Long-range repression by multiple polycomb group (PcG) proteins targeted by fusion to a defined DNA-binding domain in Drosophila.

A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, as bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repressed white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC was stronger than ZnF-SCM, which was stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression was more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produced silencing, indicating that these effects were long range. Repression by ZnF-SCM required a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo.

Expression and properties of wild-type and mutant forms of the Drosophila sex comb on midleg (SCM) repressor protein.

The Sex comb on midleg (Scm) gene encodes a transcriptional repressor of the Polycomb group (PcG). Here we show that SCM protein is nuclear and that its expression is widespread during fly development. SCM protein contains a C-terminal domain, termed the SPM domain, which mediates protein-protein interactions. The biochemical function of another domain consisting of two 100-amino-acid-long repeats, termed "mbt" repeats, is unknown. We have determined the molecular lesions of nine Scm mutant alleles, which identify functional requirements for specific domains. The Scm alleles were tested for genetic interactions with mutations in other PcG genes. Intriguingly, three hypomorphic Scm mutations, which map within an mbt repeat, interact with PcG mutations more strongly than do Scm null alleles. The strongest interactions produce partial synthetic lethality that affects doubly heterozygous females more severely than males. We show that mbt repeat alleles produce stable SCM proteins that associate with normal sites in polytene chromosomes. We also analyzed progeny from Scm mutant germline clones to compare the effects of an mbt repeat mutation during embryonic vs. pupal development. We suggest that the mbt repeat alleles produce altered SCM proteins that incorporate into and impair function of PcG protein complexes.

A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.

The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

The Drosophila Polycomb group gene Sex comb on midleg (Scm) encodes a zinc finger protein with similarity to polyhomeotic protein.

The Sex comb on midleg (Scm) gene is a member of the Polycomb group (PcG) of genes in Drosophila melanogaster. The PcG genes encode transcriptional repressors required for proper spatial expression of homeotic genes. We report the isolation of new Scm mutations and the molecular characterization of the Scm gene. Scm mRNA is expressed maternally, at peak levels in early embryos and then at lower levels throughout the remainder of development. Scm encodes a putative zinc finger protein of 877 amino acids. Scm protein is similar to polyhomeotic, another member of the PcG, both in the zinc finger region and in a separate C-terminal domain of 60 amino acids, which we term the SPM domain. Sequence analysis of an Scm mutant allele suggests a functional requirement for the SPM domain. Scm protein also bears homology in multiple domains to a mouse protein, Rae-28 (Nomura, M., Takihara, Y. and Shimada, K. (1994) Differentiation 57,39-50) and to a fly tumor suppressor protein, the product of the lethal(3)malignant brain tumor gene (Wismar, J. et al., (1995) Mech. Dev. 53, 141-154). Possible functional relationships among these proteins and potential biochemical roles for Scm protein in PcG repression are discussed.

The extra sex combs product contains WD40 repeats and its time of action implies a role distinct from other Polycomb group products.

The extra sex combs (esc) gene product is a transcriptional repressor of homeotic genes. Although it is classified in the Polycomb group (PcG) on the basis of phenotypic criteria, it is distinct from most other PcG repressors in its time of action during development. We describe the temporal profile of esc mRNA expression during embryogenesis and the stage-specific rescue of esc mutants with a heat shock-inducible esc cDNA transformation construct. Both experiments support the idea that esc product plays an early, transient role in repression of homeotic genes. We also present the sequence of a full-length esc cDNA. The predicted esc protein is composed primarily of multiple copies of a repeat motif, termed the WD40 repeat, which are likely used in protein-protein contact. We provide evidence that individual copies of the esc WD40 repeats are needed for function in vivo. We suggest that esc protein is an adaptor that binds to multiple protein partners and assists in the assembly or targeting of other PcG proteins.

[Effect of the culture technic on results following corneal preservation]. / Einfluss der Kulturtechnik auf die Ergebnisse nach Hornhautkonservierung.

Organ culture of human donor material extends post mortem storage times up to 4 weeks. If dextrane is omitted from the medium, as in the authors' current technique, a stromal swelling occurs which can be reserved preoperatively. The authors also report on their experience with organ culture of donor material in a culture medium containing dextrane. Short-term incubation of donor tissue in culture medium at 32 degrees C containing dextrane may be possible as an alternative to the MK technique, in which the donor tissue is kept at 4 degrees C. All methods have specific advantages and disadvantages, not only in laboratory handling but also in the postoperative development of the graft.