cell, vol. 32, 713-724, march 1983, copyright 0 1983 by mit four … · 2020. 1. 16. · margaret...

Cell, Vol. 32, 713-724, March 1983, Copyright 0 1983 by MIT

Four Unique Genes Required for p Tubulin Expression in Vertebrates

Margaret A. Lopata,*+ Jane C. Havercroft,* Louise T. Chow’ and Don W. Cleveland* * Department of Physiological Chemistry The Johns Hopkins University School of Medicine Baltimore, Maryland 21205 *Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724

Summary

We have isolated the four separate segments of chicken DNA which contain sequence homology to /I tubulin. With the exception of a fifth region of DNA which appears to contain only a 5’ fragment of a /I gene, these four cloned sequences represent all of the p tubulin encoding DNA in the chicken. Each gene is very similar in structure, containing three or four small intervening sequences clustered in the 5’ portion of the coding region. Using RNAs prepared from a variety of cell lines and tissues, we have found five different mRNAs which carry p tubulin sequences, two of which are encoded by the same gene. Three of these mRNAs are unexpectedly long (between 3500 and 4DDD bases). However, these large mRNAs do give authentic p tubulin translation products. Overall, we conclude that each of the four fl tubulin genes is a functional gene which is expressed in a specific program during differentiation. These data strongly suggest that four p tubulins are necessary for proper microtubule function in vertebrates.

Introduction

Microtubules, comprised principally of heterodimers of one (Y and one p tubulin polypeptide, are filamen- tous protein polymers which are utilized in a diverse number of cellular processes. For example, they are the major structural components of mitotic and meiotic spindles, of eucaryotic cilia and flagella and of elon- gated neuronal processes, and in concert with actin filaments and intermediate filaments they specify for most animal cells the characteristic cell shape and internal cytoplasmic structure. The variety of cellular events in which microtubules participate has long encouraged speculation that different tubulin gene products form microtubules which are functionally distinct. This multitubulin hypothesis, formally pro- posed by Fulton and Simpson (1976), has been af- firmed in several recent investigations employing bio- chemical and genetic analyses to identify different tubulin polypeptides. Multiple sequences for both (Y and ,f3 tubulin have been identified in a variety of species, although the precise number of genes ap-

t Present address: Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

pears to vary widely. The Chlamydomonas genome contains two (Y and two p tubulin sequences (Silflow and Rosenbaum, 1981). Drosophila melanogaster DNA contains four (Y tubulin sequences (Sanchez et al., 1980; Kalfayan and Wensink, 1981; Mischke and Pardue, 1982) and four/? tubulin sequences (Sanchez et al., 1980). Raff and coworkers (Kemphues et al., 1979, 1980; Raff et al., 1982) have demonstrated, using mutants and two-dimensional gel analyses, the expression of three different Drosophila p tubulins. Moreover, Kalfayan and Wensink (1982) have shown that each of the four Drosophila cy tubulin genes is transcribed into a stable, polysomal RNA and hence is very probably a functional gene.

In sharp contrast to these lower eucaryotic examples, the sea urchin, mouse, rat and human genomes contain lo-20 sequences for each tubulin subunit (Cleveland et al., 1980; Alexandraki and Ruderman, 1981; Cowan et al., 1981). Direct amino acid sequencing has demonstrated the presence of at least four expressed (Y and two p tubulin polypeptides in hog (Ponstingl et al., 1981; Krauhs et al., 1981). Similarly, from DNA sequence data of cloned copies of (Y tubulin mRNAs, at least two (Y tubulins have been demonstrated in rat (Lemischka et al., 1981; Ginzburg et al., 1981) and human (Cowan et al., submitted for publication). On the other hand, the considerable DNA sequence data of Cowan and his associates have clearly demonstrated that many, if not most, of the human DNA segments with tubulin sequence homology are pseudogenes which contain multiple in-phase, translation termination codons and/or deletions within the coding regions (Cowan et al., 1981; Wilde et al, 1982a, 1982b, 1982c, 1982d).

The determination of the precise number of functional tubulin genes which are necessary in higher animals thus remains a complicated and uncertain issue. We have recently determined that, unlike the mammalian examples cited above, the complexity of tubulin gene sequences in one vertebrate, the chicken, is surprisingly simple, consisting of four or five each of (Y tubulin and p tubulin sequences (Cleve- land et al., 1980, 1981 a). We present here the isolation of four of the p tubulin members of this relatively small gene family and demonstrate that each represents a functional gene which is expressed in a complicated program in differentiated cells.

Results

Our previous data (Cleveland et al., 1980, 1981 a), based on blot analysis of chicken DNA hybridized to probes corresponding to the N-terminal or C-terminal coding regions of a cloned /? tubulin mRNA indicated the probable presence in the chicken genome of four or five independent p tubulin sequences. We have unambiguously confirmed this conclusion by isolation of the authentic chicken DNA segments from a ge-

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Figure 1. Restriction Maps of the /3l,fi2, p3, and j34 Phages

Restriction maps are shown of a representative phage from each of four nonoverlapping classes of Charon 30 recombinants which contain chicken genomic DNA with sequence homology to p tubulin. Each phage contains 15-17 kb of chicken DNA. Regions of each recombinant which hybridize to the chicken fl tubulin cDNA clone pT2 are shown by the blackened bars. Direction of transcription (5’ to 3’) of each was determined by blot hybridization to probes corresponding to the N-terminal or C-terminal p tubulin coding sequences from pT2. Restriction endonucleases employed are designated in the figure as fol-

t t +t

lows: Hpa=Hpa I; Kpn=Kpn I; Xho=Xho I;

earn aam aam HIndIll ECORf Bam=Bam HI; Xba=Xba I; Sph=Sph I; EWRI Pst=Pst I; Sal=Sal I; Bgl=Bgl II. A 1 kb scale

for the expanded portions of the maps is shown at the top of the figure.

nomic DNA library constructed in Charon 30 by B. Vennstrom (Vennstrom and Bishop, 1982). Approxi- mately 2 million phages were screened, yielding about 150 recombinants which carried sequence homology to pT2, our p tubulin cDNA clone which contains the entire coding sequence of a chicken brain ,L? tubulin mRNA (Valenzuela et al., 1981). Four classes of nonoverlapping segments of chicken DNA were identified. A partial restriction map of representative phages from each of these four classes is presented in Figure 1. The regions of each isolate which contain fi tubulin sequence homology were determined by blot hybridization analysis. Orientation of the gene sequences- that is, 5’ to 3’- and verification that each recombinant contained an essentially complete coding sequence for p tubulin was achieved using probes specific for the N-terminal or C-terminal protein coding regions.

The ,f3 tubulin sequences carried by the four recombinants have been named ,&, p2, ,l?3 and p4, respectively, in ascending order of the size of the Eco RI restriction fragment derived from the corresponding fi tubulin sequence in genomic chicken DNA. These

Eco RI digestion products, identified by their homology to the full-length p tubulin cDNA clone, pT2, are displayed following blot hybridization in Figure 2A. Prominent signals are seen in DNA fragments at 2.6, 4.4, 7.5, 15 and 20 kb.

As illustrated in the restriction maps in Figure 1, the recombinant phage carrying the@ sequence consists of approximately 15 kb of chicken DNA. The majority of the p tubulin sequences in this phage are present on a 2.6 kb Eco RI fragment. This corresponds in size to the /31 sequence in genomic chicken DNA. Simi- larly, the second phage shown in the figure contains approximately 2.5 kb of DNA homologous top tubulin, and these sequences are contained within a 4.4 kb Eco RI fragment. Hence, this phage carries the /?2 gene. The third phage carries 16 kb of chicken DNA including only a single Eco RI site. This site is located in the extreme 3’ coding portion of the /3 tubulin homology region. No additional Eco RI sites are located 5’ to the tubulin sequences within at least 6 kb, indicating that this fi tubulin gene is derived from a genomic DNA region whose Eco RI digestion product is in excess of 8 kb. Finally, the fourth phage contains

Four Expressed p Tubulin Genes in Chicken 715

ABCDEF

Figure 2. Assignment of the fil, p2. p3, and p4 Genes to the Cor- responding Segment of Genomic Chicken DNA

Blot hybridization of genomic chicken DNA digested with Eco RI was performed as detailed in Experimental Procedures using 32P-labeled probes constructed from (A) the cDNA clone pT2. (B) the 2.6 kb Eco RI fragment of phage pl, (C) the Kpn I-Sph I fragment of phage p2, (D) the Sph I-Xho I fragment of phage p3. (E) the Xho I-Kpn I fragment of phage p4 or(F) the 3’ untranslated region of pT2. For B- E, the respective probes represented essentially all of the p tubulin sequence homology contained in each of the isolated p tubulin phage.

no Eco RI sites within the 14 kb of cloned chicken DNA, and thus the /3 tubulin sequences contained within this phage must derive from a genomic Eco RI fragment of more than 14 kb.

To demonstrate clearly that the p3 and ,&4 phages do indeed carry sequences representing the 15 kb and 20 kb /I tubulin Eco RI fragments shown in Figure 2A, we have subcloned into pBR322 the regions from each phage with p tubulin homology and used portions of these as hybridization probes in genomic blot analyses. These subclones are indicated by the expanded portions of the restriction maps in Figure 1. The resultant autoradiographs are shown in Figures 2D and 2E. It is clear in Figure 2E that the Xho I-Kpn I fragment which encompasses all of the region with p tubulin homology in the /I4 clone preferentially hybridizes to an Eco RI fragment of genomic chicken DNA which is indistinguishable in molecular weight from that identified as the ,&4 gene by hybridization to the cDNA clone (Figure 2A). We conclude therefore that this clone does contain the sequences corresponding to the /I4 gene. Similarly, the Sph I-Xho I segment of the /?3 phage hybridizes preferentially to a 14 kb chicken DNA Eco RI fragment which is identical in mobility with the DNA segment initially identified as p3. A second Eco RI fragment of 7.0 kb is also selected by this probe. This is not unexpected since the probe itself contains an internal Eco RI site at the extreme 3’ end of the tubulin homology region and hence when used in hybridizations should identify

both a DNA segment with strong tubulin homology and a second segment with little tubulin homology.

In addition, for further confirmation of the assign- ments of pl and p2, the equivalent hybridization experiments were performed with probes from these phages. The data are displayed in Figures 2B and 2C. For ,f32, the appropriate Kpn I-Sph I fragment preferentially hybridizes to a DNA fragment comigrating with authentic /32 (Figure 2C). Moreover, we have determined that the cDNA clone is derived from this gene. To demonstrate this, we prepared from pT2 a subclone corresponding only to the 3’ untranslated region of the original mRNA (see Experimental Procedures for details). When used as a hybridization probe, this subclone identifies only the /32 gene (Figure 2F). This result is substantiated by electron microscopic heteroduplex analysis of the cDNA and /32 clones (see below).

For pl, the probe derived from the large Eco RI fragment which contains the majority of the /3 tubulin sequence homology carried by the phage (see Figure 1) preferentially hybridizes to a 2.6 kb chicken Eco RI fragment which is indistinguishable from the pi DNA segment, although substantial cross-hybridization to ,&2 is also evident (Figure 2B). From the intensities of the genomic DNA bands in Figure 2 when probed with the various clones, it can be concluded that pi and p2 are highly homologous and that/?3 is most distantly related. A similar conclusion was reached in the electron microscopic heteroduplex studies of these clones (see below).

Overall, we conclude that we have isolated the /31, ,l32, /33 and /I4 genes from chicken. These gene sequences represent, with a single exception, the entire complement of chicken DNA sequences which have strong homology to a cloned p tubulin cDNA. The single exception (marked with an arrow in Figure 2A) is found on a 7.5 kb Eco RI fragment and contains only 5’-terminal coding sequences for /? tubulin (Cleveland et al., 1980; Lopata and Cleveland, unpublished). To date, we have been unable to detect an additional 3’ sequence in the genome. Although we cannot exclude the possibility that such a fragment may be obscured on our blots by signals from other genes, it is also possible that this fifth sequence represents either a /? tubulin highly diverged in its 3’ sequences or a remnant pseudogene deleted in 3’ coding sequences.

Electron Microscopic Heteroduplex Analysis of Genomic Clones In order to study the structure of the p tubulin genes, the subclone of each gene in pBR322 (see Figure 1) was digested with an appropriate restriction endonuclease so as to release a segment of, or the entire, insert. These were separately hybridized to the linearized P tubulin cDNA clone pT2. The resulting heteroduplexes were analyzed by electron microscopy. Rep-

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iii i 11 x.x I I 2 mm - =

il,,’ i ’ ///

I : I \ chicken 84 \

I “I chicken p4 (alternate)

I I I 0 I 2 3 ?. 7 Kb

Figure 3. The Structure of Chicken b Tubulin Genes

(A-C) Electron micrographs of heteroduplexes between the linearized cDNA clone pT2 and, respectively, the pl , /32 and p3 inserts released from pBR322 with restriction endonucleases. (D-E) Micrographs of two related heteroduplexes between pT2 and restricted /34 DNA showing different structures near the 5’ end. In (A-C), pBR322 released from the /3 clones has also hybridized to the linearized pT2 DNA causing it to circularize. In (D), a fragment of pBR322 has hybridized to the short arm of pBR322 in pT2 DNA. The 5’ and 3’ ends of each gene were determined by the asymmetrical lengths of the pBR322 sequences in pT2 DNA in the heteroduplexes (as seen in 3D) and are marked with large and small arrowheads


resentative heteroduplexes, interpretative tracings and length measurements are presented in Figure 3A-3F. To summarize, ,L?l and p2 showed three intervening sequences (P/S) at similar sites relative to the conserved regions. These IVSs ranged from 160 to 500 nucleotides long and were separated from each other by sequences homologous to the pT2 cDNA of about 130 nucleotides each (Figures 3A, 3B). The intervening sequences were located near the 5’ end of each gene as concluded from the asymmetrical disposition of the pBR322 sequence relative to the cDNA insert in pT2 DNA in the heteroduplexes (for example, Figure 3D). About 250 nucleotides of the 3’ end of the cDNA in pT2 corresponding to the untranslated region of the mRNA (Valenzuela et al., 1981) did not hybridize to ,l31, indicating sequence divergence in the 3’ untranslated region (Figure 3A). In contrast, the ,L?2 clone hybridized to the entire cDNA in pT2 (Figure 3B). This observation confirms the finding from blotting analysis (Figure 2F) that the cDNA is derived from a mRNA transcribed from the /I tubulin gene in the p2 clone. The pi and p2 genes appear highly homologous in all conserved segments since the heteroduplexes with pT2 were formed at fairly stringent conditions (50% formamide, 0.1 M Tris, pH 8, 37°C).

,LI3 hybridized to pT2 at lower frequency than pi or P2, suggesting some sequence divergence. It ap- peared to have three or four small intervening sequences of 80 to 500 nucleotides clustered at the 5’ end of the gene; three of these were located at sites similar to those in the pl and p2 genes (Figure 30 Occasionally, an additional homologous sequence was detected at the 5’ end and was possibly followed by a small intervening sequence as diagrammed in Figure 3F. The first two conserved segments (Cl and C2 in Figure 3F) hybridized to pT2 only in some of the heteroduplexes at a nonstringent condition (40% formamide, 0.1 M Tris, pH 8, 22°C). This is most likely due to sequence divergence. About 250 nucleotides of the 3’ untranslated region in the cDNA did not hybridize to p3 DNA.

in the tracing, respectively. Large and small arrowheads in the electron micrographs point to DNA deletion loops corresponding to the intervening sequences in the four /3 tubulin genes. Each micrograph is accompanied by an interpretative tracing: (-) genomic DNA; (. . .) cDNA in pT2; (- - -) vector pBR322 DNA. (F) is a diagram summarizing the structures of the four chicken clones as well as a human fi tubulin gene /35 (Cowan et al, 1981). AS discussed in the text, the first conserved segment and first intervening sequence in p3 were seen only infrequently. The regions hybridized to pT2 DNA are represented by the filled in boxes. The redundant sequence in /34 that did not hybridize to pT2 is represented as an open box. The hatched box in p2 represents the untranslated region which hybridizes to pT2 but has no homology to the other p clones. The 3’ untranslated regions Of Pl , p3 and /34 cannot be deduced by this analysis. The corresponding conserved segments in different clones are connected with dashed lines and are designated Cl through C4 or C5. starting from the 5’ end of the genes. The intervening sequences are designated I1 through I4 in the same direction. The lengths of various segments (in kilobases) and their standard deviations (numbers in parentheses) are as follows. For /?l , Cl : 0.15 (0.04). II: 0.48 (0.07). C2: 0.13(0.02). 12: 0.18 (0.04). C3: 0.13 (0.02). 13: 0.18 (0.05). C4: 1.08 (0.05). ForpP, Cl: 0.15 (0.02). II: 0.36(0.04). C2: 0.13 (0.03). 12: 0.20 (0.04). C3: 0.13 (0.02). 13: 0.16 (0.04). C4: 1.30 (0.05). Forp3, Cl: 0.07 (0.01). II: 0.08, (0.02). C2: 0.07 (0.02). 12: 0.5(0.07). C3: 0.12 (0.04). 13: 0.22(0.03). C4: 0.14(0.03). 14: 0.36(0.04). C5: 1.13(0.05). For/34 in Figure3D, Cl: 0.07(0.02). Ii: 0.16(0.02). C2: 0.07 (0.02). 12: 0.4 (0.05). C3: 0.14 (0.02). 13: 0.47 (0.04). C4: 0.14 (0.02). 14: 0.12 (0.02). C5: 1 .lO (0.05). ForP 4 in Figure 3E, Cl and C2 were contiguous and measured 0.13 (0.06) and the IVS separating it and C3 measured 0.72 (0.1). Each of the lengths given was an average Of 9 to 28 measurements except for Cl and I1 for p3 which had three measurements because of the small size of this putative intervening sequence and the low frequencies of hybridization to pT2 near the 5’ end. (Intervening sequences less than 500 nucleotides are likely to be underestimated because they tend to collapse and cannot be measured as accurately.)

,l34 and pT2 formed two related heteroduplexes that had three or four small intervening sequences, respectively, near the 5’ end of the gene (Figures 3D and 3E). The first, large IVS in the heteroduplexes with three IVSs apparently contained sequences redundant to, but separate from, the 3’ half of the first conserved segment. Branch migration led to the for- mation of a fourth IVS in the heteroduplexes. Conse- quently, the first conserved region was split into two shorter segments. In fact, most heteroduplexes had four IVSs, suggesting a better sequence homology to pT2 in this configuration. Again, the untranslated region in pT2 cDNA did not hybridize to p4 DNA.

The arrangement of conserved segments and IVS in the chicken and human /? tubulin genes are dia- grammatically summarized in Figure 3F. interestingly, all three IVS in pl and p2 and the latter three IVS in /I3 and ,L?4 were located at sites similar to each other and to those detected in a human p tubulin gene (Cowan et al., 1981). Moreover, the pT2 cDNA is more homologous to the human p gene than it is to p3 and p4, suggesting that p3 and ,L?4 may encode polypeptides of substantial sequence divergence to the ,I32 polypeptide.

Multiple /3 Tubulins in Fibroblasts Identified with Gene-Specific Probes To determine if each of the isolated p tubulin genes is a functional gene, we attempted to determine whether each gene encodes a stable, cytoplasmic mRNA. To develop specific nucleic acid probes for putative RNAs transcribed from different genes we noted the following: the cross-hybridization of the coding regions of each of the four genes might preclude the inclusion of regions of coding sequence in the prospective gene specific probes; and heteroduplex analysis (see above) indicated that the presumptive 3’ untranslated regions of individual genes did not detectably hybridize to each other. We therefore isolated from genes pl and p3 those sequences immediately 3’ to the termination of homology with the pT2 cDNA. For gene

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A B C D E

Figure 4. Assignment of p Tubulin RNAs Expressed in Chick Em- broyo Fibroblasts to Genes pi, /32, and /34

Poly (A)’ RNA was prepared from secondary chick embroyo fibroblasts as previously described (Cleveland et al. 1981 b). Aliquots of 0.5 pg were electrophoresed on a denaturing 2.2 M formaldehyde gel and transferred to nitrocellulose. The resultant replicas were hybridized to “P-labeled probes corresponding to (A) pT2, the /3 tubulin cDNA clone, (6) the pi, 3’ probe, (C)the /32. 3’ probe, (D) the 83, 3’ probe, or (E) the p4 probe. An autoradiograph of the hybridization is shown. Isolation of the template DNAs for probe preparation is detailed in Experimental Procedures.

,f32, a subclone of pT2 containing only the 3’ untranslated region of pT2 represented the analogous sequence. For gene p4, it was found that the entire coding sequence region plus presumptive 3’ untranslated region was a sufficiently specific probe.

We utilized these four probes to investigate the expression of multiple p tubulin RNAs in a variety of chicken cell types. Initially, poly(A)’ RNA was prepared from secondary chick fibroblasts, electrophoresed on a denaturing gel and transferred to nitrocellulose (Thomas, 1980). Five identical blots were prepared and hybridized to 32P-labeled probes. The resultant autoradiographs are shown in Figure 4. Lane A displays hybridization to the full-length cDNA probe. Three RNA species of markedly different sizes are clearly resolvable. These sizes are 1800, 3500, and 4000 bases, as judged by comparison with E. coli and chicken rRNA standards. The /32 3’ probe (lane C) hybridizes (even on very long exposures) only to the 1800 base RNA. The ,L?4 probe, on the other hand, preferentially selects the 3500 base species (lane E). The ,& 3’ probe hybridizes almost exclusively with the 4000 base species (lane B). Although hybridizing preferentially to a single RNA species, weak signals are detected with either the pl or /?4 probe to the other two size classes of /? tubulin RNAs. For fll , this may be the result of a small portion of coding se-

quence in the presumed 3’ probe or of a low degree of conservation of 3’ sequences between different genes. For /34, some cross-hybridization is expected since the probe contains the entire coding region as well as the presumptive 3’ untranslated region. None- theless, it is clear that we have isolated probes for pl , p2 and /34 which are selective hybridization tools and may be used for assigning different RNA species containing /3 tubulin sequence homology to their respective genes. Finally, the p3 3’ probe detects a weak signal in the 1800 base region (lane D). We cannot be sure in this experiment whether this represents authentic hybridization to a p3 transcript or contaminating cross-hybridization to the p2 mRNA. This point is clarified by additional experiments with different cell lines (see below). However, from this experiment we are able to conclude that three different p tubulin genes are expressed in chicken fibroblasts.

Expression of All Four j3 Tubulin Genes Is Determined by a Specific Program during Differentiation We next used the gene-specific probes detailed above to investigate the expression of multiple p tubulin mRNAs in a variety of chicken cell lines. Poly (A)+ RNA was prepared from 1104 cells, a cloned cell line which was derived from a bursal tumor induced by avian leukosis virus (ALV) infection and has retained or acquired morphological characteristics reminiscent of fibroblasts; secondary chicken fibroblasts; BK4408 cells, a cloned lymphoid line isolated from an ALV induced tumor; R2B cells, another clonal isolate from an ALV induced tumor; 249 cells, a clonal isolate from an MC29 virus induced hepatoma; and embryonic chick brain. Blots containing these six RNA samples were hybridized with the 3’ probes from each p tubulin gene. Autoradiograms of the hybridizations are shown in Figure 5. Several striking features should be noted:

First, four different RNA species are clearly resolvable by size when these RNAs are probed with the total cDNA (Figure 5A). In addition to the three species already identified in fibroblasts, a 3700 base species is a major hybridizing RNA in 1104 (lane I), BK4408 (lane 3) and possibly 249 (lane 5).

Second, as expected from the initial experiment with fibroblast RNA, in each sample in which the cDNA selects the 4000 base RNA (fibroblasts, 249 and brain) the /G-specific probe preferentially hybridizes to this species (Figure 5B, lanes 2, 5 and 6).

Third, the 3500 base RNA present in fibroblasts, BK4408, R2B, 249 and brain hybridizes selectively to the ,@4 probe (Figure 5E, lanes 2, 3, 4, 5 and 6). More importantly, the 3700 base transcript also selectively hybridizes to this probe (Figure 5E, lanes 1, 3 and 5). This strongly suggests that both RNAs are derived from the P4 gene. Even more unexpectedly, the pat- tern of appearance of these two sibling RNAs is quite


1 2 3 4 5 6 A

1 23456 c

1

123456 I ,,

1 23 456 D

I

complicated. The 3500 base RNA is found in fibroblasts, R2B ceils and brain; the 3700 base RNA is present in 1104 cells. Both species are expressed in BK4408 and 249 cells.

Fourth, the /?P-specific probe hybridizes exclusively to an 1800 base mRNA which is the major /3 tubulin mRNA in brain (Figure 5C, lane 6). Hence, this gene almost certainly encodes a neuronal or glial p tubulin.

Fifth, comparison of the relative intensities of hybridization of the 1800 base region of the blots probed with the cDNA (Figure 5A), /32 3’ (Figure SC) or /?3 3’ (Figure 5D) probes provides convincing evidence that the ,L?3 3’ probe does preferentially detect an RNA species distinct from that recognized by the ,f32 3’ probe despite the comigration of these two RNAs. For example, in 1104 cells (lane 1 of Figures 5C and 5D) and R2B cells (lane 4 of Figures 5C and 5D) the p3 3’ probe hybridizes strongly to an 1800 base RNA, whereas the ,I32 probe hybridizes very weakly.

The Surprisingly Large RNAs Encoded by 81 and p4 Represent Authentic /I Tubulin mRNAs The RNAs encoded by the pl and ,l34 genes (between 3500 and 4000 bases) are unexpectedly large for ,I3 tubulin RNAs since the length of RNA required to specify the coding region of ,L? tubulin is only 1338 bases (Valenzuela et al., 1981). Each of these RNAs therefore appears to contain about 2100 or 2600 bases of noncoding sequences, more than the sum of the intervening sequences in either of the p clones. The extra sequences must, therefore, originate from regions upstream and/or downstream of the coding

Figure 5. Identification of Multiple p Tubulin RNAs in a Variety of Chicken Cell Lines or Tissues

Poly (A)+ RNA was prepared from (1) 1104 cells, a cloned cell line which was derived from a bursal tumor induced by avian leukosis virus (ALV) infection and has retained or acquired morphological characteristics reminiscent of fibroblasts, (2) secondary chicken fibroblasts, (3) BK4408 cells, a cloned lymphoid line isolated from an ALV induced tumor, (4) R2B cells, another clonal isolate from an ALV induced tumor, (5) 249 cells, a clonal isolate from an MC29 virus induced hepatoma, and (6) embryonic chick brain. Blots containing these 6 RNA samples were hybridized with (A) pT2, the j3 tubulin cDNA clone, (B) the ,81, 3’ probe, (C) the p2. 3’ probe, (D) the 83, 3’ probe, or (E) the /34 probe. An autoradiogram of the resultant hybridization is shown.

segments. The possibility that either or both of these RNAs may be unprocessed nuclear precursors is certainly not the case, since each is found on polysomes and is not enriched in nuclear RNA (Lopata and Cleve- land, unpublished). Nonetheless, the extensive additional sequence suggests that although each RNA contains sequence homology to p tubulin, each may encode a polypeptide which is substantially larger than the expected p tubulin subunit. To test whether these two large RNA transcripts from pl and p4 actually encode a p tubulin-like polypeptide, we fractionated chicken fibroblast RNA on a preparative, denaturing agarose gel to separate RNAs by size. Gel slices containing RNAs between 4500 bases and 1800 bases were excised, the RNA extracted and aliquots of the recovered RNAs electrophoresed on a second agarose gel to verify purity. This second gel was blotted onto nitrocellulose and hybridized to the pT2 cDNA probe. The resultant autoradiogram is shown in Figure 6. As is evident in the figure, in RNA from gel slices 3, 5 and 9, we have isolated the pl , p4, and p2 transcripts, respectively, free from each of the other /? tubulin RNAs.

To determine whether each of these isolated RNAs was competent to encode a p tubulin polypeptide, each RNA fraction was translated in vitro in a reticulocyte cell free system in the presence of ?S-methionine (Pelham and Jackson, 1976). The translation products are shown in Figure 7A along with the corresponding translation of chick brain poly (A)+ RNA in which (Y tubulin, P tubulin and actin are the major translation products. From the fractionated fibroblast

Cdl 720

CF 1 2 3 4 5 CF 6 7 8 9

Figure 6. Isolation of the 4000 Base, 3500 base and 1600 Base b Tubulin mRNAs from Chick Embryo Fibroblast RNA

Thirty micrograms of poly (A)+ RNA from chick embryo fibroblasts was electrophoresed on an agarose gel containing methylmercury hydroxide. The resultant gel was sliced and the RNA eluted from successive gel pieces (see Experimental Procedures). Aliquots (1% of recovered RNA) from each fraction were electrophoresed on a gel containing 2.2 M formaldehyde and then blotted onto nitrocellulose. The presence of /3 tubulin RNAs was detected by hybridization to the pT2 p tubulin coding sequence probe. Fractions 3, 5 and 9 show prominent p tubulin hybridization. CF: the original unfractionated chick embryo fibroblast RNA. The numbers above each lane represent the gel slice number from the original denaturing gel (with 1 representing RNAs of slowest mobility).

RNA samples, a major polypeptide which comigrates with brain p tubulin (principally the translation product of the p2 gene-see above) is present only in fractions 3, 5 and 9. That these translation products are in fact authentic p tubulin polypeptides has been determined by immunoprecipitation with an antiserum prepared against chick brain 01 and p tubulin (Cleve- land et al., 1981 b). The immunoprecipitation products are shown in Figure 76. As expected, ,B tubulin polypeptides are precipitable only from fractions identified by blot analysis to contain RNAs with p tubulin sequence homology (Figure 6). In particular, fraction 3 containing the 4000 base pl RNA and fraction 5 containing the 3500 base p4 RNA are both translated into p tubulin subunits. Whether either of these p polypeptides differs from the ,B2-encoded polypeptide is not yet known. However, to date we have been unable to detect differences by two-dimensional gel electrophoresis (Havercroft and Cleveland, unpublished). Overall, it is clear that the large pl and fi4 mRNAs do represent translatable ,B tubulin mRNAs which unexpectedly carry very extensive noncoding sequences.

Discussion

Speculation that multiple tubulin subunits polymerize to form microtubules with different functional characteristics has provoked numerous attempts to determine precisely the number of tubulin gene products which are functionally distinct in higher eucaryotes. In this regard we have demonstrated that the genome of a highly developed vertebrate, the chicken, contains four independent p tubulin genes. With the possible exception of a fifth DNA segment which may represent

a pseudogene remnant, these four genes comprise the entire set of sequences with strong homology to p tubulin in chicken. Each of these has been found to encode a stable, polysomal RNA and three of these RNAs have been demonstrated to encode authentic p tubulin polypeptides. It is clear then from these data that four p tubulins (or at most five if the partial gene sequence proves to represent a real gene) are suffi- cient to allow development of a higher eucaryote.

These data represent determination of the complexity of functional gene sequences for ,f3 tubulin subunits in a vertebrate. In other vertebrate systems investi- gated this determination has proven to be a laborious and difficult task. On the order of lo-20 sequences homologous to either cx tubulin or p tubulin have been discovered in all mammalian genomes studied (Cleve- land et al., 1980; Cowan et al., 1981: Wilde et al., 1982a; Lemischka et al., 1981; Lemischka and Sharp, 1982). With only one exception (Lemischka and Sharp, 19821, each of these genomic sequences thus far studied in detail has been shown to be a pseudogene. Data from direct protein sequencing (Ponstingl et al., 1981; Krauhs et al., 1981) and from cDNA sequencing (Lemischka et al., 1981; Ginzburg et al., 1981; Cowan et al., submitted for publication) has indicated at least two expressed (Y or /3 tubulin genes. The demonstration that all four of the p tubulin genes in chicken are functional genes leads us to predict that four p tubulin genes will prove to be the unique number of functionally required p tubulin sequences in all higher eucaryotes. The presence in the chicken of four DNA segments homologous to (Y tubulin (Cleve- land et al., 1980, 1981 a) further suggests that four functional genes may prove to be the requisite number of (Y genes as well.

Four Expressed /3 Tubulin Genes in Chicken 721

Br 3 4 5 6 7 8 9 Bg

B

Figure 7. Characterization of the In Vitro Translation Products of the 4000 Base, 3500 Base and 1800 Base p Tubulin mRNAs

Aliquots of the gel-purified RNA fractions displayed in Figure 6 were translated in a rabbit reticulocyte system in vitro. The translation products were analysed by gel electrophoresis and fluorography. (A) Shows the translation products of RNA from fractions 3-9, along with a chick brain RNA marker translation. (B) Shows the polypeptides from these translations which are immunoprecipitable with an affinity- purified antibody to chick brain 01 and ,L? tubulin subunits. The lane marked Br represents the translation products of chick brain RNA: Bg represents the reticulocyte background, 01, p and A mark the

Another unexpected finding is the presence of multiple RNA species apparently derived from the same ,B tubulin gene. One possible interpretation of these results is that the differences in RNA size are the consequence of an allelic difference in p4. In this view, each of the two RNA species is derived from a separate allele. Alternatively, it is possible that two transcripts are produced from a single ,@4 gene as the result of differential RNA processing events. This type of phenomonon has been well documented for the mouse dihydrofolate reductase gene in which four mRNAs which differ in the site of poly (A) addition have been described (Setzer et al., 1980). A final intriguing possibility is that alternative splicing path- ways involving the redundant conserved segments in p4 which we have detected in heteroduplexes might produce multiple transcripts. If either of these latter possibilities is correct, comparison of appropriate cDNA clones for each of the two p tubulin mRNAs with the p4 gene should demonstrate the specific processing event involved.

Finally, it is noteworthy that in RNA from each migration positions of oi tubulin, p tubulin and actin, respectively. chicken cell line or whole tissue we have always

We have only begun to assess the possible functional significance of different p tubulin subunits. The RNA from gene p2 is heavily enriched in brain and must represent the dominant neuronal or glial polypeptide. Lymphoid ceils, on the other hand, express only low levels (or perhaps none at all) of this gene. Instead, these cells express roughly comparable levels of p3 and /?4. Fibroblasts express relatively equivalent levels of ,Bl , /?2 and ,B4; hepatocyte RNA (either from cultured 249 cells or from whole liver- Lopata and Cleveland, unpublished) appears to contain transcripts from all four genes. Thus the present data suggest that the expression of each p gene is probably determined by a complicated program during differentiation.

The RNA transcripts derived from genes fil and /34 are surprisingly large for p tubulin mRNAs. Since only 1338 coding nucleotides are required for j? tubulin, mRNAs between 3500 and 4000 bases must contain lengthy untranslated regions or encode a polypeptide or polypeptides whose cumulative molecular weights are substantially greater than the 50,000 dalton ,I3 tubulin subunit. In this regard, we have shown that a p tubulin chain of mobility indistinguishable from the normal P polypeptide is synthesized in vitro from both the RNAs transcribed from gene ,I31 and gene p4. We favor the possibility that the approximately 2700 additional bases present on these mRNAs are noncoding. The alternative hypothesis that these large mRNAs may encode more than one polypeptide seems unlikely, since no polycistronic mRNA encoded by a cellular gene has yet been discovered in a eucaryote. This point is under continuing investiga- tion.

Cell 722

detected at least two ,8 tubulin genes, implying that all cells require at least two such gene products. It is tempting to speculate from these data that since microtubules in every dividing cell must undergo cyclic polymerization/depolymerization, first to convert the interphase arrays of microtubules into a mitotic configuration and second to restore the interphase array, a different ,8 tubulin subunit might be required for the proper assembly of each of these arrays.

Experimental Procedures

Screening of the Lambda Library Genomic DNA segments containing p tubulin sequences were isolated by screening an amplified lambda library prepared from embryonic chicken DNA. The library, constructed by B. Vennstrom (Vennstrom and Bishop, 1982), was produced by partial digestion of chicken DNA with Mbo I followed by selection of DNA molecules approximately 15-20 kb in length. These were then ligated into Charon 30 which had been digested with Barn HI. Approximately 2.0 million phages were screened for /3 tubulin sequences by the protocol of Maniatis et al. (1978). “P-labeled probe was prepared as described by Shank et al. (1978) from an embryonic chick brain cDNA clone (pT2) containing the complete coding sequence for p tubulin (Cleveland et al., 1980). Conditions of hybridization and washing were as given below for DNA blots.

Restriction endonuclease digestions of phage DNA were performed according to the suppliers’ recommendations. Enzymes were obtained from New England BioLabs or BRL.

Gel Electrophoresis and Blotting Gel electrophoresis of DNA was carried out on vertical slab gels cast from 0.8-l .2% agarose containing 40 mM Tris-Cl, pH 8.1, 20 mM Na-acetate and 2 mM EDTA. Gels were stained with 1 pg/ml of ethidium bromideforvisualization of DNA bands. DNA was transferred to nitrocellulose filters according to the method of Southern (1975).

Gel electrophoresis of RNA was normally carried out on vertical slab gels cast from 0.8% agarose containing 2.2 M formaldehyde (Boedtker, 1971). RNA was transferred to nitrocellulose according to Thomas (1980). Filters containing bound DNA or RNA were heated to 80°C for 3 hr and prehybridized for 2-16 hr at 41’C in 50% formamide, 5X SSC (1 X SSC = 150 mM NaCI, 15 mM Na-citrate), 20 mM HEPES (pH 7.4), 0.4 mg/ml yeast tRNA, 0.06 mg/ml soni- cated, denatured salmon sperm DNA, 0.06% bovine albumin, 0.06% ficoll 400, 0.06% polyvinylpyrrolidone. The filters were hybridized in the same solution containing approximately lo6 dpm/ml of a 32P- labeled probe. Filters were washed in three changes of 0.1 X SSC. 0.1% SDS at 53’C for a total wash time of 1 hr. Hybridization was detected on RP-XOmat x-ray film using Du Pont Cronex Lightning Plus intensifying screens.

When RNA was to be recovered from a gel and translated in vitro, 1 % gels were cast from low melting temperature agarose (Sigma) containing 10 mM methylmercury hydroxide as detailed by Bailey and Davidson (1976). The resultant gels were incubated in 0.1 M 2- mercaptoethanol for 30 min and then sliced with a razor blade using ethidium-bromide-stained rRNA markers as guides. RNA in individual gel slices was recovered as follows: 0.3 ml of 50 mM Tris-Cl, pH 7.4, 0.1% SDS, 1 mM EDTA was added to each gel slice, and the aliquots were heated for 3-5 min at 65’C to melt the agarose. Each fraction was extracted twice with phenol, then twice with chloroform, and finally poly(A)+ RNA was recovered by passage over an oligo(dT) column. Ten micrograms of tRNA carrier (Miles) was added, and each fraction was twice precipitated with ethanol. Recovered RNA was then resuspended in water and stored at -80°C.

Protein samples were electrophoresed on 8.5% polyacrylamide gels containing SDS according to Laemmli (19701, with the exception that the pH of the resolving gels was titrated to pH 9.1. Radioactive

polypeptides were visualized by fluorography (Bonnet and Laskey. 1974) of dried gels.

Electron Microscopic Heteroduplex Analysis of Genomic Clones Subclones of /31 and 83 in pBR322 were digested with Hind Ill restriction endonuclease; those of fi2 and /34 were digested with Eco RI or Bst Eli, respectively (see Figure 1). The cDNA clone pT2 was linearized with Sal I. The restricted DNAs were extracted with phenol and ether, precipitated with ethanol and then redissolved in 10 mM Tris and 1 mM EDTA. pH 8.5. Each of the genomic DNA clones was then separately hybridized to the cDNA clone in the presence of 40% or 50% formamide at 22’C or 37OC. Aliquots were used to prepare electron microscope grids using the formamide technique. Grids were examined in a Zeiss IOA electron microscope. Micrographs were analyzed with a Numonics electronic planimeter. The renatured pBR322 DNA (4362 base pairs) and the single stranded cDNA loop (1681 bases) in pT2/pBR322 heteroduplexes were used as double- and single-stranded DNA length standards, respectively.

Isolation of Gene-Specific Probes The putative 3’ noncoding region corresponding to the transcription product of the pl gene was prepared by subcloning the 9.8 kb Hind Ill fragment from phage fil into the Hind Ill site of pBR322. Orientation of the /?I sequence in the plasmid was determined to be such that the transcription of the 81 gene was in the direction (5’ to 3’) of the single Barn HI site of pBR322. The hybrid plasmid was digested with Barn HI, and the 5.0 kb fragment which contained 0.2 kb of pBR322 and 4.8 kb of chicken DNA extending from the single Barn HI site near the 3’ terminus of the pl coding region to the distal Hind Ill site of the subcloned chicken DNA was then purified by electrophoresis and electroelution. This segment represented the j31 3’ probe.

For p2, we initially determined that the cDNA clone pT2 was derived from this gene (see Results and Figure 28). We and our colleagues have already established that pT2 contains the complete coding sequence and complete 3’ untranslated region of its comple- mentary mRNA (Valenzuela et al., 1981). Hence, it was possible to isolate from pT2 a 240 bp fragment which contained only 3’ untranslated region. The segment selected extended from the Dde I site 30 bp 3’ to the UGA termination codon of pT2 to the terminal Pst I site produced in the original construction of the plasmid. This fragment was isolated, treated with Si nuclease, ligated to Hind Ill linkers (Collaborative Research) and recloned into the Hind Ill site of pBR322. This subcloned Dde I-Pst I fragment represented the p2 3’ probe.

For j33, the 9.9 kb Hind Ill fragment of phage /33 was subcloned into pBR322. The resultant plasmid was digested with Barn HI, and the 4.4 kb fragment extending from the 3’ terminus of the B tubulin coding region to the adjacent Barn HI site was isolated. This segment represented the p3 3’ probe.

For 84, an initial subclone of the 9 kb Sph I fragment (the raised, expanded portion of 84 in Figure 1) from the b4 phage was constructed in the Sph I site of pBR322. This plasmid, containing a single Hind Ill site in the pBR322 portion and single Xho I and Kpn I sites in the fl4 portion, was digested with these three enzymes, and the 5.7 kb Xho I-Kpn I fragment containing the 84 coding region and some of its presumptive 3’ untranslated region was isolated. This fragment was used as the p4 probe.

In Vitro Labeling of DNA Cloned DNA sequences were labeled in vitro with 32P essentially as described by Shank et al. (1978). Briefly, a 32P-labeled deoxynucle- otide triphosphate was incorporated into short DNA segments by reverse transcriptase using cloned DNA as templates. DNA synthesis was primed with a 1 OOO-fold weight excess of oligomers derived from calf thymus DNA.

Preparation of RNA Cytoplasmic RNA from chick fibroblasts and chick brain was prepared by SDS-phenol extraction and ethanol precipitation as previously described (Cleveland et al., 1980, 1981 b). Total RNA from 1104.


R2B. BK4408 and 249 cells was prepared with the CsCl step method of Chirgwin et al. (1979). Poly (A)-containing RNA was selected by chromatography on oligo(dT)-cellulose (Collaborative Research).

In Vitro Translation and lmmunopracipitation RNA was translated in vitro using the RNA-dependent rabbit reticulocyte system (Pelham and Jackson, 1976) and 35S-methionine (Amersham, 1000-l 500 Ci/mmole). lmmunoprecipitations of in vitro translation products were performed by diluting the translation mix- ture with an equal volume of Laemmli gel sample buffer (Laemmli, 1970) and boiling for 2-3 min. The samples were then diluted to a final concentration of SDS of 0.5% with phosphate-buffered saline containing 0.05% NP40. Indirect immunoprecipitations were then performed using an affinity-purified rabbit anti-chick tubulin antiserum and cross-linked Staphylococcus aureus as previously described (Cleveland et al., 1981 b).

Acknowledgments

A big thanks to Drs. Steve Hughes and Tom Kost at Cold Spring Harbor Laboratory for familiarizing us with how to work with recombinant bacteriophage. We also wish to thank Dr. Bjorn Vennstrom for providing the chicken genomic library. This work has been supported by a grant from the National Institutes of Health to D. W. C., who is the recipient of a Research Career Development Award from the NIH.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 14. 1982; revised January 7, 1983

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