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Biomedical subjects

M Affolter

Publications and source records attributed to M Affolter.

At least 55 records · Page 3Linked to original sources
PubMed1994

Homeodomain proteins in development and therapy.

Homeobox genes encode transcriptional regulators found in all organisms ranging from yeast to humans. In Drosophila, a specific class of homeobox genes, the homeotic genes, specifies the identity of certain spatial units of development. Their genomic organization, in Drosophila, as well as in vertebrates, is uniquely connected with their expression which follows a 5'-posterior-3'-anterior rule along the longitudinal body axis. The 180-bp homeobox is part of the coding sequence of these genes, and the sequence of 60 amino acids it encodes is referred to as the homeodomain. Structural analyses have shown that homeodomains consist of a helix-turn-helix motif that binds the DNA by inserting the recognition helix into the major groove of the DNA and its amino-terminal arm into the adjacent minor groove. Developmental as well as gene regulatory functions of homeobox genes are discussed, with special emphasis on one group, the Antennapedia (Antp) class homeobox genes and a representative 60-amino acid Antennapedia peptide (pAntp). In cultured neuronal cells, pAntp translocates through the membrane specifically and efficiently and accumulates in the nucleus. The internalization process is followed by a strong induction of neuronal morphological differentiation, which raises the possibility that motoneuron growth is controlled by homeodomain proteins. It has been demonstrated that chimeric peptide molecules encompassing pAntp are also captured by cultured neurons and conveyed to their nuclei. This may be of enormous interest for the internalization of drugs.

Amino Acid Sequence↗
PubMed1994

Multiple requirements for the receptor serine/threonine kinase thick veins reveal novel functions of TGF beta homologs during Drosophila embryogenesis.

Differentiation of distinct cell types at specific locations within a developing organism depends largely on the ability of cells to communicate. A major class of signalling proteins implicated in cell to cell communication is represented by members of the TGF beta superfamily. A corresponding class of transmembrane serine/threonine kinases has recently been discovered that act as cell surface receptors for ligands of the TGF beta superfamily. The product of the Drosophila gene decapentaplegic (dpp) encodes a TGF beta homolog that plays multiple roles during embryogenesis and the development of imaginal discs. Here we describe the complex expression pattern of thick veins (tkv), which encodes a receptor for dpp. We make use of tkv loss-of-function mutations to examine the consequences of the failure of embryonic cells to respond to dpp and/or other TGF beta homologs. We find that while maternal tkv product allows largely normal dorsoventral pattering of the embryo, zygotic tkv activity is indispensable for dorsal closure of the embryo after germ band retraction. Furthermore, tkv activity is crucial for patterning the visceral mesoderm; in the absence of functional tkv gene product, visceral mesoderm parasegment 7 cells fail to express Ultrabithorax, but instead accumulate Antennapedia protein. The tkv receptor is therefore involved in delimiting the expression domains of homeotic genes in the visceral mesoderm. Interestingly, tkv mutants fail to establish a proper tracheal network. Tracheal braches formed by cells migrating in dorsal or ventral directions are absent in tkv mutants. The requirements for tkv in dorsal closure, visceral mesoderm and trachea development assign novel functions to dpp or a closely related member of the TGF beta superfamily.

Animals↗
PubMed1994

The Drosophila SRF homolog is expressed in a subset of tracheal cells and maps within a genomic region required for tracheal development.

The Drosophila homolog of the vertebrate serum response factor (SRF) was isolated by low stringency hybridization. Nucleotide sequence analysis revealed that the Drosophila SRF homolog (DSRF) codes for a protein that displays 93% sequence identity with human SRF in the MADS domain, the region required for DNA binding, dimerization and interaction with accessory factors. The DSRF gene is expressed during several phases of embryonic development. In the egg, both the RNA and the protein are maternal in origin and slowly decrease in amount during gastrulation. After germ band retraction, high levels of zygotic expression are observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. Many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues. The DSRF gene was mapped to position 60C on the second chromosome, and overlapping deficiencies which remove the gene were identified. Analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. Although the initial migration of tracheal cells was not affected in those deficient embryos, many tracheal cells appeared not to maintain their correct position and continued to migrate. Thus, the DSRF gene might play a role in the proper formation and maintenance of the trachea.

Amino Acid Sequence↗
PubMed1993

Regional repression of a Drosophila POU box gene in the endoderm involves inductive interactions between germ layers.

An induction process occurring between the mesodermal and the endodermal germ layers has recently been described in the regulation of the Drosophila homeotic gene labial (lab). We report here that proper spatial regulation of the Drosophila POU box gene pdm-1 products also involves interaction between these two germ layers. pdm-1 transcripts are initially present in both the anterior and the posterior endodermal midgut primordia. Upon fusion of the two primordia, transcripts disappear from two regions in the endoderm, a central domain and an anterior domain. The anterior repression domain of pdm-1 is independent of the expression of known homeotic genes and genes encoding secreted signalling molecules in the visceral mesoderm, both for its positioning and its repression. Repression in the central domain requires both the homeotic gene Ultrabithorax (Ubx) and the decapentaplegic (dpp) gene, which encodes a secreted protein. Both of these genes are also required for lab induction. However, the analysis of pdm-1 expression in various mutant backgrounds indicates that the regulation of lab and pdm-1 across germ layers is controlled by different genetic cascades. Our study indicates that dpp is not the signal that dictates central pdm-1 repression across germ layers and suggests that in the same midgut region, different signalling pathways result in the differential activation or repression of potential transcription factors.

Animals↗
PubMed1992

NMR structure determination reveals that the homeodomain is connected through a flexible linker to the main body in the Drosophila Antennapedia protein.

The secondary structure of an N-terminally elongated Antennapedia (Antp) homeodomain (HD) polypeptide containing residues -14 to 67, where residues 1-60 constitute the HD, has been determined by NMR in solution. This polypeptide contains the conserved motif -Tyr-Pro-Trp-Met- (YPWM) at positions -9 to -6. Despite the hydrophobic nature of this tetrapeptide motif, the N-terminal arm consisting of residues -14 to 6 is flexibly disordered, and the well-defined part of the HD structure with residues 7-59 is indistinguishable from that of the shorter Antp HD polypeptide (where positions 0, 1, and 67 are methionine, arginine, and glycine, respectively). In vitro biochemical studies showed that the stability and specificity of the DNA binding previously observed for the shorter Antp HD polypeptide is preserved in the elongated polypeptide. These results strongly support the view that the HD is connected through a flexible linker to the main body in the Antp protein and that the minor groove contacts by the N-terminal arm (residues 1-6) in the Antp HD-DNA complex are an intrinsic feature of the DNA-binding interactions of the intact Antp protein.

Amino Acid Sequence↗
PubMed1992

Distamycin-induced inhibition of homeodomain-DNA complexes.

The mobility shift assay was used to study the competition of the minor groove binder distamycin A with either an Antennapedia homeodomain (Antp HD) peptide or derivatives of a fushi tarazu homeodomain (ftz HD) peptide for their AT-rich DNA binding site. The results show that distamycin and the homeodomain peptides compete under the conditions: (i) preincubation of DNA with distamycin and subsequent addition of HD peptide; (ii) simultaneous incubation of DNA with distamycin and HD peptide; and (iii) preincubation of DNA with HD peptide and subsequent addition of distamycin. There is also competition when using a peptide which lacks the N-terminal arm of ftz HD that is involved in contacts in the minor groove. It is proposed that the protein's binding affinity is diminished by distamycin-induced conformational changes of the DNA. The feasibility of the propagation of conformational changes upon binding in the minor groove is also shown for the inhibition of restriction endonucleases differing in the AT content of their recognition site and of their flanking DNA sequences. Thus, it is demonstrated that minor groove binders can compete with the binding of proteins in the major groove, providing an experimental indication for the influence of biological activities exerted by DNA ligands binding in the minor groove.

Animals↗
PubMed1992

A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei.

The major component of the cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei is a membrane skeleton which consists of a single layer of tightly spaced microtubules. This array encloses the entire cell body, and it is apposed to, and connected with, the overlying cell membrane. The microtubules of this array contain numerous microtubule-associated proteins. Prominent among those is a family of high molecular weight, repetitive proteins which consist to a large extent of tandemly arranged 38-amino acid repeat units. The binding of one of these proteins, MARP-1, to microtubules has now been characterized in vitro and in vivo. MARP-1 binds to microtubules via tubulin domains other than the COOH-termini used by microtubule-associated proteins from mammalian brain, e.g., MAP2 or Tau. In vitro binding assays using recombinant protein, as well as transfection of mammalian cell lines, have established that the repetitive 38-amino acid repeat units represent a novel microtubule-binding motif. This motif is very similar in length to those of the mammalian microtubule-associated proteins Tau, MAP2, and MAP-U, but both its sequence and charge are different. The observation that the microtubule-binding motifs both of the neural and the trypanosomal proteins are of similar length may reflect the fact that both mediate binding to the same repetitive surface, the microtubule, while their sequence and charge differences are in agreement with the observation that they interact with different domains of the tubulins.

Alkaloids↗
PubMed1992

In vivo analysis of the helix-turn-helix motif of the fushi tarazu homeo domain of Drosophila melanogaster.

We report a systematic mutational analysis of the helix-turn-helix motif (HTH) of the fushi tarazu (ftz) homeo domain (HD) of Drosophila. We started out by testing the function of chimeric ftz proteins containing either a part of the Sex combs reduced (Scr) or the muscle segment homeobox (msh) HDs. By complementation tests in transgenic flies, cotransfection assays in cultured Drosophila cells and in vitro DNA-binding assays, we have found that the ftz activity is retained in the ftz-Scr chimera but is lost in the ftz-msh chimera, which is defective in binding to an Antennapedia (Antp)-class target site. Further studies with a series of back-mutants of the ftz-msh chimera have revealed that a set of class-specific DNA backbone-contacting residues in the HTH, particularly Arg-28 and Arg-43, are required for efficient target site recognition and, hence, full ftz activity both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1991

Primary structure of a new actin-binding protein from human seminal plasma.

Secretory actin-binding protein (SABP), a glycoprotein from human seminal plasma, was isolated according to Akiyama and Kimura [Akiyama, K. & Kimura, H. (1990) Biochim. Biophys. Acta 1040, 206-210]. The complete amino acid sequence of SABP was determined with the aid of fragments generated by trypsin, Staphylococcus aureus V8 protease and pepsin. The single polypeptide chain of SABP contains 118 amino acids with a calculated Mr of 13,506 and pyroglutamic acid as the N-terminal residue. A single N-glycosidic carbohydrate moiety is located at Asn77. The carbohydrate composition shows an unusually high amount of fucose. The arrangement of the two disulfide bonds is Cys37-Cys63 and Cys61-Cys95. Sequence comparison revealed a high degree of similarity with a 14-kDa submandibular gland protein from mouse (45% identity and 64% similarity). SABP is identical with a prolactin-inducible protein and a protein termed gross cystic disease fluid protein 15 (sequences translated from cDNA clones), both from human breast tissues. Although SABP was also detected in saliva, in extracts of the submandibular gland and seminal vesicles, little is known of its function.

Actins↗
PubMed1990

Protein--DNA contacts in the structure of a homeodomain--DNA complex determined by nuclear magnetic resonance spectroscopy in solution.

The 1:1 complex of the mutant Antp(C39----S) homeodomain with a 14 bp DNA fragment corresponding to the BS2 binding site was studied by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. The complex has a molecular weight of 17,800 and its lifetime is long compared with the NMR chemical shift time scale. Investigations of the three-dimensional structure were based on the use of the fully 15N-labelled protein, two-dimensional homonuclear proton NOESY with 15N(omega 2) half-filter, and heteronuclear three-dimensional NMR experiments. Based on nearly complete sequence-specific resonance assignments, both the protein and the DNA were found to have similar conformations in the free form and in the complex. A sufficient number of intermolecular 1H-1H Overhauser effects (NOE) could be identified to enable a unique docking of the protein on the DNA, which was achieved with the use of an ellipsoid algorithm. In the complex there are intermolecular NOEs between the elongated second helix in the helix-turn-helix motif of the homeodomain and the major groove of the DNA. Additional NOE contacts with the DNA involve the polypeptide loop immediately preceding the helix-turn-helix segment, and Arg5. This latter contact is of special interest, both because Arg5 reaches into the minor groove and because in the free Antp(C39----S) homeodomain no defined spatial structure could be found for the apparently flexible N-terminal segment comprising residues 0-6.

Animals↗
PubMed1990

The interaction with DNA of wild-type and mutant fushi tarazu homeodomains.

The in vitro DNA binding properties of wild-type and mutant fushi tarazu homeodomains (ftz HD) have been analysed. The DNA binding properties of the ftz HD are very similar to those of the Antp HD. In interference experiments with mutant ftz HDs, close approaches between specific portions of the ftz HD peptide and specific regions of the binding site DNA were mapped. A methylation interference, G7 on the beta strand of BS2, is absent from the interference pattern with a mutant ftz HD [ftz (R43A) HD] in which the Arg43 at the second position of helix III (the recognition helix) is replaced by an Ala. This indicated that Arg43 of the ftz HD is in close proximity to the N7 of G7 of the beta strand of BS2 in the major groove. The methylation and ethylation interference patterns with the ftz (NTD) HD, in which the first six amino acids of the homeodomain were deleted, were extensively altered relative to the ftz HD patterns. Methylation of A11 and G12 of the alpha strand and ethylation of the phosphate of nucleotide A12 of the alpha strand no longer interfere with binding. This indicated that the first six amino acids of the homeodomain of ftz interact with A11 of the alpha strand in the minor groove, the phosphate of the nucleotide A13 on the alpha strand and G12 of the alpha strand in the adjacent major groove of BS2. In a binding study using a change of specificity mutation [ftz (Q50K) HD], in which the Gln50 at the ninth position of the third helix is exchanged for a Lys (as in the bicoid HD), and variant binding sites, we concluded that position 50 of the ftz HD and the ftz (Q50K) HD peptides interacts with base pairs at positions 6 and 7 of BS2. These three points of contact allowed us to propose a crude orientation of the ftz HD within the protein-DNA complex. We find that the ftz HD and the Antp HD peptides contact DNA in a similar way.

Amino Acid Sequence↗
PubMed1990

The structure of the homeodomain and its functional implications.

The three-dimensional structure of the homeodomain, as determined by nuclear magnetic resonance spectroscopy, reveals the presence of a helix-turn-helix motif, similar to the one found in prokaryotic gene regulatory proteins. Isolated homeodomains bind with high affinity to specific DNA sequences. Thus, the structure-function relationship is highly conserved in evolution.

Amino Acid Sequence↗
PubMed1990

DNA binding properties of the purified Antennapedia homeodomain.

The in vitro DNA binding properties of a purified 68-amino acid Antennapedia homeodomain (Antp HD) peptide have been analyzed. Equilibrium and kinetic binding studies showed that stable DNA-protein complexes are formed with a Kd of 1.6 x 10(-9) M and 1.8 x 10(-10) M, respectively. Heterodimer analysis led to the conclusion that Antp HD interacts in vitro as a monomer with the DNA target sites used in our study. The results of methylation and ethylation interference studies indicated that the Antp HD closely approaches the target DNA primarily from one side in a region extending across three phosphate backbones. The DNA binding properties of the Antp HD and prokaryotic DNA binding domains that share a helix-turn-helix motif are compared.

Alkylation↗
PubMed1990

Analysis of the ftz upstream element: germ layer-specific enhancers are independently autoregulated.

The Drosophila fushi tarazu (ftz) upstream element is an enhancer-like element that is required for the correct expression of ftz in developing embryos and that directs transcription from a minimal promoter in a ftz-like seven-striped pattern. Using a deletion analysis, we have identified several independent cis-regulatory elements in the upstream element. A distal enhancer directs fusion gene expression in seven stripes primarily in the mesoderm. A more complex proximal enhancer contains a mesodermally active element and a second element with which it interacts to generate seven stripes in the ectoderm. Striped expression directed by each enhancer is ftz-dependent, and each contains binding sites for purified ftz homeo domain. We suggest that ftz protein acts in combination with germ layer-restricted transcription factors directly and positively to regulate the transcription of its own gene.

Animals↗
PubMed1989

The tubulin gene cluster of Trypanosoma brucei starts with an intact beta-gene and ends with a truncated beta-gene interrupted by a retrotransposon-like sequence.

In Trypanosoma brucei the genes coding for alpha- and beta-tubulin are arranged in a single cluster comprising about ten alternating alpha and beta genes. The cluster starts at its 5' end with an intact beta gene. In this area, a 1.1-kb segment upstream of the mini-exon addition site has been sequenced. Several potential transcription signals were seen. The 3' end of the cluster terminates in the middle of a beta gene, making it an inactive truncated pseudogene. The adjoining 1.7-kb sequence shows a high degree of similarity with a retrotransposon-like (RTnL) element previously characterized elsewhere in the trypanosome genome. This is the first report of an RTnL element interrupting a protein-coding gene of T. brucei.

Animals↗