Targeted disruption of the mouse Npal3 gene leads to deficits in behavior, increased IgE levels, and impaired lung function.

The non-imprinted in Prader-Willi/Angelman syndrome (NIPA) proteins are highly conserved receptors or transporters. Translocation of NIPA genes were found in patients with Prader-Willi syndrome, and loss-of-function of the NIPA1 gene was identified in hereditary spastic paraplegia. The family of NIPA-like domain containing (NPAL) proteins is closely related to the NIPA proteins, but to date nothing is known about their function. Here, we could demonstrate that both human NPAL3 and mouse NPAL3 are ubiquitously expressed and encode highly conserved proteins. To further elucidate the function of the Npal3 gene, knockout (Npal3(-/-)) mice were generated. Intensive phenotypic analyses revealed that disruption of the Npal3 gene results in a pleiotropic phenotype. The function of the nervous system was impaired in both mutant males and females which could be demonstrated in behavioral tests. In addition, in NPAL3 mutants the number of NK cells was decreased and changes in IgM, IgG(2), and IgA were observed, indicating that the immune system is also affected. Interestingly, increased IgE levels as well as impaired lung functions were observed in mutant males but not in mutant females. It should be noted that the human Npal3 gene is located at 1p36.12-->p35.1, and atopic diseases were previously linked to this genomic region. Thus, the Npal3(-/-) mice could serve as a valuable model system for studying atopic diseases.

The putative peroxisomal gene Pxt1 is exclusively expressed in the testis.

Genes reported to be crucial for spermatogenesis are often exclusively expressed in the testis. We have identified a novel male germ cell-specific expressed gene named peroxisomal testis specific 1 (Pxt1) with expression starting at the spermatocyte stage during mouse spermatogenesis. The putative amino acid sequence encoded by the cDNA of the Pxt1 gene contains a conserved Asn-His-Leu (NHL)-motif at its C-terminal end, which is characteristic for peroxisomal proteins. Pxt1-EGFP fusion protein is co-localized with known peroxisomal marker proteins in transfected NIH3T3 cells. In addition, we could demonstrate that the peroxisomal targeting signal NHL is functional and responsible for the correct subcellular localization of the Pxt1-EGFP fusion protein. In male germ cells peroxisomes were reported only in spermatogonia. The Pxt1 gene is so far the first gene coding for a putative peroxisomal protein which is expressed in later steps of spermatogenesis, namely in pachytene spermatocytes.

Histopathology of lupus-like nephritis in Dnase1-deficient mice in comparison to NZB/W F1 mice.

Deoxyribonuclease 1 (Dnase1)-deficient mice develop symptoms of Systemic lupus erythematosus (SLE). Here we analysed the renal histopathology of these animals in comparison to F1 hybrids of New Zealand black and white mice (NZB/W F1), an established model of SLE. Animals were divided into three groups according to the presence of anti-nuclear antibodies (ANA) and renal lesions. Groups 1a-1c were healthy, whereas group 2 and 3 were classified as lupus-prone and affected. Subendothelial and/or mesangial immune complex deposits, mesangial and endocapillary proliferation, haematoxylin bodies and platelet aggregation were detected in both mouse strains but were more severe in the NZB/W F1 mice. The lupus nephritis was classified as a proliferating (WHO type III or IV), which appeared to be preceded by a mesangial form (WHO type II). Subclassification of the ANA revealed a high prevalence of anti-nucleosome antibodies in Dnase1-deficient mice, whereas NZB/W F1 mice developed autoantibodies against a broad range of chromatin constituents. Mapping of the murine Dnase1 gene locus to chromosome 16A1-3 did not coincide with one of the reported susceptibility loci in the NZB/W F1 model, although a reduced Dnasel serum and urine activity has been described previously in these mice.

Identification of the human ortholog of the t-complex-encoded protein TCTE3 and evaluation as a candidate gene for primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive disease that is caused by impaired ciliary and flagellar functions. About 50% of PCD patients show situs inversus, denoted as Kartagener syndrome. In most cases, axonemal defects in cilia and sperm tails can be demonstrated by electron microscopy, i.e. PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD. In order to identify novel PCD genes we have isolated the human ortholog of the murine TCTE3 gene. The human TCTE3 gene encodes a dynein light chain and shares high similarity to dynein light chains of other species. The TCTE3 gene is expressed in tissues containing cilia or flagella, it is composed of four exons and located on chromosome 6q25-->q27. To elucidate the role of TCTE3 as a candidate gene for PCD a mutational analysis of thirty-six PCD patients was performed. We detected five polymorphisms in the coding sequence and in the 5' UTR of the TCTE3 gene. In one patient a heterozygous nucleotide exchange was identified resulting in an arginine to isoleucine substitution at the amino acid level. However, this exchange was also detected in one control DNA. Our results indicate that mutations in the TCTE3 gene are not a main cause of primary ciliary dyskinesia.

Teratozoospermia in mice lacking the transition protein 2 (Tnp2).

It is believed that the transition proteins (Tnp1 and Tnp2) participate in the removal of the nucleohistones and in the initial condensation of the spermatid nucleus. Later in spermatogenesis, Tnp1 and Tnp2 are replaced by the protamines 1 and 2. In an effort to elucidate the physiological role of Tnp2, we have disrupted its locus by homologous recombination. Breeding of the Tnp2(-/-) males on different genetic backgrounds revealed normal fertility on the mixed background C57BL/6Jx129/Sv, but total infertility on the inbred 129/Sv background. Light and electron microscopy showed that the germ cells were capable of undergoing chromatin condensation, although many spermatozoa exhibited head abnormalities with acrosomes not attached to the nuclear envelope. Furthermore, migration of Tnp2(-/-) spermatozoa from the uterus into the oviduct was reduced. These results suggest that male infertility of the Tnp2(-/-) mice is a result of sperm head abnormalities and reduced sperm motility. The increased level of the Tnp1 transcript in testes of the Tnp2-deficient mice raises the possibility that a deficiency created through the disruption of the Tnp2 gene can be compensated for by recruitment of the Tnp1.

Isolation and characterization of a novel human gene, NIF3L1, and its mouse ortholog, Nif3l1, highly conserved from bacteria to mammals.

We report the cloning and characterization of novel human and murine genes NIF3L1 and Nif3l1 which are strongly homologous to the yeast Ngg1-interacting factor 3 homolog. Mouse Nif3l1 and human NIF3L1 encode predicted proteins of 376 amino acids and 377 amino acids, respectively. Northern blot analysis on RNA from different postnatal murine tissues showed a ubiquitous expression pattern of mouse Nif3l1 with a transcript of approximately 1.85 kb. RT-PCR analysis on prenatal mouse RNA and embryonic stem cell RNA demonstrated expression of Nif3l1 throughout embryonic development. Additionally, expression analysis on cell lines revealed strong overexpression of Nif3l1 in the spermatogonia-derived cell line GC-1 spg and in the teratocarcinoma cell line F9. The mouse gene was mapped to chromosome 1, region C. Human NIF3L1 consists of seven exons spanning 14.5 kb of genomic DNA and is located on chromosome 2q33. A fusion protein consisting of the GFP (green fluorescent protein) and the ORF of human NIF3L1 showed a localization of the predicted protein in the cytoplasm. In the N-terminal and C-terminal region, mouse Nif3l1 and human NIF3L1 are strongly homologous to proteins of other species, e.g. the recently cloned Drosophila symbol=anon-35F/36F gene with 41% amino acid identity and several proteins from yeast including the yeast Ngg1-interacting factor 3 homolog with 46% amino acid identity, the hypothetical protein YGL221c and yeast Ngg1-interacting factor 3 (Nif3) with 37% amino acid identity. Other proteins from lower organisms, e.g a conserved hypothetical protein from Ureaplasma urealyticum or a hypothetical protein SCC30.09c from Streptomyces coelicolor show approximately 25-30% amino acid identity in the two flanking regions of the protein. These similarities indicate a high degree of conservation of mouse Nif3l1 and human NIF3L1 from bacteria to mammals.

Molecular cloning, chromosomal localization, and expression of the murine SALL1 ortholog Sall1.

SALL1 has been identified as one of now three human homologs of the region specific homeotic gene spalt (sal) of Drosophila, which encodes a zinc finger protein of characteristic structure. Mutations of SALL1 on chromosome 16q12.1 cause Townes-Brocks syndrome (TBS, OMIM no. 107480). In order to facilitate functional studies of this gene in a model organism, we searched for the murine homolog of SALL1. Here we report the genomic cloning, chromosome mapping, and partial expression analysis of the gene Sall1. Sequence comparison, Northern blot hybridization as well as the conserved chromosome location on the homologous mouse chromosome indicate that we have indeed isolated the murine homolog of SALL1.

Alternative splicing, chromosome assignment and subcellular localization of the testicular haploid expressed gene (THEG).

We have previously isolated and characterized the mouse Testicular Haploid Expressed Gene (Theg) that is specifically expressed in haploid germ cells. We now describe the molecular cloning and characterization of the human homologue (THEG) of mouse Theg. Expression studies by using both dot blot and Northern blot techniques revealed that human THEG is expressed specifically in the testis. Additionally, we found two alternatively spliced transcripts (THEG major and THEG minor) for THEG by using reverse transcription-polymerase chain reaction on human testicular RNA. Sequence analysis of these PCR products demonstrated that the smaller transcript (THEG minor) lacks 72 bp which was also observed for the mouse Theg. We have isolated the cDNAs of human THEG major and THEG minor, containing the complete open reading frames, which encode putative nuclear proteins of 379 amino acids and 355 amino acids, respectively. Database searches identified two genomic clones on chromosome 19 harboring the human THEG gene, which is approximately 14 kb pairs in size, contains eight exons, and comparison of the two cDNA sequences with the genomic sequence indicated that the smaller transcript lacks exon 3. Furthermore, we assigned the human THEG gene (THEG) to human chromosome 19ptel--> p13 by fluorescence in situ hybridization. Moreover, we detected mouse THEG protein prominently in the nucleus of round spermatids by using an antibody against THEG on both testicular sections and cellular suspensions. Additionally, the subcellular localization of mouse THEG was confirmed by a green fluorescent protein (GFP) fusion protein of mouse THEG which was found mainly in the nucleus of transfected NIH3T3 cells. These data suggest that both human and mouse THEG are specifically expressed in the nucleus of haploid male germ cells and are involved in the regulation of nuclear functions.

Genomic cloning, chromosomal mapping, and expression analysis of msal-2.

Mutations of SALL1 related to spalt of Drosophila have been found to cause Townes-Brocks syndrome, suggesting a function of SALL1 for the development of anus, limbs, ears, and kidneys. No function is yet known for SALL2, another human spalt-like gene. The structure of SALL2 is different from SALL1 and all other vertebrate spalt-like genes described in mouse, Xenopus, and Medaka, suggesting that SALL2-like genes might also exist in other vertebrates. Consistent with this hypothesis, we isolated and characterized a SALL2 homologous mouse gene, Msal-2. In contrast to other vertebrate spalt-like genes both SALL2 and Msal-2 encode only three double zinc finger domains, the most carboxyterminal of which only distantly resembles spalt-like zinc fingers. The evolutionary conservation of SALL2/Msal-2 suggests that two lines of sal-like genes with presumably different functions arose from an early evolutionary duplication of a common ancestor gene. Msal-2 is expressed throughout embryonic development but also in adult tissues, predominantly in brain. However, the function of SALL2/Msal-2 still needs to be determined.

SALL3, a new member of the human spalt-like gene family, maps to 18q23.

spalt (sal) of Drosophila melanogaster is an important developmental regulator gene and encodes a zinc finger protein of unusual but characteristic structure. Two human sal-like genes have been isolated so far, SALL1 on chromosome 16q12.1 and SALL2 on chromosome 14q11.1-q12.1. Truncating mutations of SALL1 have been shown to cause Townes-Brocks syndrome and are thought to result in SALL1 haploinsufficiency. Sequence comparison of SALL1 to the related genes Msal in mouse and Xsal-1 in Xenopus laevis suggested that SALL1 was not the human orthologue of Msal and Xsal-1. By database searching and genomic cloning, we isolated an EST and a corresponding human cosmid clone, which contain coding sequence of a human gene highly similar to mouse Msal. This gene, named SALL3, was found to be expressed in different regions of human fetal brain and in different adult human tissues. The chromosomal localization of SALL3 at 18q23 suggests that haploinsufficiency of this gene might contribute to the phenotype of patients with 18q deletion syndrome.

Alternative splicing produces transcripts encoding four variants of mouse G-protein-coupled receptor kinase 6.

A family of protein kinases, termed G-protein-coupled receptor kinases (GRK1-6), is known to phosphorylate agonist-occupied G-protein-coupled receptors. We have identified mRNAs encoding four distinct mouse GRK6 isoforms (mGRK6), designated mGRK6-A through mGRK6-D. Mouse GRK6-B and mGRK6-C diverge from the known human GRK6 (577 residues) at residue 560 and are 13 residues longer and 16 residues shorter, respectively, than human GRK6, while mGRK6-A very likely represents the mouse equivalent of human GRK6. Mouse GRK6-D is identical to the other mGRK6 variants in the amino-terminal region, but comprises only 59 of the 263 amino acids of the putative catalytical domain. As mGRK6-D retains the region involved in interacting with activated receptors, but most likely lacks catalytic activity, this variant might represent a naturally occurring inhibitor of other GRKs. Analysis of the genomic organization of mGRK6 gene revealed that the four mRNAs are generated by alternative RNA splicing from a single approximately 14. 5-kb gene, made up of at least 17 exons and located on mouse chromosome 13. Similar to human GRK6, mGRK6-A contains three cysteine residues within its carboxyl-terminal region known to serve as substrates for palmitoylation. Mouse GRK6-B lacks these palmitoylation sites, but carries a basic carboxyl-terminus containing consensus sequences for phosphorylation by protein kinases C and cAMP/cGMP-dependent protein kinases. Mouse GRK6-C displays none of these motifs. Thus, mGRK6-A, mGRK6-B, and mGRK6-C are predicted to differ in terms of their regulation by carboxyl-terminal posttranslational modification. Analysis of mRNA expression revealed that the four mGRK6 mRNAs are differentially expressed in mouse tissues, suggesting that the four mGRK6 isoforms are involved in regulating tissue- or cell type-specific functions in vivo.

ZOO-FISH analysis in a species of the order Chiroptera: Glossophaga soricina (Phyllostomidae).

Glossophaga soricina is a flower-visiting bat which lives in the neotropics. The diploid chromosome number is 2n = 32 with a fundamental number of autosomal arms, FN, of 60. G. soricina belongs to the Microchiroptera which have a lower diploid DNA content and a higher AT composition in their DNA compared with other mammals. By ZOO-FISH analysis with human chromosome-specific DNA probes, the human autosomes were found conserved in 41 segments. This is an arrangement similar to other mammals which have been analyzed. Several chromosomal associations already known from ZOO-FISH studies in other species were also present in G. soricina.

Detailed marker chromosome analysis in cell line U-BLC1, established from transitional-cell carcinoma of the bladder.

A permanent cell line, U-BLC1, was established from a primary transitional-cell carcinoma, TCC, of the urinary bladder. Karyotype analysis showed the line to be highly aberrant, with a near-triploid chromosome number of 68 to 73. Comparative genomic hybridization revealed some distinct differences between the primary tumor and the established cell line. Karyotype analysis showed 3 marker chromosomes with homogeneously staining regions, HSRs, in the cell line. The HSRs were isolated by microdissection and the microdissection probes were hybridized to normal metaphase chromosomes. The HSRs contain sequences known to be frequently involved in amplification in transitional-cell carcinoma of the bladder, 6p22, 7p11-p12, 9p23-pter, and one region not yet reported to be amplified in primary TCC of the bladder, 1p31-p32. A candidate-gene approach showed that in the region 7p11-p12 the EGFR locus is amplified and highly expressed.

Molecular cloning and characterization of murine Mpgc60, a gene predominantly expressed in the intestinal tract.

We have isolated from mouse intestine a full-length cDNA clone that encodes an 86-amino acid precursor protein containing a 26-amino acid signal sequence. As deduced from its sequence, the mature 60-aa protein named MPGC60 belongs to the Kazal type of secreted trypsin inhibitors. The MPGC60 peptide has 58% homology with the PEC-60 peptide isolated from pig intestine. In the gut of adult mice, an increasing rostrocaudal gradient in MPGC60 mRNA levels was observed by Northern analysis. In situ hybridization analysis demonstrated strong Mpgc60 expression in Paneth cells and in a subset of goblet cells in the differentiated gut. During postnatal differentiation of the gut, a strong increase in Mpgc60 expression was detected in both small and large intestine. However, in small intestine activation of the Mpgc60 gene occurred earlier than in the large intestine. Apart from the intestinal tract, MPGC60 mRNA was also detectable in the mesenchyme surrounding the uterine epithelium and in endothelia of some blood vessels. However, in contrast to the situation observed in pig, no Mpgc60 expression was detectable by Northern, in situ and reverse transcriptase polymerase chain reaction (RT-PCR) analysis in cells of the immune system, that is, in monocytes, macrophages, peripheral blood and in spleen. Northern blot analysis on mRNA isolated from porcine and murine intestine showed a single transcript in mouse, but several transcripts in pig. Southern blot and fluorescent in situ hybridisation (FISH) analysis demonstrated the presence of a single gene situated in band A of chromosome 4. This region is syntenic with human chromosome regions 6q, 8q and 9p. The gene responsible for human hereditary mixed polyposis syndrome has been localized to human 6q. This raises the possibility that Mpgc60 is a candidate gene for this human disorder.

Structural analysis of the amplified IFN-beta and DHFR genes in a Chinese hamster ovary cell line using multicolour FISH analysis.

Multicolour FISH was used to get insight into the structural arrangement of a homogeneously staining region which bears the co-transfected and subsequently co-amplified IFN-beta and DHFR genes in a CHO cell line. On metaphase chromosomes an arrangement of multiple bands with regular spacing is revealed. On extended chromatin fibres a cluster of directly repeated and interspersed IFN-beta and DHFR genes is visible. Up to three clusters were found arranged in tandem. The different chromosomal mechanisms leading to gene amplification are discussed.

ZOO-FISH analysis in insectivores: "Evolution extols the virtue of the status quo".

In evolutionary terms, insectivores are thought to be close to primates. Through ZOO-FISH analysis using human chromosome-specific painting probes, the syntenic relationship between the human and common shrew, Sorex araneus, karyotypes was studied. The human karyotype was found to be conserved in the shrew, with 32 autosomal segments of common synteny. Special arrangements, already known from similar studies on other species, were noted: fusions of HSA 16 and 19, HSA 3 and 21, and HSA 14 and 15. Only 10 breaks are necessary to transform the human karyotype into the karyotype of the common shrew. Together with known ZOO-FISH data from species belonging to other orders, this puts the human karyotype arrangement near the basis of all mammalian karyotypes. Human chromosome 2 was found to be conserved in its entirety as a single chromosome arm in the shrew. Evidence is presented that the same fusion of two original chromosomal segments formed the shrew chromosome ortholog of HSA 2 as the fusion that occurred during primate evolution to form human chromosome 2. This is a remarkable example of chromosomal coevolution.

Cloning and chromosomal localisation of the murine epidermal-type fatty acid binding protein gene (Fabpe).

We succeeded in cloning the gene encoding the murine epidermal-type fatty acid binding protein (E-FABP). To avoid the screening of pseudogenes, the presence of which was shown by PCR, we designed an intron-specific probe and screened a bacterial artificial chromosome library from mouse embryonic stem cells. One of the clones obtained was analysed by restriction with various enzymes and an 11-kb EcoRI fragment with the complete gene was subcloned. The gene revealed the canonical exon/intron FABP structure consisting of four exons (112, 173, 102 and 544bp, respectively) and three introns (2217, 327 and 546bp, respectively). The exon sequences were identical with the cDNA encoding mouse E-FABP (Krieg, P., Feil, S., Fürstenberger, G., Bowden, T.G., 1993. Tumor-specific overexpression of a novel keratinocyte lipid-binding protein. Identification and characterisation of a cloned sequence activated during multistage carcinogenesis in mouse skin. J. Biol. Chem. 268, 17362-17369). Of the 5' region, 2470bp were sequenced and searched for transcription factor binding sites. Putative responsive elements within the promoter region were identified that may be responsible for the wide expression observed for E-FABP in mouse tissues. The 11-kb EcoRI fragment was used to localise Fabpe on chromosome 3 in the region 3A1-3 by fluorescence in-situ hybridisation.

Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2beta.

Expression of AP-2 transcription factors has been detected previously in embryonic renal tissues. We show here that AP-2beta -/- mice complete embryonic development and die at postnatal days 1 and 2 because of polycystic kidney disease. Analyses of kidney development revealed that induction of epithelial conversion, mesenchyme condensation, and further glomerular and tubular differentiation occur normally in AP-2beta-deficient mice. At the end of embryonic development expression of bcl-X(L), bcl-w, and bcl-2 is down-regulated in parallel to massive apoptotic death of collecting duct and distal tubular epithelia. Addressing the molecular mechanism we show that transfection of AP-2 into cell lines in vitro strongly suppresses c-myc-induced apoptosis pointing to a function of AP-2 in programming cell survival during embryogenesis. The position of the human AP-2beta gene was identified at chromosome 6p12-p21.1, within a region that has been mapped for autosomal recessive polycystic kidney disease (ARPKD). Sequence analyses of ARPKD patients and linkage analyses using intragenic polymorphic markers indicate that the AP-2beta gene is located in close proximity to but distinct from the ARPKD gene.

Zoo-FISH analysis: the American mink (Mustela vison) closely resembles the cat karyotype.

The 24 human chromosome-specific DNA probes were used to visualize segments of conserved synteny on metaphase chromosomes of the American mink (Mustela vison). A comparison with the hitherto known gene mapping data shows a high degree of correspondence. The human chromosomes were found conserved in only 34 segments of common synteny. The mink arrangement proved to be very similar to the arrangement found in the cat, thus corroborating the well-known high karyotype conservation described for Feloidea.