Bioinformatic Prediction of Ultraviolet Light Mutagenesis Sensitivity of Human Genes and a Method for Genetically Engineering UVB Resistance

Living on earth, we are exposed to ultraviolet (UV) light as part of the solar radiation. UVB spectrum light exposure contributes to the development of skin cancer by interacting with pyrimidine pairs to create lesions called cyclobutane pyrimidine dimers. If these lesions are not removed by nucleotide excision repair, they often give rise to C to T transition mutations. Based on these observations, a bioinformatics approach was used to predict the vulnerability of human protein coding genes to UVB induced loss of function mutations. This data was used to evaluate in depth those genes associated with malignant melanoma. In addition, we demonstrate a method of genetically engineering genes that significantly improves resistance to UVB loss of function mutations

A petal breakstrength meter for Arabidopsis abscission studies

BACKGROUND: Abscission is the regulated dropping of plant organs, such as leaves or flower petals. This process involves a break down of the cell wall between layers of cells in the abscission zone, causing the organ to become detached. The model plant Arabidopsis thaliana undergoes floral organ abscission. Various experimental methods have been used to study Arabidopsis floral organ abscission, including measuring the petal breakstrength, or the amount of force required to pull a petal from the receptacle. Petal breakstrength provides a quantitative insight into the physical integrity of the petal abscission zone. RESULTS: We developed a petal breakstrength meter that allows rapid data acquisition on a personal computer. We present the design of the device and show its utility in measuring Arabidopsis petal breakstrength for abscission studies. CONCLUSION: This petal breakstrength meter should enable researchers to perform the petal breakstrength assay as a routine part of the characterization of environmental and genetic factors affecting abscission

The Arabidopsis Unannotated Secreted Peptide Database, a Resource for Plant Peptidomics

In the era of genomics, if a gene is not annotated, it is not investigated. Due to their small size, genes encoding peptides are often missed in genome annotations. Secreted peptides are important regulators of plant growth, development, and physiology. Identification of additional peptide signals by sequence homology searches has had limited success due to sequence heterogeneity. A bioinformatics approach was taken to find unannotated Arabidopsis (Arabidopsis thaliana) peptides. Arabidopsis chromosome sequences were searched for all open reading frames (ORFs) encoding peptides and small proteins between 25 and 250 amino acids in length. The translated ORFs were then sequentially queried for the presence of an amino-terminal cleavable signal peptide, the absence of transmembrane domains, and the absence of endoplasmic reticulum lumenal retention sequences. Next, the ORFs were filtered against the The Arabidopsis Information Resource 6.0 annotated Arabidopsis genes to remove those ORFs overlapping known genes. The remaining 33,809 ORFs were placed in a relational database to which additional annotation data were deposited. Genome-wide tiling array data were compared with the coordinates of the ORFs, supporting the possibility that many of the ORFs may be expressed. In addition, clustering and sequence similarity analyses revealed that many of the putative peptides are in gene families and/or appear to be present in the rice (Oryza sativa) genome. A subset of the ORFs was evaluated by reverse transcription-PCR and, for one-fifth of those, expression was detected. These results support the idea that the number and diversity of plant peptides is broader than currently assumed. The peptides identified and their annotation data may be viewed or downloaded through a searchable Web interface at peptidome.missouri.edu

BRS1, a serine carboxypeptidase, regulates BRI1 signaling in Arabidopsis thaliana

Brassinosteroid-insensitive 1 (BRI1) of Arabidopsis thaliana encodes a cell surface receptor for brassinosteroids. Mutations in BRI1 severely affect plant growth and development. Activation tagging of a weak bri1 allele (bri1-5) resulted in the identification of a new locus, brs1-1D. BRS1 is predicted to encode a secreted carboxypeptidase. Whereas a brs1 loss-of-function allele has no obvious mutant phenotype, overexpression of BRS1 can suppress bri1 extracellular domain mutants. Genetic analyses showed that brassinosteroids and a functional BRI1 protein kinase domain are required for suppression. In addition, overexpressed BRS1 missense mutants, predicted to abolish BRS1 protease activity, failed to suppress bri1-5. Finally, the effects of BRS1 are selective: overexpression in either wild-type or two other receptor kinase mutants resulted in no phenotypic alterations. These results strongly suggest that BRS1 processes a protein involved in an early event in the BRI1 signaling

Phosphorylation of protein kinase C-related kinase PRK2 during meiotic maturation of starfish oocytes

The resumption of meiosis in the developing starfish oocyte is the result of intracellular signaling events initiated by 1-methyladenine stimulation. One of the earliest detectable kinase activities during meiotic maturation of starfish oocytes is a protein kinase C or PKC-like activity. In this study, several isoforms of protein kinase C were cloned from the oocyte; however, the most abundant PKC-like maternal transcript corresponds to protein kinase C-related kinase 2 (PRK2). PRK2 is expressed in the immature oocyte and at least until germinal vesicle breakdown. Subcellular localization of PRK2 revealed a cytoplasmic distribution in the immature oocyte, which, during meiotic maturation, remained in the cytoplasm but also localized to the disintegrating germinal vesicle. Significantly, PRK2 is phosphorylatedin vivoin response to 1-methyladenine which precedes MPF activation, making PRK2 a candidate regulator of early signaling events of meiotic maturation

A Mutant Arabidopsis Heterotrimeric G-Protein β Subunit Affects Leaf, Flower, and Fruit Development

A genetic screen was performed to find new mutants with an erecta (er) phenotype and to identify genes that may function with ER, a receptor-like kinase. These mutants were named elk (for erecta-like) and were placed into five complementation groups. We positionally cloned ELK4 and determined that it encodes AGB1, a putative heterotrimeric G-protein β subunit. Therefore, elk4 was renamed agb1. agb1-1 plants express similar fruit phenotypes, as seen in er plants, but differ from er in that the stem is only slightly shorter than that in the wild type, the pedicel is slightly longer than that in the wild type, and the leaves are rounder than those in er mutants. Molecular analysis of agb1-1 indicates that it is likely a null allele. AGB1 mRNA is expressed in all tissues tested but is highest in the silique. Analysis of agb1-1 er double mutants suggests that AGB1 may function in an ER developmental pathway regulating silique width but that it functions in parallel pathways affecting silique length as well as leaf and stem development. The finding that AGB1 is involved in the control of organ shape suggests that heterotrimeric G-protein signaling is a developmental regulator in Arabidopsis

Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy

Background: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. Methods: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. Results: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. Conclusions: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.</p