ZYP1 is required for obligate cross-over formation and cross-over interference in Arabidopsis

The synaptonemal complex is a tripartite proteinaceous ultrastructure that forms between homologous chromosomes during prophase I of meiosis in the majority of eukaryotes. It is characterized by the coordinated installation of transverse filament proteins between two lateral elements and is required for wild-type levels of crossing over and meiotic progression. We have generated null mutants of the duplicated Arabidopsis transverse filament genes zyp1a and zyp1b using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of the zyp1 null mutants reveals loss of the obligate chiasma, an increase in recombination map length by 1.3- to 1.7-fold and a virtual absence of cross-over (CO) interference, determined by a significant increase in the number of double COs. At diplotene, the numbers of HEI10 foci, a marker for Class I interference-sensitive COs, are twofold greater in the zyp1 mutant compared to wild type. The increase in recombination in zyp1 does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% in msh5/zyp1 compared to msh5. These data suggest that ZYP1 limits the formation of closely spaced Class I COs in Arabidopsis. Our data indicate that installation of ZYP1 occurs at ASY1-labeled axial bridges and that loss of the protein disrupts progressive coalignment of the chromosome axes.

The synaptonemal complex (SC) is a proteinaceous ultrastructure that forms between homologous chromosomes (homologs) during midprophase I of meiosis and plays a critical role in coordinating the repair of programmed DNA double-strand breaks (DSBs) to form cross-over (CO) products (1, 2). At the onset of leptotene, the sister chromatids are organized into linear looped chromatin arrays conjoined at the loop bases by a protein axis that runs along the chromosomes (3, 4). Early steps in the recombination pathway enable the loose alignment of homolog axes at a distance of ∼400 nm (5). Formation of the SC then initiates and continues throughout zygotene via progressive installation of transverse filaments (TFs) that run perpendicular to the aligned homolog axes (referred to as lateral elements in the context of the SC), ultimately bringing them into close apposition along their entire length at a distance of ∼100 nm (2, 5). Installation of the TFs starts at multiple synapsis initiation sites that correspond to future Class I COs in Saccharomyces cerevisiae (6). In species with larger chromosomes such as Sordaria macrospora, synapsis initiates from CO-designated sites as well as additional sites whose distribution also appears sensitive to interference (1, 5). In Arabidopsis thaliana, 20 to 25 synapsis initiation sites per cell indicate a ∼2- to 2.5-fold excess over COs and in barley 76 synapsis initiation sites, versus 17 chiasmata reveal a ∼4.5-fold excess (7, 8). Full synapsis denotes the onset of pachytene and is maintained throughout this stage during which time CO formation is completed. As prophase I progresses to diplotene/diakinesis, the SC is disassembled.TFs have been described in a variety of organisms, and in most cases, they are composed of a single protein. These include Zip1 in budding yeast, C(3)G in Drosophila melanogaster, SYCP1 in mouse, ZYP1 in A. thaliana (encoded by duplicated genes, ZYP1a and ZYP1b), ZEP1 in rice (Oryza sativa), and ZYP1 in barley (Hordeum vulgare) (9–16). Caenorhabditis elegans is an exception that possesses six TF proteins (SYP1-6) required for normal synapsis (17–22). Despite a striking lack of homology between the TFs at the primary amino acid sequence level, they share very similar structures, comprising a globular N-terminal domain linked to another globular domain at the C terminus via a long alpha helical central region that is able to form large stretches of parallel, in-register, homodimeric coiled coils (23). Studies have shown that the TFs are oriented such that the C termini are associated with lateral elements potentially interacting with DNA, while the N-terminal domains localize to the central region (2, 24). Evidence suggests that the overall three-dimensional macromolecular organization of the SC is also somewhat conserved. Analyses in mouse, Drosophila, and H. vulgare (barley) strongly suggest that these organisms form SCs with a bilayer of TFs (25–28). A multilayered structure is also supported by studies in Blabs cribrosa (beetle) (29, 30). However, key aspects of the organization of the TFs within the SC remain a matter of debate. Initially, analysis of zip1 mutants in S. cerevisiae suggested that the TFs comprise a tetramer of two opposing Zip1 dimers with their N termini forming overlapping interactions in the central region of the SC (31). X-ray crystallographic studies of the human TF, SYCP1, report that the protein forms a tetrameric building block that self-assembles into a zipper-like lattice through “head-to-head” N-terminal interactions in the SC central region and “back-to-back” interactions between adjacent C-terminal dimers at the lateral elements (24). In contrast, analysis of the mouse SC using electron tomography has led to the proposal that the SC has a more dynamic structure with TF dimers forming a variety of less regimented interactions as part of an irregular single plane. However, this model appears inconsistent with other studies in mouse which support a more ordered structure (25, 26).Mutant analysis has demonstrated that TF proteins are essential for assembly of the SC central region and thus homolog synapsis. These also confirm an important role in the control of CO formation but with some variation between organisms. Studies of zip1 mutants in S. cerevisiae have shown that the Zip1 protein is a member of the ZMM group of proteins comprising Zip1, Zip2, Zip3, Zip4, Msh4, Msh5, and Mer3 that are required for the formation of Class I interfering COs (32). CO interference is a patterning mechanism that ensures even spacing of COs along the chromosomes (33–35). In S. cerevisiae and Arabidopsis, Class I COs account for ∼85% of total COs and the remaining Class II COs (∼15%) are randomly distributed (36–38). However, in plants, Zip1 orthologs appear to be functionally independent of the other ZMM proteins for CO formation (14–16). Genetic analysis of S. cerevisiae zip1 deletion mutants revealed a modest reduction in CO formation ∼30 to 40% with residual COs no longer exhibiting CO interference leading to the suggestion that the SC may mediate this process (10). Subsequent studies based on a molecular analysis of recombination intermediates in zip1 and other zmm mutants argue against a role for the SC in mediating interference as they indicate that the fate of DSBs is designated at an early stage in the recombination pathway prior to installation of the SC (32, 39). In female Drosophila lacking the TF protein C(3)G, DSB formation is thought to be reduced and they fail to form COs, although SC formation is independent of recombination (12). These authors also report that analysis of flies expressing a mutant version of the protein reveals that a complete SC is not required for CO interference (12). A major reduction in COs of ∼90% is also observed in mouse sycp1 mutants although DSB formation appears normal (13). Similarly in C. elegans (in which SC installation occurs at pairing centers), syp-1 and syp-2 null mutants recombination is initiated but COs do not form (17, 18). A further study in which the SC central region was partially depleted by RNA interference (RNAi)–induced SYP-1 knockdown found that CO interference was reduced leading to an increase in COs, suggesting a role for the SC in limiting COs (40).TFs have been studied in several plant species including Arabidopsis, barley, and rice (14–16). Analysis of Tos17 insertion mutants of the rice TF gene ZEP1 demonstrated that in common with other organisms, it is essential for SC formation and affects CO formation (16). However, rather than displaying a reduction in COs, analysis of the short arm of chromosome 11 revealed a more than threefold increase in COs in zep1 mutants (16). Like rice, barley is a member of the grass family (Poaceae), and in common with rice, RNAi knockdown lines of the TF protein HvZYP1 are defective in SC formation, but in contrast, CO formation is reduced to ∼25% of wild-type levels (15). In Arabidopsis, the TF protein, ZYP1, is encoded by functionally redundant duplicated genes, ZYP1a and ZYP1b, which share 93% homology and are encoded within 2 kb of each other on opposite strands of chromosome 1. Individual zyp1a and zyp1b mutants are fertile and possess only mild meiotic phenotypes, and as isolation of a double mutant has thus far proved intractable, functional analysis of ZYP1 has relied on RNAi knockdown lines (14). As expected, these lines failed to assemble an SC. Chiasma frequency was reduced by ∼20 to 30% and based on metaphase I bivalent shapes, they appeared to exhibit interference, but a proportion involved ectopic recombination with nonhomologs (14).Although existing studies imply that there may be some variation in the role of the SC in relation to CO control in plants, the studies in Arabidopsis and barley were based on RNAi knockdown lines rather than TF mutants. Hence to address this issue, we have generated CRISPR/Cas zyp1a/zyp1b mutants. This has enabled a detailed analysis of ZYP1 function in Arabidopsis, revealing that it is required for formation of the obligate CO and implementation of CO patterning. Loss of the protein also disrupts the normal program of homolog coalignment during prophase I.     

The expression of Rho proteins decreases with human brain tumor progression: Potential tumor markers

Astrocytic tumors are the most common human brain tumors. Establishment of tumor grade is a key determinant both in the choice of a therapeutic approach and in the prognosis. The diagnosis of astrocytic tumors is currently determined following histopathological analysis. The identification of molecular markers would offer a complementary tool for characterizing tumors with respect to their clinical behavior. In this study we determined the expression levels of 3 small GTP binding proteins (RhoA, RhoB and Rac1), of their inhibitor RhoGDI and of caveolin-1 in 24 human astrocytic tumors of grades I to IV. Our results demonstrated that the expression of RhoA and RhoB decreased significantly in all brain tumors studied and was inversely related with tumor of grade II to IV malignancy. The amount of caveolin-1 immunodetected was not significantly different from normal brain samples while the Rac1 expression level was diminished in astrocytic tumors of grades III and IV. Our finding that RhoA and RhoB expression levels are correlated to tumor malignancy suggests that they may serve as novel and efficient diagnostic markers for astrocytic brain tumors of histological grade II to IV and complement currently applied histopathological analysis.     

Comparison between two thoracotomy closure techniques: postoperative pain and pulmonary function

OBJECTIVE:

To compare two thoracotomy closure techniques (pericostal and transcostal suture) in terms of postoperative pain and pulmonary function.

METHODS:

This was a prospective, randomized, double-blind study carried out in the Department of Thoracic Surgery of the Luzia de Pinho Melo Hospital das Clínicas and at the University of Mogi das Cruzes, both located in the city of Mogi das Cruzes, Brazil. We included 30 patients (18-75 years of age) undergoing posterolateral or anterolateral thoracotomy. The patients were randomized into two groups by the type of thoracotomy closure: pericostal suture (PS; n = 16) and transcostal suture (TS; n = 14). Pain intensity during the immediate and late postoperative periods was assessed by a visual analogic scale and the McGill Pain Questionnaire. Spirometry variables (FEV₁, FVC, FEV₁/FVC ratio, and PEF) were determined in the preoperative period and on postoperative days 21 and 60.

RESULTS:

Pain intensity was significantly greater in the PS group than in the TS group. Between the preoperative and postoperative periods, there were decreases in the spirometry variables studied. Those decreases were significant in the PS group but not in the TS group.

CONCLUSIONS:

The patients in the TS group experienced less immediate and late post-thoracotomy pain than did those in the PS group, as well as showing smaller reductions in the spirometry parameters. Therefore, transcostal suture is recommended over pericostal suture as the thoracotomy closure technique of choice.     

Dyadic OPTION: Measuring perceptions of shared decision-making in practice

Background

Current models of the medical consultation emphasize shared decision-making (SDM), whereby the expertise of both the doctor and the patient are recognised and seen to equally contribute to the consultation. The evidence regarding the desirability and effectiveness of the SDM approach is often conflicting. It is proposed that the conflicts are due to the nature of assessment, with current assessments from the perspective of an outside observer.

Aims

To empirically assess perceived involvement in the medical consultation using the dyadic OPTION instrument.

Method

36 simulated medical consultations were organised between general practitioners and standardized- patients, using the observer OPTION and the newly developed dyadic OPTION instruments.

Results

SDM behaviours observed in the consultations were seen to depend on both members of the doctor and patient dyad, rather than each in isolation. Thus a dyadic approach to measurement is supported.

Conclusions

This current study highlights the necessity for a dyadic approach to assessment and introduces a novel research instrument: the dyadic OPTION instrument.     

BORIS,a novel male germ-line-specific protein associated with epigenetic reprogramming events,shares the same 11-zinc-finger domain with CTCF,the insulator protein involved in reading imprinting marks in the soma

CTCF, a conserved, ubiquitous, and highly versatile 11-zinc-finger factor involved in various aspects of gene regulation, forms methylation-sensitive insulators that regulate X chromosome inactivation and expression of imprinted genes. We document here the existence of a paralogous gene with the same exons encoding the 11-zinc-finger domain as mammalian CTCF genes and thus the same DNA-binding potential, but with distinct amino and carboxy termini. We named this gene BORIS for Brother of the Regulator of Imprinted Sites. BORIS is present only in the testis, and expressed in a mutually exclusive manner with CTCF during male germ cell development. We show here that erasure of methylation marks during male germ-line development is associated with dramatic up-regulation of BORIS and down-regulation of CTCF expression. Because BORIS bears the same DNA-binding domain that CTCF employs for recognition of methylation marks in soma, BORIS is a candidate protein for the elusive epigenetic reprogramming factor acting in the male germ line.     

Treatment of cardiomyopathy and rhabdomyolysis in long-chain fat oxidation disorders using an anaplerotic odd-chain triglyceride

The current dietary treatment of long-chain fatty acid oxidation defects (high carbohydrate with medium-even-chain triglycerides and reduced amounts of long-chain fats) fails, in many cases, to prevent cardiomyopathy, rhabdomyolysis, and muscle weakness. We hypothesized that the apparent defect in energy production results from a depletion of the catalytic intermediates of the citric acid cycle via leakage through cell membranes (cataplerosis). We further hypothesized that replacing dietary medium-even-chain fatty acids (precursors of acetyl-CoA) by medium-odd-chain fatty acids (precursors of acetyl-CoA and anaplerotic propionyl-CoA) would restore energy production and improve cardiac and skeletal muscle function. We fed subjects with long-chain defects a controlled diet in which the fat component was switched from medium-even-chain triglycerides to triheptanoin. In three patients with very-long-chain acyl-CoA dehydrogenase deficiency, this treatment led rapidly to clinical improvement that included the permanent disappearance of chronic cardiomyopathy, rhabdomyolysis, and muscle weakness (for more than 2 years in one child), and of rhabdomyolysis and weakness in the others. There was no evidence of propionyl overload in these patients. The treatment has been well tolerated for up to 26 months and opens new avenues for the management of patients with mitochondrial fat oxidation disorders.     

The multicenter trial SAKK 37/95 of cladribine, cyclophosphamide and prednisone in the treatment of chronic lymphocytic leukemias and low-grade non-Hodgkin's lymphomas

A multicenter trial was performed to confirm the therapeutic efficacy and the toxicity profile of the combination of cladribine, cyclophosphamide and prednisone in low-grade non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Twenty-three adults with previously treated (61%) or untreated (39%) NHL International Working Formulation A or Binet B and C CLL were administered cladribine 0.1 mg/kg/day as a subcutaneous bolus for 5 days, intravenous cyclophosphamide 500 mg/m2 on day 1, and oral prednisone 40 mg/m2 on days 1-5, every 4 weeks. Unexpected early hematological toxicities led to dose modifications for pretreated patients who received cladribine for 3 days only up to a maximum of five courses. Responses were observed in 75%, with 7 patients obtaining a complete clinical and hematological response. Median duration of complete response was 9 months. Median time to progression or relapse was 31 months. Myelosuppression and infections were dose limiting whereas posttreatment complications, including fatalities, resulted from infections. Median overall survival time from trial entry was 60 months. Activity of the combination of cladribine, cyclophosphamide and prednisone was confirmed. However, in the specific setting of a multicenter trial, unexpected fatal infectious episodes occurred in pretreated patients. Great caution is thus required in these susceptible patients and the routine use of corticosteroids should probably be abandoned.