Germ line transformation has been used to map the cis regulatory DNA elements responsible for the precise and evolutionarily stable developmental expression of the s18 chorion gene. Constructs ...containing chimeric combinations of Drosophila melanogaster and D. grimshawi DNA regions, as well as D. grimshawi sequences alone, can direct expression in the follicular epithelium, in an s18-specific temporal and spatial pattern. The results indicate that both positive and negative regulatory elements can function when transferred from D. grimshawi to D. melanogaster. The first ca. 100 bp of the 5'-flanking DNA region constitute a minimal, developmentally regulated promoter, expression of which is inhibited by the next 100-bp DNA segment and activated by positive elements located further upstream. Expression of the minimal promoter can also be enhanced by more distant chorion regulatory elements, provided the inhibitory DNA segment is absent.
The study of the lectin binding sites of ricin B chain and of other homologous members of the small gene family that make up ricin-like molecules has revealed a number of key contact residues ...involved in sugar binding. In particular, on the basis of data generated by the X-ray crystallographic structure of ricin, comparisons of sequence homologies to other ricin-like molecules and substrate binding studies with these molecules, it has been proposed that His248 of Ricinus communis agglutinin (RCA) B chain may interfere with galactose binding in the second binding domain of that lectin. To test that hypothesis, single binding domain 2 (SBD2) of ricin B chain was expressed as a gene 3 fusion protein on the surface of fd phage to measure directly the effect of mutational changes on this binding site. Replacement of tyrosine with histidine at amino acid position 248 of SBD2 of ricin B chain was shown to reduce lectin activity. The sequences of RCA and ricin B chains were aligned and compared with the tertiary structure of ricin B chain to select various mutations that were introduced as controls in the study. One of these controls, Leu247 to Val247, displayed increased affinity for galactosides. The role of sequence changes is discussed in relation to the structural and functional divergence in these molecules.
We present a total of 6.1 x 10(3) base-pairs of DNA sequences, encompassing the s18 gene and flanking regions within the autosomal chorion cluster of three Drosophila species. Against a background of ...extensive divergence in the intron and even in parts of the coding region, islands of strong sequence conservation are evident. These are particularly notable in the 5' flanking DNA where they extend to approximately -600 base-pairs from the transcription start site. The most conserved segment of the entire chorion cluster is 71 base-pairs in the s18 5' flanking DNA, which in D. melanogaster is part of a region defined functionally as containing amplification control elements (ACE3 region). Transformation analysis, using chimeric transposons of D. melanogaster and D. grimshawi DNA, revealed that amplification control elements of D. grimshawi can support amplification in D. melanogaster. The functionally defined ACE3 region of D. grimshawi includes the conserved 71 base-pair segment, but also non-conserved sequences further upstream, which apparently enhance amplification.
We present a total of 13 kb of DNA sequences, encompassing autosomal chorion genes and their flanking DNA in four species of the genus Drosophila. Against a background of extensive divergence in ...introns and even in parts of the coding regions, islands of strong conservation are evident in the proximal 5' flanking and 5' untranslated sequences. An extragenic region of strong conservation is seen downstream of the last chorion gene in the autosomal cluster. The conserved DNA elements may be related to the conserved regulatory features of this cluster, including gene amplification and tissue- and temporally regulated transcription.
Previous studies have demonstrated that mutations at amino acid position 128 of the simian virus 40 large T antigen can alter the subcellular localization of the antigen. A second domain in which ...mutations can alter localization of the nuclear antigen has been identified by mutations at amino acid positions 185, 186, and 199. Mutations in this region cause the polypeptide to accumulate in both the nucleus and cytoplasm of monkey cells. These T-antigen variants accumulate to near normal levels, but they don't bind to the simian virus 40 origin of DNA replication and are unable to mediate DNA replication. Furthermore, the altered tumor antigens can no longer transform secondary rat cells at normal efficiency, but they retain the ability to transform established mouse and rat cell lines.
