George Eggleston Professor of Biochemistry
Chair, Department of Molecular Biology, Cell Biology and Biochemistry
Ph.D., Yale University, 1970
Tel. (401) 863-2359; Fax (401) 863-2921; E-mail: [email protected]

Research Summary

Tools of molecular biology allow us to analyze the structure, function, and evolution of eukaryotic nucleic acids. Currently, there are two main projects in which we are involved:

DNA Replication. Initiation of DNA synthesis is the major check point in the cell cycle: the cell is committed to divide once the genome has been replicated. Are there specific regions and sequences where DNA synthesis will start? To address this question, we are using the DNA puffs of the giant salivary gland chromosomes of the fly Sciara coprophila. DNA puffs represent sites of intrachromosomal gene amplification and are an excellent model for study of DNA replication. We have mapped the origin of amplification by 2-D gels, by a 3-D gel method we developed, and by PCR analysis. The origin contracts from an 8 Kb zone of initiation at pre-amplification to 1 Kb at amplification stage. The left boundary is the same at both stages and contraction in the initiation zone when DNA amplification starts reflects the change in position of the right boundary which correlates with the appearance there of RNA polymerase II. We are exploring further the interplay between regulation of replication and transcription.

How does DNA amplification override the controls that ensure that an origin fires once and only once per cell cycle? To understand the regulation of initiation, we developed Replication Initiation Point (RIP) mapping to identify the start sites of DNA synthesis at the nucleotide level. We have shown that in yeast and in metazoa (Sciara) the site of initiation of replication is directly adjacent to the Origin Recognition Complex (ORC) binding site. Preliminary data suggests that the steroid hormone, ecdysone, induces DNA amplification, providing the first example of hormonal regulation of DNA replication, and may provide a useful paradigm for understanding certain cancers in humans.

Ribosomal RNA. Previously, we determined the first sequence of a multicellular eukaryotic 28S ribosomal RNA (rRNA). We demonstrated that some nucleotide stretches as well as the secondary structure are conserved between the frog Xenopus and the bacterium E. coli, thereby identifying areas of likely functional importance. Our current studies deal with rRNA biogenesis. Specifically, U3 small nucleolar RNA (snoRNA) is localized in the nucleolus, and we have shown by injection of antisense oligonucleotides into Xenopus oocytes that it plays a role in rRNA processing, the mechanism of which we are presently studying. We demonstrated that U3 and other box C/D snoRNAs require the box C/D signature motif for nucleolar localization. We have also identified the Nucleolar Localization Elements (NoLEs) for box H/ACA snoRNAs and for spliceosomal snRNAs.

Once U3 snoRNA has arrived at its nucleolar destination, it influences the order of cleavages to process pre-rRNA, may act as a chaperone to prevent premature pseudoknot formation in 18S rRNA, and is a molecular bridge to draw together the 5' and 3' ends of 18S rRNA in the precursor. U3 snoRNA docks on pre-rRNA by base-pairing interactions that are species specific, and may provide an application for design of a new class of antibiotics against eukaryotic pathogens.

Representative Publications:

DNA Replication:

A.-K. Bielinsky and S.A. Gerbi. (1998). Discrete start sites for DNA synthesis in the yeast ARSI origin. Science 279: 95-98.

A.-K. Bielinsky and S.A. Gerbi. (1999). Chromosomal ARSI has a single leading strand start site. Molecular Cell 3: 477-486.

S.A. Gerbi, A.-K. Bielinsky, C. Liang, V.V. Lunyak and F.D. Urnov. (1999). Methods to map origins of replication in eukaryotes. In Eukaryotic DNA Replication: a Practical Approach (ed., S. Cotterill), Oxford University Press, pp. 1-42.

A.-K. Bielinsky and S.A. Gerbi (2000). Where it all starts: eukaryotic origins of DNA replication. Journal of Cell Science 114:643-651.

E.H. Mok, H.S. Smith, S.M. DiBartolomeis, A.W. Kerrebrock, L.J. Rothschild, T.S. Lange and S.A. Gerbi (2000). Maintenance of the DNA puff expanded state is independent of active replication and transcription. Chromosoma (W. Beermann Memorial Issue) 110: 186-196.

A.-K. Bielinsky, H. Blitzblau, E.L. Beall, M. Ezrokhi, H.S. Smith, M.R. Botchan and S.A. Gerbi. (2001). Origin recognition complex binding to a metazoan replication origin. Current Biology 11: 1427-1431.

S.A. Gerbi and A.-K. Bielinsky (2002). DNA replication and chromatin. Current Opinion in Genetics and Development 12: 243-248.

F.D. Urnov, C. Liang, H.G. Blitzblau, H.S. Smith and S.A. Gerbi (2002). A DNase I hypersensitive site flanks an origin of DNA replication and amplification in Sciara. Chromosoma (A. Wolffe Memorial Issue) 111: 291-303.

V.V. Lunyak, M. Ezrokhi, H.S. Smith and S.A. Gerbi (2002). Developmental changes in the Sciara II/9A initiation zone for DNA replication. Molecular and Cellular Biology 22: 8426-8437.

S.A. Gerbi, Z. Strezoska and J.M. Waggener (2002). Initiation of DNA replication in multicellular eukaryotics. Journal of Structural Biology 140: 17-30.

rRNA Processing:

A.V. Borovjagin and S.A. Gerbi. (1999). U3 small nucleolar RNA is essential for cleavage at sites 1, 2 and 3 in pre-rRNA and determines which rRNA processing pathway is taken in Xenopus oocytes. Journal of Molecular Biology 286: 1347-1363.

A.V. Borovjagin and S.A. Gerbi. (2000). The spacing between functional cis-elements of U3 snoRNA is critical for rRNA processing. Journal of Molecular Biology 300: 57-74.

A.V. Borovjagin, and S.A. Gerbi (2001). Xenopus U3 snoRNA GAC-Box A' and Box A sequences play distinct functional roles in rRNA processing. Molecular and Cellular Biology 21: 6210-6221.

S.A. Gerbi, A.V. Borovjagin, M. Ezrokhi and T.S. Lange. (2002). Ribosome biogenesis: role of small nucleolar RNA in maturation of eukaryotic rRNA. Cold Spring Harbor Symposium on Quantitative Biology LXVI: 575-590.

S.A. Gerbi, A.V. Borovjagin and T.S. Lange (2003). The nucleolus - a site of ribonucleoprotein maturation. Current Opinions in Cell and Developmental Biology (in press).

S.A. Gerbi and A.V. Borovjagin (2003). Ribosomal RNA processing in vertebrates. In The Nucleolus (ed: M.D.J. Olson), Landes Biosciences Publishing (in press).

Nucleolar Localization of RNAs:

T.S. Lange, A. Borovjagin, E.S. Maxwell and S.A. Gerbi. (1998). Conserved Boxes C and D are essential nucleolar localization elements of U8 and U14 snoRNAs. EMBO Journal 17: 3176-3187.

T.S. Lange, A. V. Borovjagin and S.A. Gerbi. (1998). Nucleolar localization elements in U8 snoRNA differ from sequences required for rRNA processing. RNA 4: 789-800.

T.S. Lange, M. Ezrokhi, A. V. Borovjagin, R. Rivera-León, M.T. North and S.A. Gerbi. (1998). (including journal cover) Nucleolar localization elements of Xenopus laevis U3 small nucleolar RNA. Molecular Biology of the Cell 9:2973-2985.

T.S. Lange, M. Ezrokhi, F. Amaldi and S.A. Gerbi. (1999). Box H and Box ACA are nucleolar localization elements of U17 snoRNA. Molecular Biology of the Cell 10: 3877-3890.

T.S. Lange and S.A. Gerbi (2000). Transient localization of U6 small nucleolar RNA in Xenopus laevis oocytes. Molecular Biology of the Cell 11: 2419-2428.

S.A. Gerbi and T.S. Lange (2002). All small nuclear RNAs (snRNAs) of the [U4/U6.U5] tri-snRNP localize to nucleoli; identification of the nucleolar localization element of U6 snRNA. Molecular Biology of the Cell 13: 3123-3137.