Insect immunity. A transgenic analysis in Drosophila defines several functional domains in the diptericin promoter.

Diptericins are antibacterial polypeptides which are strongly induced in the fat body and blood cells of dipteran insects in response to septic injury. The promoter of the single-copy, intronless diptericin gene of Drosophila contains several nucleotide sequences homologous to mammalian cis-regulatory motifs involved in the control of acute phase response genes. Extending our previous studies on the expression of the diptericin gene, we now report a quantitative analysis of the contribution of various putative regulatory elements to the bacterial inducibility of this gene, based on the generation of 60 transgenic fly lines carrying different elements fused to a reporter gene. Our data definitively identify two Kappa B-related motifs in the proximal promoter as the sites conferring inducibility and tissue-specific expression to the diptericin gene. These motifs alone, however, mediate only minimal levels of expression. Additional proximal regulatory elements are necessary to attain some 20% of the full response and we suspect a role for sequences homologous to mammalian IL6 response elements and interferon-gamma responsive sites in this up-regulation. The transgenic experiments also reveal the existence of a distal regulatory element located upstream of -0.6 kb which increases the level of expression by a factor of five.     

Complex CIS-Acting Regulators and Locus Structure of Drosophila Tissue-Specific Adh Variants

Diverse patterns of tissue-specific expression of alcohol dehydrogenase (ADH) among species of the grimshawi subgroup of Hawaiian picture-winged Drosophila suggests control by complex or multiple, independently acting regulatory elements. These elements act by controlling Adh mRNA accumulation in individual tissue types. Restriction mapping of the Adh loci from these species reveals several insertion/deletion differences, one of which lies just outside the 5' end of the structural sequences and correlates with differences in larval patterns of ADH expression. No tissue-specific rearrangement of Adh sequences was observed.     

Tissue-Specific Expression Phenotypes of Hawaiian Drosophila Adh Genes in Drosophila Melanogaster Transformants

Interspecific differences in the tissue-specific patterns of expression displayed by the alcohol dehydrogenase (Adh) genes within the Hawaiian picture-winged Drosophila represent a rich source of evolutionary variation in gene regulation. Study of the cis-acting elements responsible for regulatory differences between Adh genes from various species is greatly facilitated by analyzing the behavior of the different Adh genes in a homogeneous background. Accordingly, the Adh gene from Drosophila grimshawi was introduced into the germ line of Drosophila melanogaster by means of P element-mediated transformation, and transformants carrying this gene were compared to transformants carrying the Adh genes from Drosophila affinidisjuncta and Drosophila hawaiiensis. The results indicate that the D. affinidisjuncta and D. grimshawi genes have relatively higher levels of expression and broader tissue distribution of expression than the D. hawaiiensis gene in larvae. All three genes are expressed at similar overall levels in adults, with differences in tissue distribution of enzyme activity corresponding to the pattern in the donor species. However, certain systematic differences between Adh gene expression in transformants and in the Hawaiian Drosophila are noted along with tissue-specific position effects in some cases. The implications of these findings for the understanding of evolved regulatory variation are discussed.     

Complexity in evolved regulatory variation for alcohol dehydrogenase genes in HawaiianDrosophila

Summary The alcohol dehydrogenase gene (Adh gene) ofDrosophila affinidisjuncta is expressed at a higher level in the larval midgut and Malpighian tubules than the homologous gene fromDrosophila hawaiiensis. This study analyzed thecis-acting sequences responsible for these regulatory differences in larval tissues ofDrosophila melanogaster transformants. A series of 10 chimeric and deletedAdh genes was introduced into the germ line ofD. melanogaster, and tissue-specific expression levels were quantified by gel electrophoresis of tissue extracts. Sequences in the upstream region of the two genes had the strongest influence on enzyme production in the midgut and Malpighian tubules. Other sequence elements also showed effects, some of which were tissue specific. Most gene fragments displayed context-dependent effects, thus supporting the proposed model of polygenic regulation ofAdh gene expression.     

水稻胚乳特异表达基因的挖掘及顺式元件分析

本研究旨在利用计算机方法对水稻胚乳特异性表达基因进行挖掘和功能分析,及特异性顺式调控元件的预测。我们将基因在不同组织中的表达信号谱看作多维空间内的向量,利用向量夹角余弦法计算其与理想状态下该基因在某一组织特异表达向量的相似度,以此来判断组织特异性表达基因。本文通过对水稻芯片数据的大规模分析,共挖掘出了127个在水稻胚乳中特异表达基因。并对其启动子进行顺式元件预测,发现两个与胚乳特异表达相关的顺式调控元件,其保守序列分别为CATGCATSCM和GATCGATCGR。与已知功能的顺式元件比较显示,前者为种子特异基因表达相关的RY repeat元件,而后者则与元件RNFG1相似,但其具体功能尚不明确。     

Induction and repression of the Drosophila Sgs-3 glue gene are mediated by distinct sequences in the proximal promoter.

The normal developmental expression of the Drosophila salivary gland secretion protein gene Sgs-3 requires the interaction of a distal and proximal regulatory element. A deletion/replacement analysis of the proximal promoter in stably transformed lines shows that induction of an Sgs-3/Adh fusion gene is normal if sequences from +10 to -50 are replaced by those of the hsp70 gene. Sequences between -98 and -50 are necessary for this expression but there is internal redundancy within this region as two distinct upstream sequences of 18 and 22 bp respectively are sufficient for stage- and tissue-specific expression, albeit at reduced levels. A point mutation at -53 eliminates the ecdysone-mediated repression of the Sgs-3 promoter at pupariation. We report mosaicisms of expression within the salivary gland for a number of stably transformed lines.     

Molecular Dissection of the 5' Region of no-on-transientA of Drosophila melanogaster Reveals cis-Regulation by Adjacent dGpi1 Sequences

The nonA gene of Drosophila melanogaster is important for normal vision, courtship song, and viability and lies approximately 350 bp downstream of the dGpi1 gene. Full rescue of nonA mutant phenotypes can be achieved by transformation with a genomic clone that carries approximately 2 kb of 5' regulatory material and that encodes most of the coding sequence of dGpi1. We have analyzed this 5' region by making a series of deleted fragments, fusing them to yeast GAL4 sequences, and driving UAS-nonA expression in a mutant nonA background. Regions that both silence and enhance developmental tissue-specific expression of nonA and that are necessary for generating optomotor visual responses are identified. Some of these overlap the dGpi1 sequences, revealing cis-regulation by neighboring gene sequences. The largest 5' fragment was unable to rescue the normal electroretinogram (ERG) consistently, and no rescue at all was observed for the courtship song phenotype. We suggest that sequences within the nonA introns that were missing in the UAS-nonA cDNA may carry enhancer elements for these two phenotypes. Finally, we speculate on the striking observation that some of the cis-regulatory regions of nonA appear to be embedded within the coding regions of dGpi1.     

Different cis-Regulatory DNA Elements Mediate Developmental Stage- and Tissue-specific Expression of the Human COL2A1 Gene in Transgenic Mice

Expression of the type II collagen gene (human COL2A1, mouse Col2a1) heralds the differentiation of chondrocytes. It is also expressed in progenitor cells of some nonchondrogenic tissues during embryogenesis. DNA sequences in the 5′ flanking region and intron 1 are known to control tissue-specific expression in vitro, but the regulation of COL2A1 expression in vivo is not clearly understood. We have tested the regulatory activity of DNA sequences from COL2A1 on the expression of a lacZ reporter gene in transgenic mice. We have found that type II collagen characteristic expression of the transgene requires the enhancer activity of a 309-bp fragment (+2,388 to +2,696) in intron 1 in conjunction with 6.1-kb 5′ sequences. Different regulatory elements were found in the 1.6-kb region (+701 to +2,387) of intron 1 which only needs 90-bp 5′ sequences for tissue-specific expression in different components of the developing cartilaginous skeleton. Distinct positive and negative regulatory elements act together to control tissue-specific transgene expression in the developing midbrain neuroepithelium. Positive elements affecting expression in the midbrain were found in the region from −90 to −1,500 and from +701 to +2,387, whereas negatively acting elements were detected in the regions from −1,500 to −6,100 and +2,388 to +2,855.     

Unexpected position-dependent expression of H-2 and beta 2-microglobulin/lacZ transgenes.

In order to study sequences involved in the developmentally regulated and tissue-specific expression of the class I Major Histocompatibility Complex (MHC) genes, we have constructed several H-2/lacZ transgenic lines in which the 5' regulatory sequences of the H-2Kb gene are linked to the Escherichia coli beta-galactosidase (lacZ) gene. In five H-2/lacZ lines, the pattern of lacZ expression, detected histochemically varied greatly from line to line. None of the H-2/lacZ transgenes were transcribed in cells normally expressing a high level of endogenous H-2 molecules, although these H-2 regulatory sequences have been shown to be sufficient to drive tissue-specific expression of other reporter genes. Interestingly, when constructs containing 5' beta 2-microglobulin (beta 2m) regulatory sequences linked to lacZ were used to derive transgenic lines, similar results were obtained. A survey of lacZ labeling in H-2/lacZ and beta 2m/lacZ transgenic mice strongly suggests that these transgenes are very sensitive to position effect, lacZ expression being controlled by endogenous chromosomal regulatory elements specific for each insertion site. Here we describe the complex pattern of lacZ expression in the different transgenic lines during development; we discuss the unusual properties of these transgenes and underline their potential use for developmental studies and characterization of genomic sequences involved in spatiotemporal gene expression.     

The effect of regulatory sequences of alphaS1-casein gene on the expression of the lacZ-gene in loach Misgurnus fossilis L. transgenic embryos

Transient expression of recombinant plasmids carrying the lacZ gene under the control of either bovine alphaS1-casein gene tissue-specific promoter-enhancer region or highly homologous goat alphaS1-casein gene promoter-enhancer region with supplementary regulatory sequences of the goat gene were studied in Misgurnus fossilis L. loach embryos. It has been shown previously that the expression of the constructs carrying these regulatory elements in transgenic mice occurred primarily in the mammary glands. At early developmental stages, loach embryos and early prelarvae showed nonspecific and mosaic transient expression of lacZ carrying casein regulatory sequences. Transgenic activity was the highest in 1-3-day embryos. At the same time, the efficiency of expression of lacZ gene carrying regulatory sequences of the alphaS1-casein gene of goat was higher than with the promoter-enhancer region of the bovine alphaS1-casein gene. Thus, regulatory sequences of the bovine or goat alphaS1-casein gene appeared capable of providing similar transient expression of reporter gene in the loach embryos. This model can be used for rapid testing of promoter-enhancer activity of transgenes.