The molecular evolution of the alcohol dehydrogenase and alcohol dehydrogenase-related genes in the Drosophila melanogaster species subgroup

The DNA sequences of the Adh genes of three members of the Drosophila melanogaster species subgroup have been determined. This completes the Adh sequences of the eight species of this subgroup. Two species, D. yakuba and D. teissieri, possess processed Adh pseudogenes. In all of the species of the subgroup, a gene of unknown function, Adhr, is located about 300 bp 3' to Adh. Although this gene is experiencing a higher rate of synonymous substitution than Adh, it is more constrained at the amino acid level. Phylogenetic relationships between all eight members of the melanogaster subgroup have been analyzed using a variety of methods. All analyses suggested that the D. yakuba and D. teissieri pseudogenes have a single common ancestor, rather than evolving independently in each species, and that D. melanogaster is the sister species to D. simulans, D. sechellia, and D. mauritiana. The evolutionary relationships of the latter three species remain equivocal.      

Isolation and characterization of the genomic region from Drosophila kuntzei containing the Adh and Adhr genes

The nucleotide sequences of the Adh and Adhr genes of Drosophila kuntzei were derived from combined overlapping sequences of clones isolated from a genomic library and from cloned PCR and inverse-PCR fragments. Only a proximal promoter was detected upstream of the Adh gene, indicating that D. kuntzei Adh is regulated by a one-promoter system. Further upstream of the Adh structural gene, an adult enhancer region (AAE) was found that contains most of the regulatory sequences described for AAEs of other Drosophila species. Analysis of the ADH protein showed an amino acid change from valine to threonine in the active site at position 189 which is also found in D. funebris but is otherwise unique among DROSOPHILA: This difference alone may be responsible for the very low ADH activity found in this species and may cause a difference in substrate usage pattern. Codon bias in Adh and Adhr was comparable and found to be very low compared with other species. Phylogenetic analysis showed that D. kuntzei is closest related to D. funebris and D. immigrans. The time of divergence between D. kuntzei and D. funebris was estimated to be 14.2-20.2 Myr and that between D. kuntzei-D. funebris and D. immigrans to be 30.8-44.0 Myr. An analysis of the genetic variation in the Adh gene and upstream sequences of four European strains showed that this gene was highly variable. Overall nucleotide diversity (pi) was 0.0139, which is two times higher than that in D. melanogaster.     

Organization and evolution of the alcohol dehydrogenase gene in Drosophila

The alcohol dehydrogenase (Adh) gene was isolated from Drosophila simulans and D. mauritiana, and the DNA sequence of a 4.6-kb region, containing the structural gene and flanking sequence, was determined for each. These sequences were compared with the Adh region of D. melanogaster to characterize changes that occur in the Drosophila genome during evolution and to identify conserved sequences of functional importance. Drosophila simulans and D. mauritiana Adh are organized in a manner similar to that of D. melanogaster Adh, including the presence of two promoters for the single Adh gene. This study identified conserved flanking elements that, in conjunction with other studies, suggest regions that may be involved in the control of Adh expression. Inter- and intraspecies comparisons revealed differences in the kinds of sequence changes that have accumulated. Sequence divergence in and around the Adh gene was used to assess inter- and intraspecies evolutionary relationships. Finally, there appears to be an unrelated structural gene located directly 3' of the Adh transcribed region.      

The structure of the Adh locus of Drosophila mettleri: an intermediate in the evolution of the Adh locus in the repleta group of Drosophila.

Members of species of the mulleri and hydei subgroups of the repleta group of Drosophila have duplicate Adh genes. The Adh regions of D. mojavensis, D. mulleri, and D. hydei contain three genes--a pseudogene, Adh-2, and Adh-1--arranged 5' to 3'. To understand the evolution of the triplicate Adh structure, we have cloned and sequenced the Adh locus of D. mettleri. This region consists of a 5' pseudogene and a 3' functional Adh gene. On the basis of the structure and nucleotide sequence comparisons of Adh genes of D. mettleri and other species, we propose that an initial duplication of the ancestral Adh gene generated two Adh genes arranged in tandem. The more 5' Adh gene became a pseudogene, while the more 3' gene remained functional through all the developmental stages. A second duplication of this 3' gene resulted in Adh regions with three genes--a pseudogene, Adh-2, and Adh-1.     

Deletion of a conserved regulatory element in the Drosophila Adh gene leads to increased alcohol dehydrogenase activity but also delays development

In vivo levels of enzymatic activity may be increased through either structural or regulatory changes. Here we use Drosophila melanogaster alcohol dehydrogenase (ADH) in an experimental test for selective differences between these two mechanisms. The well-known ADH-Slow (S)/Fast (F) amino acid replacement leads to a twofold increase in activity by increasing the catalytic efficiency of the enzyme. Disruption of a highly conserved, negative regulatory element in the Adh 3' UTR also leads to a twofold increase in activity, although this is achieved by increasing in vivo Adh mRNA and protein concentrations. These two changes appear to be under different types of selection, with positive selection favoring the amino acid replacement and purifying selection maintaining the 3' UTR sequence. Using transgenic experiments we show that deletion of the conserved 3' UTR element increases adult and larval Adh expression in both the ADH-F and ADH-S genetic backgrounds. However, the 3' UTR deletion also leads to a significant increase in developmental time in both backgrounds. ADH allozyme type has no detectable effect on development. These results demonstrate a negative fitness effect associated with Adh overexpression. This provides a mechanism whereby natural selection can discriminate between alternative pathways of increasing enzymatic activity.     

麻竹miR172a靶基因DlAP2的克隆及其表达

为了解开花麻竹(Dendrocalamus latiflorus)的Dl AP2基因功能,采用RT-PCR和RACE技术克隆了mi R172a靶基因AP2同源序列c DNA全长,命名为Dl AP2。结果表明,Dl AP2基因c DNA全长为1729 bp,包含5′端非编码区81 bp、开放阅读框1464 bp、3′端非编码区160 bp和24个碱基的Poly A尾巴,在编码框靠近3′端130 bp处有1个高度匹配mi R172a的结合位点(CTGCAGCATCATCAGGATTCT)。Dl AP2编码487个氨基酸的蛋白,具有两个AP2结构域,属于AP2/ERF家族AP2亚家族的AP2组,与来自其它单子叶植物的AP2蛋白均有较高同源性。RLM-5′RACE分析表明,mi R172a主要在靶序列的第11~12个碱基之间剪切靶基因Dl AP2的mi RNA。q RT-PCR结果表明,麻竹花芽中Dl AP2基因的表达规律与mi R172a表达变化正好相反,证明mi R172a对Dl AP2基因的表达具有调控作用。     

Structure and Evolution of the Adh Genes of Drosophila Mojavensis

The nucleotide sequence of the Adh region of Drosophila mojavensis has been completed and the region found to contain a pseudogene, Adh-2 and Adh-1 arranged in that order. Comparison of the sequence divergence of these genes to one another and to the Adh region of Drosophila mulleri and other species has allowed the development of a model for the evolution of the duplication of the Adh genes. There have been two major events. An initial duplication of an Adh gene whose dual promoter structure was similar to Drosophila melanogaster, resulted in a species with two Adh genes, one of which may have had only a proximal promoter. A second duplication of this gene generated an Adh region containing three genes. It is proposed that one of these is the ancestral gene having dual promoters, while the other two possess only proximal promoters. Subsequent events have resulted in both a change in the regulation of Adh-2 such that it is expressed as if it had a "distal" type promoter and the mutational inactivation of the most upstream gene resulting in the creation of a pseudogene. The sequence of the D. mojavensis Adh region has also revealed the presence of an element which is composed of juxtaposed inverted imperfectly repeated elements. There is a surprising and not fully explainable strong similarity of the nucleotide sequence of the 5' flanking region of the pseudogene in D. mojavensis and D. mulleri.     

Excess Polymorphism at the Adh Locus in DROSOPHILA MELANOGASTER

The evolutionary history of a region of DNA encompassing the Adh locus is studied by comparing patterns of variation in Drosophila melanogaster and its sibling species, D. simulans. An unexpectedly high level of silent polymorphism in the Adh coding region relative to the 5' and 3' flanking regions in D. melanogaster is revealed by a populational survey of restriction polymorphism using a four-cutter filter hybridization technique as well as by direct sequence comparisons. In both of these studies, a region of the Adh gene encompassing the three coding exons exhibits a frequency of polymorphism equal to that of a 4-kb 5' flanking region. In contrast, an interspecific sequence comparison shows a two-fold higher level of divergence in the 5' flanking sequence compared to the structural locus. Analysis of the patterns of variation suggest an excess of polymorphism within the D. melanogaster Adh locus, rather than lack of polymorphism in the 5' flanking region. An approach is outlined for testing neutral theory predictions about patterns of variation within and between species. This approach indicates that the observed patterns of variation are incompatible with an infinite site neutral model.     

Isolation and characterisation of a sucrose: sucrose 1-fructosyltransferase gene from perennial ryegrass (Lolium perenne)

A sucrose: sucrose 1-fructosyltransferase (1-SST) gene and cDNA (Lp 1-SST) from perennial ryegrass (Lolium perenne) were isolated. The Lp 1-SST gene was fully sequenced and shown to contain three exons and two introns. Nucleotide sequence analysis of the 4824 bp Lp 1-SST genomic sequence revealed 1618 bp of 5' UTR and an open reading frame of 1962 bp encoding a protein of 653 amino acids. Lp 1-SST is 95% identical to the tall fescue 1-SST and contains plant fructosyltransferase functional domains. Lp 1-SST corresponds to a single copy gene in perennial ryegrass, and is expressed in young leaf bases and mature leaf sheaths. The recombinant Lp 1-SST protein from corresponding cDNA expression in Pichia pastoris showed 1-SST activity.