DNA replication and transcription of adenovirus (Ad) have been studied extensively as a model eukaryotic system. The dissection and reconstitution of the cell-free DNA replication system using the Ad DNA terminal protein complex (Ad DNAprot) have revealed the detailed mechanism of Ad genome replication (1-3). The Ad genome is a linear DNA of '36 kbp that contains 55-kDa terminal proteins covalently attached to its 5' ends. Replication of the Ad DNA-prot initiates by a protein-priming mechanism in which the 5' terminal nucleotide of the nascent DNA, dCMP, is linked to the 80-kDa

Abstract. The Saccharomyces cerevisiae mutant strains blocked in the protein secretion pathway are not able to induce sexual aggregation. We have utilized the defect of aggregation to concentrate the secretion-deficient cells and identified a new gene which functions in the process of intracellular protein transport. The new mutant, usol, is temperature sensitive for growth and protein secretion. At the restrictive temperature (37°C), usol mutant accumulated the core-glycosylated precursor form of the exported protein invertase in the cells. Ultrastructural study of the mutant fixed by the freeze-substitution method revealed expansion of the nuclear envelope lumen and accumulation of the ER at the restrictive temperature. Abnormally oriented bundles of microtubules were often found in the nucleus. The US01 gene was cloned by complementation of the usol temperature-sensitive growth defect. DNA sequence analysis revealed a hydrophilic protein of 1790 amino acids with a COOHterminal 1,100-amino acid-long t~-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins. These observations suggest that Usol protein plays a role as a cytoskeletal component in the protein transport from the ER to the later secretory compartments.

We determined the nucleotide sequence of a 1.9-kilobase fragment of Pseudomonas paucimobilis SYK6 chromosomal DNA that included genes encoding protocatechuate 4,5-dioxygenase, the enzyme responsible for the aromatic ring fission of protocatechuate. Two open reading frames of 417 and 906 base pairs were found that had no homology with previously reported sequences, including those encoding protocatechuate 3,4-dioxygenase. Since both open reading frames were indispensable for the enzyme activity, they should encode the subunits of protocatechuate 4,5-dioxygenase. We named these genes ligA and ligB. Protocatechuate 4,5-dioxygenase was efficiently expressed in Escherichia coli with the aid of the lac promoter, and the polypeptides of the ligA and ligB gene products were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid sequencing.