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     

Restoration by the rac locus of recombinant forming ability in recB? and recC? merozygotes of Escherichia coli K-12

Summary The recombinant forming ability of recB ^– or recC ^– strains of E. coli K12 is almost totally recovered in merozygotes which are heterozygous for a genetic locus denoted rac which is located five minutes clockwise from trp on the genetic map. This transient recovery phenomenon only occurs when the donor strain is rac ⁺ (wild type) and the recipient strain is rac ^-. The recombinants derived from such crosses all have the normal phenotype characteristic of recB ^– (or recC ^–) strains, and they are almost always rac ^-. The results imply that the rac ⁺ locus (or loci) is zygotically expressed and excised from the chromosome in a manner which is analogous to the zygotic induction of a prophage.     

Measurements of the gas temperature in an oxygen plasma by spectroscopy of the O2( b 1Σ g+ ) → O2( X 3Σ g? ) transition

A method for measuring the gas temperature in an oxygen plasma by spectroscopy of the electronic transition from the O₂(b ¹Σ _g ⁺ , v = 0) metastable state of molecular oxygen into the O₂(X ³Σ _g ⁻ , v = 0) ground state is considered in detail. The method is verified experimentally for the plasma of dc glow discharge in pure oxygen. It is shown that the gas temperature can be determined by analyzing high-resolution spectra of the P branch of this transition, no matter whether its fine structure (^P P and ^P Q branches) is resolved or masked, provided that the rotational structure of the spectrum is resolved. The feasibility of the method proposed in 1999 by P. Maco and P. Veis for determining the gas temperature from the ratio between the intensity maxima of the R and P branches of the O₂(b ¹Σ _g ⁺ , v = 0) → O₂(X ³Σ _g ⁻ , v = 0) transition in a poorly resolved spectrum was studied experimentally. It is shown that, in order to use this method, it is necessary to know the spectrograph instrumental function. The effect of the spatial inhomogeneity of the temperature and concentration of O₂(b ¹Σ _g ⁺ ) molecules on the accuracy of integral (over the plasma volume) measurements of the gas temperature is investigated using spatially resolved spectroscopy of the O₂(b ¹Σ _g ⁺ , v = 0) → O₂(X ³Σ _g ⁻ , v = 0) transition. It is shown that precise measurements of the temperature require that the optical measurement system be thoroughly adjusted in order for the temperature and concentration of the emitting particles to vary insignificantly over the optically selected volume. Original Russian Text ? S.M. Zyryanov, D.V. Lopaev, 2007, published in Fizika Plazmy, 2007, Vol. 33, No. 6, pp. 563–574.     

DnaK-dependent ribosome biogenesis in Escherichia coli? : competition for dominance between the alleles dnaK756 and dnaK ?+

The Escherichia coli chaperone DnaK is vital for many cellular functions, including ribosome biogenesis at high temperature. Thus, the dnaK756-ts (λ ^R ) mutant, at the non-permissive temperature, is inhibited at a late stage of ribosome assembly, yielding 21S, 32S and 45S precursor particles. This defect, unlike the λ resistance and thermosensitivity phenotypes, is not complemented by lysogenisation with a transducing phage λ dnaK ⁺ bearing the wild-type dnaK gene. However this dominant phenotype becomes recessive when dnaK ⁺ is expressed from a medium-copy-number plasmid. On the other hand, an excess of DnaK causes an unexpected dominant-lethal effect of the dnaK756 allele near non-permissive temperatures. This interplay between the dnaK ⁺ and dnaK756 alleles supports the idea of that DnaK oligomers form in the cell. Received: 28 April 1998 / Accepted: 24 July 1998     

Effects of cytochalasin B on water, Na+ and Cl? exchanges in the gill of seawater adapted mulletMugil capito

Summary Electron microscopy study shows that cytochalasin treatment of the mullet damages the microfilaments system in the apex of gill ionocytes: the microfilaments are reduced in number and shortened. Cytochalasin causes a reduction of transgill potential difference and an increase of the Na⁺ and Cl^– blood concentration, of the diffusional water permeability of the gill, of the Na⁺ branchial influx and of Cl^– efflux. The increase of the Na⁺ influx may result in a reduction of the Na⁺ net excretion flux compared to the control. The increased permeability in cytochalasin treated fish facilitates the Cl^– entry probably leading to a reduction of the net Cl^– excretion. The partial inhibition of the K⁺ dependent components of Na⁺ and Cl^– effluxes also contributes to the reduction of Na⁺ and Cl^– excretion. The role of microfilaments in the mechanisms of ionic excretion by the gill is discussed.     

Expression of the plasmid pKM101-determined DNA repair system in recA? and lex? strains of Escherichia coli

Summary pKM101, a plasmid R factor of the N compatibility group increases methylmethane sulfonate mutagenesis and diminishes UV-killing in recA ⁺ lex ⁺ and recA ⁺ lex ^– strains, but not in recA ^– lex ⁺ strains. The induction of a reclex dependent colicin is not present in lex ^– strains carrying the pKM101 factor. These facts indicate that pKM101 acts through an error-prone DNA repair system, which is recA ⁺ dependent, but not lex ⁺ dependent.This paper is published on the occasion of Dr. C. Callerio's seventy-fifth birthday     

Overexpression of Arabidopsis thaliana Na+/H+ antiporter gene enhanced salt resistance in transgenic poplar (Populus?×?euramericana ‘Neva’)

Salinity is a major abiotic stress factor limiting plant growth and productivity. One possible method to enhance plant salt-resistance is to compartmentalize sodium ions away from the cytosol. In the present work, a vacuolar Na⁺/H⁺ antiporter gene AtNHX1 from Arabidopsis thaliana, was transferred into Populus × euramericana ‘Neva’ by Agrobacterium tumefaciens in order to enhance poplar salt-resistance. The results showed that the transgenic poplar were more resistant to NaCl than the wild-type (WT) in greenhouse condition. Compared with the WT, plant growth and photosynthetic capacity of the transgenic plants were enhanced, and the transgenic plants accumulated more Na⁺ and K⁺ in roots and leaves under the same NaCl condition, whereas malondialdehyde and relative electrical conductivity were lower. All of these properties of the transgenic poplar were likely to be a consequence of the overexpression of AtNHX1 caused Na⁺ sequestration in the vacuoles and improved K⁺ absorption, thus reducing their toxic effects. These results indicated overexpression of the AtNHX1 enhanced salt-resistance of poplar, and AtNHX1 played an important role in the compartmentation of Na⁺ into the vacuoles. Therefore, this study provides an effective way for improving salt resistance in trees.     

Characterization of the lys2 gene of Penicillium chrysogenum encoding α -aminoadipic acid reductase

A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr^ 154 859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomycespombe (51.3% identity) and Candida albicans (48.12% identity) α-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the α-aminoadipate-activating domain of the α-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5′-region and other in the 3′-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region. Received: 26 January 1998 / Accepted: 4 May 1998     

Construction of an 800-kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta? 2 using a highly representative rice BAC library

We constructed a rice Bacterial Artificial Chromosome (BAC) library from green leaf protoplasts of the cultivar Shimokita harboring the rice blast resistance gene Pi-ta. The average insert size of 155 kb and the library size of seven genome equivalents make it one of the most comprehensive BAC libraries available, and larger than many plant YAC libraries. The library clones were plated on seven high density membranes of microplate size, enabling efficient colony identification in colony hybridization experiments. Seven percent of clones carried chloroplast DNA. By probing with markers close to the blast resistance genes Pi-ta ² (closely linked to Pi-ta) and Pi-b, respectively located in the centromeric region of chromosome 12 and near the telomeric end of chromosome 2, on average 2.2 ± 1.3 and 8.0 ± 2.6 BAC clones/marker were isolated. Differences in chromosomal structures may contribute to this wide variation in yield. A contig of about 800 kb, consisting of 19 clones, was constructed in the Pi-ta ² region. This region had a high frequency of repetitive sequences. To circumvent this difficulty, we devised a “two-step walking” method. The contig spanned a 300 kb region between markers located at 0 cM and 0.3 cM from Pi-ta ² . The ratio of physical to genetic distances (> 1,000 kb/cM) was more than three times larger than the average of rice (300 kb/cM). The low recombination rate and high frequency of repetitive sequences may also be related to the near centromeric character of this region. Fluorescent in situ hybridization (FISH) with a BAC clone from the Pi-b region yielded very clear signals on the long arm of chromosome 2, while a clone from the Pi-ta ² region showed various cross-hybridizing signals near the centromeric regions of all chromosomes. Received: 14 August 1996 / Accepted: 2 December 1996     

Disulfide bonds Cys9–Cys57, Cys34–Cys88 and Cys38–Cys90 of the β-subunit of human chorionic gonadotropin are crucial for heterodimer formation with the α-subunit: experimental evidence for the conclusions from the crystal structure of hCG

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α- and β-subunits of hCG are highly cross-linked internally by disulfide bonds that seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This paper describes the results of our studies on the role of the disulfide bonds of hCG-β in heterodimer formation with the α-subunit. Six disulfide peptides incorporating each of the six disulfide bonds of hCG-β were screened, along with their linear counterparts, for their ability to competitively inhibit the recombination of α- and β-subunits. The disulfide peptides Cys (9–57), Cys (34–88) and Cys (38–90) were found to inhibit the α/β recombination whereas the remaining three disulfide peptides viz. Cys (23–72), Cys (26–110) and Cys (93–100) did not exhibit any inhibition activity. Interestingly, none of the linear peptides could inhibit the α/β recombination. Results clearly demonstrate that the disulfide bonds Cys⁹–Cys⁵⁷, Cys³⁴–Cys⁸⁸ and Cys³⁸–Cys⁹⁰ of the β-subunit of hCG are crucial for heterodimer formation with the α-subunit thus providing experimental confirmation of the conclusions from the crystal structure of the hormone.     

Over-expression of the Arabidopsis Na+/H+ antiporter gene in Populus deltoides CL?×?P. euramericana CL “NL895” enhances its salt tolerance

Soil salinity is a serious problem worldwide. It is necessary to improve the salt tolerance of plants to avoid the progressive deterioration of saline soil. We showed that the over-expression of AtNHX1 improves salt tolerance in a transgenic poplar (Populus deltoides CL × P. euramericana CL “NL895”) under mannose selection. Four transgenic poplar plants were obtained. Southern blot analysis showed that the pmi gene had integrated into the genome of the poplar. RT-PCR confirmed that AtNHX1 could be expressed normally in the transgenic plants. When tested for salt tolerance by NaCl stress, we measured a 100% increase in Na⁺ content in the three transgenic lines (T18, T50, T98) significantly higher than the 33% increase seen in wild-type plants. The chlorophyll content of the transgenic plants was not altered significantly, while the chlorophyll content in the control plants showed a small decrease. MDA content was decreased in the transgenic plants. These results show that the AtNHX1 gene may enhance salt tolerance due to increased vacuolar compartmentalization of sodium ions.     

Molecular characterization of the human protein kinase C θ * gene locus (PRKCQ)

Members of the protein kinase C (PKC) family of serine/threonine kinases, in particular PKCθ, play critical roles in the regulation of differentiation and proliferation of T lymphocytes. In this study the genomic structure of the human PRKCQ gene that encodes PKCθ was determined. Two genomic P1 clones were isolated from human P1 libraries using the PKCθ cDNA as a probe and have been used to confirm the assignment of the single PRKCQ locus to chromosome 10p15 by FISH analysis. The PRKCQ locus, the first mammalian PKC gene locus characterized so far, spans approximately 62 kb and is composed of 15 coding exons and 14 introns, varying in size between 98 and 16 000 bp. All exon-intron boundaries have been determined by long-range PCR and subsequent DNA sequence analysis. Comparison with other known genomic PKC genes reveals a high degree of homology to the genomic organization of the Drosophila melanogaster dPRKC gene. Alignment of the intron positions in the PRKCQ gene with the intron locations in the dPRKC gene indicates that the sites of seven of the 14 PRKCQ introns are exactly conserved. Exons 5 (32 bp), 11 (174 bp) and 12 (92 bp) share highest similarity in size, organization and primary structure with their counterparts in the Drosophila gene. On the basis of this knowledge of the genomic PRKCQ locus, a directed search for potential genetic polymorphisms and/or genetic abnormalities involved in human genetic disease(s) can now be initiated. Received: 6 February 1998 / Accepted: 25 May 1998     

On the interrelations of the Na+-and Cl?-influxes in the pulmonate freshwater snailLymnaea stagnalis

Summary In the freshwater snailLymnaea stagnalis the influxes of Na⁺ and Cl^– were studied at different external concentrations of these ions. The characteristies of the Na⁺- and Cl^–-influxes are similar with respect to saturation kinetics,K _m (0.1 mM) and activation by low-salt adaptation. In short-term experiments the Na⁺- and Cl^–-influxes are independent. Because of the counter-ions (H⁺ and HCO ₃ ^– ) involved, this indicates a potential acid-base regulatory capacity. Low-salt adaptation, due to either Na⁺-or Cl^–-depletion, activates both the Na⁺- and the Cl^–-influx. It is suggested that under both conditions the number of active integumental pumps, involved in Na⁺- as well as in Cl^–-uptake, is increased.     

Thrombin Specificity: Further Evidence for the Importance of the β-Insertion Loop and Trp96. Implications of the Hydrophobic Interaction Between Trp96 and Pro60B Pro60C for the Activity of Thrombin

A number of thrombin mutants have been constructed to investigate the role of Trp⁹⁶ and the -insertion loop for the specificity of thrombin. Thrombin(60D) consists of the replacement of the -insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; thrombin(GGG) is a smaller loop mutation in which the residues Tyr^60APro^60BPro^60CTrp^60D Asp^60ELys^60F of the -insertion loop were replaced by Gly-Gly-Gly; thrombin(96S) consists of a point mutation Trp⁹⁶Ser; and thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen ~3 times more slowly than thrombin, with the two -insertion loop mutants, thrombin(GGG) and thrombin(GGG/96S), reacting ~3000- and 1300-fold more slowly, respectively. The specificity constant k _cat/K _m for the cleavage of fibrinopeptide A and fibrinopeptide B by thrombin(96S) was 2.6 and 0.35 M^–1 s^–1 respectively, compared to 10 and 2.5 M^–1 s^–1 for wild-type recombinant thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant K _m increased ~6-fold for thrombin(96S) and >200-fold for thrombin(GGG) and thrombin(GGG/96S) when compared to wild-type recombinant thrombin, while the catalytic constant k _cat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type recombinant thrombin. Clearly, the -insertion loop is important for thrombin activity. But the mutation of Trp⁹⁶Ser can compensate somewhat for the loss of binding at the -insertion loop. The deletion of the hydrophobic interaction between Trp⁹⁶ and Pro^60BPro^60C appears to decrease the stability of the -insertion loop, thereby causing a decrease in binding efficiency.     

BOULTONIA属的一个新种*

(竹蜓)科Boultonia一屬係1927年李四光教授所建立,以Boultonia willsi Lee为其屬型。标本採自遼东半島五湖嘴煤田,時代屬上石炭紀太原統。在同一文中,他又描寫了另一个种Boultonia rawi Lee, B. rawi是中石炭紀本溪統的產物,後來这个种曾被許多(竹蜓)科学者歸入Wedekindella或Fusiellina。其後,陈旭教授在1934年描寫了一个採自廣西馬平石灰岩中的Boultonia新种,名为Boultonia cylindrica Chen。最近Skin-ner及Wilde根据陈旭教授对該种的描述和圖版指出該种以其隔壁褶皺不强烈,可能     

The initiation site for recombination cog is at the 3′ end of the his-3 gene in Neurospora crassa

Summary The response of Nicotiana tabacum to tentoxin (chlorosis) is inherited with chloroplasts. N. tabacum var. Xanthi, a tentoxin-resistant line, was used to pollinate tentoxin-sensitive N. tabacum line 92, an alloplasmic male-sterile line containing N. undulata plastids. The seeds were mutagenized with nitrosomethylurea and germinated in the presence of tentoxin. Two percent of the seedlings had green sectors in their first true leaves. These plants were grown to maturity under non-selective conditions. Homogeneous tentoxin-resistant lines were obtained in the third generation. DNA analysis indicated, however, that selection for paternal plastids, rather than mutagenesis of maternal ones, had occurred in the tentoxin-resistant progeny. Mitochondria, which were not under selection pressure, were inherited maternally as expected. Inheritance of tentoxin-resistant paternal plastids did not require seed mutagenesis. Normally germinated seedlings that were kept under tentoxin selection consistently produced a low level of resistant green sectors in their first true leaves. Thus, normal, low-frequency transmission of paternal plastids in N. tabacum can be directly revealed by using tentoxin.