We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained.

/pdf/the-scanning-model-for-translation-an-update-52r2in8l71.pdf

The scanning model for translation: an update.

https://www.cell.com/cell/pdf/0092-8674(92)90471-N.pdf

Homeobox genes and axial patterning

T URNOVER OF AB ERR ANT PROT EINS IN E. COLI . . . . . . . . . . . . . . . . . 445 The Lon Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446 DnaK. DnaJ. GrpE. GroEL and GroES . . . . . . . . . . . . . . . . . . . . . . . . . . 447 The Clp Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 The DegP Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

The Function of Heat-Shock Proteins in Stress Tolerance: Degradation and Reactivation of Damaged Proteins

Poliovirus RNA is naturally uncapped, therefore its translation must proceed via a cap-independent mechanism. Translation initiation on poliovirus RNA occurs by binding of ribosomes to an internal sequence within the 5' noncoding region. This novel mechanism of initiation may explain the disparate translation of several other eukaryotic messenger RNAs.

Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA

https://www.cell.com/cell/pdf/0092-8674(86)90464-2.pdf

Translational and transcriptional control elements in the untranslated leader of the heat-shock gene hsp22

Genetic and molecular studies on the expression of Antennapedia (Antp) have suggested that this gene specifies mainly the second thoracic segment. On the basis of our molecular analysis of dominant gain-of-function mutants we have postulated that the transformation of antennae into second legs is due to the ectopic overexpression of the Antp+ protein. This hypothesis was tested by inserting the complementary DNA encoding the normal Antp protein into a heat-shock expression vector and subsequent germ-line transformation. As predicted, heat induction at defined larval stages leads to the transformation of antennae into second legs. The dorsal part of the head can also be transformed into second thoracic structures (scutum) indicating that Antp indeed specifies the second thoracic segment. By ectopic overexpression of the Antp protein the body plan of the fruit fly can be altered in a predictable way.

Redesigning the body plan of Drosophila by ectopic expression of the homoeotic gene Antennapedia.

We describe new vectors suitable for P-element mediated germ line transformation of Drosophila melanogaster using passenger genes whose expression does not result in a readily detectable phenotypic change of the transformed flies. The P-element vectors contain the white gene fused to the heat shock protein 70 (hsp70) gene promoter. Expression of the white gene rescues the white phenotype of recipient flies partly or completely even without heat treatment. Transformed descendents of most founder animals (GO) fall into two classes which are distinguishable by their orange and red eye colours. The different levels of white expression are presumably due to position effects associated with different chromosomal sites of insertion. Doubling of the gene dose in orange eyed fly stocks results in an easily visible darkening of the eye colour. Consequently, the generation of homozygous transformants is easily possible by simple inbreeding due to the phenotypic distinction of homo- and heterozygous transformants. Cloning into these P-element vectors is facilitated by the presence of polylinkers with 8 and 12 unique restriction sites.

The white gene as a marker in a new P-element vector for gene transfer in Drosophila

One of the effects of a temperature increase above 35 degrees C on Drosophila melanogaster is a rapid switch in selectivity of the translational apparatus Protein synthesis from normal, but not from heat shock, mRNA is much reduced Efficient translation at high temperature might be a result of the primary sequence of heat shock genes Alternatively a mRNA modification mechanism, altered as a consequence of heat shock, might allow for efficient high temperature translation of any mRNA synthesized during a heat shock The gene for alcohol dehydrogenase (Adh) was fused to the controlling elements of a heat shock protein 70 (hsp70) gene Authentic Adh mRNA, synthesized from this fusion gene at elevated temperatures was not translated during heat shock A second Adh fusion gene in which the mRNA synthesized contained the first 95 nucleotides of the Hsp70 non-translated leader sequence gave rise, at high temperature, to mRNA which was translated during the heat shock Thus, the signal(s) in the mRNAs controlling translation efficiency at heat shock temperatures is encoded within the heat shock genes

Selective translation of heat shock mRNA in Drosophila melanogaster depends on sequence information in the leader.

The fusbi tarazu (ftz) protein of Drosopbila is required during embryogenesis for the process of body segmentation. To study the biochemical properties of the ftz protein, ftz cDNA was expressed in Escherichia coli and the protein was purified to homogeneity. Polyclonal antibodies raised against the purified protein were used to localize and quantitate the protein during embryogenesis. Three temporally and spatially distinct phases of expression were observed, which include a previously undetected period later in embryogenesis. During this last phase, the protein is localized predominantly in the developing hindgut. Analysis of embryonic ftz protein on Western blots permitted us to approximate the number of protein molecules per nucleus. During the blastoderm phase of development, when ftz protein is most abundant, we estimate that there are —20,000 molecules of protein per /tz-expressing nucleus. The embryonic ftz protein migrates more slowly on SDSpolyacrylamide gels than protein made either in E, coli or in a reticulocyte lysate system in vitro, indicating that it is modified in the embryo. To facilitate characterization of ftz protein made in embryos, an ftz overexpression system functional in Drosophila was developed. When fused to an hsp70 heat shock promoter and introduced into the germ line by P-element-mediated transformation, ftz could be overexpressed at all stages of development by heat shock. This protein is localized in the nucleus and comigrates on SDSpolyacrylamide gels with endogenous ftz protein. Two-dimensional gel electrophoresis followed by Western blotting resolves the overexpressed protein into a series of isoforms that differ in charge and electrophoretic mobility. Post-translational modification may influence the biochemical properties and functions of the ftz protein during embryogenesis.

/pdf/expression-modification-and-localization-of-the-fushi-tarazu-1awh3znd2z.pdf

Roman Klemenz

Papers

Translational and transcriptional control elements in the untranslated leader of the heat-shock gene hsp22

Redesigning the body plan of Drosophila by ectopic expression of the homoeotic gene Antennapedia.

The white gene as a marker in a new P-element vector for gene transfer in Drosophila

Selective translation of heat shock mRNA in Drosophila melanogaster depends on sequence information in the leader.

Expression, modification, and localization of the fushi tarazu protein in Drosophila embryos.