Biomedical subjects

R K Dawe

Publications and source records attributed to R K Dawe.

15 recordsLinked to original sources

PubMed2005

The simple ultrastructure of the maize kinetochore fits a two-domain model.

Light microscope observations suggest there are two discrete biochemical domains in the plant kinetochore, an inner domain containing structural proteins, and an outer domain containing proteins involved in motility. We analyzed the ultrastructure of maize meiotic kinetochores following high pressure freezing and freeze substitution, a method that provides excellent sample preservation. Data from meiosis II support previous descriptions of plant kinetochores as diffuse, nearly invisible domains, sometimes nesting in a cup of darkly staining chromatin. The ultrastructure is similar in meiosis I but there are two sister kinetochores that each protrude away from the chromosome and form their own distinct kinetochore fibers. Microtubules terminate within kinetochores where their ends are splayed in a cone-shaped configuration suggestive of microtubule disassembly. We could not detect any significant substructure within the kinetochore proper. We suggest that the diffuse structure classically defined as the kinetochore represents only the outer domain of a two-domain organelle. The inner domain, known to contain chromatin-binding proteins, probably extends into the electron-dense chromatin of the primary constriction.

Chromosomes, Plant↗

PubMed2001

Dyneins have run their course in plant lineage.

Flowering plant genomes lack flagellar and cytoplasmic dyneins as well as the proteins that make up the dynactin complex. The mechanisms for organizing the Golgi apparatus, establishing spindle poles, and moving nuclei, vesicles, and chromosomes in flowering plants must be fundamentally different from those in other systems where these processes are dependent upon dynein and dynactin.

Cytoplasm↗

PubMed2000

Functional redundancy in the maize meiotic kinetochore.

Kinetochores can be thought of as having three major functions in chromosome segregation: (a) moving plateward at prometaphase; (b) participating in spindle checkpoint control; and (c) moving poleward at anaphase. Normally, kinetochores cooperate with opposed sister kinetochores (mitosis, meiosis II) or paired homologous kinetochores (meiosis I) to carry out these functions. Here we exploit three- and four-dimensional light microscopy and the maize meiotic mutant absence of first division 1 (afd1) to investigate the properties of single kinetochores. As an outcome of premature sister kinetochore separation in afd1 meiocytes, all of the chromosomes at meiosis II carry single kinetochores. Approximately 60% of the single kinetochore chromosomes align at the spindle equator during prometaphase/metaphase II, whereas acentric fragments, also generated by afd1, fail to align at the equator. Immunocytochemistry suggests that the plateward movement occurs in part because the single kinetochores separate into half kinetochore units. Single kinetochores stain positive for spindle checkpoint proteins during prometaphase, but lose their staining as tension is applied to the half kinetochores. At anaphase, approximately 6% of the kinetochores develop stable interactions with microtubules (kinetochore fibers) from both spindle poles. Our data indicate that maize meiotic kinetochores are plastic, redundant structures that can carry out each of their major functions in duplicate.

Biomechanical Phenomena↗

PubMed2000

The plant kinetochore.

Kinetochores are large protein complexes that bind to centromeres. By interacting with microtubules and their associated motor proteins, kinetochores both generate and regulate chromosome movement. Kinetochores also function in the spindle checkpoint; a surveillance mechanism that ensures that metaphase is complete before anaphase begins. Although the ultrastructure of plant kinetochores has been known for many years, only recently have specific kinetochore proteins been identified. The recent data indicate that plant kinetochores contain homologs of many of the proteins implicated in animal and fungal kinetochore function, and that the plant kinetochore is a redundant structure with distinct biochemical subdomains.

Amino Acid Sequence↗

PubMed1999

Meiotic drive of chromosomal knobs reshaped the maize genome.

Meiotic drive is the subversion of meiosis so that particular genes are preferentially transmitted to the progeny. Meiotic drive generally causes the preferential segregation of small regions of the genome; however, in maize we propose that meiotic drive is responsible for the evolution of large repetitive DNA arrays on all chromosomes. A maize meiotic drive locus found on an uncommon form of chromosome 10 [abnormal 10 (Ab10)] may be largely responsible for the evolution of heterochromatic chromosomal knobs, which can confer meiotic drive potential to every maize chromosome. Simulations were used to illustrate the dynamics of this meiotic drive model and suggest knobs might be deleterious in the absence of Ab10. Chromosomal knob data from maize's wild relatives (Zea mays ssp. parviglumis and mexicana) and phylogenetic comparisons demonstrated that the evolution of knob size, frequency, and chromosomal position agreed with the meiotic drive hypothesis. Knob chromosomal position was incompatible with the hypothesis that knob repetitive DNA is neutral or slightly deleterious to the genome. We also show that environmental factors and transposition may play a role in the evolution of knobs. Because knobs occur at multiple locations on all maize chromosomes, the combined effects of meiotic drive and genetic linkage may have reshaped genetic diversity throughout the maize genome in response to the presence of Ab10. Meiotic drive may be a major force of genome evolution, allowing revolutionary changes in genome structure and diversity over short evolutionary periods.

Chromosome Aberrations↗

PubMed1999

A maize homolog of mammalian CENPC is a constitutive component of the inner kinetochore.

Genes for three maize homologs (CenpcA, CenpcB, and CenpcC) of the conserved kinetochore assembly protein known as centromere protein C (CENPC) have been identified. The C-terminal portion of maize CENPC shares similarity with mammalian CENPC and its yeast homolog Mif2p over a 23-amino acid region known as region I. Immunolocalization experiments combined with three-dimensional light microscopy demonstrated that CENPC is a component of the kinetochore throughout interphase, mitosis, and meiosis. It is shown that sister kinetochore separation occurs in two discrete phases during meiosis. A partial separation of sister kinetochores occurs in prometaphase I, and a complete separation occurs in prometaphase II. CENPC is absent on structures known as neocentromeres that, in maize, demonstrate poleward movement but lack other important features of centromeres/kinetochores. CENPC and a previously identified centromeric DNA sequence interact closely but do not strictly colocalize on meiotic chromosomes. These and other data indicate that CENPC occupies an inner domain of the maize kinetochore.

Amino Acid Sequence↗

PubMed1998

Plant centromeres: structure and control.

Recent work has led to a better understanding of the molecular components of plant centromeres. Conservation of at least some centromere protein constituents between plant and non-plant systems has been demonstrated. The identity and organization of plant centromeric DNA sequences are also beginning to yield to analysis. While there is little primary DNA sequence conservation among the characterized plant centromeres and their non-plant counterparts, some parallels in centromere genomic organisation can be seen across species. Finally, the emerging idea that centromere activity is controlled epigenetically finds support in an examination of the plant centromere literature.

Centromere↗

PubMed1997

Neocentromere-mediated chromosome movement in maize.

Neocentromere activity is a classic example of nonkinetochore chromosome movement. In maize, neocentromeres are induced by a gene or genes on Abnormal chromosome 10 (Ab10) which causes heterochromatic knobs to move poleward at meiotic anaphase. Here we describe experiments that test how neocentromere activity affects the function of linked centromere/kinetochores (kinetochores) and whether neocentromeres and kinetochores are mobilized on the spindle by the same mechanism. Using a newly developed system for observing meiotic chromosome congression and segregation in living maize cells, we show that neocentromeres are active from prometaphase through anaphase. During mid-anaphase, normal chromosomes move on the spindle at an average rate of 0.79 micron/min. The presence of Ab10 does not affect the rate of normal chromosome movement but propels neocentromeres poleward at rates as high as 1.4 micron/min. Kinetochore-mediated chromosome movement is only marginally affected by the activity of a linked neocentromere. Combined in situ hybridization/immunocytochemistry is used to demonstrate that unlike kinetochores, neocentromeres associate laterally with microtubules and that neocentromere movement is correlated with knob size. These data suggest that microtubule depolymerization is not required for neocentromere motility. We argue that neocentromeres are mobilized on microtubules by the activity of minus end-directed motor proteins that interact either directly or indirectly with knob DNA sequences.

Anaphase↗

PubMed1997

Conservation of the centromere/kinetochore protein ZW10.

Mutations in the essential Drosophila melanogaster gene zw10 disrupt chromosome segregation, producing chromosomes that lag at the metaphase plate during anaphase of mitosis and both meiotic divisions. Recent evidence suggests that the product of this gene, DmZW10, acts at the kinetochore as part of a tension-sensing checkpoint at anaphase onset. DmZW10 displays an intriguing cell cycle-dependent intracellular distribution, apparently moving from the centromere/kinetochore at prometaphase to kinetochore microtubules at metaphase, and back to the centromere/kinetochore at anaphase (Williams, B.C., M. Gatti, and M.L. Goldberg. 1996. J. Cell Biol. 134:1127-1140). We have identified ZW10-related proteins from widely diverse species with divergent centromere structures, including several Drosophilids, Caenorhabditis elegans, Arabidopsis thaliana, Mus musculus, and humans. Antibodies against the human ZW10 protein display a cell cycle-dependent staining pattern in HeLa cells strikingly similar to that previously observed for DmZW10 in dividing Drosophila cells. Injections of C. elegans ZW10 antisense RNA phenocopies important aspects of the mutant phenotype in Drosophila: these include a strong decrease in brood size, suggesting defects in meiosis or germline mitosis, a high percentage of lethality among the embryos that are produced, and the appearance of chromatin bridges at anaphase. These results indicate that at least some aspects of the functional role of the ZW10 protein in ensuring proper chromosome segregation are conserved across large evolutionary distances.

Animals↗

PubMed1996

Induction of centromeric activity in maize by suppressor of meiotic drive 1.

The Abnormal chromosome 10 (Ab10) in maize causes normally-quiescent blocks of heterochromatin called knobs to function as meiotic centromeres. Under these circumstances genetic markers associated with knobs exhibit meiotic drive, i.e., they are preferentially transmitted to progeny. Here we describe a mutation called suppressor of meiotic drive (smd1) that partially suppresses meiotic drive, and demonstrate that smd1 causes a quantitative reduction in the mobility of knobs on the meiotic spindle. We conclude that Smd1 encodes a product that is necessary for the activation of ectopic centromeres, and that meiotic drive occurs as a consequence of the resulting change in chromosome movement. As a genetic system, Ab10 offers a new and powerful approach for analyzing centromere/kinetochore function.

Anaphase↗

PubMed1996

Both 5' and 3' sequences of maize adh1 mRNA are required for enhanced translation under low-oxygen conditions.

Alcohol dehydrogenase-1 (ADH1) synthesis in O2-deprived roots of maize (Zea mays L.) results from induced transcription and selective translation of ADH1 mRNA. The effect of ADH1 mRNA sequences on message stability and translation was studied in protoplasts of the maize cell line P3377.5' capped and 3' polyadenylated mRNA constructs containing the firefly gene (luc) for luciferase (LUC) or the Escherichia coli gene (uidA) for beta-glucuronidase (GUS) coding region were synthesized in vitro and electroporated into protoplasts that were cultured at 40 or 5% O2. A LUC mRNA with a 17-nucleotide polylinker 5' untranslated region (UTR) was expressed 10-fold higher under aerobic conditions than under hypoxic conditions. Expression of five chimeric ADH1-GUS mRNAs was measured relative to this LUC mRNA. An mRNA containing the 5'-UTR and the first 18 codons of adh1 in a translational fusion with the GUS coding region and followed by the 3'-UTR of adh1 was expressed 57-fold higher at 5% O2. Progressive deletion of adh1 5'-UTR and coding sequences reduced expression of the GUS-mRNA at 5% O2, but had little impact on expression of 40% O2. Enhancement of expression in hypoxic protoplasts conferred by the adh1 5'-UTR and the first 26 codons decreased more than 3-fold when the adh1 3'-UTR was removed. In addition, the adh1 3'-UTR slightly inhibited expression in aerobic protoplasts. The physical half-lives of the GUS and LUC mRNAs were similar under both anaerobic and hypoxic conditions, indicating that expression levels were largely independent of mRNA stability. Thus, both adh1 5' and 3' mRNA sequences are required for enhanced translation in protoplasts under O2 deprivation.

Alcohol Dehydrogenase↗

PubMed1994

Meiotic chromosome pairing in maize is associated with a novel chromatin organization.

Three-dimensional light microscopy and the excellent cytological features of maize meiotic chromosomes are used to analyze the early events of chromosome synapsis. We demonstrate that the chromosomes undergo a dramatic structural reorganization prior to synapsis in zygotene. The unique features of prezygotene chromosomes are a partial separation of sister chromatids, an elongation of knob heterochromatin, an increase in surface complexity, a 50% increase in total chromosome volume, and a peripheral localization and alignment of telomeric, but not proximal, loci. At zygotene, chromosome volume decreases and chromosomes appear as single fibers. The specialized prezygotene chromosome morphology may facilitate homology recognition once the homologs have been brought together.

Chromatin↗

PubMed1993

Transposon-mediated mutations in the untranslated leader of maize Adh1 that increase and decrease pollen-specific gene expression.

The unstable mutation Adh1-Fm335 contains a Dissociation (Ds1) transposable element at position +53 in the untranslated leader of the maize Alcohol dehydrogenase-1 (Adh1) gene. Excision of Ds1 is known to generate new alleles with small additions and rearrangements of Adh1 DNA. We characterized 16 revertant alleles with respect to ADH1 activity levels in scutellum (nutritive tissue of the seed), anaerobic root, and pollen. Whereas gene expression was not different from the wild type in the sporophytic tissues of the scutellum and anaerobic root, there were strong allelic differences in pollen. One allele underexpressed pollen ADH1 at 48% of the wild-type level, and another overexpressed pollen ADH1 at 163% of the wild-type level. Quantitative RNase protection assays demonstrated that the mutant phenotypes reflected changes in the levels of steady state mRNA in pollen. These data provide a definitive demonstration of an overexpression mutant in plants and further show that marked increases in mRNA levels can follow minor alterations in central untranslated leader sequences. The nucleotide sequence of 12 new revertant alleles and the molecular mechanisms responsible for pollen-specific gene expression are discussed.

Alcohol Dehydrogenase↗

PubMed1992

The role of initial cells in maize anther morphogenesis.

The near absence of cell movement in plants makes clonal analysis a particularly informative method for reconstructing the early events of organ formation. We traced the patterns of cell division during maize anther development by inducing sector boundaries that preceded the earliest events of anther initiation. In doing this, we were able to estimate the smallest number of cells that are fated to form an anther, characteristic cell division patterns that occur during anther morphogenesis, and the relationship between the pre-existing symmetry of the initial cells and the final symmetry of the mature anther. Four general conclusions are made: (1) anthers are initiated from small groups of 12 or fewer cells in each of two floral meristematic layers; (2) the early growth of the anther is more like a shoot than a glume or leaf; (3) cell ancestry does not dictate basic structure and (4) the orientation of initial cells predicts the orientation of the four pollen-containing microsporangia, which define the axes of symmetry on the mature anther. The final point is discussed with other data, and an explanation involving a 'structural template' is invoked. The idea is that the orientation of initial cells within the floral meristem establishes an architectural pattern into which anther cells are recruited without regard to their cellular lineages. The structural template hypothesis may prove to be generally applicable to problems of pattern formation in plants.

Gametogenesis↗

PubMed1990

Clonal analysis of the cell lineages in the male flower of maize.

The cell lineages in the male flower of maize were characterized using X-rays and transposable elements to produce clonal sectors differing in anthocyanin pigmentation. Less than 50% of the somatic tassel mutations (caused by reversion of unstable color mutations) that were visible on the anther wall were sexually transmitted by the male gametes, unless the sectors were larger than half the tassel circumference. This result is explained by showing that: (a) both the outer (LI) and inner (LII) lineages of the shoot apical meristem form a cell layer in the bilayered anther wall, and that anther pigmentation can be derived from either cell layer; and that (b) the male germ cells are derived almost exclusively from the LII. Therefore, while reversion events in either the LI or LII are visible on the anther, only the LII events are heritable. Reversion events that occur prior to the organization of the shoot apical meristem however, produce large (usually more than one-half tassel) sectors that include both the outer and inner lineages. In contrast to the high level of cell layer invasion previously reported during leaf development, during anther development less than 10(-3) cells in the LI invade the LII to form male gametes. The strong correlation between cell lineage and cell fate in the maize anther has implications for studies on plant evolution and the genetic improvement of cereals by DNA transformation.

Anthocyanins↗