Mutation (Biology)

The inevitable aging process can be partially attributed to the accumulation of
oxidative damage that results from the action of free radicals. Methionine sulfoxide
reductases (Msr) are a class of enzymes that repair oxidized methionine residues. The
two known forms of Msr are MsrA and MsrB which reduce the R- and S- enantiomers of
methionine sulfoxide, respectively. Our lab has created the first genetic animal model
that is fully deficient for any Msr activity. Previously our lab showed that these animals
exhibit a 20 hour delay in development of the third instar larvae (unpublished data). My
studies have further shown that the prolonged third-instar stage is due to a reduced
growth rate associated with slower food intake and a markedly slower motility. These
Msr-deficient animals also exhibit decreased egg-laying that can be attributed to a lack of
female receptivity to mating.

Oxidative stress is considered a major factor in the etiology of age related diseases and the aging process itself. Organisms have developed mechanisms to protect against oxidative damage resulting from increased production of reactive oxygen species during aging. One of the major antioxidant systems is the methionine sulfoxide reductase (Msr) enzyme family. The two major Msr enzymes, MsrA and MsrB, can stereospecifically reduce the S and R epimers, respectively, of methionine sulfoxide in proteins back to methionine. This study, using Drosophila melanogaster, decribes the first animal system lacking both MsrA and MsrB. The loss of either MsrA or MsrB had no effect on lifespan in Drosophila, but loss of MsrB results in a slight decrease in locomotor activity from middle age onward. Double mutants lacking both forms of Msr have a significantly decreased lifespan and decreased locomotor activity at all ages examined. The double Msr mutants had no detectable increase in protein oxidation or decrease in mitochondrial function and were not more sensitive to oxidative stress. These results suggested that other cellular antioxidant systems were protecting the flies against oxidative damage and the decreased life span observed in the double knockouts was not due to widespread oxidative damage. However, one cannot exclude limited oxidative damage to a specific locus or cell type. In this regard, it was observed that older animals, lacking both MsrA and MsrB, have significantly reduced levels of dopamine, suggesting there might be oxidative damage to the dopaminergic neurons. Preliminary results also suggest that the ratio of F to G actin is skewed towards G actin in all mutants. The present results could have relevance to the loss of dopaminergic neurons in Parkinson’s disease.

The research efforts refer to tracking homologus loci in the chromosomes of a pair of a species. The purpose is to infer the extent of maximum syntenic correlation when an exhaustive set of orthologs of the species are searched. Relevant bioinformatic analyses use comparative mapping of conserved synteny via Oxford grid. In medical diagnostic efforts, deducing such synteny correlation can help screening chromosomal aberration in genetic disorder pathology. Objectively, the present study addresses: (i) Cytogenetic framework of syntenic correlation and, (ii) applying information-theoretics to determine entropy-dictated synteny across an exhaustive set of orthologs of the test pairs of species.

Aging is a biological process that has many detrimental effects due to the
accumulation of oxidative damage to key biomolecules due to the action of free
radicals. Methionine sulfoxide reductase (Msr) functions to repair oxidative
damage to methionine residues. Msr comes in two forms, MsrA and MsrB, each
form has been shown to reduce a specific enantiomer of bound and free oxidized
methionine. Effects of Msr have yet to be studied in the major developmental
stages of Drosophila melanogaster despite the enzymes elevated expression
during these stages. A developmental timeline was determined for MsrA mutant,
MsrB mutant, and double null mutants against a wild type control. Results show
that the Msr double mutant is delayed approximately 20 hours in the early/mid
third instar stage while each of the single mutants showed no significant difference to the wild type. Data suggests that the reasoning of this phenomenon
is due to an issue gaining mass.

Aging is a multifactoral biological process of progressive and deleterious changes partially attributed to a build up of oxidatively damaged biomolecules resulting from attacks by free radicals. Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine (Met) residues found in proteins. Oxidized Met produces two enantiomers, Met-S-(o) and Met-R-(o), reduced by MsrA and MsrB respectively. Unlike other model organisms, our MsrA null fly mutant did not display increased sensitivity to oxidative stress or shortened lifespan, suggesting that in Drosophila, having either a functional copy of either Msr is sufficient. Here, two Msr mutant types were phenotypically assayed against isogenic controls. Results suggest that only the loss of both MsrA and MsrB produces increased sensitivity to oxidative stress and shortened lifespan, while locomotor defects became more severe with the full Msr knockout fly.

Harman's theory of aging proposes that a buildup of damaging reactive oxygen species (ROS) is one of the primary causes of the deleterious symptoms attributed to aging. Cellular defenses in the form of antioxidants have evolved to combat ROS and reverse damage; one such group is the methionine sulfoxide reductases (Msr), which function to reduce oxidized methionine. MsrA reduces the S enantiomer of methionine sulfoxide, Met-S-(o), while MsrB reduces the R enantiomer, Met-R-(o). The focus of this study was to investigate how the absence of one or both forms of Msr affects locomotion in Drosophila using both traditional genetic mutants and more recently developed RNA interference (RNAi) strains. Results indicate that lack of MsrA does not affect locomotion. However, lack of MsrB drastically reduces rates of locomotion in all age classes. Furthermore, creation of an RNAi line capable of knocking down both MsrA and MsrB in progeny was completed.

Cytogenetics is a study on the genetic considerations associated with structural and functional aspects of the cells with reference to chromosomal inclusions. Chromosomes are structures within the cells containing body's information in the form of strings of DNA. When atypical version or structural abnormality in one or more chromosomes prevails, it is defined as chromosomal aberrations (CA) depicting certain genetic pathogeny (known as genetic disorders). The present study assumes the presence of normal and abnormal chromosomal sets in varying proportions in the cytogenetic complex ; and, stochastical mixture theory is invoked to ascertain the information redundancy as a function of fractional abnormal chromosome population. This bioinformatic measure of redundancy is indicated as a track-parameter towards the progression of genetic disorder, for example, the growth of cancer. Lastly, using the results obtained, conclusions are enumerated, inferences are outlined and directions for future studies are considered.

Drosophila melanogaster encounter periods of increased temperature or decreased oxygen in its native environment. One consequence of these environmental stresses is increased production of reactive oxygen species that damage major molecules within cells. Another consequence is that flies fall into a protective coma where biological functions are minimized to conserve energy expenditures. This biological phenomenon is called spreading depression. The overarching aim of this project is to determine if methionine sulfoxide reductases affect entrance or exit from the protective coma induced by acute thermal stress. The data revealed that complete deficiency of Msr in young flies causes a faster induction of the coma. In both young and old flies, Msr does not affect average recovery time but does affect the pattern of recovery from coma. Entrance into the coma is age dependent with young flies maintaining activity longer than before entering into the coma as compared to old flies.

Mitochondrial disorders resulting from defects in oxidative phosphorylation are the most common form of inherited metabolic disease. Mutations in the human mitochondrial translation elongation factor GFM1 have recently been shown to cause the lethal pediatric disorder Combined Oxidative Phosphorylation Deficiency Syndrome (COXPD1). Children harboring mutations in GFM1 exhibit severe developmental, metabolic and neurological abnormalities. This work describes the identification and extensive characterization of the first known mutations in iconoclast (ico), the Drosophila orthologue of GFM1. Expression of human GFM1 can rescue ico null mutants, demonstrating functional conservation between the human and fly proteins. While point mutations in ico result in developmental defects and death during embryogenesis, animals null for ico survive until the second or third instar larval stage. These results indicate that in addition to loss-of-function consequences, point mutations in ico appear to produce toxic proteins with antimorphic or neomorphic effects. Consistent with this hypothesis, transgenic expression of a mutant ICO protein is lethal when expressed during development and inhibits growth when expressed in wing discs. In addition, animals with a single copy of an ico point mutation are more sensitive to acute hyperthermic or hypoxic stress. Removal of the positively-charged tail of the protein abolishes the toxic effects of mutant ICO, demonstrating that this domain is necessary for the harmful gain-of-function phenotypes observed in ico point mutants.

Aconitase is an important enzyme in the citric Acid Cycle, is needed for maintenance of mitochondrial DNA, is a key regulator of iron in the cell, and is very sensitive to oxidative stress. We have isolatd the yeast ACO1 gene, which codes for aconitase, and randomly mutated it to create a mutant library of cells each expressing a different version of ACO1. We will select for oxidative stress resistant aconitase in S. cerevisiae by subjecting strains to successive rounds of heat shock and competitive growth against other mutants. The "winner" of this competition will then be analyzed for which version of aconitase it is expressing, which may lead to increased longevity.