Neuronal activity and its influence on developmentally regulated GABA(A) receptor expression in cultured mouse cerebellar granule cells.

Mouse cerebellar granule cells (CGCs) were cultured in either non-depolarising (5 mM KCl, '5K') or depolarising (25 mM KCl, '25K') media. CGCs at 5K developed an elaborate network of processes and formed compact cell aggregates, whilst at 25K, cell aggregation was rarely observed. GABA(A) receptor (GABAR) expression was significantly affected by the culture conditions. Depolarisation resulted in a approximately 55% reduction in the expression of total specific [(3)H]Ro15-4513 binding sites, largely due to a >85% loss of the flunitrazepam-insensitive (BZ-IS) subtype of binding sites. The number of flunitrazepam-sensitive (BZ-S) [(3)H]Ro15-4513 binding sites expressed and the K(D) of [(3)H]Ro15-4513 to either GABAR subtype were not significantly affected. The BZ-S subtype expressed by 5K CGCs was essentially all type I, as expected of mature CGCs, however at 25K, these were predominantly type II (approximately 70%) and zolpidem-insensitive (approximately 30%)-pharmacological finger-prints of immature CGCs. By immunoblotting we determined that CGCs cultured at 25K expressed GABAR alpha1, alpha6 and beta3 subunits at 14, 3 and 167% of 5K levels, respectively. GABAR beta2 subunits, however, were barely detectable. The changes in GABAR subunit expression were paralleled by similar changes in the steady-state levels of the subunit mRNAs. The switch from type I to type II BZ-S pharmacology upon depolarisation was mirrored by a switch in gene expression from alpha1 (12% of 5K) to alpha3 (371% of 5K). Interestingly, although depolarisation reduced beta2 subunit mRNA to 25% of 5K, beta2 protein was undetectable.Thus, we have shown that electrically active (5K) mouse CGCs differentiate in vitro to express a GABAR profile expected of adult CGCs in vivo. Chronically depolarised, electrically silent CGCs appear to be developmentally arrested, expressing some GABAR characteristics of prenatal CGCs. This model system should prove invaluable for elucidating signalling pathways orchestrating GABAR differentiation.

Narrowing of the region of allelic loss in 21q11-21 in squamous non-small cell lung carcinoma and cloning of a novel ubiquitin-specific protease gene from the deleted segment.

We examined 42 fresh non-small cell lung carcinomas for allelic loss using 4 microsatellite markers located in a 4.5 Mb region in 21q11-21, a gene-poor interval recently found by others to be homozygously deleted and exhibiting frequent allelic loss in lung cancer. We found allelic loss across the entire segment in 13/34 informative squamous carcinomas, with 2 cases showing loss in only part of the region. Analysis by fluorescence in situ hybridization of P1-derived artificial chromosomes from the region directly on paraffin sections of the tumor is in concordance with the loss of heterozygosity (LOH) results, and tentatively excludes a 2 Mb segment bearing 2 of the only 3 known genes in the area. Exon trapping in the remaining segment of loss led to identification and cloning of a novel gene spanning 150 kb within the deletion. The full-length gene encodes a protein of 1,055 amino acids with homology to ubiquitin-specific proteases across the eukaryotic evolutionary spectrum. The expressed protein acts as a de-ubiquitinating enzyme as proved by the ability to cleave ubiquitin from a model fusion protein. We found no mutations in the sequence of the functional domains of this gene in any of the LOH-exhibiting tumor DNA samples. It is, however, interesting that genes of the same superfamily have been reported on 3p21, a locus showing the most frequent allelic instability and deletions in lung cancer. Genes Chromosomes Cancer 27:153-161, 2000.

Human epidermal differentiation complex in a single 2.5 Mbp long continuum of overlapping DNA cloned in bacteria integrating physical and transcript maps.

Terminal differentiation of keratinocytes involves the sequential expression of several major proteins which can be identified in distinct cellular layers within the mammalian epidermis and are characteristic for the maturation state of the keratinocyte. Many of the corresponding genes are clustered in one specific human chromosomal region 1q21. It is rare in the genome to find in such close proximity the genes belonging to at least three structurally different families, yet sharing spatial and temporal expression specificity, as well as interdependent functional features. This DNA segment, termed the epidermal differentiation complex, contains 27 genes, 14 of which are specifically expressed during calcium-dependent terminal differentiation of keratinocytes (the majority being structural protein precursors of the cornified envelope) and the other 13 belong to the S100 family of calcium binding proteins with possible signal transduction roles in the differentiation of epidermis and other tissues. In order to provide a bacterial clone resource that will enable further studies of genomic structure, transcriptional regulation, function and evolution of the epidermal differentiation complex, as well as the identification of novel genes, we have constructed a single 2.45 Mbp long continuum of genomic DNA cloned as 45 p1 artificial chromosomes, three bacterial artificial chromosomes, and 34 cosmid clones. The map encompasses all of the 27 genes so far assigned to the epidermal differentiation complex, and integrates the physical localization of these genes at a high resolution on a complete NotI and SalI, and a partial EcoRI restriction map. This map will be the starting resource for the large-scale genomic sequencing of this region by The Sanger Center, Hinxton, U.K.

Increased levels of a chromosome 21-encoded tumour invasion and metastasis factor (TIAM1) mRNA in bone marrow of Down syndrome children during the acute phase of AML(M7).

Children with Down syndrome (DS) have a 10-20-fold increased risk of acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML), compared to non-DS children. The myeloid leukemia that accounts for nearly 50% of DS leukemias is usually the otherwise uncommon megakaryoblastic type (AML-M7). Though an etiological role of trisomy 21 in leukemogenesis has been suggested, the expression of genes on chromosome 21 in relation to trisomy, DS, and specific DS phenotypes such as leukemia is poorly understood. We used a heterologous-mimic competitive RT-PCR technique to measure the mRNA levels of a chromosome 21 tumour invasion and metastasis factor (TIAM1) directly in bone marrow samples of DS leukemic patients. In the limited number of cases analysed so far, we found TIAM1 mRNA levels in the DS AML-M7 samples of bone marrow taken in the acute phase of the disease (presentation or relapse, n=8) to be highly significantly raised, nearly threefold, compared to that measured in the remission samples or normal individuals (normals + remissions, n=10).

Bacterial contig map of the 21q11 region associated with Alzheimer's disease and abnormal myelopoiesis in Down syndrome.

We present a high-resolution bacterial contig map of 3.4 Mb of genomic DNA in human chromosome 21q11-q21, encompassing the region of elevated disomic homozygosity in Down Syndrome-associated abnormal myelopoiesis and leukemia, as well as the markers, which has shown a strong association with Alzheimer's Disease that has never been explained. The map contains 89 overlapping PACs, BACs, or cosmids in three contigs (850, 850, and 1500 kb) with two gaps (one of 140-210 kb and the second <5 kb). To date, eight transcribed sequences derived by cDNA selection, exon trapping, and/or global EST sequencing have been positioned onto the map, and the only two genes so far mapped to this cytogenetic region, STCH and RIP140 have been precisely localized. This work converts a further 10% of chromosome 21q into a high-resolution bacterial contig map, which will be the physical basis for the long-range sequencing of this region. The map will also enable positional derivation of new transcribed sequences, as well as new polymorphic probes, that will help in elucidation of the role the genes in this region may play in abnormal myelopoiesis and leukemia associated with trisomy 21 and Alzheimer's Disease.

Localisation of receptor interacting protein 140 (RIP140) within 100 kb of D21S13 on 21q11, a gene-poor region of the human genome.

Human chromosome 21 is the smallest and one of the most intensively studied autosomes. The generation of high quality genetic and physical maps for the long arm has enabled the research community to accelerate gene discovery and the identification of disease loci on the chromosome. However, the emerging pattern from large-scale transcriptional mapping from many groups suggests that the majority of the 600-1000 genes predicted to reside on the chromosome are clustered in two distinct regions of the long arm, on distal 21q22.1 and 21q22.3. Here, we report the mapping of the gene for receptor interacting protein 140 (RIP140) on 21q11 by means of YACs, PACs and hybrid cell lines. We have placed RIP140 within 100 kb of D21S13, in a region of the chromosome where only one other gene has been described to date. The association of the RIP140 protein with the superfamily of nuclear receptors may be of significance in studies of trisomy 21 (Down syndrome) and Alzheimers disease, since a modifier locus has been speculated to reside on 21q11.

A novel human phosphoglucomutase (PGM5) maps to the centromeric region of chromosome 9.

The phosphoglucomutases (PGM1-3) in humans are encoded by three genes, PGM1, PGM2, and PGM3. These enzymes are central to carbohydrate metabolism. All three isozymes show genetic variation, and PGM1 has achieved prominence as a key marker in genetic linkage mapping and in forensic science. The human PGM genes are assumed to have arisen by gene duplication since their products are broadly similar in structure and function; however, direct proof of their evolutionary relationship is not available because only PGM1 has been cloned. During a search for other members of the PGM family, a novel sequence with homology to PGM1 was identified. Mapping using fluorescence in situ hybridization and somatic cell hybrids locates this gene to the centromeric region of chromosome 9. RT-PCR and Northern analysis indicate that this is an expressed PGM gene with widespread distribution in adult and fetal tissues. We propose that this gene be designated PGM5 and that it represents a novel member of the PGM family.

Phosphoglucomutase 1: a gene with two promoters and a duplicated first exon.

In view of its central role in glycolysis and gluconeogenesis and its polymorphic genetic variability, the phosphoglucomutase 1 (PGM1) gene in man has been the target of protein structural studies and genetic analysis for more than 25 years. We have now isolated genomic clones containing the complete PGM1 gene and have shown that it spans over 65 kb and contains 11 exons. We have also shown that the sites of the two mutations which form the molecular basis for the common PGM1 protein polymorphism lie in exons 4 and 8 and are 18 kb apart. Within this region there is a site of intragenic recombination. We have discovered two alternatively spliced first exons, one of which, exon 1A, is transcribed in a wide variety of cell types; the other, exon 1B, is transcribed in fast muscle. Exon 1A is transcribed from a promoter which has the structural hallmarks of a housekeeping promoter but lies more than 35 kb upstream of exon 2. Exon 1B lies 6 kb upstream of exon 2 within the large first intron of the ubiquitously expressed PGM1 transcript. The fast-muscle form of PGM1 is characterized by 18 extra amino acid residues at its N-terminal end. Sequence comparisons show that exons 1A and 1B are structurally related and have arisen by duplication.

The classical human phosphoglucomutase (PGM1) isozyme polymorphism is generated by intragenic recombination.

The molecular basis of the classical human phosphoglucomutase 1 (PGM1) isozyme polymorphism has been established. In 1964, when this genetic polymorphism was first described, two common allelozymes PGM1 and PGM1 2 were identified by starch gel electrophoresis. The PGM1 2 isozyme showed a greater anodal electrophoretic mobility than PGM1 1. Subsequently, it was found that each of these allelozymes could be split, by isoelectric focusing, into two subtypes; the acidic isozymes were given the suffix + and the basic isozymes were given the suffix -. Hence, four genetically distinct isozymes 1+, 1-, 2+, and 2- were identified. We have now analyzed the whole of the coding region of the human PGM1 gene by DNA sequencing in individuals of known PGM1 protein phenotype. Only two mutations have been found, both C to T transitions, at nt 723 and 1320. The mutation at position 723, which changes the amino acid sequence from Arg to Cys at residue 220, showed complete association with the PGM1 2/1 protein polymorphism: DNA from individuals showing the PGM1 1 isozyme carried the Arg codon CGT, whereas individuals showing the PGM1 2 isozyme carried the Cys codon TGT. Similarly, the mutation at position 1320, which leads to a Tyr to His substitution at residue 419, showed complete association with the PGM1+/- protein polymorphism: individuals with the + isozyme carried the Tyr codon TAT, whereas individuals with the - isozyme carried the His codon CAT. The charge changes predicted by these amino acid substitutions are entirely consistent with the charge intervals calculated from the isoelectric profiles of these four PGM1 isozymes. We therefore conclude that the mutations are solely responsible for the classical PGM1 protein polymorphism. Thus, our findings strongly support the view that only two point mutations are involved in the generation of the four common alleles and that one allele must have arisen by homologous intragenic recombination between these mutation sites.