Marvin A. Smith

Fate of bacterial plasmid DNA during uptake by barley protoplasts

There has been a great deal of excitement generated about the possibility of the genetic transformation of plants in the past few years. Some success has been reported [ 1,2] but the frequency of genetic transformation has been low and the results highly variable [3]. Plant cells devoid of cell walls (protoplasts) would seem to offer an advantage in such studies since they lack the natural barrier to uptake of DNAs, can be grown under selection conditions, and can be regenerated in some cases [4-81. Successful transformation of plants should also depend on efficient introduction of exogenous DNA into plant cells. We have been studying DNA uptake in protoplasts and have found that large amounts of linear bacterial DNA can be taken up. After typical uptake reactions, part of the DNA (20%) was of average genome size while most of the DNA (80%) was depolymerized [9,10]. It seemed that covalently closed circular (CCC) duplex plasmid DNA might be more resistant to depolymerization, and also that it might eventually allow insertion of desirable genes into protoplasts since techniques for gene insertion into plasmids have been developed [ 1 l-141. We have carried out plasmid DNA uptake reactions in barley protoplasts and present the results in this paper.

Evidence of a biotin dependent acetyl-coenzyme A carboxylase in rat muscle.

Rat hindlimb muscle tissue was extracted from male Sprague-Dawley rats exsanguinated under light ether anesthesia. Muscle homogenates (50,000 x g supernatant) were incubated with ATP, bicarbonate, acetyl-CoA, and citrate. The quantity of malonyl-CoA synthesized was determined by malonyl-CoA incorporation into long acyl chains using tritiated acetyl-CoA and fatty acid synthetase. Malonyl-CoA synthesis was found to be dependent on the presence of ATP, bicarbonate, citrate, and acetyl-CoA in the incubation medium. Incubation with avidin showed near complete inhibition of carboxylation that was restored with the addition of biotin. These results represent strong evidence of a biotin containing acetyl-CoA carboxylase in skeletal muscle.

Fate of bacterial plasmid DNA during uptake by barley and tobacco protoplasts: II. Protection by poly-L-ornithine

Summary A recombinant-DNA plasmid vehicle (pBR313) was shown to be stable in solutions containing 2% Driselase, but unstable in Driselase prepared plant protoplasts. This apparent instability was assumed to be a consequence of depolymerization by DNAases of protoplast origin. Such depolymerization was significantly reduced by addition of poly-L-ornithine (PLO), kinetin, ZnSO4, or alkali (pH 9.0) to exogenous DNA uptake reactions. The inhibition of endogenous nucleases was particularly obvious in the presence of disrupted protoplasts. In addition to reducing plasmid DNA depolymerization, PLO also stimulated DNA uptake especially at short time periods. This stimulation was probably not a consequence of increased protoplast damage, since tobacco protoplast regeneration was not reduced either by direct addition of PLO to the culture medium or by pretreatment of protoplasts with PLO and subsequent plating in normal medium. A significant fraction (64% of zero time control) of barely protoplast associated plasmid DNA (mol. wt 5.8 × 106), taken up in the presence of PLO, was completely excluded from Sepharose 4B (mol. wt ⩾ 2 × 106) 8 h after uptake. This fraction remained constant up to at least 24 h. In the case of tobacco, a large fraction (40% of zero time control) of the plasmid DNA taken up by protoplasts in the presence of PLO is of potential significance to host gene expression since this DNA (mol. wt ⩾ 2 × 106) persisted for at least 72 h after uptake.

Ultrastructure of nuclei isolated from plant protoplasts

SummaryA simple method, involving selective Triton X-100 membrane solubilization, has been developed for the isolation of nuclei from barley and tobacco protoplasts which gives a high yield of essentially pure nuclei. The isolated nuclei resembled those in leaf cells and protoplasts when the isolated nuclei were fixed for short times (2 hours, Medium II), except that their chromatin appeared to be more highly condensed and barley nuclei also lacked the outer nuclear membrane. When longer times of fixation (12 hours, Medium I) were used, the isolated nuclei lacked the characteristic condensed chromatin appearance.

Scanning electron microscopy of barley protoplasts

SummaryScanning electron micrographs of barley protoplasts were compared using various preparatory techniques. Numerous features were observed which turned out to be artifactual characteristics of the processing procedure used in collecting and dehydrating the samples. The most successful technique gave protoplasts which presumably maintained their natural structural integrity, as judged by retention of sphericity and absence of holes in the plasma membrane. The relative numbers of fragmented protoplasts and cellular organelles was also greatly reduced.

Physical and genetic mapping of safflower chloroplast DNA : Location of psbA, rbcL, atpA and rrnA.

SummaryA physical map of safflower (Carthamus tinctorius L.) chloroplast DNA has been generated using SalI, PstI, KpnI and HindIII restriction endonucleases. The circular plastid genome (151 kbp) has the usual inverted repeat. Heterologous probes containing psbA, rbcL, atpA or rrnA structural genes mapped colinearly with spinach and other chloroplast genomes.

Fate of Bacterial DNA during Uptake by Barley and Tobacco Protoplasts

Summary Protoplasts enzymatically isolated from barley and tobacco leaves took up bacterial DNA, generally as a linear function of time (0–6 hr) and DNA concentration (0–200 µ g/ml). Damaged protoplasts very rapidly saturated with large amounts of DNA and most of them had to be removed before data could be accurately evaluated. Di- and polyvalent cations enhanced uptake, but only at the expense of increased protoplast damage. Under the conditions employed, efficiency of protoplast regeneration was not significantly altered by exogenous DNA after uptake or after addition to culture medium. Protoplast associated bacterial DNA was not decreased by washing with a 10-fold excess of unlabeled DNA or by DNase treatments. Nuclei, isolated after DNA uptake, contained 60–80 % of the protoplast associated exogenous DNA. About 80 % of DNA taken up by protoplasts was degraded after a 4 hr uptake period; however, the remaining 20 % was of average gene size (5–10 × 10 5 daltons) and, therefore, of potential significance to host gene expression.

Contribution of damaged protoplasts to DNA uptake by purified plant protoplasts

Abstract Purified protoplast preparations contained few (5–9%) but significant numbers of non-spherical damaged protoplasts. Such damaged protoplasts rapidly saturated with relatively large amounts of exogenous DNA which was largely associated with their nuclei. The relative fraction of damaged protoplasts was found to increase with DNA uptake time and DNA concentration. Discontinuous gradient centrifugation provided a convenient method for selective removal of damaged protoplasts and reliable evaluation of exogenous DNA uptake by intact spherical protoplasts.

Purification of Plant Protoplasts by Discontinuous Gradient Centrifugation

Summary Discontinuous gradient centrifugation provided a technique for the isolation of purified intact barley, tobacco and safflower protoplasts in high yields. In the case of barley and tobacco, purified protoplasts were shown to retain viability following the centrifugation techniques. In addition, contaminating enzymes (e.g., deoxyribonuclease) found in the crude protoplast releasing enzyme solutions, damaged (non-spherical) protoplasts, organelles and cellular debris were significantly reduced in purified protoplast preparations.

Isolation and characterization of safflower (Carthamus tinctorius L.) chloroplast DNA.

SummarySafflower (Carthamus tinctorius L.) chloroplasts were isolated and purified with the aid of commercially available nylon mesh, differential centrifugation, and DNase I treatment. These chloroplasts were free of nuclei as determined by light microscopy of aceto orcein stained preparations, and similar to those observed by electron microscopy in spinach and many other higher plants, being bounded by a double membrane layer and characterized by the presence of a lamellar system surrounded by embedding matrix, and stacked membranes or grana lamallae. DNA was isolated and purified from such chloroplasts, and characterized with respect to cesium chloride density gradient isopycnic centrifugaton, denaturation, renaturation kinetics and restriction enzyme analysis. These studies show that safflower chloroplast DNA is similar to many other higher plant DNAs having a density of 1.700 gl cm3(G + C = 40.8%), a Tm of 86°C (G + C = 40.7%) and a molecular complexity and genome size of about 108 daltons.