Independent recruitment in vivo by Gal4 of two complexes required for transcription

We use a modified form of ChIP to analyze the recruitment of seven sets of proteins to the yeast GAL genes upon induction. We resolve three stages of recruitment: first SAGA, then Mediator, and finally Pol II along with four other proteins (including TBP) bind the promoter. In a strain lacking SAGA, Mediator is recruited with a time course indistinguishable from that observed in wild-type cells. Our results are consistent with the notion that a single species of activator, Gal4, separately contacts, and thereby directly recruits, SAGA and Mediator.     

A quantitative analysis of the kinetics of Gal4 activator and effector gene expression in the zebrafish

Using a temperature-inducible hsp70:Gal4 activator and UAS:myc-notch1a-intra as effector, we determined quantitatively the kinetics of expression of both transgenes and analysed the effects of varying their expressivity on several phenotypic traits in the developing zebrafish. hsp70:Gal4 is transcribed within 15 min after temperature-mediated induction, but Gal4 RNA decays rapidly. The Gal4 protein was found to be quite stable, as functional Gal4, which was detectable 1.5 h after heat shock (HS), persisted for at least 13 h. myc-notch1a-intra RNA is expressed approximately 1.5 h after HS, but unlike the Gal4 RNA, it was found to be very stable; it continues to accumulate during the succeeding 17 h after HS. Fully penetrant phenotypic effects are obtained after a relatively long activator induction with a 30-min HS.     

Delaying Gal4-driven gene expression in the zebrafish with morpholinos and Gal80

The modular Gal4/UAS gene expression system has become an indispensable tool in modern biology. Several large-scale gene- and enhancer-trap screens in the zebrafish have generated hundreds of transgenic lines expressing Gal4 in unique patterns. However, the early embryonic expression of the Gal4 severely limits their use for studies on regeneration or behavior because UAS-driven effectors could disrupt normal organogenesis. To overcome this limitation, we explored the use of the Gal4 repressor Gal80 in transient assays and with stable transgenes to temporally control Gal4 activity. We also validated a strategy to delay Gal4-driven gene expression using a morpholino targeted to Gal4. The first approach is limited to transgenes expressing the native Gal4. The morphant approach can also be applied to transgenic lines expressing the Gal4-VP16 fusion protein. It promises to become a standard approach to delay Gal4-driven transgene expression and enhance the genetic toolkit for the zebrafish.     

Tracing the history of Galα1–4Gal on glycoproteins in modern birds

Galα1–4Gal is typically found in mammalian glycolipids in small quantities, and recognized by some pathogens, such as uropathogenic Escherichia coli. In contrast, glycoproteins containing Galα1–4Gal were rarely found in vertebrates except in a few species of birds and amphibians until recently. However, we had previously reported that pigeon (Columba livia) egg white and serum glycoproteins are rich in N-glycans with Galα1–4Gal at non-reducing termini. Our investigation with egg white glycoproteins from 181 avian species also revealed that the distribution of (Galα1–4Gal)-containing glycoproteins was not rare among avians, and is correlated with the phylogeny of birds. The differentiated expression was most likely emerged at earlier stage of diversification of modern birds, but some birds might have lost the facility for the expression relatively recently.