The synaptonemal complex (SC) is a proteinaceous complex that apparently mediates synapsis between homologous chromosomes during meiotic prophase. In Saccharomyces cerevisiae, the Zip1 protein is the integral component of the SC. In the absence of a DNA double-strand break or the SC initiation protein Zip3, Zip1 proteins aggregate to form a polycomplex (PC). In addition, Zip1 is also responsible for DSB-independent nonhomologous centromere coupling at early meiotic prophase. We report here that Zip3 is a SUMO (small ubiquitin-related modifier) E3 ligase and that Zip1 is a binding protein for SUMO-conjugated products. Our results also suggest that at early meiotic prophase, Zip1 interacts with Zip3-independent Smt3 conjugates (e.g., Top2) to promote nonhomologous centromere coupling. At and after mid-prophase, the Zip1 protein begins to associate with Zip3-dependent Smt3 conjugates (e.g., Red1) along meiotic chromosomes in the wild-type cell to form SCs and with Smt3 polymeric chains in the zip3 mutant to form PCs.[Keywords: Meiosis; synaptomenal complex; Zip1; Zip3; SUMO; Ulp2] Supplemental material is available at http://www.genesdev.org.

The aims of high-throughput (HTP) protein production systems are to obtain well-expressed and highly soluble proteins, which are preferred candidates for use in structure-function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96-well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products. All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%). To screen for well-expressed soluble fusion protein, total cell lysates of bacteria culture (∼1.5 mL) were subjected to high-speed centrifugation in a 96-tube format and analyzed by multiwell denaturing SDS-PAGE. Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. The method is well suited for automation and is applicable for the production of large numbers of proteins for genome-wide analysis. . Cloning and expression in Escherichia coli are favored in many instances because E. coli has relatively simple genetics, is well characterized, has a relatively rapid growth rate, and has few post-translational protein modifications. One disadvantage, however, of expressing heterologous proteins in E. coli is that proteins are frequently expressed as insoluble aggregated folding intermediates, known as inclusion bodies (Paul et al. 1983). Although it may be possible to increase protein solubility by optimizing expression condition or by refolding the recombinant proteins, in the interests of throughput, only a single set of growth or folding conditions can be used.Gene fusion is another approach that has been successfully used for producing soluble heterologous proteins in E. coli (Uhl'en and Moks 1990). Several carrier proteins are widely used in gene fusion, including thioredoxin (Trx), maltose-binding protein (MBP), glutathione S-transferase (GST), intein, calmodulin-binding protein (CBP), NusA, and cellulose-associated protein (CAP). Although the use of these carrier proteins has resulted in the successful overexpression of many heterologous proteins, each was tested Reprint requests to: Ting-Fang Wang, Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, Republic of China; e-mail: tfwang@ gate.sinica.edu.tw; fax: 886-2-27889759. 1These two authors contributed equally to this work.

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The structures of biomacromolecules are conventionally characterized by crystallography and cryogenic electron microscopy . The requirements of sample preparation limit the understanding of the specimens in their native states. Small-angle x-ray scattering (SAXS) has the capability of obtaining structural information from biological specimens in solution. However, resolving the structure from the acquired one-dimensional (1D) diffraction data requires the prior knowledge of the sample, and no unique solution can be guaranteed. Coherent diffraction imaging (CDI) provides excellent uniqueness in 2D/3D phase retrieval while the resolution is restricted by the poor signal-to-noise ratio at high-angle scattering. Here we combine CDI and SAXS to directly image a 19-nm-sized nodavirus particle in solution and determine the core-shell density distribution at a 1.3 nm pixel resolution. With 77,170 diffraction patterns summarized from randomly distributed nodavirus particles, the structural information can be obtained from the diffraction intensity alone without preknowledge. The hollow density distribution of a nodavirus particle revealed by our reconstruction is consistent with the structural determinations from crystallography and cryogenic electron microscopy. We believe this work represents a new protocol for characterizing the structures of macromolecules in solution from accumulated x-ray scattering data.

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