Student Theses and Dissertations

Date of Award

1972

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Rollin Hotchkiss

Keywords

merozygote formation, pneumococcus, sulfanilamide resistance, genetic segregation, tandem duplication, DNA-mediated transformation

Abstract

Unstable prokaryotic strains arise occasionally under a variety of circumstances and with a variety of properties which make them amenable to genetic and physiological analysis at different levels of precision. A review of a number of such unstable systems, initially identified on the basis of a regular segregation to phenotypically non-parental types, has led to the recognition of a series of criteria for the systematic analysis of such strains. These criteria are applied to a sulfanilamideresistant mutant of pneumococcus which regularly segregates to strains sensitive to sulfanilamide and resistant to p-nitrobenzoic acid, another analogue of paraminobenzoic acid. From a combination of information gathered by previous workers and new data, it is confirmed that this sulfanilamide-resistant mutant, sul-c, is a genetically determined variant whose segregants are stable and indistinguishable from the strain wild type at the same locus (c+) by criteria of drug-resistant phenotype, behavior as recipient and donor in DNA-mediated genetic crosses (transformation), and inability under a variety of conditions to give rise to the original sul-c type. By analysis of genetic crosses mediated by DNA from the c strain and from complex resistant strains designated cd, it is established that the segregants arise by separation of the mutant and wild type determinants pre-existing in the parental strain, rather than by some rapid reverse mutation. Thus strains containing sul-c are merozygotes, partial diploids for a region of genetic material encompassing the c locus and closely linked sites. Linkage of the c marker to the marker governing streptomycin resistance can be demonstrated although the latter marker is never seen in an unstable form. Consequently, the basis for the instability must lie within the pneumococcal chromosome, rather than on an extrachromosomal genome fragment. The complex cd types may assume a variety of properties with respect to frequency of wild type segregation and frequency of c, d, and d+ markers in the DNA of the strains. These properties provide a basis for designating three classes of cd types, which have the same properties whether generated from a transformation of a d recipient by c donor DNA or of a c recipient by d donor DNA. The various marker frequencies in the three classes probably result from a combination of an increased dosage of the genetic loci involved and an altered efficiency of integration of these loci in transformation. The segregation process itself was investigated. It was established that a given c-containing strain will always give segregants at the same frequency of the total population, accumulation of these segregants with time being impossible to demonstrate. However the segregation frequency of a given strain is characteristic of the strain, and may vary among independent isolates. Perturbations of general cellular metabolism such as nutritional supplementation, changes in growth temperature, or repeated freezing and thawing had no consistent, progressive, and readily interpretable effect on segragation frequency. However, treatment with ultraviolet light or mitomycin C at doses permitting 80 to 90% survival of the total cell population resulted in a pronounced stimulation in the number of segregants. This stimulation failed to persist as cell growth resumed, suggesting that the process stimulated was readily reversible. Treatment with acridine orange gave the opposite effect, a depression in the frequency of segregants. From the known effects of these three agents on DNA and on processes known to be mediated by recombination, it is proposed that the mechanism of segregation involves a recombination step. Two models are presented which account for the known properties of the c and cd strains. One involves a region of triple-strandedness around the c locus; the other involves a duplication in tandem of the c region. An attempt was made to distinguish the models on the basis of physical properties of the DNA of the strain: buoyant density, affinity for methylated albumin-kieselguhr, and ability to become inactivated by binding formaldehyde. In none of these tests was the c marker affected differently from markers known to be stable, although a triple-stranded structure might be expected to behave abnormally. However the presumed small size of the c region makes it possible that the inability to differentiate c was owing to insufficient sensitivity of the methods rather than to a strictly double-stranded structure. Data of my colleague, Dr. S.V.S. Kashmiri, is cited in which heteroduplex DNA molecules bearing the c marker is one strand and its wild type allele in the other showed a markedly depressed ability to transform c and markers linked to it. Heteroduplexes in which both strands contained c or both strands contained c+ did not show this depression. This evidence was interpreted as favoring the tandem duplication model, which is proposed as an hypothesis on which to base further experimentation. The specific nature of the c mutation and the mechanism by which the mutation generates merozygosis are speculated upon, as is the basis for the mutant phenotype, the detailed mechanism of segregation, and the relationship of c to other unstable strains of prokaryotes.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

License and Reuse Information

Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

Download

Included in

Life Sciences Commons

Share

COinS