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The human gene encoding the myogenic determination factor myf3 (mouse MyoD1) has been mapped to the short arm of chromosome 11. Analysis of several somatic cell hybrids containing various derivatives with deletions or translocations revealed that the human MyoD (MYF3) gene is not associated with the WAGR locus at chromosomal band 11pl3 nor with the loss of the heterozygosity region at 11p15.5 related to the Beckwith-Wiedemann syndrome. Subregional mapping by in situ hybridization with an myf3 specific probe shows that the gene resides at the chromosomal band llp14, possibly at llp14.3.
The Wilms tumor gene WTl, a proposed tumor suppressor gene, has been identifled based on its location within a homozygous deletion found in tumor tissue. The gene encodes a putative transcription factor containing a Cys/His zinc finger domain. The critical homozygous deletions, however, are rarely seen, suggesting that in many cases the gene may be inactivated by more subtle alterations. To facilitate the seareh for smaller deletions and point mutations we have established the genomic organization of the WTl gene and have determined the sequence of all 10 exons and flanking intron DNA. The pattern of alternative splicing in two regions has been characterized in detail. These results will form the basis for future studies of mutant alleles at this locus.
Transiently activating (A-type) potassium (K) channels are important regulators of action potential and action potential firing frequencies. HK1 designates the firsthuman cDNA that is highly homologous to the rat RCK4 cDNA that codes for an A-type K-channel. The HK1 channel is expressed in heart. By somatic cell hybrid analysis, the HK1 gene has been assigned to human chromosome 11p13-pl4, the WAGR deletion region (Wilms tumor, aniridia, genito-urinary abnormalities and mental retardation). Subsequent pulsed field gel (PFG) analysis and comparison with the well-established PFG map of this region localized the gene to 11p14, 200-600 kb telomeric to the FSHB gene.
The direct involvment of the Wilm's tumor suppressor gene (WTl) in Denys-Drash syndrome through mutations within exons 8 or 9 has recently been established. The absence of such alterations in three patients with Frasier syndrome provides a molecular basis for distinguishing these two syndromes that are associated with streak gonads, pseudohermaphroditism and renal failure.
Pericentric intrachromosomal insertion responsible for recurrence of del(11)(p13p14) in a family
(1993)
The combined use of qualitative and quantitative analysis of I I p I 3 polymorphic markers tagether with chromosomal in situ suppression hybridization (CISS) with biotin labeled probes mapping to I I p allowed us to characterize a complex rearrangement segregating in a family. We detected a pericentric intrachromosomal insertion responsible (or recurrence of del( I I )(p 13p 14) in the family: an insertion of band I I p 13-p 14 carrying the genes for predisposition to Wilms' tumor, WT I, and for aniridia, AN2, into the long arm of chromosome I I in II q 13-q 1<4. Asymptomatic balanced carriers were observed over three generations. Classical cytogenetics had failed to detect this anomaly in the balanced carriers, who were first considered to be somatic mosaics for del( II )(p 13). Two of these women gave birth to children carrying a deleted chromosome II. most likely resulting from the loss of the I I p 13 band inserted in I I q. Although in both cases the deletion encompassed exactly the same maternally inherited markers, there was a wide Variation in clinical expression. One child, with the karyotype 46,XY,del(ll)(pllpl4), presented the full-blown WAGR syndrome with anlridia, mental retardation, Wilms' tumor, and pseudohermaphroditism, but also had proteinuria and glomerular sclerosis reminiscent of Drash syndrome. In contrast, the other one, a girl with the karyotype 46,XX,del( I I )(p I 3), only had aniridia. Although a specific set of mutational sites has been observed in Drash patients, these findings suggest that the loss of one copy of the WTI gene can result in similar genital and kidney abnormalities.
Wilms' tumor is a childhood nephroblastoma that is postulated to arise through the inactivation of a tumor suppressor gene by a two-hit mechanism. A candidate II p 13 Wilms' tumor gene, WTI, has been cloned and shown to encode a zinc finger protein. Patients with the WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation) have a high risk of developing Wilms' tumor and they carry constitutional deletions of one chromosome II allele encompassing the WTI gene. Analysis of the remaining WTI allele in a Wilms' tumor from a WAGR patient revealed the deletion of a single nucleotide in exon 7. This mutation likely played a key role in tumor formation, as it prevents translation of the DNA-binding zinc finger domain that is essential for the function of the WTI polypeptide as a transcriptional regulator.
The human recombination activating gene 1 (RAGl) has previously been mapped to chromosomes 14q and 11 p. Here we confirm the chromosome 11 assignment by two independent approaches: autoradiographic and fluorescence in situ hybridization to metaphase spreads and analysis of human-hamster somatic cell hybrid DNA by the polymerase chain reaction (PCR) and Southern blotting. Our results unequivocally localize RAG1 to llp13.
Developmental delay or mental retardation is a frequent component of multi-system anomaly syndromes associated with chromosomal deletions. Isolation of genes involved in the mental dysfunction in these disorders should define loci important in brain formation or function. We have identified a highly conserved locus in the distal part of 11 p 13 that is prominently expressed in fetal brain. Minimal expression is observed in a number of other fetal tissues. The gene maps distal to PAX-6 but proximal to the loci for brain-derived neurotrophic factor (BDNF) and the beta subunit of follicle stimulating hormone (FSHB), within a region previously implicated in the mental retardation component of some WAGR syndrome patients. Within fetal brain, the corresponding transcript is prominent in frontal, motor and primary visual cortex as weil as in the caudate-putamen. The characteristics of this gene, including the striking evolutionary conservation at the locus, suggest that the encoded protein may function in brain development.
b-Type cytochromes
(1980)
The amino acid sequence of the proteolipid subunit of the A TP synthase was analyzed in six mutant strains from Escherichia coli K 12, selected for their increased resistance towards the inhibitor N,N'-dicyclohexylcarbodiimide. All six inhibitor-resistant mutants were found to be altered at the same position of the proteolipid, namely at the isoleucine at residue 28. Two substitutions could be identified. In type I this residue was substituted by a valine resulting in a moderate decrease in sensitivity to dicyclohexylcarbodiimide. Type II contained a threonine residue at this position. Here a strong resistance was observed. These two amino acid substitutions did not influence functional properties of the ATPase complex. ATPase as well as A TP-dependent proton-translocating activities of mutant membranes were indistinguishable from the wild type. At elevated concentrations, dicyclohexylcarbodiimide still bound specifically to the aspartic acid at residue 61 of the mutant proteolipid as in the wild type, and thereby inhibited the activity of the ATPase complex. It is suggested that the residue 28 substituted in the resistant mutants interacts with dicyclohexylcarbodiimide during the reactions leading to the covalent attachment of the inhibitor to the aspartic acid at residue 61. This could indicate that these two residues are in close vicinity and would thus provide a first hint on the functional conformation of the proteolipid. Its polypeptide chain would have to fold back to bring together these two residues separated by a segment of 32 residues.
Interleukin-4 (IL-4) und Interleukin-13 (IL-13) sind bedeutende Regulatorproteine des Immunsystems. Sie spielen eine entscheidende Rolle bei der Entstehung und dem Verlauf von allergischen Erkrankungen, wie z.B. Asthma. Um ihre Signale in die Zielzelle zu transduzieren, kann von beiden Zytokinen der gleiche Zelloberflächenrezeptor verwendet werden, wodurch sich die überlappenden, biologischen Funktionen erklären lassen. Dieser gemeinsam genutzte Rezeptor ist aus den beiden Untereinheiten IL-4Ralpha; und IL-13Ralpha1 aufgebaut. Da IL-4 und IL-13 auf Aminosäureebene nur etwa 25% Sequenzidentität besitzen und stark unterschiedliche Affinitäten zu den beiden Rezeptorketten besitzen, stellt sich die Frage, durch welchen molekularen Erkennungsmechanismus, die Affinität und die Spezifität der Ligand-Rezeptor-Interaktion unabhängig voneinander reguliert werden kann. In dieser Arbeit gelang es, rekombinante Expressions- und Aufreinigungsstrategien für IL-13 und die extrazellulären Domänen der Rezeptorketten IL-13Ralpha1 und IL-13Ralpha2 zu entwickeln. Dadurch war es mögliche, eine breite Mutations-/Interaktionsanalyse der IL-13Ralpha1-Kette durchzuführen.Es konnte gezeigt werden, dass die N-terminale FnIII-ähnliche Domäne von IL-13Ralpha1 sowohl an der Bindung von IL-13 als auch an der Interaktion mit IL-4 beteiligt ist. Im funktionellen Bindeepitop der IL-13Ralpha1-Kette wurden die Aminosäurereste Arg84, Phe253 und Tyr321 als Hauptbindungsdeterminanten für die Interaktion mit IL-13 identifiziert. Durch die Interaktionsstudien der IL-13Ralpha1-Varianten mit IL-4 wurde gezeigt, dass diese Hauptbindungsdeterminanten auch für die niederaffine Bindung von IL-4 von größter Bedeutung sind. Die funktionellen Bindeepitope für IL-4 und IL-13 auf der IL-13Ralpha1-Kette sind nahezu identisch und überlappen in einem großen Bereich. Aufgrund der Ergebnisse aus der Mutagenesestudie war es möglich, ein Strukturmodell der extrazellulären Domäne der IL-13Ralpha1-Kette zu erstellen. Darin wird eine neuartige Orientierung der N-terminalen FnIII-Domäne und deren Beteiligung an der Ligandeninteraktion dargestellt. Mit Hilfe des Strukturmodells gelang es, neue Aminosäurerest auf der Oberfläche von IL-13 zu identifizieren, die an der Bindung zu IL-13Ralpha1 beteiligt sind, was die Relevanz des Strukturmodells weiter unterstreicht. In einem weiteren Teil dieser Arbeit wurde versucht, den molekularen Mechanismus aufzuklären, durch den es den superagonistischen IL-4-Varianten T13D und F82D gelingt, mit dreifach höherer Affinität an die IL-4Ralpha-Kette zu binden, als wildtypischer Ligand. Durch strukturelle und funktionelle Untersuchungen wurde gezeigt, dass der Affinitätssteigerung ein indirekter Mechanismus zugrunde liegt, bei dem eine Konformationsänderung und die Fixierung der Arg85-Seitenkette von IL-4 zur Ausbildung von zusätzlichen Ligand-Rezeptor-Interaktionen führt. Das Bindeepitop zwischen IL-4 und der IL-4Ralpha-Kette besitzt eine modulare Architektur aus drei unabhängig voneinander agierenden Interaktionsclustern. Bei der Interaktion von wildtypischem IL-4 mit IL-4Ralpha tragen nur zwei dieser Cluster in signifikanter Weise zur freien Bindeenergie bei. Im Falle der superagonistischen IL-4-Varianten ist jedoch auch das dritte Cluster an der Generierung von zusätzlicher, freier Bindeenergie beteiligt, wodurch die Affinität zwischen Ligand und Rezeptor erhöht wird. Damit stellt der modulare Aufbau der Interaktionsfläche zwischen IL-4 und der IL-4Ralpha-Kette möglicherweise einen Mechanismus dar, über den Proteine die Affinität von Wechselwirkungen über einen großen Bereicht variieren können, ohne dabei Spezifität einzubüssen. Da IL-4 und IL-13 als interessante Zielmoleküle für die Therapie von allergischen und asthmatischen Erkrankungen erkannt worden sind, können die in der vorliegenden Arbeit gewonnenen Informationen über den Bindemechanismus und die Einblicke in den molekularen Charakter der Interaktion zwischen den beiden Zytokinen und ihren spezifischen Rezeptorketten dabei helfen, neuartige und hoch spezifische, inhibitorische Moleküle zu entwickeln.