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1.
CBE Life Sci Educ ; 20(3): ar46, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34415196

RESUMEN

Course-based undergraduate research experiences (CUREs) engage students in authentic research experiences in a course format and can sometimes result in the publication of that research. However, little is known about student-author perceptions of CURE publications. In this study, we examined how students perceive they benefit from authoring a CURE publication and what they believe is required for authorship of a manuscript in a peer-reviewed journal. All 16 students who were enrolled in a molecular genetics CURE during their first year of college participated in semistructured interviews during their fourth year. At the time of the interviews, students had been authors of a CURE publication for a year and a half. Students reported that they benefited personally and professionally from the publication. Students had varying perceptions of what is required for authorship, but every student thought that writing the manuscript was needed, and only two mentioned needing to approve the final draft. Additionally, we identified incomplete conceptions that students had about CURE publications. This work establishes student-perceived benefits from CURE publications and highlights the need for authorship requirements to be explicitly addressed in CUREs.


Asunto(s)
Estudiantes , Universidades , Autoria , Humanos , Percepción
2.
MethodsX ; 6: 1-5, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30591915

RESUMEN

The development of gene editing technologies, especially the CRISPR-Cas9 system, has been pivotal for understanding the functional role of proteins. Rapid and efficient genotyping methods are necessary to screen for generated mutations and streamline the isolation of homozygotes. CRISPR-Cas9 system targeting a single site in the gene typically results in small indels. Many genotyping methods utilize the heteroduplex that is formed when wild-type and mutant amplicons with small indels anneal during PCR creating a bubble due to mismatched strands. These methods include T7 endonuclease/Cel-I assay, high resolution melting (HRM) analysis, and heteroduplex mobility assay (HMA). Our protocol explains a simple, two step method of a mixing HMA (mHMA) to identify homozygous mutants, a modification of the previously published HMA. We have utilized the mHMA for screening and genotyping numerous CRISPR generated models. The mHMA method to differentiate homozygous wild type from homozygous mutant animals eliminates - •DNA sequencing, even with small indels that can be difficult to discern on a gel.•additional enzymatic reaction steps, such as with the T7EI/Cel-I assay.•specialized equipment and analysis tools, such as with HRM analysis.

3.
J Genet ; 97(5): 1315-1325, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30555080

RESUMEN

Nodal-related protein (ndr2) is amember of the transforming growth factor type ß superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced and the remaining alleles were obtained via random insertion, gene targeting (TALEN) or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos.We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted polymerase chain reaction amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in the loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into the structure-function of the ndr2 gene.


Asunto(s)
Sistemas CRISPR-Cas , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Ribonucleoproteínas/genética , Proteínas de Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Holoprosencefalia/genética , Péptidos y Proteínas de Señalización Intracelular/química , Modelos Moleculares , Fenotipo , Dominios Proteicos , Ribonucleoproteínas/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química
4.
NPJ Breast Cancer ; 4: 29, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30182054

RESUMEN

The key negative regulatory gene of the RAS pathway, NF1, is mutated or deleted in numerous cancer types and is associated with increased cancer risk and drug resistance. Even though women with neurofibromatosis (germline NF1 mutations) have a substantially increased breast cancer risk at a young age and NF1 is commonly mutated in sporadic breast cancers, we have a limited understanding of the role of NF1 in breast cancer. We utilized CRISPR-Cas9 gene editing to create Nf1 rat models to evaluate the effect of Nf1 deficiency on tumorigenesis. The resulting Nf1 indels induced highly penetrant, aggressive mammary adenocarcinomas that express estrogen receptor (ER) and progesterone receptor (PR). We identified distinct Nf1 mRNA and protein isoforms that were altered during tumorigenesis. To evaluate NF1 in human breast cancer, we analyzed genomic changes in a data set of 2000 clinically annotated breast cancers. We found NF1 shallow deletions in 25% of sporadic breast cancers, which correlated with poor clinical outcome. To identify biological networks impacted by NF1 deficiency, we constructed gene co-expression networks using weighted gene correlation network analysis (WGCNA) and identified a network connected to ESR1 (estrogen receptor). Moreover, NF1-deficient cancers correlated with established RAS activation signatures. Estrogen-dependence was verified by estrogen-ablation in Nf1 rats where rapid tumor regression was observed. Additionally, Nf1 deficiency correlated with increased estrogen receptor phosphorylation in mammary adenocarcinomas. These results demonstrate a significant role for NF1 in both NF1-related breast cancer and sporadic breast cancer, and highlight a potential functional link between neurofibromin and the estrogen receptor.

5.
Dis Model Mech ; 9(7): 759-67, 2016 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-27482814

RESUMEN

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by the occurrence of nerve sheath tumors and considerable clinical heterogeneity. Some translational studies have been limited by the lack of animal models available for assessing patient-specific mutations. In order to test therapeutic approaches that might restore function to the mutated gene or gene product, we developed mice harboring NF1 patient-specific mutations including a nonsense mutation (c.2041C>T; p.Arg681*) and a missense mutation (c.2542G>C; p.Gly848Arg). The latter is associated with the development of multiple plexiform neurofibromas along spinal nerve roots. We demonstrate that the human nonsense NF1(Arg681*) and missense NF1(Gly848Arg) mutations have different effects on neurofibromin expression in the mouse and each recapitulates unique aspects of the NF1 phenotype, depending upon the genetic context when assessed in the homozygous state or when paired with a conditional knockout allele. Whereas the missense Nf1(Gly848Arg) mutation fails to produce an overt phenotype in the mouse, animals homozygous for the nonsense Nf1(Arg681*) mutation are not viable. Mice with one Nf1(Arg681*) allele in combination with a conditional floxed Nf1 allele and the DhhCre transgene (Nf1(4F/Arg681*); DhhCre) display disorganized nonmyelinating axons and neurofibromas along the spinal column, which leads to compression of the spinal cord and paralysis. This model will be valuable for preclinical testing of novel nonsense suppression therapies using drugs to target in-frame point mutations that create premature termination codons in individuals with NF1.


Asunto(s)
Codón sin Sentido/genética , Mutación Missense/genética , Neurofibromatosis 1/genética , Neurofibromina 1/genética , Animales , Modelos Animales de Enfermedad , Embrión de Mamíferos/patología , Humanos , Integrasas/metabolismo , Ratones , Neurofibroma/patología , Fenotipo , Nervio Ciático/patología , Nervio Ciático/ultraestructura , Médula Espinal/patología , Médula Espinal/ultraestructura
6.
J Food Prot ; 79(6): 922-8, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27296595

RESUMEN

Salmonella bacteria may internalize into tomato pulp when warm tomatoes from the field are submerged into colder water. Several washing steps may follow the initial washing and packing of tomatoes at the packinghouses; the potential for internalization into tomatoes in subsequent washing steps when tomatoes have a cooler pulp temperature is unknown. Our objective was to evaluate Salmonella internalization into mature green and red tomatoes with ambient (21°C) and refrigeration (4°C) pulp temperatures when they were submerged into water at various temperature differentials, simulating repacking and fresh-cut operations. Red (4°C and 21°C) and mature green (21°C) tomatoes were submerged (6 cm) into a six-strain Salmonella cocktail (6 log CFU/ml) and maintained at ±5 and 0°C temperature differentials for varying time intervals, ranging from 30 s to 5 min. Following submersion, tomatoes were surface sterilized using 70% ethanol, the stem abscission zone and blossom end epidermis were removed, and cores were recovered, separated into three segments, and analyzed. Salmonella populations in the segments were enumerated by most probable number (MPN). The effects of temperature differential and maturity on Salmonella populations were analyzed; results were considered significant at a P value of ≥0.5. Internalized populations were not significantly different (P ≥0.5) across temperature differentials. Salmonella internalization was seen in tomatoes under all treatment conditions and was highest in the segment immediately below the stem abscission zone. However, populations were low (typically >1 log MPN per segment) and varied greatly across temperature differentials. This suggests that the temperature differential between tomatoes and water beyond the initial packinghouse may be less important than submersion time in Salmonella internalization.


Asunto(s)
Lycopersicon esculentum/microbiología , Temperatura , Recuento de Colonia Microbiana , Manipulación de Alimentos , Microbiología de Alimentos , Salmonella , Factores de Tiempo , Agua
7.
PLoS One ; 11(5): e0155812, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27224051

RESUMEN

Tyrosinase is a key enzyme in melanin biosynthesis. Mutations in the gene encoding tyrosinase (Tyr) cause oculocutaneous albinism (OCA1) in humans. Alleles of the Tyr gene have been useful in studying pigment biology and coat color formation. Over 100 different Tyr alleles have been reported in mice, of which ≈24% are spontaneous mutations, ≈60% are radiation-induced, and the remaining alleles were obtained by chemical mutagenesis and gene targeting. Therefore, most mutations were random and could not be predicted a priori. Using the CRISPR-Cas9 system, we targeted two distinct regions of exon 1 to induce pigmentation changes and used an in vivo visual phenotype along with heteroduplex mobility assays (HMA) as readouts of CRISPR-Cas9 activity. Most of the mutant alleles result in complete loss of tyrosinase activity leading to an albino phenotype. In this study, we describe two novel in-frame deletion alleles of Tyr, dhoosara (Sanskrit for gray) and chandana (Sanskrit for sandalwood). These alleles are hypomorphic and show lighter pigmentation phenotypes of the body and eyes. This study demonstrates the utility of CRISPR-Cas9 system in generating domain-specific in-frame deletions and helps gain further insights into structure-function of Tyr gene.


Asunto(s)
Alelos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Color del Cabello/genética , Monofenol Monooxigenasa/genética , Eliminación de Secuencia , Animales , Femenino , Masculino , Ratones
8.
Fungal Biol ; 119(5): 370-82, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25937065

RESUMEN

Recently diverged species may form complexes of morphologically similar, yet genetically distinct lineages that occur in overlapping geographic ranges and niches. Using a multilocus sequencing approach we discovered that gummy stem blight of cucurbits is caused by three genetically distinct species: Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), Stagonosporopsis citrulli, and Stagonosporopsis caricae, which had previously been considered only a pathogen of papaya. Experiments showed that all three species are pathogenic to cucurbits in the genera Cucurbita, Cucumis, and Citrullus, but only S. caricae is aggressive to papaya. Species tree estimates show that S. citrulli and S. cucurbitacearum are phylogenetically distinct sister species, and that S. caricae is the ancestral lineage. The time estimate for divergence of S. caricae from the ancestor of S. cucurbitacearum and S. citrulli at 72 900 YBP pre-dates domestication of papaya and Cucurbita species in the American tropics. The divergence estimate observed for S. cucurbitacearum and S. citrulli at 10 900 YBP suggests that diversification of Cucurbita species and domestication of gourds and squashes could have driven their divergence. This work highlights the use of molecular systematics and population genetics to elucidate genetic identity among previously unassociated fungi and to understand the patterns of pathogen diversification.


Asunto(s)
Ascomicetos/genética , Ascomicetos/fisiología , Citrus/microbiología , Evolución Molecular , Especificidad del Huésped , Magnoliopsida/microbiología , Enfermedades de las Plantas/microbiología , Ascomicetos/clasificación , Ascomicetos/aislamiento & purificación , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Filogenia
9.
Mycologia ; 106(3): 415-23, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24871592

RESUMEN

Exobasidium leaf and fruit spot of blueberry (Vaccinium section Cyanococcus) is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA. To determine whether this disease is caused by a new species of Exobasidium, we studied the morphology and phylogenetic relationship of the causal fungus compared with other members of the genus, including the type species E. vaccinii and other species that parasitize blueberry and cranberry (V. macrocarpon). Both scanning electron microscopy and light microscopy were used for morphological characterization. For phylogenetic analyses, we sequenced the large subunit of the rDNA (LSU) from 10 isolates collected from leaf or fruit spots of rabbiteye blueberry (V. virgatum), highbush blueberry (V. corymbosum) and southern highbush blueberry (Vaccinium interspecific hybrid) from Georgia and North Carolina and six isolates from leaf spots of lowbush blueberry (V. angustifolium) from Maine and Nova Scotia, Canada. LSU was sequenced from isolates causing red leaf disease of lowbush blueberry and red leaf spot (E. rostrupii) and red shoot (E. perenne) of cranberry. In addition, LSU sequences from GenBank, including sequences with high similarity to the emerging parasite and from Exobasidium spp. parasitizing other Vaccinium spp. and related hosts, were obtained. All sequences were aligned and subjected to phylogenetic analyses. Results indicated that the emerging parasite in the southeastern USA differs morphologically and phylogenetically from other described species and is described herein as Exobasidium maculosum. Within the southeastern USA, clustering based on host species, host tissue type (leaf or fruit) or geographic region was not detected; however, leaf spot isolates from lowbush blueberry were genetically different and likely represent a unique species.


Asunto(s)
Basidiomycota/aislamiento & purificación , Basidiomycota/fisiología , Arándanos Azules (Planta)/microbiología , Enfermedades de las Plantas/microbiología , Vaccinium macrocarpon/microbiología , Basidiomycota/clasificación , Frutas/microbiología , Datos de Secuencia Molecular , Filogenia , Hojas de la Planta/microbiología , Sudeste de Estados Unidos
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