Electrotransformation of intact and osmotically sensitive cells of Corynebacterium glutamicum

Summary Intact and osmotically sensitive cells of Corynebacterium glutamicum can be efficiently transformed by electroporation. This was shown by using the plasmid vector pUL-330 (5.2 kb), containing the kanamycin resistance gene of transposon Tn5. The following electric parameters yielded efficient transformation. For intact cells: one exponentially decaying field pulse with time constants and with initial field intensities of E ₀=35–40 kV cm^-1; prepulse temperature 20°C. Cell regeneration (survival) was 100%–80%. Transformation efficiency can be increased by an additional freeze and thaw cycle of the cells, prior to electroporation. Lysozyme treated cells (osmotically sensitive) were transformed with three successive pulses of E ₀=25–30 kV cm^-1. Cell regeneration under these conditions was found to be 20–30%. The optimum yield of transformants/g plasmid-DNA was 3×10³ for intact cells, 2×10⁴ for intact cells which were frozen and thawed twice and 7×10⁴ for osmotically sensitive cells if the cell suspension was pulsed at a cell density of 1–3×10⁸/ml and at a DNA concentration of 0.2 g/ml up to 2 g/ml. The data obtained for osmotically sensitive cells suggest that the temperature increase accompanying the electric field pulse enhances colony formation and transformation efficiency if the initial prepulse temperature is 20°C, although regeneration of electroporated C. glutamicum cells starts to decrease at temperatures20°C.     

Plant regeneration in vitro of South Pacific taro (Colocasia esculenta var. esculenta cv. Akalomamale,Aracea)

Summary Axillary bud expiants from South Pacific (Solomon Islands) taro, Colocasia esculenta var. esculenta cv. Akalomamale (Araceae) cultured on a modified Murashige-Skoog medium containing 1 mg NAA 1^–1 and TE formed callus and produced multiple plantlets. Explants died if NAA was present at levels lower than 0.1 mg 1^–1. BA was not required and may have been inhibitory. Plantlets developed faster and became larger following transfer to a hormone-free medium two weeks after the start of culture. Fully grown plants were established in a potting mix and are growing well in a greenhouse.Abbreviations BA benzyladenine - BM basal medium - Ca Colocasia esculenta var. antiquorum - Ce Colocasia esculenta var. esculenta - Ck cytokinin(s) - CW coconut water - HSMSM half strength Murashige Skoog macroelements - HSMS half strength Murashige and Skoog medium - IM initial medium(ia) - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - SM second medium - TE taro corm extract - UCI University of California, Irvine     

Microdissection of the Prader-Willi syndrome chromosome region and identification of potential gene sequences

The Prader-Willi syndrome chromosome region on the long arm of human chromosome 15 was microdissected and microcloned from 20 GTG-banded metaphase chromosomes, and 5000 recombinant clones were obtained. Of these clones, 39% identify single-copy human DNA sequences, most of which map to the dissected chromosome region and are evolutionarily conserved in other species. Three of eleven clones studied in detail are deleted in several patients with Prader-Willi syndrome. The microclones will be useful for the physical characterization of the Prader-Willi syndrome chromosome region and the identification of the affected genes in this disease.     

Purification and properties of theDrosophila zen protein

Summary The zen protein is encoded by the zerknullt gene required for normal early development inDrosophila. Like many regulatory proteins of this type, zen contains a 60 amino acid homeobox sequence. We have purified the zen protein and studied its solution behavior and its interaction with DNA. The zen protein exists as a monomer in solution with a molecular weight of about 40000. It binds specifically to a site about 900 bases upstream from thezen gene. Within this binding site DNase protection experiments indicate that binding is confined to two regions approximately 11 and 14 bases in length that are separated by about 30 base pairs. The protein concentration dependence of the binding curve suggests that protein binding is non cooperative.     

Regulation of cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary The two high affinity calcium binding sites of the cardiac (Ca²⁺ + Mg²⁺)-ATPase have been identified with the use of Eu³⁺. Eu³⁺ competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of H₂O molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca²⁺ sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6–8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites.     

Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells.

Hypervariable minisatellites can be amplified from human DNA by the polymerase chain reaction, using primers from DNA flanking the minisatellite to amplify the entire block of tandem repeat units. Minisatellite alleles up to 5-10 kb long can be faithfully amplified. At least six minisatellite loci can be co-amplified from the same DNA sample and simultaneously detected to provide a reproducible and highly variable DNA fingerprint which can be obtained from nanogram quantities of human DNA. The polymerase chain reaction can also be used to analyse single target minisatellite molecules and single human cells, despite the appearance of spurious PCR products from some hypervariable loci. DNA fingerprinting at the level of one or a few cells therefore appears possible.     

Thermodynamic aspects of plant cell adhesion to polymer surfaces

Summary A thermodynamic model of particle adhesion from a suspension onto a solid surface is used to predict the extent of adhesion of suspension-cultured Catharanthus roseus cells to the following polymer substrates: fluorinated ethylene-propylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), sulphonated polystyrene (SPS), and glass. According to this model, the extent of adhesion is determined by the surface tensions of the plant cells, the polymer substrates, and the suspending liquid medium. Experimentally, adhesion of the washed plant cells was found to decrease with increasing substrate surface tension, following the sequence FEP>PS>PET>SPS>glass, when the surface tension of the liquid was greater than that of the plant cells, in agreement with the model. However, adhesion increased with increasing substrate surface tension when the liquid surface tension was lower than the cellular surface tension, also in agreement with the model. When the liquid and cellular tensions were equal the extent of adhesion was independent of the substrate surface tension. This also agrees with model predictions and leads to a value for the surface tension of C. roseus cells of approximately 54 ergs/cm² which is in agreement with a value obtained from contact angle measurements on layers of cells and sedimentation volume analysis. The cellular surface tension determined by the sedimentation volume method showed a biphasic alteration during growth cycles of C. roseus cell cultures. These variations (between 55 and 58 ergs/cm²) agree with the pattern of adhesion previously described.