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[
Worm Breeder's Gazette,
1982]
We have done an in situ hybridization analysis on dissected male tissues by using a 720bp Xba genomic fragment that is complementary to 15K mRNA as a probe. Hybridization was confined to a specific region of the male gonad in the mid-proximal arm beginning approximately one fourth the distance past the loop region. This is the same region that the sperm-specific protein 15K is first detected by immunocytochemistry.Hybridization was sensitive to RNase pre-treatment and was not observed in spermatids or in any other tissues of the male. DNase pre-treatment had no effect on hybridization. The conditions for in situ hybridization were as follows: 1) Tissues were dissected from adult males in M9 salt solution on gelatinized glass slides and fixed by the addition of 45 acetic acid 2) Tissues were flattened (not squashed) by the pressure of a cleaned coverslip; coverslips were subsequently removed by dipping in liquid nitrogen 3) Post fixation was in 3:1 ethanol: acetic acid 4) Slides were dehydrated to 100% ethanol at -20 C, air dried and baked at 80 C for 2 hrs in a vacuum oven. 5) Hybridization conditions were the same as those used by Thomas ( PNAS 77, 5201-5205, 1980) for the Northern blot. Pre-treatment was in 50% formamide, 5 X SSC, 50mM NaP04 pH 6.5, 250mg/ml sheared E. coli DNA, 0.02% w/v BSA, 0.02% w/v Ficoll, 0.02% w/v polyvinylpyrolidone at 42 C for 1 hr. Hybridization solution consisted of pre-hybridization solution plus 10% dextran sulfate, 2 mg/ml of Poly A and the labeled probe ([3H]p lambda8 1 X 10+E6 cpm/slide; 0.5-1.0 X 10+E7 cpm/ug). Hybridization was carried out overnight at 42 C by adding 35-40ml of hybridization solution to each slide, covering with a cleaned coverslip and sealing with Sanford's Rubber contact cement. 6) Post-Hybridization washes were carried out by first gently removing the coverslip and washing in 2 changes of prehybridization buffer without DNA for 10 minutes each at 42 C followed by four washes in 2 X SSC for five minutes each at room temperature. The slides were then washed twice in 0.1 X SSC at 50 C for 15 minutes each followed by washing in distilled water, washing in 5% TCA at 0 C and finally in distilled water and air dried. Squashing the tissue caused severe tissue destruction and a great increase in background probably due to the leakage of the cellular contents (mRNA?) by the gonad. Pre-treatment with proteinase-K caused an increase in the loss of tissue even after baking at 80 C. Washing in 0.1 X SSC at 50 C after hybridization was necessary to reduce background. Sealing the coverslip with rubber cement was suggested by Dr. W. Jeffery and was necessary to prevent evaporation. Removal of the sealed coverslip after hybridization often caused tissue loss due to the rubbing of the coverslip on the tissue. This was prevented by suspending the coverslip over a single layer of Scotch brand tape surrounding the tissue area.
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[
Worm Breeder's Gazette,
1982]
Using a combination of 2D gels and Northern blot analysis we have been able to demonstrate that the regulation of the spermatogenic pathway in the hermaphrodite occurs at the level of transcription at least with respect to the major sperm protein 15K. 2D gel analysis of pulse-labeled synchronous cultures of N2 shows that 15K is not synthesized in L1 or L2 larvae but is synthesized in L4 larvae during spermatogenesis and is then repressed after sperm production has stopped in gravid hermaphrodites. Northern blot analysis using our cloned genomic probe for 15K mRNA has demonstrated the lack of 15K mRNA in L2 and gravid hermaphrodites and its presence only during spermatogenesis in the hermaphrodite.
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[
Worm Breeder's Gazette,
1982]
We have recently begun a search for genomic DNA sequences that are transcriptionally active only during spermatogenesis by the following procedure: We have screened triplicate copies of a C. elegans clone bank (graciously provided by Scott Emmons) with three separate probes: 1) [32P]cDNA of Poly A RNA from synchronous L2 worms; 2) [32P]cDNA of Poly A RNA from 70% adult males; and 3) [32P]cDNA of Poly A RNA from gravid hermaphrodites. Plaques were screened and those plaques showing positive hybridization to only the male cDNA probe were picked for further analysis. So far of 1600 phage screened 200 were positive but only 2 of these showed specific hybridization only to the male probe. They have been picked as candidates for being spermatogenesis specific. Interestingly, three plaques were found to be hermaphrodite specific, hybridizing only to the gravid hermaphrodite probe. Our plan is to analyze these spermatogenesis specific sequences by determining their products and how they are regulated.
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[
Biochemistry,
2012]
Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.
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[
J Infect Dis,
2015]
BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.
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[
Dev Biol,
1982]
The major protein found in nematode sperm exhibits a distinct pattern of developmental regulation. In the nematode Caenorhabditis elegans, the synthesis of the major sperm protein (15K) begins with the onset of spermatogenesis in both the male and hermaphrodite. Both spermatogenesis and 15K synthesis continue for the life of the male while in the protandrous hermaphrodite the major sperm protein is synthesized only during the fourth larval stage. Inhibitor studies using actinomycin D and a-actinin as well as Northern blot analysis have shown that the primary regulatory mechanism of this gene is at the transcriptional level. Recombinant molecules have been selected bearing the 15K genomic sequence by a positive hybridization translation assay. Using one of these cloned fragments as a probe for in situ hybridization, 15K transcripts have been localized to a specific region of the male gonad. These studies indicate that the gene for the major sperm protein is regulated by a cell-specific transcriptional control mechanism coincidental with the onset of sexual differentiation in the nematode.
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[
Aging Cell,
2011]
The roundworm C. elegans is widely used as an aging model, with hundreds of genes identified that modulate aging (Kaeberlein etal., 2002. Mech. Ageing Dev.123, 1115-1119). The development and bodyplan of the 959 cells comprising the adult have been well described and established for more than 25 years (Sulston and Horvitz, 1977. Dev. Biol.56, 110-156; Sulston et al., 1983. Dev. Biol.100, 64-119.). However, morphological changes with age in this optically transparent animal are less well understood, with only a handful of studies investigating the pathobiology of aging. Age-related changes in muscle (Herndon etal., 2002. Nature419, 808-814), neurons (Herndon etal., 2002), intestine and yolk granules (Garigan etal., 2002. Genetics161, 1101-1112; Herndon etal., 2002), nuclear architecture (Haithcock etal., 2005. Proc. Natl Acad. Sci. USA102, 16690-16695), tail nuclei (Golden etal., 2007. Aging Cell6, 179-188), and the germline (Golden etal., 2007) have been observed via a variety of traditional relatively low-throughput methods. We report here a number of novel approaches to study the pathobiology of aging C. elegans. We combined histological staining of serial-sectioned tissues, transmission electron microscopy, and confocal microscopy with 3D volumetric reconstructions and characterized age-related morphological changes in multiple wild-type individuals at different ages. This enabled us to identify several novel pathologies with age in the C. elegans intestine, including the loss of critical nuclei, the degradation of intestinal microvilli, changes in the size, shape, and cytoplasmic contents of the intestine, and altered morphologies caused by ingested bacteria. The three-dimensional models we have created of tissues and cellular components from multiple individuals of different ages represent a unique resource to demonstrate global heterogeneity of a multicellular organism.
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[
Worm Breeder's Gazette,
1976]
We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.
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[
Worm Breeder's Gazette,
1994]
cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.
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[
Mech Ageing Dev,
2009]
Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants
daf-2(
e1370),
age-1(
hx546),
clk-1(
qm30),
isp-1(
qm150) and
eat-2(
ad465) all have a lower DeltaPsi(m) than wild type animals. The lower DeltaPsi(m) of
daf-2(
e1370) is
daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on the mitochondria and lead to a decreased DeltaPsi(m). Our results are consistent with the 'uncoupling to survive' hypothesis, which states that dissipation of the DeltaPsi(m) will extend lifespan.