A novel potential cell surface receptor of the tyrosine kinase gene family has been identified and characterized by molecular cloning. Its primary sequence is very similar to that of the human epidermal growth factor receptor and the v-erbB oncogene product; the chromosomal location of the gene for this protein is coincident with the neu oncogene, which suggests that the two genes may be identical.

The RAD6 gene of the yeast Saccharomyces cerevisiae is required for a variety of cellular functions including DNA repair. The discovery that the RAD6 gene product can catalyse the covalent attachment of ubiquitin to other proteins suggests that the multiple functions of the RAD6 protein are mediated by its ubiquitin-conjugating activity.

Human Ha-ras1 cDNAs encoding normal and activated p21 polypeptides have been efficiently expressed in Escherichia coli and the biochemical activities associated with each polypeptide compared. In addition to the guanine nucleotide binding activity, normal p21 displays a GTPase activity which is selectively impaired by a mutation which activates its oncogenic potential.

Mutants in the gene CDC34 of the yeast Saccharomyces cerevisiae are defective in the transition from G1 to the S phase of the cell cycle. This gene was cloned and shown to encode a 295-residue protein that has substantial sequence similarity to the product of the yeast RAD6 gene. The RAD6 gene is required for a variety of cellular functions including DNA repair and was recently shown to encode a ubiquitin-conjugating enzyme. When produced in Escherichia coli, the CDC34 gene product catalyzed the covalent attachment of ubiquitin to histones H2A and H2B in vitro, demonstrating that the CDC34 protein is another distinct member of the family of ubiquitin-conjugating enzymes. The cell cycle function of CDC34 is thus likely to be mediated by the ubiquitin-conjugating activity of its product.

Mammalian tumours displaying multidrug resistance overexpress a plasma membrane protein (P-glycoprotein), which is encoded by the MDR1 gene and apparently functions as an energy-dependent drug efflux pump. Tissue-specific expression of MDR1 and other members of the MDR gene family has been observed in normal cells, suggesting a role for P-glycoproteins in secretion. We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a protein very similar to mammalian P-glycoproteins. Deletion of this gene resulted in sterility of MATa, but not of MAT alpha cells. Subsequent analysis revealed that the yeast P-glycoprotein is the product of the STE6 gene, a locus previously shown to be required in MATa cells for production of a-factor pheromone. Our findings suggest that the STE6 protein functions to export the hydrophobic a-factor lipopeptide in a manner analogous to the efflux of hydrophobic cytotoxic drugs catalysed by the related mammalian P-glycoprotein. Thus, the evolutionarily conserved family of MDR-like genes, including the hlyB gene of Escherichia coli and the STE6 gene of S. cerevisiae, encodes components of secretory pathways distinct from the classical, signal sequence-dependent protein translocation system.

Kirsten (Ki)-ras cDNA clones were prepared from human lung and colon carcinoma cell lines expressing an activated c-Ki-ras2 gene. DNA sequence analysis and transfection studies indicate that different point mutations at the same codon can activate the gene; that most human c-Ki-ras2 mRNA uses sequences from a fourth coding exon distinct from that of its viral counterpart; and that at least one cell line is functionally homozygous for the activated gene.

Gene transfer vectors based on the replication-defective human parvovirus, adeno-associated virus type 2 (AAV-2), are viable candidates for in vivo and ex vivo human use. However, widespread testing of AAV vectors has been limited by difficulties in generating pure, high-titer vector stocks that are fully characterized. To address these issues, we have developed a single-step purification scheme using heparin affinity chromatography. Recovery from the crude lysate starting material exceeds 70%, and the end product rAAV vector is highly purified and appears to be free of adenovirus and cellular contaminates. Importantly, purified vectors retain predicted in vivo biologic activity. Concurrently, we have developed simple and rapid approaches for vector quantification using real-time PCR. These new methods, combined with the use of stable producer cell lines for rAAV production, make the commercial production of rAAV vectors for human use truly viable and pragmatic.

Ubiquitin (Ub) activation by the Ub-activating (E1) enzyme is the initial and essential step common to all of the known processes that involve post-translational conjugation of Ub to itself or other proteins. The "activated" Ub, linked via a thioester bond to a specific cysteine residue in one of several Ub-conjugating (E2) enzymes, which catalyze the formation of isopeptide bonds between the C-terminal glycine of Ub and lysine residues of acceptor proteins. In the yeast Saccharomyces cerevisiae, a 114-kDa E1 enzyme is encoded by an essential gene termed UBA1 (McGrath, J.P., Jentsch, S., and Varshavsky, A. (1991) EMBO J. 10, 227-236). We describe the isolation and analysis of another essential gene, termed UBA2, that encodes a 71-kDa protein with extensive sequence similarities to both the UBA1-encoded yeast E1 and E1 enzymes of other organisms. The regions of similarities between Uba1p and Uba2p encompass a putative ATP-binding site as well as a sequence that is highly conserved between the known E1 enzymes and contains the active-site cysteine of E1. This cysteine is shown to be required for an essential function of Uba2p, suggesting that Uba2p-catalyzed reactions involved a transient thioester bond between Uba2p and either Ub or another protein. Uba2p is located largely in the nucleus. The putative nuclear localization signal of Uba2p is near its C terminus. The Uba1p (E1 enzyme) and Uba2p cannot complement each others essential functions even if their subcellular localization is altered by mutagenesis. Uba2p appears to interact with itself and several other S. cerevisiae proteins with apparent molecular masses of 52, 63, 87, and 120 kDa. Uba2p is multiubiquitinated in vivo, suggesting that at least a fraction of Uba2p is metabolically unstable. Uba2p is likely to be a component of the Ub system that functions as either an E2 or E1/E2 enzyme.