Homology maps of the Drosophila α-tubulin gene family: one of the four genes is different (original) (raw)
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The beta 3-tubulin gene of Drosophila melanogaster is essential for viability and fertility
Genetics, 1991
We have previously shown that the P3-tubulin gene of Drosophila melanogaster encodes a divergent isoform expressed in a complex developmental pattern. The P3 gene is transiently expressed in the embryo and again in the pupa at high levels in the developing musculature, and at lower levels in several different pupal tissues of ectodermal origin. Adult expression is confined to specific somatic cells in the gonads. In some of the cell types in which it is expressed, P3 is the sole or predominant 8tubulin, while in others the 83 protein is a minor component of the P-tubulin pool. The sites and timing of P3 expression demonstrated that P3-tubulin is utilized primarily in cytoplasmic microtubule arrays involved in changes in cell shape and tissue organization, and suggested to us that this isoform may be functionally specialized. T o determine whether the expression of the P3 gene is essential for normal development, and to examine the specific functions of this divergent isoform, we have generated mutations within the gene. We determined that the small deficiency Df(2R)PxZ, which deletes the 60C5,6-60D9,10 region of chromosome 2, removes all of the P3 coding sequences, and that the distal breakpoint of the deficiency is approximately 2 kb upstream from the start of transcription of the 83 gene. We have generated a total of 31 ethyl methanesulfonate-or diepoxybutane-induced recessive lethal or visible mutations which map within the deficiency. These mutations define 12 new lethal complementation groups, which together with two previously identified visible mutations, altogether identify 14 genes in this interval of the second chromosome. A lethal complementation group comprising mutations in the P3-tubulin gene (PTub60D) was identified by rescue of their lethality by a wild-type copy of the gene introduced into the genome via P element-mediated germ line transformation. Analysis of the homozygous and transheterozygous phenotypes of the five 03 mutations recovered (alleles designated B?t'-B?t5) demonstrates that P3-tubulin is essential for viability and fertility.
Background: The completion of 19 insect genome sequencing projects spanning six insect orders provides the opportunity to investigate the evolution of important gene families, here tubulins. Tubulins are a family of eukaryotic structural genes that form microtubules, fundamental components of the cytoskeleton that mediate cell division, shape, motility, and intracellular trafficking. Previous in vivo studies in Drosophila find a stringent relationship between tubulin structure and function; small, biochemically similar changes in the major alpha 1 or testis-specific beta 2 tubulin protein render each unable to generate a motile spermtail axoneme. This has evolutionary implications, not a single non-synonymous substitution is found in beta 2 among 17 species of Drosophila and Hirtodrosophila flies spanning 60 Myr of evolution. This raises an important question, How do tubulins evolve while maintaining their function? To answer, we use molecular evolutionary analyses to characterize the evolution of insect tubulins. Results: Sixty-six alpha tubulins and eighty-six beta tubulin gene copies were retrieved and subjected to molecular evolutionary analyses. Four ancient clades of alpha and beta tubulins are found in insects, a major isoform clade (alpha 1, beta 1) and three minor, tissue-specific clades (alpha 2-4, beta 2-4). Based on a Homarus americanus (lobster) outgroup, these were generated through gene duplication events on major beta and alpha tubulin ancestors, followed by subfunctionalization in expression domain. Strong purifying selection acts on all tubulins, yet maximum pairwise amino acid distances between tubulin paralogs are large (0.464 substitutions/site beta tubulins, 0.707 alpha tubulins). Conversely orthologs, with the exception of reproductive tissue isoforms, show little sequence variation except in the last 15 carboxy terminus tail (CTT) residues, which serve as sites for post-translational modifications (PTMs) and interactions with microtubule-associated proteins. CTT residues overwhelming comprise the co-evolving residues between Drosophila alpha 2 and beta 3 tubulin proteins, indicating CTT specializations can be mediated at the level of the tubulin dimer. Gene duplications post-dating separation of the insect orders are unevenly distributed, most often appearing in major alpha 1 and minor beta 2 clades. More than 40 introns are found in tubulins. Their distribution among tubulins reveals that insertion and deletion events are common, surprising given their potential for disrupting tubulin coding sequence. Compensatory evolution is found in Drosophila beta 2 tubulin cis-regulation, and reveals selective pressures acting to maintain testis expression without the use of previously identified testis cis-regulatory elements. Conclusion: Tubulins have stringent structure/function relationships, indicated by strong purifying selection, the loss of many gene duplication products, alpha-beta co-evolution in the tubulin dimer, and compensatory evolution in beta 2 tubulin cis-regulation. They evolve through gene duplication, subfunctionalization in expression domain and divergence of duplication products, largely in CTT residues that mediate interactions with other proteins. This has resulted in the tissue-specific minor insect isoforms, and in particular the highly diverse α3, α4, and β2 reproductive tissue-specific tubulin isoforms, illustrating that even a highly conserved protein family can participate in the adaptive process and respond to sexual selection.
Two Drosophila beta tubulin isoforms are not functionally equivalent
The Journal of Cell Biology, 1990
We have tested the functional capacity of different beta tubulin isoforms in vivo by expressing B3-tubulin either in place of or in addition to/32tubulin in the male germ line of Drosophila melanogaster. The testes-specific isoform, B2, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform,/33, is considerably divergent in sequence. B3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line, /~2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of B2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5' sequences from the/~2 gene were joined to protein coding and 3' sequences of the ~3 gene. Drosophila transformed