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O Reiner

Publications and source records attributed to O Reiner.

At least 37 records · Page 2Linked to original sources
PubMed1999

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.

Animals↗
PubMed1999

Analysis of lissencephaly-causing LIS1 mutations.

Mutations in the LIS1 gene may result in severe abnormalities of brain cortical layering known as lissencephaly. Most lissencephaly-causing LIS1 mutations are deletions that encompass the entire gene, therefore the mechanism of the disease is regarded as haploinsufficiency. So far, 13 different intragenic mutations have been reported: one point mutation, H149R; deletion of exon 9, which results in deleted acids Delta301-334; deletion of exon 4, which results in deleted amino acids Delta40-64; 10 mutations resulting in truncated proteins and one predicted to result in extra amino acids. We studied the consequences of the point mutation, deletion mutation and one of the reported truncations. In order to study LIS1 structure function, we introduced an additional point mutation and other truncations in different regions of the protein. The consequences of these mutations to protein folding were studied by gel filtration, sucrose density gradient centrifugation and measuring resistance to trypsin cleavage. On the basis of our results, we suggest that all truncation mutations and lissencephaly-causing point mutations or internal deletion result in a reduction in the amount of correctly folded LIS1 protein.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1999

Doublecortin, a stabilizer of microtubules.

X-linked lissencephaly is a severe brain malformation affecting males. Recently it has been demonstrated that the doublecortin gene is implicated in this disorder. In order to study the function of Doublecortin, we analyzed the protein upon transfection of COS cells. Doublecortin was found to bind to the microtubule cytoskeleton. In vitro assays (using biochemical methods, DIC microscopy and electron microscopy) demonstrate that Doublecortin binds microtubules directly, stabilizes them and causes bundling. In vivo assays also show that Doublecortin stabilizes microtubules and causes bundling. Doublecortin is a basic protein with an iso-electric point of 10, typical of microtubule-binding proteins. However, its sequence contains no known microtubule-binding domain(s). The results obtained in this study with Doublecortin and our previous work on another lissencephaly gene ( LIS1 ) emphasize the central role of regulation of microtubule dynamics and stability during neuronal morphogenesis.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1998

The lissencephaly gene product Lis1, a protein involved in neuronal migration, interacts with a nuclear movement protein, NudC.

Important clues to how the mammalian cerebral cortex develops are provided by the analysis of genetic diseases that cause cortical malformations [1-5]. People with Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) have a hemizygous deletion or mutation in the LIS1 gene [3,6]; both conditions are characterized by a smooth cerebral surface, a thickened cortex with four abnormal layers, and misplaced neurons [7,8]. LIS1 is highly expressed in the ventricular zone and the cortical plate [9,10], and its product, Lis1, has seven WD repeats [3]; several proteins with such repeats have been shown to interact with other polypeptides, giving rise to multiprotein complexes [11]. Lis1 copurifies with platelet-activating factor acetylhydrolase subunits alpha 1 and alpha 2 [12], and with tubulin; it also reduces microtubule catastrophe events in vitro [13]. We used a yeast two-hybrid screen to isolate new Lis1-interacting proteins and found a mammalian ortholog of NudC, a protein required for nuclear movement in Aspergillus nidulans [14]. The specificity of the mammalian NudC-Lis1 interaction was demonstrated by protein-protein interaction assays in vitro and by co-immunoprecipitation from mouse brain extracts. In addition, the murine mNudC and mLis1 genes are coexpressed in the ventricular zone of the forebrain and in the cortical plate. The interaction of Lis1 with NudC, in conjunction with the MDS and ILS phenotypes, raises the possibility that nuclear movement in the ventricular zone is tied to the specification of neuronal fates and thus to cortical architecture.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1998

Abnormal cortical development; towards elucidation of the LIS1 gene product function (review).

Lissencephaly is a relatively common brain malformation. Lissencephaly type 1 is characterized by the smooth appearance of the cortex and the presence of four abnormally positioned layers instead of the normal six. Lissencephaly is considered to be an abnormality in neuronal migration. The gene mutated in type 1 lissencephaly was cloned by us and designated LIS1. Recently, several genes involved in cortical development have been cloned in the mouse. In human an additional X-linked lissencephaly gene has been identified. We summarize here our current knowledge on the LIS1 gene and its function. It has been identified as a non-catalytic subunit of PAF-acetylhydrolase, a heterotrimeric enzyme which inactivates the platelet-activating factor (PAF). In addition, we have demonstrated that LIS1 interacts with tubulin, and affects the dynamics properties of microtubles. LIS1 contains seven WD repeats and may structurally resemble the beta-subunit of heterotrimeric G proteins. Interestingly, the catalytic subunit of PAF-acetylhydrolase was found to resemble the alpha subunit of heterotrimeric G proteins. We raise the possibility that LIS1 is part of an intracellular signaling pathway involved in neuronal migration.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1997

Reduction of microtubule catastrophe events by LIS1, platelet-activating factor acetylhydrolase subunit.

Forming the structure of the human brain involves extensive neuronal migration, a process dependent on cytoskeletal rearrangement. Neuronal migration is believed to be disrupted in patients exhibiting the developmental brain malformation lissencephaly. Previous studies have shown that LIS1, the defective gene found in patients with lissencephaly, is a subunit of the platelet-activating factor acetylhydrolase. Our results indicated that LIS1 has an additional function. By interacting with tubulin it suppresses microtubule dynamics. We detected LIS1 interaction with microtubules by immunostaining and co-assembly. LIS1-tubulin interactions were assayed by co-immunoprecipitation and by surface plasmon resonance changes. Microtubule dynamic measurements in vitro indicated that physiological concentrations of LIS1 indeed reduced microtubule catastrophe events, thereby resulting in a net increase in the maximum length of the microtubules. Furthermore, the LIS1 protein concentration in the brain, measured by quantitative Western blots, is high and is approximately one-fifth of the concentration of brain tubulin. Our new findings show that LIS1 is a protein exhibiting several cellular interactions, and the interaction with the cytoskeleton may prove to be the mode of transducing a signal generated by platelet-activating factor. We postulate that the LIS1-cytoskeletal interaction is important for neuronal migration, a process that is defective in lissencephaly patients.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

Folding of proteins with WD-repeats: comparison of six members of the WD-repeat superfamily to the G protein beta subunit.

The family of WD-repeat proteins comprises over 30 different proteins that share a highly conserved repeating motif [Neer, E. J., Schmidt, C. J., Nambudripad, R., & Smith, T. F. (1994) Nature 371, 297-300]. Members of this family include the signal-transducing G protein beta subunit, as well as other proteins that regulate signal transduction, transcription, pre-mRNA splicing, cytoskeletal organization, and vesicular fusion. The crystal structure of one WD-repeat protein (G beta) has now been solved (Wall et al., 1995; Sondek et al, 1996) and reveals that the seven repeating units form a circular, propeller-like structure with seven blades each made up of four beta strands. It is very likely that all WD-repeat proteins form a similar structure. If so, it will be possible to use information about important surface regions of one family member to predict properties of another. If WD proteins form structures similar to G beta, their hydrodynamic properties should be those of compact, globular proteins, and they should be resistant to cleavage by trypsin. However, the only studied example of a WD-repeat protein, G beta, synthesized in vitro in a rabbit reticulocyte lysate, is unable to fold into a native structure without its partner protein G gamma. The non-WD-repeat amino terminal alpha helix of G beta does not inhibit folding because G beta does not fold even when this region is removed. It is not known whether all WD-repeat proteins are unable to fold when synthesized in an in vitro system. We synthesized seven members of the family in a rabbit reticulocyte lysate, determined their Stokes radius, sedimentation coefficient, and frictional ratio, and assayed their stability to trypsin. Our working definition of folding was that the proteins from globular, trypsin-resistant structures because, except for G beta gamma, their functions are not known or cannot be assayed in reticulocyte lysates. We chose proteins that include amino and carboxyl extensions as well as proteins that are made up entirely of WD-repeats. We show that unlike G beta, several proteins with WD-repeats are able to fold into globular proteins in a rabbit reticulocyte lysate. One protein, beta Trcp, formed large aggregates like G beta, suggesting that it may also require a partner protein. Despite the presence of many potential tryptic cleavage sites, all of the proteins that did fold gave stable large products on tryptic proteolysis, as predicted on the basis of the structure of G beta. These studies suggest that other WD-repeat proteins are likely to form propeller structures similar to G beta.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

Physical mapping of the gene for juvenile nephronophthisis (NPH1) by construction of a complete YAC contig of 7 Mb on chromosome 2q13.

Familial juvenile nephronophthisis (NPH) is an autosomal recessive cystic disease of the kidney that leads to end-stage renal failure in adolescence. NPH is the most common genetic cause of end-stage renal disease in children. A gene locus for nephronophthisis (NPH1) has been mapped by linkage analysis to chromosome 2q13. We report here the construction of a complete YAC contig in the minimum genetic region for NPH1 by STS content mapping using clones of the CEPH YAC libraries. A physical map of maximum distances between 32 STS markers was constructed, thereby defining the order of a total of 27 STS markers. Since D2S340 and D2S121 have previously been identified as flanking markers to the NPH1 gene, the new contig defines on a physical map the NPH1 minimum genetic region to a 6.4-Mb interval. As a novel assignment, expressed genes, some of which may be candidates for the disease, were localized to the NPH1 region. In addition, the known interstitial telomeric repeat on chromosome 2 was physically mapped to this region. This contig assembly provides the basis for closer definition of the NPH1 critical region through identification of more narrow flanking markers and for the construction of a transcriptional map of the region towards isolation of the NPH1 gene.

Base Sequence↗
PubMed1995

LIS2, gene and pseudogene, homologous to LIS1 (lissencephaly 1), located on the short and long arms of chromosome 2.

We report here the isolation of a novel cDNA, designated LIS2, that maps to chromosome 2p11.2 by in situ hybridization and demonstrates extremely high sequence similarity to the recently identified LIS1 gene involved in Miller-Dieker lissencephaly at 17p13.3. Specific probes for LIS2 revealed a pattern of expression resembling that of LIS1, although LIS2 is less abundant. Surprisingly, LIS2 detected an additional, higher molecular weight transcript in adult skeletal muscle. Isolated YAC clones and P1 clones mapped by in situ hybridization to two loci on chromosome 2,2p11.2 and 2q13-q14. This hybridization was due to the existence of LIS2 pseudogene LIS2P on the long arm of chromosome 2.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1995

Lissencephaly gene (LIS1) expression in the CNS suggests a role in neuronal migration.

Miller-Dieker lissencephaly syndrome (MDS) is a human developmental brain malformation caused by neuronal migration defects resulting in abnormal layering of the cerebral cortex. LIS1, the gene defective in MDS, encodes a subunit of brain platelet-activating factor (PAF) acetylhydrolase which inactivates PAF, a neuroregulatory molecule. We have isolated murine cDNAs homologous to human LIS1 and mapped these to three different chromosomal loci (Lis1, Lis3, Lis4). The predicted sequences of murine Lis1 protein and its human homolog LIS1 are virtually identical. In the developing mouse and human, Lis1 and LIS1 genes were strongly expressed in the cortical plate. In the adult mouse Lis1 transcripts were abundant in cortex and hippocampus. The direct correlation between cortical defects in MDS patients and Lis1 expression in the murine cortex suggest that the mouse is a model system suitable to study the mechanistic basis of this intriguing genetic disease.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1993

Lissencephaly. A human brain malformation associated with deletion of the LIS1 gene located at chromosome 17p13.

OBJECTIVE: We review the clinical phenotype, pathological changes, and results of cytogenetic and molecular genetic studies in 90 probands with lissencephaly (smooth brain) with emphasis on patients with the classical form (type I). We also describe the recent discovery of the lissencephaly gene (LIS1), deletions of which have been implicated as the cause of this disorder in many patients. DATA SOURCES: We have performed clinical, cytogenetic, and molecular genetic studies of 25 probands with Miller-Dieker syndrome and 65 probands with isolated lissencephaly sequence (ILS). We have further subdivided patients with ILS into those with classical lissencephaly and those with lissencephaly variants. STUDY SELECTION: We consider primarily our own published and unpublished data, but include references to studies of other series of patients with lissencephaly. DATA SYNTHESIS: Visible cytogenetic deletions of 17p13.3 were detected in 14 of 25 Miller-Dieker syndrome probands, and either visible cytogenetic or submicroscopic deletions in 23 (92%) of 25. Submicroscopic deletions were detected in eight of 45 patients with all types of ILS. If only ILS patients with the classical form are considered, we detected deletions in eight (38%) of 21. CONCLUSIONS: Deletions of the lissencephaly critical region in chromosome 17p13.3, including LIS1, appear to be the most frequent cause of classical lissencephaly. Molecular cytogenetic studies, particularly fluorescence in situ hybridization, should be performed in all such patients. LIS1 shows homology to genes involved in signal transduction, which may be its function in development of the telencephalon. Other genetic causes of classical lissencephaly and genetic and nongenetic causes of other types of lissencephaly exist and are under study.

Brain↗
PubMed1993

Isolation of a Miller-Dieker lissencephaly gene containing G protein beta-subunit-like repeats.

Lissencephaly (agyria-pachygyria) is a human brain malformation manifested by a smooth cerebral surface and abnormal neuronal migration. Identification of the gene(s) involved in this disorder would facilitate molecular dissection of normal events in brain development. Type 1 lissencephaly occurs either as an isolated abnormality or in association with dysmorphic facial appearance in patients with Miller-Dieker syndrome. About 15% of patients with isolated lissencephaly and more than 90% of patients with Miller-Dieker syndrome have microdeletions in a critical 350-kilobase region in chromosome 17p13.3 (ref. 6). These deletions are hemizygous, so haplo-insufficiency for a gene in this interval is implicated. Here we report the cloning of a gene (LIS-1, lissencephaly-1) in 17p13.3 that is deleted in Miller-Dieker patients. Non-overlapping deletions involving either the 5' or 3' end of the gene were found in two patients, identifying LIS-1 as the disease gene. The deduced amino-acid sequence shows significant homology to beta-subunits of heterotrimeric G proteins, suggesting that it could possibly be involved in a signal transduction pathway crucial for cerebral development.

Amino Acid Sequence↗
PubMed1991

Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome.

Fragile X syndrome is the most frequent form of inherited mental retardation and is associated with a fragile site at Xq27.3. We identified human YAC clones that span fragile X site-induced translocation breakpoints coincident with the fragile X site. A gene (FMR-1) was identified within a four cosmid contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRI genomic fragment, containing FMR-1 exonic sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragile X chromosomes. This fragment contains a lengthy CGG repeat that is 250 bp distal of the CpG island and maps within a FMR-1 exon. Localization of the brain-expressed FMR-1 gene to this EcoRI fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome.

Alleles↗
PubMed1989

The human glucocerebrosidase gene and pseudogene: structure and evolution.

We report the sequence of the entire human gene encoding beta-glucocerebrosidase and that of the associated pseudogene. The gene contains 11 exons extending from base pair 355 to base pair 7232 in the overall sequence. The gene promoter contains TATA- and CAT-like boxes upstream of the major 5' end of the glucocerebrosidase RNA. The two TATA boxes lie between nucleotides (-23)-(-27) and (-33)-(-39) and the two possible CAT boxes reside between nucleotides (-90)-(-94) and (-96)-(-99) in relation to the major 5' end of the mRNA. The functionality of the promoter region was monitored by coupling it to the bacterial gene coding for chloramphenicol acetyltransferase (CAT) and assaying the expression of the enzyme in cells transfected with this vector. The glucocerebrosidase promoter not only directs synthesis of the bacterial enzyme but also exhibits the same pattern of tissue-specific expression as that of the endogenous gene. An apparently tightly linked pseudogene is approximately 96% homologous to the functional gene. However, introns 2, 4, 6, and 7 have large "deletions" consisting of Alu sequences 313, 626, 320, and 277 bp in length, respectively. It is entirely possible that the ancestral gene lacks these sequences and that they have been inserted into the introns of the functioning gene. There is also a 55-bp deletion from a part of exon 9 flanked by a short inverted repeat. The sequence data should facilitate development of methods for diagnosis of Gaucher disease at the molecular level.

Base Sequence↗
PubMed1989

Characterization of mutations in Gaucher patients by cDNA cloning.

Mutated cDNA clones containing the entire coding sequence of human glucocerebrosidase were isolated from libraries originated from Gaucher patients. Sequence analysis of a mutated cDNA derived from a type II Gaucher patient revealed a C-to-G transversion causing a substitution of an arginine for a proline at residue 415. This change creates a new cleavage site for the enzyme HhaI in the mutated cDNA. Allele-specific oligonucleotide hybridization made it possible to show that this mutation exists in the genomic DNA of the patient. From a cDNA library originated from a type I Gaucher patient, a mutated allele was cloned that contains a T-to-C transition causing a substitution of proline for leucine at residue 444 and creating a new NciI site. This mutation is identical to that described by S. Tsuji and colleagues in genomic DNA from type I, type II, and type III patients. Since the new NciI site generates RFLP, it was used to test the existence of this mutated allele in several Gaucher patients by Southern blot analysis. This allele was found in type I (Jewish and non-Jewish), type II, and type III Gaucher patients. These findings led us to conclude that the patient suffering from type II disease (denoted GM1260) carried both mutations described above. Any one of the amino acid changes described reduces the glucocerebrosidase activity as tested by transfection of COS cells with expression vectors harboring the mutated cDNAs. The base changes in the two mutated cDNAs do not affect the electrophoretic mobility of the corresponding polypeptides on an SDS polyacrylamide gel.

Alleles↗
PubMed1989

Human sphingolipid activator protein-1 and sphingolipid activator protein-2 are encoded by the same gene.

Mixed oligonucleotide primers complementary to the translation product of the sphingolipid activator protein (SAP)-2 were used to generate a 144-base pair (bp) complementary DNA (cDNA). This cDNA probe was used to isolate a 2,649-nucleotide-long cDNA that was sequenced and found to contain coding sequences for two known activators of lysosomal enzymes, namely, the sphingolipid activator protein (SAP)-1 and SAP-2. The cDNA contains an open reading frame of 1,482 nucleotides and 1,167 nucleotides of 3'-nontranslated region, followed by a stretch of 24 residues of adenylic acid. At 20 nucleotides upstream from the poly(A) tail there is a consensus AATAAA polyadenylation signal that is preceded by another potential polyadenylation signal. The cDNA, designed SAP-1/SAP-2 cDNA, hybridizes with two human mRNA species of approximately 3 kb in length, which most probably arise from polyadenylation at different sites. There are higher amounts of steady-state RNA levels of the SAP-1/SAP-2 mRNA in skin fibroblasts in comparison to B cells. The steady-state SAP-1/SAP-2 mRNA levels in Gaucher B cells are higher than in their normal counterparts. There is one human SAP-1/SAP-2 gene that has been cloned and is localized on two approximately 5 kb BamHI fragments.

Amino Acid Sequence↗