CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^

VERIFICATION OF DNA PREDICTED PROTEIN SEQUENCES BY ENZYME HYDROLYSIS AND MASS SPECTROMETRIC ANALYSIS

The focus of this thesis lies in the development of a sensitive method for the analysis of protein primary structure which can be easily used to confirm the DNA sequence of a protein's gene and determine the modifications which are made after translation. This technique involves the use of dipeptidyl aminopeptidase (DAP) and dipeptidyl carboxypeptidase (DCP) to hydrolyze the protein and the mass spectrometric analysis of the dipeptide products.^ Dipeptidyl carboxypeptidase was purified from human lung tissue and characterized with respect to its proteolytic activity. The results showed that the enzyme has a relatively unrestricted specificity, making it useful for the analysis of the C-terminal of proteins. Most of the dipeptide products were identified using gas chromatography/mass spectrometry (GC/MS). In order to analyze the peptides not hydrolyzed by DCP and DAP, as well as the dipeptides not identified by GC/MS, a FAB ion source was installed on a quadrupole mass spectrometer and its performance evaluated with a variety of compounds.^ Using these techniques, the sequences of the N-terminal and C-terminal regions and seven fragments of bacteriophage P22 tail protein have been verified. All of the dipeptides identified in these analysis were in the same DNA reading frame, thus ruling out the possibility of a single base being inserted or deleted from the DNA sequence. The verification of small sequences throughout the protein sequence also indicates that no large portions of the protein have been removed after translation. ^

The in vivo and in vitro formation of sticky DNA and the GAA.TTC trinucleotide repeat associated with Friedreich's ataxia

Friedreich's ataxia is caused by the expansion of the GAA•TTC trinucleotide repeat sequence located in intron 1 of the frataxin gene. The long GAA•TTC repeats are known to form several non-B DNA structures including hairpins, triplexes, parallel DNA and sticky DNA. Therefore it is believed that alternative DNA structures play a role in the loss of mRNA transcript and functional frataxin protein in FRDA patients. We wanted to further elucidate the characteristics for formation and stability of sticky DNA by evaluating the structure in a plasmid based system in vitro and in vivo in Escherichia coli. The negative supercoil density of plasmids harboring different lengths of GAA•TTC repeats, as well as either one or two repeat tracts were studied in E. coli to determine if plasmids containing two long tracts (≥60 repeats) in a direct repeat orientation would have a different topological effect in vivo compared to plasmids that harbored only one GAA•TTC tract or two tracts of < 60 repeats. The experiments revealed that, in fact, sticky DNA forming plasmids had a lower average negative supercoil density (-σ) compared to all other control plasmids used that had the potential to form other non-B DNA structures such as triplexes or Z-DNA. Also, the requirements for in vitro dissociation and reconstitution of the DNA•DNA associated region of sticky DNA were evaluated. Results conclude that the two repeat tracts associate in the presence of negative supercoiling and MgCl 2 or MnCl2 in a time and concentration-dependent manner. Interaction of the repeat sequences was not observed in the absence of negative supercoiling and/or MgCl2 or in the presence of other monovalent or divalent cations, indicating that supercoiling and quite specific cations are needed for the association of sticky DNA. These are the first experiments studying a more specific role of supercoiling and cation influence on this DNA conformation. To support our model of the topological effects of sticky DNA in plasmids, changes in sticky DNA band migration was measured with reference to the linear DNA after treatment with increasing concentrations of ethidium bromide (EtBr). The presence of independent negative supercoil domains was confirmed by this method and found to be segregated by the DNA-DNA associated region. Sequence-specific polyamide molecules were used to test the effect of binding of the ligands to the GAA•TTC repeats on the inhibition of sticky DNA. The destabilization of the sticky DNA conformation in vitro through this binding of the polyamides demonstrated the first conceptual therapeutic approach for the treatment of FRDA at the DNA molecular level. ^ Thus, examining the properties of sticky DNA formed by these long repeat tracts is important in the elucidation of the possible role of sticky DNA in Friedreich's ataxia. ^

Clinical relevance of polymorphic DNA copy number variation (CNV) in African American women with stage I-II breast cancer

Objectives. The chief goal of this study was to analyze copy number variation (CNV) in breast cancer tumors from 25 African American women with early stage breast cancer (BC) using molecular inversion probes (MIP) in order to: (1) compare the degree of CNV in tumors compared to normal lymph nodes, and (2) determine whether gains and/or losses of genes in specific chromosomes differ between pathologic subtypes of breast cancer defined by known prognostic markers, (3) determine whether gains/losses in CN are associated with known oncogenes or tumor suppressor genes, and (4) determine whether increased gains/losses in CN for specific chromosomes were associated with differences in breast cancer recurrence. ^ Methods. Twenty to 37 nanograms of DNA extracted from 25 formalin-fixed paraffin embedded (FFPE) tumor samples and matched normal lymph nodes were added to individual tubes. Oligonucleotide probes with recognition sequences at each terminus were hybridized with a genomic target sequence to form a circular structure. Probes are released from genomic DNA obtained from FFPE samples, and those which have been correctly "circularized" in the proper allele/nucleotide reaction combination are amplified using polymerase chain reaction (PCR) primers. Amplicons were fluorescently labeled and the tag sequences released from the genome homology regions by treatment with uracil-N-glycosylase to cleave the probe at the site where uracils are present, and detected using a complementary tag array developed by Affymetrix. ^ Results. Analysis of CN gains and losses from tumors and normal tissues showed marked differences in tumors with numerous chromosomes affected. Similar changes were not observed in normal lymph nodes. When tumors were stratified into four groups based on expression or lack of expression of the estrogen receptor and HER2/neu, distinct patterns of CNV for different chromosomes were observed. Gains or losses in CN for specific chromosomes correlated with amplifications/deletions of particular oncogenes or tumor suppressor genes (i.e. such as found on chromosome 17) known to be associated with aggressive tumor phenotype and poor prognosis. There was a trend for increases in CN observed for chromosome 17 to correlate inversely with time to recurrence of BC (p=0.14 for trend). CNV was also observed for chromosomes 5, 8, 10, 11, and 16, which are known sites for several breast cancer susceptibility alleles. ^ Conclusions. This study is the first to validate the MIP technique, to correlate differences in gene expression with known prognostic tumor markers, and to correlate significant increases/decreases in CN with known tumor markers associated with prognosis. The results of this study may have far reaching public health implications towards identifying new high-risk groups based on genomic differences in CNP, both with respect to prognosis and response to therapy, and to eventually identify new therapeutic targets for prevention and treatment of this disease. ^

Regulation of chromatin structure, Smad binding and AFP expression by the Forkhead box transcription factor: Foxa1

Transcription factors must be able to access their DNA binding sites to either activate or repress transcription. However, DNA wrapping and compaction into chromatin occludes most binding sites from ready access by proteins. Pioneer transcription factors are capable of binding their DNA elements within a condensed chromatin context and then reducing the level of nucleosome occupancy so that the chromatin structure is more accessible. This altered accessibility increases the probability of other transcription factors binding to their own DNA binding elements. My hypothesis is that Foxa1, a ‘pioneer’ transcription factor, activates alpha-fetoprotein (AFP) expression by binding DNA in a chromatinized environment, reducing the nucleosome occupancy and facilitating binding of additional transcription factors.^ Using retinoic-acid differentiated mouse embryonic stem cells, we illustrate a mechanism for activation of the tumor marker AFP by the pioneer transcription factor Foxa1 and TGF-β downstream effector transcription factors Smad2 and Smad4. In differentiating embryonic stem cells, binding of the Foxa1 forkhead box transcription factor to chromatin reduces nucleosome occupancy and levels of linker histone H1 at the AFP distal promoter. The more accessible DNA is subsequently bound by the Smad2 and Smad4 transcription factors, concurrent with activation of transcription. Chromatin immunoprecipitation analyses combined with siRNA-mediated knockdown indicate that Smad protein binding and the reduction of nucleosome occupancy at the AFP distal promoter is dependent on Foxa1. In addition to facilitating transcription factor binding, Foxa1 is also associated with histone modifications related to active gene expression. Acetylation of lysine 9 on histone H3, a mark that is associated active transcription, is dependent on Foxa1, while methylation of H3K4, also associated with active transcription, is independent of Foxa1. I propose that Foxa1 potentiates a region of chromatin to respond to Smad proteins, leading to active expression of AFP.^ These studies demonstrate one mechanism whereby a transcription factor can alter the accessibility of additional transcription factors to chromatin, by altering nucleosome positions. Specifically, Foxa1 exposes DNA so that Smad4 can bind to its regulatory element and activate transcription of the tumor-marker gene AFP.^

Involvement of HMGB1 in the repair of DNA adducts and the responses to DNA damage in mammalian cells

High mobility group protein B1 (HMGB1) is a multifunctional protein with roles in chromatin structure, transcription, V(D)J recombination, and inflammation. HMGB1 also binds to and bends damaged DNA, but the biological consequence of this interaction is not clearly understood. We have shown previously that HMGB1 binds cooperatively with nucleotide excision repair (NER) damage recognition proteins XPA and RPA to triplex-directed psoralen DNA interstrand crosslinks (ICLs). Based on this we hypothesized that HMGB1 is enhancing the repair of DNA lesions, and through this role, is affecting DNA damage-induced mutagenesis and cell survival. Because HMGB1 is also a chromatin protein, we further hypothesized that it is acting to facilitate chromatin remodeling at the site of the DNA damage, to allow access of the repair machinery to the DNA lesion. We demonstrated here that HMGB1 could bind to triplex-directed psoralen ICLs in a complex with NER proteins XPC-RAD23B, XPA and RPA, which occurred in the presence or absence of DNA. Supporting these findings, we demonstrated that HMGB1 enhanced repair of triplex-directed psoralen ICLs (by nucleotide incorporation), as well as removal of UVC irradiation-induced DNA lesions from the genome (by radioimmunoassay). We also explored HMGB1's role in chromatin remodeling upon DNA damage. Immunoblotting demonstrated that, in contrast to HMGB1 proficient cells, cells lacking HMGB1 showed no increase in histone acetylation after UVC irradiation. Additionally, purified HMGB1 protein enhanced chromatin formation in an in vitro chromatin assembly system. However, HMGB1 also has a role in DNA repair in the absence of chromatin, as shown by measuring UVC-induced nucleotide incorporation on a naked substrate. Upon exploration of HMGB1's effect on several cellular outcomes of DNA damage, we found that mammalian cells lacking HMGB1 were hypersensitive to DNA damage induced by psoralen plus UVA irradiation or UVC radiation, showing less survival and increased mutagenesis. These results reveal a new role for HMGB1 in the error-free repair of DNA lesions in a chromosomal context. As strategies targeting HMGB1 are currently in development for treatment of sepsis and rheumatoid arthritis, our findings draw attention to potential adverse side effects of anti-HMGB1 therapy in patients with inflammatory diseases. ^

Structure and biosynthesis of a pyruvate -dependent enzyme---phosphatidylserine decarboxylase from {\it Escherichia coli\/}

Phosphatidylserine decarboxylase of E. coli, a cytoplasmic membrane protein, catalyzes the formation of phosphatidylethanolamine, the principal phospholipid of the organism. The activity of the enzyme is dependent on a covalently bound pyruvate (Satre and Kennedy (1978) J. Biol. Chem. 253, 479-483). This study shows that the enzyme consists of two nonidentical subunits, $\alpha$ (Mr = 7,332) and $\beta$ (Mr = 28,579), with the pyruvate prosthetic group in amide linkage to the amino-terminus of the $\alpha$ subunit. Partial protein sequence and DNA sequence analysis reveal that the two subunits are derived from a proenzyme ($\pi$ subunit, Mr = 35,893) through a post-translational event. During the conversion of the proenzyme to the $\alpha$ and $\beta$ subunits, the peptide bond between Gly253-Ser254 is cleaved, and Ser254 is converted to the pyruvate prosthetic group at the amino-terminus of the $\alpha$ subunit (Li and Dowhan (1988) J. Biol. Chem. 263, 11516-11522).^ The proenzyme cannot be detected in cells carrying either single or multiple copies of the gene (psd), but can be observed in a T7 RNA polymerase/promoter and transcription-translation system. The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min. Changing of the Ser254 to cysteine (S254C) or threonine (S254T) slows the cleavage rate dramatically and results in mutants with a half-time for processing of around 2-4 h. Change of the Ser254 to alanine (S254A) blocks the cleavage of the proenzyme. The reduced processing rate with the mutations of the proenzyme is consistent with less of the functional enzyme being made. Mutants S254C and S254T produce $\sim$15% and $\sim$1%, respectively, of the activity of the wild-type allele, but can still complement a temperature-sensitive mutant of the psd locus. Neither detectable activity nor complementation is observed by mutant S254A. These results are consistent with the hydroxyl-group of the Ser254 playing a critical role in the cleavage of the peptide bond Gly253-Ser254 of the pro-phosphatidylserine decarboxylase, and support the mechanism proposed by Snell and co-workers (Recsei and Snell (1984) Annu. Rev. Biochem. 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases. ^

Mechanism of subunit interaction and DNA binding of the heterotrimeric CCAAT binding factor CBF

Many eukaryotic promoters contain a CCAAT element at a site close ($-$80 to $-$120) to the transcription initiation site. CBF (CCAAT Binding Factor), also called NF-Y and CP1, was initially identified as a transcription factor binding to such sites in the promoters of the Type I collagen, albumin and MHC class II genes. CBF is a heteromeric transcription factor and purification and cloning of two of the subunits, CBF-A and CBF-B revealed that it was evolutionarily conserved with striking sequence identities with the yeast polypeptides HAP3 and HAP2, which are components of a CCAAT binding factor in yeast. Recombinant CBF-A and CBF-B however failed to bind to DNA containing CCAAT sequences. Biochemical experiments led to the identification of a third subunit, CBF-C which co-purified with CBF-A and complemented the DNA binding of recombinant CBF-A and CBF-B. We have recently isolated CBF-C cDNAs and have shown that bacterially expressed purified CBF-C binds to CCAAT containing DNA in the presence of recombinant CBF-A and CBF-B. Our experiments also show that a single molecule each of all the three subunits are present in the protein-DNA complex. Interestingly, CBF-C is also evolutionarily conserved and the conserved domain between CBF-C and its yeast homolog HAP5 is sufficient for CBF-C activity. Using GST-pulldown experiments we have demonstrated the existence of protein-protein interaction between CBF-A and CBF-C in the absence of CBF-B and DNA. CBF-B on other hand, requires both CBF-A and CBF-C to form a ternary complex which then binds to DNA. Mutational studies of CBF-A have revealed different domains of the protein which are involved in CBF-C interaction and CBF-B interaction. In addition, CBF-A harbors a domain which is involved in DNA recognition along with CBF-B. Dominant negative analogs of CBF-A have also substantiated our initial observation of assembly of CBF subunits. Our studies define a novel DNA binding structure of heterotrimeric CBF, where the three subunits of CBF follow a particular pathway of assembly of subunits that leads to CBF binding to DNA and activating transcription. ^

Diversity of zooplankton assemblages at Station L4, Western English Channel, measured using next generation DNA sequencing

Background: Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel Next Generation Sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify the richness and diversity of a mixed zooplankton assemblage from a productive monitoring site in the Western English Channel. Methodology/Principle Findings: Plankton WP2 replicate net hauls (200 µm) were taken at the Western Channel Observatory long-term monitoring station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,042 sequences were obtained for all samples. The sequences clustered in to 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 138 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 75 taxonomic groups. Conclusions: The percentage of OTUs assigned to major eukaryotic taxonomic groups broadly aligns between the metagenetic and morphological analysis and are dominated by Copepoda. However, the metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for estimating diversity and species richness of zooplankton communities.

Shifts in the microbial community structure in different temperatures during sample storage from IODP Hole 325-M0058A

The objective of this study was to determine shifts in the microbial community structure and potential function based on standard Integrated Ocean Drilling Program (IODP) storage procedures for sediment cores. Standard long-term storage protocols maintain sediment temperature at 4°C for mineralogy, geochemical, and/or geotechnical analysis whereas standard microbiological sampling immediately preserves sediments at -80°C. Storage at 4°C does not take into account populations may remain active over geologic time scales at temperatures similar to storage conditions. Identification of active populations within the stored core would suggest geochemical and geophysical conditions within the core change over time. To test this potential, the metabolically active fraction of the total microbial community was characterized from IODP Expedition 325 Great Barrier Reef sediment cores prior to and following a 3-month storage period. Total RNA was extracted from complementary 2, 20, and 40 m below sea floor sediment samples, reverse transcribed to complementary DNA and then sequenced using 454 FLX sequencing technology, yielding over 14,800 sequences from the six samples. Interestingly, 97.3% of the sequences detected were associated with lineages that changed in detection frequency during the storage period including key biogeochemically relevant lineages associated with nitrogen, iron, and sulfur cycling. These lineages have the potential to permanently alter the physical and chemical characteristics of the sediment promoting misleading conclusions about the in situ biogeochemical environment. In addition, the detection of new lineages after storage increases the potential for a wider range of viable lineages within the subsurface that may be underestimated during standard community characterizations.

DNA alignment and Bayesian trees of partial sequences of COI, COIII and the haemocyanin functional unit f-g of 28 octopod species

Background: Octopods have successfully colonised the world's oceans from the tropics to the poles. Yet, successful persistence in these habitats has required adaptations of their advanced physiological apparatus to compensate impaired oxygen supply. Their oxygen transporter haemocyanin plays a major role in cold tolerance and accordingly has undergone functional modifications to sustain oxygen release at sub-zero temperatures. However, it remains unknown how molecular properties evolved to explain the observed functional adaptations. We thus aimed to assess whether natural selection affected molecular and structural properties of haemocyanin that explains temperature adaptation in octopods. Results: Analysis of 239 partial sequences of the haemocyanin functional units (FU) f and g of 28 octopod species of polar, temperate, subtropical and tropical origin revealed natural selection was acting primarily on charge properties of surface residues. Polar octopods contained haemocyanins with higher net surface charge due to decreased glutamic acid content and higher numbers of basic amino acids. Within the analysed partial sequences, positive selection was present at site 2545, positioned between the active copper binding centre and the FU g surface. At this site, methionine was the dominant amino acid in polar octopods and leucine was dominant in tropical octopods. Sites directly involved in oxygen binding or quaternary interactions were highly conserved within the analysed sequence. Conclusions: This study has provided the first insight into molecular and structural mechanisms that have enabled octopods to sustain oxygen supply from polar to tropical conditions. Our findings imply modulation of oxygen binding via charge-charge interaction at the protein surface, which stabilize quaternary interactions among functional units to reduce detrimental effects of high pH on venous oxygen release. Of the observed partial haemocyanin sequence, residue 2545 formed a close link between the FU g surface and the active centre, suggesting a role as allosteric binding site. The prevalence of methionine at this site in polar octopods, implies regulation of oxygen affinity via increased sensitivity to allosteric metal binding. High sequence conservation of sites directly involved in oxygen binding indicates that functional modifications of octopod haemocyanin rather occur via more subtle mechanisms, as observed in this study.

Structure Variation And Dynamics of the Nuclear Ribosomal Intergenic Spacer Region in Key Gibberella Fujikuroi Complex Species

The intergenic spacer (IGS) region of the ribosomal DNA was cloned and sequenced in eight species within the Gibberella fujikuroi species complex with anamorphs in the genus Fusarium , a group that includes the most relevant toxigenic species. DNA sequence analyses revealed two categories of repeated elements: long repeats and short repeats of 125 and 8 bp, respectively. Long repeats were present in two copies and were conserved in all the species analyzed, whereas different numbers of short repeat elements were observed, leading to species-specific IGS sequences with different length. In Fusarium subglutinans and Fusarium nygamai , these differences seemed to be the result of duplication and deletion events. Here, we propose a model based on unequal crossing over that can explain these processes. The partial IGS sequence of 22 Fusarium proliferatum isolates was also obtained to study variation at the intraspecific level. The results revealed no differences in terms of number or pattern of repeated elements and detected frequent gene conversion events. These results suggest that the homogenization observed at the intraspecific level might not be achieved primarily by unequal crossing-over events but rather by processes associated with recombination such as gene conversion events.

Mycorrhizal preferences and fine spatial structure of the epiphytic orchid Epidendrum rhopalostele

• Premise of the study: The presence of compatible fungi is necessary for epiphytic orchid recruitment. Thus, identifying associated mycorrhizal fungi at the population level is essential for orchid conservation. Recruitment patterns may also be conditioned by factors such as seed dispersal range and specific environmental characteristics. • Methods: In a forest plot, all trees with a diameter at breast height >1 cm and all individuals of the epiphytic orchid Epidendrum rhopalostele were identified and mapped. Additionally, one flowering individual of E. rhopalostele per each host tree was randomly selected for root sampling and DNA extraction. • Key results: A total of 239 E. rhopalostele individuals were located in 25 of the 714 potential host trees. Light microscopy of sampled roots showed mycorrhizal fungi in 22 of the 25 sampled orchids. Phylogenetic analysis of ITS1-5.8S-ITS2 sequences yielded two Tulasnella clades. In four cases, plants were found to be associated with both clades. The difference between univariate and bivariate K functions was consistent with the random labeling null model at all spatial scales, indicating that trees hosting clades A and B of Tulasnella are not spatially segregated. The analysis of the inhomogenous K function showed that host trees are not clustered, suggesting no limitations to population-scale dispersal. χ2 analysis of contingency tables showed that E. rhopalostele is more frequent on dead trees than expected. • Conclusions: Epidendrum rhopalostele establishes mycorrhizal associations with at least two different Tulasnella species. The analysis of the distribution patterns of this orchid suggests a microsite preference for dead trees and no seed dispersal limitation.

Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)–binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3–CEN DNA complex by atomic force microscopy. Assembly of CBF3–CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is ≈9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent ≈55° at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.

Architectural specificity in chromatin structure at the TATA box in vivo: Nucleosome displacement upon β-phaseolin gene activation

Extensive studies of the β-phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.