A Study of the Base Sequence Arrangements in DNA by Electron Microscopy

Year: 1970 DOI: 10.7907/1QZW-ZP91
The study of base sequence arrangements in DNA molecules was accomplished by coupling electron microscopy (EM) with DNA hybridization. Modifications of the basic protein film technique were used. EM methods for examining both double-stranded and single-stranded DNA were used.

Many EM studies have a tendency to be rather descriptive in nature. However, it has been my aim to approach problems from a more quantitative aspect. The major problem in quantitative EM of DNA is length fluctuations. It was discovered that the standard deviation in the length of a homogeneous sample of DNA molecules is directly proportional to the square root of the length.

An EM method was developed for determining the size and location of deletion mutations and substitutions in the λ bacteriophage DNA. Heteroduplexes with one strand from the wild type and one strand from the mutant were formed by renaturation. The location of the nonhomology regions can be accurately mapped. A number of mutants were selected that are unable to integrate and by EM studies of the heteroduplexes it was discovered that there is a specific site in the λ DNA (0.574 from the left end) and a specific site in the E. coli DNA where the integration takes place. It was also shown that this specific site in λ DNA would not form stable base pairing with the specific site in the E. coli DNA. This means that the two sites have a base sequence similarity of less than 10 base pairs. A physical gene map of the left arm of the λ DNA molecule was constructed by comparing the physical and genetic location of a large number of mutations.

The DNA isolated from a defective lysogenic phage of E. coli 15 was studied. After induction there is a closed circular DNA in vivo. However, the DNA is packaged as a linear molecule in the phage head. The mature linear DNA is 7.5% longer than the in vivo closed circular DNA. This was found to be because the mature linear DNA is both terminally repetitious (TR) and circularly permuted. Circular molecules are obtained by denaturing and renaturing the linear molecules. These in vitro circular molecules have two single-stranded DNA branches, each equal in length to the TR. The amount of the TR was found to be slightly heterogeneous. Therefore, the amount of linear DNA that is packaged in the phage head is slightly variable and there is no mechanism for packaging exactly the same amount of DNA.

The mitochondria of human leukemic leukocytes contain covalently closed circular DNA molecules of twice the molecular weight found in mitochondria of normal leukocytes. Heteroduplexes containing one dimer length strand and two complementary monomer length strands were formed by renaturation. These heteroduplex DNA molecules appear as figure eights. They were carefully examined for DNA substitutions or deletions but none were found. Therefore, the dimer molecule is composed of exactly two monomer molecules.

EM studies of the SV40 DNA showed that it is heterogeneous in length. From heteroduplex studies it was learned that this DNA is heterogeneous in base sequence as well. About half of the self-renatured molecules contained non-homology regions. The size (up to 1/2 the molecule) and the number of non-homology regions varied considerably. Unique classes of heteroduplex molecules could not be found and no reasonable conclusions could be given for the nature or origin of the heterogeneity.

The replicating intermediates in the in vivo synthesis of ØX174 DNA were studied. The single-stranded DNA is synthesized from a double-stranded template as a linear molecule. The replicating intermediate appears as a double-stranded circular molecule with a linear single-stranded branch attached to the circle. The linear branch was never found to be longer than the mature circular DNA and is presumably released after one round of replication on the template and then cyclized by an as yet undetermined means.

RNA was synthesized on T₇ DNA in vitro. The resulting RNA-RNA polymerase-DNA complex was visualized in the EM. By synchronizing initiation and mapping the location of the RNA at various times it was discovered that synthesis is always initiated close to one unique end, and thus, presumably at a unique site. At one min there is one PNA per DNA. At high RNA polymerase concentrations, closely spaced sequential initiations occur from the one active initiation site. The rate of propagation was measured as about 45 bases/sec.