Heat shock-induced repression of proteolysis: poly(A)-binding protein degradation patterns can illusorily suggest its specific loss during heat shock.

Poly(A)-binding protein (PABP) is highly susceptible to proteolysis during cell lysis of Drosophila tissue culture cells unless substantial amounts of proteolysis inhibitors are included in the extraction buffer. This intrinsic proteolytic activity is substantially reduced during heat shock. An artifactual appearance that poly(A)-binding protein is specifically degraded by heat shock can result. Several contradictory descriptions of PABP may also be related to its proteolysis. Repression of proteolysis is likely to reflect a physiologically significant regulatory event, based on recent examinations of HSP70 stability during and after heat shock.

Drosophila melanogaster poly(A)-binding protein: cDNA cloning reveals an unusually long 3'-untranslated region of the mRNA, also present in other eukaryotic species [corrected].

Two classes of cDNAs encoding the Drosophila melanogaster (poly(A)-binding protein (PABP), which differ in length due to different positions of their respective 3' ends, were isolated by screening an embryonic cDNA library. These cDNAs hybridize to a single chromosomal site at position 55B on the right arm of the second chromosome. A unique 3.8-kb PABP mRNA species was detected, indicating that 'long' cDNAs correspond to full-length cDNAs and that the 3'-untranslated region of the D. melanogaster mRNA is close to 1.5 kb long. The PABP transcript accumulates in oocytes, is maternally inherited by the embryo and present at every other developmental stage tested. The D. melanogaster PABP cDNAs contain a 1722-nt ORF encoding a 64-kDa protein. This protein contains four RNA-binding domains which show limited primary sequence divergence during evolution, in contrast to the C-terminal third of the protein. The strikingly long 3'-untranslated region of the D. melanogaster PABP mRNA is shown to exist also in other eukaryotes including vertebrate species. It suggests that important regulatory sequences intrinsic to the PABP mRNA are present within this 3'-untranslated region.

The closely related Drosophila sry beta and sry delta zinc finger proteins show differential embryonic expression and distinct patterns of binding sites on polytene chromosomes.

Serendipity (sry) beta (beta) and delta (delta) are two finger protein genes resulting from a duplication event. Comparison of their respective protein products shows interspersed blocks of conserved and divergent amino-acid sequences. The most extensively conserved region corresponds to the predicted DNA-binding domain which includes 6 contiguous fingers; no significant sequence conservation is found upstream and downstream of the protein-coding region. We have analysed the evolutionary divergence of the sry beta and delta proteins on two separate levels, their embryonic pattern of expression and their DNA-binding properties in vitro and in vivo. By using specific antibodies and transformant lines containing beta-galactosidase fusion genes, we show that the sry beta and sry delta proteins are maternally inherited and present in embryonic nuclei at the onset of zygotic transcription, suggesting that they are transcription factors involved in this process. Zygotic synthesis of the sry beta protein starts during nuclear division cycles 12-13, prior to cellularisation of the blastoderm, while the zygotic sry delta protein is not detectable before germ band extension (stage 10 embryos). Contrary to sry delta, the zygotic sry beta protein constitutes only a minor fraction of the total embryonic protein. The sry beta and delta proteins made in E. coli bind to DNA, with partly overlapping specificities. Their in vivo patterns of binding to DNA, visualised by immunostaining polytene chromosomes, differ both in the number and position of their binding sites. Thus changes in expression pattern and DNA-binding specificity have contributed to the evolution of the sry beta and delta genes.

Functional domains of bacteriophage Mu transposase: properties of C-terminal deletions.

We have generated a series of 3' deletions of a cloned copy of the bacteriophage Mu transposase (A) gene. The corresponding truncated proteins, expressed under the control of the lambda PI promoter, were analysed in vivo for their capacity to complement a super-infecting MuAam phage, both for lytic growth and lysogeny, and for their effect on growth of wild-type Mu following infection or induction of a lysogen. Using crude cell extracts, we have also examined binding properties of these proteins to the ends of Mu. The results allow us to further define regions of the protein important in replicative transposition, establishment of lysogeny and DNA binding.

The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth.

We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site-specific recombinational switches. Furthermore, we show that induction of a Mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucts62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage lambda.