Go, Hiroki (2005) Characterization of doublesex and nanos genes in Acropora millepora. PhD thesis, James Cook University.

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Abstract

Scleractinian coral, Acropora millepora is a hermaphroditic species which develops both male and female gonads in each polyp at the same time. Corals are diploblastic metazoa and have a simple body structure without well-defined organs. They lack typical reproductive system. Instead, eggs and sperms form independent clusters in the mesoglea of mesenteries.

Little has been known for the molecular mechanisms of sexdetermination in coral. Recently, a homolog of doublesex (AmDM1) was cloned for Acropora millepora. doublesex was identified as the somatic sex-determinant of Drosophila melanogaster and has been shown to be well conserved throughout the metazoan evolution. The gene product, Dsx, is a transcription factor containing the DM domain, a highly conserved DNA-binding domain. Thus it was speculated that AmDM1 took part in the sex-determination cascade by controlling the transcription of genes which regulated the female and/or male gonad formation.

In order to study the involvement of AmDM1 in the gonad development, I tried to isolate the candidates for the downstream genes of AmDM1 by applying the yeast one-hybrid screening system. This selection system enabled me to isolate the genomic DNA fragments including the potential binding elements for the DM domain. 5 clones were finally isolated by this screening. 4 of them contain the repeat sequences which are specific to genus Acropora. The interaction of these sequences and the DM domain of AmDM1 was demonstrated by the electrophoretic mobility shift assay (EMSA).

A genomic clone was isolated by using the fifth clone for the hybridization probe and identified as a coral homolog of T-type amino acid transporters 1 (TAT1) which was supposed to mediated the membrane transport of aromatic amino acids. Therefore this gene was named AmTAT1. The nucleotide sequence of the genomic clone indicated that the DNA fragment isolated by one-hybrid screening was located in the second intron of AmTAT1. According to the deduced amino acid sequence, AmTAT1 was predicted to have common features of membrane transporters with 12 transmembrane domains.

The responsive sequence to DM domain in the second intron of AmTAT1 was identified by EMSA. Comparison of this sequence with that in the repeat sequence revealed that the recognition motif was AATGT(GT)/(TG)C. The mutant study showed the AATGT pentanucleotide stretch was essential to the interaction with the DM domain. Moreover alignment of DM recognition elements of various species indicates that this stretch is absolutely conserved throughout evolution.

The regulatory function of AmDM1 on the DM recognition motif was investigated by CAT assay with Hep-2 cell line. In this system, AmDM1 acted on the recognition motif in an inhibitory manner. The transcriptional inhibition by AmDM1 was insensitive to the orientation of the responsive element. Therefore I concluded that the DM responsive element in the second intron of AmTAT1 was the cisacting on the gene expression of this gene. This observation is consistent with the result of expression analysis on AmTAT1. In adult branch tips of Acropora millepora, the expression of AmDM1 was reported to be detected in October, the season of sexual maturation. On the contrary, the expression of AmTAT1 is down-regulated in October but is detectable before and after October.

Although the biological significance of the inhibition of AmTAT1 expression in the process of sex-determination is yet to be known, it may lead to the sexual maturation of coral polyps by blocking the influx of specific amino acids or derivatives.

vitellogenin gene expression is controlled by Doublesex homologs in Caenorhabditis elegans and Bombyx mori. I isolated cDNA and genomic DNA of coral vitellogenin (AmVit). The genomic DNA was partially sequenced. However, no potential AmDM1 binding site was found in the upstream regulatory region and introns.

nanos plays crucial roles in abdominal formation and proper germline differentiation in Drosophila. Lower animals including sponges and cnidarians also have nanos homologs. To know the roles of nanos genes in the germline speciation in coral, I isolated cDNAs and genomic DNAs of nanos homologs of Acropora millepora and Nematostella vectensis. Cnidarians have two nanos homologs, cnnos1 and cnnos2. All Nanos proteins have the highly conserved Zinc finger domains. The phylogenetic analysis demonstrated that two cnnos genes segregated before the cnidarian radiation.

The sequence of 5'-flanking regions of these cnnos genes were subjected to the prediction of potential transcription factor binding sites. Their features turned out to be quite different. This finding is in agreement with their expression pattern: the expression of Cnnos1(Am) was only observed in the early embryonal stage and no expression was detected in the adult coral tips and Cnnos2(Am) expression was detected in all stages of development (eggs, prawnchip and pre-settlement) and all samples for the adult colony (September, October and December).

Although the difference in functions between cnnos1(Am) and cnnos2(Am) is suggested by these data, the roles of cnnos genes in the germline speciation is still unknown.

Item ID:	1204
Item Type:	Thesis (PhD)
Keywords:	Acropora millepora; coral reproduction; doublesex genes; gene expression; nanos; genes; scleractinia; scleractinian coral; sex determination; sex-determination
Date Deposited:	05 Aug 2015 23:21
FoR Codes:	06 BIOLOGICAL SCIENCES > 0604 Genetics > 060403 Developmental Genetics (incl Sex Determination) @ 100%
SEO Codes:	96 ENVIRONMENT > 9608 Flora, Fauna and Biodiversity > 960808 Marine Flora, Fauna and Biodiversity @ 100%
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