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At least 703 records · Page 39Linked to original sources
PubMed1998

The suprachiasmatic nucleus: a 25-year retrospective.

The suprachiasmatic nuclei (SCN) of the anterior hypothalamus contain the master circadian pacemaker in mammals. On the occasion of the 25th anniversary of the discovery of the SCN as the circadian clock, Charles A. Czeisler and Steven M. Reppert organized a meeting to review milestones and recent developments in the study of the SCN. The discovery that the SCN contain tissue necessary for generation of circadian rhythmicity was established by lesion studies published in 1972. The second phase of study demonstrated unequivocally that the SCN contain an autonomous circadian pacemaker. The principal studies in this period showed the presence of metabolic and electrical activity rhythms in the SCN in vivo and progressed to studies showing that the SCN maintain rhythmicity in vitro, demonstrating that the transplanted SCN can restore circadian function following destruction of the host SCN and ultimately showing that single SCN "clock cells" exhibit independent rhythms in firing rate. The third phase of study, aimed at identifying the biochemical and molecular mechanisms responsible for rhythmicity within the SCN, has begun with the identification of circadian mutants (tau mutant hamsters and Clock mutant mice) and the isolation of the Clock gene. This report traces the important steps forward in our understanding of the suprachiasmatic circadian clock by recounting the information presented at the SCN Silver Anniversary Celebration.

Animals↗
PubMed2004

Enhancer detection and gene trapping as tools for functional genomics in plants.

Although more than 25,000 genes of Arabidopsis thaliana have been sequenced and mapped, adequate expression or functional information is available for less than 15% of them. In the case of Oryza sativa (rice), about half of more than 55,000 predicted genes have been assigned to a vague functional category on the basis of their sequence, but fewer than 100 have been ascribed a precise, verified function after the identification of a mutant phenotype caused by the molecular disruption of the corresponding gene. Enhancer detection and gene trapping represent insertional mutagenesis strategies that report random expression of many genes and often generate loss-of-function mutations. Several trapping vectors have been designed in a limited number of species, and large-scale enhancer detection and gene trap screens that aim to generate a wide range of spatially and temporally restricted expression patterns have been initiated in both Arabidopsis and rice. These strategies are proving to be essential to the functional annotation of completely sequenced genomes, enabling the analysis of gene function in the context of the entire plant life cycle and substantially expanding our understanding of plant growth and development.

Arabidopsis↗
PubMed1990

New results of hemoglobin variant structure determinations by fast atom bombardment mass spectrometry.

Fast atom bombardment mass spectrometry has already been used for the identification of mutations in abnormal human hemoglobin chains. This paper presents new results obtained with this technique. The methodology used here is compared with more conventional biochemical techniques and automated microsequencing. In every case, a well-chosen combination of peptide-high performance liquid chromatography, mass spectrometry, amino acid analysis, and sequence analysis led rapidly to the identification of the mutant. The high sensitivity of these techniques holds great promise for the analysis of molecular abnormalities in various genetic disorders presently detectable only by the application of a molecular biological approach.

Adult↗
PubMed2000

Role of cAMP cascade in synaptic stability and plasticity: ultrastructural and physiological analyses of individual synaptic boutons in Drosophila memory mutants.

Mutations of the genes rutabaga (rut) and dunce (dnc) affect the synthesis and degradation of cAMP, respectively, and disrupt learning in Drosophila. Combined ultrastructural analysis and focal electrophysiological recording in the larval neuromuscular junction revealed a loss of stability and fine tuning of synaptic structure and function in both mutants. Increased ratios of docked/undocked vesicles and poorly defined synaptic specializations characterized dnc synapses. In contrast, rut boutons possessed fewer, although larger, synapses with lower proportions of docked vesicles. At reduced Ca(2+) levels, decreased quantal content coupled with an increase in failure rate was seen in rut boutons and reduced pair-pulse facilitation were found in both rut and dnc mutants. At physiological Ca(2+) levels, strong enhancement, instead of depression, in evoked release was observed in some dnc and rut boutons during 10 Hz tetanus. Furthermore, increased variability of synaptic transmission, including fluctuation and asynchronicity of evoked release, paralleled an increase in synapse size variation in both dnc and rut boutons, which might impose problems for effective signal processing in the nervous system. Pharmacological and genetic studies indicated broader ranges of physiological alteration by dnc and rut mutations than either the acute effects of cAMP analogs or the available mutations that affect cAMP-dependent protein kinase (PKA) activity. This is consistent with previous reports of more severe learning defects in dnc and rut mutations than these PKA mutants and allows identification of the phenotypes involving long-term developmental regulation and those conferred by PKA.

Alleles↗
PubMed1992

Osteogenesis imperfecta type I is commonly due to a COL1A1 null allele of type I collagen.

Dermal fibroblasts from most individuals with osteogenesis imperfecta (OI) type I produce about half the normal amount of type I procollagen, as a result of decreased synthesis of one of its constituent chains, pro alpha 1 (I). To test the hypothesis that decreased synthesis of pro alpha (I) chains results from mutations in the COL1A1 gene, we used primer extension with nucleotide-specific chain termination to measure the contribution of individual COL1A1 alleles to the mRNA pool in fibroblasts from affected individuals. A polymorphic MnlI restriction endonuclease site in the 3'-untranslated region of COL1A1 was used to distinguish the transcripts of the two alleles in heterozygous individuals. Twenty-three individuals from 21 unrelated families were studied. In each case there was marked diminution in steady-state mRNA levels from one COL1A1 allele. Loss of an allele through deletion or rearrangement was not the cause of the diminished COL1A1 mRNA levels. Primer extension with nucleotide-specific chain termination allows identification of the mutant COL1A1 allele in cell strains that are heterozygous for an expressed polymorphism. It is applicable to sporadic cases, to small families, and to large families in whom key individuals are uninformative at the polymorphic sites used in linkage analysis, making it a useful adjunct to the biochemical screening of collagenous proteins for OI.

Alleles↗
PubMed2004

Advances in acute lymphoblastic leukemia.

DATA SOURCES: Current literature. DATA SYNTHESIS: Acute lymphoblastic leukemia (ALL) is a stem cell disorder characterized by an overproduction of lymphoblasts in the bone marrow that eventually spill into circulation, producing lymphocytosis. As with the other acute leukemias, the most common symptoms experienced by patients include fatigue, bleeding, and recurrent infections resulting from the suppression of normal hematopoiesis in the bone marrow by the accumulating blasts. ALL primarily affects children and exhibits the best response to standard chemotherapy as compared to acute myeloblastic leukemias (AML). Further, remission rates are highest among ALL patients, many of whom are experiencing sustained remissions suggesting cure. In light of early treatment successes, researchers began to investigate modifications of standard treatment regimens to accommodate variability in weight, age, and response to therapy among children with ALL. Individualized treatment plans were implemented where some patients received a reduced intensity course of therapy to minimize drug toxicity while others received drug intensification to maximize response. More recently, research efforts have been directed at the elucidation of leukemogenic mechanisms implicated in ALL to identify specific protein mutants that can be used to design drugs tailored to interfere with the activity of these mutant protein targets. Identification of chimeric proteins produced from chromosomal translocations and gene expression profiles from microarray analyses are the primary techniques used to identify the potential therapeutic targets. CONCLUSION: Several reliable prognostic indicators have been identified and are being used to improve therapeutic planning and outcome prediction in ALL patients. Individualized treatment regimens have been developed based on the specific characteristics of each patient to minimize treatment related adverse events and maximize response. Through the use of cytogenetic, molecular, and microarray testing, ALL classification schemes have improved and potential therapeutic targets have been identified. It is anticipated that the next major advance in the treatment of ALL will involve the use of designer therapies developed to specifically interfere with particular molecular abnormalities producing the leukemogenic aberration to the normal signal transduction pathways.

Adult↗
PubMed2000

[Complete androgen insensitivity in an Icelandic family caused by mutation in the steroid binding region of the androgen receptor.].

INTRODUCTION: Androgen insensitivity syndrome (AIS) is a X-linked rescessive disorder characterized by impairment of the androgen-dependant male sexual differentiation. The cause of AIS is in most cases a mutation in the gene of the androgen receptor on the X chromosome. In this study we describe an Icelandic family with two girls with AIS. A search for mutations in the androgen receptor gene was performed in order to identify the genetical and molecular basis for AIS in this family. MATERIAL AND METHODS: Genomic DNA was isolated from two girls with complete AIS and their close relatives. PCR was used to amplify all eight exons of the androgen receptor gene of the two AIS girls and SSCP used to screen for mutations. DNA fragments showing abnormal SSCP pattern were subjected to nucleotide sequencing. PCR based diagnostic method was developed and used to detect the mutation causing AIS in the family. RESULTS AND CONCLUSIONS: Using SSCP and DNA sequencing a CGA to CAA missense mutation in exon 5 at codon 752 was identified. The mutation causes in an Arg to Gln amino acid substitution (R752Q mutation) in the ligand binding domain of the androgen receptor and a complete androgen insensitivity. Members of the family were genotyped using a PCR based method for identification of the mutant allele. The results strongly indicated a de novo mutation in a germ cell of the maternal grandmother, as the mutation was not found in her blood leucocytes. The diagnostic test provided a basis for genetic counselling for the family.

English Abstract↗
PubMed1979

A new system for studying molecular mechanisms of mutation by carcinogens.

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.

Bacteriophage phi X 174↗
PubMed1985

[Prenatal prevention of drepanocytosis: analysis of cellular DNA in 2 cases].

Direct analysis of fetal DNA using restriction endonucleases constitutes a major area of progress in prenatal diagnosis. This recent technology may permit the precise identification of a mutant allele for some diseases, whereas in others it allows the familial segregation of a pathogenic allele to be followed by its linkage to a DNA sequence polymorphism. This type of analysis, available in a few centers, is currently used, among others, for the prenatal diagnosis of hemoglobinopathies such as sickle cell anemia. After fetal cells have been obtained by choriocentesis or amniocentesis, the extracted DNA is exposed to selected restriction enzymes. In the diagnosis of sickle cell anemia the mutant codon responsible for the substitution of glutamic acid by valine in the beta hemoglobin chain is no longer cut by the enzyme Mst II, due to its variance with the normal codon; this difference in fragment length is detected by DNA electrophoresis, and the particular fragments are identified by molecular hybridization with appropriate radioactive probes. Utilizing these methods the genotype of a homozygous normal fetus can be distinguished from that of a homozygote affected or a heterozygote for the sickle mutation of the beta hemoglobin chain. We have recently applied this prenatal methodology to the pregnancies of two couples from Zaire, in which each member was a proven sickle cell carrier. Fetal material was obtained in both cases by amniocentesis at the 16th week of gestation and followed by cell culture. In the first case, a 46, XX fetus, DNA (10 mcg) revealed a heterozygous sickle cell carrier genotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Prevalence of nine mutations among Jewish and non-Jewish Gaucher disease patients.

The frequency of nine different mutated alleles known to occur in the glucocerebrosidase gene was determined in 247 Gaucher patients, of whom 176 were of Jewish extraction, 2 were Jewish with one converted parent, and 69 were of non-Jewish origin. DNA was prepared from peripheral blood, active glucocerebrosidase sequences were amplified by using the PCR technique, and the mutations were identified by using the allele-specific oligonucleotide hybridization method. The N37OS mutation appeared in 69.77% of the mutated alleles in Jewish patients and in 22.86% of the mutated alleles in non-Jews. The 84GG mutation, which has not been found so far among non-Jewish patients, existed in 10.17% of the disease alleles among Jewish patients. The IVS + 1 mutation constituted 2.26% of the disease alleles among Jewish patients and 1.43% among the non-Jewish patients. RecTL, a complex allele containing four single-base-pair changes, occurred in 2.26% of the alleles in Jewish patients and was found in two (1.43%) of the patients of non-Jewish extraction. Another complex allele, designated "RecNciI" and containing three single-point mutations, appeared in 7.8% of alleles of non-Jewish patients and in only two (0.56%) of the Jewish families. The prevalence of the L444P mutation among non-Jewish Gaucher patients was 31.43%, while its prevalence among Jewish patients was only 4.24%. The prevalence of two other point mutations--D409H and R463C--was 5.00% and 3.57%, respectively, among non-Jewish patients and was not found among the Jewish Gaucher patient population. The prevalence of the R496H mutation, found so far only among Jewish patients, was 1.13%. The results presented demonstrate that seven mutations identify 90.40% of the mutations among Jewish patients and that these seven mutations allow diagnosis of only 73.52% of the non-Jewish patients. Identification of additional mutant alleles will enhance the accuracy of carrier detection.

Base Sequence↗
PubMed1994

Genetic linkage mapping of multiple epiphyseal dysplasia to the pericentromeric region of chromosome 19.

Multiple epiphyseal dysplasia (MED) is an inherited chondrodystrophy that results in deformity of articular surfaces and in subsequent degenerative joint disease. The disease is inherited as an autosomal dominant trait with high penetrance. An MED mutation has been mapped by genetic linkage analysis of DNA polymorphisms in a single large pedigree. Close linkage of MED to 130 tested chromosomal markers was ruled out by discordant inheritance patterns. However, strong evidence for linkage of MED to markers in the pericentromeric region of chromosome 19 was obtained. The most closely linked marker was D19S215, with a maximum LOD score of 6.37 at theta = .05. Multipoint linkage analysis indicated that MED is located between D19S212 and D19S215, a map interval of 1.7 cM. Discovery of the map location of MED in this family will facilitate identification of the mutant gene. The closely linked DNA polymorphisms will also provide the means to determine whether other inherited chondrodystrophies have underlying defects in the same gene.

Centromere↗
PubMed1996

Rapid method for isolation of high molecular weight plasmids suitable for digestion with restriction endonucleases.

The described method is a modification of the hot alkaline method (Kado and Liu, 1981). The modification lies in replacement of SDS with Triton X- 100. The DNA purified by this method was readily digested with restriction endonucleases for a variety of applications, e. g. restriction analysis of plasmid DNA in epidemiological studies, restriction mapping and cloning of high molecular weight plasmids, and for identification of transpozon mutants.

DNA Restriction Enzymes↗
PubMed2001

Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements.

We have investigated the productive interaction between the four mammalian Ras proteins (H-, N-, KA- and KB-Ras) and their activators, the mammalian exchange factors mSos1, GRF1 and GRP, by using a modified Saccharomyces cerevisiae whose growth is dependent on activation of a mammalian Ras protein by its activator. All four mammalian Ras proteins were activated with similar efficiencies by the individual exchange factors. The H-Ras mutant V103E, which is competent for membrane localization, nucleotide binding, intrinsic and stimulated GTPase activity as well as intrinsic exchange, was defective for activation by all factors tested, suggesting that the integrity of this residue is necessary for catalyzed exchange. However, when other H-Ras mutants were studied, some distinct sensitivities to the exchange factors were observed. GRP-mediated, but not mSos1-mediated, exchange was blocked in additional mutants, suggesting different structural requirements for GRP. Analysis of Ras-mediated gene activation in murine fibroblasts confirmed these results.

3T3 Cells↗
PubMed1993

A mutant androgen receptor from patients with Reifenstein syndrome: identification of the function of a conserved alanine residue in the D box of steroid receptors.

Reifenstein syndrome is an eponymic term that describes partial androgen-insensitive disorders. Androgen receptor isolated from five patients with this syndrome contains a specific mutation in the DNA binding domain of the receptor. This mutation converts an alanine to a threonine at position 596 next to the zinc catenation site at the second finger. The threonine 596 mutant receptor mediated normal androgen response at promoters with closely positioned multiple regulatory elements for the androgen receptor and other transcription factors. Promoters with single isolated androgen response elements were not transactivated by the mutant receptor. In in vitro receptor-DNA binding studies, interaction with DNA by the mutant receptor was achieved only in the presence of an anti-androgen receptor antibody. Exchanging alanine 596 in the wild-type androgen receptor with serine or valine produced mutants with properties indistinguishable from those of the naturally occurring threonine 596 mutant receptor. These results indicate that an alanine residue at position 596 contributes important structural and functional activities to the androgen receptor. In the androgen receptor from the patients with Reifenstein syndrome, in which this alanine is converted to a threonine, wild-type receptor properties can be restored by exchanging an additional threonine at position 602 to an alanine. An alanine residue at position 596 or 602 in the DNA binding domain of the androgen receptor is therefore important for the full function of this receptor. In all steroid receptors that bind the core sequence AGAACANNNTGTTCT, an alanine residue is also present at a position equivalent to alanine 596 in the androgen receptor.

Alanine↗
PubMed1997

Exon 5 encoded domain is not required for the toxic function of mutant SOD1 but essential for the dismutase activity: identification and characterization of two new SOD1 mutations associated with familial amyotrophic lateral sclerosis.

Two new mutations in the gene encoding cytoplasmic Cu,Zn superoxide dismutase (SOD1) have been discovered in patients with familial amyotrophic lateral sclerosis (FALS). These mutations result in the truncation of most of the polypeptide segment encoded by exon 5, one by the formation of a stop codon in codon 126 (L126Z) and the other by inducing alternative splicing in the mRNA (splicing junction mutation). These two mutants of SOD1 result in a FALS phenotype similar to that observed in patients with missense mutations in the SOD1 gene, establishing that exon 5 is not required for the novel toxic functions of mutant SOD1 associated with ALS. These mutant enzymes are present at very low levels in FALS patients, suggesting elevated toxicity compared to mutant enzymes with single site substitutions. This increased toxicity likely arises from the extreme structural and functional changes in the active site channel, beta-barrel fold, and dimer interface observed in the mutant enzymes, including the loss of native dismutase activity. In particular, the truncation of the polypeptide chain dramatically opens the active site channel, resulting in a marked increase in the accessibility and flexibility of the metal ions and side chain ligands of the enzyme active site. These structural changes are proposed to cause a decrease in substrate specificity and an increase in the catalysis of harmful chemical reactions such as peroxidation.

Aged↗
PubMed1983

Isolation and characterization of a mutant liver aldolase in adult hereditary fructose intolerance. Identification of the enzyme variant by radioassay in tissue biopsy specimens.

Hereditary fructose intolerance (HFI) is a metabolic disorder caused by enzymic deficiency of aldolase B, a genetically distinct cytosolic isoenzyme expressed exclusively in liver, kidney, and intestine. The molecular basis of this enzyme defect has been investigated in three affected individuals from a nonconsanguineous kindred, in whom fructose-l-phosphate aldolase activities in liver or intestinal biopsy samples were reduced to 2-6% of mean control values. To identify a putative enzyme mutant in tissue extracts, aldolase B was purified from human liver by affinity chromatography and monospecific antibodies were prepared from antiserum raised in sheep. Immunodiffusion gels showed a single precipitin line common to pure enzyme and extracts of normal liver and intestine, but no reaction with extracts of brain, muscle, or HFI liver. However, weak positive staining for aldolase in hepatocyte and enterocyte cytosol was demonstrated by indirect immunofluorescence of HFI tissues. This was abolished by pretreatment with pure enzyme protein. Accordingly, a specific radioimmunoassay (detection limit 7.5 ng) was established to quantify immunoreactive aldolase B in human biopsy specimens. Extracts of tissue from affected patients gave 10-25% immunoreactive enzyme in control samples; immunoreactive aldolase in intestinal extracts from four heterozygotes was reduced (to 55%) when compared with seven samples from normal control subjects (P < 0.05). In extracts of HFI tissues, there was a sevenfold reduction in apparent absolute specific activity (1.02 vs. 8.82 U/mg) of immunoreactive fructose-l-phosphate aldolase B, but the apparent specific activity in heterozygotes (7.71 U/mg) was only slightly impaired. Displacement radioimmunotitration of aldolase B in liver supernatants showed a significant (P < 0.005) decrease in antibody avidity for immunoreactive protein in HFI tissue when compared with the pure enzyme or extract of normal control liver. Immunoaffinity chromatography on antialdolase B-Sepharose facilitated isolation and purification of enzyme from liver biopsy specimens. Active aldolase in normal liver, with substrate activity ratios and Michaelis constants identical to biochemically purified human enzyme, could be recovered from antibody columns. Chromatography on monospecific Fab' antialdolase B enabled pure enzyme protein to be retrieved quantitatively from normal control and HFI liver: direct chemical assay showed 1.88 and 1.15 mg aldolase protein/g of tissue, respectively. This confirmed that the catalytic properties of the HFI aldolase were profoundly impaired with specific activities of fructose-l-phosphate cleavage of 7.21 and 0.07 U/mg, respectively. Radioimmunoassay gave estimates of 7.66 and 1.18 U/mg, respectively. Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that immunopurified aldolase from HFI liver possessed a single subunit size similar to material from control liver extracts: M(r) 39,100 vs. 37,900+/-700 (SD) D, respectively. Electrofocusing under denaturing conditions of aldolase isolated in parallel from control and HFI liver revealed the same complement of subunits and, despite qualitative differences in distribution of bands during degradation, no additional charged species. Fructose phosphate aldolase deficiency in hereditary fructose intolerance is attended by the synthesis of an immunoreactive, but functionally and structurally modified enzyme variant that results from a restricted genetic mutation.

Adolescent↗
PubMed2005

Aminoacylation and conformational properties of yeast mitochondrial tRNA mutants with respiratory deficiency.

We report the identification and characterization of eight yeast mitochondrial tRNA mutants, located in mitochondrial tRNA(Gln), tRNA(Arg2), tRNA(Ile), tRNA(His), and tRNA(Cys), the respiratory phenotypes of which exhibit various degrees of deficiency. The mutations consist in single-base substitutions, insertions, or deletions, and are distributed all over the tRNA sequence and structure. To identify the features responsible for the defective phenotypes, we analyzed the effect of the different mutations on the electrophoretic mobility and efficiency of acylation of the mutated tRNAs in comparison with the respective wild-type molecules. Five of the studied mutations determine both conformational changes and defective acylation, while two have neither or limited effect. However, variations in structure and acylation are not necessarily correlated; the remaining mutation affects the tRNA conformation, but not its acylation properties. Analysis of tRNA structures and of mitochondrial and cytoplasmic yeast tRNA sequences allowed us to propose explanations for the observed defects, which can be ascribed to either the loss of identity nucleotides or, more often, of specific secondary and/or tertiary interactions that are largely conserved in native mitochondrial and cytoplasmic tRNAs.

Aminoacylation↗
PubMed1980

Toxin A-deficient mutants of Pseudomonas aeruginosa PA103: isolation and characterization.

An immunological assay utilizing double-diffusion principles was developed for identification of Pseudomonas aeruginosa mutants deficient in toxin A. Mutations were chemically induced, and mutants were isolated from P. aeruginosa strain PA103. Quantitative assays, both enzymatic and immunological, indicated that five mutants produced toxin A in vitro at levels of 0.3% or less of parental strain levels. Characterization indicated that the mutants fell into four classes and suggested that multiple genes are involved in the regulation of toxin A yields. Classes 1 to 3 produced less than 1% of parental levels of extracellular toxin A. Class 1 mutants are apparently specific for toxin A. Class 2 mutants are pleotropic and produced toxin A, protease, and other extracellular proteins at reduced yields. Class 3 mutants are pleotropic and in addition have relatively high levels of cell-bound toxin A. Class 4 mutants produce toxin A at levels greater than 1% of parental yields. Of 16 toxin A-deficient mutants examined, only 1 was a class 1 mutant. This mutant (PA103-29) was shown to be identical to the parental strain in all respects tested except for its marked deficiency in toxin A. The suitability of this class 1 mutant for use in virulence studies is discussed.

Bacterial Proteins↗