Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

Atrazine-resistant cauliflower obtained by somatic hybridization between Brassica oleracea and ATR-B. napus

Summary Somatic hybridization between Brassica oleracea ssp. botrytis (cauliflower, 2n=18), carrying the Ogura (R1) male-sterile cytoplasm and B. napus (2n= 38), carrying a male-fertile, atrazine-resistant (ATR) cytoplasm, yielded three hybrids (2n=56) and six cauliflower cybrids (2n=18), which were selected for resistance to the herbicide in vitro. The hybrids and cybrids were male fertile and self-compatible. They contained both chloroplasts and mitochondria from the ATR cytoplasm. We found no evidence for mtDNA recombination in any of the regenerated plants. Selfed progeny of the B. oleracea atrazine-resistant cybrids were evaluated for tolerance to the herbicide in the field. Resistant plants exposed to 0.56–4.48 kg/ha (0.5–4.0 pounds/acre) atrazine in the soil showed no damage at any herbicide level, whereas plants of a susceptible alloplasmic line were severely damaged at the lowest level of herbicide application and killed at all higher levels. These atrazine-resistant cauliflower may have potential horticultural use, especially in fields where atrazine carry over is a serious problem.     

Synthesis of male sterile,triazine-resistant Brassica napus by somatic hybridization between cytoplasmic male sterile B. oleracea and atrazine-resistant B. campestris

Summary Fusion of leaf protoplasts from an inbred line of Brassica oleracea ssp. botrytis (cauliflower, n=9) carrying the Ogura (R1) male sterile cytoplasm with hypocotyl protoplasts of B. campestris ssp. oleifera (cv Candle, n=10) carrying an atrazine-resistant (ATR) cytoplasm resulted in the production of synthetic B. napus (n=19). Thirty-four somatic hybrids were produced; they were characterized for morphology, phosphoglucose isomerase isoenzymes, ribosomal DNA hybridization patterns, chromosome numbers, and organelle composition. All somatic hybrids carried atrazine-resistant chloroplasts derived from B. campestris. The mitochondrial genomes in 19 hybrids were examined by restriction endonuclease and Southern blot analyses. Twelve of the 19 hybrids contained mitochondria showing novel DNA restriction patterns; of these 12 hybrids, 5 were male sterile and 7 were male fertile. The remaining hybrids contained mitochondrial DNA that was identical to that of the ATR parent and all were male fertile.     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.     

Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria

Lactococci are fastidious bacteria which require an external source of amino acids and many other nutrients. These compounds have to pass the membrane. However, detailed analysis of transport processes in membrane vesicles has been hampered by the lack of a suitable protonmotive force (pmf)-generating system in these model systems. A membrane-fusion procedure has been developed by which pmf-generating systems can be functionally incorporated into the bacterial membrane. This improved model system has been used to analyze the properties of amino acid transport systems in lactococci. Detailed studies have been made of the specificity and kinetics of amino acid transport and also of the interaction of the transport systems with their lipid environment. The properties of a pmf-independent, arginine-catabolism specific transport system in lactococci will be discussed.Abbreviations pmf protonmotive force - transmembrane electrical potential - pH transmembrane pH gradient - PE phosphatidylethanolamine - PC phosphatidylcholine Paper adapted from a treatise Secondary Transport of Amino Acids by Membrane Vesicles Derived from Lactic Acid Bacteria and awarded the Kluyver Prize 1988 by the Netherlands Society of Microbiology.     

Ultrastructure of poplar cortical cells during the transition from growing to wintering stages and vice versa

Summary Electron microscopic studies revealed that major cytological changes in the cortical cells of poplar (Populus euramericana cv. gelrica) began to occur in early September in conjunction with the metabolic transition from the growing to the wintering stage. During this transition, the cells became temporarily rich in endoplasmic reticulum, polysomes and vesicles. As the conspicuous formation of organelles progressed, the large vacuoles became smaller and filled with osmiophilic materials. Undefined organelles (protein-lipid bodies) also increased in number. From late October until March, organelles involved in protein synthesis were sparsely distributed in the cells, indicating that the number of these organelles is probably linked to the seasonal cycle of protein synthesis. In early February, after release from dormancy, fusion of vacuoles proceeded in the cells. The inclusion of organelles and a gradual decrease in the amount of osmiophilic materials in the vacuoles occurred at this stage. Subsequently, the structure of the cells continued to undergo changes to accommodate growth, which occurred in early May.     

A system for the inducible secretion of proteins from Bacillus subtilis during logarithmic growth

Abstract A Bacillus subtilis-Escherichia coli shuttle vector was constructed containing the B. subtilis levansucrase gene promoter and region encoding its signal sequence.
A site for the restriction enzyme Nae I was included to facilitate precise translational fusions to the DNA encoding the levansucrase signal sequence. Fusions of TEM β-lactamase to this construct displayed sucrose-inducible expression and secretion of B. subtilis .     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.