Telomere dynamics unique to meiotic prophase: formation and significance of the bouquet.

Telomeres carry out conserved and possibly ancient functions in meiosis. During the specialized prophase of meiosis I, meiotic prophase, telomeres cluster on the nuclear envelope and move the diploid genetic material around within the nucleus so that homologous chromosomes can align two by two and efficiently recombine with precision. This recombination is in turn required for proper segregation of the homologs into viable haploid daughter cells. The meiosis-specific telomere clustering on the nuclear envelope defines the bouquet stage, so named for its resemblance to the stems from a bouquet of cut flowers. Here, a comparative analysis of the literature on meiotic telomeres from a variety of different species illustrates that the bouquet is nearly universal among life cycles with sexual reproduction. The bouquet has been well documented for over 100 years, but our understanding of how it forms and how it functions has only recently begun to increase. Early and recent observations document the timing and provide clues about the functional significance of these striking telomere movements.

Construction and transposition of a 100-kilobase extended P element in Drosophila.

We have used P element deletion derivatives at defined locations in the Drosophila genome to construct a 100-kb extended P element more than twice the size of any previously available. We demonstrate that this prototypical extended P element is capable of transposition to new sites in the genome. The structural and functional integrity of a transposed extended P element was confirmed using molecular, genetic, and cytogenetic criteria. This is the first method shown to be capable of producing large, unlinked transpositional duplications in Drosophila. The ability to produce functional transposable elements from half-elements is novel and has many potential applications for the functional analysis of complex genomes.

Chromosome condensation induced by geminivirus infection of mature plant cells.

Tomato golden mosaic virus (TGMV) is a geminivirus that replicates its single-stranded DNA genome through double-stranded DNA intermediates in nuclei of differentiated plant cells using host replication machinery. We analyzed the distribution of viral and plant DNA in nuclei of infected leaves using fluorescence in situ hybridization (FISH). TGMV-infected nuclei showed up to a sixfold increase in total volume and displayed a variety of viral DNA accumulation patterns. The most striking viral DNA patterns were bright, discrete intranuclear compartments, but diffuse nuclear localization was also observed. Quantitative and spatial measurements of high resolution 3-dimensional image data revealed that these compartments accounted for 1-18% of the total nuclear volume or 2-45% of the total nuclear FISH signals. In contrast, plant DNA was concentrated around the nuclear periphery. In a significant number of nuclei, the peripheral chromatin was organized as condensed prophase-like fibers. A combination of FISH analysis and indirect immunofluorescence with viral coat protein antibodies revealed that TGMV virions are associated with the viral DNA compartments. However, the coat protein antibodies failed to cross react with some large viral DNA inclusions, suggesting that encapsidation may occur after significant viral DNA accumulation. Infection by a TGMV mutant with a defective coat protein open reading frame resulted in fewer and smaller viral DNA-containing compartments. Nevertheless, nuclei infected with the mutant virus increased in size and in some cases showed chromosome condensation. Together, these results established that geminivirus infection alters nuclear architecture and can induce plant chromatin condensation characteristic of cells arrested in early mitosis.

Evidence for the coincident initiation of homolog pairing and synapsis during the telomere-clustering (bouquet) stage of meiotic prophase.

To improve knowledge of the prerequisites for meiotic chromosome segregation in higher eukaryotes, we analyzed the spatial distribution of a pair of homologs before and during early meiotic prophase. Three-dimensional images of fluorescence in situ hybridization (FISH) were used to localize a single pair of homologs in diploid nuclei of a chromosome-addition line of oat, oat-maize9b. The system provided a robust assay for pairing based on cytological colocalization of FISH signals. Using a triple labeling scheme for simultaneous imaging of chromatin, telomeres and the homolog pair, we determined the timing of pairing in relation to the onset of three sequential hallmarks of early meiotic prophase: chromatin condensation (the leptotene stage), meiotic telomere clustering (the bouquet stage) and the initiation of synapsis (the zygotene stage). We found that the two homologs were mostly unpaired up through middle leptotene, at which point their spherical cloud-like domains began to transform into elongated and stretched-out domains. At late leptotene, the homologs had completely reorganized into long extended fibers, and the beginning of the bouquet stage was conspicuously marked by the de novo clustering of telomeres at the nuclear periphery. The homologs paired and synapsed during the bouquet stage, consistent with the timing of pairing observed for several oat 5S rDNA loci. In summary, results from analysis of more than 100 intact nuclei lead us to conclude that pairing and synapsis of homologous chromosomes are largely coincident processes, ruling out a role for premeiotic pairing in this system. These findings suggest that the genome-wide remodeling of chromatin and telomere-mediated nuclear reorganization are prerequisite steps to the DNA sequence-based homology-search process in higher eukaryotes.

DSK1, a kinesin-related protein involved in anaphase spindle elongation, is a component of a mitotic spindle matrix.

DSK1 is a kinesin-related protein that is involved in anaphase spindle elongation in the diatom Cylindrotheca fuisiformis [Wein et al., 1996: J. Cell Biol. 113:595-604]. DSK1 staining appeared to be concentrated in the gap that forms as the two half-spindles separate, suggesting that DSK1 may be part of a non-microtubule spindle matrix. We set out to investigate this possibility using three-dimensional high-resolution fluorescence microscopy, and biochemical methods of tubulin extraction. Three-dimensional fluorescence microscopy reveals that DSK1 remains in the midzone after the bulk of the microtubules from the two half-spindles have left the region. Biochemical studies show that CaCl2 extraction of tubulin from a mitotic spindle preparation does not extract similar proportions of DSK1 protein. Immunofluorescence confirms that this CaCl2 extraction leaves behind spindle-like bars that are recognized by anti-DSK1, but not by anti-tubulin antibodies. We conclude that DSK1 is part of, or attached to, a non-microtubule scaffold in the diatom central spindle. This discovery has implications for both the structural organization of the mitotic spindle and the mechanism of spindle elongation.

A defective signal peptide tethers the floury-2 zein to the endoplasmic reticulum membrane.

The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD alpha-zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD alpha-zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this alpha-zein reveals a putative mutation alanine-->valine (Ala-->Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 alpha-zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 alpha-zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 alpha-zein and the fl2 alpha-zein (Val-->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 alpha-zein (Val-->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 alpha-zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.

Telomeres cluster de novo before the initiation of synapsis: a three-dimensional spatial analysis of telomere positions before and during meiotic prophase.

We have analyzed the progressive changes in the spatial distribution of telomeres during meiosis using three-dimensional, high resolution fluorescence microscopy. Fixed meiotic cells of maize (Zea mays L.) were subjected to in situ hybridization under conditions that preserved chromosome structure, allowing identification of stage-dependent changes in telomere arrangements. We found that nuclei at the last somatic prophase before meiosis exhibit a nonrandom, polarized chromosome organization resulting in a loose grouping of telomeres. Quantitative measurements on the spatial arrangements of telomeres revealed that, as cells passed through premeiotic interphase and into leptotene, there was an increase in the frequency of large telomere-to-telomere distances and a decrease in the bias toward peripheral localization of telomeres. By leptotene, there was no obvious evidence of telomere grouping, and the large, singular nucleolus was internally located, nearly concentric with the nucleus. At the end of leptotene, telomeres clustered de novo at the nuclear periphery, coincident with a displacement of the nucleolus to one side. The telomere cluster persisted throughout zygotene and into early pachytene. The nucleolus was adjacent to the cluster at zygotene. At the pachytene stage, telomeres rearranged again by dispersing throughout the nuclear periphery. The stage-dependent changes in telomere arrangements are suggestive of specific, active telomere-associated motility processes with meiotic functions. Thus, the formation of the cluster itself is an early event in the nuclear reorganizations associated with meiosis and may reflect a control point in the initiation of synapsis or crossing over.

A maize ribosome-inactivating protein is controlled by the transcriptional activator Opaque-2.

Although synthesis of the cytosolic maize albumin b-32 had been shown to be controlled by the Opaque-2 regulatory locus, its function was unknown. We show here that b-32 is a member of the large and widely distributed class of toxic plant proteins with ribosome-inactivating activity. These ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that remove a single base from a conserved 28S rRNA loop required for elongation factor 1 alpha binding. Cell-free in vitro translation extracts were used to show that both maize and wheat ribosomes were resistant to molar excesses of b-32 but not to the dicotyledonous RIP gelonin. We extracted RIP activity from kernels during seed maturation and germination. The amount of RIP activity increased during germination, although the amount of b-32 protein remained fairly constant. Expression of a maize RIP gene under the control of an endosperm-specific transcriptional regulatory may be an important clue prompting investigation of the biological basis for RIP expression in seeds of other plants.

Allopolyploidy in bryophytes: Multiple origins of Plagiomnium medium.

Bryophytes are thought to be unique among land plants in lacking the important evolutionary process of allopolyploidy, which involves interspecific hybridization and chromosome doubling. Electrophoretic data show, however, that the polyploid moss Plagiomnium medium is an allopolyploid derivative of Plagiomnium ellipticum and Plagiomnium insigne, that P. medium has originated more than once from these progenitors, and that cross-fertilization results in interlocus genetic recombination. Evidence from restriction fragment length polymorphisms in chloroplast DNA implicates P. insigne as the female parent in interspecific hybridizations with P. ellipticum. Contrary to prevailing views, it appears that those evolutionary processes responsible for genetic differentiation and speciation in other land plants occur in the bryophytes as well.

Restriction fragment length polymorphisms in diploid and allotetraploid Gossypium: assigning the late embryogenesis-abundant (Lea) alloalleles in G. hirsutum.

We have determined the copy number of 21 genes in an allotetraploid and several diploid species of cotton by gel and dot blot hybridization with cloned cDNAs. The legumin A, legumin B, and all 18 unique Lea (late embryogenesis-abundant) cDNA sequences isolated from the AD allotetraploid Gossypium hirsutum are present in one copy in A, D, E, and F diploid species and in two copies in G. hirsutum. Gel blot analysis of DNAs digested with EcoRI or BamHI usually detects different sized fragments in A and D diploids. Conservation of these restriction fragment length polymorphisms in G. hirsutum allows most of these fragments to be assigned to their respective subgenomes. Furthermore, both subgenomes in G. hirsutum can be distinguished from those in the interfertile allotetraploid G. barbadense. These results show that physical mapping of both sets of chromosomes in an allotetraploid should be possible by segregation analysis.