Isolation and characterization of mutants of Arabidopsis thaliana with increased resistance to growth inhibition by indoleacetic acid-amino acid conjugates.

Two mutants of Arabidopsis thaliana that are resistant to growth inhibition by indole-3-acetic acid (IAA)-phenylalanine have been isolated. Both mutants were 2- to 3-fold more resistant than wild type to inhibition by IAA-phenylalanine, IAA-alanine, and IAA-glycine in root growth assays. The mutant icr1 (but not icr2) also shows some resistance to IAA-aspartate. Studies using 3H-labeled IAA-phenylalanine showed that the uptake of conjugate from the medium by icr1 was the same as wild type and was reduced by about 25% in icr2. No differences in hydrolysis of the exogenous conjugate were detected between the mutants and their wild-type parents. There was no significant metabolism of the IAA released from the [3H]IAA-phenylalanine, whereas exogenous [3H]IAA was rapidly metabolized to two unidentified products considerably more polar than IAA. Analysis of a cross between icr1 and icr2 indicated that these mutations were at distinct loci and that their effects were additive, and preliminary mapping data indicated that icr1 and icr2 were located at the top and bottom of chromosome V, respectively.     

PATTERNS OF ALLOZYME DIVERSITY IN THE WEST INDIES CYCAD ZAMIA PUMILA (ZAMIACEAE)

The West Indies cycad, Zamia pumila, is restricted to the Greater Antilles, northern Bahama Islands, Florida, and the southeastern coast of Georgia. An electrophoretic study based on nine enzymes compared 21 accessions from throughout the range of the species. Lower levels of intrapopulation variation than those reported for ferns, other gymnosperms, and angiosperms were discovered for the two more extensively sampled populations. However, this variation was similar to that found in other island taxa and in the endemic Australian cycad, Macrozamia communis. In contrast, allozyme divergence among accessions of Z. pumila appeared relatively high, mostly as a result of rare alleles restricted in geographic distribution. The age and biogeography of Z. pumila may gave contributed to population differentiation. Also, mean number of alleles per locus was low for the species (1.75). Finally, the time-since-divergence value (10.8 million years ago) between Z. pumila and its closest extant relative, Z. splendens, was much smaller than the age of Z. pumila suggested by the fossil record and historical geology of the Caribbean (30–60 million years ago). Together, these data indicate that biochemical evolution within this species, and perhaps in all cycads, is slow when compared to that of noncycad seed plants.     

Molecular heterogeneity underlying the G6PD Mediterranean phenotype

Summary As part of a study aiming to define the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency, we analysed a sample from a Portugese boy with a family history of favism. Although the biochemical properties of red-cell G6PD from this subject were similar to those of the common variant G6PD Mediterranean, the corresponding mutation (563 CT) was not present. Instead, polymerase chain reaction (PCR) amplification and sequencing of the entire gene detected a CT transition at nucleotide 592 in exon VI, changing an arginine residue to a cysteine residue only 10 amino acids downstream from the Mediterranean mutation. Single-strand conformation polymorphism analysis of a PCR-amplified DNA fragment spanning exons VI and VII of the G6PD gene has detected the same mutation, confirmed by sequencing, in a G6PD-deficient patient from Southern Italy. We name this new variant G6PD Coimbra.     

Quantification of DNA uptake by Dictyostelium discoideum amoebae and the stability of the DNA during growth and development

We have developed a simple and accurate method to determine the amount of intact plasmid DNA taken up and retained by Dictyostelium discoideum amoebae during various transformation protocols. We have used this method to compare the efficiency of three different methods for introducing foreign DNA into D. discoideum amoebae. Both a calcium phosphate and a spheroplast fusion procedure gave good uptake, but no intracellular plasmid DNA was detectable after calcium chloride treatment. The exogenous DNA was rapidly lost after transformation but was 20-fold more stable during starvation rather than growth conditions, suggesting a possible approach to improving transformation efficiency. No transient expression of neomycin phosphotransferase activity of any of the heterologous animal or plant promoters used could be detected using a sensitive gel assay procedure.     

FINE STRUCTURE OF ANNELLOPHORES. I. SCOPULARIOPSIS BREVICAULIS AND S. KONINGII

Fine-structure observations of annelloconidium production in filamentous Hyphomycetes are reported for the first time. The difference in conidium morphology between Scopulariopsis brevicaulis and S. koningii was quite distinct. In S. brevicaulis, verrucosities appeared early in conidium ontogeny and formed an integral part of the primary wall layer of mature conidia. In S. koningii, verrucosities were absent. In S. brevicaulis, annellations did not invariably result on conidiophore necks with the production of each conidium in the basipetal sequence, but alternatively could be left on subapical regions of subsequently formed conidia. In S. koningii, annellations were more distinct, and the position of a conidium-delimiting septum was variable. If a septum were formed at a position proximal to previous septa, a portion of the annellophore neck remained attached to the base f the seceding conidium. In both species, a spherical electron-dense body, perhaps analogous to septal pore plugs in vegetative hyphae, plugged the pore between conidia and conidiophores and remained embedded in the base of seceded conidia.     

Temporal Association Between Pulmonary Inflammation and Antioxidant Induction Following Hyperoxic Exposure of the Preterm Guinea Pig

The time course and nature of the pulmonary inflammatory and antioxidant responses, both during and after hyperoxic-induced acute lung injury were studied in the preterm guinea pig. Three-day preterm (65 days gestation) guinea pigs were randomly exposed to either 21% O₂ (control) or 95% O₂ (hyperoxia) for 72 hours. All pups were then maintained in ambient conditions for up to a further 11 days, during which time lung damage was monitored. In animals exposed to hyperoxia, evidence of acute lung injury and inflammation was characterized by a marked increase in microvascular permeability and elevated numbers of neutrophils in bronchoalveolar lavage fluid. Protein concentration, elastase-like activity and elastase-inhibitory capacity in lavage fluid were at a maximum at the end of the 72 hours hyperoxic exposure. Four days later, all values had returned to control levels. In contrast, increased numbers of neutrophils, macrophages and lymphocytes were recovered in the lavage fluid during this early recovery period. Coinciding with the influx of inflammatory cells, there was a significant increase in glutathione peroxidase, manganese superoxide dismutase and catalase activities in immature lung. Lung copper/zinc superoxide dismutase activity remained unchanged during both experimental periods. The strong temporal relationship between the influx of inflammatory cells to the lung and the induction of pulmonary antioxidant enzyme defences suggests that a common mechanism underlies both responses. These findings have led us to regard inflammation in the hyperoxic-injured immature lung as a beneficial event and not, as previously suggested, as part of the injurious process.     

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae

Abstract An internal fragment from each of the penicillinebinding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae , which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.     

Use of a Plasmid DNA Probe To Monitor Populations of Bacillus pumilus Inoculant Strains in Hay

We are evaluating naturally occurring isolates of Bacillus pumilus for use as microbial hay preservatives. Seven isolates of B. pumilus from hay contained a 42-kb cryptic plasmid (pMGD296). We wished to determine whether pMGD296 could be used as a molecular marker to follow populations of these isolates in hay over time. Southern blots and colony blots of 69 isolates of B. pumilus and other Bacillus spp. were probed with ³²P-labeled pMGD296. Twenty-nine probe-positive isolates were identified; of these, 28 contained a plasmid with a restriction profile identical to that of pMGD296. One isolate from untreated hay contained a 40-kb plasmid (pMGD150) that was homologous to pMGD296 but had a different restriction fragment pattern. Regions of homology between the two plasmids were identified by Southern blotting, and a 1.9-kb HindIII-PstI fragment of pMGD296 lacking strong homology to pMGD150 was cloned in pUC18. The cloned fragment hybridized only with isolates containing pMGD296 and was used to estimate populations of these isolates in treated and untreated hay.