Absence of arterial phenotype in mice with homozygous slc2A10 missense substitutions.

Arterial tortuosity syndrome (ATS, MIM# 208050) is a rare autosomal recessive connective tissue disease, mainly characterized by widespread arterial involvement with elongation, tortuosity, and aneurysms of the large and middle-sized arteries (Callewaert et al., 2008, Hum Mutat 29:150-158). Recently, mutations were identified in the SLC2A10 gene encoding the facilitative glucose transporter GLUT10 (Coucke et al., 2006, Nat Genet 38:452-457). It was hypothesized that loss-of-function of the transporter results in upregulation of the transforming growth factor beta (TGFbeta) signaling pathway (Coucke et al., 2006, Nat Genet 38:452-457). We anticipated that a mouse model would help to gain more insight in the complex pathophysiological mechanism of human ATS. Here, we report that two mouse models, homozygous respectively for G128E and S150F missense substitutions in glut10 do not present any of the vascular, anatomical, or immunohistological abnormalities as encountered in human ATS patients. We conclude that these mouse strains do not phenocopy human ATS and cannot help the further elucidation of pathogenetic mechanisms underlying this disease.

Efficient and fast targeted production of murine models based on ENU mutagenesis.

Mice with targeted genetic alterations are the most effective tools for deciphering organismal gene function. We generated an ENU-based parallel C3HeB/FeJ sperm and DNA archive characterized by a high probability to identify allelic variants of target genes as well as high efficiencies in allele retrieval and model revitalization. Our archive size of over 17,000 samples contains approximately 340,000 independent alleles (20 functional mutations per individual sample). Based on an estimated number of approximately 30,000 mouse genes, the parallel sperm/DNA archive should permit the identification and recovery of ten or more alleles per average target gene which translates to a calculated 99% success rate in the discovery of five allelic variants for any given average gene. The low rate of unrelated ENU-induced passenger mutations has no practical impact on the analysis of the allele-specific phenotype at the G3 generation because of dilution and free segregation of such unrelated passenger mutations. To date 39 mouse models representing 33 different genes have been recovered from our archive using in vitro fertilization techniques. The generation time for a murine model heterozygous for a mutation in a gene of interest is less than 2 months, i.e., three to four times faster compared with current embryonic stem-cell-based technologies. We conclude that ENU-based targeted mutagenesis is a powerful tool for the fast and high-throughput production of murine gene-specific models for biomedical research.

Point mutations of the p150 subunit of dynactin (DCTN1) gene in ALS.

The authors report mutation screening of the p150 subunit of dynactin (DCTN1) and the cytoplasmic dynein heavy chain (DNCHC1) genes in 250 patients with ALS and 150 unrelated control subjects. Heterozygous missense mutations of the DCTN1 gene were detected in one apparently sporadic case of ALS (T1249I), one individual with familial ALS (M571T), two patients with familial ALS, and two unaffected relatives in the same kindred (R785W). The allelic variants of the DCTN1 gene may represent a previously unknown genomic risk factor for ALS.

Alopecia in a novel mouse model RCO3 is caused by mK6irs1 deficiency.

Reduced coat 3 (Rco3) is a new spontaneous autosomal recessive mutation with defects in hair structure and progressive alopecia. Here we describe chromosomal mapping and molecular identification of the Rco3 mutation. The murine Rco3 locus maps to a 2-Mb interval on chromosome 15 encompassing the keratin type II gene cluster. Recently, mK6irs1 was described as a type II keratin expressed in Henle's and Huxley's layer of the murine inner root sheath. Genomic sequencing revealed a 10-bp deletion in exon 1 of mK6irs1 resulting in a frameshift after 58 amino acid residues and, therefore, the absence of 422 carboxy-terminal amino acid residues containing the complete alpha-helical rod domain. Henle's and Huxley's layers show no immunoreactivity with mK6irs1-specific antibodies and the absence of intermediate filament formation in electron microscopic images. These results indicate that the expression of functional mK6irs1 is indispensable for intermediate filament formation in the inner root sheath and highlights the importance of the keratinization of the inner root sheath in the normal formation of the hair shaft.

A mouse model for cystinuria type I.

Cystinuria, one of the most common inborn errors of metabolism in humans, accounts for 1-2% of all cases of renal lithiasis. It is caused by defects in the heterodimeric transporter system rBAT/b0,+AT, which lead to reduced reabsorption of cystine and dibasic amino acids through the epithelial cells of the renal tubules and the intestine. In an N-ethyl-N-nitrosourea mutagenesis screen for recessive mutations we identified a mutant mouse with elevated concentrations of lysine, arginine and ornithine in urine, displaying the clinical syndrome of urolithiasis and its complications. Positional cloning of the causative mutation identified a missense mutation in the solute carrier family 3 member 1 gene (Slc3a1) leading to an amino acid exchange D140G in the extracellular domain of the rBAT protein. The mouse model mimics the aetiology and clinical manifestations of human cystinuria type I, and is suitable for the study of its pathophysiology as well as the evaluation of therapeutic and metaphylactic approaches.

Persistent measles virus infection and otosclerosis.

The cause of otosclerosis is still unknown. Recently, measles virus involvement has been implicated. The aim of this study was to analyze the presence of measles virus RNA within the otosclerotic focus and to evaluate the perilymphatic antibody pattern. Bone and perilymph specimens from 40 patients with the spontaneous form of otosclerosis and from control patients were investigated by reverse transcription polymerase chain reaction (RT-PCR), Western blot techniques, and cell culture. By the use of RT-PCR, measles virus RNA could be detected in 32 patients, but not in controls. Analysis of perilymph revealed the presence of antibodies to N, F1, and M measles virus proteins in all cases, and antibodies against H protein in 2 additional cases. In preosteoblasts cultured from otosclerotic bone chips, no measles virus RNA could be amplified. We conclude that the spontaneous form of otosclerosis is, in the vast majority of cases, a measles virus-associated disease of the otic capsule.

Shift of the distribution of age in patients with otosclerosis.

Morphological and biochemical investigations have shown evidence of an association between measles virus and otosclerosis. Epidemiological analysis of age and gender distributions in the 1960s and 1970s revealed a higher incidence of otosclerosis in women, the average age of onset of clinical disturbances and need for surgery being between 15 and 40 years. In the late 1960s and early 1970s a campaign to vaccinate children against measles was started in Germany. The aim of our study was to evaluate whether this campaign has had any influence on the distributions of the age and gender of patients affected by otosclerosis over the past 20 years. The study included patients suffering from clinical otosclerosis who had undergone stapedectomy between 1978 and 1999 and whose clinical data were complete (n = 1351). Statistical analysis during the recruitment period indicated a significant increase in the average age of the otosclerosis patients (p = 0.012). With regard to the gender distribution it was found that the increase of otosclerosis in women compared to men was statistically insignificant (p = 0.418). These data strongly support the hypothesis of a measles virus involvement in otosclerosis and may reflect a decreased incidence of otosclerosis in the generation of patients vaccinated against measles virus.

Localization of a new neutralizing epitope on the mumps virus hemagglutinin-neuraminidase protein.

Four protein fragments which span the entire hemagglutinin-neuraminidase protein (HN) of mumps virus were expressed in HeLa cells and cell extracts were tested for their capability to induce neutralizing antibodies in mice. Fragment HN3 (aa 213-372) was able to induce the production of hemagglutination-inhibiting and neutralizing antibodies. When a subfragment of HN3, the synthetic peptide NSTLGVKSAREF (aa 329-340 of HN) was used for immunization, hemagglutination-inhibiting and neutralizing antibodies against mumps wild type virus but not against the Urabe Am9 vaccine virus were raised. The peptide could, therefore, contain a new epitope, which may be critical for protective host humoral immune response.

Persistent measles virus infection as a possible cause of otosclerosis: state of the art.

The etiopathogenesis of otosclerosis is still largely unexplained and remains controversial. Morphologic examinations have shown the presence of a chronic inflammation in otosclerotic tissue. Among the proposed explanations for this inflammation are an immunologic reaction against collagen, mutations of collagen gene 1A1, and a viral infection. In this paper, we focus on the role of measles virus in otosclerosis, and we review the current literature, devoting particular attention to a suspected paramyxoviral etiopathogenesis in Paget's disease. Our examination of footplate fragments by reverse transcription polymerase chain reaction testing in 95 patients with otosclerosis revealed the presence of measles virus RNA in 83% of cases. Quantification of measles virus immunoglobulin G (IgG) in otosclerosis patients indicated that the ratio of antimeasles virus IgG in total IgG was higher in perilymph than in serum. Furthermore, an almost identical incidence of otosclerosis and measles virus-caused mortality in women suggests that women are more susceptible to measles virus infection. Finally, since the introduction of the measles virus vaccination program in Europe, there has been a decline in the incidence of otosclerosis. Moreover, the average age of patients at diagnosis and surgery at our hospital has increased to 54 years. Our findings, when they are considered along with findings regarding the presence of paramyxoviral RNA in Paget's disease, support the hypothesis that measles virus is involved in the etiopathogenesis of otosclerosis.

Rapid and efficient recovery of Sendai virus from cDNA: factors influencing recombinant virus rescue.

In a comparative study the factors influencing the recovery of recombinant Sendai viruses (SeV) from plasmid based cDNA were analysed systematically in order to establish an efficient and robust method for virus rescue. The amounts and ratios of transfected helper plasmids encoding the viral N, P and L proteins proved to be crucial for virus rescue, and they were optimised step-by-step for enhanced virus release. When the C open reading frame from the P gene was expressed at low level, virus rescue was generally possible but virus release could be improved when C gene expression was abolished completely. SeV particle formation could be increased greatly when the transcription initiation site for T7 polymerase in the cDNA was modified or when the genomic ribozyme instead of the antigenomic ribozyme of hepatitis delta virus was used for processing the 3'end of the viral RNA transcript. Heterologous helper viruses vTF7-3 and MVA-T7, which are necessary for T7 polymerase production in transfected cells, were compared for their use in SeV recovery and subsequent elimination of the helper virus from recombinant SeV. Interference with SeV replication was less severe with MVA-T7, and MVA-T7 was eliminated efficiently without the need for any inhibitors by serial passages in Vero cells. Optimal combination of all parameters led to a highly efficient generation of recombinant SeV from cDNA. Titres of the released virus particles are high enough to enable analysis of the recombinant SeV directly on test cells or propagation in cell cultures without the need for amplification in embryonated chicken eggs. The system is very robust and allows rapid generation of defined SeV mutants that require specialised host cells for propagation.

Sendai virus-like particles devoid of haemagglutinin-neuraminidase protein infect cells via the human asialoglycoprotein receptor.

Virus-like particles with genetically defined envelope proteins were generated from cDNA in order to examine the requirement of Sendai virus haemagglutinin-neuraminidase (HN) protein for particle formation, and the role of fusion protein (F) in receptor binding and membrane fusion. Characterization of particles devoid of HN protein showed that particle formation was unimpaired by the absence of HN protein, indicating that HN protein is dispensable for virus assembly and budding. Infection studies further demonstrated that virus adsorption and penetration can be mediated solely by the F protein when the human asialoglycoprotein receptor is present at the surface of host cells.

Regulation of the operon responsible for broad-spectrum mercury resistance in Streptomyces lividans 1326.

The broad-spectrum mercury resistance of Streptomyces lividans 1326 is mediated by six open reading frames (orf). These are arranged in two divergently transcribed operons. The orfs mer A (mercuric reductase) and mer B (organolyase) form one of the two operons. These genes and their regulation were further studied by deletion analysis and transcriptional fusion to the reporter gene xylE in the plasmid pXE4. An increase in XylE activity in response to the presence of mercuric ions was observed. The function of ORF2 (MerT) and ORF3 (MerP) as mercury-specific transport proteins, previously postulated based on the structural features of the predicted proteins, was confirmed. Transcription of the mer genes starts within the intercistronic region and two divergent promoters were identified by S1 nuclease mapping. Expression of the genes was negatively regulated by the product of orf1, now called merR. The repressor function was confirmed by gel retardation assays. MerR, produced in Escherichia coli, bound to two sites (operators) in the fragment containing the promoter region between merA and merR. Addition of mercuric ions and phenylmercuric acetate prevented the binding of MerR.

tRNA genes of Streptomyces lividans: new sequences and comparison of structure and organization with those of other bacteria.

Three closely linked Streptomyces lividans tRNA genes encoding two tRNA(Lys)s and a tRNA(Gly) were cloned and sequences. The structure of tRNA(Gly) is unusual for eubacterial tRNAs. Including those in previous reports (R. Sedlmeier and H. Schmieger, Nucleic Acids Res. 18:4027, 1990, and R. Sedlmeier, G. Linti, K. Gregor, and H. Schmieger, Gene 132:125-130, 1993), 18 S. lividans tRNA genes were physically mapped on the chromosome of the closely related strain Streptomyces coelicolor A3(2). The structure and organization of tRNA genes of S. lividans and S. coelicolor are compared with those of Escherichia coli and Bacillus subtilis.

Sequences of tRNA-encoding genes and associated open reading frames of Streptomyces lividans.

A gene library of Streptomyces lividans has been screened for tRNA-encoding genes with labeled Streptomyces tRNA as a probe. By sequence analysis of hybridizing fragments, two single genes have been identified which code for tRNA(Asp) and tRNA(Gly). Associated with the tRNA(Gly) gene, there are three open reading frames (ORFs) which might code for gene products possibly involved in active transport processes through the bacterial membrane. The transcriptional organization of tRNAGly and the following ORFs was examined by high-resolution S1 mapping. A third clone carried a cluster of genes which encode two tRNA(Gln) and three tRNA(Glu). This cluster corresponds to a similar cluster previously described for Streptomyces rimosus [Plohl and Gamulin, Mol. Gen. Genet. 222 (1990) 129-134].

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans.

A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cms), arginine auxotrophic (Arg-) mutant of Streptomyces lividans 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cms Arg- mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.

Isolation of ribosomal RNA operons of Streptomyces lividans and sequence analysis of a 5S-rRNA gene.

Three clones containing rRNA genes have been isolated from a gene library of Streptomyces lividans. Two clones carried entire but different rRNA-operons. The third clone comprised the 3'-end of a 23S-rRNA gene, the entire 5S-rRNA gene and the spacer region between them. The nucleotide sequence starting within the 23S-rRNA gene beyond the putative transcription terminator downstream the 5S-rRNA gene has been determined (EMBL acc. no X58874).