An evolutionary analysis of cAMP-specific Phosphodiesterase 4 alternative splicing

<p>Abstract</p> <p>Background</p> <p>Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the intracellular second messengers: cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP). The cAMP-specific PDE family 4 (PDE4) is widely expressed in vertebrates. Each of the four PDE4 gene isoforms (PDE4 A-D) undergo extensive alternative splicing via alternative transcription initiation sites, producing unique amino termini and yielding multiple splice variant forms from each gene isoform termed long, short, super-short and truncated super-short. Many species across the vertebrate lineage contain multiple splice variants of each gene type, which are characterized by length and amino termini.</p> <p>Results</p> <p>A phylogenetic approach was used to visualize splice variant form genesis and identify conserved splice variants (genome conservation with EST support) across the vertebrate taxa. Bayesian and maximum likelihood phylogenetic inference indicated PDE4 gene duplication occurred at the base of the vertebrate lineage and reveals additional gene duplications specific to the teleost lineage. Phylogenetic inference and PDE4 splice variant presence, or absence as determined by EST screens, were further supported by the genomic analysis of select vertebrate taxa. Two conserved PDE4 long form splice variants were found in each of the PDE4A, PDE4B, and PDE4C genes, and eight conserved long forms from the PDE4 D gene. Conserved short and super-short splice variants were found from each of the PDE4A, PDE4B, and PDE4 D genes, while truncated super-short variants were found from the PDE4C and PDE4 D genes. PDE4 long form splice variants were found in all taxa sampled (invertebrate through mammals); short, super-short, and truncated super-short are detected primarily in tetrapods and mammals, indicating an increasing complexity in both alternative splicing and cAMP metabolism through vertebrate evolution.</p> <p>Conclusions</p> <p>There was a progressive independent incorporation of multiple PDE4 splice variant forms and amino termini, increasing PDE4 proteome complexity from primitive vertebrates to humans. While PDE4 gene isoform duplicates with limited alternative splicing were found in teleosts, an expansion of both PDE4 splice variant forms, and alternatively spliced amino termini predominantly occurs in mammals. Since amino termini have been linked to intracellular targeting of the PDE4 enzymes, the conservation of amino termini in PDE4 splice variants in evolution highlights the importance of compartmentalization of PDE4-mediated cAMP hydrolysis.</p

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Genome-Wide Gene Expression Analysis Shows AKAP13-Mediated PKD1 Signaling Regulates the Transcriptional Response to Cardiac Hypertrophy

In the heart, scaffolding proteins such as A-Kinase Anchoring Proteins (AKAPs) play a crucial role in normal cellular function by serving as a signaling hub for multiple protein kinases including protein kinase D1 (PKD1). Under cardiac hypertrophic conditions AKAP13 anchored PKD1 activates the transcription factor MEF2 leading to subsequent fetal gene activation and hypertrophic response. We used an expression microarray to identify the global transcriptional response in the hearts of wild-type mice expressing the native form of AKAP13 compared to a gene-trap mouse model expressing a truncated form of AKAP13 that is unable to bind PKD1 (AKAP13-ΔPKD1). Microarray analysis showed that AKAP13-ΔPKD1 mice broadly failed to exhibit the transcriptional profile normally associated with compensatory cardiac hypertrophy following trans-aortic constriction (TAC). The identified differentially expressed genes in WT and AKAP13-ΔPKD1 hearts are vital for the compensatory hypertrophic response to pressure-overload and include myofilament, apoptotic, and cell growth/differentiation genes in addition to genes not previously identified as affected by AKAP13-anchored PKD1. Our results show that AKAP13-PKD1 signaling is critical for transcriptional regulation of key contractile, cell death, and metabolic pathways during the development of compensatory hypertrophy in vivo

Molecular evolution of a-kinase anchoring protein (AKAP)-7: implications in comparative PKA compartmentalization

<p>Abstract</p> <p>Background</p> <p>A-Kinase Anchoring Proteins (AKAPs) are molecular scaffolding proteins mediating the assembly of multi-protein complexes containing cAMP-dependent protein kinase A (PKA), directing the kinase in discrete subcellular locations. Splice variants from the AKAP7 gene (AKAP15/18) are vital components of neuronal and cardiac phosphatase complexes, ion channels, cardiac Ca²⁺ handling and renal water transport.</p> <p>Results</p> <p>Shown in evolutionary analyses, the formation of the AKAP7-RI/RII binding domain (required for AKAP/PKA-R interaction) corresponds to vertebrate-specific gene duplication events in the PKA-RI/RII subunits. Species analyses of AKAP7 splice variants shows the ancestral AKAP7 splice variant is AKAP7α, while the ancestral long form AKAP7 splice variant is AKAP7γ. Multi-species AKAP7 gene alignments, show the recent formation of AKAP7δ occurs with the loss of native AKAP7γ in rats and basal primates. AKAP7 gene alignments and two dimensional Western analyses indicate that AKAP7γ is produced from an internal translation-start site that is present in the AKAP7δ cDNA of mice and humans but absent in rats. Immunofluorescence analysis of AKAP7 protein localization in both rat and mouse heart suggests AKAP7γ replaces AKAP7δ at the cardiac sarcoplasmic reticulum in species other than rat. DNA sequencing identified Human AKAP7δ insertion-deletions (indels) that promote the production of AKAP7γ instead of AKAP7δ.</p> <p>Conclusions</p> <p>This AKAP7 molecular evolution study shows that these vital scaffolding proteins developed in ancestral vertebrates and that independent mutations in the AKAP7 genes of rodents and early primates has resulted in the recent formation of AKAP7δ, a splice variant of likely lesser importance in humans than currently described.</p

Sky-scattered solar radiation based plume transmissivity measurement to quantify soot emissions from flares

For gas flares typical of the upstream energy industry and similar point sources, most current methods for characterizing soot emissions are based on plume opacity rather than a quantitative measure of mass flux. The absence of more quantitative approaches is indicative of the inherent complexity of soot and the difficulties in characterizing emissions in an unbounded plume. A new experimental approach has been developed for the investigation of soot emissions in industrial plumes. Referred to as sky-LOSA, the diagnostic permits evaluation of 2D spatially resolved monochromatic skylight transmissivity data over the width of a plume, where skylight intensities behind the plume are obtained via an interpolation algorithm. By using Rayleigh-Debye-Gans Fractal Aggregate theory to relate transmissivity data to soot concentrations, and with knowledge of the velocity of the plume, it is possible to quantify mass flow rates of soot in a plume. Experiments on an unconfined lab-scale soot plume were used to support a detailed uncertainty analysis under a wide range of conditions and to estimate sensitivity limits of the technique. Results suggest field measurements of soot emission from flare plumes should be possible with overall uncertainties of less than 32%. This represents a significant advancement over existing techniques based on opacity measurements.Peer reviewed: YesNRC publication: Ye

Genome-Wide Gene Expression Analysis Shows AKAP13-Mediated PKD1 Signaling Regulates the Transcriptional Response to Cardiac Hypertrophy

<div><p>In the heart, scaffolding proteins such as A-Kinase Anchoring Proteins (AKAPs) play a crucial role in normal cellular function by serving as a signaling hub for multiple protein kinases including protein kinase D1 (PKD1). Under cardiac hypertrophic conditions AKAP13 anchored PKD1 activates the transcription factor MEF2 leading to subsequent fetal gene activation and hypertrophic response. We used an expression microarray to identify the global transcriptional response in the hearts of wild-type mice expressing the native form of AKAP13 compared to a gene-trap mouse model expressing a truncated form of AKAP13 that is unable to bind PKD1 (AKAP13-ΔPKD1). Microarray analysis showed that AKAP13-ΔPKD1 mice broadly failed to exhibit the transcriptional profile normally associated with compensatory cardiac hypertrophy following trans-aortic constriction (TAC). The identified differentially expressed genes in WT and AKAP13-ΔPKD1 hearts are vital for the compensatory hypertrophic response to pressure-overload and include myofilament, apoptotic, and cell growth/differentiation genes in addition to genes not previously identified as affected by AKAP13-anchored PKD1. Our results show that AKAP13-PKD1 signaling is critical for transcriptional regulation of key contractile, cell death, and metabolic pathways during the development of compensatory hypertrophy <i>in vivo</i>.</p></div

Differentially expressed genes between WT-Sham and AKAP13-ΔPKD1 Sham hearts.

<p>Differentially expressed genes between WT-Sham and AKAP13-ΔPKD1 Sham hearts.</p

Apoptosis Gene expression stratified by WT or AKAP13-∆PKD1 mice following sham or TAC surgery.

<p>Normalized gene expression is shown for apoptosis-associated genes <b>A)</b> Fas-associated death domain (FADD), <b>B)</b> Bcl2, <b>C)</b> Nudt1 <b>D)</b> Bax, <b>E)</b> Granzyme M (Gzmm), and <b>F)</b> Dynamin-1 like protein. Gray boxes represent the interquartile range, encompassing the first through third quartiles; the horizontal bar shows the median value. Values greater than 1.5 times the interquartile range are plotted outside of the whiskers. P values are from linear regression assuming an additive model.</p