Mauricio M. Bustos

The isolation, characterization and sequence of two divergent β-tubulin genes from soybean (Glycine max L.)

Two divergent β-tubulin genes (designated Sβ-1 and Sβ-2) were isolated by screening a soybean genomic library with a Chlamydomonas reinhardtii β-tubulin cDNA probe. Restriction fragment analysis of the clones recovered, and of soybean genomic DNA, indicated that these represent two unique classes of structurally different β-tubulin genes in the soybean genome. However, it is possible that unidentified members of these classes or additional highly divergent classes of β-tubulin genes (thus far undetected) exist in the soybean genome. The Sβ-1 and Sβ-2 genomic clones were sequenced, revealing that both are potentially functional genes which would encode β-tubulins of 445 and 449 amino acids, respectively. A comparison of their derived amino acid sequences with β-tubulins from several organisms showed that they are most homologous to Chlamydomonas β-tubulin (85–87%), with lesser degrees of homology to β-tubulins of vertebrate species (79–83%), Trypanosoma brucei (80–81%) and Saccharomyces cerevisiae (66–68%). The amino acid sequences of Sβ-1 and Sβ-2 are as divergent from each other as they are from the Chlamydomonas β-tubulin. The amino acids at the diverged positions in Sβ-2 are nearly all conservative substitutions while in Sβ-1, 18 of the 69 substitutions were non-conservative. Both soybean β-tubulin genes contain two introns in exactly the same positions. The first soybean intron is located in the same position as the third intron of the Chlamydomonas β-tubulin genes. Codon usage in the two soybean β-tubulins is remarkably similar (D2=0.87), but differs from codon usage in other soybean genes.

Expression, glycosylation and secretion of phaseolin in a baculovirus system

In this report, we describe the efficient expression and glycosylation, in insect cells, of β-phaseolin polypeptides (Mr 45 and 48 kDa) from Phaseolus vulgaris, by means of a baculovirus expression vector. N-terminal sequence analysis demonstrated that the signal peptide was efficiently processed. Tunicamycin treatment suppressed both phaseolin bands seen in untreated or control cells, and resulted in a single species (Mr 43 kDa). We provide evidence that the observed size heterogeneity arises by asymmetric glycosylation of a single, high-molecular weight precursor. These results also indicate that differential glycosylation of phaseolin polypeptides can occur on the product of a single gene, and, in that sense, is not dependent on amino acid sequence variations. Phaseolin accumulates to a very high level (90 µg/106 cells), 90% of it being secreted into the culture medium. Immuno-gold staining and electron microscopy demonstrated phaseolin polypeptides in electron-dense, membrane-bound vesicles seen at the periphery of the cytoplasm of infect cells and in cytoplasmic multivesicular bodies. The effect on protein accumulation of a single-basepair transversion (G»C) at position +6 is also described. This study constitutes, to our knowledge, one of the first instances of a plant protein being expressed in insect cells and suggests possible differences in the sorting mechanisms of glycoproteins from legume seeds and those from Spodoptera frugiperda cell line Sf9.

Developmental modulation of tubulin protein and mRNA levels during somatic embryogenesis in cultured carrot cells

The number of cortical microtubules (MTs) increases considerably as cultured carrot (Daucus carota L.) cells initiate and progress through somatic embryogenesis. The basis for this increase in MT number was investigated. A radioimmune assay was used to show that tubulin-protein per cell first decreased as the undifferentiated cells initiated embryonic development, but subsequently increased approximately fivefold between the globular and torpedo/plantlet stages. The increase during the torpedo/plantlet stage was correlated with the increase in cell size that occurred during the latter stages of embryogenesis. The cellular levels of tubulin mRNA were determined by Northern blot analysis, using labeled probes derived from soybean α- and β-tubulin genomic sequences, cloned in the vectors pSP64 and pSP65. This analysis demonstrated that the levels of tubulin-gene transcripts varied with the tubulin-protein levels. Cell-free translation of polyadenylated RNA, followed by immunoprecipitation with an anti-tubulin antiserum, established that these transcripts represented functional tubulin mRNA. These results indicate that MT formation in early embryogenesis is controlled by factors other than the availability of tubulin, but that MT formation later in embryogenesis is coordinated with concomitant changes in tubulin-gene transcription and in the size of the total tubulin-heterodimer pool.

Differential accumulation of four phaseolin glycoforms in transgenic tobacco

An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical β-phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (βwti−) and mutant phaseolin glycoforms (βdgly1, βdgly2 and βdgly1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the βdgly1 and βdgly2 mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin βdgly1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.

The expression of a chimeric soybean beta-tubulin gene in tobacco

Summary. A chimeric tubulin gene has been constructed by the fusion of a genomic sequence containing a truncated soybean beta-tubulin gene with 2 kb of upstream DNA to the Y untranslated region containing the polyadenylation signal from transcription unit 7 of the octopine Ti plasmid pGV117. The chimeric gene has been incorporated into the Ti plasmid transformation vector pGV3850: :pAP2034, along with a selectable marker gene active in plants (the neomycin phosphotransferase II gene, conferring kanamycin resistance). Strains of Agrobacterium tumefaciens harboring the plasmids were used to transform Nicotiana tabacum by the leaf disk method and plants were regenerated from transformed cells. Transgenic plants were self fertilized and the segregation of kanamycin resistance was assayed. DNA and RNA were extracted from transgenic plants, fractionated by agarose gel electrophoresis, blotted to nitrocellulose and hybridized to a probe specific for the chimeric gene to assess its structure and expression in transgenic plants. The chimeric gene was stably integrated into the tobacco genome without rearrangements and it was expressed as a polyadenylated RNA of 1.7 kb in the transformants. Genetic analysis revealed that the kanamycin-resistant phenotype was inherited in a Mendelian fashion over two generations.

Changes in the relative electrophoretic mobility of higher plant tubulin subunits in SDS-polyacrylamide gels

Abstract The relative electrophoretic mobility of higher plant tubulin subunits in SDS-polyacrylamide gels varies depending upon the electrophoretic methods used to separate them. When reduced and alkylated rat and carrot tubulin heterodimers were separated by one method, the α-tubulin subunits, identified by means of a highly specific antibody, migrated more slowly than the β-tubulin subunits. However, when separated by another method, the carrot α-subunit migrated more rapidly than its β-subunit, while the relative mobility of rat brain tubulin subunits was unchanged. The two gel systems differ principally in the pH of the separating gel during electrophoresis, suggesting that pH markedly influences the interaction of SDS with the plant, but not the vertebrate α-tubulin.