The N-terminal sequence of ribosomal protein L10 from the archaebacterium Halobacterium marismortui and its relationship to eubacterial protein L6 and other ribosomal proteins

The amino-terminal sequence of ribosomal protein L10 from Halobacterium marismortui has been determined up to residue 54, using both a liquid- and a gas-phase sequenator. The two sequences are in good agreement. The protein is clearly homologous to protein HcuL10 from the related strain Halobacterium cutirubrum. Furthermore, a weaker but distinct homology to ribosomal protein L6 from Escherichia coli and Bacillus stearothermophilus can be detected. In addition to 7 identical amino acids in the first 36 residues in all four sequences a number of conservative replacements occurs, of mainly hydrophobic amino acids. In this common region the pattern of conserved amino acids suggests the presence of a beta-alpha fold as it occurs in ribosomal proteins L12 and L30. Furthermore, several potential cases of homology to other ribosomal components of the three ur-kingdoms have been found.     

Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes

Summary When a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS⁺ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS⁺ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS⁺, LacZ⁺ transformants with a Trp^– mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC⁺ transformants which, with a low frequency, had a LacZ^– phenotype. These latter transformants had also lost the AmdS⁺ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.     

Effect of pH on the kinetics of Na+-dependent phosphate transport in rat renal brush-border membranes

The kinetics of Na+-dependent phosphate uptake in rat renal brush-border membrane vesicles were studied under zero-trans conditions at 37 degrees C and the effect of pH on the kinetic parameters was determined. When the pH was lowered it turned out to be increasingly difficult to estimate initial rates of phosphate uptake due to an increase in aspecific binding of phosphate to the brush border membrane. When EDTA or beta-glycerophosphate was added to the uptake medium this aspecific binding was markedly reduced. At pH 6.8, initial rates of phosphate uptake were measured between 0.01 and 3.0 mM phosphate in the presence of 100 mM Na+. Kinetic analysis resulted in a non-linear Eadie-Hofstee plot, compatible with two modes of transport: one major low-affinity system (Km approximately equal to 1.3 mM), high-capacity system (Vmax approximately equal to 1.1 nmol/s per mg protein) and one minor high-affinity (Km approximately equal to 0.03 mM), low-capacity system (Vmax approximately equal to 0.04 nmol/s per mg protein). Na+-dependent phosphate uptake studied far from initial rate conditions i.e. at 15 s, frequently observed in the literature, led to a dramatic decrease in the Vmax of the low-affinity system. When both the extra- and intravesicular pH were increased from 6.2 to 8.5, the Km value of the low-affinity system increased, but when divalent phosphate is considered to be the sole substrate for the low-affinity system then the Km value is no longer pH dependent. In contrast, the Km value of the high-affinity system was not influenced by pH but the Vmax decreased dramatically when the pH is lowered from 8.5 to 6.2. These results suggest that the low-affinity, high-capacity system transports divalent divalent phosphate only while the high-affinity, low-capacity system may transport univalent as well as divalent phosphate. Raising medium sodium concentration from 100 to 250 mM increased Na+-dependent phosphate uptake significantly but the pH dependence of the phosphate transport was not influenced. This observation makes it rather unlikely that pH changes only affect the Na+ site of the Na+-dependent phosphate transport system.     

Transformation of Claviceps purpurea using a bleomycin resistance gene

Summary To develop a DNA-mediated transformation system for Claviceps purpurea a vector was constructed using a bleomycin-resistance gene (bleo ^R) fused in frame to the Aspergillus nidulans trp C promoter as a dominant selection marker. The construct was shown to be functional in Aspergillus nidulans and Aspergillus niger and used to transform a wild strain of Claviceps purpurea. Transformats were obtained at low frequencies; they were shown to contain transforming DNA integrated into the chromosomal DNA, probably in multimeric copies and at multiple sites. Combined Southern, Northern and resistance level analysis indicate that the A. nidulans promoter is functional in C. purpurea.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65^th birthday     

Functional cloning of BUD5, a CDC25-related gene from S. cerevisiae that can suppress a dominant-negative RAS2 mutant

By searching for genes that behave like CDC25 of S. cerevisiae in their ability to counteract a dominant-negative RAS2 mutant in a wild-type RAS-dependent manner, we have isolated a CDC25-like homolog, BUD5. BUD5 is tightly linked to the MAT locus. Although overexpressed BUD5 cannot substitute for CDC25 function, we present evidence that its gene product can bind to the guanine nucleotide binding-deficient RAS2val19ala22 gene product and thereby counteract its dominant-negative effect. We propose that BUD5 is a member of a family of CDC25-related genes that encode activators of RAS and RAS-like proteins.     

Flow cytometric evaluation of cell dispersion from human head and neck tumors

The preparation of single-cell suspensions from 25 human head and neck tumors is described. Dispersal was performed overnight at 4 degrees C under slight agitation of the tissue suspensions using various combinations of enzymes and additives. The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM). It was shown that both trypsin/dithioerythritol (TD) and collagenase/D Nase (CDse) were of value in dispersing single cells from tumor tissue. In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes. In a number of cases, DNA-FCM revealed ploidy abnormalities in a TD-suspension, which were not discernible in the concurrent CDse-suspension. Cell culture of primary cell suspensions corroborated the reliability of the DNA-FCM measurements. Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity. Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension.     

Epidemic of prosthetic valve endocarditis caused by Staphylococcus epidermidis.

In an epidemic of prosthetic valve endocarditis caused by Staphylococcus epidermidis the surgeon was found to be the source of contamination. The probable route was accidental puncture of gloves during operation. During the epidemiological investigation a second cluster of patients contaminated with Staph epidermidis during open heart surgery was found also related to one surgeon. This strain caused no detectable signs or symptoms of infection. Carriage of virulent staph epidermidis has rarely been recognised as a hazard but may have serious consequences.     

Changes in pulsatile secretion patterns of LH, FSH, progesterone, androstenedione and oestradiol in cows after superovulation with PMSG

Six heifers were injected i.m. with 2500 i.u. PMSG followed by 15 mg prostaglandin 48 h later. Serial blood samples were collected through a catheter in the caudal vena cava every 10 min for 8 h on Day 10 (7 h after PMSG administration), during luteal regression (7 h after prostaglandin administration) and on the day thereafter. Four normally cyclic heifers served as a control group. Concentrations of progesterone, androstenedione, oestradiol, LH, FSH, and PMSG in the vena cava samples were measured and the frequency and amplitudes of episodic pulses of all hormones were estimated except for PMSG. Ovaries were collected by ovariectomy at 50 h after onset of luteal regression to determine the number of preovulatory follicles (non-atretic follicles greater than or equal to 10 mm). Stimulation of follicular growth by administration of PMSG resulted in the following effects on the secretion of steroids and endogenous gonadotrophins. (1) There were no alterations in progesterone concentration and the amplitude and frequency of episodic pulses. Mean (+/- s.e.m.) concentrations were 54.1 +/- 5.8, 19.1 +/- 3.1 and 3.4 +/- 0.9 nmol/l on Day 10 (L), during luteal regression (LR) and on the day thereafter (F) respectively. (2) There were no alterations in the episodic secretion patterns of androstenedione. Mean concentrations were 0.20 +/- 0.02, 0.15 +/- 0.02 and 0.11 +/- 0.02 nmol/l for the L, LR and F periods respectively. (3) There was an increase in oestradiol concentration from 17.1 +/- 3.0 pmol/l during the L period to 233.7 +/- 86.4 pmol/l during the F period. Pulse amplitude was enhanced compared to corresponding periods in control animals whereas pulse frequency remained the same. The oestradiol concentration was significantly correlated with the number of preovulatory follicles (r = 0.82, P less than 0.05). (4) There was a suppression of the frequency of episodic LH pulses (/8 h) during the LR (3.2 +/- 0.7) and F (4.3 +/- 0.4) periods compared to corresponding periods in control heifers (9.5 +/- 0.9 and 7.0 +/- 1.5 respectively). The preovulatory LH peak occurred earlier in 4 of 6 treated heifers. (5) There was a suppression of FSH concentrations, pulse amplitude and frequency during the LR and F (17.4 +/- 0.9 mg/l, 4.7 +/- 0.8 microgram/l and 7.5 +/- 0.4 pulses/8 h) periods compared to the corresponding F-period values (35.6 +/- 6.2 mg/l, 9.8 +/- 1.6 micrograms/l and 9.3 +/- 0.3 pulses/8 h) in control heifers.(ABSTRACT TRUNCATED AT 400 WORDS)