The homeobox gene cut interacts genetically with the homeotic genes proboscipedia and Antennapedia.

The cut locus (ct) codes for a homeodomain protein (Cut) and controls the identity of a subset of cells in the peripheral nervous system in Drosophila. During a screen to identify ct-interacting genes, we observed that flies containing a hypomorphic ct mutation and a heterozygous deletion of the Antennapedia complex exhibit a transformation of mouthparts into leg and antennal structures similar to that seen in homozygous proboscipedia (pb) mutants. The same phenotype is produced with all heterozygous pb alleles tested and is fully penetrant in two different ct mutant backgrounds. We show that this phenotype is accompanied by pronounced changes in the expression patterns of both ct and pb in labial discs. Furthermore, a significant proportion of ct mutant flies that are heterozygous for certain Antennapedia (Antp) alleles have thoracic defects that mimic loss-of-function Antp phenotypes, and ectopic expression of Cut in antennal discs results in ectopic Antp expression and a dominant Antp-like phenotype. Our results implicate ct in the regulation of expression and/or function of two homeotic genes and document a new role of ct in the control of segmental identity.

Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster.

Germ line transformation of white- Drosophila embryos with P-element vectors containing white expression cassettes results in flies with different eye color phenotypes due to position effects at the sites of transgene insertion. These position effects can be cured by specific DNA elements, such as the Drosophila scs and scs' elements, that have insulator activity in vivo. We have used this system to determine whether human matrix attachment regions (MARs) can function as insulator elements in vivo. Two different human MARs, from the apolipoprotein B and alpha1-antitrypsin loci, insulated white transgene expression from position effects in Drosophila melanogaster. Both elements reduced variability in transgene expression without enhancing levels of white gene expression. In contrast, expression of white transgenes containing human DNA segments without matrix-binding activity was highly variable in Drosophila transformants. These data indicate that human MARs can function as insulator elements in vivo.

cut interacts with Notch and protein kinase A to regulate egg chamber formation and to maintain germline cyst integrity during Drosophila oogenesis.

Communications between the germline and the soma during Drosophila oogenesis have been previously shown to be essential for the formation of egg chambers and to establish polarity in the developing oocyte. In this report, we demonstrate that the function of a somatically expressed gene, cut, is critical for maintaining the structural integrity of germline-derived cells and their arrangement within an egg chamber. Genetic manipulations of cut activity resulted in defective packaging of germline-derived cysts into egg chambers and disintegration of the structural organization of oocyte-nurse cell complexes to generate multinucleate germline-derived cells. We also found that cut interacts genetically with the Notch gene and with the catalytic subunit of Protein kinase A gene during egg chamber morphogenesis. Since cut expression is restricted to the somatic follicle cells and cut mutant germline clones are phenotypically normal, we propose that the defects in the assembly of egg chambers and the changes in germline cell morphology observed in cut mutant egg chambers are the result of altered interactions between follicle cells and germline cells. cut encodes a nuclear protein containing DNA-binding motifs, and we suggest that it participates in intercellular communications by regulating the expression of molecules that directly participate in this process.

Functional analysis of Drosophila and mammalian cut proteins in files.

The cut locus acts as a bimodal switch controlling cell fate in the peripheral nervous system of Drosophila and is also required for the development of the wing margin. It encodes a protein, Cut, that contains an atypical homeodomain and three copies of a new motif which can bind DNA in vitro. The human protein CDP and the murine protein Cux have recently been isolated as DNA-binding activities and they are structurally related to Cut. We show that ectopic expression of Cut, CDP, or Cux similarly affects embryonic sensory organ development and can rescue a wing scalloping mutant phenotype associated with loss of cut expression along the prospective wing margins. This suggests that the function of Cut is evolutionarily conserved.

Postembryonic patterns of expression of cut, a locus regulating sensory organ identity in Drosophila.

The cut locus is both necessary and sufficient to specify the identity of a class of sensory organs in Drosophila embryos. It is also expressed in and required for the development of a number of other embryonic tissues, such as the central nervous system, the Malpighian tubules and the tracheal system. We here describe the expression of cut in the precursors of adult sensory organs. We also show that cut is expressed in cells of the prospective wing margin and correlate the wing margin phenotype caused by two cut mutations with altered cut expression patterns. Finally, we observe cut-expressing cells in other adult tissues, including Malpighian tubules, muscles, the central nervous system and ovarian follicle cells.

Expression of the mouse mammary tumor virus ORF gene in cultured cells.

We have recently shown that expression vectors harboring the open reading frame of the long terminal repeat region of mouse mammary tumor virus direct the synthesis of a product which acts as a superantigen in transgenic mice. The detection of the ORF protein has been hampered by the extremely low levels of expression observed in these mice, as estimated from the low levels of specific mRNA. To study the properties of the ORF protein, we attempted its expression in different cell types in culture. The experiments performed in yeast show that the ORF gene product is a glycoprotein of approximately 45 kDA. As expected from the derived primary sequence, the unglycosylated product made in the presence of tunicamycin has a molecular weight of 36 kDA. No secretion of the glycosylated protein was observed. Curiously, the full-length molecule was made in lower amounts than a truncated version which contains only the C-terminal half of the protein. Transfection experiments in different mammalian cells suggest that high expression of the ORF protein might have an adverse effect on survival of cells in culture.

Transformation of sensory organ identity by ectopic expression of Cut in Drosophila.

The loss of cut activity results in a change in neural identity in the peripheral nervous system so that the neurons and support cells of external sensory (es) organs are transformed into those of internal chordotonal (ch) organs, cut encodes a large nuclear homeo domain protein (Cut) that is expressed in the differentiated cells of es organs and their precursors but not in the cells of ch organs. We now analyze the effects of ectopic Cut expression in transformant lines of flies containing the Cut-coding sequences under inducible regulatory control. We demonstrate that ubiquitous Cut expression in embryos results specifically in the morphologic and antigenic transformation of ch organs into es organs. This effect appears to involve positive autoregulation of Cut expression. We conclude that Cut is not only necessary but sufficient for the specification of es organ identify in sensory organ precursor cells and their progeny. The specificity of Cut function to sensory organ cells involves the proneural loci daughterless and the achaete-scute complex.

Clonal deletion of V beta 14-bearing T cells in mice transgenic for mammary tumour virus.

Autoreactive T lymphocytes are clonally deleted during maturation in the thymus. Deletion of T cells expressing particular receptor V beta elements is controlled by poorly defined autosomal dominant genes. A gene has now been identified by expression of transgenes in mice which causes deletion of V beta 14+ T cells. The gene lies in the open reading frame of the long terminal repeat of the mouse mammary tumour virus.

Patterns of expression of cut, a protein required for external sensory organ development in wild-type and cut mutant Drosophila embryos.

The loss of cut activity in Drosophila results in the transformation of the neurons and support cells of external sensory (es) organs into those of chordotonal (ch) organs. The cut locus encodes a homeo domain-containing protein, which is expressed in the cells of es, but not in ch, organs. We show by Western analyses the presence of two embryonic protein species whose approximate relative molecular masses of 280 and 320 kD are compatible with that predicted from the primary sequence. We also describe the development of the Cut protein expression pattern and show that Cut is expressed in sensory precursor cells that divide to give rise to es organs. Finally, we analyze the changes in the Cut expression pattern of several mutant alleles of the complex cut locus and show that the mutations affecting es organ development are associated with either altered protein distribution in the PNS or incorrect subcellular Cut protein localization.

Primary structure and expression of a product from cut, a locus involved in specifying sensory organ identity in Drosophila.

In the absence of cut gene activity in Drosophila, external sensory organs are transformed into chordotonal organs. Here we show that the cut locus encodes a large protein containing a homoeodomain and is expressed in nuclei of cells in external sensory organs but not in cells within chordotonal organs.

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells.