Biology subjects

Hammond, T. M.

Publications and source records attributed to Hammond, T. M..

6 recordsLinked to original sources

bioRxiv2026

Molecular characterization of required for killing-2: a gene required for spore killing by Neurospora Sk-3.

The Sk-3 selfish genetic element in Neurospora fungi is transmitted to offspring in a biased manner through spore killing. In crosses between an Sk-3 strain and a Spore killing-sensitive (Sk-S) strain, nearly all offspring that fail to inherit Sk-3 are killed. Although a mutation called rfk-2UV, which disrupts spore killing by Sk-3, was isolated in an earlier work, its precise location within the Sk-3 element was not identified. Here, we demonstrate that rfk-2UV is a T>G transversion mutation within a protein coding gene called rfk-2. The rfk-2 gene is located on Chromosome III, and it encodes a transcript that undergoes RNA splicing and at least two RNA editing events. One of these editing events changes a UAG stop codon to a UGG tryptophan codon and allows for production of a 102 amino acid protein. Additionally, while our results demonstrate that rfk-2 is required for spore killing, they also show that it is insufficient for the process. Sk-3 is thus more complex than previously anticipated. Rather than using a single gene to produce its killer, Sk-3 requires multiple genes to kill spores and break the rules of Mendelian Inheritance.

genetics↗

bioRxiv2021

RNA editing controls meiotic drive by a Neurospora Spore killer

Neurospora Sk-2 is a complex meiotic drive element that is transmitted to offspring through sexual reproduction in a biased manner. Sk-2s transmission mechanism involves spore killing, and recent evidence has demonstrated that its spore killing mechanism is mediated by a gene called rfk-1. The native rfk-1 sequence, referred to as rfk-1+, encodes an early UAG stop codon and a late UAA stop codon. When translation stops at the early stop codon, a 102 amino acid protein called RFK-1A is produced, and when translation stops at the late stop codon, a 130 amino acid protein called RFK-1B is produced. We show that expression of RFK-1B occurs when the early stop codon undergoes adenosine-to-inosine (A-to-I) mRNA editing (UAG is edited to UIG), and that this editing event is required for spore killing. We also show that RFK-1B, but not RFK-1A, acts as a poison when it is ectopically expressed within the vegetative tissue of a non-Sk-2 strain. Furthermore, we show that RFK-1B toxicity can be neutralized in vegetative tissue by co-expressing RSKSk-2, which is presumed to be Sk-2s antidote protein. Finally, we show that rfk-1+s first intron, or a truncated version of this intron, when present, improves phenotypic expression of RFK-1B in vegetative tissue. Overall, our results demonstrate that Sk-2 uses A-to-I mRNA editing to control when its killer protein (poison) is produced, and that the primary killing and resistance functions of Sk-2 can be established within a non-Sk-2 strain by the ectopic expression of only two genes.

genetics↗

bioRxiv2020

Investigating expression of a human optimized cas9 transgene in Neurospora crassa

The CRISPR-associated Cas9 enzyme is used in molecular biology to engineer the genomes of a wide range of organisms. While Cas9 can be injected or transfected into a target cell to achieve the desired goal, there are situations where stable expression of Cas9 within a target organism is preferable. Here, we show that the model filamentous fungus Neurospora crassa is recalcitrant to heterologous expression of a human-optimized version of Streptococcus pyogenes cas9. Furthermore, partial optimization of cas9 by synonymous codon exchange failed to improve its expression in the fungus. Finally, we show that transgene expression can be detected when cas9Hs sequences are placed in the 3 UTR regions of transgene-derived mRNAs, but not when the same sequences are in the translated part of the transgene-derived mRNA. This finding suggests that the primary obstacle to high cas9Hs expression levels in N. crassa is translational in nature.

molecular biology↗

bioRxiv2020

An introgressed gene causes meiotic drive in Neurospora sitophila

Meiotic drive elements cause their own preferential transmission following meiosis. In fungi this phenomenon takes the shape of spore killing, and in the filamentous ascomycete Neurospora sitophila, the Sk-1 spore killer element is found in many natural populations. In this study, we identify the gene responsible for spore killing in Sk-1 by generating both long and short-read genomic data and by using these data to perform a genome wide association test. Through molecular dissection, we show that a single 405 nucleotide long open reading frame generates a product that both acts as a poison capable of killing sibling spores and as an antidote that rescues spores that produce it. By phylogenetic analysis, we demonstrate that the gene is likely to have been introgressed from the closely related species N. hispaniola, and we identify three subclades of N. sitophila, one where Sk-1 is fixed, another where Sk-1 is absent, and a third where both killer and sensitive strain are found. Finally, we show that spore killing can be suppressed through an RNA interference based genome defense pathway known as meiotic silencing by unpaired DNA. Spk-1 is not related to other known meiotic drive genes, and similar sequences are only found within Neurospora. These results shed new light on the diversity of genes capable of causing meiotic drive, their origin and evolution and their interaction with the host genome. Significance StatementIn order to survive, most organisms have to deal with parasites. Such parasites can be other organisms, or sometimes, selfish genes found within the host genome itself. While much is known about parasitic organisms, the interaction with their hosts and their ability to spread within and between species, much less is known about selfish genes. We here identify a novel selfish "spore killer" gene in the fungus Neurospora sitophila. The gene appears to have evolved within the genus, but has entered the species through hybridization and introgression. We also show that the host can counteract the gene through RNA interference. These results shed new light on the diversity of selfish genes in terms of origin, evolution and host interactions.

evolutionary biology↗

bioRxiv2019

A nonhomologous end-joining mutant for Neurospora sitophila research

Disruption of the nonhomologous end-joining (NHEJ) pathway has been shown to increase the efficiency of transgene integration into targeted genomic locations of Neurospora crassa and other fungi. Here, we report that a similar phenomenon occurs in a second Neurospora species: N. sitophila. Specifically, we show that deletion of N. sitophila mus-51 increases the efficiency of targeted-transgene integration, presumably by disrupting NHEJ. Researchers interested in obtaining the N. sitophila mus-51{Delta} strains described in this study can obtain them from the Fungal Genetics Stock Center (FGSC).

genetics↗

bioRxiv2018

Identification of a genetic element required for spore killing in Neurospora

Meiotic drive elements like Spore killer-2 (Sk-2) in Neurospora are transmitted through sexual reproduction to the next generation in a biased manner. Sk-2 achieves this biased transmission through spore killing. Here, we identify rfk-1 as a gene required for the spore killing mechanism. The rfk-1 gene is associated with a 1,481 bp DNA interval (called AH36) near the right border of the 30 cM Sk-2 element, and its deletion eliminates the ability of Sk-2 to kill spores. The rfk-1 gene also appears to be sufficient for spore killing because its insertion into a non-Sk-2 isolate disrupts sexual reproduction after the initiation of meiosis. Although the complete rfk-1 transcript has yet to be defined, our data indicate that rfk-1 encodes a protein of at least 39 amino acids and that rfk-1 has evolved from a partial duplication of gene ncu07086. We also present evidence that rfk-1s location near the right border of Sk-2 is critical for the success of spore killing. Increasing the distance of rfk-1 from the right border of Sk-2 causes it to be inactivated by a genome defense process called meiotic silencing by unpaired DNA (MSUD), adding to accumulating evidence that MSUD exists, at least in part, to protect genomes from meiotic drive.

genetics↗