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Two distinct types of Polycomb complexes have been identified in flies and in vertebrates, one containing ESC and one containing PC. Using LexA fusions, we show that PC and ESC can establish silencing of a reporter gene but that each requires the presence of the other. In early embryonic extracts, we find PC transiently associated with ESC in a complex that includes EZ, PHO, PH, GAGA, and RPD3 but not PSC. In older embryos, PC is found in a complex including PH, PSC, GAGA, and RPD3, whereas ESC is in a separate complex including EZ, PHO, and RPD3.

Polycomb Group complexes assemble at polycomb response elements (PREs) in vivo and silence genes in the surrounding chromatin. To study the recruitment of silencing complexes, we have targeted various Polycomb Group (PcG) proteins by fusing them to the LexA DNA binding domain. When LexA-PC, -PSC, -PH or -SU(Z)2 are targeted to a reporter gene, they recruit functional PcG-silencing complexes that recapitulate the silencing behavior of a PRE: silencing is sensitive to the state of activity of the target chromatin. When the target is transcriptionally active, silencing is not established but when the target is not active at syncytial blastoderm, it becomes silenced. The repressed state persists through embryonic development but cannot be maintained in larval imaginal discs even when the LexA-PcG fusion is constitutively expressed, suggesting a discontinuity in the mechanism of repression. These proteins also interact with other PC-containing complexes in embryonic nuclear extracts. In contrast LexA-PHO is neither able to silence nor to interact with PC-containing complexes. Analysis of pho mutant embryos and of PRE constructs whose PHO-binding sites are mutated suggests that, while PHO is important for silencing in imaginal discs, it is not necessary for embryonic PcG silencing.

We have used P‐mediated transformation to introduce into the Drosophilagenome transposons containing the hsp‐70heat shock promoter (hsp) placed at different distances upstream of the whitegene. Using these constructions we found a strong terminator 250 nucleotides upstream of the whitetranscription start. When all whiteupstream sequences are deleted and the hspis fused to the whiteleader region, whitetranscription is under hspcontrol, however, flies carrying this construction have red eyes even at low temperature, due to the basal level of activity of the hsp. High levels of transcription are obtained after heat shock but about half of the transcripts are incompletely spliced. One transformed line carries the transposon integrated near the heterochromatin. These flies have very pale eyes but develop strongly pigmented, variegated eyes if heat shocked in the first 2 days of the pupal stage. Heat shocks after eclosion have no effect on eye pigmentation. The properties of these transposons also indicated that the control sequences required for Zesteinteraction lie > 1 kb upstream of the whitetranscription start while those required for dosage compensation are close to the gene.

Constructions containing the Drosophila white gene and different amounts and arrangements of its regulatory region were introduced into the germ line of white mutant flies by P‐mediated transformation. The results obtained with the different transposon constructions show that different parts of the 1.8‐kb region preceding the transcription start are required for the expression of the gene in different tissues and at different developmental stages. Different sequences independently control the expression of the gene in the adult testes, in the larval and adult Malpighian tubules and in the eye. Another sequence located greater than 1080 bp upstream of the transcription start is the target of zeste interaction. The results also suggest that sequences required for dosage compensation are contained between ‐216 and the transcription start site. We show that at least some of these regulatory elements are equally functional if their distance from the promoter is varied or if their orientation is inverted. Their properties suggest that they act as enhancer‐like elements to regulate the activity of the white promoter and, at least in the case of the zeste regulatory site, that they can act also in ‘trans’ on a white promoter locked in close physical proximity by homologous chromosome pairing.

The Drosophila zeste protein binds in vitro to several sites in the white, Ultrabithorax, decapentaplegic, Antennapedia, and engrailed genes and to at least one site in the zeste gene itself. The distribution of these sites corresponds often with that of regulatory elements in these genes as defined by mutations or, in the case of white, by molecular analysis. A zeste binding site is frequently found in the immediate vicinity of the promoter. zeste binding sites are composed of two or more zeste recognition sequences T/CGAGT/CG. Isolated consensus sequences do not bind or footprint. Cooperative interactions are involved both in binding to a given site and between proteins bound at independent sites. zeste bound to one DNA molecule can in fact bind simultaneously to another DNA molecule. These results suggest a general role for zeste in bringing together distant regulatory elements controlling the activity of a target gene. In this model, transvection effects are a by‐product of normal intragenic zeste action.

Drosophilalarvae are rapidly killed by food containing the antibiotic G418. The bacterial gene for neomycin resistance introduced in the genome by P‐mediated transformation renders larvae resistant to G418 and able to grow to fertile adults. The neogene transcribed from the herpes thymidine kinase promoter gives low levels of resistance but high levels can be obtained using the hsp70heat‐shock promoter. We have constructed a vector for P‐mediated transformation which uses this finding to allow dominant selection of transformed progeny. Features of this vector also facilitate cloning and allow the rapid recovery of the inserted transposon from transformed flies. We have also constructed a cosmid vector for P‐mediated transformation that incorporates the hsp70‐neogene.

Heavy and light chain zein genes from maize, affected by different regulatory loci, are related in sequence and structure. The two kinds of genes code for a signal peptide, a head region of 67 amino acids, six and a half to eight repetitive blocks of ˜20 amino acids each and a short tail piece. This block structure would allow inter or intragenic recombination giving rise to heterogeneous zein genes. Length variation in zein polypeptides is also due to the occurrence of termination mutations within some genes. Homology between heavy and light chain zein genes extends to the flanking sequences where a short region at the 5′ end of the transcript can base pair with the 3′ end and may have regulatory implications.

We have constructed and screened cDNA libraries from total maize endosperm poly(A)+RNA or from a mRNA fraction enriched in zein sequences. From these libraries we have isolated clones representative of the major classes of zein cDNA sequences and have characterised them by crosshybridisation, by hybrid‐selected translation, by in situhybridisation to maize chromosomes, and hybridisation to genomic Southern blots. We conclude that at least four types of non cross‐hybridising zein sequences are present, two coding for light chains and two for heavy chains. At least in the case of the light zeins, there is considerable sequence diversity among the clones which hybridise to each type. Similar results are obtained by translation of the mRNAs selected by each clone. In situhybridisation shows that the light chain zein genes are located on chromosomes 4, 7, and 10, whilst genes coding for some of the heavy chain zeins are confined to the distal part of the long arm of chromosome 4.