Pamela L. Larsen

Mapping proteinDNA interactions in vivo with formaldehyde: Evidence that histone H4 is retained on a highly transcribed gene

We have used formaldehyde-mediated protein-DNA crosslinking within intact cells to examine the in vivo chromatin structure of the D. melanogaster heat shock protein 70 (hsp70) genes. In agreement with previous in vitro studies, we find that the heat shock-mediated transcriptional induction of the hsp70 genes perturbs their chromatin structure, resulting in fewer protein-DNA contacts crosslinkable in vivo by formaldehyde. However, contrary to earlier in vitro evidence that histones may be absent from actively transcribed genes, we show directly, by immunoprecipitation of in vivo-crosslinked chromatin fragments, that at least histone H4 remains bound to hsp70 DNA in vivo, irrespective of its rate of transcription. The formaldehyde-based in vivo mapping techniques described in this work are generally applicable, and can be used both to probe protein-DNA interactions within specific genes and to determine the genomic location of specific chromosomal proteins.

Structure and expression of daf-12: a nuclear hormone receptor with three isoforms that are involved in development and aging in Caenorhabditis elegans.

During Caenorhabditis elegans early larval development environmental conditions promote a cascade of signaling molecules to direct growth to the reproductive adult or to arrest development as a dauer larva. Two parallel chemosensory signal transduction pathways, one of which is transforming growth factor (TGF)-beta-like, converge on the daf-12 gene to regulate dauer formation. A third insulin-like signaling pathway interacts with the daf-12 pathway to regulate both dauer formation and adult longevity. To further understand the role of daf-12 in these processes, we have molecularly characterized this gene. We establish rescue of the mutant dauer defective phenotype with a genomic clone. We show that three transcripts of different lengths, due to differential splicing, are made from the daf-12 gene. The deduced protein isoforms are similar to both DNA- and ligand-binding domains of nuclear hormone receptors. The three daf-12 transcripts are produced throughout development and expression increases during the preparation for and execution of dauer formation. Analysis of various daf mutant strains suggests that the isoform ratios of daf-12 steady-state mRNA are not changed by reduction of function in the TGF-beta and insulin signaling components of the dauer pathway. The daf-12 promoter directs expression of GFP in the pharynx. daf-12 is a C. elegans nuclear hormone receptor with multiple isoforms, is expressed throughout development in distinct cells, and functions under a variety of environmental conditions.

Altered bacterial metabolism, not coenzyme Q content, is responsible for the lifespan extension in Caenorhabditis elegans fed an Escherichia coli diet lacking coenzyme Q

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. Caenorhabditis elegans fed Escherichia coli devoid of Q8 have a significant lifespan extension when compared to C. elegans fed a standard ‘Q‐replete’E. coli diet. Here we examine possible mechanisms for the lifespan extension caused by the Q‐less E. coli diet. A bioassay for Q uptake shows that a water‐soluble formulation of Q10 is effectively taken up by both clk‐1 mutant and wild‐type nematodes, but does not reverse lifespan extension mediated by the Q‐less E. coli diet, indicating that lifespan extension is not due to the absence of dietary Q per se. The enhanced longevity mediated by the Q‐less E. coli diet cannot be attributed to dietary restriction, different Qn isoforms, reduced pathogenesis or slowed growth of the Q‐less E. coli, and in fact requires E. coli viability. Q‐less E. coli have defects in respiratory metabolism. C. elegans fed Q‐replete E. coli mutants with similarly impaired respiratory metabolism due to defects in complex V also show a pronounced lifespan extension, although not as dramatic as those fed the respiratory deficient Q‐less E. coli diet. The data suggest that feeding respiratory incompetent E. coli, whether Q‐less or Q‐replete, produces a robust life extension in wild‐type C. elegans. We believe that the fermentation‐based metabolism of the E. coli diet is an important parameter of C. elegans longevity.

Reproductive Fitness and Quinone Content of Caenorhabditis elegans clk-1 Mutants Fed Coenzyme Q Isoforms of Varying Length

Caenorhabditis elegans clk-1 mutants lack coenzyme Q9 and accumulate the biosynthetic intermediate demethoxy-Q9. A dietary source of ubiquinone (Q) is required for larval growth and development of the gonad and germ cells. We considered that uptake of the shorter Q8 isoform present in the Escherichia coli food may contribute to the Clk phenotypes of slowed development and reduced brood size observed when the animals are fed Q-replete E. coli. To test the effect of isoprene tail length, N2 and clk-1 animals were fed E. coli engineered to produce Q7, Q8, Q9, or Q10. Wild-type nematodes showed no change in reproductive fitness regardless of the Qn isoform fed. clk-1(e2519) fed the Q9 diet showed increased egg production; however, this diet did not improve reproductive fitness of the clk-1(qm30) animals. Furthermore, animals with the more severe clk-1(qm30) allele become sterile and their progeny inviable when fed Q7-containing bacteria. The content of Q7 in the mitochondria of clk-1 animals was decreased relative to Q8, suggesting less effective transport of Q7 to the mitochondria, impaired retention, or decreased stability. Additionally, regardless of E. coli diet, clk-1(qm30) animals contain a dysfunctional dense form of mitochondria. The gonads of clk-1(qm30) worms fed Q7-containing food were severely shrunken and disordered. The differential fertility of clk-1 mutant nematodes fed Q isoforms may result from changes in Q localization, altered recognition by Q-binding proteins, and/or potential defects in mitochondrial function resulting from the mutant CLK-1 polypeptide itself.

Spontaneous reiterations of DNA sequences near the ends of adenovirus type 3 genomes

Repeated passage of adenovirus type 3 in HeLa cells has led to a novel stock of variant genomes. Most of the DNA molecules in this stock are characterized by deletions and substitutions of DNA sequences near the left end of the adenovirus type 3 genome map, as reported earlier (C.C. Robinson and C. Tibbetts (1984) Virology 137, 276-286). In this report the characterization of the variant genomes is extended and reveals elongated DNA molecules bearing tandem repetitions of viral DNA sequences near the left and right ends of the viral DNA. Evidence is also presented supporting the cellular DNA origin of short insert sequences found in substitution variants. The elongated variants are of interest because of their novel repeated DNA structures. The locations of these aberrant sequences raise questions about their potential impact on viral gene expression.

Asking the age-old questions

Control of lifespan in Caenorhabditis elegans by the DAF-2 insulin-like signaling pathway requires daf-16, which encodes a member of the forkhead family of transcription factors. A new study provides evidence that DAF-2 negatively regulates DAF-16 activity by promoting its retention in the cytoplasm. But, constitutive targeting of DAF-16 to the nucleus is not sufficient to extend lifespan, revealing a new layer of complexity in the genetic control of aging in the nematode.

Animal models in aging research: A critical examination

Understanding why different organisms show diverse rates of aging may provide useful insights into basic aging processes. Biogerontologists have converged on a few model organisms that represent only a minute fraction of the animal kingdom, but nevertheless span a considerable distance in animal evolution. Shared features of these evolutionary divergent animals have highlighted some conserved regulatory processes in animal aging. However, these traditional models are all short-lived and may have unintentionally constrained research to focus on only those areas in which their use is most appropriate. Surprisingly few studies focus on slow-aging organisms, or nontraditional model organisms that may be better suited to address successful aging and issues more relevant to long-living humans. This chapter critically assesses both traditional and nontraditional animal models used in aging research, and emphasizes the importance and judicious use of the comparative method to test the ubiquity of aging theories, mechanisms, and their potential translation for human application.

Laboratory cultivation of caenorhabditis elegans and other free-living nematodes

Publisher Summary This chapter describes the cultivation of caenorhabditis elegans and other free-living nematodes in the laboratory. An adult C. elegans is about 1.5 mm long and, under optimal laboratory conditions, has a life cycle of approximately 3 days. There are two sexes, males and self-fertile hermaphrodites that are readily distinguishable as adults. The animals are transparent throughout the life cycle, permitting the observation of cell divisions in living animals using a differential interference microscopy. Tools for manipulating individual nematodes on plates are made by anchoring a 1.5-cm piece of 32-gauge platinum wire in a 6 inch inoculating loop holder or in a Pasteur pipette. For best results the end of the wire used for manipulation should be flattened and, if necessary, rounded to remove sharp edges. Remove supernatant and re-suspend in 15 ml of M9 buffer, divide between two 15-ml tubes, and place on ice. When cold, add 7.5 ml of cold 60% sucrose to each tube. Mix by inversion and centrifuge immediately at 1500 RCF for 5 min.