Search for: All records

ABSTRACT NeurosporaSk-2is a complex meiotic drive element that is transmitted to offspring through sexual reproduction in a biased manner.Sk-2’s biased transmission mechanism involves spore killing, and recent evidence has demonstrated that spore killing is triggered by a gene calledrfk-1. However, a second gene,rsk, is also critically important for meiotic drive by spore killing because it allows offspring with anSk-2genotype to survive the toxic effects ofrfk-1. Here, we present evidence demonstrating thatrfk-1encodes two protein variants: a 102 amino acid RFK-1^Aand a 130 amino acid RFK-1^B, but only RFK-1^Bis toxic. We also show that expression of RFK-1^Brequires an early stop codon inrfk-1mRNA to undergo adenosine-to-inosine (A-to-I) mRNA editing. Finally, we demonstrate that RFK-1^Bis toxic when expressed within vegetative tissue of Spore killer sensitive (Sk^S) strains, and that this vegetative toxicity can be overcome by co-expressingSk-2’s version of RSK. Overall, our results demonstrate thatSk-2uses RNA editing to control when its spore killer is produced, and that the primary killing and resistance functions ofSk-2can be conferred upon anSk^Sstrain by the transfer of only two genes. 

Neurospora crassa is a genus of fungus that exhibits a phenomenon called Sk-3 spore killing. Sk-3 spore killing occurs when an Sk-3 killer strain mates with an Sk-3 sensitive strain, and it results in the death of half of the offspring. A DNA interval called i350, located on N. crassa Chromosome III, has previously been identified as critical for spore killing. Here, to obtain a more detailed understanding of this DNA interval, the effects of the deletion of related DNA intervals i386 and i408 on spore killing has been studied. Deletion of i386 resulted in no disruption of spore killing while deletion of i408 disrupted spore killing. These results provide a better understanding of the DNA sequences required for the spore killing process. 

Neurospora crassa is a well-known model organism for studying eukaryotic genetics, particularly non-Mendelian inheritance mechanisms such as meiotic drive. In N. crassa, meiotic drive can be observed in fungal spore killing, where Spore killer-3 (Sk-3) is a selfish genetic element transmitted to offspring through spore killing. Sk-3 is thought to contain two principal components: a killer (poison) gene and a resistance (antidote) gene. While the resistance gene (rsk) has been identified, the killer gene remains unknown. Building on previous research that identified a 1.3 kb DNA interval (i350) essential for Sk-3-based spore killing, I analyzed two subintervals, i382 and i400, to narrow down the functional components of the Sk-3 locus. Deletion of i382 does not disrupt spore killing and deletion of interval i400 partially disrupts spore killing but does not eliminate it. Future work should retest the i400 strains in spore killing assays, to determine if the partial spore killing phenotype can be detected in all crosses when larger numbers of rosettes are examined. The findings presented here help narrow down the search for the unknown poison gene involved in spore killing, which is a critical step towards understanding processes that allow for the evolution of Sk-3-type selfish genetic elements. 

Neurospora fungi are found around the world. The species N. crassa is a popular model for use in genetics research. N. crassa produces sexual spores, called ascospores, during mating between strains of opposite mating types. N. crassa also produces spore sacs called asci, and each ascus typically contains eight viable ascospores. However, some Neurospora fungi carry selfish genetic elements called Spore killers, and when a strain carrying a Spore killer mates with a spore killing-susceptible strain, asci contain four black viable ascospores and four white inviable ascospores. In this project, I investigated a Spore killer called Sk-3. To act as a selfish genetic element, Sk-3 is thought to require at least two genes, a poison gene and an antidote gene. The Sk-3 antidote gene (rsk) has been identified, but the poison gene has not. The purpose of this study is to help identify the location of the poison gene. To do this, I deleted two DNA intervals (i383 and i394) from a location of the Neurospora genome that may harbor the poison gene. My results indicate that deletion of i383 eliminates spore killing while deletion of i394 has no effect on spore killing. The possibility that i394 overlaps with the poison gene is discussed. 

Meiotic drive is a non-Mendelian inheritance phenomenon where selfish genetic elements change gene transmission in their own favor. This phenomenon occurs in the fungus Neurospora crassa during spore killing. When a strain carrying a spore killer genetic element is crossed with a non-spore killing wild type strain, the cross will produce half viable and half inviable offspring. The N. crassa Sk-3 spore killer is found on Chromosome III. Sk-3 is one of the most studied meiotic drive elements in Neurospora fungi and it is thought to require a killer gene and a resistance gene for spore killing. While the killer gene has not been identified, recent work has isolated a mutation (rfk-2UV) that disrupts spore killing. Although this mutation has been mapped to Chromosome III, its exact location is not known. In this work, I investigate the role of one DNA interval in Sk-3-based spore killing. This DNA interval, referred to as v373, is thought to reside within or near rfk-2UV. My results will contribute to future efforts to identify the Sk-3 killer gene. 

Some isolates of the fungus Neurospora crassa possess a chromosomal factor that causes spore killing, leading to death of ascospores. It has been shown that these chromosomal factors are genetic elements called spore killers. For example, if a cross is performed between a parent with an Sk-S (sensitive) allele and a parent with an Sk-K (killer) allele, the cross will produce half viable offspring and half inviable offspring, where the inviable half has been killed by spore killing. This phenomenon can be explained by meiotic drive, wherein a selfish gene disrupts the randomness of sexual transmission, favoring its own success. In this study, I focus on a Neurospora Spore killer known as Sk-3. Sk-3 is thought to possess both a killer element and a resistance element. The resistance element is rsk, a gene that keeps ascospores alive and viable when in the presence of the killer element. However, the mechanism by which the killer element kills ascospores is unknown. A major obstacle to studying the killing mechanism is that the identity of the Sk-3 killer element itself has remained elusive. My goal is to help identify the Sk-3 killer element. Preliminary results by others have narrowed the search to the left arm of Chromosome III. These results have also shown that deletion of a 1.3 kb DNA interval, called v350, causes loss of spore killing. This suggests that a regulatory element, or a hidden gene, may overlap with the v350 interval. To help determine why v350 deletion correlates with loss of spore killing, I investigated a related DNA interval, called v384. My results suggest that v384, like v350, is required for spore killing. 

Meiotic drive describes a process in which selfish alleles are recovered in more than half of a progeny generation. It is a type of gene drive and it has been discovered in strains of Neurospora, a filamentous fungus, through its spore killing mechanism. One of the most studied meiotic drive elements within N. crassa is Spore killer-3 (Sk-3). Previous studies have indicated that there is a genomic region within Sk-3 that encodes resistance to spore killing and another that encodes an element that is required for spore killing. Sk-3’s resistance gene, rsk, has been identified. However, the exact region that mediates Sk-3’s spore killing mechanism is currently unknown. In a previous study, it was found that a mutation called rfk-2UV disrupts spore killing by Sk-3. To better understand the region of Chromosome III in which rfk-2UV is located (its exact location is unknown), I constructed a deletion vector to replace a DNA interval (v374) with a hygromycin resistance gene marker (hph). Transformants were crossed to produce offspring, and offspring were tested to determine if they possess the ability to kill ascospores. These findings will contribute to future efforts to determine the molecular nature of rfk-2UV and why this mutation disrupts the ability of Sk-3 to kill spores. 

Neurospora crassa is a fungus that serves as a model organism for genetic research. N. crassa Spore killer-3 (Sk-3) is a genetic element transmitted to offspring through spore killing. Sk-3 is located on Chromosome III and it is thought to require two genes for spore killing. These two genes are the poison gene, for killing, and the antidote gene, for resistance to killing. While the Sk-3 resistance gene has been identified (rsk), the Sk-3 killer gene has not. The primary goal of this study is to help identify the killer gene by investigating the role of a DNA interval called v377 in spore killing. To determine if this interval is required for spore killing, a DNA deletion vector (Vector v377) was constructed and used to replace the v377 interval with a hygromycin resistance gene in strain RDGR170.3. My results demonstrate that v377 is required for spore killing. The possibility that v377 is within a gene required for spore killing, or a regulatory element that controls spore killing, is discussed. 

In Neurospora fungi, the ascospores formed during reproduction will most often be black and viable. Occasionally, these ascospores will end up inviable and white or yellow. The discovery of a selfish genetic element called Spore killer (Sk) in 1979 gave researchers insight into a mechanism that causes some Neurospora crosses to produce a consistent ratio of 4 black, viable ascospores and 4 inviable, white ascospores. In these 4:4 splits, the Spore killer genetic element causes the death of exactly half of the ascospores. There are now three known spore killers in Neurospora: Sk-1, Sk-2, and Sk-3. This thesis examines the role of a DNA element within Sk-3. In an Sk-3 × Sk-3-sensitive (Sk-S) cross, Sk-3 genes are transmitted to the four black, viable ascospores, and, through a poorly understood mechanism, the Sk-3 genes kill ascospores that fail to inherit these genes. The Sk-3 genes reside on Chromosome III, but the exact locations of all critical genes are unknown. Preliminary results suggest that a DNA interval called v350 may harbor a critical Sk-3 gene. For example, deletion of the v350 interval eliminates Sk-3 spore killing. Here, I explore the deletion of an additional DNA interval located within v350. Specifically, I tested the role of DNA interval v376 on Sk-3 spore killing. The research presented here should help determine why v350, and perhaps v376, are required for spore killing by Neurospora Sk-3. 

Meiotic drivers are selfish genetic elements that skew transmission in their favor. In the filamentous fungus N. crassa, one such meiotic driver is Spore killer-3 (Sk-3). In a cross between Sk-3 and a spore killer-sensitive mating partner (Sk-S), only half of the ascospores (sexual spores) survive. Nearly all of the survivors inherit the genes for spore killing. Previous studies have established that a gene called rfk-2 (required for spore killing) is essential for the spore killing activity of Sk-3. The rfk-2 gene has been mapped to Chromosome III, but its exact location is unknown. The goal of this study is to help identify the exact location of rfk-2. Towards this goal, I investigated a DNA interval called v378. Preliminary findings suggested that this interval may be important for spore killing. To determine if v378 is required for spore killing, I constructed and used a transformation vector to replace v378 with a hygromycin resistance gene (hph+) in an N. crassa Sk-3 strain. Strains deleted of v378 were then crossed with two spore killing-sensitive tester strains. The spore sacs containing ascospores from these crosses were imaged to analyze the effects of replacement of v378 on Sk-3-based spore killing. My findings demonstrate that v378 is required for spore killing. The potential implications of my findings with respect to our understanding of meiotic drive elements, and their potential applications, is discussed.