Evolutionary conservation of the fidelity of transcription.

Accurate transcription is required for the faithful expression of genetic information. However, relatively little is known about the molecular mechanisms that control the fidelity of transcription, or the conservation of these mechanisms across the tree of life. To address these issues, we measured the error rate of transcription in five organisms of increasing complexity and found that the error rate of RNA polymerase II ranges from 2.9 × 10-6 ± 1.9 × 10-7/bp in yeast to 4.0 × 10-6 ± 5.2 × 10-7/bp in worms, 5.69 × 10-6 ± 8.2 × 10-7/bp in flies, 4.9 × 10-6 ± 3.6 × 10-7/bp in mouse cells and 4.7 × 10-6 ± 9.9 × 10-8/bp in human cells. These error rates were modified by various factors including aging, mutagen treatment and gene modifications. For example, the deletion or modification of several related genes increased the error rate substantially in both yeast and human cells. This research highlights the evolutionary conservation of factors that control the fidelity of transcription. Additionally, these experiments provide a reasonable estimate of the error rate of transcription in human cells and identify disease alleles in a subunit of RNA polymerase II that display error-prone transcription. Finally, we provide evidence suggesting that the error rate and spectrum of transcription co-evolved with our genetic code.

The landscape of transcription errors in eukaryotic cells.

Accurate transcription is required for the faithful expression of genetic information. To understand the molecular mechanisms that control the fidelity of transcription, we used novel sequencing technology to provide the first comprehensive analysis of the fidelity of transcription in eukaryotic cells. Our results demonstrate that transcription errors can occur in any gene, at any location, and affect every aspect of protein structure and function. In addition, we show that multiple proteins safeguard the fidelity of transcription and provide evidence suggesting that errors that evade these layers of RNA quality control profoundly affect the physiology of living cells. Together, these observations demonstrate that there is an inherent limit to the faithful expression of the genome and suggest that the impact of mutagenesis on cellular health and fitness is substantially greater than currently appreciated.

Genomic Methods and Microbiological Technologies for Profiling Novel and Extreme Environments for the Extreme Microbiome Project (XMP).

The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the world with the intent of discovering new species, genes, and gene clusters. Once a project site is complete, the resulting data will be publically available. Sites include Lake Hillier in Western Australia, the "Door to Hell" crater in Turkmenistan, deep ocean brine lakes of the Gulf of Mexico, deep ocean sediments from Greenland, permafrost tunnels in Alaska, ancient microbial biofilms from Antarctica, Blue Lagoon Iceland, Ethiopian toxic hot springs, and the acidic hypersaline ponds in Western Australia.

Temporal genetic stability in natural populations of the waterflea Daphnia magna in response to strong selection pressure.

Studies monitoring changes in genetic diversity and composition through time allow a unique understanding of evolutionary dynamics and persistence of natural populations. However, such studies are often limited to species with short generation times that can be propagated in the laboratory or few exceptional cases in the wild. Species that produce dormant stages provide powerful models for the reconstruction of evolutionary dynamics in the natural environment. A remaining open question is to what extent dormant egg banks are an unbiased representation of populations and hence of the species' evolutionary potential, especially in the presence of strong environmental selection. We address this key question using the water flea Daphnia magna, which produces dormant stages that accumulate in biological archives over time. We assess temporal genetic stability in three biological archives, previously used in resurrection ecology studies showing adaptive evolutionary responses to rapid environmental change. We show that neutral genetic diversity does not decline with the age of the population and it is maintained in the presence of strong selection. In addition, by comparing temporal genetic stability in hatched and unhatched populations from the same biological archive, we show that dormant egg banks can be consulted to obtain a reliable measure of genetic diversity over time, at least in the multidecadal time frame studied here. The stability of neutral genetic diversity through time is likely mediated by the buffering effect of the resting egg bank.

Nematodes from galls on Myrtaceae. IX. Fergusobia rosettae n. sp. on Melaleuca quinquenervia and F. tolgaensis n. sp. on Syzygium luehmannii, from Queensland.

Two new species of Fergusobia, collected from 'rosette' shoot bud galls on Melaleuca quinquenervia, and from leaf, stem, leaf and flower bud galls on Syzygium luehmannii, both from the Cairns region of Queensland, Australia, are described. Fergusobia rosettae Davies n. sp. is characterised by the combination of a small, arcuate parthenogenetic female having a short conoid tail with a bluntly rounded tip, an arcuate, relatively slender, infective female with an almost hemispherical tail tip, and arcuate males with arcuate to angular (not heavily sclerotised) spicules and leptoderan bursa arising at 40-50% of body length from tail tip. Fergusobia tolgaensis Davies n. sp. is characterised by the combination of a small open C-shaped parthenogenetic female with a broadly conoid tail, an arcuate infective female with a broadly rounded tail tip, and arcuate males with angular (not heavily sclerotised) spicules and short to mid-length leptoderan bursa. These two species of nematodes are associated with fly larvae that have dorsal shields comprising bars of raised cuticular ridges and spicules, similar to that of fly larvae from the M. leucadendra species group. The shield morphologies of these fly larvae and their possible genetic relationships are discussed. Possible evolutionary relationships of the Fergusobia nematodes from these galls are discussed, considering their morphology, DNA sequences, and the relationships of the associated Fergusonina flies and host plants.

The Dscam homologue of the crustacean Daphnia is diversified by alternative splicing like in insects.

In insects, the homologue of the Down syndrome cell adhesion molecule (Dscam) is a unique case of a single-locus gene whose expression has extensive somatic diversification in both the nervous and immune systems. How this situation evolved is best understood through comparative studies. We describe structural, expression, and evolutionary aspects of a Dscam homolog in 2 species of the crustacean Daphnia. The Dscam of Daphnia generates up to 13,000 different transcripts by the alternative splicing of variable exons. This extends the taxonomic range of a highly diversified Dscam beyond the insects. Additionally, we have identified 4 alternative forms of the cytoplasmic tail that generate isoforms with or without inhibitory or activating immunoreceptor tyrosine-based motifs (ITIM and ITAM respectively), something not previously reported in insect's Dscam. In Daphnia, we detected exon usage variability in both the brain and hemocytes (the effector cells of immunity), suggesting that Dscam plays a role in the nervous and immune systems of crustaceans, as it does in insects. Phylogenetic analysis shows a high degree of amino acid conservation between Daphnia and insects except in the alternative exons, which diverge greatly between these taxa. Our analysis shows that the variable exons diverged before the split of the 2 Daphnia species and is in agreement with the nearest-neighbor model for the evolution of the alternative exons. The genealogy of the Dscam gene family from vertebrates and invertebrates confirmed that the highly diversified form of the gene evolved from a nondiversified form before the split of insects and crustaceans.