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S N Slilaty

Publications and source records attributed to S N Slilaty.

11 recordsLinked to original sources
PubMed1998

Accurate insertional inactivation of lacZalpha: construction of pTrueBlue and M13TrueBlue cloning vectors.

Color selection plasmid and phage vectors based on insertional inactivation of the lacZalpha gene fragment have been in wide use for nearly two decades. The originals (the pUC and M13mp series) and all subsequent derivatives of these vectors contain the same mechanism for insertional inactivation of lacZalpha placing the sites for gene interruption between the initiator ATG codon and codon 7, a region that encodes a functionally nonessential part of the lacZalpha-complementation peptide. Numerous observations found in the literature and made in our laboratory suggest that the color selection process in these vectors does not function reliably and that erroneous conclusions about the success of cloning experiments are frequently drawn. We have studied the efficiency of lacZalpha inactivation by performing insertions at different intervals along the entire length of the proximal 60-amino-acid (aa)-coding region of the lacZ gene (lacZalpha). Our results show that accurate and reliable inactivation of lacZalpha occurs only when insertions are made within the DNA region that encodes aa 11-36. Based on these results, novel color selection plasmid and phage vectors, pTrueBlue and M13TrueBlue, have been constructed. Application of these vectors for gap-free shotgun sequencing of genomic DNA is discussed.

Amino Acid Sequence↗
PubMed1991

Primary sequence of the glucanase gene from Oerskovia xanthineolytica. Expression and purification of the enzyme from Escherichia coli.

A 2.7-kilobase fragment of DNA from Oerskovia xanthineolytica containing the gene for a beta-1,3-glucanase has been isolated and its complete nucleotide sequence determined. The sequence was found to contain two large open reading frames. Purification of the mature native enzyme and subsequent amino-terminal sequencing defined the glucanase gene in one reading frame which potentially encodes a protein of 548 amino acids. We have expressed this glucanase gene in Escherichia coli under control of the lacUV5 promoter and found the product to be secreted into the periplasm as a mature enzyme of about the same molecular weight as that of the native protein. The recombinant enzyme was purified to near homogeneity by a single step of high performance liquid chromatography. The ability of the recombinant enzyme to digest beta-glucan substrates and to lyse viable yeast cells was found to be indistinguishable from that of the native protein. Deletion of the cysteine-rich carboxyl-terminal 117 amino acids of the enzyme, which also contain two duplicated segments, abolished the lytic activity but did not significantly affect the glucanase function of the protein. The possible involvement of this domain in interaction with the yeast cell wall is discussed.

Amino Acid Sequence↗
PubMed1991

The role of electrostatic interactions in the mechanism of peptide bond hydrolysis by a Ser-Lys catalytic dyad.

General-base catalysis in the active site of serine proteases is carried out by the imidazole side chain of a histidine. During formation of the transition state, an adjacent carboxylic acid group stabilizes the positive charge that forms on the general-base catalyst and as a result contributes several orders of magnitude to the catalytic efficiency of these enzymes. In the recently discovered family of self-cleaving proteins exemplified by the LexA repressor of Escherichia coli, instead of the imidazole of a histidine, the active-site general-base catalyst was found to be the epsilon-amino of a lysine. The considerably higher capacity of the lysine side chain for proton acceptance raises interesting questions concerning the role of electrostatic interactions in the mechanism of proton transfer by this highly basic group. The negative charge elimination studies described here and their effects on the kmax and pK of LexA self-cleavage are consistent with a model in which electrostatic interactions between an acidic side chain and the general-base catalyst form a barrier to proton transfer. The implications are that the epsilon-amino group, unlike the imidazole group, is capable of effecting proton transfer without the intervention of a countercharge.

Bacterial Proteins↗
PubMed1990

Independent folding of individual components in hybrid proteins. Evidence that the carboxy-terminal 135 residues of the LexA repressor constitute a single autonomous domain.

Inactivation of the Escherichia coli repressor protein, LexA, takes place through a cleavage reaction which hydrolyzes the Ala84-Gly85 peptide bond near the center of the molecule. The mechanism of cleavage has previously been shown to be an intramolecular reaction stimulated in vitro by elevated pH or by the addition of activated RecA protein. The entire self-cleavage activity of LexA has been found to lie within a 135-residue tryptic fragment extending from Leu68 to the end of the protein at Leu202. Since the activity of self-cleavage is dependent on the proper three-dimensional structure of the protein, we have used it as a probe to investigate the extend of folding autonomy and functional independence of this 135-residue carboxy-terminal domain of LexA by applying a protein fusion approach. A series of twelve different hybrid proteins, containing LexA sequences in a variety of predefined primary structural arrangements, were constructed and evaluated for whether or not self-cleavage activity has been retained. The results revealed that retention or loss of activity is independent of the nature or size of the foreign protein used. Loss of self-cleavage was found to be a function of amino- or carboxy-terminal deletions in the self-cleaving LexA component of the fusion proteins. The present findings, together with the observations of other artificial fusions proteins and the naturally occurring bifunctional and multifunctional proteins, along with the data on helix packing, provide further support for the notion of modular architecture of proteins and suggest that when these autonomous units are fused, they retain their tendency to fold independently of the remainder of the polypeptide to generate physically linked active domains, rather than to fold dependently and yield scrambled structures.

Bacterial Proteins↗
PubMed1990

Site-directed mutagenesis by complementary-strand synthesis using a closing oligonucleotide and double-stranded DNA templates.

An approach for generating structures capable of directing full-length complementary-strand synthesis for double-stranded plasmid DNA is described. The structures are formed following heat denaturation and cooling of linearized plasmid DNA molecules in the presence of what is referred to as a "closing" oligonucleotide. Consisting of a sequence complementary to the free ends of one of the two plasmid strands, the closing oligonucleotide functions as an agent for recircularization of a DNA strand and generation of a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently closed heteroduplex molecule. When combined with a mutagenic oligonucleotide and uracil-substituted DNA templates, this approach allows site-directed mutagenesis to be performed directly on double-stranded DNA with a mutant formation efficiency of about 50%, a level amenable to rapid screening by DNA sequencing.

Base Sequence↗
PubMed1989

Synthesis and secretion of hepatitis B middle surface antigen by the methylotrophic yeast Hansenula polymorpha.

The methylotrophic yeast, Hansenula polymorpha, has been developed as a host system for the synthesis of heterologous proteins. The middle surface antigen of hepatitis B virus (preS2-HBsAg) has been synthesized under the control of a methanol-regulated promoter derived from the methanol oxidase-encoding gene. The synthesized preS2-HBsAg protein was found to be secreted outside the cell membrane into the periplasm and further excreted into the culture medium following permeabilization of the cell wall with beta-1,3-glucanase (beta Glu). Cell cultures treated with beta Glu were able to continuously synthesize and secrete 22-nm particles of preS2-HBsAg into the medium for several days. The overall yield of antigen from treated cultures was found to be over threefold greater than that of untreated controls. The observation that complex supramolecular structures, such as the 22-nm particles of preS2-HBsAg, can be secreted by H. polymorpha and released into the medium, suggests the potential for these yeasts to be an alternative secretory host.

Cloning, Molecular↗
PubMed1987

Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism.

LexA repressor of Escherichia coli is inactivated in vivo by a specific cleavage reaction requiring activated RecA protein. In vitro, cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH. These two cleavage reactions have similar specificities, suggesting that RecA acts indirectly to stimulate self-cleavage, rather than directly as a protease. We have studied the chemical mechanism of cleavage by using site-directed mutagenesis to change selected amino acid residues in LexA, chosen on the basis of kinetic data, homology to other cleavable repressors, and potential similarity of the mechanism to that of proteases. Serine-119 and lysine-156 were changed to alanine, a residue with an unreactive side chain, resulting in two mutant proteins that had normal repressor function and apparently normal structure, but were completely deficient in both types of cleavage reaction. Serine-119 was also changed to cysteine, another residue with a nucleophilic side chain, resulting in a protein that was cleaved at a significant rate. These and other observations suggest that hydrolysis of the scissile peptide bond proceeds by a mechanism similar to that of serine proteases, with serine-119 being a nucleophile and lysine-156 being an activator. Possible roles for RecA are discussed.

Amino Acid Sequence↗
PubMed1986

Intramolecular cleavage of LexA and phage lambda repressors: dependence of kinetics on repressor concentration, pH, temperature, and solvent.

LexA repressor of Escherichia coli and phage lambda repressor are inactivated in vivo and in vitro by specific cleavage of an Ala-Gly peptide bond in reactions requiring RecA protein. At mildly alkaline pH, the in vitro cleavage reaction also proceeds spontaneously, suggesting that peptide bond hydrolysis is an activity of the repressors rather than of RecA. The spontaneous cleavage reaction, termed "autodigestion", has been characterized for the LexA and lambda repressors. The results show that the reaction is intramolecular. The rate of LexA autodigestion was studied over the pH range 7.15-11.77 and over the temperature range 4-46 degrees C. The logarithm of the rate constant increased linearly with pH and reached a plateau value (2.5 X 10(-3) s-1 at 37 degrees C) at pH above 10. The data closely followed a model in which a single residue side chain (apparent pK = 9.8 at 37 degrees C) must be deprotonated for the protein to show activity. Analysis of the temperature dependence gave the heat of proton dissociation as 19.9 kcal/mol and the heat of activation for hydrolysis as 15.3 kcal/mol at 25 degrees C. Autodigestion of lambda repressor, studied over the pH range 8.65-10.70 at 37 degrees C, was similar to the LexA reaction in its pH dependence, yielding a pK of 9.8. The maximum rate at 37 degrees C for lambda repressor, 6.1 X 10(-5) s-1, was 40 times slower than for LexA, a difference similar to that previously observed in vivo and in vitro for RecA-dependent cleavage reactions. There was no significant solvent deuterium isotope effect on the autodigestion of LexA. Changes in buffer composition, including high concentrations of glycine for lambda repressor and of imidazole or hydroxylamine for LexA, indicated that solvent components other than water do not participate in the rate-determining step. Removal or addition of metal ions did not significantly affect LexA autodigestion. These and other observations suggest that the deprotonated form of an amino acid side chain plays a central role in the chemistry of the cleavage reaction. The above observations establish repressor autodigestion as a member of an emerging set of biologically important self-processing reactions.

Bacterial Proteins↗
PubMed1983

Gene transfer by polyoma-like particles assembled in a cell-free system.

Empty capsids of polyoma virus interact with DNA in a cell-free system to form polyoma-like particles (PLP). The DNA in these particles is protected from the action of pancreatic deoxyribonuclease. Transfer of genetic information by PLP has been accomplished by using a restriction fragment containing the transforming sequences of polyoma DNA as a model gene. Infection of rat F111 cells by PLP containing these sequences results in DNA-mediated cellular transformation. Gene transfer by PLP is 50 to 150 times more efficient than by the calcium phosphate precipitation method.

Animals↗
PubMed1982

Polyoma-like particle: characterization of the DNA encapsidated in vitro by polyoma empty capsids.

Polyoma empty capsids and DNA interact in a cell-free system to form a polyoma-like particle (PLP). The particle is stable in high concentrations of salt and contains a DNA fragment that is protected by the capsid from the action of DNase I. The DNA extracted from PLP is heterogeneous in size. It has a mean molecular weight of 1.2 x 10(6) with a standard deviation of +/- 0.5 x 10(6). Analysis of PLP DNA with restriction endonucleases and the Southern blot technique reveals that a specific primary sequence is not required for PLP formation. Either linear, circular, or supercoiled polyoma DNA, as well as single-stranded DNA, rRNA, and the synthetic homopolymers poly(dA) . poly(dT) and poly(dG) . poly(dC) can be used for PLP formation. It is concluded that polyoma empty capsids can be used to bind and encapsidate nucleic acids differing in their primary, secondary, and superhelical tertiary structure to form, in vitro, stable polyoma-like particles.

Capsid↗