The semidominant mutation Krd (kidney and retinal defects) was identified in transgenic line Tg8052. Krd/+ mice have a high incidence of kidney defects including aplastic, hypoplastic, and cystic kidneys. Retinal defects in Krd/+ mice include abnormal electroretinograms and a reduction of cell numbers that is most extreme in the inner cell and ganglion layers. Viability of Krd/+ mice is strongly influenced by genetic background, and growth retardation is observed in young animals. Homozygosity results in early embryonic lethality. Fluorescence in situ hybridization of a transgene-specific probe localized the insertion site to the distal region of mouse Chromosome 19. The sequence of the insertion site revealed transgene insertion into a LINE element with deletion of a single nucleotide from the 3' terminus of the transgene. A polymorphic microsatellite, D19Umi1, was identified in a junction clone and mapped in several large crosses. D19Umi1 is located 1.7 +/- 1.0 cM distal to Pax2, which encodes a paired type transcription factor expressed in embryonic kidney and eye. Deletion of Pax2 from the transgenic chromosome was demonstrated by Southern analysis of genomic DNA from (Krd/+ x SPRET/Ei)F1 mice. Additional genetic and molecular data are consistent with an approximately 7-cM deletion that includes the loci stearoyl CoA desaturase (Scd1), pale ear (ep), D19Mit17, D19Mit24, D19Mit27, D19Mit11, and Pax2. This deletion, Del(19)TgN8052Mm, will be useful for genetic and functional studies of this region of mouse Chromosome 19.
Transcriptional repressor proteins play essential roles in controlling the correct temporal and spatial patterns of gene expression in Drosophila melanogaster embryogenesis. Repressors such as Knirps, Krüppel, and Snail mediate short-range repression and interact with the dCtBP corepressor. The mechanism by which short-range repressors block transcription is not well understood; therefore, we have undertaken a detailed structure-function analysis of the Knirps protein. To provide a physiological setting for measurement of repression, the activities of endogenous or chimeric Knirps repressor proteins were assayed on integrated reporter genes in transgenic embryos. Two distinct repression functions were identified in Knirps. One repression activity depends on dCtBP binding, and this function maps to a C-terminal region of Knirps that contains a dCtBP binding motif. In addition, an N-terminal region was identified that represses in a CtBP mutant background and does not bind to the dCtBP protein in vitro. Although the dCtBP protein is important for Knirps activity on some genes, one endogenous target of the Knirps protein, the even-skipped stripe 3 enhancer, is not derepressed in a CtBP mutant. These results indicate that Knirps can utilize two different pathways to mediate transcriptional repression and suggest that the phenomenon of short-range repression may be a combination of independent activities.Transcriptional repression is a critical component of genetic regulation during development, and the Drosophila melanogaster embryo has served as an important model for elucidation of basic repression mechanisms (7,19). Differential gene expression in the early embryo is controlled in large part by the activity of repressor proteins encoded by gap, pair-rule, and other genes (42, 45). Repression of transcription can involve reactions occuring off the DNA, such as the formation of inactive heteromeric complexes. Another mechanism involves competition between activators and repressors for binding sites on DNA. DNA-binding repressors that function by mechanisms other than competitive binding have been termed active repressors (24).An active repressor can repress basal promoters or enhancer elements over a short range (Ͻ100 bp) or, alternatively, over long ranges (Ͼ1,000 bp) (7,17). One model of repression in the embryo suggests that the short-range-long-range distinction results from the recruitment of distinct classes of cofactors (36, 55). Short-range repressors may interact with dCtBP, while long-range repressors interact with Groucho.Long-range repressors are typified by the Hairy protein, a transcription factor that binds the Groucho cofactor (5, 27, 40). Long-range repression complexes regulating the dpp, tld, and zen genes also recruit Groucho (7, 27), as do Engrailed, Runt, and dTCF, Drosophila repressors whose range of action has not yet been determined (3, 9, 50).Short-range repressors present in the early Drosophila em-
The regulatory properties of mouse pancreatic amylase genes include exclusive expression in the acinar cells of the pancreas and dependence on insulin and glucocorticoids for maximal expression. We have characterized a murine pancreatic amylase gene, Amy-2.2y, whose promoter sequence is 30% divergent from those of previously sequenced amylase genes. To localize sequences required for tissue-specific and hormone-dependent activation, we established two lines of transgenic mice. The first line contained a single copy of the complete Amy-2.2y gene as well as 9 kilobases of 5'-flanking sequence and 5 kilobases of 3'-flanking sequence. The second line carried a minigene which included 208 base pairs of 5'-flanking sequence and 300 base pairs of 3'-flanking sequence. In both lines the transgene was expressed at high levels exclusively in the pancreas. Both constructs were dependent on insulin and induced by dexamethasone. Thus, the transferred genes contained the sequences required for tissue-specific and hormonally regulated expression.Pancreatic amylase (Amy-2) genes are members of a multigene cluster which also includes the genes for salivary amylase (Amy-i). Although the coding regions of Amy-i and Amy-2 are 90% homologous (21), the promoters associated with each type of gene are dissimilar. Amy-i can be transcribed from two distinct promoters: a strong, parotidspecific promoter located 7 kilobases (kb) upstream of the first coding exon, and a weaker promoter active in liver (52). Each Amy-2 gene, in contrast, is associated with a single promoter adjacent to the structural gene.Amy-2 expression is restricted to the exocrine pancreas, where amylase accounts for 15 to 25% of protein synthesis and mRNA concentration (8, 44). Pancreatic amylase genes have been shown to be hormonally regulated in vivo by insulin and by glucocorticoids. Production of pancreatic amylase is reduced in human diabetic patients (11), and amylase mRNA is greatly reduced in diabetic rats and mice (14,29). Administration of insulin restores amylase expression in these diabetic animals. Induction of pancreatic amylase by glucocorticoids has been demonstrated in neonatal mice (48) and in cultured cells (13,31).Recent studies have identified gene regions which appear to be required for pancreas-specific gene expression. identified a conserved sequence element upstream of five genes which are expressed specifically in the exocrine pancreas. Deletion of this element resulted in loss of pancreatic cell specificity in transfection assays (9). This sequence is therefore a likely candidate for mediation of pancreasspecific expression in vivo.
We localized the c/s-acting sequences that mediate the regulation of a pancreatic amylase gene, Amy-2.2, in diabetic mice. We constructed three hybrid genes containing sequences from the 5'-flanking region of the amylase gene upstream of the heterologous elastase promoter linked to the CAT structural gene. These constructs were transferred to the germ line of transgenic mice by microinjection of fertilized eggs. The amylase sequences had two effects on expression of the elastase promoter: Basal expression was increased, and expression in diabetic animals was reduced to -~2% of basal levels. A 30-bp amylase fragment was sufficient to transfer both of these regulatory functions to the elastase promoter. Sequences within this 30-bp fragment are included in the binding site for the pancreatic nuclear protein PTFI. The close association of the PTFl-binding site and the regulatory functions is consistent with a mechanism based on interference with activation by PTFI in diabetic pancreas. PTFl-binding activity is not reduced in diabetic pancreas. The data presented here demonstrate that the 5'-flanking region of the pancreatic amylase gene contains a novel insulin-dependent element (IDE) that mediates the loss of expression in diabetic animals.
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