly(A)-RNA obtained following optic nerve crush was enriched in specific messenger RNAs, particularly that for tubulin. Translation of retinal poly(A)-containing RNA in a heterologous, cell-free protein synthesizing system, followed by SDS gel electrophoresis of the products, provided the necessary tools to detect the proposed changes in RNA populations. Cytoplasmic poly(A)-containing RNA preparations were isolated from both normal and from post-crush retinas, then incubated in a wheat germ protein synthesizing system. The protein labeling patterns from 4, 10 and 15 day post-crush incubations and their controls are shown in Fig. 1. A distinct increase in radioautographic density in the tubulin region of the gel was apparent in proteins translated

Nucleoside 5′-triphosphates in biological samples can be quantitated accurately to 0.1 pmol using a thin-layer chromatography (TLC)-coupled bioluminescence procedure. 32 P-Labeled nucleotides of known specific activities are added to a cell extract as internal standards and the mixture is separated by two-dimensional TLC. Isolated nucleotides are located on the TLC by autoradiography, eluted, and measured in a two-step reaction using nucleoside 5′-diphosphate kinase to catalyze an equimolar phosphorylation of ADP; the mass of resulting ATP is then determined in the firefly luciferin-luciferase reaction. This method provides a 10-fold increase in sensitivity as compared to previously described techniques for nucleotide measurement.

Accumulation of radioactivity from [3H]uridine in incubations of whole goldfish retinas is increased in the ipsilateral retina during a period of regeneration that follows unilateral optic nerve crush. Brief incubations to investigate the nature of enhanced labeling of the acid-soluble fraction showed a peak uptake 4 days following crush, with a gradual decrease to control levels by 21 days following crush. That nucleoside uptake may not mediate the effect is supported by the observation that the rate of uptake of 5'-deoxyadenosine, a nonmetabolizable nucleoside analog, is the same in post-crush (PC) and normal (N) retinal incubations. Following brief incubations of PC and N retinas with [3H]uridine, there is enhanced labeling in PC retinas relative to N retinas of recovered UMP, UDP, UTP, and uridine nucleotide sugars, whereas recovery of labeled uridine itself is slightly decreased. The results suggest that the increased accumulation of radioactivity in PC retinas following incubation with uridine reflects an increase in the activities of retinal uridine kinase and uridine nucleotide kinases.

— In goldfish, regeneration of the optic nerve following axotomy is accompanied by changes in retinal RNA metabolism. Approximately 4 days after unilateral optic nerve crush, there is an increased uptake of 3H-labeled precursors of RNA into cells of the retina as well as an increase in labeling of total retinal RNA following incubation for 3–4 h in vitro. Subfractionation indicates that the resultant labeled free ribosomai RNA has a much higher specific activity than that from membrane-bound ribosomes in post-crush, as compared to normal retinas. Total retinal RNA content also increases, reaching a peak value about 8 days after axotomy, coincident with the time of observed maximum labeling of ribosomal RNA. No change in the total protein or DNA content of the retina is detected during this period as a result of the optic nerve crush. Saturating amounts of uridine or adenosine in the medium eliminate the general effects of enhanced uptake of the precursors on RNA labeling. Elevated labeling of poly(A)-containing RNA in retinal cytoplasm is nevertheless detected 10–14 days after optic nerve crush with no change in the labeling of the polyadenylate segment. A decrease in the labeling of poly(A)-containing nuclear RNA, from a significantly greater amount than that of normal retinas to a significantly lower one, occurs during the period of observed increased labeling of cytoplasmic poly(A)-containing RNA. The results indicate that in addition to changes in RNA precursor metabolism, optic nerve regeneration is characterized by alterations in the labeling of free and membrane-bound retinal ribosomes as well as in the post-transcriptional processing of messenger RNA.

A procedure is described for obtaining the specific activity of α- 32 P-labeled nucleotides in the range where mass determination by uv spectrophotometry or by radioisotopic double-label techniques is not feasible. The procedure, based on the template-dependent synthesis of an alternating-sequence copolymer catalyzed by DNA polymerase, requires no elaborate equipment and uses commercially available materials and enzymes. Results obtained with this assay indicate that the measured specific activities of 2′-deoxyribonucleoside 5′-[α- 32 P]triphosphates synthesized according to published procedures can vary significantly from expected theoretical values.