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D Shortle

Publications and source records attributed to D Shortle.

72 records · Page 4Linked to original sources
PubMed1987

Isolation and sequencing of the gene encoding delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni: overexpression of the protein.

We describe the cloning, sequencing, and overexpression of the steroid isomerase (3-oxosteroid delta 5-delta 4-isomerase, EC 5.3.3.1) gene of Pseudomonas testosteroni. A genomic library of P. testosteroni total DNA constructed from partial EcoRI digests ligated to a lambda gtWES vector was probed with a 23-base oligonucleotide mixture [ATGAAC(T)ACC(A,T)CCG(C,A)GAG(A)CAC(T)ATGAC] corresponding to the NH2-terminal sequence of steroid isomerase. Subclones derived from a recombinant phage containing a 5400-base-pair insert were sequenced and found to contain the expected 375-nucleotide open reading frame flanked at both ends by in-frame TGA termination codons. The DNA sequence agreed with the above 125-amino acid sequence except for codons 22, 24, 33, and 38, all of which encoded Asp rather than Asn, and codon 77, which encoded Glu rather than Gln. A 1370-base-pair fragment was inserted into pUC 19 plasmid vector and used to construct a strain of Escherichia coli JM 101 that overexpressed the isomerase gene in the presence of isopropyl beta-D-thiogalactopyranoside. Cytosolic extracts of this strain contained a major soluble protein that migrated with the steroid delta-isomerase subunit on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These cytosolic extracts had 10-50% of the specific activity of crystalline isomerase, depending on the method of preparation. The recombinant enzyme was crystallized in both monoclinic (flat plates) and hexagonal (bipyramids) crystal forms, described previously for the enzyme isolated from P. testosteroni. The kinetic properties of the crystalline recombinant enzyme, including specific activity, Km for 5-androstene-3,17-dione (340 microM), and Ki for the competitive inhibitor 19-nortestosterone (11.9 microM), agreed closely with the values reported for the isolated enzyme.

Amino Acid Sequence↗
PubMed1986

Kinetic and magnetic resonance studies of effects of genetic substitution of a Ca2+-liganding amino acid in staphylococcal nuclease.

The X-ray structure of staphylococcal nuclease suggests octahedral coordination of the essential Ca2+, with Asp-21, Asp-40, and Thr-41 of the enzyme providing three of the six ligands [Cotton, F. A., Hazen, E. E., Jr., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555]. The Asp-40 codon was mutated to Gly-40 on the gene that had been cloned into Escherichia coli, and the mutant (D40G) and wild-type enzymes were both purified from E. coli by a simple procedure. The D40G mutant forms a (5 +/- 2)-fold weaker binary complex with Ca2+ as found by kinetic analysis and by Ca2+ binding studies in competition with Mn2+, a linear competitive inhibitor. Similarly, as found by electron paramagnetic resonance (EPR), Mn2+ binds to the D40G mutant with a 3-fold greater KD than that found with the wild-type enzyme. These differences in KD are increased by saturation of staphylococcal nuclease with the DNA substrate such that KmCa is 10-fold greater and KIMn is 15-fold greater for the mutant than for the wild-type enzyme, although KMDNA is only 1.5-fold greater in the mutant. The six dissociation constants of the ternary enzyme-Mn2+-nucleotide complexes of 3',5'-pdTp and 5'-TMP were determined by EPR and by paramagnetic effects on 1/T1 of water protons, and the dissociation constants of the corresponding Ca2+ complexes were determined by competition with Mn2+. Only small differences between the mutant and wild-type enzymes are noted in K3, the dissociation constant of the nucleotides from their respective ternary complexes. 3',5'-pdTp raises the affinities of both wild-type and mutant enzymes for Mn2+ by factors of 47 and 31, respectively, while 5'-TMP raises the affinities of the enzymes for Mn2+ by smaller factors of 6.8 and 4.4, respectively. Conversely, Mn2+ raises the affinities of both wild-type and mutant enzymes for the nucleotides by 1-2 orders of magnitude. Analogous effects are observed in the ternary Ca2+ complexes. Dissociation constants of Ca2+ and Mn2+ from binary and ternary complexes, measured by direct binding studies, show reasonable agreement with those obtained by kinetic analysis. Structural differences in the ternary metal complexes of the D40G mutant are revealed by a 31-fold decrease in Vmax with Ca2+ and by 1.4-3.1-fold decreases in the enhancement of 1/T1 of water protons with Mn2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1986

Guanidine hydrochloride denaturation studies of mutant forms of staphylococcal nuclease.

Several mutant forms of staphylococcal nuclease with one or two defined amino acid substitutions have been purified, and the effects of the altered amino acid sequence on the stability of the folded conformation have been analyzed by guanidine hydrochloride denaturation. Two nuc- mutations, which greatly reduced the level of enzyme activity accumulated in E coli colonies carrying a recombinant plasmid with the mutant nuc gene (ie, a NUC- phenotype), both result in protein unfolding at significantly lower guanidine hydrochloride concentrations than the wild-type protein, whereas three sup mutations isolated on the basis of their ability to suppress partially the NUC- phenotype of the above two mutations result in unfolding at significantly higher guanidine hydrochloride concentrations. Characterization of nuclease molecules with two different amino acid substitutions, either nuc- + sup pairs or sup + sup pairs, suggests that the effect of an amino acid substitution on the stability of the native conformation, as measured by the value of delta delta GD, may not be a constant, but rather a variable that is sensitive to the presence of other substitutions at distant sites in the same molecule. Surprisingly, the slopes of the log Kapp vs guanidine hydrochloride concentration plots vary by as much as 35% among the different proteins.

Amino Acid Sequence↗
PubMed1986

Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation.

Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m--the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration--these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified--interactions which appear to involve hydrophobic groups.

Circular Dichroism↗
PubMed1985

Genetic analysis of staphylococcal nuclease: identification of three intragenic "global" suppressors of nuclease-minus mutations.

A collection of 77 unique missense mutations distributed across the gene encoding staphylococcal nuclease (nuc) has been assembled. These mutations were induced by random gap misrepair mutagenesis of the cloned gene and were identified in E. coli transformants expressing reduced levels of nuclease activity. Four nuc- mutations which alter amino acid residues at positions outside of the active site region of the enzyme were submitted to a second round of mutagenesis, and characterization of several independent NUC+ isolates lead to the identification of three second-site suppressor mutations within the protein-coding sequence of the nuc gene. On separation from the mutation originally suppressed and recombination with a number of other nuc- mutations, all three suppressors displayed the property of "global" suppression, i.e., phenotypic suppression of the nuclease-minus character of multiple different alleles. A simple and generally applicable strategy was used to obtain efficient homologous recombination between plasmids for purposes of mapping nuc- mutations, mapping second-site suppressors and constructing double mutant combinations from pairs of single mutations.

Alleles↗
PubMed1984

Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene.

Two temperature-sensitive mutations have been constructed in the single actin gene (ACT1) of the yeast Saccharomyces cerevisiae by in vitro mutagenesis of the cloned gene followed by integrative transformation of mutagenized DNA into yeast cells. A strategy of allele replacement was used that allowed recessive mutations to be phenotypically expressed in the initial transformants, thus simplifying the screening of large numbers of independently transformed cells. After confirming that several ts mutations were located within the actin structural gene by genetic methods, these mutant alleles were cloned, and the altered amino acid residues were defined by DNA sequence analysis. The two unique mutations resulted in substitution of proline-32 with leucine and alanine-58 with threonine. In the course of isolating these mutations, the observation was made that a high proportion of yeast cells transformed with exogenous DNA by the spheroplast method are temperature sensitive for growth because of genetic changes unrelated to the transforming DNA.

Actins↗
PubMed1982

Lethal disruption of the yeast actin gene by integrative DNA transformation.

A mutant allele of the chromosomal locus corresponding to the cloned actin gene of the yeast Saccharomyces cerevisiae has been constructed by DNA transformation with a hybrid plasmid which integrates into, and thereby disrupts, the protein-encoding sequences of the gene. In a diploid strain of yeast, disruption of the actin gene on one chromosome results in a mutation that segregates as a recessive lethal tightly linked to a selectable genetic marker on the integrated plasmid. The actin gene, therefore, must encode an essential function for yeast cell growth.

Actins↗
PubMed1982

Single-stranded gaps as localized targets for in vitro mutagenesis.

Short single-stranded gaps in circular DNA molecules can be generated enzymatically, often at predetermined sites. These can serve as targets for in vitro mutagenesis procedures that result in alterations in nucleotide sequence within or very near the gap. Deamination of unpaired cytosine residues with sodium bisulfite has been used to induce mutations in the BglI restriction site of SV40 DNA and within defined regions of the beta-lactamase gene on pBR322. A new method of induction of mutations at gaps, called "gap misrepair," has been developed; it was used to cause changes at the HindIII and C1aI restriction sites on pBR322 DNA. Gap misrepair reactions using DNA polymerase I of Micrococcus luteus in the presence of T4 DNA ligase and three of the four deoxynucleoside triphosphates yielded all three possible substitutions for adenine and cytosine residues in the DNA.

Chemical Phenomena↗
PubMed1982

Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro.

Short single-stranded gaps can be constructed by limited exonuclease action at single-stranded breaks (nicks) placed at predetermined sites on closed circular DNA molecules. As efficient primer-templates for DNA polymerase, single-stranded gaps can be repaired in vitro to regenerate an intact DNA duplex. In this report two in vitro reaction schemes are described that produce a high frequency of errors during repair ("misrepair") of gaps and thereby allow the efficient recovery of mutations limited to the nucleotide sequence at or near the original gap. In the first of these misrepair schemes, nucleotide misincorporations are stimulated by omission of one of the four deoxynucleoside triphosphates; the misincorporations are trapped by the presence of excess DNA ligase in the reaction mixture. The second misrepair scheme involves the misincorporation of an excision-resistant alpha-thiophosphate nucleotide, followed by gap filling in the presence of all four conventional deoxynucleoside triphosphates. When applied to short gaps constructed at one of several unique restriction sites on the small plasmid pBR322, both gap misrepair methods yielded mutations within the targeted restriction site at high frequency (6--42%). A majority of the sequence changes identified were base substitutions; transversions and transitions are approximately equally represented. The remaining sequence changes were an insertion of a single base pair and deletions of one to four base pairs.

DNA Polymerase I↗
PubMed1980

Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule.

A general method for efficiently mutagenizing a predetermined segment of a closed circular duplex DNA molecule was used to construct mutations in two specific regions of the beta-lactamase (bla) gene carried by the small plasmid pBR322. The principle of segment-directed mutagenesis is the use of a single-stranded homologous DNA fragment to direct the nicking of circular duplex DNA within a segment defined by the DNA fragment in a two-step reaction. First, Escherichia coli recA protein is used to catalyze assimilation of the homologous single-stranded DNA, producing a displacement loop ("D-loop") in the circular DNA. Second, a small amount of the single-strand-specific S1 nuclease is used to nick the displaced DNA. The segment-directed nicks are converted to small gaps, which are then mutagenized specifically with sodium bisulfite. A short (128-base pair) restriction endonuclease fragment from the center of the bla gene was used to direct mutagenesis with the result that 7.5% of the recovered plasmids were bla- mutants and 49/51 of these mutants, mapped genetically, were found to lie in a deletion interval whose endpoints approximate those of the restriction fragment. Similar results were obtained when another short fragment covering the beginning of the gene was used; many of these mutations map in the region coding the "signal" sequence thought to be involved in secretion of beta-lactamase.

Base Sequence↗
PubMed1978

Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome.

DNA from simian virus 40 (SV40) was prepared for local mutagenesis by nicking the molecule at a specific site with a restriction endonuclease that recognizes one site in SV40 DNA and then extending the nick enzymatically to expose a short, single-stranded segment of DNA. The "gapped" DNA was treated with a single-strand-specific mutagen, sodium bisulfite, which converts cytosine to uracil. After mutagenesis, the gap was repaired with DNA polymerase, generating molecules resistant to the restriction enzyme used to make the initial nick. From cells infected with DNA thus modified, SV40 mutants were isolated that had enzyme-resistant genomes. In some cases, precise positions of G.C to A.T transitions could be inferred from the patterns of susceptibility of mutant DNA to other restriction endonucleases whose recognition sequences were altered by the mutagenesis procedure. One of the restriction endonuclease sites mutagenized (Bgl I) maps at the origin of SV40 DNA replication and near sequences corresponding to the 5' ends of viral mRNAs. Many of the resulting Bgl I-resistant mutants yielded small plaques, suggesting partial defectiveness in DNA replication or transcription.

DNA Polymerase I↗
PubMedDate not supplied

A genetic system for analysis of staphylococcal nuclease.

The gene encoding staphylococcal nuclease (strain Foggi) has been isolated, and its complete nucleotide sequence determined. When inserted into a derivative of plasmid pBR322, the nuc gene is expressed in Escherichia coli at a low level, and nuclease activity in individual colonies is readily assayed by a replica-plating chromogenic test. A set of plasmids with BamHI-linker "bracketed" deletions spanning the nuc gene can be used to map mutations genetically to defined segments of the gene. In combination with present methods for efficient in vitro mutagenesis, this plasmid-based genetic system can be applied to the detailed genetic analysis of this small, biochemically and biophysically well-characterized enzyme.

Amino Acid Sequence↗