PubMed Health⌕ Search

Biomedical subjects

G J Warren

Publications and source records attributed to G J Warren.

16 recordsLinked to original sources
PubMed1991

Inhibition of recrystallization in ice by chimeric proteins containing antifreeze domains.

Using synthetic DNA, we assembled a gene encoding a protein identical in sequence to one of the antifreeze proteins produced by the fish Pseudopleuronectes americanus (winter flounder). To address the relationship between structure and function, we also assembled genes encoding proteins varying in sequence and length. The synthetic genes were cloned into a bacterial expression vector to generate translational fusions to the 3' end of a truncated staphylococcal protein A gene; the chimeric proteins encoded by these fusions, varying only in their antifreeze domains, were isolated from Escherichia coli. The antifreeze domains conferred the ability to inhibit ice recrystallization, which is characteristic of naturally occurring antifreeze proteins, on the chimeric proteins. The chimeric proteins varied in their effectiveness of inhibiting ice recrystallization according to the number of 11-amino acid repeats present in the antifreeze moiety. A protein with only two repeats lacked activity, while the inhibitory activity increased progressively for proteins containing three, four, and five repeats. Some activity was lost upon removal of either the salt bridge or the carboxyl-terminal arginine, but surprisingly, not when both features were absent together.

Antifreeze Proteins↗
PubMed1991

Enhanced survival of yeast expressing an antifreeze gene analogue after freezing.

Yeast, like most organisms, survives poorly under freezing conditions. It has been proposed that after rapid cooling yeast suffers a loss in viability from the recrystallization of intracellular ice. Antifreeze proteins found in the blood of certain polar fishes have been shown to be potent inhibitors of ice recrystallization at very low concentrations. We have examined the feasibility of protecting rapidly cooled yeast cells from freezing damage by inhibiting the recrystallization of intracellular ice through in vivo expression of an antifreeze analogue gene. A chemically synthesized gene encoding a protein similar to but differing from the antifreeze proteins of the fish Pseudopleuronectes americanus (winter flounder) was genetically fused to the 3' end of a truncated staphylococcal Protein A gene. When the fused gene was expressed in the budding yeast Saccharomyces cerevisiae, its cells were shown to produce a new chimeric protein that inhibited the recrystallization of ice in vitro. Yeast cells expressing the chimeric antifreeze protein showed a twofold increase in survival after rapid freezing (95 degrees C/min to -196 degrees C) and moderate rates of warming (26 to 64 degrees C/min) compared to cells lacking the chimeric protein.

Antifreeze Proteins↗
PubMed1990

Clustering of ice nucleation protein correlates with ice nucleation activity.

Antibodies raised against a synthetic peptide specifically detect ice nucleation proteins from Pseudomonas species in Western blots. In immunofluorescent staining of whole bacteria, the antibodies reveal the protein in clusters, as indicated by patches of intense fluorescence in Escherichia coli cells heterologously expressing Pseudomonas ice nucleation genes. The abundance, size, and brightness of the clusters vary considerably from cell to cell. Their varying sizes may explain the variability in activity of bacterial ice nuclei. Growth at lower temperatures produces more ice nuclei, and gives brighter and more frequent patches, than growth at 37 degrees C. The observed clustering may thus reflect formation of functional ice nucleation sites in vivo. The presence of ice nucleation protein in clusters is also correlated with alterations in cell morphology.

Amino Acid Sequence↗
PubMed1990

Rates of assembly and degradation of bacterial ice nuclei.

The kinetics of ice-nucleus assembly from newly synthesized nucleation protein were observed following induction of nucleation gene expression in the heterologous host Escherichia coli. Assembly was significantly slower for the small proportion of ice nuclei active above -4.4 degrees C; this was consistent with the belief that these nuclei comprise the largest aggregates of nucleation protein. The kinetics of nucleus degradation were followed after inhibiting protein synthesis. Nucleation activity and protein showed a concerted decay, indicating that most of the functional ice nuclei are in equilibrium with a single cellular pool of nucleation protein. A minority of the ice nuclei decayed much more slowly than the majority; presumably their nucleation protein was distinct either by virtue of different structure or different subcellular compartmentalization, or because of its presence in a metabolically distinct subpopulation of cells.

Bacterial Proteins↗
PubMed1988

Nonlinear relationship between concentration and activity of a bacterial ice nucleation protein.

The expression level of an ice nucleation gene (inaZ) was varied in Escherichia coli to observe the relationship between activity and gene product. The ice nucleation activity increased as the 2nd to 3rd power of the membrane concentration of the inaZ gene product, implying that molecules of InaZ protein interact cooperatively in groups of two to three at the rate-limiting step of ice nucleus assembly. The 2nd to 3rd power relationship was independent of the threshold temperature at which ice nucleation was measured and was consistent over a 500-fold range of protein concentration. Such a relationship indicates that the same rate-limiting step must be common to the formation of ice nuclei displaying all the various threshold temperatures within a bacterial population. Observations of Pseudomonas syringae, expressing the inaZ gene at various levels, were consistent with a similar relationship and hence a similar mechanism of ice nucleus assembly in Pseudomonas.

Bacterial Proteins↗
PubMed1988

Deletion mutagenesis of the ice nucleation gene from Pseudomonas syringae S203.

The ice nucleation gene inaZ, from Pseudomonas syringae S203, was manipulated to produce a series of defined rearrangements in its coding sequence without changing the reading frame. The effects of these mutations on the ice nucleation phenotype were determined in a heterologous host, Escherichia coli K12. Deletions which disrupted the periodicity of 16 codons, in a repetitive region of inaZ, caused the frequencies of ice nuclei in the bacterial population to be significantly depressed; the nuclei with thresholds at warmer temperatures were most affected. In contrast, when the periodicity was left intact, deletions and duplications in the same region had only slight effects on nucleation activity. Deletions removing part or all of one of the nonrepetitive regions (that encoding the amino-terminal domain of the InaZ protein) did not abolish nucleation activity, but caused it to be limited to cooler threshold temperatures. In contrast, the non-repetitive carboxy-terminal domain of the InaZ protein was shown to be essential for ice nucleation at all temperatures. The differential requirements (for periodicity, and for the amino-terminus) in forming nuclei with different thresholds may be significant for understanding what determines the threshold temperature of an ice nucleus.

Amino Acid Sequence↗
PubMed1988

An iron-antagonized fungistatic agent that is not required for iron assimilation from a fluorescent rhizosphere pseudomonad.

Fluorescent rhizosphere Pseudomonas sp. strain NZ130 promotes plant growth, and may do so in part because of its production of a growth inhibitory factor that is active against phytopathogenic fungi. Analysis of the inhibitory factor that is active against the phytopathogen Pythium ultimum showed that its activity is antagonized at iron concentrations above 10 microM. The iron-antagonized inhibitor was separated from the fluorescent siderophore of this pseudomonad by gel filtration. Mutants that lacked either the iron-antagonized inhibitor or the fluorescent siderophore were isolated. Results of complementation analysis of these mutants by use of a cosmid library indicated that distinct DNA sequences are required for the production of each factor. Analysis of isogenic mutant strains showed that the genetic requirements for the production of the iron-antagonized inhibitor and the fluorescent siderophore are different, and that only the fluorescent siderophore is required for iron assimilation. Fusions of these same sequences to a beta-galactosidase gene were used to show that the regions required for the production of both the fluorescent siderophore and the iron-antagonized inhibitor were iron-regulated.

Antifungal Agents↗
PubMed1988

Genetic determinants for catabolite induction of antibiotic biosynthesis in Pseudomonas fluorescens HV37a.

Antibiotic biosynthesis is regulated by glucose in Pseudomonas fluorescens HV37a. Fusions between antibiotic biosynthetic operons (afu operons) and the Escherichia coli lac operon were isolated to evaluate the genetic determinants for the regulation of antibiotic biosynthesis. Four afu transcriptional units were defined, afuE, afuR, afuAB, and afuP. The afuE and afuR transcripts were promoted divergently at one locus and were catabolite induced, by 250-fold and 5-fold, respectively; the afuAB and afuP transcriptional units were not linked to the others and were not catabolite induced. Thus, regulation of afuE and afuR operon transcription is apparently the mechanism whereby glucose regulates antibiotic biosynthesis. Catabolite induction of the afuE and afuR transcriptional unit was dependent on the products of the afuA, afuB, and afuP genes. Expression of the afuE transcriptional unit was altered quantitatively in afuE mutants. Apparently the afuE transcriptional unit is regulated, at least in part, by its own gene products. Under inducing conditions, expression of the afuE, afuR, and afuP transcriptional units increased rapidly during a 6-h period.

Antifungal Agents↗
PubMed1986

Ice nucleation activity of Pseudomonas fluorescens: mutagenesis, complementation analysis and identification of a gene product.

A DNA fragment of 7.5 kb from Pseudomonas fluorescens MS1650 confers an ice nucleation phenotype when cloned in Escherichia coli. This DNA encodes a protein with an apparent mol. wt of 180 kd, which is found in both inner and outer membrane fractions of transformed E. coli cells. Insertion mutations throughout a 3.9-kb region cause deficiency in ice nucleation, and eliminate the 180-kd protein. Complementation is not observed between any pair of mutations, suggesting that the nucleating phenotype is encoded by a single transcriptional unit. Mutations in most parts of the 3.9-kb region are not completely deficient in phenotype: they still generate ice nuclei at low frequency. One insertion mutation was found to generate pseudowild revertants, which had undergone deletions of the entire insertion and some of the adjacent sequence; these could account for the incomplete deficiency. These deletions displayed depressed nucleation temperatures, but their nucleation frequencies were close to that of the wild-type gene.

Alleles↗
PubMed1986

Identification and purification of a bacterial ice-nucleation protein.

The protein product of a gene (inaZ) responsible for ice nucleation by Pseudomonas syringae S203 has been identified and purified after overexpression in Escherichia coli. The amino acid composition and the N-terminal sequence of the purified, denatured protein corresponded well with that predicted from the sequence of the inaZ gene. The product of inaZ was also found to be the major component in preparations of ice-nucleating, proteinaceous particles, obtained after extraction with and gel filtration in a mixture of urea and the nondenaturing detergent octyl beta-D-thioglucopyranoside. The activity of these preparations in the absence of added lipid implies that the protein participates directly in the nucleation process.

Amino Acids↗
PubMed1986

Molecular cloning of genetic determinants for inhibition of fungal growth by a fluorescent pseudomonad.

Pseudomonas fluorescens HV37a inhibits growth of the fungus Pythium ultimum in vitro. Optimal inhibition is observed on potato dextrose agar, a rich medium. Mutations eliminating fungal inhibition were obtained after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were classified by cosynthesis and three groups were distinguished, indicating that a minimum of three genes are required for fungal inhibition. Cosmids that contain wild-type alleles of the genes were identified in an HV37a genomic library by complementation of the respective mutants. This analysis indicated that three distinct genomic regions were required for fungal inhibition. The cosmids containing these loci were mapped by transposon insertion mutagenesis. Two of the cosmids were found to contain at least two genes each. Therefore, at least five genes in HV37a function as determinants of fungal inhibition.

Alleles↗
PubMed1985

Comparison of physical and genetic properties of palindromic DNA sequences.

Some viable palindromic DNA sequences were found to cause an increase in the recovery of genetic recombinants. Although these palindromes contained no Chi sites, their presence in cis caused apparent recA+-dependent recombination to increase severalfold. This biological property did not correlate with the physical properties of the palindromes' extrusion of cruciform structures in vitro. Thus, two unrelated palindromes with similar effects on recombination in both Escherichia coli and Pseudomonas syringae displayed quite different kinetics of cruciform formation. In plasmids of native superhelical density, one palindrome underwent rapid cruciform formation at 55 degrees C, whereas the other did not form detectable cruciforms at any temperature. A shorter palindrome with similarly rapid kinetics of cruciform formation did not affect recombination detectably. The lack of a clear relationship between physical and genetic properties was also demonstrated in the case of longer, inviable palindromes. Here we found that the degree of asymmetry required in vivo to rescue a long palindrome from inviability far exceeded that required to kinetically prohibit cruciform extrusion in vitro.

Base Sequence↗
PubMed1979

ColE1 plasmid mobility: essential and conditional functions.

Sequences essential for the conjugal transfer of ColE1 can be divided into a cis-acting site and a region encoding trans-acting products. Each of these was successively cloned into a non-transmissible plasmid vector. The resulting chimera was transmissible by the conjugative plasmids F'lac,pro (incFI) and R64drd11 (incIalpha). The sequences encoding colicin E1, immunity, and incompatibility were absent from this chimera: therefore they are not essential for the conjugal transmission of the ColE1 plasmid. In contrast to ColE1, however, the same chimera was deficient in conjugal transfer initiated by R751 (incP) and R388 (incW). This suggests that ColE1 sequences other than those cloned in the chimeric plasmid are necessary for its mobilization by R751 and R388. Three such regions were revealed by screening a series of ColE1 insertion mutants for transfer by R751 and R388. Two of these regions encode no other known function while the third is encoded by a region which overlaps the gene for colicin E1 itself.

Conjugation, Genetic↗