Mutations in the Drosophila mitochondrial tRNA amidotransferase, bene/gatA, cause growth defects in mitotic and endoreplicating tissues

Abstract

Rapid larval growth is essential in the development of most metazoans. In this article, we show that bene, a gene previously identified on the basis of its oogenesis defects, is also required for larval growth and viability. We show that all bene alleles disrupt gatA, which encodes the Drosophila homolog of glutamyl-tRNA(Gln) amidotransferase subunit A (GatA). bene alleles are now referred to as gatA. GatA proteins are highly conserved throughout eukaryotes and many prokaryotes. These enzymes are required for proper translation of the proteins encoded by the mitochondrial genome and by many eubacterial genomes. Mitotic and endoreplicating tissues in Drosophila gatA loss-of-function mutants grow slowly and never achieve wild-type size, and gatA larvae die before pupariation. gatA mutant eye clones exhibit growth and differentiation defects, indicating that gatA expression is required cell autonomously for normal growth. The gatA gene is widely expressed in mitotic and endoreplicating tissues.

Figure 1.—

gatA growth and maturation defects. All larvae are siblings hatched from eggs collected from the same vial after a 2-hr egg lay. (A) gatA larvae are much smaller than wild-type 5 days AEL. (Left) gatA/+. (Right) gatA¹¹²/Df. (B) Graph showing delayed molting into third instar larvae of gatA mutants. (Left bar) gatA/+. (Right bars) gatA¹¹²/Df. (C–E) mouth-hook morphology 5 days AEL. (C) Wild-type third instar larva. (D) gatA¹¹²/Df second instar larva. (E) gatA¹¹²/Df third instar larva.

Figure 2.—

Growth defects in mitotic tissues in 5-day-AEL gatA larvae. (A–D) 20×, DAPI staining; bars, 200 μm. (A) gatA/+ wing disc (arrow) and haltere discs (top right). (B) gatA¹¹²/Df wing disc (arrow). (C) gatA/+ brain (D) gatA¹¹²/Df brain.

Figure 3.—

Growth and DNA content of salivary glands in wild-type and gatA larvae. (A–H) 20×, DAPI staining. (A, C, E, and G) gatA/+. (B, D, F, and H) gatA¹¹²/Df. (A and B) 2 days AEL. (C and D) 3 days AEL. (E and F) 4 days AEL. (G and H) 5 days AEL. Arrow points to gatA/Df salivary gland.

Figure 4.—

Mapping and identification of gatA as the gene disrupted in all bene alleles. (A) Df(3R)Exel6182 and Df(3R)Exel6183 extend in opposite directions from a shared breakpoint at the insertion site of P{XP}d03824, which falls at bp 14,853,107 (K. Cook, FlyBase, personal communication). The P insertion P{lacW}l(3)S092902 was previously mapped by cytology to 91F1-F2. We mapped the insertion site molecularly, using inverse PCR (see materials and methods), to the third intron of gatA between codons 8 and 9 of NP15.6. Gene positions and splice forms adapted from FlyBase (Grumbling and Strelets 2006). (B–E) DAPI stainings of tissues in 5-day-AEL gatA/+ and gatA⁵⁰/gatA¹¹² trans-heterozygotes. (B and C) gatA/+. (D and E) gatA⁵⁰/gatA¹¹². (B and D) salivary gland. (C and E) Brain. (F) Northern blot showing strong NP15.6 expression in gatA/+ and gatA⁵⁰/gatA¹¹². RP49 is the loading control.

Figure 5.—

ClustalW alignment of Drosophila GatA protein sequence with GatA sequences from other species. Accession numbers are as follows: fly—NP_650775.2; human—CAB66614; yeast (S. cerevisiae)—Q03557; bacteria (the cyanobacteria, Prochlorococcus marinus)—YP_291459. Arrows signify lesions in gatA point mutants. gatA¹⁴⁵, W145STOP; gatA¹¹², W154STOP; gatA⁵⁰, T-A substitution in intron donor sequence, predicted to disrupt splicing between 174Y and 175A.

Figure 6.—

Mosaic analysis and expression of gatA. w, ey-FLP, Gla-lacZ; FRT Rps3, P{ubi-GFP, w+}/TM6B females were crossed with FRT e gatA⁵⁰/TM3, Sb males, FRT e gatA¹¹²/TM3, Sb males or FRT +/TM3, Sb males. (A–H) Adult eyes. The FRT Rps3 chromosome carries a w+ transgene. Rps3 is recessive cell lethal. Therefore, in males, cells that have not undergone recombination are yellow. Cells that have recombined are white (homozygous for + or gatA, depending on the cross). (A, B, E, and F) Female eyes are w+, aiding phenotypic analysis, but making it difficult to determine the genotype of cells. (C, D, G, and H) Male eyes are w−, permitting easy genotyping of eye clones. (A) Wild-type clone shows normal growth and development. (B) Rps3/+ eyes (no clones induced) are slightly smaller than wild type. (C) gatA¹¹²/+ eyes (no clones induced) and (G) gatA⁵⁰/+ eyes (no clones induced) are normal. (D–F) gatA¹¹² clones and (H) gatA⁵⁰ clones cause small, misshapen eyes with cuticle scars and ectopic bristles. There is a small, pale yellow region in the top portion of the eye in D, because ey-FLP induced mitotic recombination somewhat late in this eye. (I) RT–PCR of gatA from RNA extracts from larval gut (lanes 1 and 2), imaginal discs (lanes 3 and 4), fat bodies (lanes 5 and 6), salivary gland (lanes 7 and 8), brain (lanes 9 and 10), and from adult ovary (separate gel, lanes 11 and 12). “+” and “−” refer to treatment of extracts with reverse transcriptase.