Submaxillary gland of mouse: properties of a purified protein affecting muscle tissue in vitro.

A protein from the salivary gland of mice has been highly purified. It affects embryonic muscle tissue in vitro and has both esterase and peptidase activities. Addition of the pure protein to tissue culture in synthetic medium causes dissociation of muscle fibers in individual myoblasts with loss of myosin. This biological activity, as well as the esterase activity, is inhibited by low concentrations of phenylmethanesulfonyl fluoride; this suggests that the effect on the tissue is a consequence of the protein's enzymatic activities.

Conservation of substrate recognition mechanisms by tRNA splicing endonucleases.

Accuracy in transfer RNA (tRNA) splicing is essential for the formation of functional tRNAs, and hence for gene expression, in both Eukaryotes and Archaea. The specificity for recognition of the tRNA precursor (pre-tRNA) resides in the endonuclease, which removes the intron by making two independent endonucleolytic cleavages. Although the eukaryal and archaeal enzymes appear to use different features of pre-tRNAs to determine the sites of cleavage, analysis of hybrid pre-tRNA substrates containing eukaryal and archaeal sequences, described here, reveals that the eukaryal enzyme retains the ability to use the archaeal recognition signals. This result indicates that there may be a common ancestral mechanism for recognition of pre-tRNA by proteins.

Structural alterations in mutant precursors of the yeast tRNALeu3 gene which behave as defective substrates for a highly purified splicing endoribonuclease.

We have produced a highly purified preparation of the Xenopus laevis splicing endonuclease (XlaI RNase). The purified enzyme correctly cleaves tRNA precursors, creating substrates for subsequent ligation. The 5'-half molecules have a 2',3' cyclic phosphate at their 3' termini. Assuming that splicing enzymes recognize primarily structural elements in the 'mature domain', we have been studying the conformation of three splicing-defective precursors made from mutants of the yeast tRNALeu3 gene. The mutations alter base-pairing in the D-stem region and two of the mutants are absolute defectives. Enzymatic probing of the structures of the altered tRNA precursors shows that the structural perturbations in these mutants are localized on the 'inside' of the 'L'-shaped three-dimensional structure. The implications of this finding for the recognition process are discussed.

DNA-relaxing activity and endonuclease activity in Xenopus laevis oocytes.

Complex simian virus 40 DNA produced by a soluble cell-free extract derived from stage 6 oocytes of Xenopus laevis consists of fully relaxed circles (i.e., with no superhelical turns). An endonuclease and a DNA-relaxing protein, either or both of which could be responsible for the relaxation of the complex DNA, have been purified from the extract. The endonuclease(s) produces nicked circles (having a single-strand scission) and linear full-size molecules. The DNA-relaxing protein is in the nucleus, has a molecular weight of apporximately 70,000, and is able to remove both negative and positive superhelical turns.

Purification and characterization of Xenopus laevis type I topoisomerase.

A topoisomerase activity was purified from mature ovaries and from nuclei of stage 6 oocytes of Xenopus laevis. From both preparations we obtained a single polypeptide chain having an estimated molecular weight of 67,000. The enzyme purified from ovaries is active in the presence of 150 mM monovalent cation, but its activity is more than 1 order of magnitude higher in the presence of 6 mM Mg2+; the enzyme purified from nuclei requires Mg2+ through all the steps of purification. Enucleated oocytes are devoid of topoisomerase activity but are able to convert the nuclear enzyme to a species active also in the presence of monovalent cations. The difference in ionic requirement between the nuclear topoisomerase and the enzyme purified from ovaries as well as the topoisomerases from other eukaryotic sources, which are most active in the presence of monovalent cations, may depend on the source of the enzyme and/or on the extraction procedure. Ovarian and nuclear topoisomerases catalyze relaxation of both negatively and positively superhelical DNA; the relaxed isomers produced in the presence of Mg2+ have a few positive superhelical turns.

Binding and cleavage of pre-tRNA by the Xenopus splicing endonuclease: two separable steps of the intron excision reaction.

We have constructed three base-substitution mutants of the yeast tRNALeu3 gene. In two of them the ability to form an extended anticodon stem is lost. In the first mutant the bases encoding the anticodon change from TTG to GAC (positions 37, 36, 35); in the second, the nucleotides encoding the region of the intron that base-pair with the anticodon change from CAA to GTC (positions 48, 47, 46). The third is a double mutant characterized by both substitutions described above so that its ability to form an extended anticodon stem is restored. The precursors derived from the two single mutants are accurately spliced in the X. laevis germinal vesicles (GV) extract: pairing of the anticodon with the intron, therefore, is not required for the splicing reaction. The precursor derived from the double mutant is not spliced, indicating that the new extended anticodon stem exerts an inhibitory action. Since the double mutant precursor binds to the purified splicing endonuclease, binding and cleavage occur as two separable steps in the intron excision reaction.