Genetics最新文献

RNA binding proteins play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the Adenosine DeAminase acting on RNA (ADAR) family of RNA binding proteins and the sole adenosine-to-inosine RNA editing enzyme in C. elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RNA binding proteins that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogenous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals, and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and independent mechanisms in regulating embryogenesis.

Neurons are highly polarized cells with dendrites and axons. Dendrites, which receive sensory information or input from other neurons, often display elaborately branched morphologies. While mechanisms that promote dendrite branching have been widely studied, less is known about the mechanisms that restrict branching. Using the nematode Caenorhabditis elegans, we identify rabr-1 (for Rab-related gene 1) as a factor that restricts branching of the elaborately branched dendritic trees of PVD and FLP somatosensory neurons. Animals mutant for rabr-1 show excessively branched dendrites throughout development and into adulthood in areas where the dendrites overlay epidermal tissues. Phylogenetic analyses show that RABR-1 displays similarity to small GTPases of the Rab-type, although based on sequence alone, no clear vertebrate ortholog of RABR-1 can be identified. We find that rabr-1 is expressed and can function in epidermal tissues, suggesting that rabr-1 restricts dendritic branching cell-non-autonomously. Genetic experiments further indicate that for the formation of ectopic branches rabr-1 mutants require the genes of the Menorin pathway, which have been previously shown to mediate dendrite morphogenesis of somatosensory neurons. A translational reporter for RABR-1 reveals a subcellular localization to punctate, perinuclear structures, which correlates with endosomal and autophagosomal markers, but anticorrelates with lysosomal markers suggesting an amphisomal character. Point mutations in rabr-1 analogous to key residues of small GTPases suggest that rabr-1 functions in a GTP-bound form independently of GTPase activity. Taken together, rabr-1 encodes for an atypical small GTPase of the Rab-type that cell-non-autonomously restricts dendritic branching of somatosensory neurons, likely independently of GTPase activity.

Nuclear migration through narrow constrictions is important for development, metastasis, and proinflammatory responses. Studies performed in tissue culture cells have implicated linker of nucleoskeleton and cytoskeleton (LINC) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nuclei in vivo. In Caenorhabditis elegans larvae, six pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here, we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin-binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function; this and structural predictions suggest that FLN-2 does not function as a filamin. The immunoglobulin-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex component unc-84, fln-2 mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.

Epigenetics in the form of DNA methylation and other processes is an established property of genotypes and a focus of empirical research. Yet, there remain fundamental gaps in the evolutionary theory of epigenetics. To support a comprehensive understanding of epigenetics, this paper investigates theoretically the combined effects of deleterious mutation and epimutation with and without inbreeding. Both spontaneous epimutation and paramutation are considered to cover a broader range of epigenetic phenomena. We find that inbreeding generally reduces the amount of segregating deleterious genetic and epigenetic variation at equilibrium, although interestingly inbreeding can also increase the amount of deleterious genetic or epigenetic variation. Furthermore, we also demonstrate that epimutation indirectly can cause increased or decreased deleterious genetic variation at equilibrium relative to classic expectations, which is particularly evident when paramutation is occurring. With the addition of deleterious epimutation, there may be significantly increased purging of deleterious variation in more inbred populations and a significantly increased amount of segregating deleterious variation in more outbred populations, with notable exceptions.

Characterizing spatial patterns in allele frequencies is fundamental to evolutionary biology because these patterns contain evidence of underlying processes. However, the spatial scales at which gene flow, changing selection, and drift act are often unknown. Many of these processes can operate inconsistently across space, causing nonstationary patterns. We present a wavelet approach to characterize spatial pattern in allele frequency that helps solve these problems. We show how our approach can characterize spatial patterns in relatedness at multiple spatial scales, i.e. a multilocus wavelet genetic dissimilarity. We also develop wavelet tests of spatial differentiation in allele frequency and quantitative trait loci (QTL). With simulation, we illustrate these methods under different scenarios. We also apply our approach to natural populations of Arabidopsis thaliana to characterize population structure and identify locally adapted loci across scales. We find, for example, that Arabidopsis flowering time QTL show significantly elevated genetic differentiation at 300-1,300 km scales. Wavelet transforms of allele frequencies offer a flexible way to reveal geographic patterns and underlying evolutionary processes.

Although gene conversion (GC) in Saccharomyces cerevisiae is the most error-free way to repair double-strand breaks (DSBs), the mutation rate during homologous recombination is 1,000 times greater than during replication. Many mutations involve dissociating a partially copied strand from its repair template and re-aligning with the same or another template, leading to -1 frameshifts in homonucleotide runs, quasipalindrome (QP)-associated mutations and microhomology-mediated interchromosomal template switches. We studied GC induced by HO endonuclease cleavage at MATα, repaired by an HMR::KI-URA3 donor. We inserted into HMR::KI-URA3 an 18-bp inverted repeat where one arm had a 4-bp insertion. Most GCs yield MAT::KI-ura3::QP + 4 (Ura-) outcomes, but template-switching produces Ura+ colonies, losing the 4-bp insertion. If the QP arm without the insertion is first encountered by repair DNA polymerase and is then (mis)used as a template, the palindrome is perfected. When the QP + 4 arm is encountered first, Ura+ derivatives only occur after second-end capture and second-strand synthesis. QP + 4 mutations are suppressed by mismatch repair (MMR) proteins Msh2, Msh3, and Mlh1, but not Msh6. Deleting Rdh54 significantly reduces QP mutations only when events creating Ura+ occur in the context of a D-loop but not during second-strand synthesis. A similar bias is found with a proofreading-defective DNA polymerase mutation (poI3-01). DSB-induced mutations differed in several genetic requirements from spontaneous events. We also created a + 1 frameshift in the donor, expanding a run of 4 Cs to 5 Cs. Again, Ura+ recombinants markedly increased by disabling MMR, suggesting that MMR acts during GC but favors the unbroken, template strand.

Cryptococcus neoformans is a fungal pathogen of the top critical priority recognized by the World Health Organization. This clinically important fungus also serves as a eukaryotic model organism. A variety of resources have been generated to facilitate investigation of the C. neoformans species complex, including congenic pairs, well-annotated genomes, genetic editing tools, and gene deletion sets. Here, we generated a set of strains with all major organelles fluorescently marked. We tested short organelle-specific targeting sequences and successfully labeled the following organelles by fusing the targeting sequences with a fluorescence protein: the plasma membrane, the nucleus, the peroxisome, and the mitochondrion. We used native cryptococcal Golgi and late endosomal proteins fused with a fluorescent protein to label these two organelles. These fluorescence markers were verified via colocalization using organelle-specific dyes. All the constructs for the fluorescent protein tags were integrated in an intergenic safe haven region. These organelle-marked strains were examined for growth and various phenotypes. We demonstrated that these tagged strains could be employed to track cryptococcal interaction with the host in phagocytosis assays. These strains also allowed us to discover remarkable differences in the dynamics of proteins targeted to different organelles during sexual reproduction. Additionally, we revealed that "dormant" spores transcribed and synthesized their own proteins and trafficked the proteins to the appropriate subcellular compartments, demonstrating that spores are metabolically active. We anticipate that these newly generated fluorescent markers will greatly facilitate further investigation of cryptococcal biology and pathogenesis.

UV light is a potent mutagen that induces bulky DNA damage in the form of cyclobutane pyrimidine dimers (CPDs). Photodamage and other bulky lesions occurring in nuclear genomes can be repaired through nucleotide excision repair (NER), where incisions on both sides of a damaged site precede the removal of a single-stranded oligonucleotide containing the damage. Mitochondrial genomes (mtDNAs) are also susceptible to damage from UV light, but current evidence suggests that the only way to eliminate bulky mtDNA damage is through mtDNA degradation. Damage-containing oligonucleotides excised during NER can be captured with antidamage antibodies and sequenced (XR-seq) to produce high-resolution maps of active repair locations following UV exposure. We analyzed previously published datasets from Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster to identify reads originating from the mtDNA (and plastid genome in A. thaliana). In A. thaliana and S. cerevisiae, the mtDNA-mapping reads have unique length distributions compared to the nuclear-mapping reads. The dominant fragment size was 26 nt in S. cerevisiae and 28 nt in A. thaliana with distinct secondary peaks occurring in regular intervals. These reads also show a nonrandom distribution of di-pyrimidines (the substrate for CPD formation) with TT enrichment at positions 7-8 of the reads. Therefore, UV damage to mtDNA appears to result in production of DNA fragments of characteristic lengths and positions relative to the damaged location. The mechanisms producing these fragments are unclear, but we hypothesize that they result from a previously uncharacterized DNA degradation pathway or repair mechanism in mitochondria.

Gregor Mendel developed the principles of segregation and independent assortment in the mid-1800s based on his detailed analysis of several traits in pea plants. Those principles, now called Mendel's laws, in fact, explain the behavior of genes and alleles during meiosis and are now understood to underlie "Mendelian inheritance" of a wide range of traits and diseases across organisms. When asked to give examples of inheritance that do NOT follow Mendel's laws, in other words, examples of non-Mendelian inheritance, students sometimes list incomplete dominance, codominance, multiple alleles, sex-linked traits, and multigene traits and cite as their sources the Khan Academy, Wikipedia, and other online sites. Against this background, the goals of this Perspective are to (1) explain to students, healthcare workers, and other stakeholders why the examples above, in fact, display Mendelian inheritance, as they obey Mendel's laws of segregation and independent assortment, even though they do not produce classic Mendelian phenotypic ratios and (2) urge individuals with an intimate knowledge of genetic principles to monitor the accuracy of learning resources and work with us and those resources to correct information that is misleading.