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Homologous chromosomes display associations in many organisms. Drosophila melanogaster (here after, Drosophila) serves as an excellent model to study pairing interactions since chromosomes are paired in all somatic cells throughout development. For many genes, the degree of homolog association influences gene expression. These effects, collectively referred to as transvection, can promote gene activation or silencing. Requirements for transvection are poorly understood. Chapter One reviews what is known about transvection in Drosophila and chromosome interactions in mammals. Recent cell culture studies implicated a requirement for Topoisomerase 2 (Top2) in chromosome pairing. Top2 encodes an ATP dependent homodimeric enzyme that generates double stranded breaks to change DNA topology. This enzyme is a common target of anticancer drugs due to its role in DNA metabolism. To understand the in vivo role of Drosophila Top2, an EMS screen was completed. Chapter Two describes the identification and characterization of fifteen new EMS generated Top2 mutations. Fifteen null and hypomorphic alleles were obtained, including one that displays temperature sensitivity. Molecular analyses of these alleles uncovered single or multiple base pair substitutions within the coding region of each mutant gene. Even though flies carrying individual missense alleles in trans to a deficiency are inviable, heteroallelic combinations of several missense alleles produced viable flies, including two lines carrying mutations that display resistance to anti-cancer drugs. These data indicate that Top2 activity can be restored by dimerization of defective subunits. Our new Top2 alleles establish a novel allelic series and provide a platform for understanding drug resistance. In Chapter Three, the role of Top2 in chromosome associations was tested to determine whether mutations in Top2 disrupted transvection. Viable heteroallelic combinations of Top2 mutations were used to test transvection at three classically studied loci. For each gene, homologous interactions were analyzed by screening for alterations in pairing-dependent changes in phenotype involving transvecting alleles. Only one of the three genes tested displayed phenotypic changes in Top2 complementing adults that were consistent with an alteration in pairing dependent changes in expression. Transcript levels were assessed at the three genes studied that display transvection. Our studies indicate that changes in the phenotype, due to altered Top2, are likely gene specific transcriptional changes. Further investigation of gene associations in Top2 mutants employed fluorescence in situ hybridization (FISH). These studies showed that all loci examined were paired near wild type levels, suggesting that Top2 does not globally disrupt homolog associations in vivo. The differences observed in Top2 function in vivo and in vitro may be explained by two possibilities. First, the probes studied differ from those used in vitro, indicating that different genetic loci may have different sensitivities to unpairing. Second, Top2 plays a role in the segregation of sister chromatids during anaphase and loss of Top2 causes improper resolution of chromosomes resulting in aneuploidy. In cell culture, cells were allowed to go through one division and then were subsequently fixed, permitting analyses on all cells. It is possible that nuclei exhibiting aneuploidy have undergone cell death in vivo, explaining why we do not see increased amounts of unpairing. In conclusion, Top2 contributions to nuclear functions are complex. Loss of Top2 may result in subtle changes in pairing that may affect transcription and transvection.

Pairing and synapsis of homologous chromosomes are required for normal chromosome segregation and the exchange of genetic material during meiosis. Pairing is defined as the recognition and alignment of chromosomes that occurs either pre-meiotically or during early prophase I to ensure that associations via synapsis and recombination occur only between homologues. Synapsis is the intimate juxtaposition of homologous chromosomes that is complete at pachytene following formation of a tri-partite proteinaceous structure known as the synaptonemal complex (SC). In yeast, HOP1 is an essential component of the SC that localises along chromosome axes during prophase I and promotes homologous chromosome interactions. Homologues in Arabidopsis (AtASY1), Brassica (BoASY1) and rice (OsPAIR2) have been isolated through analysis of mutants that display decreased fertility due to severely reduced synapsis of homologous chromosomes. Analysis of these genes has indicated that they play a similar role to HOP1 in pairing and formation of the SC through localisation to axial/lateral elements of the SC. In this study, we have characterised the bread wheat homologue of HOP1, TaASY1, and its encoded protein. The full length cDNA and genomic DNA clones of TaASY1 have been isolated, sequenced and characterised. TaASY1 is located on chromosome group 5 and the open reading frame displays significant similarity to OsPAIR2 (84%) and AtASY1 (63%). In addition to OsPAIR2 and AtASY1, the deduced amino acid sequence also displays sequence similarity to ScHOP1, with all four proteins containing a HORMA domain. Transcript and protein analysis showed that expression is largely restricted to meiotic tissue, with elevated levels during the stages of prophase I when pairing and synapsis of homologous chromosomes occurs. Antibodies specific to TaASY1 were used in immuno-fluorescence microscopy and immuno-gold transmission electron microscopy to investigate the localisation of TaASY1 in meiotic cells. Immuno-fluorescence analysis initially detected ASY1 in pollen mother cells (PMCs) during meiotic interphase as foci randomly distributed over the chromatin. The ASY1 signal became increasingly continuous during leptotene, reflecting the changes occurring in chromosome morphology. Throughout zygotene, the signal became progressively more continuous, localising along the entire length of the axial elements as chromosomes synapsed. This signal appeared to persist until pachytene, before disappearing from the chromatin as the SC disassociated through late pachytene and early diplotene. The immuno-gold based electron microscopy displayed that TaASY1 localises to chromatin that is associated with both axial elements before SC formation as well as chromatin of lateral elements within formed SCs. Analysis of RNAi Taasy1 mutants was performed to further define the role of ASY1 in bread wheat meiosis. ASY1 localisation was disrupted in these mutants, with a diffuse and non-continuous signal observed through leptotene and zygotene. Feulgen staining of meiotic chromosomes displayed reduced synapsis during prophase I, as well as multivalents at metaphase I and abnormal chromosome segregation during anaphase I. These observations are consistent with the presence of homoeologous chromosome interactions. TaASY1 expression and localisation was also investigated in the bread wheat pairing mutant, ph1b. Quantitative real-time PCR (Q-PCR) revealed that TaASY1 is significantly up-regulated in ph1b, with greater then 20-fold expression compared to wild-type Chinese Spring, while maintaining the same pattern of expression as wild-type through progressive stages of meiosis. ASY1 localisation was significantly disrupted in ph1b, with irregular loading on axial elements during mid to late zygotene, indicative of abnormal chromatin remodelling and multiple axial element associations that have previously been reported in ph1b. Taken together, these results indicate that TaASY1 is essential for promoting homologous chromosome interactions during meiosis, and that impairment of ASY1 function in bread wheat meiosis results in reduced restriction of chromosome associations to homologues. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1340087 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

A história de domesticação da cana-de-açúcar (Saccharum spp.) é atípica. As variedades modernas derivam de um processo que inclui hibridações entre a espécie domesticada S. officinarum e a silvestre S. spontaneum, sucessivos retrocruzamentos, no sentido de recuperar o genoma de S. officinarum e a seleção de progênies superiores. Além disso, as genealogias contemplam cruzamentos entre genótipos e eventualmente espécies, todos com elevado grau de ploidia e número de cromossomos distintos, assim como aneuploidias. Frente ao exposto, este trabalho teve como objetivos estabelecer o número de cromossomos e avaliar o comportamento meiótico da cultivar IACSP93-3046, bem como, identificar as associações cromossômicas em meiose I dos genótipos IACSP93-3046, IACSP95-3018 e de um representante de S. officinarum, Caiana Fita, pela marcação dos centrômeros usando FISH. O número de cromossomos da cultivar IACSP93-3046 foi determinado a partir de preparações do meristema radicular, pré-tratado com 8-hidroxiquinolina (0,03%, 4h), e corado pelo método de Feulgen. As células metafásicas foram analisadas sob microscopia óptica, preferencialmente intactas e com o mínimo de sobreposição de cromossomos. Para a análise do comportamento meiótico utilizou-se a técnica de esmagamento, e as células foram coradas com carmim propiônico. Foram observadas as fases meióticas desde a metáfase I até a telófase II, bem como as tétrades. O pareamento cromossômico em meiose I foi analisado usando a técnica de hibridização in situ fluorescente (FISH). Para tanto, preparações dos genótipos IACSP93-3046, IACSP95-3018 e Caiana Fita foram realizadas pelo gotejamento de uma suspensão de células em diacinese. As sondas foram obtidas por PCR a partir da amplificação da região centromérica de cana-de-açúcar, marcadas com digoxigenina-11-dUTP, por nick translation, e detectadas com anti-digoxigenina-rodamina. As lâminas foram montadas em DAPI-Vectashield e analisadas sob microscopia de fluorescência. O número diplóide 2n = 112 foi observado para a cultivar IACSP93-3046, sendo caracterizado pela primeira vez neste estudo. A microsporogênese de IACSP93-3046 apresentou elevado percentual de irregularidades (68%). De modo geral, as anormalidades foram relativas à segregação dos cromossomos, e incluíram migração precoce para os polos em metáfase I e II, cromossomos retardatários em anáfase (I e II) e em telófase (I e II), cromossomos perdidos em prófase II, e micronúcleos nas tétrades. A análise dos sítios de hibridização permitiu comprovar que os cromossomos se associam predominantemente como bivalentes em IACSP93-3046, IACSP95-3018 e Caiana Fita. As irregularidades na segregação dos cromossomos conduzem a micrósporos aneuploides, como constatado em IACSP93-3046. Sugere-se que a assincronia do processo meiótico entre os genomas que compõem a cana-de-açúcar tem papel relevante na geração dessas irregularidades. / The history of the sugarcane domestication (Saccharum spp.) is atypical. Modern varieties are derived from a hybridization process between the domestic species S. officinarum and the wild species S. spontaneum, successive backcrossings to recover the genome of S. officinarum, and the selection of superior progenies. The genealogies include crossings among genotypes, and possibly Saccharum species, all with a high degree of ploidy and different numbers of chromosomes, as well as aneuploidies. The study aimed to establish the number of chromosomes and evaluate the meiotic behavior of cultivar IACSP93-3046, and identify chromosomal associations in meiosis I of genotypes IACSP93-3046, IACSP95-3018 and Caiana Fita (a representative of S. officinarum) by labeling centromeres using fluorescence in situ hybridization (FISH). The number of chromosomes in cultivar IACSP93-3046 was determined from the root meristem preparations, pretreated with 8-hydroxiquinoline and stained by the Feulgen method. Metaphasic cells, preferably intact and with minimum chromosome overlap, were analyzed under an optical microscope. Meiotic behavior was examined from the preparations by using squashing method and stained with propionic carmine. Meiotic phases were observed from metaphase I to telophase II, and tetrad stages. Chromosomal pairing in meiosis I was analyzed by using the FISH technique. The slides of genotypes IACSP93-3046, IACSP95-3018 and Caiana Fita were produced by dropping a suspension of meiocytes in diakinesis. The probes were obtained by PCR, with amplification of the centromere region, and labeled with digoxigenin-11-dUTP, by nick translation, and detected with anti-digoxigenin-rhodamine. The slides were mounted in DAPI-Vectashield and analyzed under a fluorescence microscope. The diploid number 2n = 112 was observed for cultivar IACSP93-3046 and characterized in this study for the first time. Microsporogenesis of IACSP93-3046 presented a high irregularity percentage regarding chromosome segregation, especially precocious migration to poles in metaphase I and II, laggard chromosomes in anaphase and telophase I and II, lost chromosomes in prophase II, and micronuclei in the tetrad stages. The analysis from the hybridization sites proved that the chromosomal pairing occurred predominantly as bivalents in IACSP93-3046, IACSP95-3018 and Caiana Fita. Chromosomal segregation irregularities led to aneuploid microspores, as confirmed in IACSP93-3046, suggesting the asynchrony in the meiotic process between the sugarcane genomes play an important role in producing these irregularities.

Meiosis is a specialized type of cell division essential for the production of four normal haploid gametes. In early prophase I of meiosis, the intimate synapsis between homologous chromosomes, and the formation of chiasmata, is facilitated by a proteinaceous structure known as the synaptonemal complex (SC). Ultrastructural analysis of germ cells of a number of organisms has disclosed that SC is a specialized tripartite structure composed of two lateral elements, one on each homolog, and a central element, which, in turn, are linked by transverse elements. Genetic studies have revealed that defects in meiotic chromosome alignment and/or segregation result in aneuploidy, which is the leading cause of spontaneous miscarriages in humans, hereditary birth defects such as Down syndrome, and are also, associated with the development and progression of certain forms of cancer. The mechanism(s) underlying the alignment/pairing of chromosomes at meiosis I differ among organisms. These can be divided into at least two broad pathways: one is independent of DNA double-strand breaks (DSB) and other is mediated by DSBs. In the DSB-dependent pathway, SC plays crucial roles in promoting homolog pairing and disjunction. On the other hand, the DSB-independent pathway involves the participation of telomeres, centromeres and non-coding RNAs in the pre-synaptic alignment, pre-meiotic pairing as well as pairing of homologous chromosomes. Although a large body of literature highlights the central role of SC in meiotic recombination, the possible role of SC components in homolog recognition and alignment is poorly understood. Genetic screens for Saccharomyces cerevisiae mutants defective in meiosis and sporulation lead to the isolation of genes required for interhomolog recombination, including those that encode SC components. In S. cerevisiae, ten meiosis-specific proteins viz., Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8 have been recognized as bona fide constituents of SC or associated with SC function. Mutations in any of these genes result in defective SC formation, thus leading to reduction in the rate of recombination. HOP1 (Homolog Pairing) encodes a ̴ 70 kDa structural protein, which localizes to the lateral elements of SC. It was found to be essential for the progression of meiotic recombination. In hop1Δ mutants, meiosis specific DSBs are reduced to 10% of that of wild type level and it fails to produce viable spores. It also displays relatively high frequency of inter-sister recombination over inter-homolog recombination. Bioinformatics analysis suggests that Hop1 comprises of an N-terminal HORMA domain (Hop1, Rev7 and Mad2), which is conserved among Hop1 homologs from diverse organisms. This domain is also known to be present in proteins involved in processes like chromosome synapsis, repair and sex chromosome inactivation. Additionally, Hop1 harbors a 36-amino acid long zinc finger 348374 motif (CX2CX19CX2C) which is critical for DNA binding and meiotic progression, and a putative nuclear localization signal corresponding to amino acid residues from 588-594. Previous studies suggested that purified Hop1 protein exists in multiple oligomeric states in solution and displays structure specific DNA binding activity. Importantly, Hop1 exhibited higher binding affinity for the Holliday junction (HJ), over other early recombination intermediates. Binding of Hop1 to the HJ at the core resulted in branch migration of the junction, albeit weakly. Intriguingly, Hop1 showed a high binding affinity for G4 DNA, a non-B DNA structure, implicated in homolog synapsis and promotes robust synapsis between double-stranded DNA molecules. Hop1 protein used in the foregoing biochemical studies was purified from mitotically dividing S. cerevisiae cells containing the recombinant plasmid over-expressing the protein where the yields were often found to be in the low-microgram quantities. Therefore, one of the major limitations to the application of high resolution biophysical techniques, such as X-crystallography and spectroscopic analyses for structure-function studies of S. cerevisiae Hop1 has been the non-availability of sufficient quantities of functionally active pure protein. In this study, we have performed expression screening in Escherichia coli host strains, capable of high level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed +2 for expression and purification of S. cerevisiae Hop1 protein, using Ni-NTA and double-stranded DNA-cellulose chromatography. Recombinant S. cerevisiae Hop1 protein thus obtained was >98% pure and exhibited DNA binding activity with high-affinity for Holliday junction. The availability of the bacterial HOP1 expression vector and functionally active Hop1 protein has enabled us to glean and understand several vital biological insights into the structure-function relationships of Hop1 as well as the generation of appropriate truncated mutant proteins. Mutational analyses in S. cerevisiae has shown that sister chromatid cohesion is required for proper chromosome condensation, including the formation of axial elements, SC assembly and recombination. Consistent with these findings, homolog alignment is impaired in red1hop1 strains and associations between homologs are less stable. red1 mutants fail to make any discernible axial elements or SC structures but exhibit normal chromosome condensation, while hop1 mutants form long fragments of axial elements but without any SCs, are defective in chromosome condensation, and produce in-viable spores. Using single molecule and ensemble assays, we found that S. cerevisiae Hop1 organizes DNA into at least four major distinct DNA conformations: (i) a rigid protein filament along DNA that blocks access to nucleases; (ii) bridging of non-contiguous segments of DNA to form stem-loop structures; (iii) intra-and intermolecular long range synapsis between double-stranded DNA molecules; and (iv) folding of DNA into higher order nucleoprotein structures. Consistent with B. McClintock’s proposal that “there is a tendency for chromosomes to associate 2-by-2 in the prophase of meiosis involving long distance recognition of homologs”, these results to our knowledge provide the first evidence that Hop1 mediates the formation of tight DNA-protein-DNA nucleofilaments independent of homology which might help in the synapsis of homologous chromosomes during meiosis. Although the DNA binding properties of Hop1 are relatively well established, comparable knowledge about the protein is lacking. The purification of Hop1 from E. coli, which was functionally indistinguishable from the protein obtained from mitotically dividing S. cerevisiae cells has enabled us to investigate the structure-function relationships of Hop1, which has provided important insights into its role in meiotic recombination. We present several lines of evidence suggesting that Hop1 is a modular protein, consisting of an intrinsically unstructured N-terminal domain and a core C-terminal domain (Hop1CTD), the latter being functionally equivalent to the full-length Hop1 in terms of its in vitro activities. Importantly, however, Hop1CTD was unable to rescue the meiotic recombination defects of hop1null strain, indicating that synergy between the N-terminal and C-terminal domains of Hop1 protein is essential for meiosis and spore formation. Taken together, our findings provide novel insights into the molecular functions of Hop1, which has profound implications for the assembly of mature SC, homolog synapsis and recombination. Several lines of investigations suggest that HORMA domain containing proteins are involved in chromatin binding and, consequently, have been shown to play key roles in processes such as meiotic cell cycle checkpoint, DNA replication, double-strand break repair and chromosome synapsis. S. cerevisiae encodes three HORMA domain containing proteins: Hop1, Rev7 and Mad2 (HORMA) which interact with chromatin during diverse chromosomal processes. The data presented above suggest that Hop1 is a modular protein containing a distinct N-terminal and C-terminal (Hop1CTD) domains. The N-terminal domain of Hop1, which corresponds to the evolutionarily conserved HORMA domain, although, discovered first in Hop1, its precise biochemical functions remain unknown. In this section, we show that Hop1-HORMA domain expressed in and purified from E. coli exhibits preferential binding to the HJ and G4 DNA, over other early recombination intermediates. Detailed functional analyses of Hop1-HORMA domain, using mobility shift assays, DNase I footprinting and FRET, have revealed that HORMA binds at the core of Holliday junction and induces marked changes in its global conformation. Further experimental evidence also suggested that it causes DNA stiffening and condensation. However, like Hop1CTD, HORMA domain alone failed to rescue the meiotic recombination defects of hop1 null strain, indicating that synergy between the N-and C-terminal domains of Hop1 is essential for meiosis as well as for the formation of haploid gametes. Moreover, these results strongly implicate that Hop1 protein harbours a second DNA binding motif, which resides in the HORMA domain at its N-terminal region. To our knowledge, these findings also provide the first insights into the biochemical mechanism underlying HORMA domain activity. In summary, it appears that the C-terminal (CTD) and N-terminal (HORMA) domains of Hop1 may perform biochemical functions similar (albeit less efficiently) to that of the full-length Hop1. However, further research is required to uncover the functional differences between these domains, their respective interacting partners and modulation of the activity of these domains.