Lynn C. Klotz

Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes.

Abstract The DNA content of the nucleus of the yeast Saccharomyces cerevisiae has been determined by both renaturation kinetics and DNA per cell measurements. Renaturation kinetics experiments were performed by following the decrease of optical hyperchromicity at 260 nm and by hydroxyapatite chromatography. DNA per cell measurements were made by the diaminobenzoic acid method and by the ethidium bromide method of Klotz & Zimm (1972 b ). The conclusion from the above experiments is that the S. cerevisiae nucleus contains 9 × 10 9 ± 2 × 10 9 daltons of DNA. Previously we (Lauer & Klotz, 1975) had measured the molecular weight of the largest piece of DNA in the yeast nucleus to be 2 × 10 9 ± 0.2 × 10 9 . Here we extend this work by using a more highly protein-denaturing buffer system and conclude that the largest piece of DNA in the S. cerevisiae nucleus contains 1.5 × 10 9 to 2.2 × 10 9 daltons of DNA in both haploid and diploid cell lysates. From genetics, the largest yeast chromosome should contain 13% of the genome, or 0.9 × 10 9 to 1.5 × 10 9 daltons of DNA (using our DNA per cell range). Thus, the large DNA we measure contains from one to two times the amount of the DNA predicted from genetics to be in the largest chromosome. In light of these new data, viscoelastic measurements on yeast DNA are now consistent with the idea that each chromosome contains one piece of DNA.

Characterization of the DNA from the dinoflagellate crypthecodinium cohnii and implications for nuclear organization

Although dinoflagellates are eucaryotes, they possess many bacterial nuclear traits. For this reason they are thought by some to be evolutionary intermediates. Dinoflagellates also possess some unusual nuclear traits not seen in either bacteria or higher eucaryotes, such as a very large number of identical appearing, permanently condensed chromosomes suggesting polyteny or polyploidy. We have studied the DNA of the dinoflagellate Crypthecodinium cohnii with respect to DNA per cell, chromosome counts, and renaturation kinetics. The renaturation kinetic results tend to refute extreme polyteny and polyploidy as the mode of nuclear organization. This organism contains 55-60% repeated, interspersed DNA typical of higher eucaryotes. These results, along with the fact that dinoflagellate chromatin contains practically no basic protein, indicate that dinoflagellates may be organisms with a combination of both bacterial and eucaryotic traits.

Computer comparison of new and existing criteria for constructing evolutionary trees from sequence data

SummaryThree new methods for constructing evolutionary trees from molecular sequence data are presented. These methods are based on a theory for correcting for non-constant evolutionary rates (Klotz et al. 1979; Klotz and Blanken 1981). Extensive computer simulations were run to compare these new methods to the commonly used criteria of Dayhoff (1978) and Fitch and Margoliash (1967). The results of these simulations showed that two of the new methods performed as well as Dayhoffs criterion, significantly better than that of Fitch and Margoliash, and as well as a simple variation of the latter (Prager and Wilson 1978) where any topology containing negative branch mutations is discarded. However, no method yielded the correct topology all of the time, which demonstrated the need to determine confidence estimates in a particular result when evolutionary trees are determined from sequence data.

Determination of the molecular weight of Saccharomyces cerevisiae nuclear DNA

Abstract Viscoelastic retardation-time experiments on the DNA released from spheroplasts of the yeast Saccharomyces cerevisiae yield a molecular weight of 2 × 10 9 for the largest DNA, assuming linear unbranched DNA, and of 4.3 × 10 9 assuming circular unbranched DNA. Both log and stationary-phase cells give the same results. Comparison of these results with the nuclear DNA content of S. cerevisiae determined by renaturation kinetics suggests that the largest piece of DNA in the yeast nucleus may, at least during part of the cell cycle, consist of from one-fourth to all of the yeast genome.

Characterization of a blue-green algal genome

The following properties of the genomic DNA of the unicellular blue-green alga Agmenellum quadruplicatum have been determined: (1) buoyant density in neutral CsCl (1·701 2 g/cm 3 ); (2) thermal denaturation profile ( T m =89·2°C); (3) kinetic complexity (2·2×10 9 to 2·8×10 9 daltons); (4) quantity per cell (8×10 9 to 13×10 9 daltons); and (5) molecular weight (3·9×10 9 ). These experiments indicate that blue-green algal DNA is similar to that of bacteria in the following ways: (1) there is little base composition heterogeneity present; (2) repeated sequences are below the level of detection; and (3) the size of the chromosomal DNA is most likely equal to the kinetic complexity. Our studies do suggest, however, that Agmenellum unlike common bacteria, may have a basal DNA content of two or more chromosomes per cell.

Physical Studies on DNA From “Primitive” Eucaryote

AbstractINTRODUCTIONStudies carried out over the years on bacteria and higher eucaryotes show several major differences between the two types of organisms in the nature of the DNA and chromosomes. Some of the differences are1. The existence of a large percentage of interspersed, repetitive sequences in higher eucaryotes but not in bacteria.2. The existence of chromatin containing large amounts of basic (histone) and nonhistone chromosomal proteins in higher eucaryotes but not in bacteria.3. Bidirectional DNA replication from many initiation sites found in higher eucaryotes as compared to the single initiation site found in bacteria.

A unified theory of nucleation-rate-limited DNA renaturation kinetics

DNA renaturations under nucleation-rate-limiting conditions on simple DNA such as bacterial and bacteriophage DNA show significant deviation from ideal second-order kinetics when followed by optical density measurements at 260 nm. Ideal second-order kinetics yield linear plots when the data is plotted in the standard reciprocal second-order (RSO) manner. The observed deviations from ideal second-order behavior take the form of steadily downward-curving RSO plots. In this paper, experiments are presented for E. coli and T2 DNA documenting this non-ideal behavior. Since experiments using T4, T5 and B, subtilis DNA yield identical non-ideal behavior, this behavior appears to be a property of DNA renaturation followed by optical density, not a peculiarity of a particular DNA. Identical non-ideal behavior is also seen in kinetics followed by S1 nuclease assay. A theory is developed to explain this deviation from ideal second-order kinetics. The theory also explains why kinetics followed by hydroxyapatite chromatography show nearly ideal second-order kinetics. In contrast to the approach taken by others in developing equations that describe S1 nuclease monitored reactions, our view is that nonideal second-order kinetics are fundamentally due to the reacton of free single strands to yield partially helical duplex species. Later reactions of these species tend to reduce the deviations from non-ideal second-order kinetics.

A more complete kinetic theory of DNA renaturation

We develop here a more complete theory of DNA renaturation kinetics which differs from the Wetmur and Davidson theory mainly in the assumption that nucleation is not necessarily the rate−limiting step. Thus, the theory includes the several first order zipping steps. Our assumption that zipping is much slower than previously thought appears to be due to the presence of secondary structure in reacting single strands. The theory explains several anomalies seen in renaturation kinetic experiments followed by optical density. Specifically, it explains quantitatively (1) the deviations from linearity in reciprocal−second−order (RSO) plots, (2) the variation with concentration of rate constants obtained from the early−time linear region of an RSO plot, and (3) the high values of complexity calculated for viral DNAs using bacterial DNAs as the known standard. Renaturation kinetic experiments followed by hydroxypatite chromatography do not show these anomalies, since hydroxyapatite presumably ’’sees’’ only nucleat...

Retardation time measurements on replicating Bacillus subtilis chromosomes: Effect of EDTA concentration

We have found that high concentrations of EDTA (greater than 0.024 M) are necessary to produce large, constant numbers of intact replicating Bacillus subtilis chromosomes in lysates of log phase cells. The retardation time of replicating chromosomes in log phase cell lysates is about double that for chromosomes in stationary phase cell lysates, thus making measurement of retardation time a sensitive way to detect and study replicating chromosomes. A theory is developed to predict retardation times for many possible models of DNA replication. The retardation time data on log phase cells is sufficient to eliminate many replication models, but many possibilities remain.