The Tcl element of Caenorhabditis elegans is a member of the most widespread class of DNA transposons known in nature. Here, we describe efficient and precise transposition of Tcl in a cell-free system. Tcl appears to jump by a cut-and-paste mechanism of transposition. The terminal 26 bp of the Tcl terminal repeats together with the flanking TA sequence are sufficient for transposition. The target site choice in vitro is similar to that in vivo. Transposition is achieved with an extract prepared from nuclei of transgenic nematodes that overexpress Tcl transposase but also by recombinant transposase purified from Escherichia coli. The simple reaction requirements explain why horizontal spread of Tcl/mariner transposons can occur.They also suggest that Tcl may be a good vector for transgenesis of diverse animal species.

We have described recently the purification and cloning of PP2A (protein phosphatase 2A) leucine carboxylmethyltransferase. We studied the purification of a PP2A-specific methylesterase that co-purifies with PP2A and found that it is tightly associated with an inactive dimeric or trimeric form of PP2A. These inactive enzyme forms could be reactivated as Ser/Thr phosphatase by PTPA (phosphotyrosyl phosphatase activator of PP2A). PTPA was described previously by our group as a protein that stimulates the in vitro phosphotyrosyl phosphatase activity of PP2A; however, PP2A-specific methyltransferase could not bring about the activation. The PTPA activation could be distinguished from the Mn2+ stimulation observed with some inactive forms of PP2A, also found associated with PME-1 (phosphatase methylesterase 1). We discuss a potential new function for PME-1 as an enzyme that stabilizes an inactivated pool of PP2A.

We have tried to rind out why Ascaris hemoglobin has such an exceptionally high oxygen affinity (P5o (2) showed that this high affinity was due to an extremely slow dissociation (off rate, koff 0.0041/s-1), whereas the association rate (on rate, kon) was normal. By contrast, the affinity for carbon monoxide is similar to that of vertebrate hemoglobins. Synthetic chemistry has indicated no way of strengthening the Fe2+-02 bond by the equivalent of nearly 3 kcal/mol. The Fe2+{-2 bond is polar with partial Fe3+2-O character, whereas the Fe2+0-C bond is nonpolar. In consequence, the iron-bound oxygen, but not the carbon monoxide, accepts a hydrogen bond from the distal histidine in myoglobin and in the a subunit of human hemoglobin (in the , ( subunit Fe and 02 are too far apart) (3). In Ascaris hemoglobin the slow off rate of oxygen, in contrast to the normal offrate of carbon monoxide, suggested that the bound oxygen might be stabilized by an additional hydrogen bond from the globin.The amino acid sequence of Ascaris hemoglobin showed that glutamine takes the place of the distal histidine (4, 5).This could serve equally well as a hydrogen bond donor to the bound oxygen, but that bond would be no stronger than the bond from histidine. Initially, the sequence gave no clue to the possible nature of a second hydrogen bond donor.Such a clue did come from the observation of Olson and collaborators (6) that the replacement of leucine B10 (residue 29 of the polypeptide) in sperm whale myoglobin by phenylalanine raised its oxygen affinity 10-fold, because a hydrogen atom in the benzene ring of the phenylalanine made a favorable contact with the bound oxygen (6). In Ascaris hemoglobin this position is occupied by tyrosine. However, the myoglobin mutant B10 leucine -+ tyrosine (L -+ Y) had a lowered oxygen affinity, with an off rate 50 times higher than that of the wild type, suggesting that tyrosine B10 was unlikely to contribute the high oxygen affinity of Ascaris hemoglobin (7).Ascaris hemoglobin consists of eight identical subunits, each containing two myoglobin-like domains in tandem, followed by a repetitive sequence ofpolar residues suggestive of a "zipper" (4,8). Kloek et al. (9) cloned into an M13mpl9 vector (10). Site-directed mutagenesis was carried out by the dut, ung-method with E. coli BW313 (11). After the DNA sequences of these clones had been confirmed by single-stranded sequencing (12) using Sequenase (United States Biochemical), the Xba 1-HindI1 fragment was excised and cloned between the Xba I and HindIll sites of a T7 RNA polymerase expression vector, pRK172 (13). The BL21(DE3) strain was transformed with these vectors, and a large-scale culture was carried out at 370C in 2x TY medium containing ampicillin (100 pg/ml).Cells were harvested after induction with 1 mM isopropyl f3-D-thiogalactopyranoside for 3 hr and suspended in 10 mM 1594The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance wi...

The extracellular hemoglobin of Ascaris has an extremely high oxygen affinity (P5. = 0.004 mmflg). All retain the heme-linked proximal histidine (F8) and the phenylalanine (CD1) that wedges the heme into its pocket. Most of them also show the distal histidine (E7) and valine or isoleucine (Ell), but more important, all retain a pattern ofabout 33 internal sites from which all polar residues except serine and threonine residues are excluded (6, 7). Ascaris is a parasitic nematode that lives in the intestines of animals where oxygen is scarce. It contains a hemoglobin with an oxygen affinity two orders of magnitude higher than that of any vertebrate myoglobin or hemoglobin or any synthetic iron porphyrin made so far (P50 = 0.004 mmHg). We know of no way by which the oxygen affinity offerrous heme could be raised to that level. As a first step toward the solution of this chemical mystery, we have determined the amino acid sequence of Ascaris hemoglobin.tt MATERIALS AND METHODSThe perienteric fluid hemoglobin of Ascaris suum, obtained in a local slaughterhouse, was prepared essentially according to ref. 8. The final preparation, which was passed over a Superose 6 column in 50 mM Tris HC1, pH 7.5/100 mM NaCl, had an A412/A28w = 1.3 and showed a single but broad spot on two-dimensional SDS/PAGE. The protein was split chemically or enzymatically. Chemical cleavage was done by CNBr, 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine, or partial acid hydrolysis (9). Enzymatic cleavage was done on the denatured, unmodified maleylated protein, by either trypsin, chymotrypsin, or Staphylococcus V8 protease. Conformation-dependent cleavage was done with subtilisin (9). Specific cleavage after lysine was done with endoproteinase Lys-C (10). Analysis with carboxypeptidase P was done at pH 5.6 and 250C with an enzyme-to-substrate ratio of 1:200. Peptide mixtures from chemical or enzymatic cleavage were separated by reversed-phase HPLC on C4 (Vydac) or C18 (uBondapak; Waters) columns, and the peptides were eluted with acetonitrile in 0.1% trifluoroacetic acid at or near neutral conditions (50 mM triethylamine/acetic acid, pH 6.5). Several CNBr peptides were purified by preparative SDS/PAGE and electroelution according to refs. 11 and 12. Coomassie stain was displaced from the peptide with 1% SDS and extracted with four volumes of acetone.