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S Elledge

Publications and source records attributed to S Elledge.

6 recordsLinked to original sources
PubMed2000

The multifunctional character of a geminivirus replication protein is reflected by its complex oligomerization properties.

Tomato golden mosaic virus (TGMV), a member of the geminivirus family, encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its plant host. TGMV AL1 is an oligomeric protein that binds double-stranded DNA and catalyzes cleavage and ligation of single-stranded DNA. The oligomerization domain, which is required for DNA binding, maps to a region that displays strong sequence and structural homology to other geminivirus Rep proteins. To assess the importance of conserved residues, we generated a series of site-directed mutations and analyzed their impact on AL1 function in vitro and in vivo. Two-hybrid experiments revealed that mutation of amino acids 157-159 inhibited AL1-AL1 interactions, whereas mutations at nearby residues reduced complex stability. Changes at positions 157-159 also disrupted interaction between the full-length mutant protein and a glutathione S-transferase-AL1 oligomerization domain fusion in insect cells. The mutations had no detectable effect on oligomerization when both proteins contained full-length AL1 sequences, indicating that AL1 complexes can be stabilized by amino acids outside of the oligomerization domain. Nearly all of the oligomerization domain mutants were inhibited or severely attenuated in their ability to support AL1-directed viral DNA replication. In contrast, the same mutants were enhanced for AL1-mediated transcriptional repression. The replication-defective AL1 mutants also interfered with replication of a TGMV A DNA encoding wild type AL1. Full-length mutant AL1 was more effective in the interference assays than truncated proteins containing the oligomerization domain. Together, these results suggested that different AL1 complexes mediate viral replication and transcriptional regulation and that replication interference involves multiple domains of the AL1 protein.

Amino Acid Sequence↗
PubMed1995

Fas-activated serine/threonine kinase (FAST) phosphorylates TIA-1 during Fas-mediated apoptosis.

We have identified a serine/threonine kinase that is rapidly activated during Fas-mediated apoptosis. Fas-activated serine/threonine kinase (FAST) is phosphorylated on serine and threonine residues in Jurkat cells. In response to Fas ligation, it is rapidly dephosphorylated and concomitantly activated to phosphorylate TIA-1, a nuclear RNA-binding protein that has been implicated as an effector of apoptosis. Phosphorylation of TIA-1 precedes the onset of DNA fragmentation, suggesting a role in signaling downstream events in the apoptotic program. Our results introduce Fast and TIA-1 as components of a molecular cascade involved in signaling Fas-mediated apoptosis.

Amino Acid Sequence↗
PubMed1994

Isolation of a cDNA encoding a metal response element binding protein using a novel expression cloning procedure: the one hybrid system.

A new method for isolation of cDNA clones encoding sequence-specific DNA-binding proteins is described. This method, the one-hybrid system, is based on the use of reporter genes whose transcription can be activated through synthetic cis elements recognized by the sought-after DNA-binding protein. These reporter genes are used for in vivo screening of a library of cDNAs fused to a DNA fragment encoding the GAL4 activation domain. cDNA clones expressing the appropriate fusion proteins lead to activation of these reporter genes in transformed yeast cells. We have used this approach to isolate a mammalian cDNA clone encoding a sequence-specific DNA-binding protein that recognizes the metal response elements (MREs) of the metallothionein (MT) genes. The protein encoded by this cDNA, M96, shows similarity to the trithorax proteins. Expression of a functional DNA-binding form of M96 requires Zn2+ ions. The recombinant protein binds to several different MREs but fails to recognize nonfunctional mutant MREs. M96 may be involved in the activation of MT genes in response to heavy-metal ions.

Amino Acid Sequence↗
PubMed1992

Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle.

Human cyclin E, originally identified on the basis of its ability to function as a G1 cyclin in budding yeast, associated with a cell cycle-regulated protein kinase in human cells. The cyclin E-associated kinase activity peaked during G1, before the appearance of cyclin A, and was diminished during exit from the cell cycle after differentiation or serum withdrawal. The major cyclin E-associated kinase in human cells was Cdk2 (cyclin-dependent kinase 2). The abundance of the cyclin E protein and the cyclin E-Cdk2 complex was maximal in G1 cells. These results provide further evidence that in all eukaryotes assembly of a cyclin-Cdk complex is an important step in the biochemical pathway that controls cell proliferation during G1.

Animals↗
PubMed1986

Rapid mapping of antigenic coding regions and constructing insertion mutations in yeast genes by mini-Tn10 "transplason" mutagenesis.

A "transplason" mutagenesis procedure was developed for the dual purposes of low resolution mapping of antigenic coding regions (using transposons) and constructing insertion mutations in yeast genes (by transplacement). Mini-Tn10 transposon derivatives containing both Escherichia coli and yeast selectable markers have been constructed. These elements are used to mutagenize lambda gt11 clones that express foreign antigens in E. coli. The transposition events are first selected in E. coli, and the effect of these insertions on antigen expression is used to locate the antigenic coding regions on the cloned DNA. Insertion mutations located within a desired yeast sequence are then substituted for the genomic copies by one-step gene transplacement. This provides a powerful method for rapidly mapping antigenic coding sequences of cloned genes and inactivating these genes in yeast to help determine their function. Several examples using this technique are presented.

Antibodies↗