BIOLOGICAL OVERVIEW

R7 is the last of eight photoreceptor cells to differentiate in the fly's compound eye. The regulation of R7 fate determination is one of the best studied differentiation processes in Drosophila or in any developmental system. This is due to the many advantages it affords the researcher. First, it is a process clearly localized in time and space. Second, it takes place post mitotically, thus distinctly separating fate determination from cell cycle control. Finally, the inputs are relatively well defined and a precise geometry is involved in the cell-cell interactions.

sevenless expression is not limited to R7 cells: it has been detected in the R1 and R6 pair, the R3 and R4 pair, four cone cell precursors and mystery cells (cells that are left out of the differentiation process and ultimately under programmed cell death). Some sevenless expressing cells such as cone cell precursors are competent to become R7 cells but fail to do so since they do not contact R8 cells (expressing the ligand for Sevenless known as BOSS). In addition, there is a refractoriness of R1 and R6 cells in response to BOSS (Bride of Sevenless) signals; neither R1 nor R6 internalize BOSS. R1 and R6 can, however, respond to Sevenless signals when the SEV tyrosine kinase is activated constitutively (Basler, 1991) The refractoriness of R1 and R6 is relieved in Notch and rough mutant backgrounds, where multiple cells in individual clusters internalize Boss (Van Vactor, 1991). Furthermore, the spatiotemporal pattern of seven-up expression, confined as it is to the R1/R6 and R3/R4 fate, plays an essential role in controlling the number and cellular origin of the R7 neuron in the ommatidium (Hiromi, 1993).

Activation of Sevenless by BOSS results in the phosphorylation of Sevenless and the transduction of the Sevenless signal, through the ras pathway, into the nucleus. The docking protein DRK (Downstream of receptor kinase) binds to phosphorylated Sevenless, and participates in building a protein complex associated with Sevenless that includes the SOS (Son of sevenless) and Ras1 proteins. RAS (a GTP exchange factor) and its target RAF (a serine/threonine kinase) initiate a phosphorylation cascade that transduces and amplifies the Sevenless signal, resulting in activation of pathway targets.

What are the targets of the ras pathway? The list is long and varied. JUN cooperates with the ETS domain transcription factor Pointed to induce R7 fate (Treier, 1995). Acting counter to this activation is Yan, a negative regulator of R7 fate. Two novel proteins, Seven in absentia (SINA) (Carthew, 1994) and Phyllopod (Chang, 1995) are downstream targets of the ras pathway, but neither required the ras pathway for expression. Activation of the ras pathway also activates a gene coding for a basal transcription factor (dTFIIA-S, the small subunit of TFIIA) (Zeidler, 1996). Also involved in Sevenless signaling are Hsp83 (a Drosophila chaperone protein) and cdc37, which interacts with cell cycle genes (Cutforth, 1994). Cdc37, in concert with the mammalian homolog of Hsp83, has been shown to be required for Raf-1 function (Grannatikakis, 1999).

The Sevenless signaling which initiates R7 photoreceptor maturation, triggers a phosphorylation cascade that activates transcription factors, modifies cell cycle regulation, affects basal transcription factors and involves protein chaperones. Even the Notch pathway is brought into play to restrict the effects of a ligand-receptor interaction. The R7 pathway continues to yield new information in what has become an important model for cell differentiation (Yamamoto, 1994).

GENE STRUCTURE

Bases in 5' UTR -798

Exons - 12

Bases in 3' UTR - 322

PROTEIN STRUCTURE

Amino Acids - 3554

Structural Domains

The transmembrane domain stretches from residues 2042-2051. The C-terminal region shows a high degree of homology to all known protein tyrosine kinase domains (Bowtell, 1988).

Sevenless protein possesses protein tyrosine kinase activity. The protein is first synthesized as a 280-kDa glycoprotein precursor. This subsequently cleaves into two subunits that remain associated by noncovalent interations: a 220-kDa amino-terminal and a 58-kDa carboxyl-terminal. The 220-kDa subunit is glycosylated and contains most of the extracellular portion of the protein, and the 58-kDa subunit is composed of a small portion of the extracellular sequences and the intracellular protein tyrosine kinase domain, that includes an ATP binding site (Mullins, 1991). This complex is subsequently cleaved into either 49- or 48-kDa carboxyl-terminal fragments with concomitant degradation of the rest of the protein (Simon, 1989).

EVOLUTIONARY HOMOLOGS

The DNA sequence of the sevenless gene from Drosophila melanogaster has been compared with that of D. virilis. The two species diverged approximately 60 million years ago. There is 60% conservation in the extracellular domain and complete homology in the transmembrane domain. The kinase domains are 83% identical. 42 of the 43 cysteines in DM are conserved in DV, while DV has an additional 9 cysteine residues (Michael, 1990)

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