Initial characterization of Aspergillus nidulans mutants blocked in the nuclear replication cycle

Several hundred temperature-sensitive mutants of Aspergillus nidulans were screened for ability of their conidia to produce germ tubes at the nonpermissive temperature while still remaining with the original single conidial nucleus.     

Active site mutants of human herpesvirus-6 proteinase

OBJECTIVES: To assess the comparability of the Hybritech Tandem-R and Abbott AxSYM PSA assays in the setting of a hospital laboratory changing methods of PSA assay. METHODS: A total of 115 serum samples were tested simultaneously with both reagent kits. These include samples from patients evaluated for screening, benign prostatic hyperplasia, and follow-up of prostate cancer. RESULTS: The outcomes of the Hybritech Tandem-R PSA test ranged from 0.0 to 48.3 ng/mL with a median value of 2.4 ng/mL (mean 3.48, SD 5.46). The outcomes of the Abbott AxSYM PSA test ranged from 0.0 to 49.33 ng/mL with a median of 2.22 ng/mL (mean 3.82, SD 5.59). The outcomes of the two assays were found to be highly correlated by the Pearson correlation coefficient (r = 0.9942). When samples were divided according to PSA levels of 0.0 to less than 2.5, 2.5 to less than 4.0, 4.0 to less than 10.0, and 10.0 to less than 25.0 ng/mL, the outcomes were also highly correlated in all PSA level ranges (r = 0.9619, 0.8094, 0.9167, and 0.9081, respectively). CONCLUSIONS: The PSA values of the Tandem-R and Abbott AxSYM assays are highly correlated in the PSA level ranges of 0.0 to less than 2.5, 2.5 to less than 4.0, 4.0 to less than 10.0, and 10.0 to less than 25.0 ng/mL.     

Purification and characterization of cysteine proteinase from a baculovirus gene

To analyze the degradation of product proteins at the late stage of virus infection in the baculovirus expression system, a cysteine proteinase was purified from hemolymph of Bombyx mori infected with wild-type B. mori nuclear polyhedorosis virus (BmNPV). The purified cysteine proteinase preparation had two protein bands (major 35-kDa active protein and 28-kDa inactive protein) on SDS-PAGE. Based on the N-terminal amino acid sequences of them, it was found that both proteins originated in the cysteine proteinase gene of BmNPV. The purified cysteine proteinase had an optimum pH at 4.0, and also had activities at neutral pHs. When recombinant luciferase was used as a natural substrate, it was degraded rapidly by the cysteine proteinase at the physiological pH of hemolymph. These results suggest that the cysteine proteinase from a BmNPV gene participates in the degradation of foreign protein expressed by the baculovirus system.     

Purification and characterization of human topoisomerase I mutants

A system for rapid purification and characterization of eukaryotic topoisomerase-I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates. Relaxation-deficient mutants were obtained by site-directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50-fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu comparable to that of wild-type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo-dUTP-substituted DNA oligonucleotides carrying a topoisomerase-I-binding site, indicating that the deficiency of Tyr723Phe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently.     

Purification and partial characterization of an acid proteinase from Dirofilaria immitis

Tumour necrosis factor-alpha (TNF-alpha) is a pluripotent cytokine with its receptors distributed throughout many different cell types. Because of the diverse effects of the cytokine, it is difficult to clearly define its role in infection and immunity, and appreciate its clinical therapeutic value. We have identified peptides derived from the primary amino acid sequence of human TNF-alpha that have neutrophil-stimulating activity, as measured by enhanced chemiluminescence and superoxide production, and peptides which are both directly cytotoxic for tumour cells (WEHI-164) in vitro and also prevent TNF binding to tumour cells. However, only one of these neutrophil-stimulating peptides was toxic for tumour cells in vitro. Our results indicate that the region of amino acids 54-94 of human TNF-alpha has previously undescribed human neutrophil-stimulatory activity, while peptides encompassing the regions 43-68 and 132-150, which are in close proximity, as indicated in the recently determined three-dimensional structure of human TNF-alpha, have in vitro anti-tumour activity. These peptides also slowed tumour growth or induced tumour regression in WEHI-164 tumour-bearing mice. The peptide 73-94, which activated neutrophils but which was not cytotoxic for tumour cells in vitro, also caused in vivo tumour regression, presumably by activating neutrophils with the consequent release of free radicals at the tumour site. Peptide 63-83, which was able to activate neutrophils in vitro, did not possess tumour regression activity in vivo. The TNF peptides described in this report did not elicit procoagulant activity in cultured bovine aortic endothelial cells and as such are devoid of at least one of the potentially lethal side-effects of elevated TNF levels in vivo.     

Isolation and characterization of high-osmolarity-sensitive mutants of fission yeast

For the fission yeast Schizosaccharomyces pombe, adaptation to high-osmolarity medium is mediated by a mitogen-activated protein (MAP) kinase cascade, involving the Wis1 MAP kinase kinase and the Sty1 MAP kinase. The MAP kinase pathway transduces an osmotic signal and accordingly regulates the expression of the downstream target gene (gpd1(+)) that encodes NADH-dependent glycerol-3-phosphate dehydrogenase, in order to adaptively accumulate glycerol inside the cells as an osmoprotectant. We previously characterized a set of high-osmolarity-sensitive S. pombe mutants, including wis1, sty1, and gpd1. In this study, we attempted to further isolate novel osmolarity-sensitive mutants. For some of the mutants isolated, profiles of glycerol production in response to the osmolarity of the growth medium were indistinguishable from that of the wild-type cells, suggesting that they are novel types. They were classified into three distinct types genetically and, thus, were designated hos1, hos2, and hos3 (high osmolarity sensitive) mutants. One of them, the hos1 mutant, was characterized in detail. The hos1 mutant was demonstrated to have a mutational lesion in the known ryh1(+) gene, which encodes a small GTP-binding protein. Disruption of the ryh1(+) gene results not only in osmosensitivity but also in temperature sensitivity for growth. It was also found that the delta ryh1 mutant is severely sterile. These results are discussed with special reference to the osmoadaptation of S. pombe.     

Initial characterization of a protochordate histocompatibility locus

The colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction which is controlled by a single, highly polymorphic locus called the Fu/HC. We are using map-based cloning to identify Fu/HC gene(s), and have currently delineated their location to an approximately 1-cM region of the B. schlosseri genome. The Fu/HC physical map currently consists of 85 sequence-tagged sites mapped on a minimum tiling path of 800 kb which consists of five contigs, with four gaps remaining to be crossed, and is estimated to be 75% completed. Approximately half this region has been sequenced throughout the locus, allowing the first analysis of a metazoan histocompatibility locus outside of vertebrates. This has resulted in the identification of 18 predicted genes, a number of which have been found to be expressed. Several of these genes are well conserved among the chordates; however, none of the predicted or expressed genes are linked within the genome of any organism in the databases. In addition, the Fu/HC is one of the most polymorphic loci ever described, and physical mapping has revealed that the locus is quite dynamic, and includes features such as hotspots of recombination.     

Molecular characterization and rescue of acatalasemic mutants of Drosophila melanogaster

The enzyme catalase protects aerobic organisms from oxygen-free radical damage by converting hydrogen peroxide to molecular oxygen and water before it can decompose to form the highly reactive hydroxyl radical. Hydroxyl radicals are the most deleterious of the activated oxygen intermediates found in aerobic organisms. If formed, they can react with biological molecules in their proximity; the ensuing damage has been implicated in the increasing risk of disease and death associated with aging. To study further the regulation and role of catalase we have undertaken a molecular characterization of the Drosophila catalase gene and two potentially acatalasemic alleles. We have demonstrated that a previously existing allele, Catn4, likely contains a null mutation, a mutation which blocks normal translation of the encoded mRNA. The Catn1 mutation appears to cause a significant change in the protein sequence; however, it is unclear why this change leads to a nonfunctioning protein. Viability of these acatalasemic flies can be restored by transformation with the wild-type catalase gene; hence, we conclude that the lethality of these genotypes is due solely to the lack of catalase. The availability of flies with transformed catalase genes has allowed us to address the effect of catalase levels on aging in Drosophila. Though lack of catalase activity caused decreased viability and life span, increasing catalase activity above wild-type levels had no effect on normal life span.     

Selection and characterization of Toxoplasma gondii mutants resistant to artemisinin

Toxoplasma gondii infection, like malaria, is sensitive to inhibition by artemisinin (ART). Mechanisms of action for ART in malaria treatment have been proposed, but little is known about its effects in T. gondii infection. To better understand its inhibitory effects on T. gondii, mutants resistant to ART were selected by progressive culture in permissive levels of the drug. Five clonal isolates were established and characterized. The isolates were approximately 65-fold less sensitive to ART than is the parental RH and showed cross-resistance to the ART derivatives dihydroartemisinin and artemether. In addition to ART resistance, 1 clone (C9) formed morphologically unusual parasitophorous vacuoles and another (A2) was avirulent for mice and protected mice from challenge with the wild type. These clonal T. gondii mutant isolates will be useful for the study of not only the mechanism of action of ART but also parasite vacuole biology and virulence factors.     

Construction and characterization of avian Escherichia coli cya crp mutants

We constructed delta cya delta crp mutants of two avian septicemic Escherichia coli strains and evaluated their attenuation in virulence. The P1 phage was used to transfer cya::Tn10 from an E. coli K-12 strain into virulent avian O78 and O2 E. coli isolates. Tetracycline-resistant transductants were plated on Bochner-Maloy Medium, and tetracycline-sensitive colonies were selected, then tested by polymerase chain reaction to confirm that they had deletions of the cya gene. Deletions of crp were created by the same technique in isolates with deletions in cya. The delta cya and delta cya delta crp derivatives had slower growth rates, smaller colonies, and impaired fermentation of carbohydrates compared with their wild parents, and they did not revert. Attenuation of the mutant strains was evaluated by subcutaneous (s.c.) inoculation of day-old chicks and by intratracheal (i.t.) inoculation of 9-day-old chicks previously inoculated intranasally with infectious bronchitis virus. For the wild O78 strain and its delta cya and delta cya delta crp derivatives, the percentages of chicks that died within 6 days of s.c. injection of approximately 5 x 10(7) organisms were 100, 60, and 0, respectively. The corresponding percentages for wild-type O2 and its delta cya and delta cya delta crp mutants were 100, 70, and 20 at a dose of approximately 2 x 10(5) organisms. Following i.t. inoculation, group scores based on pathologic and bacteriologic findings were 51%, 15%, and 9% for wild, delta cya, and delta crp O78 strains (inoculum approximately 2 x 10(7) organisms) and 98%, 31%, and 11%, respectively, for the corresponding O2 strains (inoculum approximately 4 x 10(6) organisms). This study demonstrated reduced virulence and stability of the double mutant, which may useful as a live attenuated vaccine against poultry colibacillosis.     

Genetic and biochemical characterization of Saccharomyces cerevisiae mutants resistant to trifluoroleucine

Eighteen mutants resistant to 5',5',5'-trifluoroleucine (TFL), a leucine analog, were isolated in Saccharomyces cerevisiae strains YNN281 and YNN282. The mutants were characterized genetically and clustered in two groups, one comprising all the dominant (TFL1) and the other one all the recessive (tfl2) mutations. Genetic and biochemical data suggested that the dominant mutations are located on the LEU4 gene, coding for alpha-isopropylmalate synthase I. These mutations resulted in accumulation of leucine as a consequence of the synthesis of an enzyme insensitive to the feedback inhibition by leucine. Leucine excretion in the TFL1 mutants appeared to be affected by the genetic background of the strain and was greatly influenced by lysine metabolism. The measurement of intra- and extracellular amino acid concentrations in prototrophic strains carrying TFL1 or tfl2 genes showed that both were leucine overproducers. Some of the TFL-resistant mutants were tested in alcoholic fermentation of grape must: analysis of the fermentation secondary metabolites showed that the major effect of the TFL-resistant strains was an increased production of isoamyl alcohol compared to that of the parental strain.     

Biochemical characterization of primary peritoneal carcinoma cell adhesion, migration, and proteinase activity

The incidence and time course of arm morbidity after sector resection and axillary dissection with or without postoperative radiotherapy to the breast was assessed in a prospective randomised trial among 381 patients with stage I breast cancer. At 3-12 months, arm symptoms were reported by 59/110 of the patients who had > or = 10 lymph nodes found in the axillary specimen versus 85/253 in whom < 10 lymph nodes were found (P = 0.002); at 13-36 months, the corresponding figures were 35/106 versus 44/225 (P = 0.001). Postoperative wound complications increased the incidence of arm symptoms at 3-12 months from 104/283 to 39/79 at 3-12 months (P = 0.03). Employed patients and patients < 65 years of age reported arm symptoms at 3-12 months in 86/161 and 94/191 compared to 58/207 and 50/177 among retired patients and patients > or = 65 years of age, respectively (P = 0.0001 and P = 0.0002, respectively). In a multivariate logistic regression analysis at 3-12 months, only young age (relative risk = 0.93 per year of increasing age, 95% CI 0.91-0.97) and the number of lymph nodes found in the axillary specimen (relative risk = 1.11 per lymph node found, 95% CI 1.05-1.18) remained statistically significant. No negative impact on arm morbidity was found by the addition of postoperative radiotherapy only to the breast, either in univariate or multivariate models. We conclude that factors directly related to the extent of the surgical procedure and young age are determinants of arm morbidity after breast preserving treatment for stage I breast cancer. Arm symptoms are most common during the first year after treatment and are reduced over the subsequent 2-3 years by around 40-50%.     

Isolation and characterization of melanin-producing (mel) mutants of Vibrio cholerae

Vibrio cholerae strain Htx-3, a hypertoxinogenic mutant of V. cholerae 569B Inaba, produces a dark brown pigment under certain growth conditions, whereas strain 569B does not. We investigated the biochemical basis for this pigment production and the possible relationships between pigmentation and other phenotypic properties in V. cholerae. After mutagenesis of V. cholerae 569B with N-methyl-N'-nitro-N-nitrosoguanidine, 28 independently derived pigment-forming (mel) mutants were isolated and characterized. The mel mutants frequently differed from wild type in toxinogenicity or motility and occasionally differed in other phenotypic traits. Individual mel mutants differed from wild type both in the amount of toxin produced and in the growth conditions optimal for toxin production. It has not yet been established whether multiple phenotypic changes in individual mel mutants represent pleiotropic effects of single mutations or induction of multiple mutations by N-methyl-N'-nitro-N-nitrosoguanidine or both. Production of pigment by mel mutants occurred at temperatures from 22 to 40 degrees C, was inhibited by anaerobiosis, and was stimulated by supplementation of growth media with the amino acid precursors of melanin (l-phenylalanine, l-tyrosine, or l-tyrosine plus l-cysteine). The pigment possessed several other properties reported for microbial melanins. We conclude that a biochemical pathway for melanin production is present in V. cholerae, that this pathway cannot be fully expressed in wild-type strain 569B, and that mutations in the gene(s) which we have designated mel can permit hyperproduction of melanin under appropriate conditions.     

Isolation and characterization of Rhizobium etli mutants altered in degradation of asparagine

Rhizobium etli mutants unable to grow on asparagine as the nitrogen and carbon source were isolated. Two kinds of mutants were obtained: AHZ1, with very low levels of aspartase activity, and AHZ7, with low levels of asparaginase and very low levels of aspartase compared to the wild-type strain. R. etli had two asparaginases differentiated by their thermostabilities, electrophoretic mobilities, and modes of regulation. The AHZ mutants nodulated as did the wild-type strain and had nitrogenase levels similar to that of the wild-type strain.     

Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding

Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates.     

Cadmium binding by bacteria: screening and characterization of new isolates and mutants

A fast and simple methodology was developed that enables screening of microbial strains for their ability to bind cadmium. It is based on the use of a radioisotope of cadmium (109Cd) for screening colonies and for evaluation of cadmium binding. The methods described here can be used to screen new environmental isolates or to obtain mutants with altered ability to bind cadmium. Examples for the two uses are described in the paper.     

Characterization of human immunodeficiency virus type 1 mutants with decreased sensitivity to proteinase inhibitor Ro 31-8959

A human immunodeficiency virus type 1 (HIV-1) variant with highly reduced susceptibility to Ro 31-8959, an inhibitor of the viral proteinase, has been selected by repeated passage of wild-type virus in CEM cells in the presence of increasing concentrations of the inhibitor. Peptide sequences of the proteinase of selected virus were obtained from proviral DNA. Sequence comparison to wild-type (wt) proteinase demonstrated two amino acid substitutions in the resistant virus, a Gly to Val exchange at position 48 and a Leu to Met exchange at position 90. Furthermore, sequences of intermediate passage virus suggest contributions from positions 12, 36, 57, and 63 in early steps of resistance development. The selected virus showed a ca. 40-fold increase in 50% inhibitory concentration of Ro 31-8959. Growth kinetics of resistant virus were comparable to wild-type virus and the resistant genotype proved to be stable in the absence of inhibitor. Directed mutagenesis of the HIV-1 HXB2 proteinase at positions 48 and 90 suggested that each mutation alone led to a moderate decrease in sensitivity of the recombinant virus to proteinase inhibitor. However, a recombinant virus carrying both mutations in the proteinase gene showed a significant reduction in its sensitivity to Ro 31-8959 thus proving the importance of these exchanges for the resistance phenotype.