A rapid assay for chloroplast-encoded triazine resistance in higher plants

We have designed a simple and rapid assay for chloroplast-based triazine resistance in higher plants using PCR amplification of thepsbA gene coupled toMaeI digestion of the amplified product to distinguish triazine resistant from sensitive biotypes. Our assay is universal and avoids the need of lengthy procedures of previously published assays, which either required spraying of seedlings in a controlled environment, quantification of chlorophyll fluorescence of leaf discs after incubation in triazine solution, DNA sequencing of thepsbA gene, or Southern-blot analysis. Our diagnostic system is qualitative, reliable, fast and simple. More than 100 seedlings taken directly from the field can be analyzed in one day. This system has a direct application towards a more rational use of herbicides in production fields. It also represents a valuable tool to monitor spreading of resistant biotypes through time and space and can serve as a model system applicable to other gene monitoring needs.     

东方粉蝶幼虫马氏管的超微构造分化

东方粉蝶幼虫有6条马氏管,每条管可以简易地分为四个部分:直肠导,迴肠纲,黄色段和白色段,所有这四个部分首要细胞的顶面都折叠形成微绒毛(特别是迴肠纲,其顶面进一步折叠形成微道),线粒体几乎延伸到微绒毛顶端。在基部,大量基膜折叠形成细胞内管道,向细胞顶部延伸。在细胞质中,有许多线粒体,糙面内质纲和空泡。每一个细胞的细胞核中有一些散的染色质物质,并且外形呈不规则形。在黄色段首要细胞中,有大量证明是由矿物质沉积形成的电子密集颗粒,及猜疑为微孢子原虫的细胞质囊球。上述每段可能具有的功能会在这报告中讨论。     

Differential scanning calorimetric study of the thermal unfolding of myosin rod,light meromyosin,and subfragment 2

The thermal unfolding of myosin rod, light meromyosin (LMM), and myosin subfragment 2 (S-2) was studied by differential scanning calorimetry (DSC) over the pH range of 6.5–9.0 in 0.5M KCl and either 0.20M sodium phosphate or 0.15M sodium pyrophosphate. Two rod samples were examined: one was purified by Sephadex G-200 without prior denaturation (native rod), and the other was purified by a cycle of denaturation-renaturation followed by Sephacryl S-200 chromatography (renatured rod). There were clearly distinguishable differences in the calorimetric behavior of these two samples. At pH 7.0 in phosphate the DSC curves of native rod were deconvoluted into six endothermic two-state transitions with melting temperatures in the range of 46–67°C and a total enthalpy of 4346 kJ/mol. Under identical conditions the melting profile of LMM was resolved into five endothermic peaks with transition temperatures in the range of 45–66°C, and the thermal profile of long S-2 was resolved into two endotherms, 46 and 57°C. Transition 4 observed with native rod was present in the deconvoluted DSC curve for long S-2, but absent in the DSC curve for LMM. This transition was identified with the high-temperature transition detected with long S-2 and attributed to the melting of the coiled-coil α-helical segment of subfragment 2 (short S-2). The low-temperature transition of long S-2 was attributed to the unfolding of the hinge region. The smallest transition temperatures observed for all three fragments were 45–46°C. It is suggested that the most unstable domain in rod (domain 1) responsible for the 46°C transition includes both the hinge region, which is the C-terminal segment of long S-2, and a short N-terminal segment of LMM. This domain, accounting for 21% of the rod structure, contains the S-2/LMM junction, and upon proteolytic cleavage yields the C-terminal and N-terminal ends of long S-2 and LMM, respectively. Over the pH range of 6.5–7.5, the observed specific heat of denaturation of rod was approximately equal to the sum of the specific heats of LMM and S-2. This finding provides an additional argument for the existence of independent domains in myosin rod.     

Insertional inactivation of a chromosomal locus that modulates expression of potential virulence determinants in Staphylococcus aureus.

A single insertion of transposon Tn551 into a unique chromosomal locus of Staphylococcus aureus ISP479C has resulted in a pleiotropic effect on the expression of both extracellular and cell wall proteins. In particular, the expression of cell wall protein A and clumping activity with fibrinogen were rendered undetectable in the mutant 1E3 compared with the parent. The secretion of alpha-hemolysin in mutant 1E3 was modestly increased. Southern blot and phenotypic analyses indicated that this locus is distinct from agr, xpr, and sar, three previously described global regulatory loci. Transduction experiments demonstrated that the genotype associated with mutant 1E3 could be transferred back into the parental strain ISP479C. The transductant 1E3-2 displayed a phenotypic profile similar to that of the original mutant. Northern (RNA) blot studies showed that this locus may be involved in modulating target genes at the mRNA level. In the rabbit endocarditis model, there was a significant decrease in both the infectivity rate and intravegetation bacterial density with mutant 1E3 compared with the parent at an inoculum of 10(3) CFU. Since protein A and the fibrinogen-binding protein(s) are major surface proteins that may mediate bacterial adhesion to host tissues, this locus may be an important genetic element involved in the expression of virulence determinants in S. aureus.     

Nonreductive release of O-linked oligosaccharides from mucin glycoproteins for structure/function assignments as neoglycolipids: application in the detection of novel ligands for E-selectin

The neoglycolipid technology comprises several microproceduresinvolving the generation of lipid-linked oligosaccharide probesfor carbohydrate recognition studies in conjunction with oligosaccharidesequence determination by mass spectrometry. Although applicableto any desired oilgosaccharides, procedures are greatly facilitatedif the ohgosaccharides are nonreduced, as conjugation is byreductive amination of a reducing end aldehyde to a phosphatidylethanolamine.Using bovine submaxillary mucin as a model for release of O-glycansin the reducing state, and based on yields of neoglycolipidsand side-products from "peeling" reactions and degradation,aqueous ethylamine 70% w/v at 22°C for 48 h has been selectedin preference to other conditions, triethylainine, sodium hydroxide,and bydrazine. The integrity of the main acidic and neutraloligosaccharides released under these conditions, di- to octasaccharides,was established by analyses of free oligosaccharides by liquidsecondary ion mass spectrometry (LSIMS) and of the derived neoglycolipidsby TLCLSIMS; the repertoire compared favorably with that ofthe oligosaccharide alditols generated by conventional reductivealkaline borohydride treatment. More forcing conditions of ethylamine70% w/v at 65°C for 6 h were required to release oligosaccharidesfrom porcine gastric mucin; di- to nonasaccharides were obtainedof which about one-third had an intact core GalNAc. Relativeto yields after reductive alkaline hydrolysis, the overall yieldsfor these two glycoproteins were 20% and 40–50% for acidicand neutral oligosaccharides, respectively. Among O-glycansreleased from an ovarian cystadenoma glycoprotein using ethylamine,three variants of the sulfated Le^a/x sequences were identifiedas ligands for the endothelial adhesion molecule E-selectin,one of which is based on the unusual backbone Gal-3/4GlcNAc-3Gal-3Gal. mucins O-linked oligosaccharides TLC-LSIMS neoglycolipids E-selectin     

Homopolymer mutational hot spots mediate herpes simplex virus resistance to acyclovir.

In the majority of cases, the mechanism underlying the resistance to acyclovir (ACV) of herpes simplex viruses (HSVs) is thymidine kinase (TK) deficiency. Plaque isolates from eight ACV-resistant (ACVr) clinical isolates from AIDS patients, of which five reactivated, were sequenced to determine the genetic lesion within the tk gene conferring resistance and whether this may have correlated with reactivation potential. Mutations were clustered within two homopolymer nucleotide stretches. Three plaque isolates (1737-14, 90-150-3, and 89-650-5) had insertion mutations within a stretch of 7 guanosines, while two isolates (89-063-1 and 89-353-1) had frameshift mutations within a stretch of 6 cytosines (a deletion and an insertion, respectively). Mutations resulted in premature termination codons, and the predicted 28- and 32-kDa truncated TK products were detected by Western blot analysis of virus-infected cell extracts. The repair of one homopolymer frameshift mutation (in isolate 1737-14) restored TK activity, demonstrating that this mutation is the basis of TK deficiency. Of the five reactivated isolates, four were TK deficient and contained frameshift mutations while the fifth retained TK activity because of its altered-TK or Pol- phenotype. These data demonstrate that the majority of ACVr clinical isolates contain frameshift mutations within two long homopolymer nucleotide stretches which function as hot spots within the HSV tk gene and produce nonfunctional, truncated TK proteins.     

Nucleotide sequence of the VP1 gene of the foot-and-mouth disease virus strain A Venceslau

The VP1 coat protein of FMDV strain A Venceslau (Aven) consists of 213 amino acid residues. Serum neutralization tests demonstrated that strain Aven is closely related to strain A Argentina/79 (A₇₉) but significantly different from strain A₂₄ Cruzeiro (A₂₄). There is a strong correlation between the amino acid sequences and the serological data. Nucleotide and amino acid sequence analyses of VP1 showed that serologically related viruses (Aven and A₇₉) differ less in this region of the genome than those of serologically distinct viruses (Aven vs. A₂₄). The most significant variation between Aven and A₂₄ occurs at amino acid positions 43 to 46, in which all four residues are different.     

Persistence of Common Alleles in Two Related Populations or Species

Mathematical studies are conducted on three problems that arise in molecular population genetics. (1) The time required for a particular allele to become extinct in a population under the effects of mutation, selection, and random genetic drift is studied. In the absence of selection, the mean extinction time of an allele with an initial frequency close to 1 is of the order of the reciprocal of the mutation rate when 4Nv << 1, where N is the effective population size and v is the mutation rate per generation. Advantageous mutations reduce the extinction time considerably, whereas deleterious mutations increase it tremendously even if the effect on fitness is very slight. (2) Mathematical formulae are derived for the distribution and the moments of extinction time of a particular allele from one or both of two related populations or species under the assumption of no selection. When 4Nv << 1, the mean extinction time is about half that for a single population, if the two populations are descended from a common original stock. (3) The expected number as well as the proportion of common neutral alleles shared by two related species at the tth generation after their separation are studied. It is shown that if 4Nv is small, the two species are expected to share a high proportion of common alleles even 4N generations after separation. In addition to the above mathematical studies, the implications of our results for the common alleles at protein loci in related Drosophila species and for the degeneration of unused characters in cave animals are discussed.