Characterization of a novel meiosis-specific protein within the central element of the synaptonemal complex

ABSTRACT AS-48 is a 70-residue, α-helical, cationic bacteriocin produced by Enterococcus faecalis and is very singular in its circular structure and its broad antibacterial spectrum. The AS-48 preprotein consists of an N-terminal signal peptide (SP) (35 residues) followed by a proprotein moiety that undergoes posttranslational modifications to yield the mature and active circular protein. For the study of the specificity of the region of AS-48 that is responsible for maturation, three single mutants have been generated by site-directed mutagenesis in the as-48A structural gene. The substitutions were made just in the residues that are thought to constitute a recognition site for the SP cleavage enzyme (His-1, Met1) and in those involved in circularization (Met1, Trp70). Each derivative was expressed in the enterococcal JH2-2 strain containing the necessary native biosynthetic machinery for enterocin production. The importance of these derivatives in AS-48 processing has been evaluated on the basis of the production and structural characterization of the corresponding derivatives. Notably, only two of them (Trp70Ala and Met1Ala derivatives) could be purified in different forms and amounts and are characterized for their bactericidal activity and secondary structure. We could not detect any production of AS-48 in JH2-2(pAM401-81 His-1Ile ) by using the conventional chromatographic techniques, despite the high efficiency of the culture conditions applied to produce this enterocin. Our results underline the different important roles of the mutated residues in (i) the elimination of the SP, (ii) the production levels and antibacterial activity of the mature proteins, and (iii) protein circularization. Moreover, our findings suggest that His-1 is critically involved in cleavage site recognition, its substitution being responsible for the blockage of processing, thereby hampering the production of the specific protein in the cellular culture supernatant.

Download Full-text

Characterization of an activated human ros gene.

Molecular and Cellular Biology ◽

10.1128/mcb.6.9.3109 ◽

1986 ◽

Vol 6 (9) ◽

pp. 3109-3116 ◽

Cited By ~ 61

Author(s):

C Birchmeier ◽

D Birnbaum ◽

G Waitches ◽

O Fasano ◽

M Wigler

Keyword(s):

Gene Transfer ◽

Protein Kinases ◽

Transmembrane Domain ◽

Extracellular Domain ◽

Kinase Domain ◽

Specific Protein ◽

Normal Human

A human oncogene, mcf3, previously detected by a combination of DNA-mediated gene transfer and a tumorigenicity assay, derives from a human homology of the avian v-ros oncogene. Both v-ros and mcf3 can encode a protein with homology to tyrosine-specific protein kinases, and both mcf3 and v-ros encode a potential transmembrane domain N terminal to the kinase domain. mcf3 probably arose during gene transfer from a normal human ros gene by the loss of a putative extracellular domain. There do not appear to be any other gross rearrangements in the structure of mcf3.

Download Full-text

Structural analysis of the human SYCE2–TEX12 complex provides molecular insights into synaptonemal complex assembly

Open Biology ◽

10.1098/rsob.120099 ◽

2012 ◽

Vol 2 (7) ◽

pp. 120099 ◽

Cited By ~ 33

Author(s):

Owen R. Davies ◽

Joseph D. Maman ◽

Luca Pellegrini

Keyword(s):

Structural Analysis ◽

Synaptonemal Complex ◽

Recurrent Miscarriage ◽

Central Element ◽

Biological Data ◽

Structural Basis ◽

Structural Framework ◽

Chromosome Synapsis ◽

Heterotypic Interactions ◽

Physical Features

The successful completion of meiosis is essential for all sexually reproducing organisms. The synaptonemal complex (SC) is a large proteinaceous structure that holds together homologous chromosomes during meiosis, providing the structural framework for meiotic recombination and crossover formation. Errors in SC formation are associated with infertility, recurrent miscarriage and aneuploidy. The current lack of molecular information about the dynamic process of SC assembly severely restricts our understanding of its function in meiosis. Here, we provide the first biochemical and structural analysis of an SC protein component and propose a structural basis for its function in SC assembly. We show that human SC proteins SYCE2 and TEX12 form a highly stable, constitutive complex, and define the regions responsible for their homotypic and heterotypic interactions. Biophysical analysis reveals that the SYCE2–TEX12 complex is an equimolar hetero-octamer, formed from the association of an SYCE2 tetramer and two TEX12 dimers. Electron microscopy shows that biochemically reconstituted SYCE2–TEX12 complexes assemble spontaneously into filamentous structures that resemble the known physical features of the SC central element (CE). Our findings can be combined with existing biological data in a model of chromosome synapsis driven by growth of SYCE2–TEX12 higher-order structures within the CE of the SC.

Download Full-text

The synaptonemal complexes of Achlya recurva (Oomycetes): karyotype analysis and three-dimensional reconstruction of pachytene nuclei in antheridia and oogonia

Canadian Journal of Botany ◽

10.1139/b91-178 ◽

1991 ◽

Vol 69 (6) ◽

pp. 1384-1395 ◽

Cited By ~ 2

Author(s):

Hobart R. Williamson ◽

Pesach Ben Yitzchak

Keyword(s):

Synaptonemal Complex ◽

Organizer Region ◽

Nucleolar Organizer Region ◽

Nucleolus Organizer Region ◽

Three Dimensional ◽

Central Element ◽

Three Dimensional Reconstruction ◽

Synaptonemal Complexes ◽

Sequence Of Events ◽

Dimensional Reconstruction

Fifteen synaptonemal complexes, as determined by three-dimensional reconstruction of serial, ultrathin sections, were present within both antheridial and oogonial zygotene and pachytene nuclei of the oomyceteous fungus Achlya recurva, thus n = 15. The present study represents the first complete reconstruction of synaptonemal complexes in the genus Achlya. The occurrence of both zygonema and pachynema was simultaneous in antheridia and oogonia. Pachytene nuclei of antheridia and oogonia are small, 13 μm3 in volume, and the average length of the synaptonemal complexes ranged from 1.9 to 4.4 μm. Lateral elements at zygotene ranged from 1.2 to 4.7 μm. Both ends of each synaptonemal complex were attached randomly to the nuclear envelope, so a bouquet formation was not observed at pachytene. In A. recurva, the dimensions of the synaptonemal complex were as follows: overall width = 270 nm; the lateral elements = 75 nm each in width and the central region = 120 nm. There was no central element and associated transverse filaments, which may be associated with development of alternative reproductive strategies other than amphimixis, as in nematodes. Of the 15 synaptonemal complexes present, only the one carrying the nucleolus organizer region could be clearly identified from one nucleus to the next. The nucleolar organizer region was on the average 0.75 μm from the telomere in both zygotene and pachytene nuclei. There were an average of three recombination nodules in each nucleus. Synaptonemal complexes have been reported in over 80 different species of fungi and related protista. Karyotypic evolution in the oomycetes and fungi may be the result of poly-ploidization, followed by cytogenetic diversification involving aneuploidy and differing degrees of polyploidy. Such a sequence of events could explain the apparent polyphyletic formation of this group. Key words: karyotype, Oomycetes, pachytene, synaptonemal complexes, three-dimensional reconstruction.

Download Full-text

A Novel Chromodomain Protein, Pdd3p, Associates with Internal Eliminated Sequences during Macronuclear Development inTetrahymena thermophila

Molecular and Cellular Biology ◽

10.1128/mcb.20.11.4128-4134.2000 ◽

2000 ◽

Vol 20 (11) ◽

pp. 4128-4134 ◽

Cited By ~ 47

Author(s):

Mikhail A. Nikiforov ◽

Martin A. Gorovsky ◽

C. David Allis

Keyword(s):

De Novo ◽

Germ Line ◽

Chromosome Breakage ◽

Specific Protein ◽

Developmentally Regulated ◽

Dna Elimination ◽

Internal Eliminated Sequences ◽

Subsequent Elimination ◽

Rearrangement Processes

ABSTRACT Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.

Download Full-text