ABSTRACT
The plethora of genome sequences produced in the postgenomic age has not resolved many of our most pressing biological questions. Correlating gene expression with an interrogatable and easily observable characteristic such as the surrogate phenotype conferred by a reporter gene is a valuable approach to gaining insight into gene function. Many reporters including lacZ, amdS, and the fluorescent proteins mRuby3 and mNeonGreen have been used across all manners of organisms. Described here is an investigation into the creation of a robust, synthetic, fusion reporter system for Cryptococcus neoformans that combines some of the most useful fluorophores available in this system with the versatility of the counter-selectable nature of amdS. The reporters generated include multiple composition and orientation variants, all of which were investigated for differences in expression. Evaluation of known promoters from the TEF1 and GAL7 genes was undertaken, elucidating novel expression tendencies of these biologically relevant C. neoformans regulators of transcription. Smaller than lacZ but providing multiple useful surrogate phenotypes for interrogation, the fusion ORF serves as a superior whole-cell assay compared to traditional systems. Ultimately, the work described here bolsters the array of relevant genetic tools that may be employed in furthering manipulation and understanding of the WHO fungal priority group pathogen C. neoformans.
ABSTRACT
Puberty is a whole-body event, driven by the hypothalamic integration of peripheral signals such as leptin or IGF-1. In the process of puberty, reproductive development is simultaneous to growth, including muscle growth. To enhance our understanding of muscle function related to puberty, we performed transcriptome analyses of muscle samples from six pre- and six post-pubertal Brahman heifers (Bos indicus). Our aims were to perform differential expression analyses and co-expression analyses to derive a regulatory gene network associate with puberty. As a result, we identified 431 differentially expressed (DEx) transcripts (genes and non-coding RNAs) when comparing pre- to post-pubertal average gene expression. The DEx transcripts were compared with all expressed transcripts in our samples (over 14,000 transcripts) for functional enrichment analyses. The DEx transcripts were associated with "extracellular region," "inflammatory response" and "hormone activity" (adjusted p < .05). Inflammatory response for muscle regeneration is a necessary aspect of muscle growth, which is accelerated during puberty. The term "hormone activity" may signal genes that respond to progesterone signalling in the muscle, as the presence of this hormone is an important difference between pre- and post-pubertal heifers in our experimental design. The DEx transcript with the highest average expression difference was a mitochondrial gene, ENSBTAG00000043574 that might be another important link between energy metabolism and puberty. In the derived co-expression gene network, we identified six hub genes: CDC5L, MYC, TCF3, RUNX2, ATF2 and CREB1. In the same network, 48 key regulators of DEx transcripts were identified, using a regulatory impact factor metric. The hub gene TCF3 was also a key regulator. The majority of the key regulators (22 genes) are members of the zinc finger family, which has been implicated in bovine puberty in other tissues. In conclusion, we described how puberty may affect muscle gene expression in cattle.