Zebra finch (Taeniopygia guttata) is a representative songbird species that has been widely studied to investigate the neurobiological basis of vocal learning, a rare trait shared in only a few species including human. In December 2019, an updated zebra finch genome annotation (bTaeGut1_v1.p) was released from Ensembl database and is substantially more comprehensive than the first version published in 2010. In this study, we utilized the publicly available RNA-seq data generated from Illumina-based short-read method and PacBio single-molecule real-time (SMRT) long-read method to assess the bird transcriptome. To analyze the high-throughput RNA-seq data, we adopted a hybrid bioinformatic approach combining short and long read pipelines to investigate the new bird annotation for the first time. From our analysis, we added 220 novel genes and 8,134 transcript variants to the Ensembl annotation, and predicted a new proteome based on the refined annotation. Our results provide additional resources for future studies of zebra finches and other songbirds utilizing this improved annotation.

Recent studies have shown that rapid modulation of the estrogen synthesizing enzyme, aromatase, regulates estrogen production in neurons to affect behavior and cognition. In songbirds, aromatase is expressed at high levels in the hippocampus (HP), hypothalamus (HYP), and caudomedial nidopallium (NCM), where estrogens have been shown to affect learning and memory (HP), reproductive behavior (HYP), and auditory processing (NCM). Previous studies, largely in the HYP of quail, show that aromatase activity is down-regulated by Ca2+-dependent phosphorylation. Here, using zebra finches (Taeniopygia guttata), we ask if similar mechanisms are at work in the songbird HYP and if there are sex differences and differences in aromatase modulation between the HYP, HP and NCM. We quantified the conversion of [3H]androstenedione to estrone with a well-established in vitro assay to measure the effects of Ca2+, Mg2+, ATP, and an inhibitor of kinase activity in homogenates and partially purified brain fractions. We report a rapid down-regulation of aromatase activity in the presence of phosphorylating conditions across all three brain regions and both sexes. However, regional differences were seen in response to individual cofactor concentrations, some of which were improved by partial purification of the homogenates. Furthermore, while low concentrations of ATP inhibited aromatase activity, unexpectedly, inhibition was no longer seen with high ATP concentrations. These results provide evidence for a regional and temporal specificity in aromatase activity that has not been observed in songbirds.

Lactose permease (LacY) catalyzes oligosaccharide/H+ symport across the membrane. Until recently, all LacY structures demonstrate inward-open conformations with closed periplasmic side. We sought to obtain LacY crystals that diffract at higher resolution and/or provide previously unsolved conformation. The strategy was to employ hybrid proteins available in the laboratory in which cytochromeb562 or flavodoxin-1 of E. coli was fused to LacY. Of the sixteen constructs, large-scale expression/purification of two LacY/cytochromeb562 constructs was successful but crystallization proved difficult. The crystal structure of LacY double-Trp (G46W/G262W) mutant with B-D-galactopyranosyl-1-thio-B-D-galactopyranoside (TDG) was recently reported. This structure has almost occluded, outward-open conformation with a rotation at the thioester linkage in TDG as compared to the previous structures. We obtained crystals of this double-mutant with octyl B-D-galactopyranoside that diffracted to ~20 Å. A mutagenesis strategy is proposed to obtain LacY in fully outward-open conformation. Further, a transport mechanism involving a configurational change(s) in TDG is postulated.