Evidence forCis- andTrans-acting element coevolution of the 2-μm circle genome inSaccharomyces cerevisiae

Summary We compared the DNA sequence of the yeas 2-μm plasmidcis-actingSTB andtrans-actingREP1 partition loci of laboratory haploid and industrial amphiploid strains. Several industrial strains had a uniqueSTB sequence (type 1) sharing only 70% homology with laboratorySTB (type 2). Type 1 plasmids had a REP1 protein with 6–10% amino acid substitutions when compared to REP1 of type 2 plasmids. All 2-μm variants that shared a similarSTB consensus sequence exhibited a high degree ofREP1 nucleotide and amino acid sequence conservation. These observations suggest molecular coevolution oftrans-acting elements with cognate target DNA structure. Based on DNA sequencing and Southern hybridization analyses, we classified 2-μm variants into two main evolutionary lineages that differ atSTB as well asREP1 loci. The role of molecular coevolution in yeast intra- and interspecies plasmid evolution was discussed.     

Splenic extramedullary hemopoiesis caused by a dysfunctional mutation in the NF-κB-inducing kinase gene

NF-κB-inducing kinase (NIK) plays critical roles in the development of lymph nodes and Peyer’s patches, and microarchitecture of the thymus and spleen via NF-κB activation. Alymphoplasia (aly/aly) mice have a point mutation in the NIK gene that causes a defect in the activation of an NF-κB member RelB. Here, we developed a novel method to determine the aly mutation by genetic typing using PCR. This method facilitated the easy establishment of a congeneic aly/aly mouse line. Indeed, we generated a mouse line with aly mutation on a BALB/cA background (BALB/cA-aly/aly). BALB/cA-aly/aly mice showed significant splenomegaly with extramedullary hemopoiesis, which was not significant in aly/aly mice on a C57BL/6 background. Interestingly, the splenomegaly and extramedullary hemopoiesis caused by the aly mutation was gender-dependent. These data together with previous reports on extramedullary hemopoiesis in RelB-deficient mice suggest that NIK–RelB signaling may be involved in the suppression of extramedullary hemopoiesis in adult mice.     

A 3′ splice site consensus sequence mutation in the cystic fibrosis gene

Summary In the cystic fibrosis (CF) gene, recently cloned, a three base pair deletion (ΔF508) has been identified in a majority of CF patients. This deletion has been found in 80% of CF chromosomes in families from north west Brittany. In order to identify new mutations we have selected 43 chromosomes negative for the three base pair deletion from these families and directly sequenced exon 11 after DNA amplification by the polymerase chain reaction. We have detected a base change (G→A) at the 3′ end of the consensus sequence of intron ten (namely 1717-1). This mutation destroys a splice site in the cystic fibrosis gene which probably produces a mutant allele. This single nucleotide mutation has been reported on two other CF chromosomes.     

Synthesis of the 2′-,3′-Didehydro-2′-,3′-dideoxy and 2′-,3′-Dideoxy Derivatives of 6-Azauridine and a New Route to 2′-,3′-Didehydro-2′-,3′-dideoxy-5-chlorouridine

Abstract

The 5′-O-(4,4′-dimethoxytrityl) and 5′-O-(tert-butyldimethylsilyl) derivatives of 2′-,3′-O-thiocarbonyl-6-azauridine and 2′,3′-O-thiocarbonyl-5-chlorouridine were synthesized from the parent nucleosides by reaction with 4, 4′-dimethoxytrityl chloride and tert-butyldimethylsilyl chloride, respectively, followed by treatment with 1,1′-thiocarbonyldiimidazole. Introduction of a 2′-,3′-double bond into the sugar ring by reaction of the 5′-protected 2′-,3′-O-thionocarbonates with 1, 3-dimethyl-2-phenyl-1, 3, 2-diazaphospholidiine was unsuccessful, but could be accomplished satisfactorily with trimethyl phosphite. Reactions were generally more successful with the 5′-silylated than with the 5′-tritylated nucleosides. Formation of 2′-,3′-O-thiocarbonyl derivatives proceeded in higher yield with 5′-protected 6-azauridines than with the corresponding 5-chlorouridines because of the propensity of the latter to form 2,2′-anhydro derivatives. In the reaction of 5′-O-(tert-butyldimethylsilyl)-2′-,3′-O-thiocarbonyl-6-azauridine with trimethyl phosphite, introduction of the double bond was accompanied by N³-methylation. However this side reaction was not a problem with 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-O-thioarbonyl-5-chlorouridine. Treatment of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-6-azauridine with tetrabutylammonium fluoride followed by hydrogenation afforded 2′-,3′-dideoxy-6-azauridine. Deprotection of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-5-chlorouridine yielded 2′-,3′-didehydro-2′-,3′-dide-oxy-5-chlorouridine.     

Formation of N-(1-Oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine by the Reaction of 2′-Deoxyguanosine with 3-Deoxyglucosone

3-Deoxyglucosone (3DG) has weaker mutagenicity than methylglyoxal by the Ames test. 3DG reacted readily with 2′-deoxyguanosine (dG) in nucleosides. Two major products (G-A and G-B) were isolated and purified from the reaction mixture of 50 mM 3DG and 50 mM dG at 50°C and pH 7.4 for 6d. G-A was identified as N-(1-oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine. G-B was identified as a diastereomer of G-A.     

Chimeric evolution of the 2-μm genome in Saccharomyces cerevisiae

We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 m) plasmid in Saccharomyces cerevisiae. The DNA sequence of the left-unique region is identical among 2-m variants, while the right-unique region shows substantial divergence. This chimeric pattern cannot be explained by neutral or Darwinian selection models. We propose that horizontal transmission of the 2-m plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection. Correspondence to: G.H. Rank     

Determination of the Group Electronegativity of CF3 Group in 3′-O-CF3-Thymidine by 1-NMR

Abstract

The interplay of enthalpy of the gauche effect (ΔH°_GE) of the [X3′-C3′-C4′-O4′] fragment in various 3′-substituted (X) 2′,3′-dideoxythymidine derivatives 1–7 and the inherent anomeric effect drives the two-state North ? South equilibrium in the constituent sugar moiety. The group electronegativity of 3′-OCF₃ substituent in Marriott's, Inamoto's and Mullay's scales has been determined from simple calibration graphs correlating the group electronegativity of various 3′-substituents (X) in 2′,3′-dideoxythymidine derivatives 1–7 with the experimental strength (ΔH°_GE) of the [X3′-C3′-C4′-O4′] gauche effect. ΔH°_GE has been experimentally determined from pseudorotational analyses of temperature-dependent ³J_HH coupling constants, and can be used as an unambiguous tool for direct experimental estimation of the group electronegativity of a specific substituent covalently attached to 3′-carbon of 2′,3′-dideoxythymidine, which can be compared, in turn, with the theoretical estimation carried out according to Marriott's or Inamoto's procedure. Inconsistency found between theoretical values in Marriott's and Inamoto's scales, on the one hand, and between our experimental estimate and the theoretical value in Marriott's scale, on the other, have been solved by refining the electronegativity scale using our experimental data for 1–7.     

Structure and Conformation of the Branch Core Triribonucleotide Containing 2′-5′ and 3′-5′ Phosphodiester Linkages (A2′p5′G 3′p5′C) in Solution,Essential for Yeast mRNA Splicing,Deduced from 1H-NMR

Abstract

The non-exchangeable ¹H-NMR signals of the branch core trinucleotide of the lariat branch site (A^2′p5′G _3′p5′C), 1) and its derivatives 2 and 3 are completely assigned using one- and two- dimensional NMR techniques including NOE, COSY, NOESY, ¹H-¹HINADEQUATE and 2D-J-resolved spectroscopy. From the vicinal coupling constants in the individual ribose rings, NOE data and T₁ measurements, the following properties of the trimers are deduced.(i)The unique stacking behavior of the trimers is S^1′N _3′N, and the sugar rings exist predominantly in the N-conformation (3′-endo-2′-exo).(ii)The sugar-base orientations appear to be anti.(iii) The branched trimers exist in solution as single-stranded right-handed conformations resembling A-RNA with stacking between the adenine and guanine residues in aqueous solution at 21°C and pH 7.2.(iv) The calculated values for the torsion angles ε^t andγ⁺ for the trimers are 201–203° and 71–86%, respectively, while the percent β¹ values are higher for the guanine (87–92%) than the cytosine residues (73–77%). The computer generated depiction of the triribonucleotide 1 is also shown. These subtle structural features may act as recognition signals for this critical lariat branch site which is essential for the second step in yeast mRNA splicing.     

Substantial decrease in cell wall α-1,3-glucan caused by disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae

Disruption of the kexB encoding a subtilisin-like processing protease in Aspergillus oryzae (ΔkexB) leads to substantial morphological defects when the cells are grown on Czapek-Dox agar plates. We previously found that the disruption of kexB causes a constitutive activation of the cell wall integrity pathway. To understand how the disruption of the kexB affects cell wall organization and components, we analyzed the cell wall of ΔkexB grown on the plates. The results revealed that both total N-acetylglucosamine content, which constitutes chitin, and chitin synthase activities were increased. Whereas total glucose content, which constitutes β-1,3-glucan and α-1,3-glucan, was decreased; this decrease was attributed to a remarkable decrease in α-1,3-glucan. Additionally, the β-1,3-glucan in the alkali-insoluble fraction of the ΔkexB showed a high degree of polymerization. These results suggested that the loss of α-1,3-glucan in the ΔkexB was compensated by increases in the chitin content and the average degree of β-1,3-glucan polymerization.     

Stannylation Approach to the Synthesis of 2′- and 3′-Substituted Analogues of 2′,3′-Didehydro-2′,3′-dideoxynucleosides

Abstract

Three methods are described for the introduction of a tributylstannyl group to the sp²-carbon of 2′,3′-didehydro-2′,3′-dideoxy nucleosides (d44Ns). The resulting stannylated products serve as versatile intermediates for the synthesis of d4Ns having various types of carbon-substituent.     

Overlapping and distinct functions of CstF64 and CstF64τ in mammalian mRNA 3′ processing

mRNA 3′ processing is dynamically regulated spatially and temporally. However, the underlying mechanisms remain poorly understood. CstF64τ is a paralog of the general mRNA 3′ processing factor, CstF64, and has been implicated in mediating testis-specific mRNA alternative polyadenylation (APA). However, the functions of CstF64τ in mRNA 3′ processing have not been systematically investigated. We carried out a comprehensive characterization of CstF64τ and compared its properties to those of CstF64. In contrast to previous reports, we found that both CstF64 and CstF64τ are widely expressed in mammalian tissues, and their protein levels display tissue-specific variations. We further demonstrated that CstF64 and CstF64τ have highly similar RNA-binding specificities both in vitro and in vivo. CstF64 and CstF64τ modulate one another''s expression and play overlapping as well as distinct roles in regulating global APA profiles. Interestingly, protein interactome analyses revealed key differences between CstF64 and CstF64τ, including their interactions with another mRNA 3′ processing factor, symplekin. Together, our study of CstF64 and CstF64τ revealed both functional overlap and specificity of these two important mRNA 3′ processing factors and provided new insights into the regulatory mechanisms of mRNA 3′ processing.     

Increasing βB1-crystallin sensitivity to proteolysis caused by the congenital cataract-microcornea syndrome mutation S129R

Congenital hereditary cataract, which is mainly caused by the deposition of crystallins in light-scattering particles, is one of the leading causes of newborn blindness in human beings. Recently, an autosomal dominant congenital cataract-microcornea syndrome in a Chinese family has been associated with the S129R mutation in βB1-crystallin. To investigate the underlying molecular mechanism, we examined the effect of the mutation on βB1-crystallin structure and thermal stability. Biophysical experiments indicated that the mutation impaired the oligomerization of βB1-crystallin and shifted the dimer–monomer equilibrium to monomer. Molecular dynamic simulations revealed that the mutation altered the hydrogen-bonding network and hydrophobic interactions in the subunit interface of the dimeric protein, which resulted in the opening of the tightly associated interacting sites to allow the infiltration of the solvent molecules into the interface. Despite the disruption of βB1-crystallin assembly, the thermal stability of βB1-crystallin was increased by the mutation accompanied by the reduction of thermal aggregation at high temperatures. Further analysis indicated that the mutation significantly increased the sensitivity of βB1-crystallin to trypsin hydrolysis. The digested fragments of the mutant were prone to aggregate and unable to protect βA3-crystallin against aggregation. These results indicated that the thermal stability-beneficial mutation S129R in βB1-crystallin provided an excellent model for discovering molecular mechanisms apart from solubility and stability. Our results also highlighted that the increased sensitivity of mutated crystallins towards proteases might play a crucial role in the pathogenesis of congenital hereditary cataract and associated syndrome.