Alberto Bernardi

Studies on acid hydrolases: I. A procedure for the preparation of acid deoxyribonuclease and other acid hydrolases

Abstract A procedure for the preparation of spleen acid deoxyribonuclease (deoxyribonucleate 3′-oligonucleonucleotidohydrolase, EC 3.1.4.6; deoxyribonuclease II) is described. The procedure can be used, with only slight modifications, to prepare other acid hydrolases from spleen, as well as from other tissues and organs. Two enzyme components, called A and B, have been separated by chromatographic fractionation of acid deoxyribonuclease on CM-Sephadex. The two components have the same enzymological, macromolecular and spectroscopic properties. Preliminary evidence suggests that the component, A, is derived from the minor component, B, by a deamidation process occurring during the acidic extraction of the enzyme from the tissue.

Studies on acid hydrolases: IV. Isolation and characterization of spleen exonuclease

Abstract 1. 1. A new procedure for the preparation of spleen exonuclease is described. The enzyme preparation obtained is free from phosphomonoesterase, deoxyribonuclease, ribonuclease and adenosine deaminase activities. 2. 2. Native DNA is degraded at a rate 25 times lower than alkali-denatured DNA. Poly (A), poly (U) and poly (I) are all degraded by the enzyme at comparable rates, whereas poly (C) is resistant. Bis -(p- nitrophenyl ) phosphate is a very poor substrate. Using acid deoxyribonuclease digest as the substrate, the pH optimum of the enzyme is close to 5.5 in succinate buffer, whereas a higher value is found in acetate buffer; pH-activity curves are barely affected by the presence of Mg2+. 3. 3. The enzyme has a sedimentation constant of 4.6 S. Heating at 56° destroys 50 % of the enzyme activity.

Studies on acid hydrolases. II. Isolation and properties of spleen acid phosphomonoesterase.

Abstract A procedure for the isolation of spleen acid phosphomonoesterase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) is reported. Several of the physical and catalytic properties of the enzyme have been studied and shown to be extremely similar to those of prostatic acid phosphomonoesterase. The sedimentation coefficient of spleen acid phosphomonoesterase is close to 5.6 S. The pH-activity curve shows a broad maximum between pH 3.0 and 4.8.

Studies on acid hydrolases: III. Isolation and properties of spleen acid ribonuclease

Abstract A procedure for the preparation of spleen acid ribonuclease is described. The enzyme has a sedimentation constant close to 2.3 S. It is thermolabile and has a pH optimum of 5.3. The hydrolysis of soluble RNA by the enzyme proceeds only slightly more slowly than that of ribosomal RNA. Among synthetic polyribonucleotides, polyuridylic acid is hydrolyzed rapidly, polyinosinic acid slowly; polycytidylic and polyadenylic acids are highly resistant to the enzyme.

Site-specific deletions in the recombinant plasmid pSC101 containing the redB-ori region of phage λ

Large deletions occur in the hybrid plasmid formed by pSC101 and the EcoRI fragment f2 of phage lambda (redB-ori region) under well defined growth conditions (Bernardi and Bernardi, 1980). We have sequenced the novel joints of the four deletions so obtained and shown that they have one endpoint in pSC101, identical in all four cases, the other endpoint being located in four different lambda sequences. Furthermore, the nucleotide sequences of the novel joints show homologies between the conserved pSC101 sequence and the lambda sequences both conserved and deleted. The presence of an IS-type element in pSC101 is postulated; however, this element is unrelated to the 200 bp element already described in pSC101 (Ravetch et al., 1976).

Studies on acid hydrolases: V. Isolation and characterization of spleen nucleoside polyphosphatase

Abstract 1. 1. A new procedure is described for the purification of spleen nucleoside polyphosphatase, an enzyme able to split both the terminal phosphates of nucleoside tri-and di-phosphates and bis -(p- nitrophenyl ) phosphate. The enzyme was eluted from the final CM-Sephadex column at a fairly constant specific activity. 2. 2. Using bis -(p- nitrophenyl ) phosphate as the substrate, the enzyme showed a pH optimum close to 6.8, and a Michaelis constant equal to 0.95 mM. Phosphate ions are competitive inhibitors. The sedimentation constant of the enzyme was found to be equal to 3.2 S. 3. 3. A comparison of the hydrolytic activities on bis -(p- nitrophenyl ) phosphate of 3 enzymes, acid deoxyribonuclease, spleen exonuclease, and nucleoside polyphosphatase, is presented.

Studies on ACID hydrolases: VI. Isolation and characterization of spleen ACID phosphomonoesterase B

Abstract A procedure for the isolation of an acid phosphomonoesterase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) from hog spleen is reported. This enzyme, called acid phosphomonoesterase B, appears to be an ideal tool for the determination of terminal nucleotides in oligonucleotides because of its extremely high purity and stability. Several properties of the enzyme have been investigated and shown to be very similar to those of another spleen acid phosphomonoesterase previously described by A. Chersi, A. Bernardi and G. Bernardi ( Biochim. Biophys. Acta , 129 (1966) 12). The two enzymes show, however, very large differences in their chromatographic behavior on different columns.

14 Spleen Acid Exonuclease

Publisher Summary This chapter discusses the Isolation and Purification techniques of spleen acid exonuclease. It also discusses its physical properties and catalytic properties. Spleen acid exonuclease is an enzyme particularly useful in sequence studies of oligonucleotides, derived from both ribonucleic acid and deoxyribonucleic acid, as it splits off, in a sequential way, nucleoside-3′-phosphates starting from the 5′OH end. The enzyme has also been called spleen phosphodiesterase and phosphodiesterase II. It has been claimed that spleen acid exonuclease has its counterpart in other tissues. It is very likely that this claim is correct, in spite of the fact that it was based on the wrong assumption that a hydrolytic activity at pH 6.0 on p-nitrophenyl thymidine-3′-phosphate could be equated with acid exonuclease activity. In fact, an acid exonuclease activity has been shown in fish muscle and, using as a substrate an acid DNase digest, in rat liver. In this latter case, it was checked that the activity on p-nitrophenyl thymidine-3′-phosphate was not inhibited by S042- and that it was lower than on thymidylyl thymidine. As far as the intracellular localization of the acid exonuclease activity is concerned, Razzell, using the synthetic substrate, found it in both the mitochondrial-lysosomal fraction and in the supernatant. Van Dyck and Bernardi found that an enzymic activity on acid DNase hydrolysates could be extracted from rat liver lysosomes and that the sedimentation coefficient of this activity, as determined by sucrose density gradient centrifugation, was the same as for spleen acid exonuclease.

Intramolecular transposition of IS102

It has been postulated that deletions mediated by transposable elements are intramolecular transposition events. An implication of this hypothesis is that the deleted fragment may be recovered if it is capable of autonomous replication. We report here the characterization of the products of intramolecular transposition of the element IS102 in bireplicons. We show that when two origins (oris) (of pSC101 and R6-5) generate the same copy numbers, two dissociated replicons are recovered as well as the inversions. On the contrary, when two oris (of pSC101 and pBR322) have different copy numbers, intramolecular transposition results essentially in inversions. However, the very low frequency (5 X 10(-8)) at which intramolecular transpositions in the bireplicons occurs, as compared to the single replicon (10(-4)), suggests that a complete transposition reaction may not be necessary to generate deletions.

Atypical deletions generated by mutated IS102 elements

SummaryThe element IS102 potentially codes for two polypeptide chains. We have introduced several mutations in the larger one near the COOH terminus and determined the residual ability of the mutated elements to generate deletions in order to assign a role to this polypeptide chain. We show that in these elements, deletions still occur, although at a reduced level, but that in all cases examined so far the ends of the element are no longer recognized as the fixed endpoint of IS-mediated deletions, even though some other structural features of normal deletions formation are still present.