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All sourcesNASA NTRSDOE OSTI
NASA NTRSDate not supplied

Space Algae: Understanding the Genomic Impacts on Microalgae After Growth in the International Space Station

Plants and microbes can be used for biological support of crewed space missions. The radiation and microgravity environment of spaceflight is expected to increase genetic mutation of all organisms. It is essential to understand how spaceflight impacts mutation rates in photosynthetic organisms to enable appropriate countermeasures and ensure productivity during long duration and deep space missions. The Space Algae flight experiments to the International Space Station (ISS) are studying the genomic stability of microalgae that could potentially be used in biological life support systems. Space Algae-1 grew ultraviolet light mutagenized Chlamydomonas reinhardtii in the VEGGIE plant growth chamber for approximately 40 mitotic generations over one month on the ISS. Whole genome sequencing from pooled cell samples every 10 generations revealed that spaceflight cultures had an ~50% increase in DNA polymorphisms relative to ground controls. These mutations had a novel base substitution signature and suggested a risk that microalgae may be unstable for long-term production in space. Space Algae-2 is focusing on the edible cyanobacterium Arthrospira platensis, commonly known as Spirulina. This experiment seeks to grow serial cultures to allow the organism to evolve in long-term spaceflight. Biological responses of the cells to spaceflight will be assessed with multi-omics analyses to determine mutation load, gene/protein expression, metabolic/nutritional composition, and cell morphology.

Algae↗
DOE OSTI2025

Combining computational modeling and experimental library screening to affinity-mature VEEV-neutralizing antibody F5

Engineered monoclonal antibodies have proven to be highly effective therapeutics in recent viral outbreaks. However, despite technical advancements, an ability to rapidly adapt or increase antibody affinity and by extension, therapeutic efficacy, has yet to be fully realized. We endeavored to stand-up such a pipeline using molecular modeling combined with experimental library screening to increase the affinity of F5, a monoclonal antibody with potent neutralizing activity against Venezuelan Equine Encephalitis Virus (VEEV), to recombinant VEEV (IAB) E1E2 antigen. We modeled the F5/E1E2 binding interface and generated predictions for mutations to improve binding using a Rosetta-based approach and dTERMen, an informatics approach. The modeling was complicated by the fact that a high-resolution structure of F5 is not available and the H3 loop of F5 exceeds the length for which current modeling approaches can determine a unique structure. A subset of the predicted mutations from both methods were incorporated into a phage display library of scFvs. This library and a library generated by error-prone PCR were screened for binding affinity to the recombinant antigen. Results from the screens identified favorable mutations which were incorporated into 12 human-IgG1 variants. The best variant, containing eight mutations, improved KD from 0.63 nM (parental) to 0.01 nM. While this did not improve neutralization or therapeutic potency of F5 against IAB, it did increase cross-reactivity to other closely related VEEV epizootic and enzootic strains, demonstrating the potential of this method to rapidly adapt existing therapeutics to emerging viral strains.

affinity-maturation↗
DOE OSTI2026

Genomic analysis of Klebsiella aerogenes circulating in New Mexico

Klebsiella aerogenes is an opportunistic pathogen and a growing cause of healthcare-associated infections, characterized by multidrug resistance and the emergence of global high-risk clones. However, regional genomic surveillance data remain limited. Here, we sought to characterize the population structure, transmission dynamics and resistance mechanisms of clinical K. aerogenes in Albuquerque, New Mexico. We sequenced 177 clinical isolates collected between 2021 and 2023. We also developed a novel, species-specific PopPUNK database to facilitate rapid, high-resolution typing. The New Mexico K. aerogenes population was diverse but dominated by two global pandemic lineages, ST93 (47.5%) and ST4 (7.9%), which were significantly enriched for the virulence factors yersiniabactin and colibactin. Genomic evidence for recent local transmission was rare, with only four putative transmission pairs identified. The resistome was characterized by intrinsic and adaptive mutations. Nearly all isolates possessed gyrA mutations associated with decreased fluoroquinolone susceptibility. Mutations in the AmpC regulator AmpD and the outer membrane porin Omp36 were common, particularly within the dominant ST93 lineage. These mutations have been associated with increased AmpC-mediated carbapenem resistance. Our findings underscore the critical importance of genomic surveillance to monitor the transmission and evolution of adaptive resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for Promoter Deletion in the Soybean Compact Mutant Leads to Overexpression of a Gene with Homology to the C20-Gibberellin 2-Oxidase Family

Height is a critical component of plant architecture, significantly affecting crop yield. The genetic basis of this trait in soybean remains unclear. In this study, we report the characterization of the Compact mutant of soybean, which has short internodes. The candidate gene was mapped to chromosome 17, and the interval containing the causative mutation was further delineated using biparental mapping. Whole-genome sequencing of the mutant revealed an 8.7 kb deletion in the promoter of the Glyma.17g145200 gene, which encodes a member of the class III gibberellin (GA) 2-oxidases. The mutation has a dominant effect, likely via increased expression of the GA 2-oxidase transcript observed in green tissue, as a result of the deletion in the promoter of Glyma.17g145200. We further demonstrate that levels of GA precursors are altered in the Compact mutant, supporting a role in GA metabolism, and that the mutant phenotype can be rescued with exogenous GA3. We also determined that overexpression of Glyma.17g145200 in Arabidopsis results in dwarfed plants. Thus, gain of promoter activity in the Compact mutant leads to a short internode phenotype in soybean through altered metabolism of gibberellin precursors. These results provide an example of how structural variation can control an important crop trait and a role for Glyma.17g145200 in soybean architecture, with potential implications for increasing crop yield.

Biomass Analytics↗
DOE OSTI2024

In Vitro Evolution and Computational Approaches to Predict, Prevent and Control Future Pandemics

The natural tendency of virus to mutate and the under-sampling of the environment makes it difficult to become aware of the emergence of new viral strains with pandemic potential. Being able to predict what mutations make a virus more infective might allow to spot such strains with minimal sampling and potentially allow to predict/prevent the next pandemic. The team attempted to mimic natural viral mutations and recombination through computational and experimental methods producing a variety of mutants of a SARS-COV-2 protein (receptor binding domain, RBD, of spike protein) responsible for viral entry in mammalian cells. The library of mutants was then interrogated for ability and lack-there-of to interact with the host cell receptor ushering viral entry, Angiotensin-converting enzyme 2 (ACE2). The negative and positive data set is intended to “teach the rules” of virus-host receptor interaction. Additionally, the positive clones were used to screen a set of antibody mutants designed to widen the breadth of viral mutants recognition, to demonstrate that this kind of libraries could also be a tool to produce antibody therapeutics impervious to viral mutation, even before a pandemic strain is discovered.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparative Uptake Patterns of Radioactive Iodine and [18F]-Fluorodeoxyglucose (FDG) in Metastatic Differentiated Thyroid Cancers

Background: Metastatic differentiated thyroid cancer (DTC) represents a molecularly heterogeneous group of cancers with varying radioactive iodine (RAI) and [ 18 F]-fluorodeoxyglucose (FDG) uptake patterns potentially correlated with the degree of de-differentiation through the so-called “flip-flop” phenomenon. However, it is unknown if RAI and FDG uptake patterns correlate with molecular status or metastatic site. Materials and Methods: A retrospective analysis of metastatic DTC patients (n = 46) with radioactive 131-iodine whole body scan (WBS) and FDG-PET imaging between 2008 and 2022 was performed. The inclusion criteria included accessible FDG-PET and WBS studies within 1 year of each other. Studies were interpreted by two blinded radiologists for iodine or FDG uptake in extrathyroidal sites including lungs, lymph nodes, and bone. Cases were stratified by BRAF V600E mutation status, histology, and a combination of tumor genotype and histology. The data were analyzed by McNemar’s Chi-square test. Results: Lung metastasis FDG uptake was significantly more common than iodine uptake (WBS: 52%, FDG: 84%, p = 0.04), but no significant differences were found for lymph or bone metastases. Lung metastasis FDG uptake was significantly more prevalent in the papillary pattern sub-cohort (WBS: 37%, FDG: 89%, p = 0.02) than the follicular pattern sub-cohort (WBS: 75%, FDG: 75%, p = 1.00). Similarly, BRAF V600E+ tumors with lung metastases also demonstrated a preponderance of FDG uptake (WBS: 29%, FDG: 93%, p = 0.02) than BRAF V600E- tumors (WBS: 83%, FDG: 83%, p = 1.00) with lung metastases. Papillary histology featured higher FDG uptake in lung metastasis (WBS: 39%, FDG: 89%, p = 0.03) compared with follicular histology (WBS: 69%, FDG: 77%, p = 1.00). Patients with papillary pattern disease, BRAF V600E+ mutation, or papillary histology had reduced agreement between both modalities in uptake at all metastatic sites compared with those with follicular pattern disease, BRAF V600E- mutation, or follicular histology. Low agreement in lymph node uptake was observed in all patients irrespective of molecular status or histology. Conclusions: The pattern of FDG-PET and radioiodine uptake is dependent on molecular status and metastatic site, with those with papillary histology or BRAF V600E+ mutation featuring increased FDG uptake in distant metastasis. Further study with an expanded cohort may identify which patients may benefit from specific imaging modalities to recognize and surveil metastases.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1992

Evolution of catalytic RNA in the laboratory

We are interested in the biochemistry of existing RNA enzymes and in the development of RNA enzymes with novel catalytic function. The focal point of our research program has been the design and operation of a laboratory system for the controlled evolution of catalytic RNA. This system serves as working model of RNA-based life and can be used to explore the catalytic potential of RNA. Evolution requires the integration of three chemical processes: amplification, mutation, and selection. Amplification results in additional copies of the genetic material. Mutation operates at the level of genotype to introduce variability, this variability in turn being expressed as a range of phenotypes. Selection operates at the level of phenotype to reduce variability by excluding those individuals that do not conform to the prevailing fitness criteria. These three processes must be linked so that only the selected individuals are amplified, subject to mutational error, to produce a progeny distribution of mutant individuals. We devised techniques for the amplification, mutation, and selection of catalytic RNA, all of which can be performed rapidly in vitro within a single reaction vessel. We integrated these techniques in such a way that they can be performed iteratively and routinely. This allowed us to conduct evolution experiments in response to artificially-imposed selection constraints. Our objective was to develop novel RNA enzymes by altering the selection constraints in a controlled manner. In this way we were able to expand the catalytic repertoire of RNA. Our long-range objective is to develop an RNA enzyme with RNA replicase activity. If such an enzyme had the ability to produce additional copies of itself, then RNA evolution would operate autonomously and the origin of life will have been realized in the laboratory.

Joyce, Gerald F.↗
NASA NTRS1994

Estimates of cellular mutagenesis from cosmic rays

A parametric track structure model is used to estimate the cross section as a function of particle velocity and charge for mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in human fibroblast cell cultures. Experiments that report the fraction of mutations per surviving cell for human lung and skin fibroblast cells indicate small differences in the mutation cross section for these two cell lines when differences in inactivation rates between these cell lines are considered. Using models of cosmic ray transport, the mutation rate at the HGPRT locus is estimated for cell cultures in space flight and rates of about 2 to 10 x 10(exp -6) per year are found for typical spacecraft shielding. A discussion of how model assumptions may alter the predictions is also presented.

Cucinotta, Francis A.↗
NASA NTRS1994

Kinetics model for initiation and promotion for describing tumor prevalence from HZE radiation

A kinetics model for cellular repair and misrepair for multiple radiation-induced lesions (mutation-inactivation) is coupled to a two-mutation model of initiation and promotion in tissue to provide a parametric description of tumor prevalence in the Harderian gland in a mouse. Dose-response curves are described for gamma-rays and relativistic ions. The effects of nuclear fragmentation are also considered for high-energy proton and alpha particle exposures The model described provides a parametric description of age-dependent cancer induction for a wide range of radiation fields. We also consider the two hypotheses that radiation acts either solely as an initiator or as both initiator and promoter and make model calculations for fractionation exposures from gamma-rays and relativistic Fe ions. For fractionated Fe exposures, an inverse dose-rate effect is provided by a promotion hypothesis using a mutation rate for promotion typical of single-gene mutations.

Cucinotta, Francis A.↗
NASA NTRS1994

Nematode radiobiology and development in space. Results from IML-1

The Radiat experiment was one of 17 investigations which used the ESA Biorack on IML-1 (International Microgravity Laboratory) and it had two objectives. The first objective was to isolate and characterize mutations induced by cosmic rays; the second was to assess the fidelity of development in 0-gravity over two consecutive generations. Two strategies were used to isolate mutations in a set of essential genes or a specific gene and to correlate the genetic events with the passage of charged particles. The results were isolation of 60 lethal mutations whose phenotypes are related to the local pattern of energy deposition. 12 mutations in the unc-22 gene include large deletions as characterized by DNA hybridization studies. Development of nematodes proceeded through two consecutive generations with no obvious defects. Cytoplasmic determinants in embryos, nuclear location and symmetry of cellular anatomy were normal as were Mendelian segregation and recombination of genetic markers.

Nelson, Gregory A.↗
NASA NTRS1999

Genetic Regulation of Charged Particle Mutagenesis in Human Cells

Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

Kronenberg, Amy↗
NASA NTRS2002

Molecular Genetics of Root Thigmoresponsiveness in Arabidopsis thaliana

The molecular mechanisms that allow plant roots to use gravity and touch as growth guides are investigated. We are using a molecular genetic strategy in Arabidopsis thaliana to study these processes. When Arabidopsis thaliana seedlings grow on tilted hard-agar surfaces, their roots develop a wavy pattern of growth which appears to derive from a succession of left-handed and right-handed circumnutation-like processes triggered by gravity and touch stimulation (Okada and Shimura, 1990; Rutherford et al., 1998; Rutherford and Masson, 1996). Interestingly, mutations that affect root waving on tilted hard-agar surfaces can be identified and characterized. Some of these mutations affect root gravitropism, while others appear to be responsible for the production of abnormal waves (no waves, compressed or square waves, coils) without affecting gravitropism. The specific objectives of this project were to functionally characterize two genes (WVD2 and WVD6) which are required for root waving on tilted agar surfaces, but not for root gravitropism. Specific objectives included a physiological and cytological analysis of the mutants, and molecular cloning and characterization of the corresponding genes. As summarized in this paper, we have reached these objectives. We have also identified and partially characterized other mutations that affect root skewing on hard-agar surfaces (sku5-1 and ago1), and have completed our work on the root-wave phenotype associated with mutations in genes of the tryptophan biosynthesis pathway (Lynn et al., 1999; Rutherford et al., 1998; Sedbrook et al., 2000, 2002). We briefly describe our progress on the cloning and characterization of WVD6, WVD2 and SKU5, and provide a list of papers (published, or in preparation) that derived from this grant. We also discuss the biological implications of our findings, with special emphasis on the analysis of WVD2.

Masson, Patrick H.↗
NASA NTRS2003

Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia

The molecular and cellular origin of the primary neurons of the inner ear, the vestibular and spiral neurons, is reviewed including how they connect to the specific sensory epithelia and what the molecular nature of their survival is. Primary neurons of the ear depend on a single basic Helix-Loop-Helix (bHLH) protein for their formation, neurogenin 1 (ngn1). An immediate downstream gene is the bHLH gene neuronal differentiation (NeuroD). Targeted null mutations of ngn1 results in absence of primary neuron formation; targeted null mutation of NeuroD results in loss of almost all spiral and many vestibular neurons. NeuroD and a later expressed gene, Brn3a, play a role in pathfinding to and within sensory epithelia. The molecular nature of this pathfinding property is unknown. Reduction of hair cells in ngn1 null mutations suggests a clonal relationship with primary neurons. This relationship may play some role in specifying the identity of hair cells and the primary neurons that connect with them. Primary neuron neurites growth to sensory epithelia is initially independent of trophic factors released from developing sensory epithelia, but becomes rapidly dependent on those factors. Null mutations of specific neurotrophic factors lose distinct primary neuron populations which undergo rapid embryonic cell death.

Review↗
NASA NTRS2001

p53 deficiency alters the yield and spectrum of radiation-induced lacZ mutants in the brain of transgenic mice

Exposure to heavy particle radiation in the galacto-cosmic environment poses a significant risk in space exploration and the evaluation of radiation-induced genetic damage in tissues, especially in the central nervous system, is an important consideration in long-term manned space missions. We used a plasmid-based transgenic mouse model system, with the pUR288 lacZ transgene integrated in the genome of every cell of C57Bl/6(lacZ) mice, to evaluate the genetic damage induced by iron particle radiation. In order to examine the importance of genetic background on the radiation sensitivity of individuals, we cross-bred p53 wild-type lacZ transgenic mice with p53 nullizygous mice, producing lacZ transgenic mice that were either hemizygous or nullizygous for the p53 tumor suppressor gene. Animals were exposed to an acute dose of 1 Gy of iron particles and the lacZ mutation frequency (MF) in the brain was measured at time intervals from 1 to 16 weeks post-irradiation. Our results suggest that iron particles induced an increase in lacZ MF (2.4-fold increase in p53+/+ mice, 1.3-fold increase in p53+/- mice and 2.1-fold increase in p53-/- mice) and that this induction is both temporally regulated and p53 genotype dependent. Characterization of mutants based on their restriction patterns showed that the majority of the mutants arising spontaneously are derived from point mutations or small deletions in all three genotypes. Radiation induced alterations in the spectrum of deletion mutants and reorganization of the genome, as evidenced by the selection of mutants containing mouse genomic DNA. These observations are unique in that mutations in brain tissue after particle radiation exposure have never before been reported owing to technical limitations in most other mutation assays.

Non-NASA Center↗
NASA NTRS1997

Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

Non-NASA Center↗
NASA NTRS1995

Mutant quantity and quality in mammalian cells (AL) exposed to cesium-137 gamma radiation: effect of caffeine

We examined the effect of caffeine (1,3,7-trimethylxanthine) on the quantity and quality of mutations in cultured mammalian AL human-hamster hybrid cells exposed to 137Cs gamma radiation. At a dose (1.5 mg/ml for 16 h) that reduced the plating efficiency (PE) by 20%, caffeine was not itself a significant mutagen, but it increased by approximately twofold the slope of the dose-response curve for induction of S1- mutants by 137Cs gamma radiation. Molecular analysis of 235 S1- mutants using a series of DNA probes mapped to the human chromosome 11 in the AL hybrid cells revealed that 73 to 85% of the mutations in unexposed cells and in cells treated with caffeine alone, 137Cs gamma rays alone or 137Cs gamma rays plus caffeine were large deletions involving millions of base pairs of DNA. Most of these deletions were contiguous with the region of the MIC1 gene at 11p13 that encodes the S1 cell surface antigen. In other mutants that had suffered multiple marker loss, the deletions were intermittent along chromosome 11. These "complex" mutations were rare for 137Cs gamma irradiation (1/63 = 1.5%) but relatively prevalent (23-50%) for other exposure conditions. Thus caffeine appears to alter both the quantity and quality of mutations induced by 137Cs gamma irradiation.

NASA Discipline Radiation Health↗
NASA NTRS2017

Exploring Connectivity in Sequence Space of Functional RNA

Emergence of replicable genetic molecules was one of the marking points in the origin of life, evolution of which can be conceptualized as a walk through the space of all possible sequences. A theoretical concept of fitness landscape helps to understand evolutionary processes through assigning a value of fitness to each genotype. Then, evolution of a phenotype is viewed as a series of consecutive, single-point mutations. Natural selection biases evolution toward peaks of high fitness and away from valleys of low fitness. whereas neutral drift occurs in the sequence space without direction as mutations are introduced at random. Large networks of neutral or near-neutral mutations on a fitness landscape, especially for sufficiently long genomes, are possible or even inevitable. Their detection in experiments, however, has been elusive. Although a few near-neutral evolutionary pathways have been found, recent experimental evidence indicates landscapes consist of largely isolated islands. The generality of these results, however, is not clear, as the genome length or the fraction of functional molecules in the genotypic space might have been insufficient for the emergence of large, neutral networks. Thorough investigation on the structure of the fitness landscape is essential to understand the mechanisms of evolution of early genomes. RNA molecules are commonly assumed to play the pivotal role in the origin of genetic systems. They are widely believed to be early, if not the earliest, genetic and catalytic molecules, with abundant biochemical activities as aptamers and ribozymes, i.e. RNA molecules capable, respectively, to bind small molecules or catalyze chemical reactions. Here, we present results of our recent studies on the structure of the sequence space of RNA ligase ribozymes selected through in vitro evolution. Several hundred thousands of sequences active to a different degree were obtained by way of deep sequencing. Analysis of these sequences revealed several large clusters defined such that every sequence in a cluster can be reached from any other sequence in the same cluster through a series of single point mutations. Sequences in a single cluster appear to adopt more than one secondary structure. The mechanism of refolding within a single cluster was examined. To shed light on possible evolutionary paths in the space of ribozymes, the connectivity between clusters was investigated. The effect of length of RNA molecules on the structure of the fitness landscape and possible evolutionary paths was examined by way of comparing functional sequences of 20 and 80 nucleobases in length. It was found that sequences of different lengths shared secondary structure motifs that were presumed responsible for catalytic activity, with increasing complexity and global structural rearrangements emerging in longer molecules.

Wei, Chenyu↗
NASA NTRSDate not supplied

The maize rough endosperm6 (rgh6) mutant encodes a predicted DEAD-box RNA helicase

Maize rough endosperm (rgh) mutants have defective seeds with a rough, etched, or pitted endosperm surface. Molecular genetic analysis of this mutant class has identified multiple RNA processing proteins critical to endosperm development. We isolated the rgh6 locus from the UniformMu transposon tagging population. Mutant kernels have reduced grain-fill with defective embryos that fail to germinate. Self-pollination of rgh6 heterozygotes produces mutant seeds at frequency consistent with a single recessive mutation. Molecular mapping localized rgh6 to a 60 kbp interval on chromosome 5. PCR analysis of gene models within the fine-map interval identified a Mutator (Mu) transposon insertion within a predicted DEAD-box RNA helicase gene. A second allele of rgh6 was identified from UniformMu population. Reciprocal crosses between rgh6-umu1 and rgh6-umu2 heterozygotes produces mutant seeds at a frequency consistent with a 3:1 ratio indicating these mutants are allelic. Both rgh6-umu1 and rgh6-umu2 have Mu element insertions at the same insertion site within the DEAD-box RNA helicase gene. However, the terminal inverted repeat sequences are polymorphic between the alleles showing the alleles are independent mutations. We conclude that mutation of the DEAD-box RNA helicase causes the rgh6 phenotype. Further characterization of the rgh6 mutant phenotype and biochemical investigation of the RGH6 protein is expected to give further insights on the roles of RNA processing in endosperm development.

maize↗