Isolation and structural analysis of two lipid A precursors from a KDO deficient mutant of Salmonella typhimurium differing in their hexadecanoic acid content

The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy-d-manno-octulosonic acid region of lipopolysaccharides.Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. Teh precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy-d-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue.The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy-d-manno-octulosonic acid and/or other polar substituents.Abbreviations LDMS laser desorption mass spectrometry - KDO 3-Deoxy-d-manno-octulosonic acid - Ts5 Salmonella typhimurium mutant Ts5 - PCP phenol/chloroform/petroleum ether - H₂F₂ hydrogen fluoride This work is dedicated to Prof. Dr. Drews, Freiburg, on the occasion of his 60th birthday     

Pigment protein complexes and functional properties of tetratype resulting from crosses between CP1 and CP2 less Chlamydomonas mutants

A chlorophyll b-less mutant of Chlamydomonas reinhardtii (Pg ₂₇) was isolated after UV irradiation of the wild type cells. This photosynthetically competent mutant totally lacks chlorophyll b and the CP₂ chlorophyll-protein complex. However, SDS-PAGE, proteolytic digestions and immunodetections demonstrated that the 24–25 Kd apoproteins of the lacking CP₂ complex are still present in thylakoids of the Pg₂₇ mutant. It is concluded that this CP₂-less mutant is affected in the biosynthesis pathway of chlorophyll b.This CP₂-less mutant was crossed with a CP₁-less mutant (Fl₅) Fluorescence emission spectra and fluorescence inductions in the presence of DCMU were analysed in the resulting (cp ₂ ^– , cp ₁ ⁺ ), (cp ₂ ⁺ , cp ₁ ^– ), (cp ₂ ⁺ , cp ₁ ⁺ ), cp ₂ ^– , cp ₁ ^– )tetratype. Differences in PS 2 optical cross section and in the relative amplitude or localisation of fluorescence emission peaks fit well with a quadripartite model where PS1 and PS2 would each correspond to a reaction centre core complex (CP₁ and CP₂ respectively) associated to a light harvesting antenna (LHC1 and LHC2 respectively). The occurrence of energy transfers from PS1 peripheral antenna to PS2 in the Fl ₅ mutant shows that, in absence of CP₁, at least a part of its associated PS1 light harvesting antenna migrates in the PS2 containing appressed thylakoids.Abbreviations Chl Chlorophyll - LHC Light harvesting chl a/b complex - CP₂ Predominant form of LHC or SDS polyacrylamide gels - WT Wild type - DM Double mutant (cp ₁ ^– , cp ₂ ^– ) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - DOC-PAGE Deoxycholate polyacrylamide gel electrophoresis     

Internode length and anatomical changes in Pisum genotypes crys and cryc in response to extended daylength and applied gibberellin A1

Dwarf pea (Pisum sativum L.) plants with genotypes cry^c and cry^s responded differently when an 8 h photoperiod (8 h daylight, 16 h dark) was extended to 24 h (8 h daylight, 16 h incandescent light). Genotype cry^c showed up to a 4-fold increase in internode length, sustained by increases in both cell length (particularly of epidermal cells) and cell number (particularly of cortical cells) while cry^s plants showed up to a 2-fold increase in internode length sustained mostly by an increase in cell number. Under an 8 h (daylight) photoperiod the two genotypes did not differ in their sensitivity to applied gibberellin A₁ (GA₁) and they showed a similar pattern of response. GA₁ significantly increased internode length, cell length and cell number in both genotypes. Incandescent light did not increase the size of the response to GA₁ except for cry^s plants at high dose rates of GA₁ (29–58 nmol). At saturating doses of GA₁ the two genotypes attained a similar peak internode length; incandescent light increased the peak by about 40%. GA₁ increased the rate of leaf appearance by up to 33% while incandescent light reduced the rate by 4–7%. The elongation response of the more mature internodes of cry^c plants to GA₁ or incandescent light was due primarily to an increase in cell length whereas increased cell number made a significant contribution in the case of internodes which were relatively immature at the time the stimulus was applied. The progressive increase in internode length of both genotypes during ontogeny was due primarily to an increase in cell number. In conclusion, alleles cry^c and cry^s (background le La) do not confer a difference in sensitivity to GA₁ and the increase in internode length in response to incandescent light is probably not the result of a real or perceived increase in GA₁ level. Allele cry^s may partially block a phytochrome mediated response to light and the key difference between genotypes cry^s and cry^c may lie in the greater elongation (extensibility?) of cry^c epidermal cells in incandescent light.     

Enhanced N2-fixing ability of a deletion mutant of arctic rhizobia with sainfoin (Onobrychis viciifolia)

Summary Mutagenesis provoked by exposure at elevated temperature of the cold-adapted, arctic Rhizobium strain N31 resulted in the generation of five deletion mutants, which exhibited loss of their smaller plasmid (200 kb), whereas the larger plasmid (> 500 kb) was still present in all mutants. Deletion mutants did not show differences from the wild type in the antibiotic resistance pattern, the carbohydrates and organic acids utilization, and the growth rate at low temperature. However, deletion mutants differed from the wild type and among themselves in the ex planta nitrogenase activity, the nodulation index, and the symbiotic effectiveness. The deletion mutant N31.6rif ^r showed higher nodulation index and exhibited higher nitrogenase activity and symbiotic efficiency than the other deletion mutants and the wild type. The process of deletion mutation resulted in the improvement of an arctic Rhizobium strain having an earlier and higher symbiotic nitrogen fixation efficiency than the wild type.     

Genetic characterization of a double-flowered tobacco plant obtained in a transformation experiment

Summary A leaf-disk transformation experiment was performed with tobacco (Nicotiana tabacum L.) using a binary vector and a strain of Agrobacterium tumefaciens that carried a wild-type Ti-plasmid, pTiBo542. Although the majority of kanamycin-resistant, transgenic plants was morphologically normal, one of the plants was double-flowered and had a slightly wavy stem and leaves whose edges were bent slightly upwards. The abnormal morphology was controlled by a single, dominant Mendelian gene. Young plants that carried this gene were distinguishable from normal plants at the stage of cotyledons. The homozygotes, with respect to this gene, were more seriously deformed than the heterozygotes. DNA segments derived from the binary vector and from the T_L-and T_R-DNA of pTiBo542 were detected in the double-flowered plant, but the T-DNA genes involved in biosynthesis of phytohormones were absent from the plant. The abnormal morphology, the resistance to kanamycin, and the segments of foreign DNA were genetically linked, and the linkage was very tight, at least between the abnormal morphology and the resistance to kanamycin; the meiotic recombination frequency was less than 0.02%, if recombination occurred at all.     

用光谱技术鉴定植物色素突变体

1983年我国报道了从γ-射线处理的“矮杆齐”大麦中得到了一株黄绿色的突变体1832C[1]。本文用光谱技术对该突变体的光合色素成分进行了鉴定。1　材料和方法　　材料为六棱裸大麦“矮杆齐”和由该品种大麦诱变形成的黄绿色突变体1832C(Mb1832C),以及作为对照的缺失Chlb的突变体大麦Chlorina-f2[2]都于3月初播种于实验田中。　　每个样品取30g新鲜的叶片,先用自来水后用蒸馏水冲洗干净。把洗净的叶片摊放在干净的纱布上吸干表面水分,剪碎,加入100mL预冷的含有0.4mol/L山梨醇、0.1mol/LTris-HCl(pH7.6)的缓冲液,用组织捣碎机先慢速捣碎1…     

利用标记基因选配褐壳蛋鸡配套杂交亲本

应用本实验室研制的抗鸡红细胞抗原单价血清(4个位点, 14个等位基因)和DNA指纹技术,对我们组配成功的一个褐壳蛋鸡配套系统的5个亲本进行了群体遗传学分析。结果表明,由标记基因测定所提供的亲本品系遗传差异的大小, 与这些品系实际杂交效果的优劣相一致,证实了标记辅助选种方法有的效性。     

Selection for mutants with low nitrate uptake ability in rice (Oryza sativa)

Screening for mutants deficient in the high affinity system of nitrate uptake was performed using mutagenized M₂ population of rice ( Oryza sativa , cv. Nipponbare or Kinmaze). For selecting mutants, M₂ seedlings were transferred individually to 10 ml solution containing 250 μ M potassium nitrate and 500 μ M calcium sulphate at 20 or 28°C. After 6 or 24 h, nitrate concentration of the solution was determined with a nitrate selective electrode and the seedlings showing impaired nitrate uptake were selected as nitrate uptake deficient variants. Of 74 variants, three were confirmed to be mutants with low nitrate uptake ability in the M₃ generation. Potassium uptake ability also decreased in the mutants. Three mutants were divided into two groups based on the analysis of nitrate reductase (NR, EC 1.6.6.1) activity and chlorate resistance. Two, NUE13 and NUE36 , had a lower level of NR activity than the original cultivar and were not resistant to chlorate, while the seedlings of NUE50 had the same level of NR activity as the original cultivar and were more resistant to chlorate than the original cultivar. All mutants were resistant to cesium, a toxic ion analogue for potassium, suggesting that the decreased levels of both nitrate and potassium uptake were coupled to the change of plasma membrane H⁺-ATPase activity.     

The dosage effect of the wildtype GBSS allele is linear for GBSS activity but not for amylose content: absence of amylose has a distinct influence on the physico-chemical properties of starch

A gene-dosage population was obtained by crossing two genotypes that were duplex for the GBSS allele. Nulliplex, simplex, duplex or triplex/quadruplex plants could be identified by monitoring the segregation of red and blue microspores after staining with iodine. GBSS activity was significantly different for all groups and showed an almost linear dosage effect for the wildtype GBSS gene. A dosage effect was found for amylose content that was not linear. The amylose content was similar for both the duplex and triplex/quadruplex group. Within the simplex group, differences in amylose content were found, which might be due to a different genetic background. There was no linear correlation between GBSS activity and amylose content. A certain level of GBSS activity led to a maximum amount of amylose, and further increase in GBSS activity did not result in a further increase in amylose content. The presence of one or more wildtype GBSS allele(s), and therefore the presence of amylose in the starch granules, had a great influence on the physico-chemical properties of the starch suspensions.     

丝孢酵母高甲硫氨酸突变株的选育及营养调控

以丝孢酵母(Trichosporon Behr)ST851为原始菌株,经紫外线诱变,在含乙硫氨酸的双层平板上筛选到多株抗乙硫氨酸突变株。其中ST851-10株抗乙硫氨酸浓度达到350μg/ml,其菌体蛋白质含量由40.5％提高到44.3％,菌体甲硫氨酸含量由20.45mg/g-DCW增加到29.32mg/g-DCW。在以苹果渣为碳源、尿素为氮源、硫酸镁作硫源的最适培养条件下,固态发酵24h后,蛋白质和甲硫氨酸含量较原始菌株分别提高了15.8％和44.9％。培养基中C/N值低有利于甲硫氨酸的合成,C/N值高则适合于菌体生长。在苹果渣固态发酵过程中,适当补加氮源既有利于菌体生长和甲硫氨酸的合成,又可起到调节培养基pH值的作用。