The effects of polylysine (PLL) and PLL-asialoorosomucoid (AsOR) on DNA condensation have been analyzed by AFM. Different types of condensed DNA structures were observed, which show a sequence of conformational changes as circular plasmid DNA molecules condense progressively. The structures range from circular molecules with the length of the plasmid DNA to small toroids and short rods with approximately 1/6 to 1/8 the contour length of the uncondensed circular DNA. Single plasmid molecules of 6800 base pairs (bp) condense into single toroids of approximately 110 nm diameter, measured center-to-center. The results are consistent with a model for DNA condensation in which circular DNA molecules fold several times into progressively shorter rods. Structures intermediate between toroids and rods suggest that at least some toroids may form by the opening up of rods as proposed by Dunlap et al. [(1997) Nucleic Acids Res. 25, 3095]. Toroids and rods formed at lysine:nucleotide ratios of 5:1 and 6:1. This high lysine:nucleotide ratio is discussed in relation to entropic considerations and the overcharging of macroions. PLL-AsOR is much more effective than PLL alone for condensing DNA, because several PLL molecules are attached to a single AsOR molecule, resulting in an increased cation density.

The atomic force microscope (AFM) was used to assay the extent of DNA condensation in approximately 100 different complexes of DNA with polylysine (PL) or PL covalently attached to the glycoproteins asialoorosomucoid (AsOR) or orosomucoid (OR). The best condensation of DNA was obtained with 10 kDa PL covalently attached to AsOR, at a lysine:nucleotide (Lys:nt) ratio of 5:1 or higher. These conditions produce large numbers of toroids and short rods with contour lengths of 300-400 nm. Some DNA condensation into shortened thickened structures was seen with 10 kDa PL attached to AsOR at Lys:nt ratios of 1.6:1 and 3:1. Some DNA condensation was also seen with 4 kDa PL at Lys:nt ratios of 3:1 and higher. Little DNA condensation was seen with PL alone or with PL convalently attached to OR at Lys:nt ratios up to 6:1. AsOR-PL enhanced gene expression in the mouse liver approximately 10- to 50-fold as compared with PL alone.

A finite mixture model using the Student's t distribution has been recognized as a robust extension of normal mixtures. Recently, a mixture of skew normal distributions has been found to be effective in the treatment of heterogeneous data involving asymmetric behaviors across subclasses. In this article, we propose a robust mixture framework based on the skew t distribution to efficiently deal with heavy-tailedness, extra skewness and multimodality in a wide range of settings. Statistical mixture modeling based on normal, Student's t and skew normal distributions can be viewed as special cases of the skew t mixture model. We present analytically simple EM-type algorithms for iteratively computing maximum likelihood estimates. The proposed methodology is illustrated by analyzing a real data example

MST3 is a member of the sterile-20 protein kinase family with a unique preference for manganese ion as a cofactor in vitro; however, its biological function is largely unknown. Suppression of endogenous MST3 by small interference RNA enhanced cellular migration in MCF-7 cells with reduced expression of E-cadherin at the edge of migrating cells. The alteration of cellular migration and protruding can be rescued by RNA interference-resistant MST3. The expression of surface integrin and Golgi apparatus was not altered, but phosphorylation on tyrosine 118 and tyrosine 31 of paxillin was attenuated by MST3 small interfering RNA (siRNA). Threonine 178 was determined to be one of the two main autophosphorylation sites of MST3 in vitro. Mutant T178A MST3, containing alanine instead of threonine at codon 178, lost autophosphorylation and kinase activities. Overexpression of wild type MST3, but not the T178A mutant MST3, inhibited migration and spreading in Madin-Darby canine kidney cells. MST3 could phosphorylate the protein-tyrosine phosphatase (PTP)-PEST and inhibit the tyrosine phosphatase activity of PTP-PEST. We conclude that MST3 inhibits cell migration in a fashion dependent on autophosphorylation and may regulate paxillin phosphorylation through tyrosine phosphatase PTP-PEST.

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 Ϸ9% 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 Ϸ55°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.Accurate chromosome segregation in mitosis and meiosis depends on the correct assembly of kinetochores on centromeric DNA (CEN DNA). During cell division, these structures attach chromosomes to the microtubules of the mitotic spindle and move the replicated chromosomes to opposing spindle poles.The minimal functional centromere in the budding yeast Saccharomyces cerevisiae contains a 125-base-pair sequence (CEN) present once on each chromosome, which is organized into three domains: CDEI, CDEII, and CDEIII ( In this paper, we describe the use of atomic force microscopy (AFM; also called scanning force microscopy) to probe the organization of the CBF3-CEN DNA complexes. We find that binding of the CBF3 proteins is associated with DNA shortening and bending. The location of the center of the CBF3 complex relative to the CDEIII site was found to be quite asymmetric, corroborating previous findings from DNase protection and DNA-protein crosslinking studies (2, 3). Additionally, we encountered evidence of joining or pairing of CEN DNA molecules by the CBF3 proteins. AFM has been successfully used to image biological samples in air or in liquid (4-8), and our findings further demonstrate that this imaging method is a powerful tool for the study of macromolecular assemblies and their interactions (9-16). MATERIALS AND METHODSPreparation of CBF3 Proteins. Cbf3a, Cbf3b, and Cbf3c͞d complex proteins tagged with 6-10 histidine residues were overexpressed and purified from Pichia pastoris, Escherichia coli, and S. cerevisiae, respectively, according to the procedure described in ref. 23.Preparation of CEN DNA Fragments and pUC19 DNA Beads. A linear DNA restriction fragment con...