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Analysis of four achaete-scute homologs in Bombyx mori reveals new viewpoints of the evolution and functions of this gene family.

Zhou Q, Zhang T, Xu W, Yu L, Yi Y, Zhang Z - BMC Genet. (2008)

Bottom Line: Functions besides neural development controlling have also been found in Drosophila AS-C genes.Proteins encoded by them contained the characteristic bHLH domain and the three proneural ones were also found to have the C-terminal conserved motif.AS-C gene duplication in insects occurs after or parallel to, but not before the taxonomic order formation during evolution.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Biotechnology Research Institute, National Engineering of crop germplasm and genetic improvement, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Qingxiang_Zhou@meei.harvard.edu

ABSTRACT

Background: achaete-scute complexe (AS-C) has been widely studied at genetic, developmental and evolutional levels. Genes of this family encode proteins containing a highly conserved bHLH domain, which take part in the regulation of the development of central nervous system and peripheral nervous system. Many AS-C homologs have been isolated from various vertebrates and invertebrates. Also, AS-C genes are duplicated during the evolution of Diptera. Functions besides neural development controlling have also been found in Drosophila AS-C genes.

Results: We cloned four achaete-scute homologs (ASH) from the lepidopteran model organism Bombyx mori, including three proneural genes and one neural precursor gene. Proteins encoded by them contained the characteristic bHLH domain and the three proneural ones were also found to have the C-terminal conserved motif. These genes regulated promoter activity through the Class A E-boxes in vitro. Though both Bm-ASH and Drosophila AS-C have four members, they are not in one by one corresponding relationships. Results of RT-PCR and real-time PCR showed that Bm-ASH genes were expressed in different larval tissues, and had well-regulated expressional profiles during the development of embryo and wing/wing disc.

Conclusion: There are four achaete-scute homologs in Bombyx mori, the second insect having four AS-C genes so far, and these genes have multiple functions in silkworm life cycle. AS-C gene duplication in insects occurs after or parallel to, but not before the taxonomic order formation during evolution.

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Bm-ASH genes enhance ASH2P Promoter activity via E-boxes. ASH2P, the promoter segment of Bm-ASH2; 194Em, ASH2P mutated in the E-box 188–194 bp upstream from the start codon; 797Em, ASH2P mutated in the E-box 791–797 bp upstream from the start codon; 194&797Em, ASH2P mutated in both of the E-boxes. Forks show the mutated E-box site (A). B, C, D and E are the results co-transfected of promoters and da with ASH1, ASH2, ASH3 and ase, respectively. E-boxes mutation significantly reduced the promoter activity in B, C and D (p < 0.01), but had no effect in E (p > 0.05).
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Figure 5: Bm-ASH genes enhance ASH2P Promoter activity via E-boxes. ASH2P, the promoter segment of Bm-ASH2; 194Em, ASH2P mutated in the E-box 188–194 bp upstream from the start codon; 797Em, ASH2P mutated in the E-box 791–797 bp upstream from the start codon; 194&797Em, ASH2P mutated in both of the E-boxes. Forks show the mutated E-box site (A). B, C, D and E are the results co-transfected of promoters and da with ASH1, ASH2, ASH3 and ase, respectively. E-boxes mutation significantly reduced the promoter activity in B, C and D (p < 0.01), but had no effect in E (p > 0.05).

Mentions: In order to study whether the bHLH transcriptional factors regulate the promoter by the E-box region, we changed one or both of the deduced E-boxes to "aAGG/CcG" by site-directed mutation (Fig. 5A). Then the mutated promoters were cloned into pGL3-Basic vector as described above, and co-transfected Bm-N cells with each Bm-ASH and da genes' transient expression vectors, respectively. The results showed that E-box mutation significantly reduced the activity of the promoter when co-transfected with silkworm proneural genes (Fig. 5B, C, D; p < 0.01). However, the activation of Bm-ase did not seem to be related with either of the two deduced E-boxes (Fig. 5E; p > 0.05). These data suggest that proteins encoded by Bm-ASH genes have similar functions to the AS-C transcriptional factors in Drosophila, and they synergize with Da protein.


Analysis of four achaete-scute homologs in Bombyx mori reveals new viewpoints of the evolution and functions of this gene family.

Zhou Q, Zhang T, Xu W, Yu L, Yi Y, Zhang Z - BMC Genet. (2008)

Bm-ASH genes enhance ASH2P Promoter activity via E-boxes. ASH2P, the promoter segment of Bm-ASH2; 194Em, ASH2P mutated in the E-box 188–194 bp upstream from the start codon; 797Em, ASH2P mutated in the E-box 791–797 bp upstream from the start codon; 194&797Em, ASH2P mutated in both of the E-boxes. Forks show the mutated E-box site (A). B, C, D and E are the results co-transfected of promoters and da with ASH1, ASH2, ASH3 and ase, respectively. E-boxes mutation significantly reduced the promoter activity in B, C and D (p < 0.01), but had no effect in E (p > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2315653&req=5

Figure 5: Bm-ASH genes enhance ASH2P Promoter activity via E-boxes. ASH2P, the promoter segment of Bm-ASH2; 194Em, ASH2P mutated in the E-box 188–194 bp upstream from the start codon; 797Em, ASH2P mutated in the E-box 791–797 bp upstream from the start codon; 194&797Em, ASH2P mutated in both of the E-boxes. Forks show the mutated E-box site (A). B, C, D and E are the results co-transfected of promoters and da with ASH1, ASH2, ASH3 and ase, respectively. E-boxes mutation significantly reduced the promoter activity in B, C and D (p < 0.01), but had no effect in E (p > 0.05).
Mentions: In order to study whether the bHLH transcriptional factors regulate the promoter by the E-box region, we changed one or both of the deduced E-boxes to "aAGG/CcG" by site-directed mutation (Fig. 5A). Then the mutated promoters were cloned into pGL3-Basic vector as described above, and co-transfected Bm-N cells with each Bm-ASH and da genes' transient expression vectors, respectively. The results showed that E-box mutation significantly reduced the activity of the promoter when co-transfected with silkworm proneural genes (Fig. 5B, C, D; p < 0.01). However, the activation of Bm-ase did not seem to be related with either of the two deduced E-boxes (Fig. 5E; p > 0.05). These data suggest that proteins encoded by Bm-ASH genes have similar functions to the AS-C transcriptional factors in Drosophila, and they synergize with Da protein.

Bottom Line: Functions besides neural development controlling have also been found in Drosophila AS-C genes.Proteins encoded by them contained the characteristic bHLH domain and the three proneural ones were also found to have the C-terminal conserved motif.AS-C gene duplication in insects occurs after or parallel to, but not before the taxonomic order formation during evolution.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Biotechnology Research Institute, National Engineering of crop germplasm and genetic improvement, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Qingxiang_Zhou@meei.harvard.edu

ABSTRACT

Background: achaete-scute complexe (AS-C) has been widely studied at genetic, developmental and evolutional levels. Genes of this family encode proteins containing a highly conserved bHLH domain, which take part in the regulation of the development of central nervous system and peripheral nervous system. Many AS-C homologs have been isolated from various vertebrates and invertebrates. Also, AS-C genes are duplicated during the evolution of Diptera. Functions besides neural development controlling have also been found in Drosophila AS-C genes.

Results: We cloned four achaete-scute homologs (ASH) from the lepidopteran model organism Bombyx mori, including three proneural genes and one neural precursor gene. Proteins encoded by them contained the characteristic bHLH domain and the three proneural ones were also found to have the C-terminal conserved motif. These genes regulated promoter activity through the Class A E-boxes in vitro. Though both Bm-ASH and Drosophila AS-C have four members, they are not in one by one corresponding relationships. Results of RT-PCR and real-time PCR showed that Bm-ASH genes were expressed in different larval tissues, and had well-regulated expressional profiles during the development of embryo and wing/wing disc.

Conclusion: There are four achaete-scute homologs in Bombyx mori, the second insect having four AS-C genes so far, and these genes have multiple functions in silkworm life cycle. AS-C gene duplication in insects occurs after or parallel to, but not before the taxonomic order formation during evolution.

Show MeSH