Signal sequence mutations as tools for the characterization of LamB folding intermediates

lamBA23DA25Y and lamBA23YA25Y tether LamB to the inner membrane by blocking signal sequence processing. We isolated suppressors of lamBA23DA25Y and lamBA23YA25Y, all of which mapped within the LamB signal sequence. Most interesting were mutations that changed an amino acid with a strong positive charge to an amino acid with no charge. Further characterization of two such suppressors revealed that they produce functional LamB that is localized to the outer membrane with its entire signal sequence still attached. Biochemical analysis shows that mutant LamB monomer chases into an oligomeric species with properties different from those of wild-type LamB trimer. Because assembly of mutant LamB is slowed, these mutations provide useful tools for the characterization of LamB folding intermediates.     

The α1 subunit of laminin-1 promotes the development of neurons by interacting with LBP110 expressed by neural crest-derived cells immunoselected from the fetal mouse gut

A plasmalemmal protein, LBP110, which binds to the α1 chain of laminin-1, is acquired by the neural crest-derived precursors of enteric neurons after they colonize the gut. We tested the hypothesis that laminin-1 interacts with LBP110 to promote enteric neuronal development. The effects of laminin-1 on neuronal development were studied in cultures of cells immunoselected from fetal mouse gut (E14–15) with antibodies to LBP110 or p75^NTR, a marker for enteric crest—derived cells. No matter which antibody was used, the development of cells expressing neuronal markers was increased three- to fourfold by culturing the cells on a laminin-1—containing substrate. To determine whether this effect of laminin-1 is due to the selective adherence of a neurocompetent subset of precursors, immunoselected cells were permitted to preadhere to poly-D-lysine. Addition of soluble laminin-1 24 h later promoted neuronal but not glial development. The laminin-1 induced increment in neuronal development was abolished both by a peptide containing the sequence of the LBP110-binding domain, IKVAV, and by antibodies to laminin α1 that recognize the IKVAV domain. Neither reagent affected the total number of cells. In contrast, the response to laminin-1 was not affected by control peptides, preimmune sera, or antibodies to laminin β1. Laminin-1 transiently induced the expression of nuclear Fos immunoreactivity; this action was blocked specifically by the IKVAV peptide. These data are consistent with the hypothesis that LBP110 interacts with the IKVAV domain of laminin α1 to promote the differentiation of neurons from enteric crest—derived precursors. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 118–138, 1997     

Rat Chromosome 1: Regional localization of seven genes (Slc9a3, Srd5a1, Esr, Tcp1, Grik5, Tnnt3, Jak2) and anchoring of the genetic linkage map to the cytogenetic map

Seven genes were regionally localized on rat Chromosome (Chr) 1, from 1p11 to 1q42, and two of these genes were also included in a linkage map. This mapping work integrates the genetic linkage map and the cytogenetic map, and allows us to orient the linkage map with respect to the centromere, and to deduce the approximate position of the centromere in the linkage map. These mapping data also indicate that the Slc9a3 gene, encoding the Na⁺/H⁺ exchanger 3, is an unlikely candidate for the blood pressure loci assigned to rat Chr 1. These new localizations expand comparative mapping between rat Chr 1 and mouse or human chromosomes. Received: 21 March 1997 / Accepted: 3 May 1997     

A candidate mouse model for Hartnup Disorder deficient in neutral amino acid transport

The mutant mouse strain HPH2 (hyperphenylalaninemia) was isolated after N-ethyl-N-nitrosourea (ENU) mutagenesis on the basis of delayed plasma clearance of an injected load of phenylalanine. Animals homozygous for the recessive hph2 mutation excrete elevated concentrations of many of the neutral amino acids in the urine, while plasma concentrations of these amino acids are normal. In contrast, mutant homozygotes excrete normal levels of glucose and phosphorus. These data suggest an amino acid transport defect in the mutant, confirmed in a small reduction in normalized values of ¹⁴C-labeled glutamine uptake by kidney cortex brush border membrane vesicles (BBMV). The hyperaminoaciduria pattern is very similar to that of Hartnup Disorder, a human amino acid transport defect. A subset of Hartnup Disorder cases also show niacin deficiency symptoms, which are thought to be multifactorially determined. Similarly, the HPH2 mouse exhibits a niacin-reversible syndrome that is modified by diet and by genetic background. Thus, HPH2 provides a candidate mouse model for the study of Hartnup Disorder, an amino acid transport deficiency and a multifactorial disease in the human. Received: 16 May 1996 / Accepted: 25 September 1996     

Linkage of genes for laminin B1 and B2 subunits on chromosome 1 in mouse

Summary We have used cDNA clones for the B1 and B2 subunits of laminin to find restriction fragment length DNA polymorphisms for the genes encoding these polypeptides in the mouse. Three alleles were found forLamB2 and two forLamB1 among the inbred mouse strains. The segregation of these polymorphisms among recombinant inbred strains showed that these genes are tightly linked in the central region of mouse Chromosome 1 betweenSas-1 andLy-m22, 7.4±3.2 cM distal to thePep-3 locus. There is no evidence in the mouse for pseudogenes for these proteins. This work was supported by U. S. Public Health Service Grant GM28464 to R.W.E. Editor's Statement Investigation into the biosynthesis of laminin indicates that several different polypeptides are assembled to form the intact molecule. This paper represents an extension of previous work which takes a genetic approach to further define the relationships among the polypeptides involved. Gordon H. Sato     

Genetic mapping of the mouse Rab7 gene and pseudogene and of the human RAB7 homolog

Rab proteins are small GTP-ases localized to distinct membrane compartments in eukaryotic cells and regulating specific steps of intracellular vesicular membrane traffic. The Rab7 protein is localized to the late endosomal compartment and controls late steps of endocytosis. We have isolated, by library screening, the 5′ region, including the promoter, of the mouse Rab7 gene and a Rab7 pseudogene. We have mapped, by genetic linkage analysis, the mouse Rab7 gene on Chromosome (Chr) 6 and the Rab7-ps1 pseudogene on Chr 9, where the Rab7 gene has been previously reported to map. By radiation hybrid mapping, we have located the human RAB7 gene on Chr 3, in a region homologous to the mouse Chr 6, where the Rab7 gene maps. Received: 27 October 1997 / Accepted: 1 January 1998     

Embryonic Heart and Skin Defects in Mice Lacking Plakoglobin

Plakoglobin is the only component common to both the desmosomal plaque and the cadherin–catenin cell adhesion complex in the adherens junction. It is highly homologous to vertebrate β-catenin and toDrosophilaarmadillo protein and may—like these proteins—be also involved in signaling pathways. To analyze the role of plakoglobin during mouse development we inactivated theplakoglobingene by homologous recombination in embryonic stem cells and generated transgenic mice.Plakoglobinnull-mutant embryos died from Embryonic Day 10.5 onward, due to severe heart defects. Some mutant embryos developed further, especially on a C57BL/6 genetic background, and died around birth, presumably due to cardiac dysfunction, and with skin blistering and subcorneal acantholysis. Ultrastructural analysis revealed that here desmosomes were greatly reduced in number and structurally altered. Thus, using reversed genetics we demonstrate that plakoglobin is an essential structural component for desmosome function. The skin phenotype in plakoglobin-deficient mice is reminiscent of the human blistering disease, epidermolytic hyperkeratosis.     

A frameshift mutation within LAMC2 is responsible for Herlitz type junctional epidermolysis bullosa (HJEB) in black headed mutton sheep

Junctional epidermolysis bullosa (JEB) is a hereditary mechanobullous skin disease in humans and animals. A Herlitz type JEB was identified in German Black Headed Mutton (BHM) sheep and affected lambs were reproduced in a breeding trial. Affected lambs showed skin and mucous membranes blistering and all affected lambs died within the first weeks of life. The pedigree data were consistent with a monogenic autosomal recessive inheritance. Immunofluorescence showed a reduced expression of laminin 5 protein which consists of 3 subunits encoded by the genes LAMA3, LAMB3 and LAMC2. We screened these genes for polymorphisms. Linkage and genome-wide association analyses identified LAMC2 as the most likely candidate for HJEB. A two base pair deletion within exon 18 of the LAMC2 gene (FM872310:c.2746delCA) causes a frameshift mutation resulting in a premature stop codon (p.A928*) 13 triplets downstream of this mutation and in addition, introduces an alternative splicing of exon 18 LAMC2. This deletion showed a perfect co-segregation with HJEB in all 740 analysed BHM sheep. Identification of the LAMC2 deletion means an animal model for HJEB is now available to develop therapeutic approaches of relevance to the human form of this disease.     

Sequence and Chromosomal Localization of Mouse Annexin XI

Mouse annexin XI (anx11)²was cloned from a macrophage cDNA library and characterized by genetic linkage mapping, DNA sequencing, and structural comparison with other annexins. TheAnx11gene localized to mouse chromosome 14 in close linkage with theRarb, Plau,andWnt5agenes near the centromere and 1.8 cM distal from theAnx7gene. The open reading frame was flanked by long, untranslated regions and encoded a 503-amino-acid protein with 93.1% identity to its human orthologue. Its 189-aa amino terminus corresponded to the widely expressed variant 1 of two possible, alternatively spliced forms. A previously described peptide fromAplysia brasilianawas identified as a closely related invertebrate homologue. Since annexin XI is known to be localized in the nucleus at certain stages of development, the identification of a region in tetrad repeats 3 and 4 resembling the “chromo box” domain may be relevant to a nuclear regulatory function of annexin XI. Knowledge of the mouse cDNA sequence and genetic map location will assist in the analysis of genomic organization and expression and provide a useful animal model to investigate gene function and hereditary phenotype for annexin XI.     

Characterization of Mutations at the Mouse Phenylalanine Hydroxylase Locus

Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. InPAH^ENU1,the phenotype is mild. ThePah^enu1mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. InPAH^ENU2,the phenotype is severe. ThePah^enu2mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. InPAH^ENU2,the sequence information was used to devise a direct genotyping system based on the creation of a newAlw26I restriction endonuclease site.     

Novel and recurrent mutations in the laminin-5 genes causing lethal junctional epidermolysis bullosa: molecular basis and clinical course of Herlitz disease

Herlitz disease (H-JEB), the lethal form of junctional epidermolysis bullosa, is a rare genodermatosis presenting from birth with widespread erosions and blistering of skin and mucosae because of tissue cleavage within the epidermal basement membrane. Mutations in any of the three genes encoding the 3, 3 and 2 chains of laminin-5 underlie this recessively inherited disorder. Here, we report the molecular basis and clinical course of H-JEB in 12 patients. Two novel nonsense mutations in the gene LAMA3 (E281X and K1299X) and a novel frame-shift mutation in the gene LAMB3 (1628insG) leading to a premature termination codon were identified by DNA sequencing and confirmed by restriction fragment length polymorphism analysis. In the four patients affected, neither the resulting truncated polypeptide chains nor assembled laminin-5 protein were detectable by immunofluorescence. Three patients were found to be heterozygous for the known hotspot mutation R635X and the recurrent mutations Q373X or 29insC in the gene LAMB3, whereas five others were homozygous for R635X. Significant variations in the disease progression and survival times between 1 and 30 months in this group of H-JEB patients emphasised the impact of modifying factors and the importance of immunostaining or mRNA assessment as parallel diagnostic methods. Interestingly, the only patients who survived for longer than 6 months were four females carrying the mutation R635X homozygously. In one of them, the clinical course may have been improved by treatment with artificial skin equivalents. These data may stimulate further investigation of genotype–phenotype correlations and facilitate mutation analysis and genetic counselling of affected families.     

Is laminin-1 a guidance cue for cerebellar granule cell migration?

Laminin-1 is a glycoprotein found in the basement membrane of many tissues. In the cerebellum of rodents, it has also been localized along Bergmann glial fibers, where it is thought to be involved in promoting granule cell migration by enhancing adhesion and neurite outgrowth along these fibers. Recent reports, however, indicate that laminin-1 is not present on Bergmann fibers, but instead is associated with blood vessels and meninges. Furthermore, attempts to block granule cell migration using antibodies against laminin-1 have yielded conflicting results. In this report, we provide further evidence that laminin-1 is associated exclusively with blood vessels and meninges in the cerebellum of postnatal rats. In addition, we show that adhesion and neurite outgrowth of granule cells was impeded on laminin-coated surfaces. In fact, cerebellar cells dramatically and consistently avoided laminin-1 regions of patterned surfaces. Cells did adhere to laminin regions if it was coadsorbed with polylysine or tested in serum-containing medium. Avoidance of laminin-1 regions in culture was not, however, blocked by pretreatment with laminin-1 antibodies. By comparison, mouse neuroblastoma cells adhered preferentially to laminin-1 regions in serum-free medium, a response which was blocked by laminin-1 antibodies. These results indicate that laminin-1 is not involved in granule cell migration along Bergmann glial fibers. Instead, they suggest that laminin-1 may function as a repulsive guidance cue preventing granule cells from following inappropriate pathways during development. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 72–84, 1997     

Aromatic amino acid metabolism in brindled (Mo br) and viable-brindled (Mo vbr), two alleles at the mottled locus in the mouse

The central and peripheral levels of aromatic amino acids and monoamines have been studied in the X-linked mutants brindled (Mo ^br) and viable-brindled (Mo ^vbr) in the mouse. Significant differences were found in brain tyrosine (Mo ^br, Mo^vbr) and tryptophan (Mo ^br) levels, which cannot be accounted for by observed changes in the systems of synthesis and degradation of these amino acids. Central norepinephrine is depressed to 30–45% (Mo ^br) and 55% (Mo ^vbr) normal level, but central dopamine and 5-hydroxy-tryptamine and peripheral norepinephrine are normal. These new data support the view that the effects of the mottled locus are not localized in the hair follicle, and the wider aspects of the mutant syndrome are discussed in relation to a possible neuroendocrine defect.     

Ferrochelatase Structural Mutant (Fech) in the House Mouse

The molecular basis of an inherited defect of ferrochelatase in mouse (Fech^m1Pas/Fech^m1Pas, described by Tutois et al., 1991, J. Clin. Invest. 88: 1730-1736) was investigated. cDNA clones encoding ferrochelatase, isolated by amplification of the mRNA from the liver of a mutant mouse using the polymerase chain reaction, were sequenced by the dideoxynucleotide chain-termination method. All the clones carried a T to A transversion at nucleotide 293, leading to a methionine to lysine substitution at position 98 in the protein (mutation M98K). Hybridization with allele-specific oligonucleotides (ASOs) confirmed the mutation at the cDNA and genomic levels. Finally, expression of the mutant ferrochelatase protein in E. coli demonstrated a marked deficiency in activity in agreement with the activity of the deficient enzyme in vivo. This Fech^m1Pas/Fech^m1Pas mutant mouse represents a useful model for studying the pathophysiological feature of the human disease and the first accessible model for gene therapy in the field of porphyrias.     

Autosomal Dominant Hypercalciuria in a Mouse Model Due to a Mutation of the Epithelial Calcium Channel,TRPV5

Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5 ^682P/+) and homozygous (Trpv5 ^682P/682P) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and ∼10% of males developed tubulointerstitial nephritis. Trpv5 ^682P/682P mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D₃ concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5 ^682P/+ and Trpv5 ^682P/682P mice consistent with a trafficking defect. In addition, Trpv5^682P/682P mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D_28K, consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria.     

Mouse uroporphyrinogen decarboxylase: CDNA cloning,expression, and mapping

Uroporphyrinogen decarboxylase (URO-decarboxylase; EC 4.1.1.37), the heme biosynthetic enzyme responsible for the conversion of uroporphyrinogen III to coproporphyrinogen III, is the enzymatic defect in porphyria cutanea tarda, the most common porphyria. The mouse URO-decarboxylase cDNA was isolated from a mouse adult liver cDNA library. The longest clone of 1.5 kb, designated pmUROD-1, had 5′ and 3′ untranslated sequences of 281 and 97 bp, respectively, and an open reading frame of 1104 bp encoding a 367-amino acid polypeptide with a predicted molecular mass of 40,595 Da. The mouse and human coding sequences had 87.8% and 90.0% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active enzyme in Escherichia coli. In addition, the analysis of two sets of multilocus genetic crosses localized the mouse gene, Urod, on Chromosome (Chr) 4, consistent with the map location of the human gene to a position of conserved synteny on Chr 1. The availability of the mouse URO-decarboxylase should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of this inherited porphyria. Received: 27 November 1995 / Accepted: 17 January 1996     

Kinetic analysis of lamB mutants suggests the signal sequence plays multiple roles in protein export

We have developed a quantitative assay to measure the rate of processing of precursor LamB into mature protein and have used this assay to characterize 10 previously isolated and 3 new lamB signal sequence mutants. The data suggest that the LamB signal sequence serves a complex function. Our assay has revealed five types of signal sequence defect: 1) a strong kinetic defect resulting from alteration of the secondary structure in the putative alpha-helical region in the hydrophobic core, 2) a strong, or 3) a weak kinetic defect due to placement of a charged residue in the hydrophobic core, 4) decreased synthesis of LamB, and 5) both a decrease in synthesis and a strong kinetic defect. The effect of an extragenic suppressor, prlA4 on the rate of processing pLamB containing signal sequence mutations was also examined and compared to the rates in wild-type strains. It was found that prlA4 increases the rate of processing in some, but not all, mutants having a kinetic defect while having no effect on the decreased synthesis seen in mutants of types 4 and 5.     

Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon

In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSF^ts1. The mutations that resulted in a temperature-sensitive phenotype in mSF^ts1 were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42 °C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 h of growth. In order to facilitate its utilization, the mSF^ts1 was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSF^ts1 for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.