The colR4 and colR15 beta-tubulin mutations in Chlamydomonas reinhardtii confer altered sensitivities to microtubule inhibitors and herbicides by enhancing microtubule stability.

The colR4 and colR15 beta 2-tubulin missense mutations for lysine-350 in Chlamydomonas reinhardtii (Lee and Huang, 1990) were originally isolated by selection for resistance to the growth inhibitory effects of colchicine. The colR4 and colR15 mutants have been found to be cross resistant to vinblastine and several classes of antimitotic herbicides, including the dinitroanilines (oryzalin, trifluralin, profluralin, and ethafluralin); the phosphoric amide amiprophos methyl; and the dimethyl propynl benzamide pronamide. Like colchicine and vinblastine, the antimitotic effects of these plant-specific herbicides have been associated with the depolymerization of microtubules. In contrast to their resistance to microtubule-depolymerizing drugs, the mutants have an increased sensitivity to taxol, a drug which enhances the polymerization and stability of microtubules. This pattern of altered sensitivity to different microtubule inhibitors was found to cosegregate and corevert with the beta-tubulin mutations providing the first genetic evidence that the in vivo herbicidal effects of the dinitroanilines, amiprophos methyl, and pronamide are related to microtubule function. Although wild-type like in their growth characteristics, the colR4 and colR15 mutants were found to have an altered pattern of microtubules containing acetylated alpha-tubulin, a posttranslational modification that has been associated with stable subsets of microtubules found in a variety of cells. Microtubules in the interphase cytoplasm and those of the intranuclear spindle of mitotic cells, which in wild-type Chlamydomonas cells do not contain acetylated alpha-tubulin, were found to be acetylated in the mutants. These data taken together suggest that the colR4 and colR15 missense mutations increase the stability of the microtubules into which the mutant beta-tubulins are incorporated and that the altered drug sensitivities of the mutants are a consequence of this enhanced microtubule stability.     

Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function.

The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.     

CHO mutants resistant to colchicine,colcemid or griseofulvin have an altered β-tubulin

Single-step mutants of Chinese hamster ovary (CHO) cells have been isolated which are resistant to killing by the anti-mitotic drugs colchicine, colcemid or griseofulvin. Two-dimensional gel analysis showed that two mutants resistant to griseofulvin, one resistant to colcemid and one resistant to colchicine carry an alteration in the β-tubulin subunit. Most of the remaining isolates are believed to be permeability mutants on the basis of their cross resistance to drugs which do not interfere with microtubular polymerization or function (Ling and Thompson, 1974; Bech-Hansen, Till and Ling, 1976). A reduced amount of the wild-type β-tubulin protein remained in each of the β-tubulin mutants, but a β-tubulin protein with a more basic isoelectric point also appeared. Messenger RNAs coding for both wild-type and variant β-tubulins were found in at least one mutant as assayed by in vitro translation in a reticulocyte lysate. This indicates that the altered tubulin does not arise as the result of a post-translational modification.     

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.     

Molecular cloning and selection of genes regulated in Aspergillus development

Over 350 clones homologous to poly(A)+ RNAs that are significantly more prevalent in conidiating cultures of Aspergillus nidulans than in somatic cells have been selected from a recombinant DNA library formed between nuclear DNA and lambda Charon 4A. The procedure used for this selection involved in situ hybridization to a cDNA probe which had been selectively depleted of sequences represented in somatic cells by complement hybridization. Five of these clones have been characterized further. All but one encoded poly(A)+ RNAs that were at least ten times more prevalent in conidiating cultures than in somatic cells. One clone hybridized to a single, developmentally regulated RNA. The three others were complementary to several RNAs having different molecular weights, each of which was more prevalent in condiating cultures than in vegetative cells. These results and quantitative aspects of the selection procedure suggest that developmentally controlled poly(A)+ RNA coding regions may not be distributed randomly in the Aspergillus genome.     

Xenopus laevis serum albumins are encoded in two closely related genes.

cDNA clones containing sequences complementary to Xenopus laevis albumin mRNA have been identified in a collection of cDNA clones made from poly(A)+ RNA prepared from male Xenopus laevis liver. Although all the albumin cDNA clones crosshybridise, restriction enzyme and heteroduplex analysis show that there are 2 closely related albumin mRNA sequences. The 2 albumin mRNAs are only mismatched by 8% but could be isolated by positive selection using stringent hybridization conditions. Oocytes injected with the 2 purified mRNAs, secreted either the 68,000 or 74,000 dalton albumin into the culture medium showing that the 2 albumins of X. laevis serum are encoded in the 2 closely related mRNAs. Measurements of the abundance of albumin mRNA show that the 2 albumin mRNAs together account for about 9% of total poly(A)+ RNA in male Xenopus laevis liver but the mRNA coding for the 74,000 dalton mRNA is about twice as abundant as that coding for the 68,000 dalton mRNA.     

Involvement of a particular species of beta-tubulin (beta 3) in conidial development in Aspergillus nidulans

Strains of Aspergillus containing the benA22 mutation are resistant to benomyl for vegetative growth but do not produce conidia. To test whether conidiation involved an additional benomyl-sensitive tubulin (i.e., was mediated by a tubulin other than the tubulins coded for by the benA locus), a collection of mutants was produced that formed conidia in the presence of benomyl, i.e., were conidiation-resistant (CR-) mutants. We analyzed the tubulins of these CR- mutants using two-dimensional gel electrophoresis and found that the mutants lacked one species of beta-tubulin (designated beta 3). We have examined two of these mutants in detail. In crosses with strains containing wild-type tubulins, we found that the absence of the beta 3-tubulin co-segregated perfectly with the CR- phenotype. In diploids containing both the benA22 and CR- mutations, we found that the CR- phenotype was recessive and that beta 3-tubulin was present on two-dimensional gels of tubulins prepared from these diploids. In another set of crosses, these two CR- strains and seven others were first made auxotrophic for uridine and then crossed against strains that had homologously integrated a plasmid containing an incomplete internal fragment of the beta 3-tubulin gene and the pyr4 gene of Neurospora crassa (which confers uridine prototrophy on transformants). If the CR- phenotype were produced by a mutation in a gene distinct from the structural gene for beta 3-tubulin (designated the tubC gene), then crossing over should have produced some CR+ segregants among the uridine auxotrophic progeny of the second cross. All of the uridine auxotrophs from this type of cross, however, showed the CR- phenotype, suggesting that the mutation in these strains is at or closely linked to the tubC locus. The most obvious explanation of these results is that beta 3-tubulin is ordinarily used during conidiation and the presence of this species of beta-tubulin renders conidiation sensitive to benomyl. In the CR- mutants, beta 3-tubulin is absent, and in the presence of the benA22 mutation the benomyl-resistant beta 1-and/or beta 2-tubulin substitutes for beta 3 to make conidiation benomyl resistant. We discuss these results and give two models to explain the interactions between these beta-tubulin species.     

Contact-dependent regulation of vinculin expression in cultured fibroblasts: a study with vinculin-specific cDNA probes.

Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin-specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin-specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0-7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin-specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin-rich focal contacts, while cells grown on poorly adhesive substrate poly(2-hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5-fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5-fold decrease in the levels of vinculin-specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Copia RNA levels are elevated in dunce mutants and modulated by cAMP

Clones carrying sequences expressed at altered abundance levels in dunce mutants were isolated by differentially screening a genomic library with cDNA probes representing the RNA population from dunce+ flies and the RNA population from dunce mutant flies. These mutants have an elevated cAMP content, so some isolates potentially contain cAMP responsive genes. Two classes of clones were isolated. One class contains genes expressed at a higher steady state abundance level in dunce mutants compared to dunce+ flies and the other contains genes expressed at a lower steady state level in the mutants. The recovery of clones from the differential screen demonstrates that in addition to altering normal behavior, fertility, and cAMP metabolism, dunce mutation confers an alteration in the level of expression of certain genes. The class of clones carrying sequences which are overexpressed in the mutants have been characterized. These clones carry a common repetitive sequence which codes for a 5.5 kb poly(A)+ RNA - the RNA species found to be overexpressed in the mutants. Restriction analysis and hybridization experiments show these repetitive sequences to be members of the copia family of transposable elements. Administration of pharmacological agents to normal flies to increase cAMP levels leads to an increased steady state level of copia RNA. Thus, copia RNA metabolism appears to be influenced by cAMP levels.     

mRNA from mutant Y1 adrenal cells directs the synthesis of altered regulatory subunits of type 1 cAMP-dependent protein kinase

The molecular basis for altered cyclic AMP-dependent protein kinase activity was examined in three different mutant clones (Kin-1, Kin-7, and Kin-8) derived from the Y1 mouse adrenocortical cell line. Parental Y1 cells and the Kin mutants were labeled with L-[35S] methionine and the regulatory subunit of the type 1 cAMP-dependent protein kinase isozyme (RI) was immunoprecipitated from each clone with a specific guinea pig antiserum. When analyzed by electrophoresis on isoelectric focusing gels, the immunoprecipitates from mutant clones exhibited parental forms of RI plus an additional acidic variant form which likely accounted for altered cAMP-dependent protein kinase activity. Poly(A+) RNA was isolated from Y1 and Kin mutant cells and was translated in a cell-free, reticulocyte lysate system in the presence of L-[35S]methionine. The RI synthesized from poly(A+) RNA was immunoprecipitated from the translation mixture and analyzed on isoelectric focusing gels. The poly(A+) RNA from the Kin mutant clones directed the synthesis of parental and acidic variant forms of RI. These results suggest that the altered electrophoretic forms of RI arise from mutations in one of two RI genes rather than from post-translational modifications of the protein. The coexistence of parental and variant forms of RI in the Kin mutants indicate that the mutations are codominant.     

The sequence and expression of the divergent beta-tubulin in chicken erythrocytes

We report here the complete sequence of a highly divergent chicken erythrocyte beta-tubulin, c beta 6, which appears to represent a major exception to the observation that the primary sequences and sites of expression of beta-tubulin isotypes are conserved within vertebrates. The amino acid sequence was deduced from overlapping cloned cDNAs identified in a chicken erythroblast cDNA library contained in the expression vector, lambda gt11. Compared with other chicken beta-tubulins, among which the maximum sequence divergence is only 8%, c beta 6-tubulin is more hydrophobic, contains seven fewer net negative charges, and exhibits a surprising 17% overall divergence in its amino acid sequence. DNA and RNA blot analyses show that c beta 6-tubulin is present as a single gene copy in the chicken genome and is specifically expressed in the bone marrow. Comparisons of RNA blots and immunoblots of various cells and tissues confirm that this beta-tubulin isotype is contained specifically in erythrocytes and thrombocytes and accounts for 75% of the beta-tubulin mRNA species contained in developing erythroblasts. Interestingly, c beta 6-tubulin exhibits 18% amino acid sequence divergence relative to MB1, the analogous hematopoietic beta-tubulin contained in mouse.     

Aspergillus contains multiple tubulin genes

Previous work with benomyl-resistant mutants of Aspergillus nidulans has demonstrated that the benA locus is a structural gene for beta-tubulin. Two of the benA mutants, benA22 and benA85, show altered electrophoretic mobilities on two-dimensional gels for two beta-tubulins (designated beta 1 and beta 2). These shifts of the beta 1- and beta 2-tubulins uncover a spot in the region where wild-type beta-tubulins migrate that is occluded on gels of wild-type extracts by the beta 1- and beta 2-tubulins. Evidence has now been obtained indicating that this spot represents an additional beta-tubulin (designated beta 3). Tubulin was partially purified from Aspergillus and run on one- and two-dimensional gels and the band or spot uncovered by the shift of the beta 1- and beta 2-tubulins was identified as a beta-tubulin by immunoblotting with monoclonal and affinity-purified polyclonal anti-tubulin antibodies and by one-dimensional peptide mapping. These observations show that Aspergillus contains at least two structural genes for beta-tubulins. Similar techniques have also been applied to a mutant showing altered alpha-tubulins to confirm and modify earlier observations suggesting that at least two structural genes for alpha-tubulins are also present.     

Sequence and expression of the chicken beta 5- and beta 4-tubulin genes define a pair of divergent beta-tubulins with complementary patterns of expression.

We have determined the nucleotide sequence of the chicken beta 5 (c beta 5)-tubulin gene. The gene displayed the coding structure common to all previously studied vertebrate beta-tubulin genes and was divided into four exon sequences interrupted by three intervening sequences (located between codons 19 and 20, within codon 56, and within codon 93). Comparison of the predicted polypeptide sequence encoded by c beta 5 with those of four other available chicken beta-tubulin sequences revealed that c beta 5 encoded a highly divergent beta-tubulin polypeptide isotype which was distinguished from previously known sequences primarily by two discrete variable sequence domains. However, c beta 5 uniquely shared identity in 16 residue positions with another divergent chicken beta-tubulin gene, c beta 4. These common sequences distinguished c beta 4 and c beta 5 from the remaining three chicken beta-tubulin genes. Analysis of the expression of c beta 5 and c beta 4 revealed a strikingly complementary pattern of gene expression: c beta 5 was expressed in a wide variety of cell and tissue types but not in neurons, whereas c beta 4 expression was detected uniquely in neuronal cells. Overall, these findings suggest the existence of two divergent families of beta-tubulin sequences in the chicken and further raise the possibility that the complementary expression of the c beta 4 and c beta 5 genes may fulfill a requirement for the presence of a divergent beta-tubulin polypeptide isotype in all cell types.