Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G 1 -phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G 1 -phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-␤). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-␤ was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-␤. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.Origins of DNA replication in animal viruses and single-cell eukaryotic organisms consist of specific cis-acting sequences that function independently (autonomously replicating sequences) when transferred to the appropriate cells or cell extract (25,26,58). However, attempts to identify similar sequences in multicellular eukaryotes have resulted in a paradox: while DNA synthesis is initiated at specific, genetically determined sites in cellular chromosomes, identification of cis-acting DNA sequences that control replication has proven elusive.Mapping initiation sites for DNA replication at 17 different locations in the chromosomes of mammals and flies has revealed that DNA synthesis initiates not randomly throughout cellular chromosomes but at specific DNA sites. These sites consist of a primary initiation locus referred to as the origin of bidirectional replication (OBR) surrounded by many secondary initiation loci distribute...

The yeast Candida albicans has a distinguishing feature, dimorphism, which is the ability to switch between two morphological forms: a budding yeast form and a multicellular invasive filamentous form. This ability has been postulated to contribute to the virulence of this organism. Studies on the morphological transition from a filamentous to a budding yeast form in C. albicans have shown that this organism excretes an autoregulatory substance into the culture medium. This substance was extracted and purified by normal-phase and reversed-phase HPLC. The autoregulatory substance was structurally identified as 3,7,11-trimethyl-2,6,10-dodecatrienoate (farnesoic acid) by NMR and mass spectrometry. Growth experiments suggest that this substance does not inhibit yeast cell growth but inhibits filamentous growth. These findings have implications for developmental signaling by the fungus and might have medicinal value in the development of antifungal therapies.

The DNA polymerase ␣-primase complex is the only enzyme that provides RNA-DNA primers for chromosomal DNA replication in eukaryotes. Mouse DNA polymerase ␣ has been shown to consist of four subunits, p180, p68, p54, and p46. To characterize the domain structures and subunit requirements for the assembly of the complex, we constructed eukaryotic polycistronic cDNA expression plasmids expressing pairwise the four subunits of DNA polymerase ␣. In addition, the constructs contained an internal ribosome entry site derived from poliovirus. The constructs were transfected in different combinations with vectors expressing single subunits to allow the simultaneous expression of three or four of the subunits in cultured mammalian cells. We demonstrate that the carboxyl-terminal region of p180 (residues 1235 to 1465) is essential for its interaction with both p68 and p54-p46 by immunohistochemical analysis and coprecipitation studies with antibodies. Mutations in the putative zinc fingers present in the carboxyl terminus of p180 abolished the interaction with p68 completely, although the mutants were still capable of interacting with p54-p46. Furthermore, the aminoterminal region (residues 1 to 329) and the carboxyl-terminal region (residues 1280 to 1465) were revealed to be dispensable for DNA polymerase activity. Thus, we can divide the p180 subunit into three domains. The first is the amino-terminal domain (residues 1 to 329), which is dispensable for both polymerase activity and subunit assembly. The second is the minimal core domain (residues 330 to 1279), required for polymerase activity. The third is the carboxyl-terminal domain (residues 1280 to 1465), which is dispensable for polymerase activity but required for the interaction with the other three subunits. Taken together, these results allow us to propose the first structural model for the DNA polymerase ␣-primase complex in terms of subunit assembly, domain structure, and stepwise formation at the cellular level.In mammalian cells, six distinct DNA polymerases, ␣, ␤, ␥, ␦, ε, and , have been cloned so far (3,13,42). Among these, DNA polymerases ␣, ␦, and ε are considered to be involved in chromosomal DNA replication. DNA polymerase ␣ is the only enzyme that is tightly coupled to DNA primase. Therefore, DNA polymerase ␣ has been considered to provide RNA-DNA primers for the initiation of leading-strand synthesis as well as Okazaki fragment synthesis on the lagging strand (12,34,42). By use of the simian virus 40 (SV40) in vitro DNA replication system, it was shown that DNA polymerase ␣ plays a role in the initiation of DNA synthesis by providing RNA-DNA primers for both leading-strand synthesis and laggingstrand synthesis and that DNA polymerase ␦ extensively elongates these primers through a polymerase switch mechanism (40). However, even though the precise roles of DNA polymerases ␣ and ␦ have been established for the SV40 DNA replication system, the way in which these enzymes function during replication of the chromosome is still not clear. Namely, we are ign...

A human homologue (GST1‐Hs) of the yeast GST1 gene that encodes a new GTP‐binding protein essential for the G1‐to‐S phase transition of the cell cycle was cloned from the cDNA library of human KB cells. The GST1‐Hs cDNA contained a 1497 bp open reading frame coding for a 499 amino acid protein with mol. wt 55,754 and with the amino acid sequence homologies of 52.3 and 37.8% to the GST1 protein and polypeptide chain elongation factor EF1 alpha respectively. The regions potentially responsible for GTP binding and GTP hydrolysis were conserved in the GST1‐Hs protein as well. When expressed in yeast cell, the GST1‐Hs gene could complement the ts phenotype of yeast gst1 mutant. GST1‐Hs and its mouse homologue were expressed in human fibroblasts and in various mouse cell types respectively, at relatively low levels in their quiescent states, and the level of those expressions increased rapidly, prior to the onset of DNA replication and the total RNA synthesis, when human or mouse fibroblasts were progressed out of the growth‐arrested state by the addition of serum. A possible role of GST1‐Hs in mammalian cell growth is discussed.