Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.

Plasmodium falciparum is a causative agent of human malaria. Sixty percent of mRNAs from its extremely AT-rich (81%) genome harbor long polyadenosine (polyA) runs within their ORFs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate polyA runs cause ribosome stalling and frameshifting, triggering mRNA surveillance pathways and attenuating protein synthesis. Here, we show that P. falciparum is an exception to this rule. We demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately translated in P. falciparum cells. We show that polyA runs do not elicit any response from No Go Decay (NGD) or result in the production of frameshifted proteins. This is in stark contrast to what we observe in human cells or T. thermophila, an organism with similar AT-content. Finally, using stalling reporters we show that Plasmodium cells evolved not to have a fully functional NGD pathway.

Background Wallerian degeneration (WD) following peripheral nerve injury (PNI) is an area of growing focus for pharmacological developments. Clinically, WD presents challenges in achieving full functional recovery following PNI, as prolonged denervation of distal tissues for an extended period of time can irreversibly destabilize sensory and motor targets with secondary tissue atrophy. Our objective is to improve upon histological assessments of WD. Methods Conventional methods utilize a qualitative system simply describing the presence or absence of WD in nerve fibers. We propose a three-category assessment that allows more quantification: A fibers appear normal, B fibers have moderate WD (altered axoplasm), and C fibers have extensive WD (myelin figures). Analysis was by light microscopy (LM) on semithin sections stained with toluidine blue in three rat tibial nerve lesion models (crush, partial transection, and complete transection) at 5 days postop and 5 mm distal to the injury site. The LM criteria were verified at the ultrastructural level. This early outcome measure was compared with the loss of extensor postural thrust and the absence of muscle atrophy. Results The results showed good to excellent internal consistency among counters, demonstrating a significant difference between the crush and transection lesion models. A significant decrease in fiber density in the injured nerves due to inflammation/edema was observed. The growth cones of regenerating axons were evident in the crush lesion group. Conclusion The ABC method of histological assessment is a consistent and reliable method that will be useful to quantify the effects of different interventions on the WD process.

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During differentiation of the Tetrahymena thermophila somatic nucleus, its germline-derived DNA undergoes extensive reorganization including the removal of ∼50 Mb from thousands of loci called internal eliminated sequences (IESs). IES-associated chromatin is methylated on lysines 9 and 27 of histone H3, marking newly formed heterochromatin for elimination. To ensure that this reorganized genome maintains essential coding and regulatory sequences, the boundaries of IESs must be accurately defined. In this study, we show that the developmentally expressed protein encoded by Lia3-Like 1 (LTL1) (Ttherm_00499370) is necessary to direct the excision boundaries of particular IESs. In ΔLTL1 cells, boundaries of eliminated loci are aberrant and heterogeneous. The IESs regulated by Ltl1 are distinct from those regulated by the guanine-quadruplex binding Lia3 protein. Ltl1 has a general affinity for double stranded DNA (Kd ∼ 350 nM) and binds specifically to a 50 bp A+T rich sequence flanking each side of the D IES (Kd ∼ 43 nM). Together these data reveal that Ltl1 and Lia3 control different subsets of IESs and that their mechanisms for flanking sequence recognition are distinct.