Molecular cloning and expression during development of the Drosophila gene for the catalytic subunit of DNA polymerase epsilon.

We have cloned the genomic DNA and cDNA of Drosophila DNA polymerase epsilon (pol-epsilon) catalytic subunit (GenBank No. AB035512). The gene is separated into four exons by three short introns, and the open reading frame consists of 6660 base pairs (bp) capable of encoding a polypeptide of 2220 amino acid residues. The calculated molecular mass is 255018, similar to that of mammalian and yeast homologues. The deduced amino acid sequence of the pol-epsilon catalytic subunit shares approximately 41% identity with human and mouse homologues as well as significant homology those of C. elegans, S. cerevisiae and S. pombe. Similar to the pol-epsilon catalytic subunits from other species, the pol-epsilon catalytic subunit contains domains for DNA polymerization and 3'-5' exonuclease in the N-terminal region, and two potential zinc-finger domains in the C-terminal regions. Interestingly, a 38 amino acid sequence in the C-terminal region from amino acid positions 1823 to 1861 is similar to the site for Mycoplasma ATP binding and/or ATPase domain (GenBank No. P47365). Northern hybridization analysis indicated that the gene is expressed at the highest levels in unfertilized eggs, followed by zero to 4h embryos and adult females, and then embryos at other embryonic stages, instar larva stages and adult males. Low levels of the mRNA were also detected at the pupa stage. This pattern of expression is similar to those of DNA replication-related enzymes such as DNA polymerase alpha and delta except for the high level of expression in adult males.

A new DNA polymerase species from Drosophila melanogaster: a probable mus308 gene product.

Harris et al. [P.V. Harris, O.M. Mazina, E.A. Leonhardt, R.B. Case, J.B. Boyd, K.C. Burtis, Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes, Mol. Cell. Biol., 16 (1996) 5764-5771.] reported the molecular cloning of Drosophila mus308 gene, and its nucleotide and protein sequences similar to DNA polymerase I. In the present study, we attempted to find and isolate the gene product by purifying a DNA polymerase fraction not present in mus308 flies. A new DNA polymerase with properties different from those of any known polymerase species was identified and partially purified from the wild-type fly embryos through ten column chromatographies. The enzyme was resistant to aphidicolin, but sensitive to ddTTP and NEM. Human proliferating cell nuclear antigen (PCNA) and Drosophila replication protein A (RP-A) did not affect the polymerase activity. It preferred poly(dA)/oligo(dT) as a template-primer. The molecular mass was about 230 kDa with a broad peak region of 200 to 300 kDa in HiPrep16/30 Sephacryl S-300 gel filtration. These properties a different from those of all reported Drosophila polymerase classes such as alpha, beta, gamma, delta, epsilon and zeta and closely resemble those of the gene product expected from the nucleotide sequence. The new polymerase species appears to have ATPase and 3'-5' exonuclease activities as shown by the chromatographies.

DNA polymerase epsilon from Drosophila melanogaster.

We identified a DNA polymerase species in Drosophila melanogaster embryos, and purified it. This polymerase shared some common properties with DNA polymerase epsilon from mammals and yeast as follows; it has a preference for poly(dA)/oligo(dT) as a template/primer, it is highly processive in DNA synthesis, it co-fractionates with 3'-5' exonuclease activity, it is sensitive to aphidicolin and is resistance to ddTTP. The polymerase activity was inhibited in the immuno-precipitation assay with anti-pol-epsilon antibodies, which were produced against a polypeptide coded on the cDNA of a putative Drosophila pol-epsilon we isolated previously. Using these antibodies, Western blot analysis revealed that this polymerase is a 250kDa polypeptide, which is the same size as observed in mammals and yeast. These results indicate that Drosophila produces the epsilon-class of DNA polymerase, and like mammals or yeast, possesses the 5 typical classes of DNA polymerases (alpha to epsilon) in its embryos.

Drosophila mitochondrial transcription factor A: characterization of its cDNA and expression pattern during development.

We cloned a cDNA for Drosophila mitochondrial transcription factor A (D-mtTFA) and characterized the recombinant protein. In Drosophila Kc cells, D-mtTFA was localized in the mitochondria, but not in the nucleus. By repetitive precipitation with His-tag and PCR amplification, the consensus nucleotide sequence for D-mtTFA-binding was determined to be 5'-TTATC/G. The binding sequence was found to be clustered in the A + T region of mitochondrial DNA which is suggested to be a replication origin and promoter region for light strand and heavy strand. We found a DNA replication-related element (DRE)-like sequence located upstream of the transcription initiation site of the D-mtTFA gene and obtained results indicating that DRE-binding factor (DREF) can bind to the DRE-like sequence of the D-mtTFA gene. The data suggest that transcription of the D-mtTFA gene is under control of the DRE/DREF regulatory system. Based on these results, the functions of D-mtTFA were discussed in relation to mitochondrial biogenesis of Drosophila melanogaster.

Purification and characterization of a 100 kDa DNA polymerase from cauliflower inflorescence.

A DNA polymerase from cauliflower (Brassica oleracea var. botrytis) inflorescence has been purified to near homogeneity through five successive column chromatographies, and temporally designated cauliflower polymerase 1. Cauliflower polymerase 1 is a monopolypeptide with a molecular mass of 100 kDa. The enzyme efficiently uses synthetic DNA homopolymers and moderately activated DNA and a synthetic RNA homopolymer as template-primers. The enzyme is strongly sensitive to dideoxythymidine triphosphate and N-ethylmaleimide, but it is insensitive to aphidicolin. It was stimulated with 250 mM KCl. Its mode of DNA synthesis is high-processive with or without proliferating-cell nuclear antigen. A 3'-->5' exonuclease activity is associated with cauliflower polymerase 1. The enzyme is clearly different from cauliflower mitochondrial polymerase and does not resemble the four different types of wheat DNA polymerase, designated wheat DNA polymerases A, B, CI and CII. In the present paper the role of the enzyme in plant DNA synthesis is discussed.