Search

In this article, I review how our research on RNA began, how it led us to demonstrate the single-stranded nature of RNA, and the ways in which it differs from double-stranded DNA. It was based on the development of a method for the isolation of undegraded rRNA and the observation that in rRNA preparations due to their viscosity behavior resemble a flexible, contractile coil. In support of this assumption, birefringence of flow measurements showed that rRNA solutions gave moderate positive values, which disappeared upon addition of salt. This is in contrast with DNA solutions where considerable negative birefringence persists even in the presence of salt. Further studies on RNA showed a close correlation of the ionic strength dependencies of optical rotation, optical density and hydrodynamic properties. These early results indicated that rRNA and tRNA possess a significant secondary structure. I then review the basis of the hairpin model for the secondary structure of RNA and finally, summarize current understanding of the tertiary structure of RNA.

We have examined the mechanism of signaling by the 67 kDa YIGSR binding protein of laminin and its properties in neuroblastoma cells. Ligand displacement analysis showed that the interaction with the C(YIGSR)3-NH2 peptide amide is of intermediate affinity (1.5×10−7 M). Cross-linking experiments with sulfo-MBS detected an additional protein with a molecular mass of 116 kDa that binds the YIGSR sequence. Incubation of neuroblastoma cells with C(YIGSR)3-NH2 peptide amide or antibody directed against the 67 kDa laminin binding protein induces tyrosine phosphorylation of proteins with a molecular mass ranging from 115 to 130 kDa and another heterogeneous protein group of 32 kDa.

Differentiated human neuroblastoma LA-N1 cells that were exposed to dibutyryl adenosine 3',5'-cyclic monophosphate for 5 days (primed cells) showed increased adhesion to laminin-, fibronectin-, and collagen type I-coated plates as compared to unprimed cells. Moreover, primed cells seemed to adhere best to laminin. The binding site in laminin, mediating cell attachment, was identified as containing the YIGSR sequence, a known cell binding motif, located in the short arm of the B1 chain of laminin. The synthetic peptide amide, C(YIGSR)3-NH2, containing a repeat of this binding motif, inhibited the attachment of neuroblastoma cells to laminin in a competitive manner, and its inhibitory activity was inversely dependent on laminin concentrations. Affinity chromatography of membrane-extracted proteins over an Affi-Gel 10 column conjugated to C(YIGSR)3-NH2, revealed a major YIGSR-binding protein with an apparent molecular mass of 67 kDa. The 67-kDa surface membrane protein was specifically eluted from the column with the soluble C(YIGSR)3-NH2 peptide, but not with an unrelated peptide. Furthermore, no 67-kDa laminin-binding protein was recovered from an unrelated peptide matrix with the free C(YIGSR)3-NH2 peptide. Ligand blot overlay assays with biotin-labeled C(YIGSR)3-NH2 peptide demonstrated that the 67-kDa receptor is indeed a YIGSR-binding protein. This 67-kDa laminin-binding protein appeared to be down-regulated upon differentiation of LA-N1 cells, as indicated by the level of this protein and its mRNA.

The appearance of neurofibrillary tangles (NFT) is one of the major structural changes that occur in neurons during Alzheimer’s disease. They are composed almost entirely of paired helical filaments (PHF), intermixed with some straight filaments. Immunocytochemical studies of NFT have revealed that they have antigenic determinants in common with noncytoskeletal elements, neurofilaments (Perry et al., 1984) and microtubule associated tau proteins (Brion et al., 1984; Wood et al., 1986; Kosik et al., 1986, 1988a,b; Nukina and Ihara, 1986; Goedert et al., 1988), while the presence of microtubule-associated protein 2 (MAP2) in NFT has yet to be established (Rosemblatt et al., 1989). Both MAP2 and tau proteins have been shown to bind to peptides derived from the carboxyl-terminal region of β-tubulin. In addition, tau proteins but not MAP2 display a strong interaction with a peptide derived from the amino-terminal domain of α-tubulin (Littauer et al., 1986). Recently, it was found that tau proteins contain three 18 amino acid repeated sequences which appear to be involved in the binding to tubulin (Lee et al., 1988; Goedert et al., 1988, 1989; Lee et al., 1989; Kosik, 1989). It was also observed that MAP2 shares the tau repeated regions (Lewis et al., 1988). However, this is not the case for a recently cloned tubulin binding protein designated neuraxin, which does not contain the tau and MAP2 repeated sequences. Neuraxin was found to be immunologically related to MAPS and is perhaps identical to this high molecular weight MAP (Rienitz et al., 1989).

Modulation of a membrane glycoprotein, approximate molecular weight 200,000, in concert with active ionic flux has been shown in a human neuroblastoma cell line. The modulating agent was 2% dimethyl sulfoxide. Other neuronal properties, acetylcholinesterase and choline acetyltransferase, were also modulated but to a lesser extent. The appearance of this glycoprotein on the surface of both human and mouse neuroblastoma cells only under conditions of differentiation leads to the suggestion that it is directly involved with the active Na+ channels.

The nucleotide sequence of a complete rat brain beta-tubulin T beta 15 has been determined from three overlapping cDNA clones. The overall length of the T beta 15 sequence is 1589 bp and shows between 84.5% and 88.6% homology within the coding region as compared with chick and human beta-tubulin sequences. On the other hand, the 3'-non-coding region is highly divergent. Comparison of the derived amino acid sequences from different species demonstrates that the amino acid changes are not randomly distributed, but rather there are several conserved and two highly variable regions common to beta-tubulin polypeptides from various sources. The T beta 15 sequence encodes a dominant neuronal 1.8-kb beta-tubulin mRNA species. Two other minor beta-tubulin mRNA species of 2.6 and 2.9 kb are present in rat brain. By using two synthetic oligonucleotide probes complementary to the carboxyl-terminal divergent region and to the amino-terminal conserved region, we have shown that the three mRNAs are distinct species, which are developmentally regulated. The level of the 1.8-kb mRNA species increases till the age of 12 days thereafter its level decreases. The 2.9-kb mRNA is an early neuronal mRNA species, while the 2.6-kb mRNA is a late neuronal species which is detected at 30 days of rat brain development. The data illustrate that there is a differential expression of the beta-tubulin multigene family during rat brain development which may suggest different functions for the various beta-tubulin isotopes.

A system was established in which nuclear preparations from rat brains were capable of protein synthesis under cell-free conditions. The electrophoretic pattern of the synthesized proteins was similar to that found in vivo provided that the reaction mixture contained pH 5 precipitated factors derived from the high speed supernatant fraction of brain. In the absence of the pH 5 factors, using nuclear preparations from brains of 2-day-old rats, approximately 1.5% and 2% of the newly synthesized proteins were identified as tubulin and actin, respectively. In the presence of pH 5 factors, protein synthesis was stimulated and the proportion of the newly synthesized tubulin and actin increased to 26% and 11%, respectively. In contrast to nuclear fractions from 2-day-old rats, when nuclei from brains of 1-month-old rats were tested in the presence of pH 5 factors, the proportion of tubulin and actin synthesized was lower and amounted to 10% and 4%, respectively. The age-dependent change in the relative amount of the tubulin and actin synthesized is in good agreement with the translational pattern shown by brain polyribosomes in a brain cell-free system as well as with the pattern obtained with brain mRNA translated in a wheat germ cell-free system. Nuclei enriched for either neuronal or glial populations synthesized similar proportions of tubulin and actin in vitro. We conclude that the reduction in the synthesis of tubulin and actin during the postnatal development of the rat brain occurs in both neuronal and glial cells.

The heterogeneity of tau microtubule-associated proteins from rat brain is developmentally determined. Newborn rat brain contains two tau polypeptides (tau 0) with somewhat different molecular weights than the five tau components associated with microtubules from 12-day-old brain (tau 12). tau 0 and tau 12 are immunologically related and crossreact with antibodies against tau 12 proteins. Enrichment of the tau mRNA was achieved by prior hybridization of unfractionated poly(A)-containing mRNA to cDNA preparations containing tubulin and actin sequences. The remaining unhybridized mRNA was further fractionated by electrophoresis on methylmercury hydroxide agarose gels. Experiments involving cell-free translation of mRNA indicated that the major differences in the composition of tau proteins from newborn and developing brain are controlled at the mRNA level. The mRNA from newborn rat brain directed the synthesis of five tau proteins, two of which are specific for newborn brain, whereas the other three forms are characteristic of the developing brain. Thus, the appearance in newborn brain of mRNA species specific for three tau 12 forms precedes the phase of the synthesis of these proteins in the cell. By contrast, mRNA from 12-day brain directed the synthesis of four tau proteins specific for the developing brain, one of which is not synthesized by mRNA from newborn brain. None of the newborn tau 0 forms were synthesized with mRNA isolated from 12-day brain.

The molecular regulation of tubulin synthesis was investigated in the regenerating goldfish retina. Previous in vivo studies pointed to an increase in tubulin synthesis in the retina during regeneration of the injured goldfish optic nerve. Using labeled cDNA probes, we showed that this increase occurs as a result of enhanced tubulin mRNA levels. Analysis of labeled in vivo products revealed enhanced β2-tubulin synthesis accompanied by an increase in the level of the low-Mr microtubule-associated proteins identified as TAU factors. The results are discussed with respect to the possible involvement of these proteins in the process of nerve regeneration.

Rabbit globin mRNA species containing poly(A) segments of different lengths were prepared by partial phosphorolysis of mRNA with Escherichia coli polynucleotide phosphorylase. By varying the salt concentration and the time of incubation of the phosphorolysis mixture, as well as performing oligo(dT)-cellulose chromatography at 22 degrees C and at 4 degrees C, globin mRNA preparations containing poly(A) segments of approximately 122, 95, 68, 39, 32, 21, and 16 adenylate residues were obtained. It was found that the functional stability of the mRNA species containing 32 or more adenylate residues after injection into Xenopus laevis oocytes equaled that of the native globin mRNA. On the other hand, the functional stability of mRNA containing an average number of 21 adenylate residues was about 30% of the native mRNA, while that of mRNA containing 16 adenylate residues was as low as poly(A)-free globin MRNA.

Cloned neuroblastoma cell lines derived from the spontaneous mouse tumor C-1300 were used to study nerve cell differentiation. Our findings included a) morphologic and electrical differentiation was induced by the addition of dimethyl sulfoxide to the culture medium of some of the neuroblastoma clonal lines; b) a contrasting difference existed between the percentage of the phenylalanine-specific, tRNA species deficient in the peroxy Y-nucleoside in the mouse embryo or rat brain (6-10%) and that of mouse neuroblastoma cells (85%); c) the assembly of neuroblastoma microtubules and neurofilaments that are necessary for neurite outgrowth proceeded from preexisting pools of tubulin and actin, but a sustained level of phosphorylated tubulin was not required for this regulation; and d) the in vitro translation of tubulin and actin was accomplished with mRNA from rat brains in a wheat-germ cellfree system.

Using an ATP:RNA adenyltransferase from Escherichia coli, a polyadenylic sequence was resynthesized onto rabbit globin mRNA from which the poly (A) segment had been previously removed. Conditions for obtaining a homogenous reconstituted globin mRNA preparation containing 30 adenylic residues per message molecule were determined. The reconstituted globin mRNA was microinjected into Xenopus laevis oocytes. Its stability was very similar to that of native mRNA.

Addition of dimethylsulfoxide at concentrations of 1% and 2% (vol/vol) to cells of mouse neuroblastoma clone NIE-115 in the confluent phase of growth resulted in the production of morphologically differentiated cultures with extensive process formation. Cell maintained in 2% dimethylsulfoxide remained in a stable nondividing condition for periods of up to 4 weeks. A high degree of electrical excitability was found in these cells, but there was no clear correlation of this property with the level of induction of either acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) or tyrosine hydroxylase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2]. In addition, intracellular levels of cyclic 3':5'-AMP were not elevated in fully morphologically and electrically differentiated cells. While cell division was markedly inhibited by 2% or higher concentrations of dimethylsulfoxide, at 1% growth continued at a somewhat slowed rate and such cultures exhibited enhanced process formation and electrical activity for a relatively short period. High concentrations (3% or 4%) of dimethylsulfoxide totally suppressed process formation and did not result in increased excitability, but cells maintained high resting potentials. The results suggest that the development of the excitable membrane in neuroblastoma cells may be expressed independently of neurospecific enzyme induction, and does not require a sustained elevation of cyclic 3':5'-AMP levels.

A specific binding assay was developed that monitors the interaction of 125I-labeled microtubule-associated proteins (MAPs) with tubulin or its fragments bound to nitrocellulose membrane. To identify the tubulin-binding domains for MAPs we have examined the binding of rat brain 125I-labeled MAP2 or 125I-labeled tau factors to 60 peptides derived from porcine alpha- and beta-tubulin. MAP2 and tau factors specifically interacted with two peptides derived from the carboxyl-terminal region of beta-tubulin, which are located between positions 392-445 and 416-445. In addition, there is a distinct tau-binding site at the amino-terminal region of alpha-tubulin. tau factors but not MAP2 displayed strong interaction with a peptide derived from the amino-terminal domain of alpha-tubulin between positions 1 and 75. To narrow down the location of the beta-tubulin binding site that is common to MAP2 and tau factors, we have synthesized five peptides that are homologous to the corresponding sequence from the porcine or rat carboxyl-terminal region. Binding studies with the synthetic peptides suggest that amino acid residues 434-440 of beta-tubulin are crucial for the interaction of MAP2 and tau factors.

The mRNA of the brain shrimp Artemia salina has two types of blocked methylated 5'-terminal structures (caps). About 75% of the mRNA molecules have the 5'-end structure of m7G5'ppp5'-AmpGp and about 25% have the structure of m7G5'ppp5'GmpGp. The only other type of methylated residue found in Artemia mRNA is N6-methyladenosine and which is located at internal positions along the mRNA chain. Translation of Artemia cyst or nauplius poly(A)-rich mRNA in wheat-germ extracts was found to be inhibited by 7-methylguanosine 5'-monophosphate, a chemical analog of the cap, as well as by snythetic caps such as m7G5'ppp5'Gm. On the other hand, the elongation activity on endonegous mRNA in an Artemia cell-free system was not sensitive to 7-methylguanosine 5'-monophosphate.

Mouse neuroblastoma cells in stationary phase of growth display partially developed electrical properties. Addition of the K+ selective carrier valinomycin to these cells causes rapid enhancement of electrical excitability. We suggest that the appearance of molecules with properties similar to valinomycin is essential for the full expression of electrical excitability in differentiating neuroblastoma.

A specific method has been developed for the removal of polyadenylic acid-rich sequences from messenger RNA. The method is based on the processive phosphorolysis of mRNA using molar excess of Escherichia coli polynucleotide phosphorylase at 0 ° C in the presence of 1 m -NaCl. It also enables the determination of the location, length and gross base composition of the poly(A)-rich segment. Under these conditions, it was established that the poly(A)-rich sequence of rabbit globin mRNA is located at the 3 ′ -OH terminus and has an average size of 149 nucleotide residues. After removal of the poly(A)-rich sequence from the mRNA, the remainder of the molecule fails to bind to oligo(dT)-cellulose columns. The poly(A)-rich sequence of rabbit globin mRNA does not interact strongly with other regions of the molecule, as it is phosphorolyzed at the same rate as free poly (A). On the other hand, phosphorolysis of the molecule beyond the poly(A)-rich sequence is rather slow, indicating that this region of the mRNA has a stable secondary structure. The sequence beyond the poly(A)-rich segment was found to be rich in guanosine and uridine residues. The poly(A)-rich sequence of the mRNA present as part of a specific polysomal ribonucleoprotein complex, is protected from the action of polynucleotide phosphorylase, indicating that the attachment of the protecting protein(s) extends to the 3 ′ end of the mRNA. The poly(A)-free mRNA can still be translated in a Krebs ascites tumor cellfree extract. The initial rate of translation of poly(A)-free mRNA, is virtually identical to that found with poly(A)-containing mRNA, in the presence or absence of reticulocyte ribosomal wash fluid. The only difference in the template activity of the mRNA preparations, appears at longer periods of incubation and in the presence of reticulocyte ribosomal wash fluid, when the rate of protein synthesis tends to level off more strongly with poly(A)-free mRNA than with native poly(A)-containing mRNA. On electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate or on cellulose acetate strips the product of the reaction has the same mobility as that of free globin.

3/-labeled N,N,N,trimethylamino-beta-alanyl-N-hydroxysuccinimido ester ([3H]TMAS), a new cationic membrane reagent, was synthesized. TMAS was shown to be impermeant through human erythrocyte membranes. Under mild physiological conditions TMAS reacted primarily with amino groups of the membrane proteins and lipids. The pattern of erythrocyte proteins labeled with [3H]TMAS was examined by polyacrylamide gel electrophoresis under denaturing conditions. Externally oriented labeling of intact erythrocytes revealed a major radioactive protein with an apparent molecular weight of 90,000. By labeling ghost preparations with [3H]TMAS the radioactivity incorporated into all the major Coomassie Brilliant Blue bands resolved by gel electrophoresis. The agreement between the results obtained with anionic and cationic amino reactive probes indicates that the ionic character of the reagent has a minor effect on the pattern of labeled exterior polypeptides observed in erythrocytes.

Prenatal rat brain tubulin was resolved by isoelectric focusing into five to six components (isotubulins 1--6) while in mature brain nine distinct forms were evident (isotubulins 1--9). Tubulin, isolated from various brain regions, displayed different proportions of these nine isotubulins which may result from the heterogeneity in brain cell population. Mature brain mRNA, when translated in vitro, in the reticulocyte lysate cell-free system, directed the synthesis of five tubulin forms, namely isotubulins 1, 3, 4 (or 5), 6 and 7. The mRNA species coding for isotubulins 3 and 7 could be partially separated on formamide/sucrose gradients, while in the absence of formamide the mRNA species directing the synthesis of isotubulins 1, 4 (or 5) and 6 showed differences in mobility. It therefore appears that brain mRNA may consist of five different species coding for distinct tubulin forms. Moreover, a marked age-dependent enhancement in the relative translation of the mRNA coding for isotubulin 7, which is not apparent among the translation products directed by the prenatal mRNA, was detected. Our results indicate that some of the age-dependent variations in tubulin microheterogeneity may be controlled at the mRNA level.

The cytoplasmic and particulate tubulin content of postnatal rat brains was determined at various stages of development. The amount of tubulin in the soluble fraction was found to increase after birth and levels off at the age of 10–15 days, while the total protein content is still increasing. Indeed, the percentage of tubulin in the soluble fraction is about 33% at birth, stays at this value until day 10, and then decreases to 20% between days 10 and 15. On the other hand, the rate of increase in the level of the particulate tubulin parallels that of the total particulate proteins, and hence there is no change in the percentage of particulate tubulin during brain development. There was close agreement between the tubulin values obtained by the [ 3 H]-colchicine binding assay and those obtained by electrophoretic resolution in sodium dodecylsulfate-polyacrylamide gels. Polyacrylamide gel electrophoresis was also utilized to determine actin levels in developing brains. The percentage of cytoplasmic brain actin also decreased with the age of the rats, from a value of 20% at birth to 10% at day 30, while the percentage of the particulate actin remained constant. The decline in the percentage of cytoplasmic tubulin and actin during brain development can be accounted for by reduction in the proportions of the respective mRNA species. Translation of poly (A)-rich brain mRNA in a wheat-germ cell-free system showed that the percentages of tubulin and actin synthesized decreased gradually with age. Similar results were obtained by analyzing the proteins produced by isolated brain polysomes in a brain cell-free system.

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.

Blot hybridization analysis that used labeled tubulin cDNA probes revealed that N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate [dibutyryl cyclic AMP (Bt2cAMP)] initially increases and later decreases the level of tubulin mRNA in a neuroblastoma-glioma hybrid cell line as well as in the parent cells. A significant increase in tubulin mRNA sequences is already evident 1 hr after the addition of Bt2cAMP to the neuroblastoma cells, and a maximal induction of 2-fold is seen after 12 hr. Continued treatment with Bt2cAMP for 4 days results in a down-regulation of the initial tubulin mRNA level independently of cell density. In the glioma cells Bt2cAMP also rapidly increases the level of tubulin mRNA sequences, reaching a maximum within 6 hr. However, in these cells the subsequent decrease in tubulin mRNA content depends on the culture's phase of growth: cells at the logarithmic growth phase do not down-regulate the tubulin mRNA content even after prolonged treatment with Bt2cAMP, whereas confluent cells do. The hybrid cell line manifests intermediate characteristics in Bt2cAMP regulation of tubulin mRNA level. The time course of induction and down-regulation of tubulin mRNA content observed in the hybrid cells is similar to that of the parent neuroblastoma, whereas the sensitivity to induction is glioma-like and is 8-fold over the initial level. Blot hybridization with labeled actin cDNA probes showed a similar but not identical induction of actin mRNA synthesis with the hybrid and glioma cells, whereas no significant change was observed with the neuroblastoma cells. Moreover, prolonged treatment with Bt2cAMP of all these cell lines did not result in down-regulation of actin mRNA sequences below the initial control value. It was also observed that the level of tubulin sequences in mRNA isolated from 12-day-old rat brain was higher than that in the newborn brain. However, at an age of 30 days, the level of tubulin sequences decreases to about 75% of the newborn level.

Successive stages in the development of the brine shrimp cyst were used as a model for studying differentiation at the level of mRNA transcription and translation. The poly (A)-containing mRNA from dormant cysts and free-swimming larvae (nauplii) was found to be efficiently translated in a wheat-germ cell-free system, and electrophoretic patterns of translation products in vitro resembled those of the endogenous proteins extracted from the equivalent developmental stages. Each stage, however, exhibits a characteristic protein pattern. Two low-molecular-weight proteins prominent in the cyst disappeared almost completely in the nauplius stage, whereas the proportion of actin increased 3-fold. Parallel patterns were observed upon translation in vitro of the respective mRNA preparations. The percentage of the acidic protein, tubulin, decreased somewhat during development.

Tubulin, the subunit protein of microtubules is found in all eukaryotic cells. Microtubules are functionally important for a wide variety of cellular activities such as mitosis, cell shaping, secretion and motility. They are also abundant in the nervous tissue where neurite outgrowth as well as axoplasmic transport are thought to be dependent on microtubules integrity [l] . Detection and quantitation of tubulin is, thus, of importance in trying to understand differentiation processes. It has recently been shown that during postnatal development of the rat brain, there is a decline in the rate of tubulin synthesis as compared to that of the total protein (2-5). The decrease in the relative amounts of this protein occurs in the soluble fraction and is accompanied by a comparable decline in the percentage of its mRNA (3-5). The relative amounts of rat brain [2,5] or chick brain [6] iubulin have been determined by colchicine binding as well as by electrophoretic resolution on polyacrylamide gels followed by densitometric scanning of the stained gels. An alternative method is the detection and quantitation of tubulin by specific anti-tubulin antibodies, which allows a more sensitive assay as tubulin determinations can be carried out also on membrane bound protein not necessarily in its functional form. Specific tubulin antibodies have been used mainly for visualization of the mitotic spindle, as well as of the microtubular network in the cell [7,8] by immunofluorescence

The structure and sequence of rat alpha-tubulin cDNA clone is being described. The 3'-end of the coding region contains the codon for a C-terminal tyrosine, which was previously considered to be post-translationally added to the completed polypeptide chain. A close homology in the coding sequence is observed when a-tubulin from rat and chick are compared, while the 3-non-translated region had diverged considerably.

Antibodies against a single minor base, inosine, have been prepared by immunizing rabbits with a conjugate of inosine and bovine serum albumin. The antisera obtained were fairly specific for inosine, only guanosine among the major nucleosides being capable of cross-reacting with them. By adsorption with guanosine-bovine serum albumin, antibodies with a specificity directed almost exclusively against inosine were obtained. The anti-inosine sera were able to cross-precipitate with polyinosinic acid if the latter was in single-stranded form. Inosine residues in polyinosinic acid paired with cytidine residues of polycytidylic acid, forming a double-stranded helix, were unable to react with the anti-inosine antibodies. The triple-stranded form of polyinosinic acid under stable conditions was also unable to form an immune precipitate with anti-inosine antibodies.

Methionine-starved tRNA was extracted from a “relaxed” methionine-requiring mutant of Escherichia coli grown in a medium deficient in methionine. The methyl-deficient tRNAPhe and normal methylated tRNAPhe species, found in unfractionated methionine-starved tRNA, were separated and extensively purified employing chromatography on reversed-phase columns, benzoylated DEAE-cellulose and methylated albumin silicic acid columns. The purified tRNAPhe species were characterized by their amino acid accepting capacity and extent of methylation. Exposure of normal E. coli tRNAPhe to pH 2.9 led to changes in its chromatographic pattern on methylated albumin-silicic acid columns.

A general chemical method for the purification of amino acid-specific tRNA's is described. The desired tRNA species are enzymatically esterified with a specific aminoacyl-tRNA synthetase. The resulting aminoacyl-tRNA is then separated from the bulk of unesterified tRNA by reacting this mixture in alkaline dioxane-water (0.6:1, v/v) with N-carboxy-β-benzyl-l-aspartate anhydride to yield the corresponding (β-benzyl-l-aspartyl)n-aminoacyl-tRNA((Bzl-Asp)n-aminoacyl-tRNA)). The latter precipitates out from the reaction mixture as a coadsorbate with free poly-β-benzyl-l-aspartate—produced as a result of the action of water—whereas uncharged tRNA chains, which do not react with N-carboxy-β-benzyl-l-aspartate anhydride, remain in solution. A substantial purification of the reacted aminoacyl-tRNA is thus achieved: between 50 and 70% of the starting aminoacyl-tRNA is recovered in the precipitate with a 10–25-fold purification. The (Bzl-Asp)n-aminoacyl-tRNA is separated from the free poly-β-benzyl-l-aspartate by dissolution in phenol and subsequent extraction with a mixture of aq. NaClO4 and chloroform. Once the free poly-β-benzyl-l-aspartate is removed, remaining (Bzl-Asp)n-aminoacyl-tRNA becomes soluble in water. The preferential precipitation of aminoacyl-tRNA is due to specific interaction (during poly-amino acid chain synthesis) between the peptidyl moiety of (Bzl-Asp)n-aminoacyl-tRNA and free poly-β-benzyl-l-aspartate. It was also shown that the peptidyl moiety of (Bzl-Asp)n-Val-tRNA must reach a minimum chain length of about five Bzl-Asp residues before any part of it can be entrapped in the dioxane-water insoluble fraction. The polypeptidyl ester is hydrolyzed by pronase to liberate purified tRNA. About 70% of the biological activity of the enriched tRNA is recovered. The success of the purification depends on various requirements, including the availability of tRNA preparations completely free of nuclease activity and devoid of polypeptide contamination. A procedure for the isolation of tRNA that meets these requirements is described.

Methods for the preparation of various peptidyl-aminoacyl-tRNA derivates are described. Aminoacyl-tRNA's are allowed to initiate the polymerization of different N-carboxy amino acid anhydrides in alkaline dimethylsulfoxide-water (97:3, v/v) to yield the corresponding peptidyl-aminoacyl-tRNA's. Near quantitative yields of ( dl -Ala)n-Val-tRNA, (sarcosyl)n-Val-tRNA, ( l -Asp-β-OBzl)n-Val-tRNA, ( l -Glu-γ-OBzl)n-Val-tRNA, and ( l -Phe)n-Val-tRNA were so obtained. In addition to peptidyl-aminoacyl-tRNA products, the polymerization reaction generates free peptides. The latter products are due to water-initiated polymerizations. Depending on the solubility properties of the particular free polyamino acid involved, one of three procedures can be used to separate it from peptidyl-aminoacyl-tRNA and tRNA. Fractionation of peptidyl-aminoacyl-tRNA away from tRNA is not, however, obtained and the two are recovered together as an aqueous mixture. The length of the petidyl moiety was found to be heterogenous in all the peptidyl-aminoacyl-tRNA derivatives tested. It was never very long relative to its tRNA carrier. The stability of the ester linkage between peptidyl-aminoacyl and polynucleotide in several peptidyl-aminoacyl-tRNA's was also studied. Although different derivatives evidenced different stabilities, all were appreciably more stable then their parent aminoacyl-tRNA compound.

Values of viscosity, sedimentation coefficient, absorbancy at 258 mμ, and optical rotatory dispersion of E. coli ribosomal RNA were measured at different sodium chloride concentrations at 25°. It was found that increasing the sodium chloride concentration led to a decrease in viscosity and absorbancy, and to an increase in sedimentation coefficient and optical rotatory dispersion. Over the range of 5·10 −4 to 1·10 −2 M NaCl the ionic-strength dependence of the viscosity of RNA was found to be greater than could be attributed to the normal polyelectrolyte nature of the molecule, indicating an anomalous contraction of the RNA molecules. The changes in absorbancy and optical rotatory dispersion were also found to take place over this same range of sodium chloride concentrations. It is possible to infer from these results that as organised secondary structure is formed the hydrodynamic volume is diminished. This is consistent with the stabilisation of ordered regions by intramolecular bonds. In support of this interpretation it was found that as the temperature was increased the dependence of the viscosity upon sodium chloride concentration approached normal polyelectrolyte behaviour. An increase in absorbancy at 258 mμ was also noticed. Furthermore when other electrolytes such as magnesium chloride and tetramethylammonium chloride were added to E. coli RNA solutions it was found that changes in absorbancy at 258 mμ and anomalous changes in viscosity were concomitant.

Several criteria have been used to compare mitochondrial ribosomal RNA from the fungus, Aspergillus nidulans, with ribosomal RNA from its corresponding cytoplasm and from Escherichia coli cells. Sedimentation velocity values of mitochondrial rRNA (23.5 S; 15.5 s) resembled those of E. coli rRNA and not of the homologous cytoplasmic rRNA (26.5 s; 17.0 s). However, when samples were analyzed by polyacrylamide gel electrophoresis, mitochondrial rRNA appeared to be larger than bacterial rRNA and similar to cytoplasmic rRNA in size. An explanation for the apparent variance in molecular weight estimation by sedimentation and electrophoretic methods was sought on the basis of configurational differences among the various rRNA species. Thermal denaturation studies were carried out at 260 mμ and 280 mμ in order to assess, separately, the contribution of A.U and G.C residues to the ordered structure of the ribosomal RNA's. Mitochondrial rRNA differed from cytoplasmic and bacterial rRNA's in several features. (1) Over the temperature range 10 to 95 °C, mitochondrial rRNA showed a greater percentage change in relative absorbance at 260 mμ than at 280 mμ while cytoplasmic and E. coli rRNA samples exhibited quite the opposite pattern. Based on these data, the G + C content of the ordered regions in the RNA chain were calculated to be 27 and 32% for the heavy and light mitochondrial rRNA components, 55.5 and 51% for the corresponding cytoplasmic ones and 54.5 and 54% for the bacterial rRNA peaks. (2) Thermal denaturation mid-points occurred at considerably lower temperatures for mitochondrial rRNA (Tm260 = 47 °C; Tm280 = 48.5 °C) than for cytoplasmic rRNA (Tm260 = 54.5 °C; Tm280 = 61 °C) or E. coli rRNA (Tm260 = 56 °C; Tm280 = 60 °C). This is interpreted on the basis of differences in the average number of G.C residues per ordered region of the respective molecules. Nucleotide base composition studies were also carried out. They showed mitochondrial rRNA to have a lower G + C content (32%) than the corresponding cytoplasmic rRNA (51%). It is concluded that Aspergillus mitochondrial rRNA represents a unique and novel molecular species differing profoundly from both cytoplasmic and bacterial rRNA types.

Mitochondria isolated from cysts of Artemia salina (brine shrimp) were found to be devoid of cristae and to possess a low respiratory capability. Hydration of the cysts induces marked biochemical and morphological changes in the mitochondria. Their biogenesis proceeds in two stages. The first stage is completed within 1 h and is characterized by a rapid increase in the respiratory capability of the mitochondria, their cytochrome oxidase, cytochrome b, cytochrome c and perhaps some morphological changes. In the second stage there is an increase in the protein-synthesizing capacity of the mitochondria as well as striking changes in mitochondrial morphology leading to the formation of cristae.

Polynucleotide phosphorylase from Escherichia coli cells catalyzes the transfer of a single nucleotidyl residue from each of the 2′(3′)-O-isovalerylesters of ADP, GDP, CDP and UDP to the initiator oligonucleotide, A-A-A. The products of these reactions are identified as the 2′(3′)-O-isovaleryl esters of the corresponding tetranucleotides A-A-A-A, A-A-A-G, A-A-A-C and A-A-A-U. The 2′(3′)-O-isovaleryl residue can be removed from the product by treatment with aqueous methanolic ammonia under conditions that do not significantly damage the oligonucleotide chains. The monoaddition of 2′(3′)-O-acyl esters of nucleoside diphosphates to an oligonucleotide acceptor is proposed as a method for the stepwise synthesis of oligoribonucleotides of defined sequence.

Snake venom phosphodiesterase, bovine spleen phosphodiesterase and polynucleotide phosphorylase are currently used for sequence analysis of oligoribonucleotides. These exonucleases remove mononucleotide residues in a sequential order from either the 3’or 5’-end of an oligonucleotide. The nucleotide sequence of a given oligonucleotide is then derived by analyzing the partially degraded oligonucleotides which appear in the course of the reaction (cf. [l-3] ). These procedures, while very useful for analyzing labeled oligonucleotides, are inadequate when only trace amounts of non-labeled material are available. In the present report we describe a novel and sensitive method for sequence analysis of non-labeled oligoribonucleotides. The method is based on the property of polynucleotide phosphorylase to phosphorolyze short oligonucleotides from the 3’-end in a stepwise fashion by a nonprocessive mechanism [4-61. The final products of the reaction consist of a mixture of nucleoside diphosphates (NDP’s) and a limit oligonucleotide that is not further degraded by the enzyme. Depending on its base composition, this oligonucleotide is a dior trinucleotide. By following the order in which the released NDP’s appear during the phosphorolysis of a given oligonucleotide, it is possible to determine the nucleotide sequence from the 3’-end up to 2-3 residues from the 5’-terminus. The high sensitivity of this method is achieved by including 32Pi in the phosphorolysis medium. Upon digestion, the label is incorporated in the P-phosphate moieties of the generated NDP’s which can be easily

A method for the purification of polynucleotide phosphorylase from Escherichia coli has been developed. The purified enzyme has a specific activity 700-fold higher than the crude extract. When enzyme fractions obtained at different stages of the purification were assayed by phosphorolysis of polyadenylic acid (poly A) or 32P-orthophosphate exchange with the 5'-diphosphates of adenosine, uridine, cytidine, guanosine, and inosine, the degree of purification at each step was approximately the same. Various factors affecting the exchange reactions between labeled orthophosphate and different ribonucleoside diphosphates were examined. An activating factor which enhances the rate of the ADP-Pi exchange reaction 3- to 6-fold was isolated during the purification of the enzyme. It affects only the exchange reaction and not the phosphorolysis of poly A. It was found to be susceptible to proteolytic enzyme action, but not to RNase degradation. The effect of the dinucleoside monophosphate ApA on the polymerization and exchange reactions was studied. At low Mg++ concentration, it could overcome the lag period in the polymerization of ADP and also stimulate the rate of the ADP-Pi exchange reaction. The results presented in this report support the hypothesis that the polymerization and exchange reactions are catalyzed by the same enzyme. Their relation to the mechanism of the exchange reaction is discussed.