Mutations That Improve the Binding of Yeast Flp Recombinase to Its Substrate

When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.     

Water hyacinth productivity and detritus accumulation

Water hyacinth [Eichhornia crassipes (Mart) Solms] productivity and detritus accumulation were evaluated in eutrophic lake water with and without added nutrients (fertilized and control reservoirs, respectively). Seasonal changes in plant productivity and detritus accumulation were determined at monthly intervals for one year. Significant differences were observed in plant productivity between seasons and nutrient additions. Seasonal plant productivity ranged from 1.9 to 23.1 mg (dry wt) ha⁻¹ for the fertilized reservoir and −0.2 to 10.2 mg ha⁻¹ for the control reservoir. Detritus accumulation was not significantly different between seasons or nutrient additions. Seasonal N assimilation by plants ranged from 34 to 242 kg N ha⁻¹ for plants in the fertilized reservoir and < 0 to 104 kg N ha⁻¹ for plants in the control reservoir. Annual net N recovered in detritus represented 21 and 28% of the total N removed by plants in the fertilized and control reservoirs, respectively. Net N loading to the reservoirs from detritus was 92 to 148 kg N ha⁻¹ yr⁻¹.     

Characterization of the glycosylation sites in yeast external invertase. I. N-linked oligosaccharide content of the individual sequons

External invertase is the product of the SUC2 gene of Saccharomyces cerevisiae. The deduced sequence of this enzyme (Taussig, R., and Carlson, M. (1983) Nucleic Acid Res. 11, 1943-1954) reveals it to contain 14 potential N-linked glycosylation sites, or sequons, although only 9-10 appear to be glycosylated (Trimble, R. B., and Maley, F. (1977) J. Biol. Chem. 252, 4409-4412). To determine the location of the glycosylated sequons, external invertase was deglycosylated with endo-beta-acetylglucosaminidase H and its component peptides analyzed by both fast atom bombardment mass spectrometry (FABMS) and classical peptide isolation procedures. By use of the former technique most of the glucosamine-containing sequons could be located and by the latter sufficient amounts of small glucosamine-containing peptides were isolated to enable their quantitation. From the combined FABMS and glucosamine analyses, it was established that eight of the sequons in a subunit of invertase are either completely or almost completely glycosylated, while five others are glycosylated to the extent of about 50% or less. In the case of two overlapping sequons (4 and 5), which include Asn92-Asn93-Thr-Ser, only the first Asn was glycosylated. Thus, all but one of the sequons of external invertase are glycosylated to some extent, giving an appearance of only 9-10 N-linked oligosaccharides/subunit. The sequence identity of both external and internal invertase was verified by FABMS and by peptide sequence analysis. In only one site was an amino acid found to differ from that deduced from the DNA sequence of the SUC2 gene. This occurred at position 390 where a proline was found in place of alanine, which could result from a single base change in the triplet specifying the latter amino acid.     

Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans

The characteristics of the uptake of lipophilic cations tetraphenylphosphonium (TPP+) into Candida albicans have been investigated to establish whether TPP+ can be used as a membrane potential probe for this yeast. A membrane potential (delta psi, negative inside) across the plasma membrane of C. albicans was indicated by the intracellular accumulation of TPP+. The steady-state distribution of TPP+ was reached within 60 min and varied according to the expected changes of delta psi. Agents known to depolarize membrane potential caused a rapid and complete efflux of accumulated TPP+. The initial influx of TPP+ was linear over a wide range of TPP+ concentrations (2.5-600 microM), indicating a non mediated uptake. Thus, TPP+ is a suitable delta psi probe for this yeast.     

Diurnal rhythm in levels of haemolymph sugar and hyperglycaemic activity of eyestalk extract in the fresh water rice field crab (Oziotelphusa senex senex Fabricius)

The concentration of haemolymph sugar and the hyperglycaemic activity of eyestalk extract was measured six times (8, 12, 16, 20 and 4 h) over a 24-h period. The concentration of haemolymph sugar and hyperglycaemic activity of eyestalk extract was higher during the night (0 h through 8 h) than that noted in day time (12 h). The variations are closely related to the activity of the animal.     

Identification of cDNA clones for ligninase from Phanerochaete chrysosporium using synthetic oligonucleotide probes

Four cDNA clones for ligninase were isolated from the cDNA library (constructed into the PstI site of E. coli vector pUC9) representing 6 day-old lignin degrading culture of Phanerochaete chrysosporium by the use of three synthetic oligonucleotide probes corresponding to partial amino acid sequences of tryptic peptides of the ligninase. Each of the three probes, 14.1, 14.2 and 25, represents a mixture of 32 12- or 14-base long oligonucleotides. Three cDNA clones hybridized with probe 14.1 but not with probe 25 or 14.2, but one cDNA clone hybridized with all of the three probes. Differential hybridization studies showed that these clones are unique to 6-day poly(A) RNA, but not to 2-day poly(A) RNA.     

An intermediate polymer in the assembly of clathrin baskets

Clathrin (8 S) is known to polymerize into two varieties of basket structures (150 S or 300 S) under the normal buffer conditions [100 mM 2-(N-morpholino)ethanesulfonic acid (Mes), pH 5.9-6.7] used for the isolation of coated vesicles. However, it is now observed that under very low salt conditions (2 mM Mes, pH 5.9), it forms a homogeneous species with a sedimentation coefficient of 27 S. Increasing the salt concentration to 50 mM Mes completely converts all the 27S species into 150S baskets. Sedimentation equilibrium data show that this 27S species has a molecular weight that is 6 times that of the clathrin protomer and is the result of highly cooperative reversible self-association of the 8S protomer. Light-scattering studies show that the stabilities of 27S species and baskets (150 S or 300 S) are comparable. Fluorescent labeling of sulfhydryl groups with N-(1-anilinonaphthalenyl)maleimide indicates that the conformation of clathrin in 27S species and baskets (150 S or 300 S) is similar. Trypsin digestion reveals that in the 27S species clathrin has a conformation differing from that in both the 8S species and baskets.     

Inactivation of D-(-)-beta-hydroxybutyrate dehydrogenase by modifiers of carboxyl and histidyl groups

Data presented in this paper suggest that D-(-)-beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains an essential carboxyl group and an essential histidyl residue at or near the active site. Lactate and malate dehydrogenases, which catalyze reactions analogous to that catalyzed by BDH, also contain an aspartyl and a histidyl residue at the active site [Birktoft, J.J., & Banaszak, L.J. (1983) J. Biol. Chem. 258, 472-482]. In addition, all three enzymes contain an essential arginyl residue, apparently concerned with electrostatic interaction with their respective carboxylic acid substrates, and promote ternary adduct formation involving the enzyme, NAD, and sulfite.     

Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates

Oligodeoxyribonucleoside methylphosphonates of defined sequence of the type d-Np(NP)nN, where n is 6-13, are readily prepared on insoluble polystyrene supports by use of protected 5'-(dimethoxytrityl)deoxyribonucleoside 3'-(methylphosphonic imidazolides) as synthetic intermediates. The imidazolides are prepared in situ by reaction of protected 5'-(dimethoxytrityl)deoxyribonucleoside with methylphosphonic bis(imidazolide) and can be stores in the reaction solution for up to 2 weeks at 4 degrees C with no loss in activity. The condensation reaction is accelerated by the presence of tetrazole, which appears to act as an acid catalyst. The half-life for dimer formation on the polystyrene support is 5 min, and the reaction is 95% complete after 60 min. Although similar kinetics are observed when controlled pore glass is used as the support, the extent of the reaction does not go beyond 78%, even after prolonged incubation. In order to simplify purification and sequence analysis of the oligomer, the 5'-terminal nucleoside unit is linked via a phosphodiester bond. This linkage may be introduced by either an o-chlorophenyl phosphotriester method or a cyanoethyl phosphoramidite method. The latter procedure simplifies the deprotection step, since the cyanoethyl group is readily cleaved by ethylenediamine, which also removes the base protecting groups and cleaves the oligomer from the support. The singly charged oligomers are easily purified by affinity chromatography on DEAE-cellulose. The chain lengths of the oligomers were confirmed after 5'-end labeling with polynucleotide kinase by partial hydrolysis of the methylphosphonate linkages with 1 M aqueous piperidine followed by polyacrylamide gel electrophoresis of the hydrolysate.(ABSTRACT TRUNCATED AT 250 WORDS)