Gail D. Lauer

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.

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.

[34] Maximizing gene expression on a plasmid using recombination in Vitro

Publisher Summary This chapter discusses a method for maximizing gene expression on a plasmid using recombination in vitro . The amount of a gene product produced in transformed cells by plasmids carrying a given gene promoter fusion is dependent on the separation between the promoter and the gene. The chapter describes a method, utilizing a combination of restriction endonuclease cleavage and digestion with Escherichia coli ( E. coli ) exonuclease III (Exo III) and S1 nuclease, which allows to position a restriction fragment bearing the promoter of the lac Z gene of E. coli and the region coding for the SD sequence of the lac Z gene at virtually any distance in front of any cloned gene. This technique has been used to produce E. coli strains, which can provide up to 200,000 monomers of cro protein per transformed cell. It is useful in constructing plasmids that direct the production of large amounts of various E. coli proteins and proteins from other prokaryotic and even eukaryotic cells.

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.