Student Theses and Dissertations

Date of Award

1963

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Bronk laboratory

Abstract

Pneumococcal transformation has been used to study intramolecular genetic recombination. A kinetic technique is developed whereby the cumulative number of multiply-marked transformants is measured as a function of the duration of exposure to a multiply-marked DNA. Transformants accumulate with time at a rate characteristic of the particular markers and the number of DNA particles involved, thus singly-marked transformants accumulate linearly with time of exposure to DNA, as do some multiply-marked transformants. Other unlinked pairs of markers accumulate exponentially with time, by a second-order process, at a rate very close to that expected from random interaction of one cell with two DNA particles. Linked markers can be shown to reside in one DNA particle by this method, because the pattern of accumulation is undistinguishable from that observed for single markers. Linkage frequency can be expressed as the ratio of the linear rate of accumulation of multiply-marked transformants relative to singly-marked transformants. Kinetic analysis has been made of the fate of each factor in a linear array of three genetic markers within one DNA particle. The linear rate of accumulation in time of each of the several kinds of recombinant progeny acquiring any one, two, or three of these markers demonstrates the frequent and regular occurrence of complex intramolecular events in pneumococcal transformation. One such complex event results in acquisition of the outer two or "bracketing" markers but not the central factor from the linear array present in the single donor DNA particle which initiated the transformation. The frequency with which these particular recombinations appear suggests that there is very little interference between events occurring over the entire interval. Furthermore, as judged by clonal analysis, the recombination is a unique event within one division cycle of the cell and gives rise to pure clones of transformants after growth in non-selective medium; heterozygous cells are not detected within these clones. The redistribution of alleles between donor DNA and recipient cells can affect the relative frequency of complex to simpler intramolecular recombinations over a range of about two-fold. iv By changing the conditions under which a cell population is brought to competence for transformation and then transformed, the complex intramolecular recombinational events can be essentially eliminated even though the DNA preparation has not been changed in any way. Such events are, therefore, a reflection of cellular processes and not alone attributable to the state of a DNA preparation. The effect of in vitro modification of DNA structure on the behavior of linked marker groups and on the frequency of occurrence of complex intramolecular recombinations is examined by the kinetic method. The rate of loss of biological activity during subcritical heat inactivation (giving localized submolecular lesions) is determined by the number of markers being introduced into the transformants and not by the genetic (and physical) relationship of these markers within DNA particles. Collapse of secondary structure in DNA, due to critical heat denaturation, causes only a slight change in adsorption affinity of the preparation for competent cells, but markedly reduces the ability of the DNA to penetrate into cells, and may also lead to some inert particles unable to adsorb to the cell surface. Nevertheless, linkage relationships of markers can be only slightly affected in otherwise good preparations. Renaturation restores some of the ability to penetrate cells and also much of the biological activity to previously denatured DNA preparations, but does not increase linkage frequencies above those of the denatured material. Annealing of mixtures containing genetically distinct DNA's creates no new biologically active hybrid particles, as determined quantitatively by kinetic experiments and by clonal analysis of transformant progeny. These data are discussed and interpreted as showing that secondary structure of DNA influences not its adsorption but primarily its penetration into the cell, whereas the specific structure along polynucleotide strands probably influences the frequency of recombinational events at individual sites.

Comments

A thesis submitted to the Faculty of The Rockefeller Institute in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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