Albrecht E. Sippel

Tissue specific and position independent expression of the complete gene domain for chicken lysozyme in transgenic mice.

A 21.5 kb DNA fragment carrying the entire chicken lysozyme gene locus was introduced into the germ line of mice. The fragment contains the transcribed region plus 11.5 kb 5′‐flanking and 5.5 kb 3′‐flanking sequences including all known cis‐regulatory elements and the 5′ and 3′ attachment elements (A‐elements) which define the borders of the DNase I sensitive chromatin domain. All sequences which adopt a DNase I hypersensitive chromatin conformation in vivo are present on the construct. Seven founder mice were analysed. All of these expressed chicken lysozyme RNA at high levels specifically in macrophages, as is the case in the donor species. Expression levels are dependent on the copy number of integrated genes indicating that a complete gene locus, as defined by its chromatin structure, functions as an independent regulatory unit when introduced into a heterologous genome.

A progesterone responsive element maps to the far upstream steroid dependent DNase hypersensitive site of chicken lysozyme chromatin.

We have investigated the influence of the 5′‐flanking region of the chicken lysozyme gene on steroid dependent gene expression. By transient transfection of lysozyme‐CAT fusion genes into the human breast cancer cell line T‐47D, a DNA element was identified which stimulates CAT expression when transfected cells are treated with progesterone. This element is distinct from a second hormone responsive element (HRE) located in the lysozyme promoter region; it activates the lysozyme and the TK promoter, irrespective of orientation and distance, and is therefore referred to as hormone responsive element on its own. The location of this newly discovered HRE between −2250 and −1815 relative to the transcriptional start site, corresponds to the position of a steroid inducible DNase I‐hypersensitive site in chromatin of oviduct cells. This observation suggests a physiological role for the upstream element. In vitro DNase I protection experiments revealed six binding sites for both progesterone and glucocorticoid receptors within the sequences of the upstream HRE. The three distal binding sites are not required for hormonal stimulation of the TK promoter, while the three proximal binding sites, which are contiguously arranged, work in a cooperative manner.

Drosophila and vertebrate myb proteins share two conserved regions, one of which functions as a DNA-binding domain

We report the nucleotide sequence of a cDNA clone of the Drosophila melanogaster homologue of c‐myb, a member of the class of vertebrate transforming genes encoding nuclear proteins. We predict the mol. wt of the Drosophila myb (D‐myb) protein to be 74,000. The D‐myb protein contains two clusters of sequences homologous to vertebrate myb proteins, surrounded by sequences lacking homology. These results extend previous evidence for the existence of a D. melanogaster homologue of c‐myb and identify two highly conserved and therefore presumably functionally important domains of c‐myb proteins. DNA‐binding experiments indicate that the NH2‐proximal of the two homology regions functions as a DNA‐binding domain. Based on the absence of the COOH‐proximal homology region in truncated oncogenic derivatives of c‐myb it is likely that this homology region encodes a function whose loss is involved in activating the oncogenic potential of c‐myb.

Identification and characterization of the protein encoded by the human c-myb proto-oncogene.

We have identified the product of the human c‐myb proto‐oncogene as a 80,000‐Mr protein, p80c‐myb, by using polyclonal and monoclonal antibodies raised against a bacterially synthesized polypeptide from the amino terminus of the viral myb protein. p80c‐myb shares at least two distinct antigenic sites with the amino terminal region of the v‐myb protein. p80c‐myb is found only in hematopoietic cells or in cells that contain amplified c‐myb genes. Like the chicken myb proteins, p80c‐myb is a nuclear DNA‐binding protein that is predominantly associated with chromatin and exhibits a short half‐life of approximately 1 hour.

Functional homology between the sequence-specific DNA-binding proteins nuclear factor I from HeLa cells and the TGGCA protein from chicken liver.

Nuclear factor I from HeLa cells, a protein with enhancing function in adenovirus DNA replication, and the chicken TGGCA protein are specific DNA‐binding proteins that were first detected by independent methods and that appeared to have similar DNA sequence specificity. To test whether they are homologous proteins from different species we have compared (i) their DNA binding properties and (ii) their function in reconstituted adenovirus DNA replication systems. Using deletion and substitution mutants derived from the DNA binding site on the adenovirus 2 inverted terminal repeat, it was found that the two proteins protect the same 24‐nucleotide region of both strands against DNase I digestion and that they have identical minimal recognition sequences of 15 bp containing dyad symmetry. Like nuclear factor I, the TGGCA protein enhances the initiation reaction of adenovirus 2 DNA replication in vitro in a DNA recognition site‐dependent manner.

The Structural and Functional Domain Organization of the Chicken Lysozyme Gene Locus

Chromatin, the nuclear form in which eukaryotic DNA is packaged with protein, is most commonly described as a regular structure of DNA wrapped around nucleosomal histone octamers connected by histone H1—covered linker DNA regions (Richmond et al. 1983). For higher order structures this 10-nm fiber of “beads on the string chromatin”is wound into “solenoids” with six nucleosomes per turn forming a 30 nm filament (McGhee et al. 1980). In metaphase chromosomes and in parts of the interphase nucleus even higher DNA compaction must exist. Metaphase or interphase chromatin was shown to be organized in large loops at their base attached to either chromosomal protein scaffolds or nuclear matrix material, respectively (Paulson and Laemmli 1977; Vogelstein et al. 1980). How are genes organized in respect to these structures? Which chromatin conformation permits the dynamic changes that are necessary for activation and specific regulation of transcription and replication? What is the biological relevance of the loop-domain organization of chromatin? These questions must be answered if we are to understand chromatin function on the level of molecules.

Chromatin Structure and Protein-DNA Interactions in the 5′-Flanking Region of the Chicken Lysozyme Gene

The exact mechanisms which ensure that a eukaryotic gene is expressed at a specific time in a specific cell are presently unknown. It is, however, generally assumed that the structural organization of chromatin determines the state of differentiation and activity of eukaryotic genes. Our work is aimed at understanding the molecular details of the processes which prepare genes to be differentially and coordinately expressed.

Rat antibodies as probes for the characterization of progesterone receptor A and B proteins from laying hen oviduct cytosol

The chicken oviduct contains two different hormone binding forms of the progesterone receptor, A and B. We have prepared rat antisera against both forms of the receptor partially purified from laying hen oviduct. The anti-progesterone receptor A antiserum reacts with both receptor forms on Western blots, while the anti-progesterone receptor B antiserum reacts mainly with the B form. Both antisera also react with the native progesterone receptor proteins as shown by sedimentation analysis of the antibody-receptor complexes. Receptors A and B are recognized on Western blots of total protein from dissolved tissue, indicating that both forms are likely to be physiological components. Epitope mapping experiments show that immunogenicity of both receptor molecules is restricted to structurally related protein domains of 28 kDa in receptor A and of 52 kDa in receptor B.