Susan H. Hardin

Replication Factor C Interacts with the C-terminal Side of Proliferating Cell Nuclear Antigen

Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and repair. It is a molecular matchmaker required for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymerases δ and ε. To elucidate the mode of RF-C binding to the PCNA clamp, modified forms of human PCNA were used that could be 32P-labeled in vitro either at the C or the N terminus. Using a kinase protection assay, we show that the heteropentameric calf thymus RF-C was able to protect the C-terminal region but not the N-terminal region of human PCNA from phosphorylation, suggesting that RF-C interacts with the PCNA face at which the C termini are located (C-side). A similar protection profile was obtained with the recently identified PCNA binding region (residues 478-712), but not with the DNA binding region (residues 366-477), of the human RF-C large subunit (Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messner, H., Khastilba, S., Hübscher, U., and Fotedar, A., (1996) EMBO J., 15, 4423-4433). Furthermore, we show that the RF-C 36 kDa subunit of human RF-C could interact independently with the C-side of PCNA. The RF-C large subunit from a third species, namely Drosophila melanogaster, interacted similarly with the modified human PCNA, indicating that the interaction between RF-C and PCNA is conserved through eukaryotic evolution.

Nucleotide modification at the γ-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase

The mechanism by which HIV-1 reverse transcriptase (HIV-RT) discriminates between the correct and incorrect nucleotide is not clearly understood. Chemically modified nucleotides containing 1-aminonaphthalene-5-sulfonate (ANS) attached to their γ-phosphate were synthesized and used to probe nucleotide selection by this error prone polymerase. Primer extension reactions provide direct evidence that the polymerase is able to incorporate the gamma-modified nucleotides. Forward mutation assays reveal a 6-fold reduction in the mutational frequency with the modified nucleotides, and specific base substitutions are dramatically reduced or eliminated. Molecular modeling illustrates potential interactions between critical residues within the polymerase active site and the modified nucleotides. Our data demonstrate that the fidelity of reverse transcriptase is improved using modified nucleotides, and we suggest that specific modifications to the γ-phosphate may be useful in designing new antiviral therapeutics or, more generally, as a tool for defining the structural role that the polymerase active site has on nucleotide selectivity.

Survey of transcripts in the adult Drosophila brain

BackgroundClassic methods of identifying genes involved in neural function include the laborious process of behavioral screening of mutagenized flies and then rescreening candidate lines for pleiotropic effects due to developmental defects. To accelerate the molecular analysis of brain function in Drosophila we constructed a cDNA library exclusively from adult brains. Our goal was to begin to develop a catalog of transcripts expressed in the brain. These transcripts are expected to contain a higher proportion of clones that are involved in neuronal function.ResultsThe library contains approximately 6.75 million independent clones. From our initial characterization of 271 randomly chosen clones, we expect that approximately 11% of the clones in this library will identify transcribed sequences not found in expressed sequence tag databases. Furthermore, 15% of these 271 clones are not among the 13,601 predicted Drosophila genes.ConclusionsOur analysis of this unique Drosophila brain library suggests that the number of genes may be underestimated in this organism. This work complements the Drosophila genome project by providing information that facilitates more complete annotation of the genomic sequence. This library should be a useful resource that will help in determining how basic brain functions operate at the molecular level.

[29]Spec proteins: Calcium-binding proteins in the embryonic ectoderm cells of sea urchins

Since their initial characterization several years ago, the Spec genes have served as models for the study of gene activation during sea urchin embryogenesis (Bruskin et al. 1981,1982). Seven or eight related Spec genes1 of Strongylocentrotus purpuratus are activated a few hours after fertilization and are expressed exclusively in cell lineages giving rise to aboral ectoderm (Hardin et al. 1988; Tomlinson and Klein 1990). This cell type is a squamous epithelium of the dorsal/lateral surfaces of the late-stage embryo and larva. Because they are representative of differentiation in aboral ectoderm, these genes have proven to be valuable markers for examining the ontogeny of this embryonic cell type (Nemer 1986; Hurley et al. 1989; Stephens et al. 1989; Wessel et al. 1989). Thus, it should be possible to trace the origins of Spec gene activation and hence aboral ectoderm specification back to maternal factors present in the egg. To this end, recent experiments have begun to dissect the cis elements and trans factors responsible for activating the Spec genes (Gan et al. 1990a,b; Tomlinson et al. 1990).

Macromolecular technologies: applications and improvements

A report on the Association of Biomolecular Resource Facilities (ABRF) meeting, San Diego, USA, 24-27 February, 2001.