Most studies of cytoskeletal organelles have concentrated on molecular analyses of abundant and biochemically accessible structures. In many of the classical cases, however, the nature of the system ...chosen has precluded a concurrent genetic analysis. The mitotic spindle of the yeast Saccharomyces cerevisiae is one example of an organelle that can be studied by both classical and molecular genetics. We show here that this microtubule structure also can be examined biochemically. The spindle can be isolated by selective extractions of yeast cells by using adaptations of methods successfully applied to animal cells. In this way, microtubule-associated proteins of the yeast spindle are identified.
The role of divergent primary sequences in restricting tubulin function was tested in vivo by a gene transfection experiment. A chicken-yeast chimeric beta-tubulin DNA was introduced into 3T3 cells ...using the transfection vector pSV2. The 5' end of this gene, from chicken, is similar but not identical with that of mouse beta-tubulins; the 3' end, from yeast, contains a carboxyl terminus that is very different from other known beta-tubulin sequences. The chimeric protein is incorporated efficiently into each of the microtubule structures and each of the microtubules in the host cells. The presence of the protein has no apparent effect on either growth rate or cell morphology. The results suggest that the divergent sequences in this chimeric tubulin molecule place no restrictions on its activities in mouse cells.
Gene silencing is required to stably maintain distinct patterns of gene expression during eukaryotic development and has been correlated with the induction of chromatin domains that restrict gene ...activity. We describe the isolation of human (EZH2) and mouse (Ezh1) homologues of the Drosophila Polycomb‐group (Pc‐G) gene Enhancer of zeste E(z), a crucial regulator of homeotic gene expression implicated in the assembly of repressive protein complexes in chromatin. Mammalian homologues of E(z) are encoded by two distinct loci in mouse and man, and the two murine Ezh genes display complementary expression profiles during mouse development. The E(z) gene family reveals a striking functional conservation in mediating gene repression in eukaryotic chromatin: extra gene copies of human EZH2 or Drosophila E(z) in transgenic flies enhance position effect variegation of the heterochromatin‐associated white gene, and expression of either human EZH2 or murine Ezh1 restores gene repression in Saccharomyces cerevisiae mutants that are impaired in telomeric silencing. Together, these data provide a functional link between Pc‐G‐dependent gene repression and inactive chromatin domains, and indicate that silencing mechanism(s) may be broadly conserved in eukaryotes.
This chapter highlights key findings concerning the assembly of silent chromatin and the regulation of silent chromatin spreading in budding yeast, focusing on the function of Sir proteins in ...silencing. In particular, the proteins and histone modifications that positively influence silent chromatin formation or restrict silent chromatin spreading are discussed. In Saccharomyces cerevisiae (S. cerevisiae) or budding yeast, silent chromatin is cytologically distinct from heterochromatin in other eukaryotes, but many of the proteins and histone modifications involved are conserved through humans. In particular, the process of silent chromatin formation and its regulation in yeast has led to an increased understanding of how chromosomal position can affect gene expression. Numerous proteins have been implicated in silencing, linking diverse cellular processes to silencing, including cell cycle progression, DNA repair, and DNA replication, among other functions. Sir proteins themselves have also been implicated in other processes, including aging, chromosome stability, DNA repair, and DNA replication. Many recent studies have addressed the establishment and maintenance of silent chromatin and the control of silent chromatin spreading. Histone modifications and histone modifying enzymes have been identified that are required for promoting or restricting silent chromatin spreading.
The entire framework of microtubules (MTs) in the mitotic apparatus of Ochromonas danica is reconstructed (except at the spindle poles) from transverse serial sections. Eleven spindles were sectioned ...and used for numerical data, but only four were reconstructed: a metaphase, an early anaphase, a late anaphase, and telophase. Four major classes of MTs are observed: (a) free MTs (MTs not attached to either pole); (b) interdigitated MTs (MTs attached to one pole which laterally associate with MTs from the opposite pole); (c) polar MTs (MTs attached to one pole); (d) kinetochore MTs (kMTs). Pole-to-pole MTs are rare and may be caused by tracking errors. During anaphase, the kMTs, free MTs, and polar MTs shorten until most disappear, while interdigitated MTs lengthen. In the four reconstructed spindles, the number of MTs decreases between early anaphase and telophase from 881 to 285, while their average length increases from 1.66 to 4.98 μm. The total length of all the MTs in the spindle (placed end to end) remains at 1.42 ± 0.04 mm between these stages. At late anaphase and telophase the spindle is comprised mainly of groups of interdigitated MTs. Such MTs from opposite poles from a region of overlap in the middle of the spindle. During spindle elongation (separation of the poles), the length of the overlap region does not decrease. These results are compatible with theories that suggest that MTs directly provide the force that elongates the spindle, either by MT polymerization alone or by MT sliding with concomitant MT polymerization.
Two α-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and ...TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to α-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences. Each gene had a single intervening sequence, located at an identical position in codon 9. Cell fractionation studies showed that both gene products were present in yeast microtubules. These two genes, along with the TUB2 β-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.
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