Taghert P H, Schneider L E
Department of Anatomy and Neurobiology, Washington University School of Medicine, Saint Louis, Missouri 63110.
J Neurosci. 1990 Jun;10(6):1929-42. doi: 10.1523/JNEUROSCI.10-06-01929.1990.
In order to identify functionally important regions of a neuropeptide gene in Drosophila melanogaster, we have studied its occurrence in related species and have characterized the structure of a homologous gene in Drosophila virilis. The melanogaster gene encodes a precursor that contains 13 neuropeptides related to the molluscan tetrapeptide FMRFamide (Nambu et al., 1988; Schneider and Taghert, 1988). Using the melanogaster gene as a probe in Southern blot analysis, related sequences were detected in DNA from each of 7 species tested. D. virilis, which is estimated to have diverged from D. melanogaster between 60 and 80 million years ago (Throckmorton, 1975), was chosen for more detailed study. Immunocytochemical staining using an antibody to authentic FMRFamide revealed a similar set of immunoreactive neurons in the CNS of larvae from the 2 Drosophila species. Using a melanogaster gene probe, overlapping clones were isolated from a virilis genomic library; DNA sequence analysis indicated the presence of a homologous gene. Comparisons of the genes and deduced proteins between the 2 species revealed the following points. (1) Both genes are divided into 2 exons: in D. melanogaster the exons are 106 and 1352 bp long; in D. virilis, they are 169 and at least 1232 bp long; in both species, the intron is approximately 2.5 kb long. (2) The sequence of exon I has largely diverged, and in neither species are exon I sequences translated. In this vicinity of the gene, sequence conservation is limited to a 67 bp region that spans the TATA box and the RNA start site. (3) The deduced neuropeptide precursors have very similar sizes (347 vs 339 amino acids) and the presumed signal sequences are perfectly conserved. (4) While the melanogaster precursor contains 13 FMRFamide-related peptides, the virilis precursor contains only 10. (5) The sequences of some but not all of the FMRFamide-like peptides are perfectly conserved. (6) In the rest of the precursor, significant sequence conservation is found only in the N-terminal portion; immediately downstream of the final FMRFamide-like peptide, the protein sequences are highly divergent. (7) 5' to the RNA start sites (1.2 kb of melanogaster DNA and 1.8 kb of virilis DNA), 17 small (9-52 base pairs) regions are evolutionarily conserved (greater than 80% sequence conservation). We discuss neuropeptide biosynthesis, the functions and evolution of FMRFamide-like neuropeptides in insects, and the cell-specific regulation of neuropeptide gene expression in the contexts of these results.
为了鉴定黑腹果蝇中一种神经肽基因的功能重要区域，我们研究了它在相关物种中的存在情况，并对粗壮果蝇中同源基因的结构进行了表征。黑腹果蝇基因编码一种前体，该前体包含13种与软体动物四肽FMRF酰胺相关的神经肽（Nambu等人，1988年；Schneider和Taghert，1988年）。在Southern印迹分析中使用黑腹果蝇基因作为探针，在测试的7个物种的DNA中均检测到相关序列。粗壮果蝇估计在6000万至8000万年前与黑腹果蝇分化（Throckmorton，1975年），因此被选作更详细的研究对象。使用针对 authentic FMRF酰胺的抗体进行免疫细胞化学染色，发现在这两种果蝇幼虫的中枢神经系统中有一组相似的免疫反应性神经元。使用黑腹果蝇基因探针，从粗壮果蝇基因组文库中分离出重叠克隆；DNA序列分析表明存在同源基因。对这两个物种的基因和推导的蛋白质进行比较，发现了以下几点。（1）两个基因均分为2个外显子：在黑腹果蝇中，外显子长度分别为106和1352 bp；在粗壮果蝇中，它们分别为169和至少1232 bp；在这两个物种中，内含子长度约为2.5 kb。（2）外显子I的序列有很大差异，并且在这两个物种中，外显子I序列均不被翻译。在该基因的这一区域附近，序列保守性仅限于跨越TATA盒和RNA起始位点的67 bp区域。（3）推导的神经肽前体大小非常相似（347对339个氨基酸），并且推测的信号序列完全保守。（4）虽然黑腹果蝇前体包含13种与FMRF酰胺相关的肽，但粗壮果蝇前体仅包含10种。（5）一些但不是所有的FMRF酰胺样肽的序列完全保守。（6）在前体的其余部分，仅在N端部分发现了显著的序列保守性；在最后一个FMRF酰胺样肽的紧邻下游，蛋白质序列高度发散。（7）在RNA起始位点的5'端（黑腹果蝇DNA的1.2 kb和粗壮果蝇DNA的1.8 kb），17个小区域（9 - 52个碱基对）在进化上保守（序列保守性大于80%）。我们结合这些结果讨论了神经肽生物合成、昆虫中FMRF酰胺样神经肽的功能和进化以及神经肽基因表达的细胞特异性调控。
