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Background: Non-traditional model systems need new tools that will enable them to enter the field of functional genetics. These tools should enable the exploration of gene function, via knock-downs of endogenous genes, as well as overexpression and ectopic expression of transgenes. Methodology: We constructed a new vector called Pogostick that can be used to over-express or down-regulate genes in organisms amenable to germ line transformation by the piggyBac transposable element. Pogostick can be found at www. addgene.org, a non-profit plasmid repository. The vector currently uses the heat-shock promoter Hsp70 from Drosophila to drive transgene expression and, as such, will have immediate applicability to organisms that can correctly interpret this promotor sequence. We detail how to clone candidate genes into this vector and test its functionality in Drosophila by targeting a gene coding for the fluorescent protein DsRed. By cloning a single DsRed copy into the vector, and generating transgenic lines, we show that DsRed mRNA and protein levels are elevated following heat-shock. When cloning a second copy of DsRed in reverse orientation into a flanking site, and transforming flies constitutively expressing DsRed in the eyes, we show that endogenous mRNA and protein levels drop following heat-shock. We then test the over-expression vector, containing the complete cDNA of Ultrabithorax (Ubx) gene, in an emerging model system, Bicyclus anynana. We produce a transgenic line and show that levels of Ubx mRNA expression rise significantly following a heat-shock. Finally, we show how to obtain genomic sequence adjacent to the Pogostick insertion site and to estimate transgene copy number in genomes of transformed individuals. Significance: This new vector will allow emerging model systems to enter the field of functional genetics with few hurdles. [ABSTRACT FROM AUTHOR]

Positive Darwinian selection on advantageous point substitutions has been demonstrated in many genes. We here provide empirical evidence, for the first time, that positive selection can also act on insertion/deletion (indel) substitutions in the evolution of a protein. CATSPER1 is a voltage-gated calcium channel found exclusively in the plasma membrane of the mammalian sperm tail and it is. essential for sperm motility. We determined the DNA sequences of the first exon of the CATSPER1 gene from 15 primates, which encodes the intracellular N terminus region of ≈400 aa. These sequences exhibit an excessively high frequency of indels. However, all indels have lengths that are multiples of 3 nt (3n indels) and do not disrupt the ORF. The number of indel substitutions per site per year in CATSPER1 is five to eight times the corresponding rates calculated from two large-scale primate genomic comparisons, which represent the neutral rate of indel substitutions. Moreover, CATSPER1 indels are considerably longer than neutral indels. These observations strongly suggest that positive selection has been promoting the fixation of indel mutations in CATSPER1 exon 1. It has been shown in certain ion channels that the length of the N terminus region affects the rate of channel inactivation. This finding suggests that the selection detected may be related to the regulation of the CATSPER1 channel, which can affect sperm motility, an important determinant in sperm competition. [ABSTRACT FROM AUTHOR]

Identifies two genes (PRM2 and FOXP2) with significantly enhanced evolutionary rates in the hominid lineage, using comparative analysis of human, chimpanzee, and mouse protein sequences. PRM2 is a histone-like protein essential to spermatogenesis and was previously reported to be a likely target of sexual selection in humans and chimpanzees. FOXP2 is a transcription factor involved in speech and language development. Human FOXP2 experienced a >60-fold increase in substitution rate and incorporated two fixed amino acid changes in a broadly defined transcription suppression domain. A survey of a diverse group of placental mammals reveals the uniqueness of the human FOXP2 sequence and a population genetic analysis indicates possible adaptive selection behind the accelerated evolution. Taken together, our results suggest an important role that FOXP2 may have played in the origin of human speech and demonstrate a strategy for identifying candidate genes underlying the emergences of human-specific features.