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Brain-Specific Expression of MAP2 Detected Using a Cloned Cdna Probe Sally A

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Brain-Specific Expression of MAP2 Detected Using a Cloned Cdna Probe Sally A View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Brain-specific Expression of MAP2 Detected Using a Cloned cDNA Probe Sally A. Lewis,* Alfredo Villasante,* Peter Sherline,* and Nicholas J. Cowan* *Department of Biochemistry, New York University Medical Center, New York, New York 10016; *Division of Endocrinology, Cornell University Medical College, New York, New York 10021 Abstract. We describe the isolation of a set of over- consistent with the coding capacity required by a lapping cDNAs encoding mouse microtubule associ- 250,000-D protein. ated protein 2 (MAP2), using an anti-MAP antiserum The MAP2-specific cloned cDNA probes were used to screen a mouse brain cDNA expression library in RNA blot transfer experiments to assay for the cloned in bacteriophage hgt 11. The authenticity of presence of MAP2 mRNA in a variety of mouse tis- these clones was established by the following criteria: sues. Though brain contained abundant quantities of (a) three non-identical clones each expressing a MAP2 mRNA, no corresponding sequences were de- MAP2 immunoreactive fusion protein were independ- tectable in RNA prepared from liver, kidney, spleen, ently isolated from the expression library; each of stomach, or thymus. We conclude that the expression these clones cross-hybridized at the nucleic acid level; of MAP2 is brain-specific. (b) anti-MAP antiserum was affinity purified using Use of the MAP2 specific cDNA probes in genomic nitrocellulose-bound fusion protein; these antibodies Southern blot transfer experiments showed the pres- detected only MAP2 in an immunoblot experiment of ence of a single gene encoding MAP2 in mouse. The whole brain microtubule protein; (c) a series of cDNA microheterogeneity of MAP2 is therefore ascribable "walking" experiments was done so as to obtain a either to alternative splicing within a single gene, or to non-overlapping cloned fragment corresponding to a posttranslational modification(s), or both. Under con- different part of the same mRNA molecule. Upon ditions of low stringency, the mouse MAP2 cDNA subcloning this non-overlapping fragment into plas- probe cross-hybridizes with genomic sequences from mid expression vectors, a fusion protein was synthe- rat, human, and (weakly) chicken, but not with se- sized that was immunoreactive with an anti-MAP2 quences in frog, Drosophila, or sea urchin DNA. specific antiserum. Thus, a single contiguous cloned Thus, there is significant interspecies divergence of mRNA molecule encodes at least two MAP2-specific MAP2 sequences. The implications of the above ob- epitopes; (d) the cloned cDNA probes detect an servations are discussed in relationship to the poten- mRNA species in mouse brain that is of a size (-9 kb) tial biological function of MAP2. EMPERATURE-DEPENDENT polymerization of micro- of molecular weight: the high molecular weight MAP (in the tubules in vitro results in the co-assembly, together range 240,000-350,000 D) and the low molecular weight tau T with a- and #-tubulin, of a group of proteins known proteins (55,000-62,000 D) (8). The high molecular weight collectively as the microtubule associated proteins (MAPs). group contains two members, MAP1 (>300,000 D) and These proteins promote the microtubule assembly process in MAP2 (250,000-300,000 D) (24), that are biochemically and vitro (8, 18, 19, 24, 25, 32) and at least some of them have immunologically distinct. Each group exhibits heterogeneity been localized as projections from the surface of the micro- on one-dimensional SDS polyacrylamide gels: in the case of tubule polymer (1, 11, 14). Thus, while the precise biological MAP1, three discrete but closely migrating bands, named function of MAP remains to be clearly demonstrated, they MAPIa, lb, and lc, have been identified, whereas MAP2 can could potentially mediate the interaction ofmicrotubules with be resolved into two components termed MAP2a and 2b (30). other cellular elements as well as serve in the control of The molecular basis for the heterogeneity of the high mo- microtubule polymerization. lecular weight MAP is unclear. While protein chemical char- Two major groups of MAP have been defined on the basis acterization (for example, analysis of proteolytic digestion 1. Abbreviations usedin this paper." MAP, microtubule associated protein; SSC, products) has been useful in showing the relatedness of some 150 mM NaC1, 15 mM Na citrate; TBST, 50 mM Tris-HC1, pH 7.5, 150 mM MAP (e.g., MAP2a and 2b [ 10, 21 ]), such experiments cannot NaCI, 0.05% Tween 20. distinguish between the expression of two similar but non- © The Rockefeller University Press, 0021-9525/86/06/2098/08 $1.00 The Journal of Cell Biology, Volume 102, June 1986 2098-2105 2098 identical genes, and the expression of one gene to yield a ovedaying with a nitrocellulose filter saturated with 10 mM isopropyl fl-~ single protein that could then undergo posttranslational mod- thiogalactopyranoside (35). The filters were removed, rinsed in 50 mM Tris- ification(s). To investigate the heterogeneity of the high mo- HCI, pH 7.5, 150 mM NaCl, 0.05% Tween 20 (TBST), and blocked for 30 rain lecular weight MAPs at the genetic level, we decided to at room temperature in TBST that contained 3% bovine serum albumin (BSA). Each filter was then immersed for 1 h in 0.1 ml of affinity purified anti-rat construct MAP-specific cDNA clones that could be used as MAP antibody (23) diluted 1:100 in TBST that contained 3% BSA. ARer three probes to assay the number of genes encoding each MAP 5-rain rinses in TBST, the bound antibody was eluted for 15 rain at room group. Such clones will also serve as a basis for understanding temperature with 3 ml of 100 mM glycine-HCl, pH 2.6, 150 mM NaCI. The the structure, expression, evolution, and function of these nitrocellulose was removed, the solution adjusted to pH 7.5 with I M Tris, and proteins. Here we report the isolation of cDNA clones encod- stabilized by the addition of 2 ml of TBST that contained 3% BSA. ing mouse MAP2. These clones have been used to show the RNA Preparation and DNA and RNA Blot existence of a single mouse gene encoding the MAP2 poly- peptide, with expression restricted exclusively to brain tissue. Transfer Experiments RNA for blot transfer analysis on 0.8% gels that contained formaldehyde (6) was prepared by the guanidine isothiocyanate procedure (3). Genomic DNAs Materials and Methods from mouse, rat, human, chicken, frog, Drosophila, and sea urchin were digested with restriction enzymes under the conditions specified by the manu- Construction and Screening of a Mouse Brain facturer (New England Biolabs). Restriction digests of DNA were resolved on cDNA Library 0.8% agarose gels and transferred to nitrocellulose as described (27). Mouse brain polyA÷ mRNA was prepared from 8-d-old mice by the guanidine isothiocyanate procedure (3). Double-stranded eDNA was synthesized from this material and inserted into the bacteriophage Xgtl I vector (35) as described Results (16). Approximately 5 x 105 recombinant plaques were screened using a rabbit anti-rat MAP antiserum (23) at a dilution of 1:500. Clones giving a positive Isolation of Clones from a cDNA Expression Library signal with the avidin/biotin/peroxidase complex procedure (Vector Labora- Using a MAP-specific Antiserum tories, Burlingame, CA) were picked, purified to homogeneity, and the eDNA inserts subcloned into plasmid pUC8 for further amplification and study. To isolate cDNA clones encoding MAP, we constructed a cDNA expression libary in ;kgtl 1 using polyA + mRNA from Identification of Overlapping Clones by the brains of 8-d-old mice. Approximately 5 x 105 clones cDNA "Walking" were screened for the expression of MAP using an affinity- Excised inserts from clones that express immunoreactive fusion proteins, or purified polyclonal rabbit anti-rat MAP-specific antibody restriction fragments derived from such clones, were 32P-labeled by nick trans- (23). Seven clones that yielded a positive signal with the anti- lation and used to screen the cDNA library (2) for cross-hybridizing sequences. After hybridization, the filters were washed to a final stringency of 2 x SSC MAP antiserum were obtained. Each positive plaque was (150 mM NaC1, 15 mM Na citrate), 68"C. Clones giving positive signals were picked as 5-mm plugs using the open end of a Pasteur pipet, and the bacterio- phage eluted in 1 ml of 0.1 M NaCI, 10 mM Tris-Hcl, pH 8.0, 10 mM MgSO4. An aliquot (30 #1) of the eluted bacteriophage was amplified in a small (1 ml) culture without further purification, and DNA prepared from each lysate by the method described (17). The final product was dissolved in 40 t~l of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 20 #1 of this solution digested with EcoRI under the conditions specified by the suppher (New England Biolabs, Beverly, MA). The digestion products were resolved on a 1% agarose gel, the gel content transferred to nitrocellulose (27), and the blot hybridized with ~ 107 cpm of the same probe(s) used to screen the cDNA library (see above). After washing to 2 × SSC, 68"C, the blots were exposed to film. This procedure served to identify those plugs that contained bacteriophage with large recombinant inserts ho- mologous to the probes used to screen the library. Eluates from plugs identified in this manner were subjected to two rounds of plaque purification; the recombinant insert from a single isolated plaque giving a positive hybridization signal with the labeled probe was then subeloned into pUC8 and amplified for further study by restriction mapping and analysis in RNA blot transfer experi- merits. Production of Anti-Mouse MAP1 and MAP2 Antisera and Immunoblotting Two-cycle purified microtubules were prepared from the brains of adult mice (22).
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