Identification and characterization of a novel human brain-specific gene, homologous to S. scrofa tmp83.5, in the chromosome 10q24 critical region for temporal lobe epilepsy and spastic paraplegia.

We describe the structure, genomic organization, and some transcription features of a human brain-specific gene previously localized to the genomic region involved in temporal lobe epilepsy and spastic paraplegia on chromosome 10q24. The gene, which consists of six exons disseminated over 16 kb of genomic DNA, is highly homologous to the porcine tmp83.5 gene and encodes a putative transmembrane protein of 141 amino acids. Unlike its porcine homolog, from which two mRNAs with different 5'-sequences are transcribed, the human gene apparently encodes three mRNA species with 3'-untranslated regions of different sizes. Mutation analysis of its coding sequence in families affected with temporal lobe epilepsy or spastic paraplegia linked to 10q24 do not support the involvement of this gene in either diseases.

neuralized Encodes a peripheral membrane protein involved in delta signaling and endocytosis.

Activation of the Notch (N) receptor involves an intracellular proteolytic step triggered by shedding of the extracellular N domain (N-EC) upon ligand interaction. The ligand Dl has been proposed to effect this N-EC shedding by transendocytosing the latter into the signal-emitting cell. We find that Dl endocytosis and N signaling are greatly stimulated by expression of neuralized (neur). neur inactivation suppresses Dl endocytosis, while its overexpression enhances Dl endocytosis and Notch-dependent signaling. We show that neur encodes an intracellular peripheral membrane protein. Its C-terminal RING domain is necessary for Dl accumulation in endosomes, but may be dispensable for Dl signaling. The potent modulatory effect of Neur on Dl activity makes Neur a candidate for establishing signaling asymmetries within cellular equivalence groups.

ZNF232: structure and expression analysis of a novel human C(2)H(2) zinc finger gene, member of the SCAN/LeR domain subfamily.

We have identified a novel zinc finger gene, ZNF232, mapped to human chromosome 17p12. The coding region of the gene is organized in three exons corresponding to a 417 amino acid long polypeptide containing a SCAN/LeR domain and five C(2)H(2)-type zinc fingers. ZNF232 is possibly a nuclear protein, as suggested by expression analysis of GFP/ZNF232 chimeric constructs. ZNF232 transcripts were detected in a wide collection of adult human tissues. The gene is possibly subjected to tissue-specific post-transcriptional regulation by means of alternative splicing.

Detection and incidence of cryptic Y chromosome sequences in Turner syndrome patients.

The presence of Y chromosome sequences in Turner syndrome (TS) patients may predispose them to gonadoblastoma formation with an estimated risk of 15-25%. The aim of this study was to determine the presence and the incidence of cryptic Y chromosome material in the genome of TS patients. The methodology involved a combination of polymerase chain reaction (PCR) and nested PCR followed by Southern blot analysis of three genes the sex determining region Y (SRY), testis specific protein Y encoded (TSPY) and RNA binding motif protein (RBM) (previously designated as YRRM) and nine additional STSs spanning all seven intervals of the Y chromosome. The methodology has a high sensitivity as it detects one 46,XY cell among 10(5) 46,XX cells. Reliability was ensured by taking several precautions to avoid false positive results. We report the results of screening 50 TS patients and the identification of cryptic Y chromosome material in 12 (24%) of them. Karyotypes were divided in four groups: 5 (23.8%) patients out of the 21 TS patients which have the 45,X karyotype (group A) also have cryptic Y sequences; none (0%) of the 7 patients who have karyotypes with anomalies on one of the X chromosomes have Y mosaicism (group B); 1 (6.3%) of the 16 patients with a mosaic karyotype have Y material (group C); and 6 (100%) out of 6 patients with a supernumerary marker chromosome (SMC) have Y chromosome sequences (group D). Nine of the 12 patients positive for cryptic Y material were recalled for a repeat study. Following new DNA extraction, molecular analysis was repeated and, in conjunction with fluorescent in situ hybridization (FISH) analysis using the Y centromeric specific probe Yc-2, confirmed the initial positive DNA findings. This study used a reliable and sensitive methodology to identify the presence of Y chromosome material in TS patients thus providing not only a better estimate of a patient's risk in developing either gonadoblastoma or another form of gonadal tumor but also the overall incidence of cryptic Y mosaicism.

Human hepatocyte nuclear factor-4 (hHNF-4) gene maps to 20q12-q13.1 between PLCG1 and D20S17.

Human hepatocyte nuclear factor 4 (hHNF-4) is a member of the nuclear hormone receptor superfamily and an important transcription factor and developmental regulator of liver-specific genes. Distinct hHNF-4 cDNAs corresponding to various HNF-4 isoforms have been recently characterised. Three cDNAs, hHNF-4A, B and C, are considered splice variants of a single hHNF-4 gene. We have mapped hHNF-4 to 20q12-q13.1 between PLCG1 and D20S17 by genetic linkage analysis, taking advantage of an adjacent PstI restriction fragment length polymorphism, (RFLP), and by fluorescence in situ hybridisation. hHFN-4 maps to chromosome 20 in a region syntenic with mouse chromosome 2 where the hnf-4 homologue has been assigned.

Map integration at human chromosome 10: molecular and cytogenetic analysis of a chromosome-specific somatic cell hybrid panel and genomic clones, based on a well-supported genetic map.

Well-characterized, chromosome-specific somatic cell hybrid panels are powerful tools for the analysis of the human genome. We have characterized a panel of human x hamster somatic cell hybrids retaining fragments of human chromosome 10 by fluorescence in situ hybridization and associated them to genetic markers. Most of the hybrids were generated by the radiation-reduction method, starting from a chromosome 10-specific monochromosomal hybrid, whereas some were collected from hybrids retaining chromosome 10-specific fragments as a result of spontaneous in vitro rearrangements. PCR was used to score the retention of 57 microsatellite markers evenly distributed along a well-supported framework genetic map containing 149 loci uniquely placed at 69 anchor points (odds exceeding 1,000:1), with an average spacing of 2.8 cM. As an additional resource for genomic studies involving human chromosome 10, we report the cytogenetic localization of a series of YAC and PAC clones recognized by at least one genetic marker. Somatic cell hybrids provide a powerful source of partial chromosome paints useful for detailed clinical cytogenetic and primate chromosome evolution investigations. Furthermore, correlation of the above physical, genetic, and cytogenetic data contribute to an emerging consensus map of human chromosome 10.

European Gene Mapping Project (EUROGEM): breakpoint panels for human chromosomes based on the CEPH reference families. Centre d'Etude du Polymorphisme Humain.

Meiotic breakpoint panels for human chromosomes 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14, 15, 17, 18, 20 and X were constructed from genotypes from the CEPH reference families. Each recombinant chromosome included has a breakpoint well-supported with reference to defined quantitative criteria. The panels were constructed at both a low-resolution, useful for a first-pass localization, and high-resolution, for a more precise placement. The availability of such panels will reduce the number of genotyping experiments necessary to order new polymorphisms with respect to existing genetic markers. This paper shows only a representative sample of the breakpoints detected. The complete data are available on the World Wide Web (URL http:/(/)www.icnet.uk/axp/hgr/eurogem++ +/HTML/data.html) or by anonymous ftp (ftp.gene.ucl.ac.uk in/pub/eurogem/maps/breakpoints).

Isolation and characterization of a third isoform of human hepatocyte nuclear factor 4.

Hepatocyte nuclear factor 4 (HNF-4) is an essential positive regulator of a large number of liver-specific genes. We report here the isolation of three HNF-4 isoforms from a human liver cDNA library. hHNF-4A and hHNF-4B, differing by the insertion of 10 amino acids in the C-terminal region, have been previously identified in mouse, rat and human liver. The novel isoform, hHNF-4C, is identical to hHNF-4A and B in the regions encompassing the DNA-binding and dimerization domains, but contains a different C-terminal domain. Similar to the other isoforms, hHNF-4C is produced in a limited number of tissues and represents 2.6-13% of the total hHNF-4 mRNA population, depending on the cell type. The chromosomal origin of all three isoforms has been localized to human chromosome 20. hHNF-4C can form heterodimers with hHNF-4A and B in vitro, and exhibits similar transactivation potential as hHNF-4A or B in transient transfection assays, suggesting that the divergent C-terminal region is not part of the transactivation domain.

Tetra-/di-nucleotide repeat polymorphism upstream of the human alpha 2-globin gene locus at 16p13.3.

A highly polymorphic tetra-/di-nucleotide repeat sequence was identified upstream of the human alpha 2/alpha 1-globin gene pair on chromosome 16p13.3. This microsatellite marker should be useful in alpha-thalassemia genotype-phenotype correlations and in respective population genetics studies.

Sequences located 3' to the breakpoint of the hereditary persistence of fetal hemoglobin-3 deletion exhibit enhancer activity and can modify the developmental expression of the human fetal A gamma-globin gene in transgenic mice.

Expression of fetal gamma-globin genes in individuals with the deletion forms of hereditary persistence of fetal hemoglobin (HPFH) has been attributed either to enhancement by 3' regulatory elements juxtaposed to gamma-globin genes or to deletion of gamma-gene silencers normally residing within the beta-globin gene cluster. In the present study, we tested the hypothesis of imported enhancers downstream of beta-globin gene using the HPFH-3 deletion as a model. The abnormal bridging fragment of 13.6 kilobases (kb) containing the A gamma-gene with its flanking sequences and 6.2 kb of the juxtaposed region was microinjected into fertilized mouse eggs. Twelve transgenic mice positive for the fragment were generated. Samples from 11.5-day yolk sacs, 16-day fetal liver, and adult blood were analyzed for A gamma-mRNA using RNase protection assays. Three mice lacked A gamma expression in the yolk sac indicating non-optimal integration site. Four expressed A gamma-mRNA at the embryonic stage only, while two expressed A gamma-mRNA in both embryonic and fetal liver erythroid cells. Since the A gamma-gene with its normal flanking sequences and in the absence of the locus control region is expressed only in embryonic cells of transgenic mice, these data suggest that the juxtaposed sequences have altered the developmental specificity of the fetal gamma-globin gene. These sequences were further tested for the presence of an enhancer element, by their ability to activate a fusion reporter gene consisting of the CAT gene linked to the gamma-globin gene promoter, in erythroid (K562) and non-erythroid (HeLa) cells. A 0.7-kb region located immediately 3' to the breakpoint, enhanced chloramphenicol acetyltransferase activity by 3-fold in erythroid cells. The enhancer also activated the embryonic epsilon-globin gene promoter by 2-fold but not the adult beta- or delta-globin gene promoters. The enhancer represents a region of previously known complex tandem repeats; in this study we have completed the sequencing of the region encompassing the 0.7-kb enhancer element. Multiple areas of the enhancer region exhibit homology to the core element of the simian virus 40 enhancer and to the sequences of the human 3' A gamma- and the chicken 3' beta-globin enhancers. A consensus binding site for the erythroid specific GATA-1 transcription factor and seven consensus sites for the ubiquitous CP1 transcription factor are also included within the enhancer. These data suggest that these sequences located immediately 3' to the breakpoint of the HPFH-3 deletion, exhibit both the structure and the function of an enhancer, and can modify the developmental specificity of the fetal gamma-globin genes, resulting in their continued expression during adult life.

[A normalized cDNA library from human erythroleukemia cells]. / Normalizovannaia klonoteka kDNK iz éritroleikoznykh kletok cheloveka.

Normalized cDNA library from human erythroleukemia cells has been constructed. For equalizing frequencies of different cDNAs denaturation was carried out followed by partial reassociation. Single-stranded cDNAs separated by hydroxyapatite chromatography were transformed into double-stranded form by PCR and cloned in lambda gt11. Frequencies of 10 control nucleotide sequences were estimated. After normalization frequencies of the abundant sequences decreased. The normalized cDNA library may be used for search of the clones corresponding to the rare mRNAs and for human genome mapping.

PKU in Slovakia: mutation screening and haplotype analysis.

The restriction fragment length polymorphism haplotypes and seven common mutations in the phenylalanine hydroxylase gene were analysed in 49 unrelated Slovak phenylketonuria (PKU) families of Caucasian origin. The predominant mutation in this population sample is R408W, with a frequency of 45.9%. In addition, four other mutations have been identified at relatively high frequencies: IVS12nt1, 10.2%; R158Q, 7.1%; R261Q, 7.1%; R252W, 2.0%. The mutation-haplotype associations correspond to those described in other European populations. The high proportion of mutations (72.4%) amenable to simple rapid detection based on the polymerase chain reaction provides a good basis for direct DNA-diagnosis of PKU in the Slovak population.

Chromosomal mapping of two members of the human glutamate dehydrogenase (GLUD) gene family to chromosomes 10q22.3-q23 and Xq22-q23.

Glutamate dehydrogenase (GLUD) is an important mitochondrial enzyme that participates in neuronal transmission by catalyzing the deamination of L-glutamate, which serves as a potent excitatory neurotransmitter. The direct involvement of GLUD in the pathogenesis of certain human neurodegenerative disorders has been suggested recently. To investigate its possible role in the induction and progression of these disorders, we have initiated studies focusing on the chromosomal organization of the several members of the GLUD family and their functional status. In the present study using a panel of human x rodent somatic cell hybrids and in situ hybridization to metaphase chromosomes, we documented that the members of the GLUD gene family are dispersed in the human genome. The functional GLUD1 gene was mapped to chromosome 10q22.3-q23, and an intronless processed gene (GLUDP1) to chromosome Xq22-q23, while the truncated intron-containing GLUD pseudogene GLUDP2 was also assigned on chromosome 10, but not closely linked to the GLUD1 gene. These results provide novel information concerning the chromosomal organization of the human GLUD gene family.

Three human glutamate dehydrogenase genes (GLUD1, GLUDP2, and GLUDP3) are located on chromosome 10q, but are not closely physically linked.

Yeast artificial chromosomes (YACs) of 340 and 370 kb that contain the functional human glutamate dehydrogenase gene (GLUD1) and the pseudogene GLUDP2, respectively, were isolated. These genes were not physically linked to each other nor to any other sequences homologous to the exons of GLUD1. No additional GLUD sequences were found within at least 70 kb of the 5' and 175 kb of the 3' end of GLUD1 or 150 kb of either end of GLUDP2. By in situ hybridization, GLUD1 was located at 10q23.3, GLUDP2 at 10q11.2, and another pseudogene of the GLUD gene family, GLUDP3, at 10q22.1. DNA fragments of these three genes showed cross-hybridization to the loci assigned to the other two genes, but not to any other chromosomal locus. Thus, these three genes are located at distinct positions on chromosome 10q.