Amino acid alignment of Ntl with human and <i>A.aeolicus</i> SLC6 transporters.

<p>Amino acid sequence alignment of <i>Drosophila melanogaster</i> Ntl (Ntl; NP_609135.1) with A.aeolicus Leu T_Aa (Leu; NP_214423), human homologues for Serotonin (5HT;P31645), Glycine (Gly, I57956), GABA (GABA; P30531), Dopamine (DOPA; Q01959) using Clustal W alignment [<a href="http://www.ebi.ac.uk/Tools/clustalw2/index.html" target="_blank">http://www.ebi.ac.uk/Tools/clustalw2/index.html</a>]. Strictly conserved residues are highlighted in red; α coils and β sheets are depicted as blocks and arrows respectively. Open and filled green circles represent putative cationic gates at extra and intra cellular surfaces (EL and IL) respectively. Open and filled blue triangles indicate sites that interact with sodium ions in the <i>LeuA</i> structure. Tyrosine in TM3 is a critical residue present in Ntl, which is indispensable for substrate binding and transport. Adapted from Yamashita <i>et al</i>, 2005.</p

<i>Ntl</i> expression is testis specific and limited to the germline.

<p>A) From the left: <i>Ntl</i> RT-PCR products from wt whole males, wt male heads, wt male testis, 129A whole male; <i>Ntl</i> RT-PCR products from wt whole females, wt female heads and wt female ovaries. The last lane is a negative control without RNA. B) <i>Ntl</i> RT-PCR products from <i>Ntl/Ntl</i> mutant males carrying a pTMR-<i>Ntl</i> cDNA construct (Lane 1; <i>yw; Ntl^129A/Ntl^129A; pTMR-Ntl1/TM3,Sb</i>) and a genomic pCaSpeR4 construct (Lane 2; <i>yw; Ntl^129A/Ntl^129A</i>; pCaSpeR 4- Ntl1/<i>TM3,Sb</i>). Lane 3: <i>Ntl</i> RT-PCR product from testes of male offspring of <i>tud/tud</i> females, which lack germ cells.</p

<i>Ntl</i> mutant sperm are immotile and are not transferred into seminal vesicles.

<p>Panels A, B, E, and F: Phase contrast images of testes from <i>Ntl⁺</i> (A, E) and <i>Ntl^-</i> (B, F) males. The major phenotypic feature of the mutants is the accumulation of coiled cysts at the base of the testis (asterisks), and the empty/shrunken state of the seminal vesicle (arrows). Panels, C, D, G, and H: <i>don juan-</i>GFP fluorescence images corresponding to phase images immediately above them, showing the disposition of elongated cysts and mature sperm in the testis and seminal vesicle. Note the complete absence of fluorescence from the seminal vesicle of <i>Ntl/Ntl</i> mutants (arrows) compared to the accumulated fluorescence in wild type seminal vesicles (arrowheads), and accumulation of coiled cysts in the base of the mutant testes (asterisks). In panels A and C, the letter M demotes dense masses of mature motile sperm which is not seen in the mutants. Left hand panels: wild type (<i>Ntl/+</i>); right hand panels: <i>Ntl</i>^129A/<i>Ntl</i>^129A mutants. Bars, 20 µm.</p

Deletion analysis of <i>Ntl</i> alleles.

<p>Numbers correspond to coordinates of Ntl genomic sequence.</p><p>+/− Refer to presence or absence of the band respectively.</p>a<p>Non specific bands.</p

Primers.

<p>Primers.</p