Generation of heteroplastidic Nicotiana cybrids by protoplast fusion: analysis for plastid recombinant types

Summary Protoplasts of a mutant line of Nicotiana tabacum having a maternally-transmitted chlorophyll deficiency were fused with protoplasts of two alloplasmic-male-sterile Nicotiana lines by the donor-recipient technique. In both fusion experiments variegated plantlets were regenerated which were shown to contain cytoplasms of mixed chloroplast nature. This confirms that with the donor-recipient method one can obtain mixed cytoplasms of genetically different chloroplasts. We present a convenient system to assay for genetic recombination between chloroplasts by combining use of several cytoplasmic markers: vis. chlorophyll pigmentation, chloroplast DNA restriction patterns, tentoxin resistance and male sterility. Within the limits of the experiment no recombinant types were recovered.     

Proximo-distal pattern regulation in deficient avian limb buds

Summary Deficient limb buds composed of prospective stylopod and autopod are able to regulate the missing intercalary zeugopod, the origin of which was investigated by heterospecific quail/chick recombinants. The associations of quail prospective autopod and chick prospective stylopod failed to regulate. The reverse combination of chick prospective autopod grafted onto a quail prospective stylopod gave rise to a three-segmented limb. In 13 out of 16 cases the regulated zeugopod was made up of both chick and quail cells. Chick cells were located predominantly along the postaxial half of the zeugopod, while the quail cells made up most of its preaxial half. In two cases, the intercalary zeugopod consisted exclusively of chick cells originating from the tip and in one case of quail cells originating from the base.These results demonstrate that during the regulative processes, the prospective values of some of the original stylopodial and autopodial cells have been shifted along the proximo-distal axis, towards the expression of more distal as well as of more proximal structures.Heteropolar stylo-autopodial or zeugo-autopodial recombinants, in which the proximo-distal axis of the base was reversed with respect to that of the tip, were unable to regulate the pattern defects and thus revealed the importance of concordant p-d polarity for regulative processes to take place between abutted tissues.     

Pilot scale production of a recombinant human epidermal growth factor, secreted by Bacillus brevis, using expanded bed adsorption

Recombinant Bacillus brevis which carried an expression plasmid encoding the human epidermal growth factor (EGF) gene on a cryptic high-copy number plasmid, pHT926, extracellularly produced EGF in its biologically active form at a concentration of over 1.5 g L⁻¹ in the culture broth in a 30-L jar fermenter. The culture broth also contained some other EGF compounds, which mainly consisted of oligomeric and polymeric forms with disulfide bonds. We developed a simple purification method for EGF, without prior cell removal from the culture broth, comprising cation exchange expanded bed adsorption followed by ultrafiltration with UF 10 000 and 3000 membranes. The EGF compounds were efficiently separated from the EGF in its native form in the expanded bed adsorption step. With this purification method, only EGF in its native form was recovered from the culture broth, with a yield of nearly 80%, and 90% purity. This efficient and economic system has made it possible to use EGF as a pharmaceutical in the livestock industry. Received 10 June 1998/ Accepted in revised form 10 September 1998     

Analysis of organelle genomes in a somatic hybrid derived from cytoplasmic male-sterile Brassica oleracea and atrazine-resistant B. campestris

Summary An atrazine-resistant, male-fertile Brassica napus plant was synthesized by fusion of protoplasts from the diploid species B. oleracea and B. campestris. Leaf protoplasts from B. oleracea var. italica carrying the Ogura male-sterile cytoplasm derived from Raphanus sativus were fused with etiolated hypocotyl protoplasts of atrazine-resistant B. campestris. The selection procedure was based on the inability of B. campestris protoplasts to regenerate in the media used, and the reduction of light-induced growth of B. oleracea tissue by atrazine. A somatic hybrid plant that differed in morphology from both B. oleracea and B. campestris was regenerated on medium containing 50 M atrazine. Its chromosome number was 36–38, approximately that of B. napus. Furthermore, nuclear ribosomal DNA from this hybrid was a mixture of both parental rDNAs. Southern blot analyses of chloroplast DNA and an assay involving tetrazolium blue indicated that the hybrid contained atrazine-resistant B. campestris chloroplasts. The hybrid's mitochondrial genome was recombinant, containing fragments unique to each parent, as well as novel fragments carrying putative crossover points. Although the plant was female-sterile, it was successfully used to pollinate B. napus.     

Isolation and characterization of wheat-rye recombinants involving chromosome arm 1DS of wheat

Summary The introgression of genetic material from alien species is assuming increased importance in wheat breeding programs. One example is the translocation of the short arm of rye chromosome 1 (1RS) onto homoeologous wheat chromosomes, which confers disease resistance and increased yield on wheat. However, this translocation is also associated with dough quality defects. To break the linkage between the desirable agronomic traits and poor dough quality, recombination has been induced between 1RS and the homoeologous wheat arm IDS. Seven new recombinants were isolated, with five being similar to those reported earlier and two havina new type of structure. All available recombinantsw ere characterized with DNA probes for the loci Nor-R1, 5SDna-R1, and Tel-R1. Also, the amount of rye chromatin present was quantified with a dispersed rye-specific repetitive DNA sequence in quantitative dot blots. Furthermore, the wheat-rye recombinants were used as a mapping tool to assign two RFLP markers to specific regions on chromosome arms 1DS and 1RS of wheat and rye, respectively.     

The effect of antibiotic treatment on the in vivo selection of resistant haemolytic Escherichia coli clones in mice

Abstract The appearance of resistant haemolytic Escherichia coli clones was studied in mice treated with a synergistic combination of antibiotics. The R plasmid donor and the haemolytic recipient were given orally. Ten animals were treated i.p. with 2.5 mg carbenicillin and 2.5 mg kanamycin pro die for 7 days. Ten control mice were injected with saline. Out of 80 faecal samples of animals receiving antibiotic treatment resistant haemolytic transconjugants were isolated from 13 samples by direct plating, and from 27 samples after selective enrichment. Out of 80 parallel samples of control animals a single one yielded a positive culture — after enrichment.     

构建稳定表达RFP及嘌呤霉素抗性的K562细胞株

目的构建稳定表达红色荧光蛋白（red fluorescent protein,RFP）和嘌呤霉素（puromycin）抗性的K562．PM．RFP细胞株,便于慢性粒细胞性白血病研究中K562细胞的观察和筛选。方法采用PCR法获得RFP片段,将其插入到慢病毒pGC-FU-3FLAG-IRES—Puromycin载体中获得pGC—PM—RFP重组质粒,经脂质体转染到293T细胞中获得慢病毒LV—PM—RFP,有限稀释法检测慢病毒在293T细胞中的转染效率,用包装获得的慢病毒感染K562细胞,经嘌呤霉素筛选获得RFP阳性的K562-PM—RFP细胞株。结果PCR及测序结果证实目的基因RFP正确克隆至慢病毒质粒中,经慢病毒LV—PM-RFP感染的K562细胞能在嘌呤霉素抗性培养基中存活,并稳定表达RFP。结论成功构建了慢病毒重组质粒pGC—PM-RFP,并获得了携带RFP及嘌呤霉素抗性基因的K562-PM—RFP细胞株。     

一种碱裂解菌液直接电泳快速筛选重组子的方法

目的：从碱裂解法提取质粒DNA的原理．经过实验摸索获得一种快速、经济、结果可靠的菌液直接碱裂解电泳筛选重组子的方法。方法：不需提取质粒DNA．只需将细菌培养液碱裂解后直接进行普通琼脂糖凝胶电泳分析,就可以快速筛选出转化重组子。结果：结果和提取质粒酶切鉴定鉴定结果一致。结论：经实验证明菌液直接碱裂解电泳筛选重组子是一种快速、经济、可靠的方法。     

Susceptibility of Oncomelania hupensis formosana recombinants and hybrids with Oncomelania hupensis nosophora to infection with Schistosoma japonicum

Chiu J.-K., Ong S.-J., Yu J.-C., Kao C.-Y. and Iuima T. 1981. Susceptibility of Oncomelania hupensis formosana recombinants and hybrids with Oncomelania hupensis nosophora to infection with Schistosoma japonicum. International Journal for Parasitology11: 391–397. Three generations of Oncomelania hupensis formosana recombinants were produced by mating the Kaohsiung race with the Ilan and Changhua races of the snails. F₂ and F₃ recombinants were produced by back-crossing F₁ and F₂ with Kaohsiung O. h. formosana. Subsequently, susceptibility of recombinants to infection with the original strain of Schistosoma japonicum, Ilan or Changhua strain, was investigated. Results indicated that susceptibility of recombinants declined steadily generation by generation. Marked decline of infectivity was observed for Kaohsiung-Ilan recombinants as compared with Kaohsiung-Changhua recombinants. For example, the overall infection rate of Kaohsiung-Ilan F₁ recombinants was 7.3 % with a 51.4 % of control snails. The same figures for F₂ were 4.2 and 52.6%, and 1 and 40.3 % for F₃. On the other hand, the overall infection rate of Kaohsiung-Changhua F₁ recombinants was 21.9% with a 46.9% of control snails; and 11.9 and 50.3 % for F₂; and 7.6 and 33.2% for F₃. The F₃ hybrids of Oncomelania hupensis nosophora from Japan and O. h. formosana from Kaohsiung and Ilan were also produced, and susceptibility with the Japanese strain of S. japonicum was studied. A highly significant decline of susceptibility was observed among hybrids (4.4%) in contrast with control snails (85.6 %).Feasibility of applying O. h. formosana in biological control of S. japonicum was discussed. One must determine in the laboratory, prior to application, which race of O. h. formosana should be used depending on the Oncomelania snails of an endemic area. For S. japonicum prevalent in Yamanashi, Japan, the Ilan race of O. h. formosana was found to be better choice than the Kaohsiung race of the snails.     

Phytotron experiments in Pisum

Summary The flowering behaviour of 17 Pisum mutants and 20 recombinants was studied under three different temperatures using long-day phytotron conditions. A constant low temperature of 12.5 ° C led to a strong delay in flowering in all the genotypes tested but distinct relative differences could be found between them. Relative differences were also present with regard to speed of ontogenetic development under a permanent high temperature of 25.5 °C or under an alternating change between low and high temperature. Under the low temperature, recombinants R 20D and R 20E, carrying gene efr for earliness, entered the flowering period more than 4 weeks later than the donor of efr, demonstrating thereby a negative influence of one of the other mutant genes on efr. The high temperature of 25 °C influenced the flowering behaviour of 4 fasciated genotypes negatively — in contrast to the other strains studied. The plants of recombinant R 405 produced only tiny flower buds under these conditions. None of the plants of recombinant R 142F flowered under either the constant low or high temperature — they need the change of low and higher temperature for normal flower formation. The experiments show that most of the genotypes tested react specifically to the three temperature conditions offered to them.