A wat1 mutant of fission yeast is defective in cell morphology

The organization of the actin cytoskeleton plays an integral role in cell morphogenesis of all eukaryotes. We have isolated a temperature-sensitive mutant in Schizosaccharomyces pombe, wat1-1, in which acting patches are delocalized, resulting in an elliptically shaped cell phenotype. Molecular cloning and DNA sequencing of wat1 ⁺ showed that the gene encodes a 314 residue protein containing WD-40 repeats. Cells lacking wat1 ⁺ are slow growing but viable at 25° C and temperature-sensitive for growth above 33° C. At restrictive temperature, wat1-d strains are phenotypically indistinguishable from wat1-1. When combined with a deletion for the wat1 ⁺ gene, cdc mutants failed to elongate at restrictive temperature and exhibited alterations in actin patch localization. This analysis suggests that wat1 ⁺ is required directly or indirectly for polarized cell growth in S. pombe. Wat1p and a functional, epitope-tagged, version of Wat1p can be overproduced without inducing alterations in cell morphology. Received: 18 September 1996 / Accepted: 22 October 1996     

A novel fission yeast gene, kms1 +, is required for the formation of meiotic prophase-specific nuclear architecture

In the meiotic prophase nucleus of the fission yeast Schizosaccharomyces pombe, chromosomes are arranged in an oriented manner: telomeres cluster in close proximity to the spindle pole body (SPB), while centromeres form another cluster at some distance from the SPB. We have isolated a mutant, kms1, in which the structure of the meiotic prophase nucleus appears to be distorted. Using specific probes to localize the SPB and telomeres, multiple signals were observed in the mutant nuclei, in contrast to the case in wild-type. Genetic analysis showed that in the mutant, meiotic recombination frequency was reduced to about one-quarter of the wild-type level and meiotic segregation was impaired. This phenotype strongly suggests that the telomere-led rearrangement of chromosomal distribution that normally occurs in the fission yeast meiotic nucleus is an important prerequisite for the efficient pairing of homologous chromosomes. The kms1 mutant was also impaired in karyogamy, suggesting that the kms1 ⁺ gene is involved in SPB function. However, the kms1 ⁺ gene is dispensable for mitotic growth. The predicted amino acid sequence of the gene product shows no significant similarity to known proteins. Received: 5 September 1996 / Accepted: 21 November 1996     

DNA sequence analysis of the C4 antigen WH: evidence for two mechanisms of expression

 Amino acid and protein analyses have allowed the construction of a model for the C4-based Rodgers and Chido blood group antigens. The single low-frequency allele (WH) in this blood group system, however, has not been characterized at the molecular level. Two WH⁺ donors were studied by C4 agarose gel electrophoreses, immunoblot studies using monoclonal anti-Rg: 1 or anti-Ch: 1, serological phenotyping, polymerase chain reaction-restriction fragment length polymorphism of their C4 genes, and DNA sequencing of the WH allele. The first donor had the C4A1, A3 phenotype; the C4A1 carried Ch: 1, 3, 6 (thus exhibiting reversed antigenicity) and the C4A3 carried the WH antigen. The amino acid sequence of the WH allele was PCPVLD at positions 1101 – 1106, S at position 1157, and VDLL at positions 1188 – 1191. A second donor typed as C4A2, A4, B1 and was also WH⁺. Immunoblot analysis showed that a C4B1 protein expressed Rg: 1. Sequence analysis of the C4B genes showed the amino acids LSPVIH at positions 1101 – 1106, S at position 1157, and ADLR at positions 1188 – 1191. Thus, the WH antigen is a conformational epitope that can arise through different mechanisms on either a C4A or C4B gene. Received: 22 November 1995 / Revised: 19 February 1996     

The RHC21 gene of budding yeast, a homologue of the fission yeast rad21 +gene, is essential for chromosome segregation

The Saccharomyces cerevisiae gene RHC21 is a homologue of the fission yeast rad21 ⁺gene, which affects the sensitivity of cells to γ-irradiation and is essential for cell growth in S. pombe. Disruption of the RHC21 gene showed that it is also essential in S. cerevisiae. To examine its function in cell growth further, we have isolated temperature-sensitive mutants for the RHC21 gene and characterized one of them, termed rhc21-sk16. When this mutant was incubated at 36° C, the percentage of large-budded cells was increased. Most of the large-budded cells had aberrant nuclear structures, such as unequally extended nuclear DNA with incompletely elongated spindles across the mother-daughter neck or only in a mother cell. Furthermore, a circular minichromosome is more unstable in the mutant than in the wild-type, even at 25° C. Flow cytometry showed that the bulk of DNA replication takes place normally at the restrictive temperature in the mutant. These results indicated that the RHC21 gene is required for proper segregation of the chromosomes. In addition, we found that the mutant is sensitive not only to UV radiation and γ-rays but also to the antimicrotubule agent nocodazole at 25° C. This suggests that the RHC21 gene is involved in the microtubule function. We discuss how the RHC21 gene product may be involved in chromosome segregation and microtubule function. Received: 10 March 1997 / Accepted: 1 September 1997     

Catabolite-repressor-like protein regulates the expression of a gene under the control of the Escherichia coli lac promoter in the plant pathogen Xanthomonas campestris pv. campestris

  Xanthomonas campestris pv. campestris, the causal agent of black-rot disease of cruciferous plants, and an important industrial microbe, was able to express the Escherichia coliβ-glucuronidase reporter gene (uidA) when fused to the E. coli lactose operon promoter on a wide-host-range plasmid vector. The gene fusion is expressed constitutively at high levels in both complex and defined media using a wide range of carbon sources, and is not repressible by glucose or inducible by the gratuitous lac inducer isopropyl β-d-thiogalactoside. An X. campestris campestris strain with a lesion in the clp (catabolite-repressor-like protein) locus, and containing the plac/uidA fusion, was tested for β-glucuronidase activity. We found that the expression of the plac/uidA fusion gene is dependent on the presence of catabolite-repressor-like protein, with an approximately 75% reduction of expression in the clp -deficient mutant. Received: 1 April 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996     

Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome

To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ ⁻transposable element was constructed to insert the lacZ ⁻(Trp570 → Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop → LacZ⁺ candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14 000 colonies, and named plm-1 (preferential lacZmutagenesis) and plm-2. The frequency of LacZ⁻→ LacZ⁺ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac⁺ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan^r gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ⁺→ LacZ⁻and Kan^r→ Kan^s mutant frequencies of these Lac⁺ revertants were in the range of 10⁻³ to 10⁻², indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome. Received: 1 August 1996 / Accepted: 3 April 1997     

Inactivation of OGG1 increases the incidence of G?·?C→T?·?A transversions in Saccharomyces cerevisiae : evidence for endogenous oxidative damage to DNA in eukaryotic cells

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. To investigate the biological role of the OGG1 gene, mutants were constructed by partial deletion of the coding sequence and insertion of marker genes, yielding ogg1::TRP1 and ogg1::URA3 mutant strains. The disruption of the OGG1 gene does not compromise the viability of haploid cells, therefore it is not an essential gene. The capacity to repair 8-OxoG has been measured in cell-free extracts of wild-type and ogg1 strains using a 34mer DNA fragment containing a single 8-OxoG residue paired with a cytosine (8-OxoG/C) as a substrate. Cell-free extracts of the wild-type strain efficiently cleave the 8-OxoG-containing strand of the 8-OxoG/C duplex. In contrast, cell-free extracts of the Ogg1-deficient strain have no detectable activity that can cleave the 8-OxoG/C duplex. The biological properties of the ogg1 mutant have also been investigated. The results show that the ogg1 disruptant is not hypersensitive to DNA-damaging agents such as ultraviolet light at 254 nm, hydrogen peroxide or methyl methanesulfonate. However, the ogg1 mutant exhibits a mutator phenotype. When compared to those of a wild-type strain, the frequencies of mutation to canavanine resistance (Can^R) and reversion to Lys⁺ are sevenfold and tenfold higher for the ogg1 mutant strain, respectively. Moreover, using a specific tester system, we show that the Ogg1-deficient strain displays a 50-fold increase in spontaneously occurring G · C→T · A transversions compared to the wild-type strain. The five other base substitution events are not affected by the disruption of the OGG1 gene. These results strongly suggest that endogeneous reactive oxygen species cause DNA damage and that the excision of 8-OxoG catalyzed by the Ogg1 protein contributes to the maintenance of genetic stability in S. cerevisiae. Received: 6 September 1996 / Accepted: 22 October 1996     

Simultaneous interpretation of Mössbauer, EPR and 57Fe ENDOR spectra of the [Fe4S4] cluster in the high-potential iron protein I Ectothiorhodospira halophila

4 S₄]^3 + and the reduced [Fe₄S₄]^2 + clusters in the high-potential iron protein I from Ectothiorhodospira halophila were measured in a temperature range from 5 K to 240 K. EPR measurements and ⁵⁷Fe electron-nuclear double resonance (ENDOR) experiments were carried out with the oxidized protein. In the oxidized state the cluster has a net spin S = 1/2 and is paramagnetic. As common in [Fe₄S₄]^3 + clusters, the M?ssbauer spectrum was simulated with two species contributing equally to the absorption area: two Fe^3 + atoms couple to the “ferric-ferric” pair, and one Fe^2 + and one Fe^3 + atom give the “ferric-ferrous pair”. For the simulation of the M?ssbauer spectrum, g-values were taken from EPR measurements. A-tensor components were determined by ⁵⁷Fe ENDOR experiments that turned out to be a necessary source of estimating parameters independently. In order to obtain a detailed agreement of M?ssbauer and ENDOR data, electronic relaxation has to be taken into account. Relaxing the symmetry condition in a way that the electric field gradient tensor does not coincide with g- and A-tensors yielded an even better agreement of experimental and theoretical M?ssbauer spectra. Spin-spin and spin-lattice relaxation times were estimated by pulsed EPR; the former turned out to be the dominating mechanism at T = 5 K. Relaxation times measured by pulsed EPR and obtained from the M?ssbauer fit were compared and yield nearly identical values. The reduced cluster has one additional electron and has a diamagnetic (S = 0) ground state. All the four irons are indistinguishable in the M?ssbauer spectrum, indicating a mixed-valence state of Fe^2.5 + for each. Received: 15 February 1999 / Accepted: 31 August 1999     

Production of synthetic spider dragline silk protein in Pichia pastoris

The methylotrophic yeast Pichia pastoris was tested as a host for the production of long, repetitive protein polymers. Synthetic genes for a designed analog of a spider dragline silk protein were readily expressed at high levels under control of the methanol-inducible AOX1 promoter. Transformants containing multiple gene copies produced elevated levels of silk protein, but of a variety of altered sizes as a result of gene rearrangements at the time of transformation. Genes up to 3000 codons in length or longer could be expressed with no evidence of the prevalent truncated synthesis observed for similar genes in Escherichia coli, though genes longer than 1600 codons were expressed less efficiently than shorter genes. Silk-producing P. pastoris strains were stable without selection for at least 100 doublings. Received: 4 March 1996 / Received revision: 26 June 1996 / Accepted: 12 August 1996     

Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K⁺ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (E_m) was not sensitive to the external K⁺ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K⁺-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (E_rev) of the s-ORC conductance. The s-ORC was blocked by Ba²⁺ (K_1/2 = 0.3–1.3mM) and verapamil (K_1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba²⁺. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K⁺ concentration. The slow activation of the IRC (t_1/2 ≈ 0.5 s) and its negative activation potential (V_threshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes. Received: 28 March 1996 / Accepted: 11 November 1996     

Controlled secretion into the culture medium of a hybrid -glucanase by Acetobacter methanolicus mediated by the kil gene of Escherichia coli located on a Tn5 -derived transposon

A Tn5-based transposon bearing the kil gene (killing protein), mediating controlled export of periplasmic proteins into the culture medium, was constructed (Tn5-KIL3). This transposon contained the kil gene of the ColE1 plasmid under the growth-phase-dependent promoter of the fic gene (filamentation induced by cAMP) of Escherichia coli, an interposon located upstream of kil, a kanamycin/neomycin-resistance gene, a multiple cloning site and the mob site. The transposition of Tn5-KIL3 to Acetobacter methanolicus showed a moderate transposition frequency (10⁻⁵–10⁻⁶). By insertion of a Bacillus hybrid β-glucanase (bgl ) as a model protein into the transposon (Tn5-LF3) it was shown that the secretion function as well as the gene of the target protein had been transferred to and stably integrated into the chromosome of A. methanolicus, and that the transposition of Tn5-LF3 was non-specific. β-Glucanase was highly overexpressed and secreted into the medium during stationary phase. Total and extracellular production of β-glucanase varied depending on the integration site of the transposon. The viability of the bacterial cells was not affected, and cell lysis did not occur. Received: 17 October 1996 / Received revision: 23 December 1996 / Accepted: 4 January 1997     

The relationship between plasma potassium concentration and muscle torque during recovery following intense exercise

The present study investigated the relationship between plasma potassium ion concentration ([K⁺]) and skeletal muscle torque during three different 15-min recovery periods after fatigue induced by four 30-s sprints. Four males and one female completed the multiple sprint exercise on three separate days; recovery was passive, i.e. no cycling exercise (PRec), active cycling at 30% peak oxygen consumption O_2peak (30% Rec) and active cycling at 60% O_2peak (60% Rec). Plasma [K⁺] was measured from blood sampled from an antecubital vein of subjects at rest and at 0, 3, 5, 10 and 15 min into each recovery. Isokinetic leg strength was measured at rest and at 1, 6, 11 and 16 min during each recovery. Following the exhaustive sprints, [K⁺] increased significantly from an average mean (SEM) resting value of 3.81 (0.07) mmol · l⁻¹ to 4.48 (0.19) mmol · l⁻¹ (P < 0.01). In all recovery conditions, plasma [K⁺] returned to resting levels within 3 min following the fourth sprint. However, in the two active recovery conditions plasma [K⁺] increased over the remainder of the recovery periods to 4.36 (0.12) mmol · l⁻¹ in the 30% Rec condition and 4.62 (0.12) mmol · l⁻¹ in the 60% Rec condition, the latter being significantly higher than the former (P < 0.01). The maximum torque measured following the sprints decreased significantly, on average, to 61.1 (8.36)% of peak levels (P < 0.01). After 15 min of recovery, maximum torque was highest in the 30% Rec condition at 92.13 (3.06)% of peak levels (P < 0.01), compared to 85.23 (3.64)% and 85.71 (0.82)% for the PRec and 60% Rec conditions, respectively. In contrast to the significant differences in plasma [K⁺] across all three recovery conditions, muscle torque recovery was significantly different in only the 30% Rec condition. In summary, recovery of peak levels of muscle torque following fatiguing exercise does not appear to follow changes in plasma [K⁺]. Accepted: 18 October 1996     

A large open reading frame ( orf1995 ) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein

An open reading frame potentially encoding a protein of 1995 amino acids (orf1995) has been found in the chloroplast genome of the green alga Chlamydomonas reinhardtii. Besides having a short hydrophobic N-terminal domain with five putative transmembrane helices, the predicted orf1995 product is highly basic. orf1995 might be a homologue of the ycf1 gene in land plants, whose function has not yet been determined. Mutants of C. reinhardtii transformed with a disruption of orf1995 remain heteroplasmic for the wild-type and disrupted alleles of this gene, indicating that the orf1995 product is essential for cell survival. Received: 18 August 1996 / Accepted: 24 September 1996     

The S?/?M checkpoint at 37°C and the recovery of viability of the mutant pol δ ts3 require the crb2 + /rhp9 + gene in fission yeast

We have isolated a mutant in fission yeast, in which mitosis is uncoupled from completion of DNA replication when DNA synthesis is impaired by a thermosensitive mutation in the gene encoding the catalytic subunit of DNA polymerase δ. By functional complementation, we cloned the wild-type gene and identified it as the recently cloned checkpoint gene crb2 ⁺ /rhp9 ⁺ . This gene has been implicated in the DNA damage checkpoint and acts in the Chk1 pathway. Unlike the deleted strain dcrb2, cells bearing the crb2-1 allele were not affected in the DNA repair checkpoint after UV or MMS treatment at 30° C, but were defective in this checkpoint function when treated with MMS at 37° C. We analysed the involvement of Crb2 in the S/M checkpoint by blocking DNA replication with hydroxyurea, by using S phase cdc mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37° C. Furthermore, the crb2 ⁺ gene was required, together with the cds1 ⁺ gene, for the S/M checkpoint at 30° C. Finally, both the crb2 deletion and the crb2-1 allele induced a rapid death phenotype in the polδts3 background at both 30° C and 37° C. The rapid death phenotype was independent of the checkpoint functions. Received: 25 May 1998 / Accepted: 21 September 1998     

Homologous and heterologous expression of a ribosomal protein gene in Podospora anserina requires an intron

Although the role of introns in eucaryotic nuclear genes has been much debated, it remains underinvestigated in fungi. The AS1 gene of Podospora anserina contains three introns and encodes a ribosomal protein (S12) belonging to the well-conserved bacterial S19 family. We attempted to complement the highly pleiotropic mutation AS1-4 with a cDNA encoding the homologous human (S15) protein (rig gene) under the control of the AS1 promoter. In a control experiment, the AS1 ⁺ cDNA was unable to complement fully the AS1-4 mutation. It was assumed that the AS1 cDNA was not well expressed and that the AS1 gene needed intron(s) to be efficiently expressed. Addition of the first intron of the AS1 gene to the AS1 and rig cDNAs did indeed allow complementation of all the phenotypic defects of the AS1-4 mutation. These data lead to two main conclusions. First, the human S15 ribosomal protein is functional in Podospora. Second, full expression of the Podospora AS1 gene requires at least one intron. Received: 26 April 1996 / Accepted: 22 August 1996     

Molecular cloning of a gene required for DNA replication in Ustilago maydis

TSD2, a gene necessary for DNA synthesis in Ustilago maydis, was cloned by complementation of the temperature sensitive growth defect of a mutant known previously as pol1-1 and renamed here tsd2-1. Linkage analysis established that the cloned fragment contained an allele of tsd2-1 and not a suppressor. DNA sequence determination of the cloned DNA fragment indicated the presence of a single large uninterrupted open reading frame capable of encoding a protein of 845 amino acids without homology to any known gene involved in DNA synthesis. TSD2 was found to be cell cycle-regulated and mRNA levels peaked in early S or G₁ phase. Received: 27 March 1996 / Accepted: 28 August 1996     

Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris

Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Mut^s clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut⁺ clones. The mixed-feeding/Mut^s strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l. Received: 19 November 1996 / Received revision: 29 January 1997 / Accepted: 7 March 1997