High Gene Count Research Articles

Limited diagnostic impact of duplications

PurposeCopy-number variants (CNVs) of uncertain clinical significance are routinely reported in a clinical setting only when exceeding predetermined reporting thresholds, typically based on CNV size. Given that very few genes are associated with triplosensitive phenotypes, it is not surprising that many interstitial duplications <1Mb are found to be inherited and anticipated to be of limited or no clinical significance. MethodsIn an effort to further refine our reporting criteria to maximize diagnostic yield while minimizing the return of uncertain variants, we performed a retrospective analysis of all clinical microarray cases reported in a 10-year window. A total of 1112 reported duplications had parental follow-up, and these were compared by size, RefSeq gene content, and inheritance pattern. De novo origin was used as a rough proxy for pathogenicity. ResultsApproximately 6% of duplications 500 kb–1 Mb were de novo observations, compared with approximately 14% for 1–2Mb duplications (p=0.0005). On average, de novo duplications had higher gene counts than inherited duplications. ConclusionOur data reveal limited diagnostic utility for duplications of uncertain significance <1Mb. Considerations for revised reporting criteria are discussed and are applicable to CNVs detected by any genome-wide exploratory methodology, including exome/genome sequencing.

Moderate sex between protocells can balance between a decrease in assortment load and an increase in parasite spread

Sexual reproduction is widespread in nature despite the different kinds of cost that it entails. We do not know exactly when the first sexual process took place and especially why it was beneficial at first. It is clearer why sex is advantageous for the prokaryotes and eukaryotes but the benefit of sex for protocells with individually replicating ribozymes is not yet fully understood. In this context sex is the simple horizontal gene transfer among two protocells that undergo transient fusion. Many authors argue that horizontal gene transfer (HGT) was very common in the early stage of evolution. However, HGT is a risky mechanism considering both the disruption of optimal compositions and the spread of parasites among protocells. In order to test the effects of HGT on the fitness of a protocell population, we explored by numerical simulations those conditions under which fusion might have been beneficial. We investigated multiple conceivable types of fusion in the stochastic corrector model framework and we considered the spread of parasites in every case. Protocells contain up to five species of unlinked, essential ribozymes; if a protocell has the same amount of each, it reaches maximum fitness. Fusion is dangerous not only due to the spread of parasites but also because it can ruin the cells with balanced ribozyme composition. We show that fusion can restore the ribozyme composition of the protocells under certain circumstances (high gene count, intermediate split size and low rate of fusion) and thus it can decrease the effect of the genetic load. Fusion could have been a useful early mechanism in contributing to the reliable coexistence of the different ribozymes before the spread of the chromosomes.

Reassessment of the succession of lactic acid bacteria in commercial cucumber fermentations and physiological and genomic features associated with their dominance

A compositional re-assessment of the microbiota present in commercial cucumber fermentation using culture independent and dependent methods was conducted, with emphasis on lactic acid bacteria (LAB). Two commercial cucumber fermentation tanks were monitored by measuring pH, dissolved oxygen and temperature, and used as sources of samples for microbial plating, genomic DNA extraction and measurement of organic acids and carbohydrates by HPLC. Six additional commercial tanks were included to identify the dominant microorganisms using molecular methods. A comparative analysis of the publically available genome sequences corresponding to the LAB found in cucumber fermentations was completed to gain an understanding of genomic features possibly enabling dominance. Analyses of the microbiota suggest Lactobacillales prevail in cucumber fermentations, including in order of prevalence Lactobacillus pentosus, Lb. plantarum, Lb. brevis, Weissella spp., Pediococcus ethanolidurans, Leuconostoc spp. and Lactococcus spp. It was observed that Lb. pentosus and Lb. plantarum have comparatively larger genomes, higher gene counts, uniquely distribute the ribosomal clusters across the genome as opposed to close to the origin of replication, and possess more predicted amino acids prototrophies and selected biosynthesis related genes. It is theorized that Lb. pentosus and Lb. plantarum dominance in cucumber fermentations is the result of their genetic make-up.

Gut microbiota: in sickness and in health.

Gut microbiota: in sickness and in health.

Development of a microinjection system for RNA interference in the water flea Daphnia pulex

BackgroundThe ubiquitous, freshwater microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have a well annotated, reference genome assembly that revealed an unusually high gene count highlighted by a large gene orphanage,-i.e., previously uncharacterized genes. Daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic manipulation, but the establishment of gene manipulation techniques is needed to accurately define gene functions. Although previous investigations developed an RNA interference (RNAi) system for one congener D. magna, these methods are not appropriate for D. pulex because of the smaller size of their early embryos. In these studies, we develop RNAi techniques for D. pulex by first determining the optimum culture conditions of their isolated embryos and then applying these conditions to the development of microinjection techniques and proof-of-principle RNAi experiments.ResultsWe found that isolated embryos were best cultured on a 2% agar plate bathed in 60 mM sucrose dissolved in M4 media, providing optimal conditions for microinjections. Then, we injected double-stranded (ds)RNA specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for limb development in invertebrates and vertebrates. Injected embryos presented with defects in the second antenna and appendage development, and dsRNA induced the degradation of Dll mRNAs, indicating that this technique successfully inhibited transcription of the target gene.ConclusionsWe developed a microinjection system for RNAi studies in D. pulex. These techniques add to the growing genomic toolbox and enhance the genetic tractability of this important model for environmental, evolutionary, and developmental genomics.

The ecoresponsive genome of Daphnia pulex.

We describe the draft genome of the microcrustacean Daphnia pulex, which is only 200 megabases and contains at least 30,907 genes. The high gene count is a consequence of an elevated rate of gene duplication resulting in tandem gene clusters. More than a third of Daphnia's genes have no detectable homologs in any other available proteome, and the most amplified gene families are specific to the Daphnia lineage. The coexpansion of gene families interacting within metabolic pathways suggests that the maintenance of duplicated genes is not random, and the analysis of gene expression under different environmental conditions reveals that numerous paralogs acquire divergent expression patterns soon after duplication. Daphnia-specific genes, including many additional loci within sequenced regions that are otherwise devoid of annotations, are the most responsive genes to ecological challenges.

Design and analysis of a robust genetic Muller C-element

This paper presents results on the design and analysis of a robust genetic Muller C-element. The Muller C-element is a standard logic gate commonly used to synchronize independent processes in most asynchronous electronic circuits. Synthetic biological logic gates have been previously demonstrated, but there remain many open issues in the design of sequential (state-holding) logic operations. Three designs are considered for the genetic Muller C-element: a majority gate, a toggle switch, and a speed-independent implementation. While the three designs are logically equivalent, each design requires different assumptions to operate correctly. The majority gate design requires the most timing assumptions, the speed-independent design requires the least, and the toggle switch design is a compromise between the two. This paper examines the robustness of these designs as well as the effects of parameter variation using stochastic simulation. The results show that robustness to timing assumptions does not necessarily increase reliability, suggesting that modifications to existing logic design tools are going to be necessary for synthetic biology. Parameter variation simulations yield further insights into the design principles necessary for building robust genetic gates. The results suggest that high gene count, cooperativity of at least two, tight repression, and balanced decay rates are necessary for robust gates. Finally, this paper presents a potential application of the genetic Muller C-element as a quorum-mediated trigger.