No mutations found in candidate genes for dystocia.

Dystocia is a disorder characterized by prolonged or dysfunctional labour. Delivery that starts late or not at all, leads to an increased risk for Caesarean section, infant morbidity and mortality. Familial aggregations of dystocia suggest a polygenic background. We have studied three candidate genes for dystocia, i.e. the genes for testosterone 5-alpha reductase type 1, prostaglandin F2alpha receptor and endothelin 1 and performed mutational screening in 23 women with dystocia, of which 12 have affected relatives. No mutations were found, making it unlikely that any of these genes represent a major cause of dystocia in man.

Restricted expression pattern of vegf-d in the adult and fetal mouse: high expression in the embryonic lung.

Endothelial growth factors have become the target of intense research since the initial discovery of vascular endothelial growth factor (VEGF/VPF). At present, VEGF is established as a major inducer of angiogenesis in normal and pathological conditions. Recently several new members in the VEGF family have been described; VEGF-B/VRF, VEGF-C and VEGF-D. VEGF-D is most closely related to VEGF-C by virtue of the presence of N- and C-terminal extensions that are not found in other VEGF family members. We have here examined the expression pattern of vegf-d mRNA with in situ hybridization in developing and adult mice. This shows a restricted expression pattern, with high levels mainly in lung tissue. The expression in embryonic lung is upregulated prior to birth. Expression of vegf-d in other tissues, as well as in lung tissue of the E14 embryo, was either low or absent. This suggests that VEGF-D may be of special relevance for the vascularization of lung tissue during the last trimester of fetal development.

Genomic structure, 5' flanking sequences, and precise localization in 1P31.1 of the human prostaglandin F receptor gene.

This paper describes the genomic structure of the human Prostaglandin F receptor gene (FP) with its exon-intron borders and 5' flanking sequences. Furthermore, the location of the gene has been localized to a very small region on 1p31.1 using FISH and radiation hybrids analysis. The PGF receptor (FP) is highly expressed in mouse tissues especially in the corpora lutea in ovaries and in the kidney. Recently, it has been shown that homozygous knockout-mice lacking the gene for this receptor are unable to deliver normal fetuses at term. It might be speculated that the lack of the FP gene has the same effect in human as in mouse. Mutation analysis in families with difficulties in parturition would therefore be of high interest. The results presented here provides data necessary for further investigations of the FP gene.

Characterization of the mouse Men1 gene and its expression during development.

The gene responsible for multiple endocrine neoplasia type 1 (MEN1), a heritable predisposition to endocrine tumours in man, has recently been identified. Here we have characterized the murine homologue with regard to cDNA sequence, genomic structure, expression pattern and chromosomal localisation. The murine Men1 gene spans approximately 6.7 kb of genomic DNA and is comprised of 10 exons with similar genomic structure to the human locus. It was mapped to the pericentromeric region of mouse chromosome 19, which is conserved with the human 11q13 band where MEN1 is located. The predicted protein is 611 amino acids in length and overall is 97% homologous to the human orthologue. The 45 reported MEN1 mutations which alter or delete a single amino acid in human all occur at conserved residues, thereby supporting their functional significance. Two transcripts of approximately 3.2 and 2.8 kb were detected in both embryonal and adult murine tissues, resulting from alternative splicing of intron 1. By RNA in situ hybridization and Northern analysis the spatiotemporal expression pattern of Men1 was determined during mouse development. Men1 gene activity was detected already at gestational day 7. At embryonic day 14 expression was generally high throughout the embryo, while at day 17 the thymus, skeletal muscle, and CNS showed the strongest signal. In selected tissues from postnatal mouse Men1 was detected in all tissues analysed and was expressed at high levels in cerebral cortex, hippocampus, testis, and thymus. In brain the menin protein was detected mainly in nerve cell nuclei, whereas in testis it appeared perinuclear in spermatogonia. These results show that Men1 expression is not confined to organs affected in MEN1, suggesting that Men1 has a significant function in many different cell types including the CNS and testis.

A comparative study of the expression patterns for vegf, vegf-b/vrf and vegf-c in the developing and adult mouse.

With the goal of better understanding the function and regulation of the different members of the VEGF family this study reports mapping of vegf, vegf-b and vegf-c mRNA expression in developing and adult mice. On embryonic day 14 (E14) there is a high expression of vegf and vegf-b, vegf-b being exceptionally high in heart and CNS. The vegf-c expression is lower with distinct signals in CNS and heart. Prior to birth (E17), vegf and vegf-b expression is moderately downregulated. Overlapping expression is present in intrascapular fat and heart. vegf dominates in thyroid and lung, while vegf-b appears to be the only VEGF member expressed at detectable levels in the CNS. In young adult mouse vegf and vegf-b show partly overlapping expression patterns particularly in kidney, heart and in the thymus, vegf displays higher levels in lung and liver, vegf-b appears to be dominating in brain, heart, testis and kidney. In brain the highest levels of vegf-b is present in the hippocampus. No vegf-c mRNA expression could be detected in the adult. Taken together, these results illustrate, in detail, the different regulations of the members of the VEGF gene family. There are at present at least three specific effectors of vascular proliferation with clear differences in their expression.

Expression and chromosomal localization of the Requiem gene.

Apoptosis in murine myeloid cell lines requires the expression of the Requiem gene, which encodes a putative zinc finger protein. We detected the protein in both cytoplasmic and nuclear subcellular fractions of murine myeloid cells and human K562 leukemia cells, which suggests that the protein might have a function distinct from a transcription factor. This distribution did not alter upon apoptosis induction by IL-3 deprivation. As an approach to investigate its role in development, we determined the spatio-temporal expression pattern in the mouse. Expression was detected in various tissues in earlier gestational age; however, confined to testes, spleen, thymus, and part of the hippocampus in the adult mouse. The expression profile is consistent with a functional role during rapid growth and cell turnover, and in agreement with a regulatory function for hematopoietic cells. The human cDNA clone sequenced showed high homology to its murine counterpart and extended the open reading frame by 20 codons upstream. The gene is located in the proximal region of mouse Chromosome (Chr) 19. In the homologous human region at 11q13, it is located at about 150 kb centromeric from MLK3.

Expression of the BCL6 gene in the pre- and postnatal mouse.

Human BCL6, also called LAZ3, is a protein involved in gene regulation and abnormal expression of BCL6 and has been implicated in the tumorigenesis of non-Hodgkin lymphoma. We have analyzed the expression of murin bcl6 in pre- and postnatal mouse using in situ hybridization histochemistry and Northern blotting. The developing olfactory epithelium in the nasal cavity was the only tissue displaying a positive bcl6 mRNA signal in the day 14 embryo. At gestational day 17, expression was primarily seen in skeletal muscle, olfactory epithelium, and thymus, and also in the epithelium lining the upper airways and esophagus. In selected tissues from postnatal mouse, bcl6 expression was detected in brain, renal cortex, spleen, and thymus. The expression in brain was restricted to the pyramidal cell layer of the cerebral cortex and the hippocampus regions CA1 and CA2, and the dentate gyrus. Our results show that bcl6 expression is not confined only to organs of the lymphatic system, such as spleen and thymus. Thus, bcl6 may be active as a regulator of gene transcription in many different cell types, including epithelial and nerve cells.

A sequence highly similar to PNG is located on chromosome 22q12 in intron 15 of the LIMK-2 gene.

In this report we describe a sequence (PNG22) highly similar to the Phospholipase C beta3 Neighboring Gene (PNG). We also report that PNG22 is located in the q12 region on chromosome 22 between markers D22S1144 and D22S280. This finding explains that PNG probes cross hybridize to sequences on chromosome 22. Fine mapping using our sequence data and the complete sequence of a PAC clone (DJ515N1), located in this region, determined that PNG22 is located in intron 15 of the LIMK-2 gene. PNG22 is 93% homologous to PNG, however it do not have the introns described for the PNG gene, instead matching the cDNA sequence. This leads us to suggest that PNG22 probably represents a PNG pseudogene. In this report we also list the exon intron borders and the genomic structure of LIMK-2 and place it on the Sanger Center chromosome 22 Physical map. It also explains the finding that PNG probes cross hybridize to sequences on chromosome 22.

Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern.

The estrogen receptor (ER) is a ligand-activated transcription factor that mediates the effects of the steroid hormone 17 beta-estradiol, in both males and females. Since the isolation and cloning of ER, the consensus has been that only one such receptor exists. The finding of a second subtype of ER (ER beta) has caused considerable excitement amongst endocrinologists. In this article, we present data regarding the genomic structure and chromosomal localization of the human ER beta gene, demonstrating that two independent ER genes do exist in the human. Furthermore, we present data regarding the tissue distribution of human ER beta, showing that this receptor is expressed in multiple tissues. For instance, ER beta is found in developing spermatids of the testis, a finding of potential relevance for the ongoing debate on the effects of environmental estrogens on sperm counts. In addition, we find ER beta in ovarian granulosa cells, indicating that estrogens also participate in the regulation of follicular growth in the human.

The germinal center kinase gene and a novel CDC25-like gene are located in the vicinity of the PYGM gene on 11q13.

Multiple endocrine neoplasia type 1 (MEN1) is tightly linked to the muscle-type glycogen phosphorylase (PYGM) gene in 11q13. This region of the human genome contains additional disease-related loci implicated in the development of insulin-dependent diabetes mellitus, familial paraganglioma type 2, spinocerebellar ataxia type 5, Bardet-Biedl syndrome and translocation t(11;17) described in B-cell non-Hodgkin's lymphoma. We approached cloning of candidate disease genes from 11q13 by large-scale genomic sequencing. We obtained > 106 kb of sequence around the PYGM gene and established a transcriptional map that includes: (i) two genes previously localized to 11q13, PYGM and a zinc-finger protein (ZFM1) gene; (ii) the germinal center kinase (GCK, human B-lymphocyte serine/threonine protein kinase) gene; (iii) a novel human CDC25-like (HCDC25L) gene; (iv) a dystrophia myotonica protein kinase-like (DMPKL) gene; and (v) a novel ubiquitously expressed gene of unknown function (germinal center kinase- neighboring gene, GCKNG).

Expression of LAZ3/BCL6 in follicular center (FC) B cells of reactive lymph nodes and FC-derived non-Hodgkin lymphomas.

Chromosomal translocation resulting in abnormal expression of the LAZ3/BCL6 gene in B cells has been implicated in the tumorigenesis of non-Hodgkin lymphoma (NHL). Therefore we studied the expression pattern of LAZ3/BCL6 by in situ hybridization with synthetic oligonucleotide probes in frozen tissue sections from five reactive lymph nodes and 38 B cell and non-B NHL. In addition, we investigated the expression of LAZ3/BCL6 by Northern blot analysis on multiple human tissues. The LAZ3/BCL6 transcript was found in a variety of tissues, including skeletal muscle, peripheral blood leukocytes, and weakly in normal lymph nodes. In the tumor samples, expression of LAZ3/BCL6 was observed in 68% of all B cell NHL and none of the non-B lymphomas. All cases of follicular, mixed small and large cell lymphomas showed LAZ3/BCL6 expression confined to the neoplastic follicles. A follicular expression pattern was also found in all non-malignant reactive lymph nodes. Hence, the expression of LAZ3/BCL6 does not correlate to malignancy, but reflects the origin of B cells from the germinal centers.

Multiple endocrine neoplasia type 1 and the search for the genetic trigger.

Multiple endocrine neoplasia type 1 (MEN-1) is characterized by primary hyperparathyroidism, endocrine pancreatic-duodenal and anterior pituitary tumors. The diagnosis is challenging and involves the exclusion of other endocrine neoplasia syndromes with overlapping features. The predisposing genetic defect was assigned to chromosomal region 11q13 based on linkage analysis. Combined tumor and pedigree genotype analysis showed that allele losses in pancreatic, parathyroid and pituitary tumors eliminated the wild-type allele at the 11q13 loci, suggesting inactivation of a tumor suppressor gene in this region. A 5-Mb integrated map of the region has been established by the European consortium on MEN-1. Based on this mapping the critical interval was restricted to 2 Mb, a region within which eight candidate genes are located.

Exclusion of the phosphoinositide-specific phospholipase C beta 3 (PLCB3) gene as a candidate for multiple endocrine neoplasia type 1.

The predisposing genetic defect in multiple endocrine neoplasia type 1 has been assigned to chromosomal region 11q13. Our previous attempts to identify the MEN1 gene have resulted in the isolation of the phospholipase C beta 3 gene from the actual region. PLCB3 plays an important role in signal transduction and, moreover, shows loss of expression in some endocrine tumors, in accordance with a putative tumor suppressor gene function, and thus appears to be an excellent candidate for MEN1. We have therefore undertaken screening for constitutional mutations in individuals from MEN1 families. Several sequence alterations have been discovered, none of them however fulfilling the criteria for a disease-related mutation. We can now exclude PLCB3 from candidacy as the MEN1 gene.

Primary structure of human 11-cis retinol dehydrogenase and organization and chromosomal localization of the corresponding gene.

The universal chromophore of visual pigments in higher animals is 11-cis retinaldehyde. The final step in the biosynthetic pathway generating this compound is catalyzed by 11-cis retinol dehydrogenase, a membrane-bound enzyme abundantly expressed in the retinal pigment epithelium of the eye. In this work we demonstrate that the primary structure of human 11-cis retinol dehydrogenase is highly conserved with 91% identity to the bovine enzyme. The gene encoding 11-cis retinol dehydrogenase spans over approximately 4.1 kb of DNA and is divided into four translated exons. Analysis of a panel of somatic cells hybrids and fluorescence in situ hybridization on metaphase chromosomes revealed that the gene is located on chromosome 12q13-q14. Due to the unique role of 11-cis retinol dehydrogenase in the generation of visual pigments, it is a candidate gene for involvement in hereditary eye disease.

Sequence and expression of the mouse homologue to human phospholipase C beta3 neighboring gene.

We describe the isolation and expression of a murine homologue of the Phospholipase C beta3 Neighboring Gene (PNG), located in the MEN1 region on chromosome 11q13. The PNG cDNA was isolated using a human PNG cDNA clone (SOM172). Human and mouse PNG do not have any marked similarity to other known genes on the DNA level, but the predicted protein display similarity to the C-terminal part of Phospholipase C beta2. Northern blots with mouse PNG probes revealed expression of a 1 kb message in multiple tissues, and an additional 2.3 kb band in testis. The predicted murine protein contains 203 amino acids. In situ hybridization histochemistry displayed png mRNA expression in several tissues of the midstage mouse embryo, including the central nervous system. In late stage embryos, png was highly expressed in skeletal muscle, retina and neocortex. In the adult animal, expression was restricted to testis and thymus.

Nucleotide sequence of a cDNA clone (SUBT1) partly homologous to a human subtelomeric repeat sequence.

Telomeres contains short tandem repeats (TTAGGG)n that are usually several kilobases in size. DNA sequences located next to the repeats are called telomere-associated or subtelomeric sequences. In this report we describe isolation and characterization of a new cDNA sequence isolated with a telomere specific, chromosome 17 genomic clone. No open reading frame of notable length could be detected. However comparison with sequences in the EMBL/Genbank revealed a 897 bp of close to perfect match, in the 3'end with the centromeric end of a genomic clone containing subtelomeric repeat sequences. The potential function of the SUBT1 transcript remains to be elucidated.

The gene for human glutaredoxin (GLRX) is localized to human chromosome 5q14.

Glutaredoxin is a small protein (12 kDa) catalyzing glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH, and glutathione reductase. A cDNA encoding the human glutaredoxin gene (HGMW-approved symbol GLRX) has recently been isolated and cloned from a human fetal spleen cDNA library. The screening of a human genomic library in Charon 4A led to the identification of three genomic clones. Using fluorescence in situ hybridization to metaphase chromosomes with one genomic clone as a probe, the human glutaredoxin gene was localized to chromosomal region 5q14. This localization at chromosome 5 was in agreement with the somatic cell hybrid analysis, using DNA from a human-hamster and a human-mouse hybrid panel and using a human glutaredoxin cDNA as a probe.