High resolution electron microscope studies of chromosomal fibers

Techniques are described for mounting and visualizing biological macromolecules for high resolution electron microscopy. Standard techniques are included in a discussion of new methods designed to provide the highest structural resolution. Methods are also discussed for handling samples on the grid, for making accurate size measurements at the 20 Å level, and for photographically enhancing image contrast.

The application of these techniques to the study of the binding of DNA polymerase to DNA is described. It is shown that the electron micrographs of this material are in agreement with the model proposed by Dr. Arthur Kornberg. A model is described which locates several active sites on the enzyme.

The chromosomal material of the protozoan tetrahymena has been isolated and characterized by biochemical techniques and by electron microscopy. This material is shown to be typical of chromatin of higher creatures.

Comparison with other chromatins discloses that the genome of tetrahymena is highly template active and has a relatively simple genetic construction.

High resolution electron microscope procedures developed in this work have been combined with standard biochemical techniques to give a comprehensive picture of the structure of interphase chromosome fibers. The distribution of the chromosomal proteins along its DNA is discussed.

Part I. Synchronization of the cell division cycle of HeLa cells in suspension cultures. Part II. Studies on chromosomal proteins of HeLa cells during the cell division cycle

Part I

These studies investigate the potential of single and double treatments with either 5-fluorodeoxyuridine of excess thymidine to induce cell division synchrony in suspension cultures of HeLa cells. The patterns of nucleic acid synthesis and cell proliferation have been analyzed in cultures thus synchronized. Several changes in cell population during long incubation with 5-fluorodeoxyuridine or excess thymidine are also described. These results are subjected to detailed evaluation in terms of the degree and quality of synchrony finally achieved.

Part II

Histones and non-histone proteins associated with interphase and metaphase chromosomes of HeLa cells have been qualitatively and quantitatively analyzed. Histones were fractionated by chromatography on Amberlite CG-50 and further characterized by analytical disc electrophoresis and amino acid analysis of each chromatographic fraction. It is concluded that histones of HeLa cells are comprised of only a small number of major components and that these components are homologous to those of other higher organisms. Of all the histones, arginine-rich histone III alone contains cysteine and can polymerize through formation of intermolecular disulfide bridges between histone III monomers.

A detailed comparison by chromatography and disc electrophoresis established that interphase and metaphase histones are made up of similar components. However, certain quantitative differences in proportions of different histones of interphase and metaphase cells are reported. Indirect evidence indicates that a certain proportion of metaphase histone III is polymerized through intermolecular disulfide links, whereas interphase histone III occurs mainly in the monomeric form.

Metaphase chromosomes are associated with an additional acid-soluble protein fraction which is absent from interphase chromosomes. All of these additional acid-soluble proteins of metaphase chromosomes are shown to be non-histones and it is concluded that the histone/DNA ratio is identical in interphase and metaphase chromosomes. The bulk of acid-soluble non-histone proteins of metaphase chromosomes were found to be polymerized through disulfide bridges; corresponding interphase non-histone proteins displayed no evidence of similar polymerization.

The factors responsible for the condensed configuration and metabolic inactivity of metaphase chromosomes are discussed in light of these findings.

The relationship between histone and DNA synthesis in nondividing differentiated chicken erythrocyte cells and in rapidly dividing undifferentiated HeLa cells is also investigated. Of all the histones, only arginine-rich histones are synthesized in mature erythrocytes. Histone synthesis in HeLa cells was studied in both unsynchronized and synchronized cultures. In HeLa cells, only part of the synthesis of all histone fractions is dependent on concurrent DNA synthesis, whereas all histones are synthesized in varying degrees even in the absence of DNA synthesis.

Investigations of nonhistone chromosomal proteins

The major nonhistone chromosomal proteins (NHC proteins) are a group of 14-20 acidic proteins associated with DNA in eukaryotic chromatin. In comparisons by SDS gel electrophoresis (molecular weight sieving) one observes a high degree of homology among the NHC protein fractions of different tissues from a given species. Tissue-specific protein bands are also observed. The appearance of a new NHC protein, A, in the NHC proteins of rat liver stimulated to divide by partial hepatectomy and of rat ascites cells suggests that this protein may play a role in preparing the cell for division. The NHC proteins of the same tissue from different species are also very similar. Quantitative but not qualitative changes in the NHC proteins of rat uterus are observed on stimulation (in vivo) with estrogen. These observations suggest that the major NHC proteins play a general role in chromatin structure and the regulation of genome expression; several may be enzymes of nucleic acid and histone metabolism and/or structural proteins analogous to histones. One such enzyme, a protease which readily and preferentially degrades histones, can be extracted from chromatin with 0.7 N NaCl.

Although the NHC proteins readily aggregate, they can be separated from histone and fractionated by ion exchange chromatography on Sephadex SE C-25 resin in 10 M urea-25% formic acid (pH 2.5). Following further purification, four fractions of NHC protein are obtained; two of these are single purified proteins, and the other two contain 4-6 and 4-7 different proteins. These NHC proteins show a ratio of acidic to basic amino acids from 2.7 to 1.2 and isoelectric points from apparently less than 3.7 to 8.0. These isolated fractions appear more soluble and easier to work with than any whole NHC protein preparation.

Molecular genetics of the Drosophila eyes absent gene

The Drosophila compound eye has provided a genetic approach to understanding the specification of cell fates during differentiation. The eye is made up of some 750 repeated units or ommatidia, arranged in a lattice. The cellular composition of each ommatidium is identical. The arrangement of the lattice and the specification of cell fates in each ommatidium are thought to occur in development through cellular interactions with the local environment. Many mutations have been studied that disrupt the proper patterning and cell fating in the eye. The eyes absent (eya) mutation, the subject of this thesis, was chosen because of its eyeless phenotype. In eya mutants, eye progenitor cells undergo programmed cell death before the onset of patterning has occurred. The molecular genetic analysis of the gene is presented.

The eye arises from the larval eye-antennal imaginal disc. During the third larval instar, a wave of differentiation progresses across the disc, marked by a furrow. Anterior to the furrow, proliferating cells are found in apparent disarray. Posterior to the furrow, clusters of differentiating cells can be discerned, that correspond to the ommatidia of the adult eye. Analysis of an allelic series of eya mutants in comparison to wild type revealed the presence of a selection point: a wave of programmed cell death that normally precedes the furrow. In eya mutants, an excessive number of eye progenitor cells die at this selection point, suggesting the eya gene influences the distribution of cells between fates of death and differentiation.

In addition to its role in the eye, the eya gene has an embryonic function. The eye function is autonomous to the eye progenitor cells. Molecular maps of the eye and embryonic phenotypes are different. Therefore, the function of eya in the eye can be treated independently of the embryonic function. Cloning of the gene reveals two cDNA's that are identical except for the use of an alternatively-spliced 5' exon. The predicted protein products differ only at the N-termini. Sequence analysis shows these two proteins to be the first of their kind to be isolated. Trangenic studies using the two cDNA's show that either gene product is able to rescue the eye phenotype of eya mutants.

The eya gene exhibits interallelic complementation. This interaction is an example of an "allelic position effect": an interaction that depends on the relative position in the genome of the two alleles, which is thought to be mediated by chromosomal pairing. The interaction at eya is essentially identical to a phenomenon known as transvection, which is an allelic position effect that is sensitive to certain kinds of chromosomal rearrangements. A current model for the mechanism of transvection is the trans action of gene regulatory regions. The eya locus is particularly well suited for the study of transvection because the mutant phenotypes can be quantified by scoring the size of the eye.

The molecular genetic analysis of eya provides a system for uncovering mechanisms underlying differentiation, developmentally regulated programmed cell death, and gene regulation.

Gene enrichment using antibodies to DNA/RNA hybrids : mapping the ribosomal DNA of slime mold and rat

A novel method for gene enrichment has been developed and applied to mapping the rRNA genes of two eucaryotic organisms. The method makes use of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the synthetic hybrid poly(rA)•poly(dT). Antibodies which cross-react with non-hybrid nucleic acids were removed from the purified IgG fraction by adsorption on columns of DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent purification of the specific DNA/RNA hybrid antibody was carried out on a column of oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these antibodies to CNBr-activated Sepharose produced an affinity resin which specifically binds DNA/RNA hybrids.

In order to map the rDNA of the slime mold Dictyostelium discoideum, R-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs of this organism. This mixture was passed through a column containing the affinity resin, and bound molecules containing R- loops were eluted by high salt. This purified rDN A was observed directly in the electron microscope. Evidence was obtained that there is a physical end to Dictyostelium rDN A molecules approximately 10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding is consistent with reports of other investigators that the rRNA genes exist as inverse repeats on extra-chromosomal molecules of DNA unattached to the remainder of the nuclear DNA in this organism.

The same general procedure was used to map the rRNA genes of the rat. Molecules of DNA which contained R-loops formed with the 188 and 288 rRNAs were enriched approximately 150- fold from total genomal rat DNA by two cycles of purification on the affinity column. Electron microscopic measurements of these molecules enabled the construction of an R-loop map of rat rDNA. Eleven of the observed molecules contained three or four R-loops or else two R-loops separated by a long spacer. These observations indicated that the rat rRNA genes are arranged as tandem repeats. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent repeating units of exactly the same length within the errors of the measurements, although a certain degree of length heterogeneity cannot be ruled out. If significantly shorter or longer repeating units exist, they are probably much less common than the 37.2 kbp unit.

The last section of the thesis describes the production of antibodies to non-histone chromosomal proteins which have been exposed to the ionic detergent sodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did not seem to affect either production of antibodies or their general specificity. Also, a technique is described for the in situ immunofluorescent detection of protein antigens in polyacrylamide gels.

Organization and function of the phage Lambda chromosome

The process of prophage integration by phage λ and the function and structure of the chromosomal elements required for λ integration have been studied with the use of λ deletion mutants. Since att^φ, the substrate of the integration enzymes, is not essential for λ growth, and since att^φ resides in a portion of the λ chromosome which is not necessary for vegetative growth, viable λ deletion mutants were isolated and examined to dissect the structure of att^φ.

Deletion mutants were selected from wild type populations by treating the phage under conditions where phage are inactivated at a rate dependent on the DNA content of the particles. A number of deletion mutants were obtained in this way, and many of these mutants proved to have defects in integration. These defects were defined by analyzing the properties of Int-promoted recombination in these att mutants.

The types of mutants found and their properties indicated that att^φ has three components: a cross-over point which is bordered on either side by recognition elements whose sequence is specifically required for normal integration. The interactions of the recognition elements in Int-promoted recombination between att mutants was examined and proved to be quite complex. In general, however, it appears that the λ integration system can function with a diverse array of mutant att sites.

The structure of att^φ was examined by comparing the genetic properties of various att mutants with their location in the λ chromosome. To map these mutants, the techniques of heteroduplex DNA formation and electron microscopy were employed. It was found that integration cross-overs occur at only one point in att^φ and that the recognition sequences that direct the integration enzymes to their site of action are quite small, less than 2000 nucleotides each. Furthermore, no base pair homology was detected between att^φ and its bacterial analog, att^B. This result clearly demonstrates that λ integration can occur between chromosomes which have little, if any, homology. In this respect, λ integration is unique as a system of recombination since most forms of generalized recombination require extensive base pair homology.

An additional study on the genetic and physical distances in the left arm of the λ genome was described. Here, a large number of conditional lethal nonsense mutants were isolated and mapped, and a genetic map of the entire left arm, comprising a total of 18 genes, was constructed. Four of these genes were discovered in this study. A series of λdg transducing phages was mapped by heteroduplex electron microscopy and the relationship between physical and genetic distances in the left arm was determined. The results indicate that recombination frequency in the left arm is an accurate reflection of physical distances, and moreover, there do not appear to be any undiscovered genes in this segment of the genome.