Genetic variation at storage protein-coding loci of common wheat (cv ‘Chinese Spring’) induced by nitrosoethylurea and by the cultivation of immature embryos in vitro

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

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Advancing RNA Virus Discovery and Biology with Whole Genome Sequencing

10.21007/etd.cghs.2021.0551 ◽

2021 ◽

Author(s):

◽

Mariah Taylor ◽

Keyword(s):

Genetic Variation ◽

Amino Acid ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Rna Virus ◽

Animal Health ◽

Functional Domains ◽

Whole Genome ◽

Coding Region

Two RNA virus families that pose a threat to human and animal health are Hantaviridae and Coronaviridae. These RNA viruses which originate in wildlife continue and will continue to cause disease, and hence, it is critical that scientific research define the mechanisms as to how these viruses spillover and adapt to new hosts to become endemic. One gap in our ability to define these mechanisms is the lack of whole genome sequences for many of these viruses. To address this specific gap, I developed a versatile amplicon-based whole-genome sequencing (WGS) approach to identify viral genomes of hantaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within reservoir and spillover hosts. In my research studies, I used the amplicon-based WGS approach to define the genetic plasticity of viral RNA within pathogenic and nonpathogenic hantavirus species. The standing genetic variation of Andes orthohantavirus and Prospect Hill orthohantavirus was mapped out and amino acid changes occurring outside of functional domains were identified within the nucleocapsid and glycoprotein. I observed several amino acid changes in functional domains of the RNA-dependent RNA polymerase, as well as single nucleotide polymorphisms (SNPs) within the 3’ non-coding region (NCR) of the S-segment. To identify whether virus adaptation would occur within the S- and L-segments we attempted to adapt hantaviruses in vitro in a spillover host model through passaging experiments. In early passages we identified few mutations in the M-segment with the majority being identified in the S-segment 3’ NCR and the L-segment. This work suggests that hantavirus adaptation occurs in the S- and L-segments although the effect of these mutants on pathology is yet to be determined. While sequencing laboratory isolates is easily accomplished, sequencing low concentrations of virus within the reservoir is a formidable task. I further translated our amplicon-based WGS approach into a pan-oligonucleotide amplicon-based WGS approach to sequence hantavirus vRNA and mRNA from reservoir and spillover hosts in Ukraine. This approach successfully identified a novel Puumala orthohantavirus (PUUV) strain in Ukraine and using Bayesian phylogenetics we found this strain to be associated with the PUUV Latvian lineage. Early during the SARS-CoV-2 pandemic, I applied the knowledge gained in the hantavirus WGS efforts to sequencing of SARS-CoV-2 from nasopharyngeal swabs collected in April 2020. The genetic diversity of 45 SARS-CoV-2 isolates was evaluated with the methods I developed. We identified D614G, a notable mutation known for increasing transmission, in over 90% of our isolates. Two major lineages distinguish SARS-CoV-2 variants worldwide, lineages A and B. While most of our isolates were found within B lineage, we also identified one isolate within lineage A. We performed in vitro work which confirmed A lineage isolates as having poor replication in the trachea as compared to the nasal cavity. Five of these isolates presented a unique array of mutations which were assessed in the keratin 18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model for its response immunologically and pathogenically. We identified a distinction of pathogenesis between the A and B lineages with emphysema being common amongst A lineage isolates. Additionally, we discovered a small cohort of likely SNPs that defined the late induction of eosinophils during infection. In summary, this work will further define the dynamics of genetic variation and plasticity within virus populations that cause disease outbreaks and will allow a deeper understanding of the virus-host relationship.

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Selectivity of auxin for induction and growth of callus from excised embryo of spring and winter wheat

Canadian Journal of Botany ◽

10.1139/b84-189 ◽

1984 ◽

Vol 62 (7) ◽

pp. 1393-1397 ◽

Cited By ~ 6

Author(s):

M. D. Zhou ◽

T. T. Lee

Keyword(s):

Winter Wheat ◽

Chinese Spring ◽

Shoot Growth ◽

Callus Formation ◽

Triticum Aestivum L ◽

Dichlorophenoxyacetic Acid ◽

Naphthaleneacetic Acid ◽

2,4 Dichlorophenoxyacetic Acid ◽

Methoxybenzoic Acid

The callus-promoting activity of most commonly known as well as some rarely tested auxins was compared with that of 2,4-dichlorophenoxyacetic acid (2,4-D) for in vitro culture of the excised embryo of spring and winter wheat (Triticum aestivum L.), cv. Chinese Spring and cv. Fredrick. Different auxins in a concentration range from 1 to 50 μM showed widely different activities. Also the two wheat cultivars responded differently to the auxins. When rapid callus formation with limited root growth was used as the basis for comparison, 2-(2-methyl-4-chlorophenoxy)propionic acid (2-MCPP), α-naphthaleneacetic acid, 3,6-dichloro-2-methoxybenzoic acid (dicamba), 4-amino-3,5,6,trichloropicolinic acid (picloram), γ-(2,4-dichlorophenoxy)butyric acid, 2,4,5-trichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxypropionic acid, in the order of effectiveness, were superior to 2,4,-D for callus induction from the embryo of 'Chinese Spring,' although the concentration required was higher than that of 2,4-D. For the winter wheat 'Fredrick,' however, only picloram, dicamba, and 2-MCPP performed as well as 2,4-D. All auxins tested promoted shoot growth; 2-methyl-4-chlorophenoxypropionic acid was most effective for 'Chinese Spring,' whereas picloram was most effective for 'Fredrick.'

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Chilling injury and chlorotic reactions of euploids and aneuploids of the group 6 homoeologues of common wheat

Genome ◽

10.1139/g92-069 ◽

1992 ◽

Vol 35 (3) ◽

pp. 468-473 ◽

Cited By ~ 1

Author(s):

Ernest D. P. Whelan ◽

G. B. Schaalje

Keyword(s):

Triticum Aestivum ◽

Common Wheat ◽

Chinese Spring ◽

Prolonged Exposure ◽

Chilling Injury ◽

Triticum Aestivum L ◽

Recessive Trait ◽

Cytoplasmic Effects ◽

Group 6 ◽

Winter Wheat Cultivars

Aneuploid seedlings of the common wheat (Triticum aestivum L.) cv. Chinese Spring (CS) that are nullisomic or telosomic for the long arm of chromosome 6D are susceptible to chilling injury under prolonged exposure to 6 °C; normal euploids or telosomics for the short arm are not. Studies of seedling grown for various durations at 20 °C prior to growth at 6 °C showed that chilling injury was a juvenile phenomenon and that the extent of injury was inversely proportional to the duration of growth at 20 °C to a maximum of about 14 days. When reciprocal crosses were made between susceptible 6D nullisomics or long-arm ditelocentrics of CS and resistant 6D nullisomics of three spring and one winter wheat cultivars, progenies from aneuploid F1 hybrids all segregated for susceptibility as a recessive trait and at a frequency approximating a dihybrid ratio; no cytoplasmic effects were detected. Aneuploids of the group 6 homoeologues of the spring wheat cvs. Cadet and Rescue were resistant, as were group 6 whole-chromosome substitutions of eight different donor wheats in the recipient parent CS and 56 other euploids tested. Genes for resistance to chilling injury appear to involve the group 6 chromosomes and the short arm of 6D in Chinese Spring. In contrast with chilling injury, all aneuploid lines with only four doses of the "corroded" loci on group 6 chromosomes exhibited chlorotic symptoms.Key words: Triticum aestivum, chilling injury.

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CRISPR Gene-Editing Models Geared Toward Therapy for Hereditary and Developmental Neurological Disorders

Frontiers in Pediatrics ◽

10.3389/fped.2021.592571 ◽

2021 ◽

Vol 9 ◽

Author(s):

Poh Kuan Wong ◽

Fook Choe Cheah ◽

Saiful Effendi Syafruddin ◽

M. Aiman Mohtar ◽

Norazrina Azmi ◽

...

Keyword(s):

Genetic Variation ◽

Neurological Disorders ◽

Gene Editing ◽

Fragile X ◽

Genetic Diseases ◽

Basic Principles ◽

Short Palindromic Repeat

Hereditary or developmental neurological disorders (HNDs or DNDs) affect the quality of life and contribute to the high mortality rates among neonates. Most HNDs are incurable, and the search for new and effective treatments is hampered by challenges peculiar to the human brain, which is guarded by the near-impervious blood-brain barrier. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), a gene-editing tool repurposed from bacterial defense systems against viruses, has been touted by some as a panacea for genetic diseases. CRISPR has expedited the research into HNDs, enabling the generation of in vitro and in vivo models to simulate the changes in human physiology caused by genetic variation. In this review, we describe the basic principles and workings of CRISPR and the modifications that have been made to broaden its applications. Then, we review important CRISPR-based studies that have opened new doors to the treatment of HNDs such as fragile X syndrome and Down syndrome. We also discuss how CRISPR can be used to generate research models to examine the effects of genetic variation and caffeine therapy on the developing brain. Several drawbacks of CRISPR may preclude its use at the clinics, particularly the vulnerability of neuronal cells to the adverse effect of gene editing, and the inefficiency of CRISPR delivery into the brain. In concluding the review, we offer some suggestions for enhancing the gene-editing efficacy of CRISPR and how it may be morphed into safe and effective therapy for HNDs and other brain disorders.

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