Publications by authors named "Diego Fajardo"

16 Publications

  • Page 1 of 1

Effects of Altering HSPG Binding and Capsid Hydrophilicity on Retinal Transduction by AAV.

J Virol 2021 Mar 3. Epub 2021 Mar 3.

Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida, Gainesville, FL, USA

Adeno-associated viruses (AAVs) have recently emerged as the leading vector for retinal gene therapy. However, AAV vectors which are capable of achieving clinically relevant levels of transgene expression and widespread retinal transduction are still an unmet need. Using rationally designed AAV2-based capsid variants, we investigate the role of capsid hydrophilicity and hydrophobicity as it relates to retinal transduction. We show that hydrophilic, single amino acid (aa) mutations (V387R, W502H, E530K, L583R) in AAV2 negatively impact retinal transduction when heparan sulfate proteoglycan (HSPG) binding remains intact. Conversely, addition of hydrophobic point mutations to an HSPG binding deficient capsid (AAV2ΔHS) lead to increased retinal transduction in both mouse and macaque. Our top performing vector, AAV2(4pMut)ΔHS, achieved robust rod and cone photoreceptor (PR) transduction in macaque, especially in the fovea, and demonstrates the ability to spread laterally beyond the borders of the subretinal injection (SRI) bleb. This study both evaluates biophysical properties of AAV capsids that influence retinal transduction, and assesses the transduction and tropism of a novel capsid variant in a clinically relevant animal model.Rationally guided engineering of AAV capsids aims to create new generations of vectors with enhanced potential for human gene therapy. By applying rational design principles to AAV2-based capsids, we evaluated the influence of hydrophilic and hydrophobic amino acid (aa) mutations on retinal transduction as it relates to vector administration route. Through this approach we identified a largely deleterious relationship between hydrophilic aa mutations and canonical HSPG binding by AAV2-based capsids. Conversely, the inclusion of hydrophobic aa substitutions on a HSPG binding deficient capsid (AAV2ΔHS), generated a vector capable of robust rod and cone photoreceptor (PR) transduction. This vector AAV2(4pMut)ΔHS also demonstrates a remarkable ability to spread laterally beyond the initial subretinal injection (SRI) bleb, making it an ideal candidate for the treatment of retinal diseases which require a large area of transduction.
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http://dx.doi.org/10.1128/JVI.02440-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8139652PMC
March 2021

Novel AAV44.9-Based Vectors Display Exceptional Characteristics for Retinal Gene Therapy.

Mol Ther 2020 06 11;28(6):1464-1478. Epub 2020 Apr 11.

Department of Ophthalmology, University of Florida, Gainesville, FL, USA. Electronic address:

The majority of inherited retinal diseases (IRDs) are caused by mutations in genes expressed in photoreceptors (PRs). The ideal vector to address these conditions is one that transduces PRs in large areas of retina with the smallest volume/lowest titer possible, and efficiently transduces foveal cones, the cells responsible for acute, daylight vision that are often the only remaining area of functional retina in IRDs. The purpose of our study was to evaluate the retinal tropism and potency of a novel capsid, AAV44.9, and rationally designed derivatives thereof. We found that AAV44.9 and AAV44.9(E531D) transduced retinas of subretinally injected (SRI) mice with higher efficiency than did benchmark AAV5- and AAV8-based vectors. In macaques, highly efficient cone and rod transduction was observed following submacular and peripheral SRI. AAV44.9- and AAV44.9(E531D)-mediated GFP fluorescence extended laterally well beyond SRI bleb margins. Notably, extrafoveal injection (i.e., fovea not detached during surgery) led to transduction of up to 98% of foveal cones. AAV44.9(E531D) efficiently transduced parafoveal and perifoveal cones, whereas AAV44.9 did not. AAV44.9(E531D) was also capable of restoring retinal function to a mouse model of IRD. These novel capsids will be useful for addressing IRDs that would benefit from an expansive treatment area.
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http://dx.doi.org/10.1016/j.ymthe.2020.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264435PMC
June 2020

A Novel Mouse Model of USH1B Reveals Auditory and Visual System Haploinsufficiencies.

Front Neurosci 2019 22;13:1255. Epub 2019 Nov 22.

Department of Ophthalmology, University of Florida, Gainesville, FL, United States.

Usher's syndrome is the most common combined blindness-deafness disorder with USH1B, caused by mutations in , resulting in the most severe phenotype. The existence of numerous, naturally occurring mice harboring variable mutations on different genetic backgrounds has complicated the characterization of MYO7A knockout (KO) and heterozygote mice. We generated a novel MYO7A KO mouse ( ) that is easily genotyped, maintained, and confirmed to be null for MYO7A in both the eye and inner ear. Like USH1B patients, mice are profoundly deaf, and display near complete loss of inner and outer cochlear hair cells (HCs). No gross structural changes were observed in vestibular HCs. mice exhibited modest declines in retinal function but, unlike patients, no loss of retinal structure. We attribute the latter to differential expression of MYO7A in mouse vs. primate retina. Interestingly, heterozygous mice had reduced numbers of cochlear HCs and concomitant reductions in auditory function relative to controls. Notably, this is the first report that loss of a single allele significantly alters auditory structure and function and suggests that audiological characterization of USH1B carriers is warranted. Maintenance of vestibular HCs in mice suggests that gene replacement could be used to correct the vestibular dysfunction in USH1B patients. While mice do not exhibit sufficiently robust retinal phenotypes to be used as a therapeutic outcome measure, they can be used to assess expression of vectored on a null background and generate valuable pre-clinical data toward the treatment of USH1B.
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http://dx.doi.org/10.3389/fnins.2019.01255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883748PMC
November 2019

Somatic Gene Editing of by AAV-CRISPR/Cas9 Alters Retinal Structure and Function in Mouse and Macaque.

Hum Gene Ther 2019 05 20;30(5):571-589. Epub 2018 Dec 20.

1 Department of Ophthalmology, University of Florida, Gainesville, Florida.

Mutations in , the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of autosomal dominant cone-rod dystrophy (CORD6). Significant progress toward clinical application of gene replacement therapy for Leber congenital amaurosis (LCA) due to recessive mutations in (LCA1) has been made, but a different approach is needed to treat CORD6 where gain of function mutations cause dysfunction and dystrophy. The CRISPR/Cas9 gene editing system efficiently disrupts genes at desired loci, enabling complete gene knockout or homology directed repair. Here, adeno-associated virus (AAV)-delivered CRISPR/Cas9 was used specifically to edit/disrupt this gene's early coding sequence in mouse and macaque photoreceptors , thereby knocking out retGC1 expression and demonstrably altering retinal function and structure. Neither preexisting nor induced Cas9-specific T-cell responses resulted in ocular inflammation in macaques, nor did it limit editing. The results show, for the first time, the ability to perform somatic gene editing in primates using AAV-CRISPR/Cas9 and demonstrate the viability this approach for treating inherited retinal diseases in general and CORD6 in particular.
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http://dx.doi.org/10.1089/hum.2018.193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534089PMC
May 2019

Topological Data Analysis as a Morphometric Method: Using Persistent Homology to Demarcate a Leaf Morphospace.

Front Plant Sci 2018 25;9:553. Epub 2018 Apr 25.

Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, United States.

Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied as a filtration across simplicial complexes (or more simply, a method to measure topological features of spaces across different spatial resolutions), to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families are made. The approach predicts plant family above chance. The application of a persistent homology method, using topological features, to measure leaf shape allows for a unified morphometric framework to measure plant form, including shapes, textures, patterns, and branching architectures.
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http://dx.doi.org/10.3389/fpls.2018.00553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996898PMC
April 2018

Strategies for optimizing BioNano and Dovetail explored through a second reference quality assembly for the legume model, Medicago truncatula.

BMC Genomics 2017 08 4;18(1):578. Epub 2017 Aug 4.

National Center for Genome Resources, 2935 Rodeo Park Drive East, Santa Fe, NM, 87505, USA.

Background: Third generation sequencing technologies, with sequencing reads in the tens- of kilo-bases, facilitate genome assembly by spanning ambiguous regions and improving continuity. This has been critical for plant genomes, which are difficult to assemble due to high repeat content, gene family expansions, segmental and tandem duplications, and polyploidy. Recently, high-throughput mapping and scaffolding strategies have further improved continuity. Together, these long-range technologies enable quality draft assemblies of complex genomes in a cost-effective and timely manner.

Results: Here, we present high quality genome assemblies of the model legume plant, Medicago truncatula (R108) using PacBio, Dovetail Chicago (hereafter, Dovetail) and BioNano technologies. To test these technologies for plant genome assembly, we generated five assemblies using all possible combinations and ordering of these three technologies in the R108 assembly. While the BioNano and Dovetail joins overlapped, they also showed complementary gains in continuity and join numbers. Both technologies spanned repetitive regions that PacBio alone was unable to bridge. Combining technologies, particularly Dovetail followed by BioNano, resulted in notable improvements compared to Dovetail or BioNano alone. A combination of PacBio, Dovetail, and BioNano was used to generate a high quality draft assembly of R108, a M. truncatula accession widely used in studies of functional genomics. As a test for the usefulness of the resulting genome sequence, the new R108 assembly was used to pinpoint breakpoints and characterize flanking sequence of a previously identified translocation between chromosomes 4 and 8, identifying more than 22.7 Mb of novel sequence not present in the earlier A17 reference assembly.

Conclusions: Adding Dovetail followed by BioNano data yielded complementary improvements in continuity over the original PacBio assembly. This strategy proved efficient and cost-effective for developing a quality draft assembly compared to traditional reference assemblies.
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http://dx.doi.org/10.1186/s12864-017-3971-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5545040PMC
August 2017

Alternative Watson-Crick Synthetic Genetic Systems.

Cold Spring Harb Perspect Biol 2016 Nov 1;8(11). Epub 2016 Nov 1.

The Westheimer Institute for Science and Technology, The Foundation for Applied Molecular Evolution, Alachua, Florida 32615.

In its "grand challenge" format in chemistry, "synthesis" as an activity sets out a goal that is substantially beyond current theoretical and technological capabilities. In pursuit of this goal, scientists are forced across uncharted territory, where they must answer unscripted questions and solve unscripted problems, creating new theories and new technologies in ways that would not be created by hypothesis-directed research. Thus, synthesis drives discovery and paradigm changes in ways that analysis cannot. Described here are the products that have arisen so far through the pursuit of one grand challenge in synthetic biology: Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using genetic and catalytic biopolymers different from those that have been delivered to us by natural history on Earth. The outcomes in technology include new diagnostic tools that have helped personalize the care of hundreds of thousands of patients worldwide. In science, the effort has generated a fundamentally different view of DNA, RNA, and how they work.
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http://dx.doi.org/10.1101/cshperspect.a023770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088529PMC
November 2016

Amylose content decreases during tuber development in potato.

J Sci Food Agric 2016 Oct 6;96(13):4560-4. Epub 2016 Apr 6.

Vegetable Crops Research Unit, USDA-ARS, 1575 Linden Drive, Madison, WI 53706, USA.

Background: Potato starch is composed primarily of amylopectin and amylose in an approximately 3:1 ratio. Amylose is considered to be nutritionally desirable in North American and European markets, so there is interest in finding strategies to increase the amylose content of potato starch. There is also interest in marketing 'baby' potatoes, which are harvested when they are physiologically immature. This study was carried out to determine weekly changes in amylose content in potato tubers of 11 North American cultivars during the growing season. The trial was repeated across 3 years.

Results: We determined that amylose content is highest early and it decreases in a linear fashion as the growing season progresses. Mean amylose content across cultivars and years declined from 30.0% in late June to 26.8% in late August. The rate of decrease varied across years, with slopes of linear regression plots ranging from -0.17 in 2012 to -0.74 in 2011. Amylose content in tuber starch varied among cultivars, with the highest levels observed in Ranger Russet (30.7%) and White Pearl (31.6%); it was lowest in Kennebec (25.7%) and Langlade (25.6%).

Conclusions: This study adds to a growing body of literature on the nutritional value of immature potato tubers. In addition to having higher levels of some phytonutrients, as reported in other studies, immature tubers have a higher proportion of amylose in the starch. This is nutritionally desirable in affluent regions where high fiber content is more important than calories from carbohydrates. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.
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http://dx.doi.org/10.1002/jsfa.7673DOI Listing
October 2016

Development and validation of 697 novel polymorphic genomic and EST-SSR markers in the American cranberry (Vaccinium macrocarpon Ait.).

Molecules 2015 Jan 27;20(2):2001-13. Epub 2015 Jan 27.

Department of Horticulture, University of Wisconsin-Madison, 1575 Linden Dr. Madison, WI 53706, USA.

The American cranberry, Vaccinium macrocarpon Ait., is an economically important North American fruit crop that is consumed because of its unique flavor and potential health benefits. However, a lack of abundant, genome-wide molecular markers has limited the adoption of modern molecular assisted selection approaches in cranberry breeding programs. To increase the number of available markers in the species, this study identified, tested, and validated microsatellite markers from existing nuclear and transcriptome sequencing data. In total, new primers were designed, synthesized, and tested for 979 SSR loci; 697 of the markers amplified allele patterns consistent with single locus segregation in a diploid organism and were considered polymorphic. Of the 697 polymorphic loci, 507 were selected for additional genetic diversity and segregation analyses in 29 cranberry genotypes. More than 95% of the 507 loci did not display segregation distortion at the p < 0.05 level, and contained moderate to high levels of polymorphism with a polymorphic information content >0.25. This comprehensive collection of developed and validated microsatellite loci represents a substantial addition to the molecular tools available for geneticists, genomicists, and breeders in cranberry and Vaccinium.
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http://dx.doi.org/10.3390/molecules20022001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272188PMC
January 2015

Tuber starch amylose content is associated with cold-induced sweetening in potato.

Food Sci Nutr 2014 Nov 30;2(6):628-33. Epub 2014 Jun 30.

USDA-ARS, Vegetable Crops Research Unit and Department of Horticulture, University of Wisconsin-Madison Madison, Wisconsin.

Cold-induced sweetening (CIS) is the accumulation of reducing sugars in potato tubers at low storage temperatures. It is undesirable because it results in dark fry products. Our study evaluated the relationship between genetic resistance to CIS and two starch parameters, amylose content and starch granule size. We found that the amylose content in four CIS-resistant varieties was higher than that in five susceptible varieties. Amylose content was influenced not only by variety but also storage, production year, and field location. However, interactions between amylose content and environmental variables were not detected. In contrast, starch granule size was not associated with CIS resistance. No effect of storage on starch granule size was detected, and interactions among variety, production year, and field location were observed. Tuber starch amylose content should be considered a source of variability for CIS.
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http://dx.doi.org/10.1002/fsn3.137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4256565PMC
November 2014

The American cranberry: first insights into the whole genome of a species adapted to bog habitat.

BMC Plant Biol 2014 Jun 13;14:165. Epub 2014 Jun 13.

USDA, Agricultural Research Service, Genetic Improvement of Fruits and Vegetables Lab, 125A Lake Oswego Rd,, Chatsworth, New Jersey 08019, USA.

Background: The American cranberry (Vaccinium macrocarpon Ait.) is one of only three widely-cultivated fruit crops native to North America- the other two are blueberry (Vaccinium spp.) and native grape (Vitis spp.). In terms of taxonomy, cranberries are in the core Ericales, an order for which genome sequence data are currently lacking. In addition, cranberries produce a host of important polyphenolic secondary compounds, some of which are beneficial to human health. Whereas next-generation sequencing technology is allowing the advancement of whole-genome sequencing, one major obstacle to the successful assembly from short-read sequence data of complex diploid (and higher ploidy) organisms is heterozygosity. Cranberry has the advantage of being diploid (2n = 2x = 24) and self-fertile. To minimize the issue of heterozygosity, we sequenced the genome of a fifth-generation inbred genotype (F ≥ 0.97) derived from five generations of selfing originating from the cultivar Ben Lear.

Results: The genome size of V. macrocarpon has been estimated to be about 470 Mb. Genomic sequences were assembled into 229,745 scaffolds representing 420 Mbp (N50 = 4,237 bp) with 20X average coverage. The number of predicted genes was 36,364 and represents 17.7% of the assembled genome. Of the predicted genes, 30,090 were assigned to candidate genes based on homology. Genes supported by transcriptome data totaled 13,170 (36%).

Conclusions: Shotgun sequencing of the cranberry genome, with an average sequencing coverage of 20X, allowed efficient assembly and gene calling. The candidate genes identified represent a useful collection to further study important biochemical pathways and cellular processes and to use for marker development for breeding and the study of horticultural characteristics, such as disease resistance.
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http://dx.doi.org/10.1186/1471-2229-14-165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076063PMC
June 2014

The American cranberry mitochondrial genome reveals the presence of selenocysteine (tRNA-Sec and SECIS) insertion machinery in land plants.

Gene 2014 Feb 14;536(2):336-43. Epub 2013 Dec 14.

USDA, Agricultural Research Service, Vegetable Crops Research Unit, 1575 Linden Drive, Madison, WI 53706, USA. Electronic address:

This is the first de novo assembly and annotation of a complete mitochondrial genome in the Ericales order from the American cranberry (Vaccinium macrocarpon Ait.). Moreover, only four complete Asterid mitochondrial genomes have been made publicly available. The cranberry mitochondrial genome was assembled and reconstructed from whole genome 454 Roche GS-FLX and Illumina shotgun sequences. Compared with other Asterids, the reconstruction of the genome revealed an average size mitochondrion (459,678 nt) with relatively little repetitive sequences and DNA of plastid origin. The complete mitochondrial genome of cranberry was annotated obtaining a total of 34 genes classified based on their putative function, plus three ribosomal RNAs, and 17 transfer RNAs. Maternal organellar cranberry inheritance was inferred by analyzing gene variation in the cranberry mitochondria and plastid genomes. The annotation of cranberry mitochondrial genome revealed the presence of two copies of tRNA-Sec and a selenocysteine insertion sequence (SECIS) element which were lost in plants during evolution. This is the first report of a land plant possessing selenocysteine insertion machinery at the sequence level.
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http://dx.doi.org/10.1016/j.gene.2013.11.104DOI Listing
February 2014

Rapid high throughput amylose determination in freeze dried potato tuber samples.

J Vis Exp 2013 Oct 14(80). Epub 2013 Oct 14.

USDA-ARS and Department of Horticulture, University of Wisconsin - Madison.

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones. 
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http://dx.doi.org/10.3791/50407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940341PMC
October 2013

Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa.

Proc Natl Acad Sci U S A 2012 Feb 23;109(6):2132-7. Epub 2012 Jan 23.

Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Hereditary retinal blindness is caused by mutations in genes expressed in photoreceptors or retinal pigment epithelium. Gene therapy in mouse and dog models of a primary retinal pigment epithelium disease has already been translated to human clinical trials with encouraging results. Treatment for common primary photoreceptor blindness, however, has not yet moved from proof of concept to the clinic. We evaluated gene augmentation therapy in two blinding canine photoreceptor diseases that model the common X-linked form of retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, which encodes a photoreceptor ciliary protein, and provide evidence that the therapy is effective. After subretinal injections of adeno-associated virus-2/5-vectored human RPGR with human IRBP or GRK1 promoters, in vivo imaging showed preserved photoreceptor nuclei and inner/outer segments that were limited to treated areas. Both rod and cone photoreceptor function were greater in treated (three of four) than in control eyes. Histopathology indicated normal photoreceptor structure and reversal of opsin mislocalization in treated areas expressing human RPGR protein in rods and cones. Postreceptoral remodeling was also corrected: there was reversal of bipolar cell dendrite retraction evident with bipolar cell markers and preservation of outer plexiform layer thickness. Efficacy of gene therapy in these large animal models of X-linked retinitis pigmentosa provides a path for translation to human treatment.
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http://dx.doi.org/10.1073/pnas.1118847109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277562PMC
February 2012

Maize rough endosperm3 encodes an RNA splicing factor required for endosperm cell differentiation and has a nonautonomous effect on embryo development.

Plant Cell 2011 Dec 2;23(12):4280-97. Epub 2011 Dec 2.

Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, USA.

Endosperm and embryo development are coordinated via epigenetic regulation and signaling between these tissues. In maize (Zea mays), the endosperm-embryo signals are not known, but endosperm cellularization is a key event for embryos to form shoots and roots. We screened seed mutants for nonautonomous functions in endosperm and embryo development with genetically nonconcordant seeds and identified the recessive mutant rough endosperm3 (rgh3). The wild-type Rgh3 allele is required in the endosperm for embryos to develop and has an autonomous role in embryo and seedling development. Endosperm cell differentiation is defective in rgh3. Results from endosperm cell culture indicate that rgh3 mutants remain in a proliferative state through mid-seed development. Rgh3 encodes the maize U2AF(35) Related Protein (URP), an RNA splicing factor involved in both U2 and U12 splicing. The Rgh3 allele produces at least 19 alternative splice variants with only one isoform encoding a full-length ortholog to URP. The full-length RGH3α isoform localizes to the nucleolus and displays a speckled pattern within the nucleoplasm, and RGH3α colocalizes with U2AF(65). A survey of alternatively spliced transcripts found that, in the rgh3 mutant, a fraction of noncanonical splicing events are altered. Our findings suggest that differentiation of maize endosperm cell types is necessary for embryos to develop. The molecular cloning of Rgh3 suggests that alternative RNA splicing is needed for cell differentiation, development, and plant viability.
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http://dx.doi.org/10.1105/tpc.111.092163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3269866PMC
December 2011

Sequence-indexed mutations in maize using the UniformMu transposon-tagging population.

BMC Genomics 2007 May 9;8:116. Epub 2007 May 9.

Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA.

Background: Gene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations.

Results: Transposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill.

Conclusion: We show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.
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http://dx.doi.org/10.1186/1471-2164-8-116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1878487PMC
May 2007
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