Characterization of isolates and clones of leishmania by analysis of kinetoplast DNA

The genetic characterization of pathogenic isolates of Leishmania was attempted by analysis of the molecular properties of kinetoplast DNA (kDNA) minicircles. Unit minicircle size is not conserved during speciation of Leishmania since the minicircles of strains and clones of L t major are smaller (700 bp) than those found in certain strains of L mexicana ssp (820 bp), L donovani (850 bp) or L t tropica (900 bp). Schizodeme analysis of minicircles reveals a high degree of sequence divergence in kDNA of Leishmania with the degree of microheterogeneity varying between species. This sequence divergence allows the discrimination of species, strains, and clones of Leishmania into schizodemes. Southern blot hybridization experiments reveal that at high stringency overall minicircle sequence homology is conserved among clones and strains of one species (L t major) but not between different species. This property of minicircle DNA permits the use of kDNA probes as a species-specific diagnostic test for the identification of unknown Leishmania isolates. The properties of kDNA from an L t tropica strain LRC-L32 (a “recidiva” organism) are so diverged from those of L t major strains as to support the classification [22,23] of L t tropica and L t major as separate species of Leishmania rather than subspecies of L tropica.     

Physical map of DNA from a new cauliflower mosaic virus strain

The restriction enzymes AluI, BamHI, BglII, EcoRI, HindIII, and SalI have been used to characterize and map a new cauliflower mosaic virus strain (Cabb-S). These fragments have been ordered by examining their overlapping regions after double enzymatic digestion. The single SalI cleavage site was chosen as the point of origin. We compare this strain with those already described.     

The isolation of DNA from agarose gels by electrophoretic elution onto malachite green-polyacrylamide columns

Electrophoretic elution of DNA coupled with direct adsorption onto malachite green-polyacrylamide columns was used to isolate double- and single-stranded DNA from agarose gels. Subsequently, DNA was eluted with a high salt buffer and filtered through Sephadex which permitted recovery of the DNA in a low salt buffer at concentrations suitable for heteroduplex analysis by electron microscopy. This method was tested by examining hetero-duplexes formed from the isolated complementary single strands of T7 wild type DNA and a T7 deletion mutant. More than 80% of the reannealed molecules were intact heteroduplexes showing the deletion loop. Irradiation of single-stranded DNA with 254 nm light resulted in distorted, convoluted heteroduplexes while 366 nm light did not show this effect.     

Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA.

The susceptibility of E. coli strain chi1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0--6.0 . 10(7) cfu/ml, followed by washing twice in cold 0.1 M NaCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6. Cells grown in the presence of D-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6 . 10(8) cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37 degrees C or 42 degrees C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell + DNA mixtures were plated on precooled (4 degrees C) Tc agar plates. The efficiency of plating was optimum when only 5 microliter of cell + DNA mixture was spread per plate, suggesting that non-viable background chi1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of chi1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli chi1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 10(7) transformants/microgram pBR322 plasmid DNA.     

Molecular and cytological characteristics of nuclear DNA and chromatin for angiosperm systematics: DNA diversification in the evolution of four orchids

The species- and genus-specific DNA content, average base composition of nuclear DNA, presence or absence of satellite DNA, the percentage of heterochromatin and other characteristics of nuclear DNA and nuclear structure allow to deduce the molecular changes which accompanied, or more probably caused, cladogenesis in the orchids studied. It is suggested that saltatory replication (generative amplification) of certain DNA sequenes, diversification of reiterated DNA sequences, and loss of DNA play an important role in the evolution of orchids.—The relationship between changes of genome composition and of nuclear structure and ultrastructure is discussed on the basis of cot curves, heterochromatin staining with Giemsa (C banding), electron microscopy of nuclei, and molecular hybridization in situ.Some aspects of this paper have been presented at the Helsinki Chromosome Conference, August 1977 (Nagl & Capesius 1977).     

The role of DNA polymerase I-associated 5'-exonuclease in replication of coliphage M13 replicative-form DNA.

The conversion of both parental- and progeny-nascent open circular M13 RF DNA into covalently closed RF I is drastically reduced in an $\text{E. coli}$ mutant deficient in the 5′ → 3′ exonuclease associated with DNA polymerase I. The nascent progeny RF DNA also contains a significant proportion of fragments of smaller than unit length.     

Interaction of purified DNA with plant protoplasts of different cell cycle stage: The concept of a competent phase for plant cell transformation

Summary The polycation mediated attachment of purified tritiated DNA to plant protoplasts has been measured by quantitative microautoradiography. The automated grain counting technique used, also provides information on the cell cycle stage of individual protoplasts, which circumvents the need to synchronize the plant cell population before preparation of protoplasts. With protoplasts from asynchronously dividing suspension cultures of Nicotiana syhestris (NS-1), S-phase protoplasts appear to be inefficient binders of ³H-DNA, as compared with G₁ or G₂ protoplasts. Protoplasts derived from a tumour line of Crepis capillaris (CAPT) exhibit ³H-DNA binding at all cell cycle phases, but Sphase protoplasts appear to be preferential binders. These differences are discussed with reference to cell cycle kinetics, membrane charge variation and the possibility of increasing the efficiency of genetic transformation of higher plant cells in culture.     

Changes occurring during aging and senescence in a submerged aquatic angiosperm (Potamogeton pectinatus)

Changes occurring during aging and senescence of leaves of a submerged aquatic angiosperm ( Potamogeton pectinatus L.) were studied. Total chlorophyll and chlorophylls a and b were maximal in mature, and minimal in old leaves. The chlorophyll a to b ratio was highest in mature leaves. During senescence, the chlorophyll content and the ratio of chlorophyll a to b decreased. The content of DNA, RNA, protein and dry weight, and the activity of alkaline pyrophosphatase decreased while free amino acids, the activity of protease, RNase and acid pyrophosphatase, and the ratio of acid to alkaline pyrophosphatase activity increased during aging and senescence. Kinetin (0.23 m M ) deferred leaf senescence by delaying the loss of chlorophyll, protein, nucleic acids and dry weight, and reducing the rise in free amino acids, the activity of protease, RNase and acid pyrophosphatase and the ratio of acid to alkaline pyrophosphatase activity; while both 0.69 m M ethrel and 0.075 m M ABA hastened senescence. Kinetin pretreatment for an optimum period (12 h) followed by ethrel or ABA treatment partially erased the senescence-promoting effect of the latter. But treatments in a reverse order markedly reduced the delaying effect of kinetin on senescence.     

Hereditary variability of plants under the action of exogenous DNA

Summary Injection of exogenous barley donor DNA into grains of barley recipient plants at the milk maturity stage, with a specially designed syringe, led to the appearance of transformed plants. The transformation (in rare cases) was caused by the unsheared DNA since the DNA passing through the syringe needle remained relatively stable (10⁶ to 10⁷ daltons) as was confirmed by DNA sedimentation analysis.14 plants grown from seeds injected with highly polymeric DNA containing close to 30 per cent protein had transformed pollen grains. In the 2nd generation only 2 plants from the 8 studied preserved these changes. In the progeny of these two plants, i.e., in the 3rd seed generation after injection, 82.1 per cent of plants preserved the transformed characters. The next, 4th generation, preserved a transformed phenotype in 89.6 per cent of plants.It was also shown that reversion to a recipient-like state was not always constant. We found the reversion of transformed properties (i.e., normal starch and two-rowed spikes) in 40 per cent of the 4th generation descendants of one of the plants which had lost the phenotypical expression of these properties in the 3rd generation but had them in the 2nd generation.The study of the morphological properties of transformed plants showed that with respect to phenotypic expression some characters were changed towards the donor type, some remained as in the recipients and some were of the intermediate type.     

Incorporation of3H-thymidine into DNA and the activity of alkaline phosphatase in zinc-deficient fetal rat brains

The incorporation of³H-thymidine into DNA in the brains of the 17-day and 20-day old rat fetuses was significantly reduced by maternal zinc restriction during pregnancy. The activity of the enzyme thymidine kinase (EC 2.7.1.21) was similarly reduced in the zine-deprived fetal brains on days 14 and 20 of gestation, but not on day 17. Fetal brain alkaline phosphatase (EC 3.1.3.1) was significantly depressed by maternal zinc deprivation on days 17 and 20 of pregnancy. The data suggest an association between thymidine kinase and the reduced incorporation of³H-thymidine into DNA in the brains of 20-day old fetuses but not in animals on day 17. Alkaline phosphatase was however depressed at this stage. The suggestion is made that because of the complexity of brain development, future biochemical studies in this area should concern specific structures in the brain at particular critical stages during neurogenesis.