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Enhancer activities have been observed in DNA fragments up to 1.36 kb long located on the 3'-side of the cluster of the three alpha-type globin-encoding genes in duck [Kretsovali et al., C.R. Acad. Sci. Paris 307 (1988) 563-568] and chicken [Knezetic and Felsenfeld, Mol. Cell. Biol. 9 (1989) 893-901]. We report here the identification of a chicken silencer element placed upstream from the three GATA-1 sites which constitute the core enhancer element in both species. This silencer element can autonomously reduce the activity of promoters for thymidine kinase and alpha D globin. Band shifts and DNase I footprinting experiments using nuclear extracts from thermosensitive avian erythroblastosis virus-transformed chicken erythroblasts led to the delineation of three sites for DNA-binding proteins within the silencer element.

The genomic DNA of cloned recombinants containing the duck globin genes was compared to that of the analogous domains of the chicken. A 36 kb insert including the three alpha-type globin genes was isolated from a newly prepared duck genomic library in the cosmid PJB8; another recombinant contained a 45 kb insert with the four beta globin genes. In the alpha globin gene domain, the relative positions of genes, of repetitive sequences, and of the A + T-rich segments (AT-rich linkers, ATRLs) which frame the gene cluster (Moreau et al. 1982), were found to be closely maintained between duck and chicken. Although ATRLs and repetitive sequences also frame the gene cluster in the beta globin domains of duck and chicken, there is more genetic drift in their relative positions than in the alpha domain. It is of interest that several repetitive DNA segments were detected in the chicken beta globin domain which do not exist in corresponding positions in the duck. In view of the strict conservation in both species of genes and their relative positions in the cluster, this observation seems to exclude a simple function of repetitive sequences in the control of individual genes. The data are discussed with regard to the possible significance of repetitive and AT-rich DNA segments in genome organisation and function.

We have detected a transcriptional enhancer sequence downstream from the adult beta-globin (beta A-globin) genes of chicken and duck. DNA segments from the beta-globin coding and flanking sequences were cloned into expression vectors containing the SV40 promoter linked to either the T antigen gene or the cat gene. The expression of these genes was measured in a chicken erythroid cell line transfected with the recombinant plasmids. We found that segments located about 400 bp downstream from the poly(A) site of both the chicken and duck beta A-globin genes (and about 1.5 kb upstream from the embryonic epsilon-globin gene) stimulate transcription of the test genes about five-fold. In chicken essentially the same segment was also found by others to act as an erythroid cell-specific enhancer [Hesse et al., Proc. Natl. Acad. Sci. USA 83 (1986) 4312-4316; Choi and Engel, Nature 323 (1986) 731-734]. The sequence containing this enhancer is conserved in evolution. A high degree of homology, reaching 84% in a segment 180 bp in length, was found between chicken and duck despite an evolutionary divergence of 70 myr.

Having previously found a reduced transcription of globin genes and an abortive processing of the already transcribed globin pre-mRNA in Avian Erythroblastosis Virus (AEV) transformed cells (1), we compared the genomic DNA of these cells with that of normal chicken erythroblasts, using 32-P-labelled cDNA probes specific for the beta, alpha A and alpha D globin sequences. Restriction endonuclease digestion, electrophoresis of digests in agarose gels, Southern blotting and hybridization were carried out. Our results show that the overall genome organization is not disturbed in the immediate neighbourhood of the adult globin genes; the observed restriction fragments are identical for both DNAs after EcoRI, HindIII, BamHI and XbaI digestion, using the beta, alpha A and alpha D globin cDNA probes. However, we observe specific modifications at some methylation sites in the beta, beta-like and alpha D regions: after HpaII or MspI digestion in the alpha D region and after HhaI digestion in the beta and beta-like region, heavier bands appear in the transformed cell DNA in addition to the ones observed in normal DNA. This implies that, at some specific sites, the transformed cell DNA is more methylated than the normal erythroblast DNA. The possible significance of this observation is discussed.

We found an enhancer element placed at the 3' side of the adult duck alpha A globin gene. The duck alpha globin gene cluster contains three genes from the 5' to 3' side: the pi embryonic gene, the alpha D minor adult gene and the alpha A adult major gene. We analyzed a 16 kb genomic domain extending from 2 kb upstream of the pi gene to 5 kb downstream of the alpha A gene. This enhancer is active in AEV transformed chicken erythroblasts. Its is inactive both in HeLa cells and in the human erythroid cells K562 which express only embryonic genes. These findings are discussed in relation to previous results concerning the duck beta globin enhancer located at the 3' side of the beta A globin gene.

An erythroid-specific enhancer was previously identified in the 3'-flanking region of the beta adult gene in chicken and duck, by transfection into AEV transformed chicken erythroblasts. Here we show that the duck enhancer is equally active in erythroid human K562 cells, presenting an embryonic/fetal program of globin gene expression. Furthermore, no other enhancer was found within the 20 kb of DNA including four beta-like globin genes as well as a 1.5 kb upstream and a 3 kb downstream sequence.

Tetrahymena pyriformis which has been starved for 20 h by incubation in buffer, and then deciliated, can regenerate its cilia in about 90 min while still in suspension in non-nutrient medium. The process of reciliation is accompanied by protein synthesis which begins a few minutes after deciliation and by synthesis of ribosomal and messenger RNAs during a period extending from about 1 h to about 3 h after deciliation. Although net synthesis of RNA remains at a very low level until 1 h after deciliation, a qualitative change in the translatable poly(A)-containing messenger RNA content of deciliated cells, and in particular, formation of beta-tubulin mRNA can be detected almost immediately after deciliation.