Increased reliability of selective PCR by using additionally mutated primers and a commercialTaq DNA polymerase enhancer

A reliable selective PCR procedure that combines the use of additionally mutated primers with the specificity-enhancing properties of a commercial preparation (Perfect Match, Stratagene) is described. The human immunodeficiency virus type 1pol gene point mutations known to confer in vitro resistance to azidothymidine were examined as a model for optimization of the assay. The usual strategy of deliberately introducing an additional mismatch 1 residue from the 3′ end in the wild-type and mutant primers did not allow reproducible discrimination between wild-type and mutant target sequences. Addition of minimal amounts of Perfect Match to the same PCR mixtures resulted in a significantly enlarged range of selective annealing temperatures, providing a valuable and cost-effective means for reliable detection of known mutations by selectivePCR.     

Contrasting rates of nucleotide substitution in the X-Linked and Y-Linked zinc finger genes

We have sequenced the entire exon (1,180 bp) encoding the zinc finger domain of the X-linked and Y-linked zinc finger genes (ZFX and ZFY, respectively) in the orangutan, the baboon, the squirrel monkey, and the rat; a total of 9,442 by were sequenced. The ratio of the rates of synonymous substitution in the ZFY and ZFX genes is estimated to be 2.1 in primates. This is close to the ratio of 2.3 estimated from primate ZFY and ZFX intron sequences and supports the view that the male-to-female ratio of mutation rate in humans is considerably higher than 1 but not extremely large. The ratio of synonymous substitution rates in ZFY and ZFX is estimated to be 1.3 in the rat lineage but 4.2 in the mouse lineage. The former is close to the estimate (1.4) from introns. The much higher ratio in the mouse lineage (not statistically significant) might have arisen from relaxation of selective constraints. The synonymous divergence between mouse and rat ZFX is considerably lower than that between mouse and rat autosomal genes, agreeing with previous observations and providing some evidence for stronger selective constraints on synonymous changes in X-linked genes than in autosomal genes. At the protein level ZFX has been highly conserved in all placental mammals studied while ZFY has been well conserved in primates and foxes but has evolved rapidly in mice and rats, possibly due to relaxation of functional constraints as a result of the development of X-inactivation of ZFX in rodents. The long persistence of the ZFY-ZFX gene pair in mammals provides some insight into the process of degeneration of Y-linked genes.Correspondence to: W.-H. Li     

Drosophila melanogaster acetylcholinesterase: Identification and expression of two mutations responsible for cold- and heat-sensitive phenotypes

Ace ^IJ29 and Ac ^IJ40 are cold- and heat-sensitive variants of the gene coding for acetylcholinesterase in Drosophila melanogaster. In the homozygous condition, these mutations are lethal when animals are raised at restrictive temperatures, i.e., below 23° C for Ace ^IJ29 or above 25° C for Ace ^IJ40. The coding regions of the gene in these mutants were sequenced and mutations changing Ser³⁷⁴ to Phe in Ace ^IJ29 and Pro⁷⁵ to Leu in Ace ^IJ40 were found. Acetylcholinesterases bearing these mutations were expressed in Xenopus oocytes and we found that these mutations decrease the secretion rate of the protein most probably by affecting its folding. This phenomenon is exacerbated at restrictive temperatures decreasing the amount of secreted acetylcholinesterase below the lethality threshold. In parallel, the substitution of the conserved Asp²⁴⁸ by an Asn residue completely inhibits the activity of the enzyme and its secretion, preventing the correct folding of the protein in a non-conditional manner.     

Anatomy of the stimulative sequences flanking the ARS consensus sequence of chromosome VI in Saccharomyces cerevisiae

We have analyzed the relationship between autonomously replicating sequence (ARS) structure and function for three ARS (ARS605, ARS607 and ARS609) from chromosome VI of Saccharomyces cerevisiae by systematic XhoI-linker mutation in the ARS consensus sequence (ACS) and flanking sequences. All mutations that encroached upon the ACS destroyed ARS activity. DNA sequences stimulative for ARS function were identified on either side of the ACS of ARS605 and only on the 3'-side of the ACS of ARS607. In ARS609, however, no such stimulative sequences were observed. Base substitutions complementary to the wild-type sequence of those stimulative regions, in ARS605 and ARS607, that did not change the AG of unwinding nor affected ARS activity suggests that these regions have, at least, a function as DNA-unwinding elements (DUE). ARS605, ARS607 and ARS609 DNA are of low AG value and showed hypersensitivity to single-strand-specific nuclease when inserted in negatively supercoiled plasmid. Linker mutations inhibitory for ARS activity (5L11 and 7L14) also caused significant changes in local nucleotide (nt) sensitivity within the ACS and its adjoining regions. Complementary base substitutions, however, did not affect these changes in local nt sensitivity. These results imply that the stimulative regions flanking the ACS are necessary to produce an optimum conformation around the ACS which may be important for full ARS activity.     

非同位素PCR－单链构象多态性技术的建立和应用

ＰＣＲ－单链构象多态性技术问世以来,成为研究基因突变的工具,特别是在分子肿瘤学研究中,广泛应用于癌基因,抑癌基因突变的研究,常规ＰＣＲ－ＳＳＣＰ采用同位素标记ＰＣＲ产物,测序板电泳分离突变,在操作和费用上有种种局限,文章建立了一种非同位素ＰＣＲ－ＳＳＣＰ技术;通过不对称ＰＣＲ获得单链,普通ＰＡＧＥ分离,经银染检出突变,用这种方法,还研究了四株鼻咽癌细胞株ＣＮＥ１,ＣＮＥ２,ＨＫ１和ＳＵＮＥ１中肿瘤     

A gene, SMP2, involved in plasmid maintenance and respiration in Saccharomyces cerevisiae encodes a highly charged protein

Summary The smp2 mutant of Saccharomyces cerevisiae shows increased stability of the heterologous plasmid pSR1 and YRp plasmids. A DNA fragment bearing the SMP2 gene was cloned by its ability to complement the slow growth of the smp2 smp3 double mutant (smp3 is another mutation conferring increased stability of plasmid pSR1). The nucleotide sequence of SMP2 indicated that it encodes a highly charged 95 kDa protein. Disruption of the genomic SMP2 gene resulted in a respiration-deficient phenotype, although the cells retained mitochondrial DNA, and showed increased stability of pSR1 like the original smp2 mutant. The fact that the smp2 mutant is not always respiration deficient and shows increased pSR1 stability even in a rho ⁰ strain lacking mitochondrial DNA suggested that the function of the Smp2 protein in plasmid maintenance is independent of respiration. The SMP2 locus was mapped at a site 71 cM from lys7 and 21 cM from ilv2/SMR1 on the right arm of chromosome XIII.     

Conformational properties of the —35 region of the trp promoter in solution: comparison of the wild-type sequence with an AT transversion

The majority of the ¹H NMR resonances of the protons in a tetradecamer containing the —35 region of the trp promoter d(GCTGTTGACAATTA): d(TAATTGTCAACAGC) and in the TA transversion have been assigned. The conformational properties of the nucleotides have been determined and compared in the two duplexes. Analysis of spin-spin coupling and NOES shows that all sugar puckers are in the south domain (i.e. near C2 endo) and the glycosidic torsion angles are anti (110°). The NMR data are consistent with the duplex being in the B family of conformations. Significant differences in chemical shifts between the two molecules were observed only for nearest neighbours to the transversion site, suggesting the absence of long range conformational effects. This was confirmed by the similarity of coupling constants and NOEs. Other properties are also not greatly affected at positions more than two base pairs from the mutation site. These results are consistent with the hypothesis that unconstrained oligonucleotides are highly flexible, and can readily accommodate significant perturbations of the local structure, such as a transversion.     

Isolation of a new paramutagenic allele of thesulfurea locus in the tomato cultivar Moneymaker following in vitro culture

A new allele, SC148, of thesulfurea locus inLycopersicon esculentum was detected in a line derived after repeated selfing of plants that had been regenerated from tissue culture. Like the originalsulf mutant, SC148 displayed two mutant phenotypes: green-yellow speckled plants in which thesulf ^vag allele is present and pure yellow plants homozygous for thesulf ^tpura allele. Although the mutant alleles are recessive to wild-type, an unpredictable number of variegated and pura plants appeared in F₁ progenies that had been derived from crosses between SC148 and wild-type tomato plants. The presence of the wild-typesulf ⁺ allele in these variegated heterozygotes was demonstrated using a cytological marker that is linked tosulf. It is concluded that the mutantsulf allele of SC148, imposes its variegated expression state on the wild-typesulf ⁺ allele present insulf ⁺/sulf^vag heterozygotes. This behaviour, known as paramutation, has also been described for the originalsulf allele. The SC148 allele, however, seems to induce changes at an earlier stage in development. The analogy of this paramutagenic system to dominant position effect variegation inDrosophila is discussed.     

Melanocytes Fail to Survive in Hair Bulbs of the Shiba Goat (Capra hircus) With the Dominant Black-Eyed White Phenotype

Dominant black-eyed white phenotypes are one of the most commonly observed traits in domestic animals. Their genetic control mechanisms, however, have not been elucidated. As the first step to approach the problem, we examined histologically the patterns of the distribution of pigment cells in Shiba goats (two each of day-73-postcoitum and day-112-postcoitum fetuses, and a 15-week-old kid) with the dominant black-eyed white phenotype. Melanocytes were present and fully pigmented in the choroid and the sclera of eyes, as well as in dorsal skin epidermis of the fetuses and of the kid. Melanocytes were also found in approximately 6% of the hair bulbs in the fetal dorsal skin, while the rest (94%) lacked them. Hair follicles of the kid did not harbor melanocytes except for some in the early anagen stage. The results suggest that the survival of melanocytes was inhibited specifically in the hair follicles of the Shiba goat with the dominant black-eyed white phenotype and that the ostensibly similar phenotypes in the Shiba goat and in the SI or W mutants of the mouse, where melanocytes die en route to the hair bulbs, are regulated by different mechanisms.     

Allosterism and Na++-d-glucose cotransport kinetics in rabbit jejunal vesicles: Compatibility with mixed positive and negative cooperativities in a homo- dimeric or tetrameric structure and experimental evidence for only one transport protein involved

Summary We first present two simple dimeric models of cotransport that may account for all of the kinetics of Na⁺⁺-d-glucose cotransport published so far in the small intestine. Both the sigmoidicity in the Na⁺⁺ activation of transport (positive cooperativity) and the upward deviations from linearity in the Eadie-Hofstee plots relative to glucose concentrations (negative cooperativity) can be rationalized within the concept of allosteric kinetic mechanisms corresponding to either of two models involving sequential or mixed concerted and sequential conformational changes. Such models also allow for 2 Na⁺⁺ 1 S and 1 Na⁺⁺ 1 S stoichiometries of cotransport at low and high substrate concentrations, respectively, and for partial inhibition by inhibitors or substrate analogues. Moreover, it is shown that the dimeric models may present physiological advantages over the seemingly admitted hypothesis of two different cotransporters in that tissue. We next address the reevaluation of Na⁺⁺-d-glucose cotransport kinetics in rabbit intestinal brush border membrane vesicles using stable membrane preparations, a dynamic approach with the Fast Sampling Rapid Filtration Apparatus (FSRFA), and both nonlinear regression and statistical analyses. Under different conditions of temperatures, Na⁺⁺ concentrations, and membrane potentials clamped using two different techniques, we demonstrate that our data can be fully accounted for by the presence of only one carrier in rabbit jejunal brush border membranes since transport kinetics relative to glucose concentrations satisfy simple Michaelis-Menten kinetics. Although supporting a monomeric structure of the cotransporter, such a conclusion would conflict with previous kinetic data and more recent studies implying a polymeric structure of the carrier protein. We thus consider a number of alternatives trying to reconcile the observation of Michaelis-Menten kinetics with allosteric mechanisms of cotransport associated with both positive and negative cooperativities for Na⁺⁺ and glucose binding, respectively. Such models, implying energy storage and release steps through conformational changes associated with ligand binding to an allosteric protein, provide a rational hypothesis to understand the long-time debated question of energy transduction from the Na⁺⁺ electrochemical gradient to the transporter.This research was supported by grant MT-7607 from the Medical Research Council of Canada. One of the authors (A.B.) was supported by a scholarship from the Fonds de la Recherche en Santé du Québec and C. C. was supported by a fellowship from the GRTM. The technical assistance of Mrs. C. Leroy has been greatly appreciated. The authors also thank D.D. Maenz and C. Malo for insightful discussions and C. Gauthier for the art work.