Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi.

Tungsten microprojectiles coated with nucleic acid and accelerated to velocities of approximately 500 m/s, can penetrate living cells and tissues with consequent expression of the introduced genes (Klein et al. 1987). Saccharomyces cerevisiae is used here as a model system to define the basic parameters governing the biolistic (biological-ballistic) delivery of DNA into cells. Among the physical factors affecting the efficiency of the process in yeast are the microprojectile's constitution, size, concentration and amount, and the procedure used for binding DNA to it. The biological parameters that affect the process include the cell's genotype, growth phase, plating density, and the osmotic composition of the medium during bombardment. By optimizing these physical and biological parameters, rates of transformation between 10(-5) and 10(-4) were achieved. Stable nuclear transformants result primarily from penetration of single particles of 0.5-0.65 micron in diameter, delivering on average 10-30 biologically active plasmids into the cell. The tungsten particles detectably increase the buoyant density of the transformants' progenitors.

Structure and evolution of prokaryotic and eukaryotic RNA polymerases: a model.

A comparative overview of the subunit taxonomy and sequences of eukaryotic and prokaryotic RNA polymerases indicates the presence of a core structure conserved between both sets of enzymes. The differentiation between prokaryotic and eukaryotic polymerases is ascribed to domains and subunits peripheral to the largely conserved central structure. Possible subunit and domain functions are outlined. The core's flexible shape is largely determined by the elongated architecture of the two largest subunits, which can be oriented along the DNA axis with their bulkier amino-terminal head regions looking towards the 3' end of the gene to be transcribed and their more slender carboxyl-terminal domains at the tail end of the enzyme. The two largest prokaryotic subunits appear originally derived from a single gene.

Purification of the three nuclear RNA polymerases from Neurospora crassa.

Nuclear RNA polymerases I, II, and III from Neurospora crassa have been purified 3,000-, 1,500-, and 10,000-fold, respectively, by a procedure that minimizes proteolysis of the 220-kDa subunit of polymerase II. The Neurospora enzymes resemble, in polypeptide composition, the corresponding polymerases isolated from other eukaryotes. The 220-kDa subunit of Neurospora polymerase II cross-reacts with antisera directed against the 220-kDa subunits of type II polymerases from Drosophila and wheat germ. However, the proteolyzed 180-kDa derivative of the Neurospora 220-kDa subunit fails to cross-react with the heterologous antisera, suggesting that the region removed by proteolysis contains or contributes to structural features of the enzyme that have been highly conserved during evolution. A 700-kDa complex of 12 polypeptides was found associated with polymerase II during purification. The complex was eventually separated from the enzyme, and its properties suggest that it might be associated with polymerase II in the nucleus. We describe two additional examples of polypeptides associated in variable amounts with Neurospora polymerase II.

Structural studies on Neurospora RNA polymerases and associated proteins.

Extensive structural homology between the three nuclear RNA polymerases of Neurospora crassa has been observed by peptide and immunological analyses. Within each polymerase, we found structural similarity between subunits in the 65- to 75-kDa range and one of the two large subunits. We observed also that polymerase II, as isolated, is associated with a 700-kDa complex of 12 polypeptides which is localized in the nucleus. A 75-kDa subunit of the 700-kDa complex was found to be structurally related to the 220-kDa subunit of polymerase II. We suggest, on the basis of the in vitro association, the common nuclear localization and the structural homology, that the 700-kDa complex and polymerase II may be associated in vivo. Evidence is also presented which suggests that polymerase III may interact with chromatin via two of its smallest subunits. A simple procedure for isolating nuclei from Neurospora is described.

Effect of alpha-isopropylmalate on the synthesis of RNA and protein in Neurospora.

The leu-3/alpha-IPM (alpha-isopropylmalate) regulatory system, previously shown to control several genes of leucine, isoleucine, valine, and histidine biosynthesis, appears likely to be involved also in the regulation of overall RNA and protein synthesis in Neurospora. Upon addition of alpha-IPM the synthesis of all major species of stable RNA was found to be transiently inhibited by approximately 50%. A similar reduction was observed in overall protein synthesis. The inhibition was dependent in both cases on a functional leu-3 gene product, in conformance with previously established patterns of alpha-IPM dependent gene regulation. The overt resemblance of the phenomenon described here to the 'stringent response' of bacteria is noted but neither the mechanism of inhibition nor the precise role of alpha-IPM in the process has been established.