Divergent structure and function of the bicoid gene in Muscoidea fly species

SUMMARY We have investigated the evolution of the bicoid ( bcd ) gene in fly species of the Muscoidea Superfamily. We obtained the complete bcd sequence from the housefly Musca domestica and found polymorphism in the coding region among Musca strains. In addition to Musca , we cloned most of the bcd coding sequences from two blowfly species Calliphora vicina and Lucilia sericata . The 5' and 3' regulatory regions flanking the Musca bcd gene are widely diverged in sequence from Drosophila; however, some important sequence motifs identified in Drosophila bcd are present. The predicted RNA secondary structures of the 3' UTRs are similar, despite sequence divergence. Comparison of Bicoid (Bcd) proteins shows a serine-rich domain of unknown function is present in the Muscoidea species, but is absent in other species. The in vivo function of bcd in Musca was tested by RNAi to mimic loss of function phenotype. We obtained a head defect phenotype similar to weak bcd alleles of Drosophila . Although our comparisons initially suggest functional conservation between species, closer inspection reveals significant differences. Divergence of structural motifs, such as regulatory elements in flanking regions and conservation of protein domains in some species but not in others, points to functional divergence between species. We suggest that the larger embryonic size in Muscoidea species restricts the morphogenetic activity of a weak Bcd activator, which has evolved a more specialized role in head determination and lost some functions in thoracic development.     

The Azorhizobium caulinodans nitrogen-fixation regulatory gene, nifA, is controlled by the cellular nitrogen and oxygen status

The nucleotide sequence of the Azorhizobium caulinodans ORS571 nifA locus was determined and the deduced NifA amino acid sequence compared with that of NifA from other nitrogen-fixing species. Highly conserved domains, including helix-turn-helix and ATP-binding motifs, and specific conserved residues, such as a cluster of cysteines, were identified. The nifA 5' upstream region was found to contain DNA sequence motifs highly homologous to promoter elements involved in nifA/ntr-mediated control and a consensus element found in the 5' upstream region of the Bradyrhizobium japonicum 5-aminolevulinic acid synthase (hemA) gene and of Escherichia coli genes activated during anaerobiosis via the fnr (fumarate nitrate reduction) control system. A nifA-lac fusion was constructed using miniMu-lac and its activity measured in different genetic backgrounds and under various physiological conditions (in culture and in planta). NifA expression was found to be negatively autoregulated, repressed by rich nitrogen sources and high oxygen concentrations, and controlled (partially) by the ntrC gene, both in culture and in planta. DNA supercoiling was also implicated in nifA regulation, since DNA gyrase inhibitors severely repressed nifA-lac expression.     

Nucleotide variation at the no-on-transient A gene in Drosophila littoralis

The no-on-transient A (nonA) gene encodes a putative RNA-binding protein, and mutations in this gene are known to affect vision, male courtship song and viability in Drosophila melanogaster. Here we have sequenced the coding region of the nonA gene of Drosophila littoralis and compared it with those of Drosophila virilis and D. melanogaster. All portions of nonA appeared to be conserved between D. littoralis and D. virilis, while the 5' region of the gene of these two species showed high divergence from that of a more distantly-related species, D. melanogaster. The same was true for the glycine repeat regions. No significant deviation from neutrality was observed in the analysis of intraspecific nucleotide variation in 5' or 3' region of the nonA gene in D. littoralis population. Also, comparison of D. littoralis sequences with homologous sequence of D. virilis suggests that the gene is evolving neutrally in D. virilis group. Divergence of the 5' regions between D. virilis group species and D. melanogaster could be a result of positive selection, but this finding is obscured by the long divergence time of the species groups.     

山葡萄几丁质酶基因VCH3启动子的分离及鉴定

利用接头PCR技术首次分离了长度为1 216bp的"双优"山葡萄(Vitis amurensis Rupr.)classⅢ几丁质酶基因VCH3上游启动子序列(GenBank登录号AF441123),并用引物延伸方法鉴定了该启动子的转录起始位点,为5'-ATCAAGCAC-31序列中的第二个A.序列分析结果表明,代表真核基因启动子特征的CAAT盒和TATA盒分别位于VCH3启动子转录起始位点上游-122和-29处.另外,在转录起始位点上游-1 181 bp和-293 bp处各有一个水杨酸(SA)响应的顺式作用元件TGACG.为了鉴定该启动子的功能,将该启动子连接到β-葡糖苷酸酶基因(GUS)编码区的上游构建了VCH3启动子-GUS融合基因,并用农杆菌介导叶盘转化法将该融合基因转入烟草栽培品种NC89中.SA处理的转基因烟草根系和叶片GUS酶活性的荧光和组织化学检测结果表明VCH3启动子的驱动作用被SA诱导,因而该启动子在基因工程中将具有潜在的应用价值.     

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.     

Cooperative enhancement at the Drosophila Sgs-3 locus

The Drosophila glue gene Sgs-3 is specifically expressed in the secretory cells of the salivary glands of third instar larvae. We have assayed the expression of gene fusions to determine the role of cis-acting Sgs-3 sequences in conferring this pattern of expression. These experiments define two regulatory regions required for expression of reporter genes from the Sgs-3 promoter. One region, between 106 and 56 bp upstream of the Sgs-3 mRNA 5' end is sufficient for low but correct tissue- and stage-specific expression. A second region, lying between 629 and 130 bp 5' of the RNA start site is functionally equivalent; that is, it alone will also direct low level, specific expression. These two regions act synergistically to give high level expression. More distant upstream regions function to further increase levels of expression. These two regulatory elements can confer a salivary gland-specific pattern of expression on a heterologous promoter and are also sufficient to drive gene expression in other Drosophila species, implying conservation of regulators.     

Molecular evolution of the histone 3 multigene family in the Drosophila melanogaster species subgroup

Molecular evolution of the histone multigene family was studied by cloning and sequencing regions of the histone 3 gene in the Drosophila melanogaster species subgroup. Analysis of the nucleotide substitution pattern showed that in the coding region synonymous changes occurred more frequently to A or T in contrast to the GC-rich base composition, while in the 3' region the nucleotide substitutions were most likely in equilibrium. These results suggested that the base composition at the third codon position of the H3 gene, i.e., codon usage, has been changing to A or T in the Drosophila melanogaster species subgroup.     

Molecular cloning and characterization of canine metalloproteinase-9 gene promoter

This paper describes the cloning and characterization of the canine matrix metalloproteinase-9 (MMP-9) gene promoter. The 5' untranslated region was obtained by genome walking upstream of the canine MMP-9 translation start site using canine genomic DNA as template. A DNA fragment of 1894 bp was isolated and on analysis demonstrated regions of sequence homology with the MMP-9 promoter sequences already determined for other species. In general, conserved regions correlated with DNA binding motifs such as a TATA-like box, AP-1 sites, GC boxes and a nuclear factor-kappaB binding domain. The DNA promoter fragment was sufficient to drive basal expression of a luciferase reporter gene in Madin Darby canine kidney (MDCK) cells and to a lesser extent in feline embryonic fibroblast (FEA) cells. Activity of the promoter was enhanced by the treatment of transfected MDCK cells with phorbol 12-myristate 13-acetate but no effect was observed in the FEA cells. Promoter deletion studies revealed that regions of promoter were necessary for induction of reporter gene expression.     

Nucleotide sequence analysis of the delta beta-globin gene region in humans

The continuous DNA sequence of a 16.5-kilobase pair region encompassing the linked delta beta-globin gene cluster in humans is presented with a detailed restriction endonuclease map. There are 38 differences (0.5%) in comparison with published sequence data, corrected for errors in sequencing, resulting in polymorphic rates of 0.2% in exons and 0.76% in 5'-gene flanking regions. Fifteen changes result in the generation or elimination of restriction sites which may be useful in linkage disequilibrium studies. Two pairs of inverted Alu repeats, a pyrimidine-rich region 5' to delta, and (TG)n, (Pu/Py)n, and (ATTTT)n tracts 5' to beta are described. Dinucleotide frequencies and deviation from expected values approximated those found in total human genomic DNA. Regions of less than 50% A + T content were found associated with Alu sequences, a 150-base pair region immediately 5' to the beta gene, exon regions from both genes, and an area 3' to the beta gene. These regions also contained significantly lower than expected CpG levels compared to other regions, suggesting a possible relationship between DNA organizational patterns and functionally important regions. In addition, strand asymmetries in base composition in this region differ from those associated with the fetal globin genes.     

Cloning of the genes for the pituitary glycoprotein hormone alpha and follicle-stimulating hormone beta subunits in the Japanese crested ibis,Nipponia nippon

We have isolated a part of the gene for the pituitary glycoprotein hormone common alpha subunit (PGHalpha) and the whole gene for the follicle-stimulating hormone beta subunit (FSHbeta) in the Japanese crested ibis (Nipponia nippon), a critically endangered bird species in East Asia. The nucleotide sequence of a part of the PGHalpha gene (5026 bp) contained three exons holding the whole coding and 3' untranslated regions, but lacked a 5' untranslated region. Its exon-intron structure was similar to that in mammals, but different from that in teleosts in the location of the second intron. For the FSHbeta gene, the nucleotide sequence of 7633 bp was assembled from two phage clones. The exon-intron structure of three exons and two introns was similar to that observed in mammals and teleosts. In the putative promoter region of the ibis FSHbeta gene, a progesterone responsive element (PRE)-like sequence and two AP-1 responsive element-like sequences reported in the ovine FSHbeta gene were not conserved in complete form. The increased number of ATTTA motifs in the putative 3' untranslated region in comparison with those in Japanese quail and chicken FSHbeta cDNA suggested that more rapid degradation of FSHbeta mRNA occurs in this species. Deduced amino acid sequences of the ibis PGHalpha and FSHbeta showed high similarities with those of the corresponding subunits of other avian species. This is the first report on the genomic sequences of the PGHalpha and FSHbeta in an avian species.