The Drosophila virilis dopa decarboxylase gene is developmentally regulated when integrated into Drosophila melanogaster.

The dopa decarboxylase gene (Ddc) has been isolated from Drosophila virilis and introduced into the germ-line of Drosophila melanogaster by P-element mediated transformation. The integrated gene is induced at the correct stages during development with apparently normal tissue specificity, indicating that cis-acting elements required for regulation are functionally conserved between the two species. A comparison of the DNA sequences from the 5' flanking regions reveals a cluster of small (8-16 bp) conserved sequence elements within 150 bp upstream of the RNA startpoint, a region required for normal expression of the D. melanogaster Ddc gene.     

果蝇中顺式调控序列的进化

顺式调控序列(cis-regularory sequences)是与基因表达调控相关、能够被调控因子特异性识别和结合的非编码DNA序列。顺式调控序列可以通过增减所含转录因子结合位点的数目,重构转录调控网络,以精准调控基因的时空表达模式,从而调控动物的生理和形态表型。顺式调控假说认为顺式调控序列进化是自然界丰富的动物表型进化的主要遗传机制。本文首先简述了顺式调控序列的结构和功能,然后重点讨论了近年来顺式调控序列进化调控果蝇表型进化如刚毛表型进化、色素沉积表型进化和胚胎发育方面的研究进展,阐释了顺式调控序列进化是动物表型进化的主要遗传机制之一。最后本文展望了顺式调控序列未来研究方向,例如应用功能基因组研究、开展ENCODE计划等,为进化发育生物学中顺式调控序列的研究提供了参考。     

The genetics of dopa decarboxylase in Drosophila melanogaster

Of 204 mutations located in the 8–12 band Df(2L)130 region, 37B9-C1,2;37D1-2, 199 have been assigned to twelve lethal genes and one visible gene (hook). The 13 genes are not evenly distributed. Twelve, (possibly all thirteen) are in the seven band region 37B10-C4 giving a gene-to-band ratio of almost two. Only one gene, 1(2)37Cf, may be in the four band region 37C5-7, and none are localized in band 37D1. In situ hybridization places the dopa decarboxylase structural gene, Ddc, in or very close to band 37C1,2 (Hirsh and Davidson, 1981). The methyl dopa hypersensitive gene, 1(2) amd, is 0.002 map units distal to Ddc. Df(2L)VA17, 37C1,2; 37F5-38A1 may actually break in the 37C1,2 singlet. It places six genes, hook, 1(2)amd, and four lethal genes, in a maximum of five bands, 37B10, 11, 12, 13 and perhaps part of the 37C1,2 singlet and localizes six genes, Ddc plus five lethal genes, in a maximum of three bands; probably part of the 37C1,2 singlet plus bands, C3, and C4. Wild type activity of five of twelve lethal genes is necessary for female fertility. — Band 37C5 puffs at the time of pupariation; Puff Stages 8–10. Twelve of eighteen alleles of 1(2)37Cf havs been examined as heterozygotes over CyO and none affect the appearance of a homozygous 37C5 puff. — Of the 204 mutations considered here only one Ddc ^p1, affects the function of more than one gene. It eliminates Ddc ⁺ and l(2) 37Ca ⁺ function and at 30 ° C reduces l(2)37Ce ⁺ function. It is not a deficiency but could be a polar mutant.Prof. Beermann's co-authors are very pleased to dedicate this paper to him in honor of his sixtieth birthday and in recognition of his seminal, most significant, extensive, and authoritive contributions on the functional organization of chromosomes     

A kinetic analysis of Drosophila melanogaster dopa decarboxylase

The kinetic mechanism of dopa decarboxylase (3,4-dihydroxy-L-phenylalanine carboxy-lyase, EC 4.1.1.28) was investigated in Drosophila melanogaster. Based on initial velocity and product inhibition studies, an ordered reaction is proposed for dopa decarboxylase. This kinetic mechanism is interpreted in the context of measured enzyme activities and the catecholamine pools in Drosophila. The 1(2)amd gene is immediately adjacent to the gene coding for dopa decarboxylase (Ddc) and determines hypersensitivity to alpha-methyldopa in Drosophila. Dopa decarboxylase does not decarboxylate alpha-methyldopa and hence does not generate a toxic product capable of inhibiting 1(2)amd gene function. We propose that the 1(2)amd gene is involved with an unknown catecholamine pathway involving dopa but not dopamine.     

The cloned dopa decarboxylase gene is developmentally regulated when reintegrated into the Drosophila genome

The Drosophila dopa decarboxylase gene, Ddc, functions normally when reintroduced into flies. DNA containing a cloned Ddc gene inserted into a P element transposon was injected into early embryos. Transformants were identified by suppression of the cuticular phenotype of a Ddc mutant allele. The reintegrated genes are expressed in the proper tissue and at the proper stages during development even though their positions within the genome are different from that of the wild-type Ddc gene. Absolute levels of DDC enzyme activity are within 35% of that found in wild-type Canton S flies, the source of the transforming DNA. The transformants' Ddc RNA is indistinguishable from that of wild type. One reintegrated Ddc gene, inserted on the X chromosome, is affected by the dosage compensation mechanism that leads to sex-specific differences in the expression of many X-chromosome genes.     

A cis-acting element and associated binding factor required for CNS expression of the Drosophila melanogaster dopa decarboxylase gene.

A cis-acting sequence from the Drosophila melanogaster dopa decarboxylase (Ddc) gene is selectively required for Ddc expression in the central nervous system. We analyze several parameters influencing the function of the sequence element and describe a factor which interacts with it and mediates CNS expression of Ddc. The element, element I, can function in vivo when included on a synthetic oligonucleotide inserted near its normal location, or closer to the RNA startpoint. It displays partial activity when inverted. Two different 2-bp mutations in element I abolish its ability to stimulate neuronal Ddc expression in the CNS. A factor present in embryonic nuclear extracts specifically protects element I in DNase I footprinting assays. The binding affinity of this factor is reduced by each alteration of element I that inhibits neuronal expression, indicating a role in mediating CNS expression of Ddc. Element I alone has no detectable activity when placed adjacent to a heterologous promoter, although 2.2 kb of 5' Ddc sequences direct correct cell-specific expression of a heterologous promoter.     

The genetics of dopa decarboxylase in Drosophila melanogaster. III. Effects of a temperature sensitive dopa decarboxylase deficient mutation on female fertility

The mutation Ddc^ts1 effects female sterility when homozygous, hemizygous, or heterozygous over a series of Ddc null alleles (Ddc^x) indicating that some aspect of Ddc gene function is necessary for female fertility. Ovary transplant experiments demonstrate that the female sterility phenotype is ovary autonomous. Two to 3% of the total DDC activity measurable in newly hatched females is localized in their previtellogenic ovaries. The degree to which females heterozygous for Ddc^ts1 over different Ddc null alleles are fertile at 22°C reflects a continuous spectrum of allelic complementation similar to that observed for the effects of these genotypes on viability at 30°C. Fertility of all the Ddc^ts1/Ddc^x females tested is significantly depressed at 30 vis-a-vis 22°C providing evidence that it is the DDC enzyme activity itself which is required for female fertility. Ddc^ts1/Ddc^ts1 homozygous and Ddc^ts1/Df hemizygous females are nonconditionally, completely sterile at 18, 20, 22, 25, and 30°C. Although all homo- and hemizygous females do lay some eggs, no evidence of embryogenesis or fertilization has ever been detected. The absolute, nonconditional sterility of Ddc^ts1 homo- and hemizygous females stands in stark contrast to the conventional temperature dependent effects of these same genotypes on viability and to the temperature sensitive effects of Ddc^ts1/Ddc^x heterozygous females on both fertility and viability. Reasons for these tissue-specific and genotypic differences are discussed.