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.

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.

MICROTUBULES are present in all eukaryotic cells and have been found to have a variety of structural and dynamic roles in cell shape, division, motility, transport and secretion1. In nervous tissue neurite outgrowth and axoplasmic transport are also thought to depend on microtubule integrity2. The micro-tubule subunit protein, tubulin, is a heterodimer composed of two polypeptides α and β (refs 3,4). The α and β subunits show microheterogeneity and both have been resolved into two or three components5–8. The question therefore arises as to whether changes occur in the relative proportions of the multiple forms of tubulin upon assumption of different roles within the nerve cell. We show here that cytoplasmic rat brain tubulin, as resolved by isoelectric focusing, is highly heterogeneous. Moreover, tubulin microheterogeneity seems to be developmentally determined, increasing from seven to nine distinct components during early postnatal rat brain maturation. However, extensive tubulin microheterogeneity is prominent in the brain, as tubulin from other organs is less heterogeneous.

Dormant Artemia salina cysts are almost devoid of polysomal structures but contain appreciable quantities of mRNA that sediments mainly as a 40S complex in sucrose gradients. The mRNA can be isolated from this complex and efficiently translated in a wheat germ cell-free system, although the 40S complex itself is inactive. During rehydration of the cysts, mRNA becomes increasingly involved in polysomal complexes which can be actively translated in the cell-free system.

Hexamethylene bisacetamide (HMBA), a potent inducer of erythroid differentiation in murine erythroleukemia cells (1), induces differentiation in mouse neuroblastoma cells, as indicated by the extension of neurites and the development of an excitable membrane. HMBA is effective at concentrations 50-fold lower than dimethylsulfoxide (2), another inducer of differentiation in both mouse neuroblastoma and murine erythroleukemia cells.

Affinity chromatography on anti-Y (Y is a tricyclic imidazopurine to which is attached a complex four-carbon side chain) antibody immobilized to Sepharose was used to determine the proportion of rat liver tRNAPhe species containing the peroxy Y-nucleoside. Unfractionated Unfractionated mammalian tRNA was aminoacylated with labeled phenylalanine. The phenylalanyl-tRNA was then chemically acetylated to yield N-acetylphenylalanyl-tRNA. When this preparation was applied to the antibody column, between 6-10% of the radioactivity was not bound to the column, indicating a deficiency of peroxy Y-nuceloside in a minor isoaccepting tRNAPhe species. In contrast to normal tissues (including embryonic tissue), about 85% of the tRNAPhe from mouse neuroblastoma C-1300 or N-18 tumors lack the peroxy Y-base, a property which is not affected by tumor age. Rat liver labeled N-acetylphenylalanyl-tRNA preparations were resolved on Plaskon chromatography (RPC-5) into two minor peaks closely followed by a mojor component. A high proportion of the two minor tRNAPhe species was unable to bind to anti-Y antibodies. Upon mild acid treatment, the minor and major tRNAPhe species eluted simultaneously from Plaskon columns, at a much reduced salt concentration. These results would indicate that the two minor tRNAPhe species from rat liver as well as the major component contain a tricyclic imidazopurine base that differs from each other in its side chain. About 85% of the N-acetylphenylalanyl-tRNA from neuroblastoma was resolved by Plaskon chromatography as an early eluting peak. The position of this major neuroblastoma tRNAPhe species was not altered by mild acid treatment, and its elution position from the column almost coincides with that of acid-treated normal rat liver tRNAPhe. The latter results would suggest that most of the tRNAPhe chains from neuroblastoma lack the tricyclic imidazopurine of normal rat liver tRNAPhe, but are very close if not identical in primary nucleotide sequence.

A method has been developed for the removal of the 3′-poly(A) tail from mRNA chains. It is based on synchronous processive phosphorolysis of mRNA using a molar excess of polynucleotide phosphorylase at 0°. This method enabled to determine the location, length and cytoplasmic role of the poly(A) tail of globin mRNA. When injected into Xenopus oocytes, deadenylated rabbit globin mRNA was shown to be translated for a relative short period and then rapidly degraded. Native poly(A)-containing mRNA, however, is considerably more stable in the same conditions and is translated for extended periods of time. The poly(A) stretch must contain a minimal number of about 30 adenylate residues to ensure its protective function. The increased stability conferred upon globin mRNA by the presence of the poly(A) tail is not a general phenomenon. Thus the translational stability in oocytes of poly(A)-containing mengovirus RNA is indistinguishable from that which is poly(A)-deficient. A regulatory role for the heterogenous 3′-end sequence in the translation of carnation mottle virus (CarMV) RNA was revealed. CarMV RNA seems to be polycistronic as it is translated in vitro into three discrete polypeptide chains (CP-I CP-II and CP-III) of which CP-II is identical with the viral coat protein. A selective reduction in the translation efficiency of CP-I could be elicited by controlled phosphorolysis of the 3′-terminus of CarMV RNA chains. This was paralleled with a gradual loss of infectivity. On the other hand phosphorolysis of only 5-10 nucleoside residues from the 3′-terminus of tobacco mosaic virus RNA eliminated its infectivity in tobacco leaves.

Murine neuroblastoma cultures were labeled externally with the cationic reagent N,N,N-[3H]-trimethylamino-beta-alanyl-N-hydroxy-succinimide ester ([3H]Me3N-beta Ala-NSuc) or with 125I/lactoperoxidase. The cells were labeled in the logarithmic and confluent growth phases as well as in a highly differentiated state following treatment with 2% dimethylsulfoxide. The labeled exterior membrane proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Major changes in the exterior membrane proteins were observed during maturation and differentiation of the cells. Most of these changes were clonal-specific, while others were common to several clones. Two proteins of Mr 55,000 and 65,000 were labeled by both 125I/lactoperoxidase and Me3N-[3H]-beta Ala-NSuc. The level of labeling was dependent on the clonal lines used and the state of the cell maturation. A group of proteins displaying a molecular weight between 150,000 and 200,000 was found to be related to the transition of a culture from logarithmic to confluent growth phases. An additional protein, with an apparent molecular weight of 95,000, was common to differentiated cells of the two inducible clones used. In general the maturation of logarithmic phase cells into confluent cells resulted in a less complex electrophoretic distribution of the pattern of labeling. After dimethyl-sulfoxide treatment, further reduction in the complexity of the externally labeled proteins was observed.

The distribution and subcellular localization of microtubules in rat brain cerebellum was analyzed by immunohistochemistry with antibodies prepared against 3 synthetic peptides corresponding to the C-terminal region of beta-tubulin. The peptides used correspond to amino acid positions 416–425 (peptide 1), 416–431 (peptide 2), and 426–445 (peptide 4). The antibodies thus obtained displayed a remarkable specificity in reacting with different cell types in the rat cerebellum. Antibodies directed against peptide 1 primarily stained Purkinje cells and their dendrites and axons. Peptide 2 antibodies preferentially stained the glomeruli, while antibodies directed against peptide 4 preferentially stained Bergmann glial fibers. These results are discussed in terms of dissimilarities in microtubule organization and masking of epitopes by microtubule-associated proteins (MAPs) in individual cerebellar cells, which may be related to specific functional properties.

The increased rate of synthesis of the “estrogen-induced protein” (IP) first detected by Notides and Gorski is one of the earliest macromolecular responses to estrogen by the rat uterus. The IP detected by autoradiography of sodium dodecyl sulfate polyacrylamide gel electropherograms was shown to correspond to a stainable protein band in electropherograms of cytosol from uteri of both untreated and estrogen-treated rats. The uteri of 9–10-day-old rats showed a maximum rate of IP synthesis 1 h after estrogen-treatment, followed by a decrease to constitutive levels by 24 h. The rate of synthesis of IP was doubled by estrogen administration to 10–30-day-old rats. The improved sensitivity of the autoradiographic method permitted the detection of some stimulation of IP synthesis in uteri of 5-day-old rats, in contrast to our previous findings in Wistar rats using a double isotope labelling technique.

Nucleotide sequence analysis of two rat alpha-tubulin cDNA clones showed a marked divergence in their 3'-untranslated regions. However, each of the alpha-tubulin isotypes shows a high interspecies homology in this region, when compared with an isotubulin sequence from human and Chinese hamster. In situ hybridization of rat cerebellum with alpha-tubulin cDNA revealed differential expression in various cell layers. The mitotically active cells in the external granular layer show the highest level of alpha-tubulin mRNA, while lower levels are observed in the migrating cells in the molecular layer and in the differentiating cells in the internal granular layer. Very low levels of the mRNA are observed in the prenatally differentiated Purkinje cells.

Carnation mottle virus (CarMV) RNA was translated in a wheat-germ, cell-free system into three discrete polypeptide chains of 77,000 (CP-I), 38,000 (CP-II) and 30,000 M r (CPIII). The size of the three polypeptide products represents the translation of almost the entire length of the viral RNA. The CP-II in vitro product was identified as the viral coat protein by gel electrophoresis under denaturing conditions, inununoprecipitation with antibodies directed against the disrupted viral particles, and by peptide mapping. The discrete nature of the three proteins was shown by peptide analysis of the proteolytic products, the absence of cross-reaction between antibodies against CarMV-disrupted particles and CP-I or possibly CP-III, and by the order of their appearance during in vitro translation. Limited phosphorolysis of the 3′-terminus of the viral RNA chains by Escherichia coli polynucleotide phosphorylase caused the selective disappearance of the 77,000 protein product, paralleled with a gradual loss of infectivity. These observations might indicate that the viral RNA is polycistronic, and suggest that the structure at the 3′-terminus of the carnation mottle virus RNA may have a regulatory role in the translation of the viral RNA chains.

Monoclonal antibodies against microtubule-associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by alpha-chymotrypsin to generate a series of high-molecular-mass fragments ranging between 270 and 140 kDa. The precursor-product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270-kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215-kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270-kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule-binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60-kDa domain(s) distal to the microtubule-binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.

A method for the purification of rat liver histidyl-tRNA synthetase was developed. The enzyme, freed of trace nuclease contamination by affinity chromatography on DNA-Sepharose, could catalyze the aminoacylation of tobacco mosaic virus (TMV) RNA with histidine. The aminoacylation reaction of TMV RNA was inhibited by KCl, while that of rat liver tRNA exhibited a maximum rate at 140 m M KCl. TMV N -acetyl [ 3 H]histidyl-RNA was prepared in order to stabilize the TMV histidyl-RNA ester bond. The TMV N -acetyl [ 3 H]histidyl-RNA showed a migration identical to that of unacylated TMV RNA upon electrophoresis in polyacrylamide agarose gels. Removal of 5 to 10 nucleoside residues from the 3′-terminus using Escherichia coli polynucleotide phosphorylase or oxidation of the 3′-terminal ribose with periodate (to produce TMV RNA ox ) eliminated the aminoacylation capacity of TMV RNA as well as its infectivity in tobacco leaves. It is concluded that the aminoacylation site is located at the 3′-terminal adenosine of TMV RNA. Fragmentation of TMV RNA ox with a crude rat liver enzyme did not expose additional sites for acylation with histidine, nor did it produce acylation capacity for other amino acids. The reduction of TMV RNA ox with sodium borohydride converted the terminal dialdehyde group to a dialcohol-lacking covalent bond between the C 2 ′ and C 3 ′ of the terminal adenosine, but did not restore its ability to accept histidine or its infectivity. Similar reduction of rat liver tRNA ox to tRNA ox-red , however, did restore its ability to accept phenylalanine while activity for histidine was not regained.

The physical and functional properties of the mRNA population from developing embryos of the brine shrimp Artemia salina were characterized. About 20% of the total poly(A)-rich mRNA in these embryos appears to be specifically associated with the membrane fraction throughout early development, and physically differs markedly from the free cytoplasmic mRNA. The membrane-associated mRNA fraction consists of two well-defined populations of molecular weight of 5.2x10(5) and 3.6x10(5), whose relative amount changes during the various stages of embryo development. The size of the poly(A) tail at the 3'-end of the mRNA molecules, as estimated by processive phosphorolysis, was found to consist of 180 and 210 adenosine residues for the two respective mRNA species. The in vitro translation products of the membrane-bound mRNA molecules are apparently similar to those of the free mRNA molecules.

Tubulin, the subunit protein of microtubules, is a heterodimer composed of two polypeptides OLand /3of -55 000 1M, each [ 1,2]. The LYand @bunits show microheterogeneity and both have been resolved into several components [3-71. We have recently shown that cytoplasmic tubulin microheterogeneity is most prominent in the brain and increases with rat brain maturation [8]. Tubulin is not only confined to the cytoplasm, as it is found to be associated with various membranes [9-131 including the presynaptic membranes [ 14,151. It was therefore of interest to determine the presence and properties of membrane bound tubulin from brain and compare it to tubulin from the cytoplasmic fraction. By the criteria of molecular weight, isoelectric point, peptide mapping and vinblastine binding we show, in the present study, that synaptosomal membranes isolated from rat cerebral cortex contain significant amounts of tubulin. Furthermore, we find that the (Yand /3-tubulins differ in their association with these membranes.

A partially purified fraction of poly(a)-rich brain mRNA coding for tubulin and actin was obtained by stepwise elution from an oligo(deoxythymidylate)-cellulose column and was efficiently translated in a wheat-germ cell-free system. The newly synthesized tubulin and actin migrated along with the authentic proteins on sodium dodecyl sulfate polyacrylamide gels and on sodium dodecyl sulfate-urea polyacrylamide gels, where tubulin alpha- and beta-subunits are separated. The two proteins synthesized in vitro were found to be biologically active; they could be induced to polymerize and were both precipitated by vinblastine. In addition, specific binding of tubulin to an affinity column of colchicine-Sepharose and actin to myosin were demonstrated.