Blattella germanica has two HMG-CoA synthase genes. Both are regulated in the ovary during the gonadotrophic cycle.

C Buesa,J Martínez-Gonzalez,N Casals,D Haro,M.D Piulachs,X Bellés,F.G Hegardt

doi:10.1016/s0021-9258(17)32630-3

C Buesa, J Martínez-Gonzalez + Show 5 more

Open Access

https://doi.org/10.1016/s0021-9258(17)32630-3

Copy DOI

Abstract

The isoprenoid pathway leads to various essential non-sterol products in insects. These end products have a crucial role in growth, differentiation, sexual maturation, and reproduction. 3-Hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) synthase (EC 4.1.3.5.) has generally been considered one of the committed steps of the pathway. We had previously reported the cloning of a cytosolic HMG-CoA synthase cDNA in Blattella germanica; we have now isolated and characterized a new cDNA clone for HMG-CoA synthase in this insect. Analysis of this 1716-base pair cDNA reveals a deduced protein of 455 residues with a molecular mass of 51,424 Da. The two HMG-CoA synthases have 69% identical amino acid residues, and both lack an N-terminal leader peptide to target the protein into mitochondria. This HMG-CoA synthase cDNA can revert the Chinese hamster ovary-K1-derived cell line, Mev-1, which is a defective mutant for HMG-CoA synthase. Both HMG-CoA synthase genes are expressed differently throughout development. Analysis of adult tissues shows higher expression in ovary and fat body. The expression of HMG-CoA synthase (EC 4.1.3.5.) and reductase (EC 1.1.1.34) genes during the gonadotrophic cycle in B. germanica shows that the three genes of the isoprenoid pathway are developmentally regulated in the ovary.

Highlights

From the $Unitof Biochemistry, University of Barcelona, School of Pharmacy, 08028 Barcelona and the **Departament of Agrobiology, Centro de Investigacidn y Desarrollo-ConsejoSuperior de Investigaciones Cientificas 08034 Barcelona, Spain
Analysis of B. germanica mRNA in isolated heads, whole adult bodies, and last instar larvuaseing this cDNA as a probe revealed a transcript size (1.7 kilobases), which was similar to the HMGS-1 previously reported (Fig. 1).this analysis showed that the expression of this new hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) synthase gene (HMGS-2) was higher in adults than in sixth instar larvae, in contrast withdata obtained for HMGS-1gene
The PCR approach to the isolation of B. genanicaHMG CoA synthase resulted in the amplification of an additional HMGCoA synthase cDNA, which is different from that previously reported, HMGS-1 [14]

Summary

11 Recipient of a Formacion de Personal Investigador fellowship from

We have analyzed the HMG-CoAreductase mRNAlevels in the ovarian development, as well as the relationship between the Ministerio de Educaci6n y Ciencia. Hybridizations were carriedout a s described [17] using either pST-365 or hBgST1-18 a s a probe, and washes were performed a t 68 "C with 0.2 x SSC and 0.1x SDS (1x SSC is 0.15 M NaCl, 0.015 M Na citrate, pH 7.0) In these conditions, we established that there was no cross-hybridization with the mRNAof the other B. germanica HMG-CoA synthase previously reported [14]. Assay of HMG-CoASynthase Activity-Three pooled ovary pairs from each day of the gonadotrophic cycle were processed a s above, and two aliquots of 50 pl were assayed. HMG-CoA synthase activity was determined in the ovarian tissue by the radiometric method described by Clinkenbeard et al [23], a s modified by Gil et al [24] except using 5-fold higher specific radioactivity. HMG-CoA synthase activity was determined in the ovarian tissue by the radiometric method described by Clinkenbeard et al [23], a s modified by Gil et al [24] except using 5-fold higher specific radioactivity. 1 unit ofHMG-CoA synthase is defined as the amount of enzyme that catalyzes the formation of 1pmol of HMG-CoA in 1min a t 37 "C

RESULTS

HMGS- I

DISCUSSION