Developmentally regulated cDNA expressed exclusively in neural tissue

Molecular Brain Research, 10 (1991) 33-41 Elsevier

33

BRESM 70278

Developmentally regulated cDNA expressed exclusively in neural tissue David E Wieczorek and Stephen R. Hughes Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Oncinnati, OH 45267-0524 (U.S.A.) (Accepted 6 November 1990) Key words: Brain-specific cDNA; Developmentally regulated; Tropomyosin related

A cDNA clone, labeled C1-13, isolated from an adult rat brain cDNA library, has been characterized and found by Northern blot and S1 nuclease mapping experiments to be solely expressed in neuronal tissue, principally, but not exclusively, in the brain. The associated mRNA is first detected in embryonic life, reaches maximum levels of expression at birth, and remains expressed in the adult. Northern blot analysis shows the transcript is not localized to one particular area of the brain, but is present in numerous regions. Low message levels of this transcript are also found in the peripheral nervous system, demonstrating that the expression of the associated gene is not restricted to the central nervous system. In addition, results indicate expression is limited to neuronal cells, and is not detected in glia. The identification of the cDNA clone C1-13, which possesses limited nucleotide homology to tropomyosin, is exciting, particularly considering the neural-specific expression that it manifests and the unique cytoskeletal and motile properties exhibited by neurons. INTRODUCTION The growth, development, and differentiation of eukaryotic cells and tissues are regulated by complex mechanisms of gene expression which govern the assembly, maintenance, and disaggregation of numerous proteins comprising the cytoskeletal architecture. The eukaryotic cell ultrastructure consists of three major elements: microtubules, microfilaments, and intermediate filaments. These three filamentous systems, in association with both c o m m o n and cell/tissue-specific proteins, are necessary and responsible for the assembly and maintenance of cytoskeletal architecture and cellular movements. These cellular movements include intracellular movements and positioning of organelles (i.e. cytokinesis and axoplasmic transport), in addition to intercellular positioning which occurs during cellular growth and differentiation. Tropomyosin (TM) plays an important role in the regulation of contractility of muscle and nonmuscle cells. This ubiquitous protein forms a coiled-coil dimer and exists in several different forms (a, fl, TM4 and TM30) 14' 23.33 with molecular weights ranging from - 3 0 to 50 kDa. Although cytoskeletal TM is an essential actin-binding protein associated with microfilament structures of the cytoskeleton, its precise function in determining the

architecture of the cell and modulating its contractile activities is unknown. The nervous system is composed of a great variety of different cell types which exhibit a complex array of morphological and physiological properties. This diversity of cell structure and function is achieved through a genetic regulatory system which generates approximately 145,000 different R N A transcripts 28. Because of the unique cellular ultrastructures associated with many of the different cell types in the nervous system, we probed a c D N A library made from rat brain R N A with a highly conserved region of a-TM. Results show the gene corresponding to one of the c D N A s we isolated is expressed solely in nervous tissue, predominantly in the central nervous system, but to a lesser degree, also in the peripheral nervous system. The associated transcript is initially detected during fetal development, peaks in expression at birth, and is maintained in the adult. Furthermore, studies demonstrate that this m R N A is found in numerous regions of the brain, and is not restricted in its expression to one particular anatomical site. Nucleotide sequence analysis of this c D N A shows it possesses an open reading frame capable of encoding 267 amino acids with a 3' untranslated region of 560 nucleotides. Although nucleotide homology between this c D N A and defined TM c D N A s is limited, the identification and

Correspondence: D.F. Wieczorek, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0524, U.S.A. 0169-328X/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

34 characterization of a brain-specific TM-related c D N A may help to unravel u n k n o w n features associated with the unique ultrastructure and intracellular movements of the nervous system.

MATERIALS AND METHODS

Screening of brain cDNA library A rat brain cDNA library (courtesy of G. Shull) was constructed in PstI-digested, G-tailed, pBR322 vector as described34. Duplicate filters from each plate were screened with radioactively labeled22 a-TM-purified cDNA insert corresponding to amino acids 120-284 and 3' untranslated sequence of the nonmuscle/cytoskeletal isoform23'3t. Filters were washed 3 times at 55 °C with 2 × SSC, 0.2% SDS. Only colonies which were positive on both of the duplicate filters were chosen for further analysis. Each clone was subsequently screened another 3 consecutive times to insure single colony isolation and hybridization.

Restriction endonuclease mapping and DNA sequence

80% deionized formamide-400 mM NaCI-10 mM PIPES (piperaacid)) (pH 6.4)-0,05% sodium dodecyl sulfate-1 mM EDTA. The hybridization mixture was incubated at 65 °C for 1 h, the temperature was adjusted to 42 °C, and the incubation continued for 16 h. S1 nuclease (100 U; New England Nuclear Corp., Boston, MA) in 300/~1 of 200 mM NaCI-30 mM sodium acetate (pH 4.5)-3 mM ZnSO4 was added to each sample and incubated at 25 °C for 1 h. The reaction was terminated with 10 mM EDTA and precipitated with ethanol. Dried pellets were dissolved in 80% formamide and electrophoresed on a 6% polyacrylamide-8 M urea sequencing gel. The gel was dried and exposed for autoradiography on Kodak X-Omat AR film.

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RESULTS

Isolation o f c D N A Cl-13 A radiolabeled probe was developed consisting of purified insert from an a - T M c D N A corresponding to amino acid 120 and extending into the 3' untranslated sequence of the cytoskeletal/smooth muscle isoform 23'3~. Using this probe, we screened duplicate filters of an adult rat c D N A library prepared from brain tissue. The hybridizing c D N A clones were screened 3 successive times to insure hybridization and individual plasmid colony purification. Based u p o n restriction map and

cDNA clone C1-13 DNA was isolated and digested with the restriction endonucleases PstI, EcoRl, HindIII, BamHI, SacI, PvulI, and AvaI. The relative position of each restriction site was determined by enzyme double digests separated on a 0.8% agarose gel and a 6% polyacrylamide gel. DNA sequence analysis was performed as described by Maxam and GilbertTM and by the dideoxy chain termination method of Sanger24. Nucleotide and deduced amino acid analysis was conducted using the DNANALYZE sequence analysis29 and Intelligenetics' PCGene systems. Both the nucleotide and the deduced amino acid sequences were compared with sequences reported in GenBank.

Southern blot analyses, we isolated 5 sets of overlapping clones. From nucleotide sequence analysis, 4 of these sets

Cell culture conditions

display significant homology to known T M isoforms, including a - T M , TM4, and TM3014'23'33. However, one

Cell cultures were established for RNA isolation and analysis according to standard procedures2°'21'31. The cell density was allowed to become 95% confluent prior to RNA isolation. Cell lines which were cultured included muscle (L6E9 myoblasts and myotubes2°), fibroblast (rat 3T3), kidney fibroblast (NRKTs371), hepatoma (HaAzC2), and PC12 pheochromocytoma cells. Primary rat brain neuronal and glial cell cultures were established separately from 13.5 day embryos and 1 day old neonates, respectively, using standard procedures z'32. Dissociated cell cultures of 13.5 day fetal rat forebrain were established and grown in 10% horse serum in DMEM; dissociated glial cell cultures were established from the cerebral cortex and serially passaged 3 successive times in DMEM containing 10% fetal calf serum, prior to RNA isolation.

RNA isolation and analysis Total cytoplasmic RNA was isolated from in vitro cultured cells according to established methods7. Total RNA was obtained from rat tissues using modifications of the hot phenol procedure 26. The procedure of Thomas27 was used for the blotting and hybridization of RNAs electrophoresed in formaldehyde-agarose gels. Spectrophotometric measurements and agarose gels were run to ensure that the quantitative amounts of the RNAs from the various samples were identical. Each sample lane was loaded with 10 ,ug of total RNA, except in Fig. 2, where the various brain RNA sample lanes contain 15 #g each of total RNA. Radioactive cDNA probes used in Northern blot hybridizations were prepared by nick-translation of purified cDNA insert sequences22. RNA-DNA hybridization followed by S1 nuclease mapping analysis was performed according to the method of Berk and Sharp3, under the conditions used previously3°. In brief, the probe was generated by digestion with restriction endonucleases (PstI and SacI) and labeled at the 3' termini with [a-32P]ddATP (Amersham) and terminal transferase (Bethesda Research Labs). The DNA strands were separated, and the strand complementary to the mRNA was purified. Total cellular RNA (25/~g) was hybridized to 2 × 104 cpm of probe in 25 al of

clone set, C1-13, was selected for further analysis because its endonuclease restriction pattern (Fig. 1A), nucleotide sequence (Fig. 1B), and tissue specificity (based on Northern blot analysis (Fig. 2A)) differed from previously identified T M c D N A clones.

Nucleotide and amino acid sequence analysis o f Cl-13 To determine the nucleotide and corresponding amino acid sequences encoded within C1-13, D N A sequence analysis was conducted on this c D N A clone. The entire clone was sequenced by the Maxam-Gilbert method, and by the dideoxy chain termination method after subcloning into M13 phage. As seen in Fig. 1B, the nucleotide sequence of C1-13 is 1475 nt long, containing an open reading frame of 267 amino acids, beginning at nucleotide 112 and extending to nucleotide 910. The 3' untranslated region is 560 nt long and contains a polyadenylation signal sequence (double underlined) at the 3' end of the c D N A . Also, the nucleotides encoding and immediately flanking amino acid codon 1 (met) demonstrate similarity to the Kozak consensus sequence for the initiation of translation ( C C A C C A A G A U G G vs

C C G C C ( A / G ) C C A UGG) 12. Since the c D N A clone C1-13 was identified and isolated by its hybridization to a purified c D N A insert of a - T M , we conducted a nucleotide and amino acid comparison

35 between C1-13 sequences and a-TM sequences. The nucleotide comparison of C1-13 surprisingly revealed only limited nucleotide homology with the a-TM sequences used to initially identify the clone. Southern blot analysis of C1-13 cDNA sequences demonstrates that a-TM sequences hybridize to the internal PstI-SacI fragment; by computer analysis, the homology in this region ranges from 64 to 76%, but is limited to stretches of 18-30 nucleotides. Using a computer analysis, a comparison was conducted at the amino acid level between the coding regions of C1-13 and defined TMs. No significant amino acid homology was found between C1-13 and the various tropomyosin proteins, although both the size of the message (1475 nt) and the deduced protein (267 aa) are similar to those of defined TMs. Thus, the homology exhibited at the nucleotide level between C1-13 and a-TM is limited, but significant enough to permit the isolation of C1-13, together with other well-defined TM cDNA clones. An analysis conducted on the Cl-13-derived amino acid sequence revealed some very interesting features. A nucleotide and amino acid comparison of C1-13 sequences to sequences reported in Genbank revealed no similarities. As previously mentioned, there is an open reading frame capable of encoding 267 amino acids (Fig. 1B). Within this coding region, according to the Eisenberg, Schwarz, Komaromy, and Walk Hydrophobic Moment Plot 5, there are two potential transmembrane domains (Fig. 1B, underlined). These regions are located between codons 26-43 and 128-148. Based upon the ChouFasman Hydropathy Analysis 4, the amino acid sequences both flanking and between these transmembrane domain regions exhibit a-helicies. In addition, two potential N-linked glycosylation sites are found in the amino acid sequence (Fig. 1B, boxed), located at codons 107-109 and 217-219, which positions the first site between the transmembrane domains and the second site to the carboxy side of the molecule. No consensus sites for phosphorylation were detected. The significance and use of the potential a-helical, transmembrane, and glycosylation sites in the associated protein are the subject of future studies. Expression of Cl-13 is restricted to brain tissue To determine the tissue specificity of C1-13 expression, total RNA was isolated from a variety of cells and tissues and blotted to nitrocellulose after agarose gel electrophoresis. 32p radiolabeled insert from C1-13 was prepared by nick-translation and used as probe in Northern blot analysis. As seen in Fig. 2A, a single prominent band is detected only in the 'brain' RNA lane, and not in other muscle or nonmuscle tissues. This band corresponds to a message size slightly less than 18S, which is in accord with

the size of C1-13, namely 1475 nt. In addition to detecting this prominent transcript, a faint band of higher molecular weight is also visible. This band (visible in several of the Northern blot autoradiograms) might represent a processing intermediate, an alternatively spliced transcript or cross-hybridization to another brain-specific message. Experiments are currently in progress to resolve this issue. To demonstrate that equivalent amounts of RNA were loaded for each sample and that the RNA was of high quality, this same Northern blot was erased of radioactive probe and the blot was re-hybridized with a cDNA probe (pA50) which encodes a message from a 'housekeeping' gene 8. As shown in Fig. 2B, all of the RNAs hybridized well with the pA50 probe with the exception of the 'Fibroblast' RNA sample. This RNA is obtained from the rat 3T3 fibroblast cell line and apparently expresses this message only at low levels. The ethidium bromide stained gel confirmed the integrity and the loading of an equivalent amount (10/zg) of rat 3T3 fibroblast RNA with the other samples (data not shown). Combined with the Northern blot analysis using the pA50 probe, these results confirm the integrity and loading of equivalent amounts of RNA for the various samples. To confirm the tissue specificity of C1-13 expression, an S1 nuclease mapping experiment was conducted. The probe used in this analysis was 564 nt long, 3' end-labeled at nt 414 (corresponding to codon 102), and extended into the 3' untranslated region of the cDNA sequence. The probe was hybridized to RNA from various cells and tissues, including brain, and the hybridization products were digested with S1 nuclease (Fig. 2C). Full protection of this probe occurs only in brain tissue, and not in any other cell or tissue, thus confirming the results obtained by Northern blot analysis. The faint signal in the uterus RNA sample is due to leakage of brain-protected message into the lane. The results of the S1 nuclease analysis (Fig. 2C) and the Northern blot hybridization (Fig. 2A) demonstrate that the gene represented by C1-13 shows a narrow tissue-specific expression that is brainspecific. The developmental profile of expression for C1-13 was addressed by probing RNA isolated from brain of various developmental stages. Total RNA from fetal (16 day embryos), newborn (3 h), neonatal (7 days postpartum), and adult (3 month) rats was isolated and hybridized in a Northern blot analysis to 32p-radiolabeled C1-13 nucleotide sequences. As seen in Fig. 3A, initial expression of these transcripts occurs during embryonic development, with maximum expression of C1-13 message detected during the newborn stage. As noted previously, this message continues to be expressed in the adult. From these results, it is reasonable to conclude that the gene

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Fig. 1. Restriction-endonuclease map (A) and nucleotide and amino acid sequences (B) of C1-13. A: restriction map analysis and sequencing strategy of the cDNA insert in C1-13. The left-to-right (5'- to-Y) orientation corresponds to the mRNA strand. The horizontal arrows indicate the direction of each sequenced fragment. The restriction sites are P (PstI), R (RsaI), HC (HinclI), S (SacI), and D (DralII). B: the coding and untranslated nucleotide sequences of cDNA C1-13and its derived amino acid sequence are shown. The nucleotide and derived amino acid sequences are written in 5' to 3' (NH2 to COO--) direction. The two potential hydrophobic transmembrane regions are underlined and the two potential glycosylation sites are boxed. The polyadenylation signal sequence is double underlined. The cDNA nucleotide sequence had been given accession number X52817 by the EMBL data library. represented by C1-13 is constitutively expressed during all developmental stages of the rat brain. Confirmation that equivalent amounts of the different brain R N A samples were present in each lane was conducted by hybridizing this same filter with radiolabeled pA50 c D N A insert, a probe for a 'housekeeping' gene s. Results shown in Fig. 3B demonstrate equal expression of this 'housekeeping' message in the brain samples from the different developmental stages. This expression was greater than that observed for the other non-neuronal R N A samples because 15/~g of total R N A per sample was employed as opposed to 10 g g for the other samples (which serve as negative controls). As previously mentioned, expression of pA50 in fibroblast R N A (rat 3T3 cells) is very low, yet visible with longer exposure of the film. A n experiment designed to analyze the regional localization of C1-13 transcripts within the brain was con-

ducted. Total R N A was prepared from various regions of the rat brain (cortex, hypothalamus, diencephelon, corpus striatum) to ascertain whether one specific area of the brain solely expresses the C1-13 associated gene or whether C1-13 is expressed in many different regions. These R N A s were probed in Northern blot analysis for C1-13 expression (Fig. 4). Results from these studies demonstrate that C1-13 transcripts are expressed in numerous regions of the brain and are not confined in its expression to one specific location. The level of expression varies among the different areas, however, expression of the associated gene does occur in all regions which were examined. To ascertain whether Ci-13 expression was restricted to the central nervous system, R N A hybridization experiments were conducted on peripheral nervous system (PNS) tissue, whose neuronal composition differs from that of the central nervous system. Spinal root ganglia of

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the PNS from adult rats were isolated and the RNA analyzed in Northern blot analysis. Upon long exposure of the autoradiogram, results indicate that C1-13 is expressed at low, but significant levels, in the spinal root ganglia (Fig. 5). This result demonstrates that in vivo expression is not restricted to the central nervous system, but also occurs in the PNS. PC12 cells, an immortalized cell line derived from rat neural crest cells, exhibit morphological and biochemical properties similar to neuronal cells of the PNS in culture 9. When RNA isolated from PC12 cells, grown in either the presence or absence of nerve growth factor, was analyzed in Northern blot analysis for expression of C1-13, the transcription and accumulation of this message was clearly visible with long exposure (7 days with an intensifying screen) (Fig. 5). Thus, the results of Fig. 5 clearly demonstrate that the expression of the gene represented by C1-13 is predominantly, but not exclusively, restricted to the central nervous system. Studies were conducted to distinguish which major cell type within the brain expresses the C1-13 associated gene: neuronal or glial cells. Primary neuronal cell cultures from 13.5 day rat brain embryos were established using standard procedures 2. Under the established conditions, these cultures differentiate and morphologically demonstrate the appearance of a predominantly neuronal cell population (see below). Glial cell cultures, established from 1 day old rat pups 32, were serially passaged and morphologically appeared to be exclusively glial cells. Total RNA was harvested from both sets of cultures and analyzed in Northern blots. Results show that C1-13 transcripts are solely expressed in the neuronal cell population and are not detected in the RNA from pure glial cell cultures (Fig. 6). From this result, it is

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Fig. 2. Tissue-specific expression of C1-13 with various tissue and cellular RNAs. A: Northern blot hybridization of C1-13; 10/tg of total RNA from rat tissues were size-fractionated in 1% agarose, 3% formaldehyde (v/v) denaturing gels, transferred to nitrocellulose filters, and hybridized to 32p-labeled C1-13 cDNA. Myoblast and myotube RNAs are from rat L6E9 muscle cells~S; RSM is adult rat skeletal muscle; cardiac, uterus, kidney, brain, and liver represent RNAs from the respective tissues obtained from adult animals; kidney fibroblast is RNA from NRKTs371 cells; hepatoma is RNA from H4AzC2 cells; fibroblast is RNA from rat 3T3 cells; and primary fibroblast is RNA from fetal rat skin fibroblasts. B: Northern blot autoradiogram of pA50, a 'housekeeping' cDNA with tissue and cellular RNAs. The nitrocellulose filter seen in A was stripped of radioactive signal and re-probed with a purified cDNA insert of pA50, which hybridizes to a 'housekeeping' transcript. RNA samples are as labeled in A. C: S1 nuclease protection analysis of cDNA CI-I3 with mRNAs from various cells and tissues. The 564 nt long probe was end-labeled at nucleotide 414 (corresponding to codon 102) and extends into the 3' untranslated region. The faint signal in the uterus RNA sample is due to leakage of brain-protected message into the lane and/or slight reannealing of the probe to itself. HaelII-digested ~X nucleotide size markers are indicated on the left.

39

reasonable to assume that expression of the gene represented by C1-13 is neuronal specific. Additional support for this conclusion resides in the fact that cell cultures prepared under similar conditions and stained with antibodies demonstrated that greater than 75% of cells in the glial cell cultures stained positive with an antibody to GFAP (glial fibrillary acidic protein), and greater than 90-95% of cells in the neuronal cell cultures stained positive with a neurofilament antibody (R. Akeson, personal communication). By integrating the results obtained from Fig. 6 with those from Fig. 3, it is reasonable to conclude that the developmental profile of C1-13 expression mimics the development and differentiation which occurs with neu-

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Fig. 3. Developmental regulation of brain-specific C1-13 expression. A: Northern blot autoradiogram of RNAs hybridized to C1-13 cDNA. RNAs are as listed in Fig. 2 with the following additions: fetal brain RNA is from embryonic rat 16 day fetuses; newborn brain RNA is from 3 h old rat pups; and neonatal brain RNA is from 7 day old rat neonates. B: Northern blot autoradiogram of pA50 with rat brain RNAs of different developmental stages. The nitrocellulose membrane of A was stripped of radioactive signal and re-hybridized with the pA50 'housekeeping' probe. The different brain samples each contained 15/~g of total RNA; the non-neuronal samples contained 10/zg of total RNA.

Fig. 5. Blot-hybridization analysis of C1-13 cDNA to peripheral nervous system (PNS) RNAs. RNAs were prepared from adult rat spinal ganglia, adult mouse brain, and PC12 cells (neural crest -derived cell line7) cultured in the absence or presence of nerve growth factor (NGF). Autoradiographic exposure was for 7 days with an intensifying screen to maximize detection in PNS RNAs. Hybridization to mouse brain RNA illustrates an analogous crossspecies transcript.

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Fig. 6. Differential expression of C1-13 in rat primary brain cultures. Brain primary cell cultures from 13.5-day rat embryos and 1 day old rat pups were established and allowed to differentiate (neuronal) or serially passaged (glial) (see text). Shown is the Northern blot autoradiograph of total RNA from these cultures, blot-hybridized to radiolabeled C1-13 cDNA probe.

ronal cells of the CNS. The lower level of expression seen in the adult brain RNA sample (Fig. 3) is probably due to the increased ratio of glial to neuronal RNA observed in adult versus newborn brain tissue and not due to a decrease in the expression of this gene in adult tissue. In situ hybridization and/or antibody staining would be necessary to confirm this cell type specificity. DISCUSSION The results presented here clearly demonstrate that C!-13 is a brain-specific cDNA, primarily expressed in the central nervous system with significantly reduced expression in the peripheral nervous system. Transcripts of the associated gene are initially detected during embryonic development, reach maximum levels at birth, and persist in the adult. These transcripts are expressed in multiple regions of the brain and the data suggest this gene is transcribed exclusively in neuronal cells and not in glia. The development, differentiation, and maturation of the nervous system is extremely complex. The increased heterogeneity of the numerous cell types and their transcription patterns illustrates an RNA complexity which is several-fold higher than other organs 19. Despite this increased complexity, relatively few brain-specific genes

have been identified. From the results presented here, C1-13 does appear to represent a brain-specific transcript. The pattern of RNA expression detected by the C1-13 cDNA appears to mimic the development and differentiation which occurs with neuronal cells of the central nervous system (CNS). In the CNS, neurogenesis primarily occurs prior to birth in most regions of the brain. Gliagenesis of oligodendrocytes is principally a postnatal event and for astrocytes occurs during both late fetal development and postnatally 11. Furthermore, when dissociated embryonic rat brain cells are cultured, the astrocytes, ependymal cells, and oligodendrocytes develop on schedule, suggesting that biological clocks are more important than positional information during gliagenesis 1. The results from this study on the in vivo and in vitro expression of C1-13 transcripts strongly suggest the associated gene is transcribed in neuronal, and not glial, cells. Furthermore, since this expression is initially detected during embryonic neuronal development and remains constitutively expressed in the adult brain, the associated protein may be essential for neuronal differentiation or function. All TM genes which have been characterized to date exhibit their expression in multiple cell types. Tissue specificity of TM isoforms is achieved by alternatively splicing tissue-specific exons in a highly regulated manner. The gene associated with C1-13, however, demonstrates a single tissue-specific expression which would be unique for TM regulation. However, since there is only limited homology to known TM sequences, and the deduced amino acid structure does not exhibit the characteristic TM a-helical coiled-coil structure, classification of the C1-13 cDNA should be labeled as being 'TM-related'. Thus, the identification of a clone whose corresponding protein might function in an analogous structural or contractile manner to TM, but in a neuralspecific environment, is exciting. Actin and actin-binding proteins, such as TM, are thought to play an integral role in numerous neuronal cytoskeletal structures and physiological properties. For example, brain-specific a-TM transcripts have recently been identified 13. Growth cones 25, dendritic spines 16, pre- and postsynaptic terminals l°'i7, and axoplasmic space 6 have all been associated with actin filaments. Furthermore, actin-based structures are involved with the neuronal motile processes of axoplasmic transport, growth-cone extensions, and synaptic formation and movements. If the protein associated with C1-13 transcription is an actin-binding protein, then morphological and/or physiological processes may be regulated by this molecule. Future studies will entail defining more precisely the cellular location and neuronal cell type in which the associated protein functions, in addition to the

41 m o l e c u l a r m e c h a n i s m w h i c h r e g u l a t e s its e x p r e s s i o n .

Acknowledgements. We wish to acknowledge Dave Eling for his technical assistance and Drs. K. Blumenthal, R. Thompson, and M.

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