Evidence for a transposon in caenorhabditis elegans

The C. elegans genome contains a 1.7 kb repeated DNA sequence (Tc1) that is present in different numbers in various strains. In strain Bristol and 10 other strains analyzed, there are 20 ± 5 copies of Tc1, and these are located at a nearly constant set of sites in the DNA. In Bergerac, however, there are 200 ± 50 interspersed copies of Tc1 that have arisen by insertion of Tc1 elements into new genomic sites. The interspersed copies of Tc1 have a conserved, nonpermuted structure. The structure of genomic Tc1 elements was analyzed by the cloning of a single Tc1 element from Bergerac and the comparison of its structure with homologous genomic sequences in Bristol and Bergerac. Tc1 elements at three sites analyzed in Bergerac undergo apparently precise excision from their points of insertion at high frequency.     

Evidence for protein-tyrosine-phosphatase catalysis proceeding via a cysteine-phosphate intermediate.

A recombinant protein-tyrosine-phosphatase has been expressed in Escherichia coli and purified to a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using affinity chromatography. When the phosphatase was allowed to react with 32P-labeled substrates and then rapidly denaturated, a 32P-labeled phosphoprotein could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transient formation of a 32P-labeled phosphoprotein was observed, and the 32P-labeled protein disappeared as substrate was consumed. In the presence of 32P-labeled p-nitrophenyl phosphate, 0.27 mol of phosphate was incorporated per mol of protein-tyrosine-phosphatase. Site-directed mutagenesis of a catalytically essential cystine residue (position 215) in the recombinant protein resulted in an inactive enzyme, and no phosphoprotein was formed. The 32P-labeled phosphoprotein showed a maximum lability between pH 2.5 and 3.5 and was rapidly decomposed in the presence of iodine. These properties, along with additional site-directed mutations, suggest that the protein-tyrosine-phosphatase forms a covalent thiol phosphate linkage between Cys215 and phosphate.     

Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells

We describe an experimental approach for generating mutant alleles in rat spermatogonial stem cells (SSCs) using Sleeping Beauty (SB) transposon-mediated insertional mutagenesis. The protocol is based on mobilization of mutagenic gene-trap transposons from transfected plasmid vectors into the genomes of cultured stem cells. Cells with transposon insertions in expressed genes are selected on the basis of activation of an antibiotic-resistance gene encoded by the transposon. These gene-trap clones are transplanted into the testes of recipient males (either as monoclonal or polyclonal libraries); crossing of these founders with wild-type females allows the insertions to be passed to F(1) progeny. This simple, economic and user-friendly methodological pipeline enables screens for functional gene annotation in the rat, with applicability in other vertebrate models where germ line-competent stem cells have been established. The complete protocol from transfection of SSCs to the genotyping of heterozygous F(1) offspring that harbor genomic SB gene-trap insertions takes 5-6 months.     

Evidence for a phosphoenzyme intermediate in the reaction pathway of rat hepatic fructose-2,6-bisphosphatase

We re-examined the kinetics of the bisphosphatase reaction of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase after depleting the enzyme of bound fructose 6-phosphate and found a hyperbolic dependence on fructose 2,6-bisphosphate at concentrations below 100 nM. The Michaelis constant was 4 nM, the Vmax was about 12 nmol X mg-1 X min-1 at 22 degrees C but the substrate inhibited at concentrations above 100 nM. Both phosphate and alpha-glycerol phosphate strongly inhibited phosphoenzyme formation and hydrolytic rate below 100 nM, but relieved the inhibition by substrate at higher concentrations probably by antagonizing substrate binding. A number of observations support the proposition that the phosphoenzyme is a necessary participant in catalysis. 1) The amount of phosphoenzyme measured during steady-state hydrolysis as a function of substrate concentration correlated with the velocity profile. 2) Rapid mixing experiments demonstrated that over a broad range of substrate concentrations phosphoenzyme formation was faster than the net rate of hydrolysis. 3) Both phosphate and alpha-glycerol phosphate inhibited the rate of phosphoenzyme formation and, at low substrate concentrations, reduced the steady-state phosphoenzyme levels. The latter correlated with inhibition of substrate hydrolysis. 4) Both phosphate and alpha-glycerol phosphate stimulate the rate of phosphoenzyme breakdown, consistent with their stimulation of substrate hydrolysis at high substrate concentrations. 5) The fractional rate of phosphoenzyme breakdown, which was pH and substrate dependent, multiplied by the amount of phosphoenzyme obtained in the steady state at that pH and substrate concentration approximated the observed rate of hydrolysis. We conclude that the phosphoenzyme is a reaction intermediate in the hepatic fructose-2,6-bisphosphatase reaction.     

Evidence in a nematode for regulation of transposon excision by tissue-specific factors

Summary The transposable element Tc1 in Caenorhabditis elegans undergoes an excision reaction, which can be detected in a Southern hybridization as the appearance of empty chromosomal insertion sites. This excision reaction is under tissue-specific regulation in that it occurs at much higher frequency in somatic cells than in the germ line. We show here that this regulation is likely to be due to the action of tissue-specific factors that either promote excision in somatic tissues or repress it in the germ line. The rate of excision of elements at five distinct chromosomal sites has been measured by a method that avoids ambiguities due to cell division. All these elements are found to undergo excision at closely similar rates during the L1 larval stage. No distinct difference exists among the elements at different sites that would suggest regulation by flanking sequences.     

Biogenesis of a photosystem I light-harvesting complex. Evidence for a membrane intermediate.

CAB-7p is a chlorophyll a/b binding protein of photosystem I (PSI). It is found in light-harvesting complex I 680 (LHCI-680), one of the chlorophyll complexes produced by detergent solubilization of PSI. Two types of evidence are presented to indicate that assembly of CAB-7p into PSI proceeds through a membrane intermediate. First, when CAB-7p is briefly imported into chloroplasts or isolated thylakoids, we initially observe a fast-migrating membrane form of CAB-7p that is subsequently converted into PSI. The conversion of the fast-migrating form into PSI does not require stroma or ATP. Second, trypsin treatment of thylakoids containing radiolabeled CAB-7p indicates that there are at least two membrane forms of the mature 23-kD protein. The predominant form is completely resistant to proteolysis; a second form of the protein is cleaved by trypsin into 12- and 7-kD polypeptides. We interpret this to mean that the intermediate is a cleavable form that becomes protease resistant during assembly. This notion is supported by the observation that CAB-7p in LHCI-680 is largely cleaved by trypsin into 12- and 7-kD polypeptides, whereas CAB-7p in isolated PSI particles is trypsin resistant. In vitro, we generated a mutant form of CAB-7p, CAB-7/BgI2p, that was able to integrate into thylakoid membranes but was unable to assemble into PSI. The membrane form of CAB-7/BgI2p, like LHCI-680, was predominantly cleaved by trypsin into 12- and 7-kD fragments. We suggest that the mutant protein is arrested at an intermediate stage in the assembly pathway of PSI. Based on its mobility in nondenaturing gels and its susceptibility to protease cleavage, we suggest that the intermediate form is LHCI-680. We propose the following distinct stages in the biogenesis of LHCI: (a) apoprotein is integrated into the thylakoid, (b) chlorophyll is rapidly bound to apoprotein forming LHCI-680, and (c) LHCI-680 assembles into the native PSI complex.     

Alteration of ganglioside composition and metabolism in doxorubicin-resistant rat tumoral cells

We have investigated the ganglioside levels, composition and metabolism in two lines of doxorubicin-resistant cells and in the corresponding wild strains, the C6 rat glioblastoma and the HTC rat hepatoma. The only ganglioside present was GM3, and its level was increased 2-fold in C6 resistant cells and decreased nearly 2-fold in HTC resistant cells. A decrease of cytidine 5'-monophospho-N-acetylneuraminic acid:galactosylglucosylceramide sialyltransferase activity was observed in both resistant lines as compared to sensitive ones, and could not, therefore, explain the increase in the GM3 level observed in the C6 resistant line. Alterations of acid neuraminidase activity were also observed; a 5-fold decrease was noticed in the C6 resistant line and could account for the increase in the GM3 level observed in these cells; in contrast, a 2-fold increase of acid neuraminidase activity was noticed in the HTC resistant cells: together, with reduced synthesis, it could explain the decrease in the GM3 level observed in these cells. No alterations of exogenous ganglioside transport was exhibited by the C6 resistant cells.     

Evidence for a hydroxide intermediate in cytochrome c oxidase

A transient intermediate of cytochrome c oxidase has been generated by exposing the enzyme to a laser beam in the presence of oxygen. This intermediate develops when the enzyme is simultaneously reduced photoreductively and oxidized chemically, thereby forcing it to turn over. Under these conditions a form of the enzyme is generated with a line at 477 cm-1 in the resonance Raman spectrum, which we attribute to an Fe-OH stretching mode based on oxygen and hydrogen isotopic substitution. This hydroxide intermediate relaxes back to the resting state of the enzyme upon removal from the laser beam. Hydroxide intermediates have been postulated many times in the past in proposed catalytic mechanisms. The data reported here supply the first evidence for the existence of such an intermediate and a method for stabilizing it.     

Evidence for extensive methylation of ribosomal RNA genes in a rat XC cell line

The effects of extracellular Na+, K+ and Cl- on neurite outgrowth of PC12 pheochromocytoma cells were studied. Nerve growth factor (NGF)-induced neurite formation was inhibited upon substitution of choline chloride for NaCl under normal culture conditions. It was found that neurite formation increased proportionately with the concentration of Na+ in medium up to 150 mM. When PC12 cells were exposed to NGF in suspension culture followed by transfer to new dishes, they showed neurite extention in response to NGF in an RNA- and protein synthesis-independent manner. Under these conditions, neurite outgrowth occurred normally in 60-150 mM Na+, whereas it decreased significantly at lower concentrations of Na+. Na+ dependency was also observed for cyclic AMP-mediated neurite formation of PC12 cells. In contrast neurite outgrowth was independent of K+ in the range 5-106 mM, suggesting that membrane potential did not play a role in this process. No alterations were observed in neurite outgrowth with Cl- replaced by NO3-, SO2-4, or 2-hydroxyethanesulfonate. Thus, extracellular Na+ plays a role in controlling neurite formation of these cells. An attempt was made to relate this effect to a decrease in cytoplasmic Ca2+ concentration monitored by a fluorescent dye sensitive to Ca2+.