Developmentally regulated transcription factors in Drosophila melanogaster

During early stages of Drosophila development the heat shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo, it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a nuclear transport protein Drosophila karyopherin-α3(dKap- α3). Biochemical methods demonstrate that the dKap-α3 protein binds specifically to the dHSF's nuclear localization sequence (NLS). Furthermore, the dKap-α3 protein does not associate with NLSs that contain point mutations which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-α3.Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-α3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.

The pair-rule gene fushi tarazu (ftz) is a well-studied zygotic segmentation gene that is necessary for the development of the even-numbered parasegments in Drosophila melanogastor. During early embryogenesis, ftz is expressed in a characteristic pattern of seven stripes, one in each of the even-numbered parasegments. With a view to understand how ftz is transcriptionally regulated, cDNAs that encode transcription factors that bind to the zebra element of the ftz promoter have been cloned. Chapter Ill reports the cloning and characterization of the eDNA encoding zeb-1 (zebra element binding protein), a novel steroid receptor-like molecule that specifically binds to a key regulatory element of the ftz promoter. In transient transfection assays employing Drosophila tissue culture cells, it has been shown that zeb-1 as well as a truncated zeb-1 polypeptide (zeb480) that lacks the putative ligand binding domain function as sequencespecific trans-activators of the ftz gene.

The Oct factors are members of the POU family of transcription factors that are shown to play important roles during development in mammals. Chapter IV reports the eDNA cloning and expression of a Drosophila Oct transcription factor. Whole mount in-situ hybridization experiments revealed that the spatial expression patterns of this gene during embryonic development have not yet been observed for any other gene. In early embryogenesis, its transcripts are transiently expressed as a wide uniform band from 20-40% of the egg length, very similar to that of gap genes. This pattern progressively resolves into a series of narrower stripes followed by expression in fourteen stripes. Subsequently, transcripts from this gene are expressed in the central nervous system and the brain. When expressed in the yeast Saccharomyces cerevisiae, this Drosophila factor functions as a strong, octamer-dependent activator of transcription. The data strongly suggest possible functions for the Oct factor in pattern formation in Drosophila that might transcend the boundaries of genetically defined segmentation genes.

Enzyme induction in Neurospora crassa

I. Studies on Nicotinamide Adenine Dinucleotide Glycohydrase (NADase)

NADase, like tyrosinase and L-amino acid oxidase, is not present in two day old cultures of wild type Neurospora, but it is coinduced with those two enzymes during starvation in phosphate buffer. The induction of NADase, like tyrosinase, is inhibited by puromycin. The induction of all three enzymes is inhibited by actinomycin D. These results suggest that NADase is synthesized de novo during induction as has been shown directly for tyrosinase. NADase induction differs in being inhibited by certain amino acids.

The tyrosinaseless mutant ty-1 contains a non-dialyzable, heat labile inhibitor of NADase. A new mutant, P110A, synthesizes NADase and L-amino acid oxidase while growing. A second strain, pe, fl;cot, makes NADase while growing. Both strains can be induced to make the other enzymes. These two strains prove that the control of these three enzymes is divisible. The strain P110A makes NADase even when grown in the presence of Tween 80. The synthesis of both NADase and L-amino acid oxidase by P110A is suppressed by complete medium. The theory of control of the synthesis of the enzymes is discussed.

II. Studies with EDTA

Neurospora tyrosinase contains copper but, unlike other phenol oxidases, this copper has never been removed reversibly. It was thought that the apo-enzyme might be made in vivo in the absence of copper. Therefore cultures were treated with EDTA to remove copper before the enzyme was induced. Although no apo-tyrosinase was detected, new information on the induction process was obtained.

A treatment of Neurospora with 0.5% EDTA pH 7, inhibits the subsequent induction during starvation in phosphate buffer of tyrosinase, L-amino acid oxidase and NADase. The inhibition of tyrosinase and L-amino acid oxidase induction is completely reversed by adding 5 x 10^-5M CaCl₂, 5 x 10^-4M CuSO₄, and a mixture of L-amino acids (2 x 10^-3M each) to the buffer. Tyrosinase induction is also fully restored by 5 x 10^-4M CaCl₂ and amino acids. As yet NADase has been only partially restored.

The copper probably acts by sequestering EDTA left in the mycelium and may be replaced by nickel. The EDTA apparently removes some calcium from the mycelium, which the added calcium replaces. Magnesium cannot replace calcium. The amino acids probably replace endogenous amino acids lost to the buffer after the EDTA treatment.

The EDTA treatment also increases permeability, thereby increasing the sensitivity of induction to inhibition by actinomycin D and allowing cell contents to be lost to the induction buffer. EDTA treatment also inhibits the uptake of exogenous amino acids and their incorporation into proteins.

The lag period that precedes the first appearance of tyrosinase is demonstrated to be a separate dynamic phase of induction. It requires oxygen. It is inhibited by EDTA, but can be completed after EDTA treatment in the presence of 5 x 10^-5M CaCl₂ alone, although no tyrosinase is synthesized under these conditions.

The time course of induction has an early exponential phase suggesting an autocatalytic mechanism of induction.

The mode of action of EDTA, the process of induction and the kinetics of induction are discussed.