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A novel DNA replicon in Escherichia coli was identified. It is the smallest natural isolate (1282 bp) found so far. In the presence of phage M13 it grows as a filamentous single‐stranded DNA phage. Contrary to previously identified mini‐phages this replicon displays sequence homology only to parts of the M13 viral and complementary strand origin. In the absence of M13 this DNA replicates autonomously. The only gene (arp) of the replicon encodes a 32‐kd protein, which is essential for autonomous replication. The host rep gene required for replication of single‐stranded DNA phages is dispensable. Distinct replication mechanisms are thus involved during growth as defective phage or as autonomous plasmid.

The start sites for leading and lagging DNA strands were determined in vitro with minichromosomes as templates. Fragments from replication intermediates were analyzed by hybridization to single‐stranded probes. Leading strand synthesis in the counterclockwise direction was found to originate in or close to (position 248 to ‐44) the minimal origin. Complementary lagging strand synthesis started several positions to the left outside of oriC. The results suggest in addition a concerted synthesis of leading and lagging strands following the dnaA directed assembly of initiation proteins at double‐stranded oricC DNA (pre‐replisome). In addition, DNA synthesis could initiate at protein n' recognition sequences located within and clockwise to the asnA gene. Initiation at n' sites was dependent on protein i activity, whereas leading and lagging strand initiation in the oriC region was not affected by protein i. Our results argue against an involvement of the phi X174‐type primosome in the initiation of discontinuous DNA synthesis at oriC. An alternative function is suggested.

In vitro replication of mini‐chromosomes in the absence of DNA ligase activity resulted in replication products with single‐strand breaks at specific sites. The occurrence of these nicks was coupled to an active replication process, therefore we expect them to represent start sites for DNA replication. Two positions within oriC for each of the two leading strands of bidirectional replication were found. Within each position are one or two start sites. Counterclockwise synthesis started at positions 194/199 and 265/272, clockwise synthesis at positions 209/219 and 254. The start positions are located close to DnaA protein binding sites. A model for initiation accommodating this observation is discussed.

We analyzed the functionality of different dnaA protein binding sites by assaying in vitro dnaA-dependent replication of pBR322 derivatives. Single dnaA sites from oriC and from the mioC and dnaA gene promoters were active when combined with the primer generating element of pBR322 in a proper distance. Prereplisome assembly did not require sequences in addition to the 9-base pair consensus dnaA binding site. Inversion of the structurally asymmetric dnaA site relative to its orientation in wild type pBR322 resulted in a marked reduction in replication efficiency, as observed with five different dnaA sites studied. The direction of DNA replication was not affected.

The UBC9 gene of the yeast Saccharomyces cerevisiae is essential for cell viability and encodes a soluble protein of the nucleus that is metabolically stable. Products of mutant alleles selected to confer temperature-sensitive in vivo function were found to be extremely short-lived at the restrictive but long-lived at the permissive condition. An extragenic suppressor mutation was isolated which increased thermoresistance of a ubc9-1 strain. This suppressor turned out to stabilize the mutated gene product, indicating that the physiological activity of ubc9-1 protein is primarily controlled by conditional proteolysis. The labile ubc9-1 protein appears to be a substrate for ubiquitination, and its turnover was substantially reduced by expression of a ubiquitin derivative that interferes with formation of multi-ubiquitin chains. Stabilization resulted also from competitive inhibition of Ubc4-related ubiquitin-conjugating enzymes. Activity of the proteasome complex was crucial to rapid breakdown, whereas vacuolar proteases were dispensable. Thus, the heat-denatured ubc9-1 protein is targeted for proteolysis by the ubiquitin-proteasome pathway and may serve as a useful tool to further define the process by which a misfolded polypeptide is recognized.

We report a novel human gene whose product specifically associates with the negative regulatory domain of the Wilms' tumor gene product (WT1) in a yeast two-hybrid screen and with WT1 in immunoprecipitation and glutathione S-transferase (GST) capture assays. The gene encodes a 17-kDa protein that has 56% amino acid sequence identity with yeast ubiquitin-conjugating enzyme (yUBC) 9, a protein required for cell cycle progression in yeast, and significant identity with other subfamilies of ubiquitin-conjugating enzymes. The human gene fully complements yeast that have a temperature-sensitive yUBC9 gene mutation to fully restore normal growth, indicating that we have cloned a functionally conserved human (h) homolog of yUBC9. Transcripts of hUBC9 of 4.4 kilobases (kb), 2.8 kb, and 1.3 kb were found in all human tissues tested. A single copy of the hUBC9 gene was found and localized to human chromosome 16p13.3. We conclude that hUBC9 retains striking structural and functional conservation with yUBC9 and suggest a possible link of the ubiquitin/proteosome proteolytic pathway and the WT1 transcriptional repressor system.