Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1997-01-28
2001-06-26
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S252300, C435S320100, C536S023500
Reexamination Certificate
active
06251664
ABSTRACT:
FIELD OF THE INVENTION
The minibrain (mnb) gene of
Drosophila melanoaster
encodes a serine-threonine protein kinase with an essential role in post-embryonic neurogenesis. A corresponding human gene with similar function to mnb could provide important. insights into both normal brain development and the abnormal. brain development and mental retardation observed in many congenital disorders. Trisomy 21 or Down syndrome (DS) is the most frequent human birth defect. It is associated with mental retardation and a broad spectrum of physical abnormalities. A region on human chromosome 21 has been designated the Down syndrome critical region (DSCR) and when present in three copies, this is responsible for many of the characteristic features of DS, including mental retardation. We have isolated a human homologue of mnb from the DSCR. Using a human probe of MNB, expression was detected in situ in several regions of the mouse brain, including the olfactory bulb, the cerebellum, the cerebral cortex, the pyramidal cell layer of the hippocampus and several hypothalamic nuclei. This expression pattern corresponds to the regions of the brain that are abnormal in individuals with DS and suggests that overexpression of MNB could have detrimental consequences in DS patients.
PRIOR ART
Down syndrome (DS) is one of the most frequent congenital defects. There is a broad spectrum of physical abnormalities associated with the syndrome, including anomalies of the gastrointestinal tract, increased risk of leukemia, defects of the immune and endocrine systems, early onset of Alzheimer's dementia and distinct facial and physical features, but perhaps the most debilitating is a rather severe mental retardation. In most cases DS is due to three full copies of human chromosome 21 which arise primarily during maternal non-disjunction, but occasionally DS occurs in people carrying unbalanced translocations, which result in the triplication of only a part of chromosome 21. By correlating phenotype with genotype in patients with partial trisomies a region has been defined, named the DSCR (Down syndrome critical region) which, when present in three copies, is responsible for many of the characteristic features of DS including mental retardation (1).
The phenotypic consequences of DS presumably result from the overexpression and subsequent interactions of a subset of chromosome 21 genes and the future challenge is to correlate overexpression of these genes, singly or in combination, with the presence of the DS phenotype. The first step is to identify the genes in the DSCR and then assess their potential contributions to the pathophysiology of DS. Assigning a function to a gene, particularly in humans, is not simple. Investigators rely on finding clues to function by analyzing the expression pattern of a gene, by looking for protein domains or motifs with known functions or by extrapolating from another species in which the function of the homologous gene is known.
The deduced amino acid sequence of MNB cDNA exhibits structural features which are shared with Dyrk (the rat homologue of mnb). The core domains which contain amino acids found in the catalytic sites of protein kinases are identical with the exception of two residues and both proteins have a potential nuclear translocation signal (FIG.
1
).
Any gene with a role in early neurogenesis is potentially important with respect to the abnormal brain development and mental retardation seen in DS. Genes which show temporal or high levels of expression during the development of the central nervous system may be of special importance in DS, especially in the pathogenesis of mental retardation. In Drosophila, the mnb gene appears to play an essential role during post-embryonic neurogenesis in regulating the numbers of distinct types of neuronal cells (2). Mutant mnb flies are characterized by a marked size reduction of the adult optic lobes and the central brain hemispheres. This is caused by the abnormal spacing of neuroblasts and hence a reduction in the production of neuronal progeny. The mnb gene encodes a serine-threonine protein kinase which is expressed in distinct neuroblast proliferation centers during Drosophila post-embryonic neurogenesis. We have isolated a human homologue of mnb from the DSCR and we show, by in situ RNA hybridization studies in mouse brains, that Mhb is normally expressed in regions of the brain which are abnormal in individuals with DS. The minibrain kinases (mnb, MNB, and Dyrk) share sequence similarity with the cyclin-dependent kinases, which are known to regulate cellular proliferation, suggesting a role for mnb in the correct mitosis of neuroblast progeny (2). Although the overall scheme of neuronal development is quite different in invertebrates and vertebrates, molecular studies on vertebrate neurogenesis have revealed a remarkable evolutionary conservation of the cellular mechanisms of neuronal development. Moreover, cyclin-dependent kinases are known to regulate cellular proliferation in various species, suggesting a more universal regulatory mechanism. It is conceivable that MNB has a role in the processes which generate neuronal cells in the brain during post-embryonic development.
The detection of MNB in the DSCR on chromosome 21 suggests that it may be involved in the altered neuronal development observed in DS. Although in Drosophila the mnb phenotype was due to a reduction in the level of expression of the mnb gene, we expect that in DS, MNB is overexpressed. At a gross morphological level, DS brains are smaller than normal and there is a decrease in the number of neurons. Neuronal number is reduced in distinct regions, including the cochlear nuclei, cerebellum, hippocampus, the cholinergic neurons of the basal forebrain, the granular layers of the cerebral cortex, and in areas of the brain stem. These abnormalities occur in regions where the Mnb gene is normally expressed and are consistent with the view that altered expression is in some way detrimental.
It is presumed that the structural alterations observed in the brain, together with the accompanying functional changes may account for the subsequent physiological and cognitive abnormalities associated with DS. Therefore, it is likely that genes involved in neurogenesis and which have altered expression in DS might account, at least partly, for the alterations that lead to mental retardation. The location of MNB in the DSCR together with its probable function in neurogenesis, supports MNB as a strong candidate gene to produce some of the neurological abnormalities present in DS patients. With the help of neuropathological, neurochemical and behavioral studies in transgenic animals, we may be able to dissect the components contributing to the mental retardation and to the complexity of the DS phenotype.
SUMMARY OF THE INVENTION
We have isolated a new human gene sequence, MNB, located in the 21q22.2 region. The expression of MNB was evident in the olfactory bulb, the cerebellum, the cerebral cortex, the pyramidal cell layer of the hippocampus and several hypothalamic nuclei, coding for a serine-threonine protein kinase. The overexpression of MNB may be involved in pathogenic abnormalities of mental retardation and/or other defects in patients with Down syndrome.
DETAILED DESCRIPTION OF THE INVENTION
Isolation of a mnb Homologue
In preparation for isolating human chromosome 21 expressed sequences we constructed contiguous cosmid sub-libraries from YACs from various regions of chromosome 21, including YACs from the DSCR. Pools of these cosmids were used for the isolation of partial cDNAs by direct selection (3) and for exon trapping experiments (4, 5). A total of 576 clones were isolated and arrayed. Of these 576 selected clones, 107 mapped back to human chromosome 21. In total we have 41 non-redundant putative cDNAs, of which 24 are novel, i.e. do not match known genes or ESTs in the databases (manuscript in preparation). We have isolated two known chromosome 21 genes, DSC1 and GIRK2 and another 13 partial cDNAs have significant matches with entries in the data
Estivill Palleja Xavier
Guimera Vilaro Jordi
Pritchard Melanie
Estivill Palleja Xavier
Hayes Robert C.
Kunz Gary L.
Ladas and Parry
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