Faithful tissue-specific expression of the human chromosome 21-linked <Emphasis Type="Italic">COL6A1</Emphasis> gene in BAC-transgenic mice

We created transgenic mice with a bacterial artificial chromosome (BAC) containing the human COL6A1 gene. In high-copy and low-copy transgenic lines, we found correct temporal and spatial expression of COL6A1 mRNA, paralleling the expression of the murine Col6a1 gene in a panel of nine adult and four fetal organs. The only exception was the fetal lung, in which the transgene was expressed poorly compared with the endogenous gene. Expression of COL6A1 mRNA from the transgene was copy number-dependent, and the increased gene dosage correlated with increased production of collagen VI alpha 1 in skin and heart, as indicated by Western blotting and immunohistochemistry. COL6A1 maps to Chromosome 21 and this gene has been a candidate for contributing to cardiac defects and skin abnormalities in Down syndrome. The low-copy and high-copy COL6A1 transgenics were born and survived in normal Mendelian proportions, without cardiac malformations or altered skin histology. These data indicate that the major promoter and enhancer sequences regulating COL6A1 expression are present in this 167-kb BAC clone. The lack of a strong cardiac or skin phenotype in the COL6A1 BAC-transgenic mice suggests that the increased expression of this gene does not, by itself, account for these phenotypes in Down syndrome.     

Targeted knockout and <Emphasis Type="Italic">lacZ</Emphasis> reporter expression of the mouse <Emphasis Type="Italic">Tmhs</Emphasis> deafness gene and characterization of the <Emphasis Type="Italic">hscy-2J</Emphasis> mutation

The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhs ^tm1Kjn ) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of β-galactosidase activity in Tmhs ^tm1Kjn heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene, hscy-2J, that causes abnormal splicing from a cryptic splice site within exon 2 and is predicted to produce a functionally null protein lacking 51 amino acids of the wild-type sequence.     

Human BAC-mediated rescue of the Friedreich ataxia knockout mutation in transgenic mice

Three independent transgenic mouse lines were generated with the human Friedreich ataxia gene, FRDA, in an 188-kb bacterial artificial chromosome (BAC) genomic sequence. Three copies of the transgene per diploid mouse genome were integrated in a single site in each mouse line. Transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Frda gene, to generate mice homozygous for the Frda knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Frda knockout mutation was observed in all three lines. Rescued mice displayed normal behavioral and biochemical parameters. RT-PCR analysis demonstrated that human FRDA mRNA is expressed in all the lines. The relative expression of the human FRDA and mouse Frda genes showed a similar pattern in different tissues in all three lines, indicating position-independent control of expression of the human FRDA transgene. However, large differences in the human:mouse mRNA ratio were observed between different tissues in all three lines. The human transgene is expressed at much higher levels in the brain, liver, and skeletal muscle than the endogenous gene, while expression of the human transgene in blood is only 25–30% of the mouse gene. These studies will facilitate the development of humanized mouse models of Friedreich ataxia through introduction of a GAA trinucleotide expansion or specific known point mutations in the normal human FRDA locus and the study of the regulation of gene expression from the FRDA locus.     

<Emphasis Type="Italic">Gsdma3</Emphasis> gene is needed for the induction of apoptosis-driven catagen during mouse hair follicle cycle

Gsdm is a newly found gene family, which is restricted in its expression to the gastrointestinal tract and the skin epithelium. As a main member of the Gsdma subfamily, Gsdma3 is expressed specifically in the hair follicle of mouse skin, but its function remains largely unclear. By hematoxylin and eosin staining, we showed that Gsdma3 gene mutation caused an abnormal catagen phase with unshortened length and unshrunk structure of the hair follicle, in which the development of catagen phase was inhibited. TUNEL staining further revealed that the apoptosis of the hair follicle was obviously decreased in mutant mice. Caspase-3 downregulation was also detected by immunofluorescence, Western blot and RT-PCR in the hair follicle of the mutant mice. After intradermal injection of Gsdma3 gene expression plasmid, apoptosis as well as Caspase-3 expression in the hair follicle of mutant mice was enhanced, and so the catagen retardation of Gsdma3-mutant mice was rescued. Our results confirmed that Gsdma3 gene mutation interfered with catagen formation during mouse hair follicle cycle and, by upregulation of Caspase-3 expression and promotion of apoptosis, Gsdma3 gene could play an essential role in normal catagen induction.     

Tomato <Emphasis Type="Italic">fruit weight 11.3</Emphasis> maps close to <Emphasis Type="Italic">fasciated</Emphasis> on the bottom of chromosome 11

Fruit weight is an important character in many crops. In tomato (Solanum lycopersicum), fruit weight is controlled by many loci, some of which have a major effect on the trait. Fruit weight 11.3 (fw11.3) and fasciated (fas) have been mapped to the same region on chromosome 11. We sought to determine whether these loci represent alleles of the same or separate genes. We show that fas and fw11.3 are not allelic and instead represent separate genes. The fw11.3 locus was fine-mapped to a 149-kb region comprised of 22 predicted genes. Unlike most fruit weight loci, gene action at fw11.3 indicates that the mutant allele is partially dominant over the wild allele. We also investigate the nature of the genome rearrangement at the fas locus and demonstrate that the mutation is due to a 294-kb inversion disrupting the YABBY gene known to underlie the fas locus.     

Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the <Emphasis Type="Italic">Foxn1</Emphasis> locus

Background  

Thymic epithelial cells (TECs) promote thymocyte maturation and are required for the early stages of thymocyte development and for positive selection. However, investigation of the mechanisms by which TECs perform these functions has been inhibited by the lack of genetic tools. Since the Foxn1 gene is expressed in all presumptive TECs from the early stages of thymus organogenesis and broadly in the adult thymus, it is an ideal locus for driving gene expression in differentiating and mature TECs.     

Positional cloning of <Emphasis Type="Italic">ds1</Emphasis>, the target leaf spot resistance gene against <Emphasis Type="Italic">Bipolaris sorghicola</Emphasis> in sorghum

Target leaf spot is one of the major sorghum diseases in southern Japan and caused by a necrotrophic fungus, Bipolaris sorghicola. Sorghum resistance to target leaf spot is controlled by a single recessive gene (ds1). A high-density genetic map of the ds1 locus was constructed with simple sequence repeat markers using progeny from crosses between a sensitive variety, bmr-6, and a resistant one, SIL-05, which allowed the ds1 gene to be genetically located within a 26-kb region on the short arm of sorghum chromosome 5. The sorghum genome annotation database for BTx623, for which the whole genome sequence was recently published, indicated a candidate gene from the Leucine-Rich Repeat Receptor Kinase family in this region. The candidate protein kinase gene was expressed in susceptible plants but was not expressed or was severely reduced in resistant plants. The expression patterns of ds1 gene and the phenotype of target leaf spot resistance were clearly correlated. Genomic sequences of this region in parental varieties showed a deletion in the promoter region of SIL-05 that could cause reduction of gene expression. We also found two ds1 alleles for resistant phenotypes with a stop codon in the coding region. The results shown here strongly suggest that the loss of function or suppression of the ds1 protein kinase gene leads to resistance to target leaf spot in sorghum.     

The <Emphasis Type="Italic">Idd6.2</Emphasis> diabetes susceptibility region controls defective expression of the <Emphasis Type="Italic">Lrmp</Emphasis> gene in nonobese diabetic (NOD) mice

The identification of genes mediating susceptibility to type 1 diabetes (T1D) remains a challenging task. Using a positional cloning approach based on the analysis of nonobese diabetic (NOD) mice congenic over the Idd6 diabetes susceptibility region, we found that the NOD allele at this locus mediates lower mRNA expression levels of the lymphoid restricted membrane protein gene (Lrmp/Jaw1). Analysis of thymic populations indicates that Lrmp is expressed mainly in immature thymocytes. The Lrmp gene encodes a type 1 transmembrane protein that localizes to the ER membrane and has homology to the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate gene, which negatively regulates intracellular calcium levels. We hypothesize that the observed decrease in expression of the Lrmp gene in NOD mice may constitute a T1D susceptibility factor in the Idd6 region.     

The endothelial-specific regulatory mutation, <Emphasis Type="Italic">Mvwf1</Emphasis>, is a common mouse founder allele

Mvwf1 is a cis-regulatory mutation previously identified in the RIIIS/J mouse strain that causes a unique tissue-specific switch in the expression of an N-acetylgalactosaminyltransferase, B4GALNT2, from intestinal epithelium to vascular endothelium. Vascular B4galnt2 expression results in aberrant glycosylation of von Willebrand Factor (VWF) and accelerated VWF clearance from plasma. We now report that 13 inbred mouse strains share the Mvwf1 tissue-specific switch and low VWF phenotype, including five wild-derived strains. Genomic sequencing identified a highly conserved 97-kb Mvwf1 haplotype block shared by these strains that encompasses a 30-kb region of high nucleotide sequence divergence from C57BL6/J flanking B4galnt2 exon 1. The analysis of a series of bacterial artificial chromosome (BAC) transgenes containing B4galnt2 derived from the RIIIS/J or C57BL6/J inbred mouse strains demonstrates that the corresponding sequences are sufficient to confer the vessel (RIIIS/J) or intestine (C57BL6/J)-specific expression patterns. Taken together, our data suggest that the region responsible for the Mvwf1 regulatory switch lies within an approximately 30-kb genomic interval upstream of the B4galnt2 gene. The observation that Mvwf1 is present in multiple wild-derived strains suggests that this locus may be retained in wild mouse populations due to positive selection. Similar selective pressures could contribute to the high prevalence of von Willebrand disease in humans. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Multiple impairments in male reproduction 1 (mimr1)</Emphasis>, a novel male-sterile mutant of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>, shows several defects in male reproductive development

We have characterized a new male-sterile mutant in Arabidopsis that exhibits conditional sterility but has restored fertility when drought-stressed. This mutant, multiple impairments in male reproduction 1 (mimr1), shows pleiotropic defects in both vegetative and reproductive development. Examination with dissecting and scanning electron microscopes revealed that its pollen grains are not effectively released from the anther locule after dehiscence, and anther differentiation is defective. Growth of the style and stamen filaments are also abnormal. Histological analysis demonstrated that these phenomena are due not only to a noticeably reduced extension of the stamen but also greater elongation of the pistil. Genetic analysis indicated that mimr1 is a single locus recessive nuclear mutant. The mutation can be mapped to a locus strongly linked to a 1200-kb region on Chromosome 3. Meta-analysis of expression patterning presented several candidate genes in that region. No mutants with similar phenotypes have previously been reported, suggesting that mimr1 is a novel male-sterile locus. Characterization of MIMR1 will provide further insights into the molecular basis for the development of plant reproductive organs.     

A new tool for conditional gene manipulation in a subset of keratin‐expressing epithelia

Megsin is a serine protease inhibitor (Serpin) that has known expression in kidney mesangial cells. Here, we report the generation and characterization of a bacterial artificial chromosome (BAC) transgene expressing Cre under the control of Megsin regulatory elements. When crossed to the ROSA26R‐lacZ reporter mice, the Megsin‐Cre transgene mediates loxP recombination primarily in the skin, forestomach, and esophagus, but surprisingly not in the mesangial cells. Within the skin, cells in all epidermal layers and the hair follicle cells expressed Cre. This transgene also has uniform expression in the epithelium of the forestomach and esophagus. Conditional deletion of Adam10, a gene known to have important functions in skin development, by using this Megsin‐Cre transgene led to severe skin defects. In addition, these mutants appear to have reduced folds and surface area in the forestomach. These results show that the Megsin‐Cre transgene can mediate loxP‐recombination in all epidermal layers of the skin, the hair follicle cells, as well as in the epithelium of the forestomach and esophagus, all of which have known expression of various keratins. This Megsin‐Cre transgene can serve as a new tool for conditional genetic manipulation to study development and diseases in the skin and the upper digestive tract. genesis 50:899–907, 2012. © 2012 Wiley Periodicals, Inc.     

Identification of transposons,retroelements, and a gene family predominantly expressed in floral tissues in chromosome 3DS of the hexaploid wheat progenitor <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

A multigene family expressed during early floral development was identified on the short arm of wheat chromosome 3D in the region of the Ph2 locus, a locus controlling homoeologous chromosome pairing in allohexaploid wheat. Physical, genetic and molecular characterisation of the Wheat Meiosis 1 (WM1) gene family identified seven members that localised within a region of 173-kb. WM1 gene family members were sequenced and they encode mainly type Ia plasma membrane-anchored leucine rich repeat-like receptor proteins. In situ expression profiling suggests the gene family is predominantly expressed in floral tissue. In addition to the WM1 gene family, a number of other genes, gene fragments and pseudogenes were identified. It has been predicted that there is approximately one gene every 19-kb and that this region of the wheat genome contains 23 repetitive elements including BARE-1 and Wis2-1 like sequences. Nearly 50% of the repetitive elements identified were similar to known transposons from the CACTA superfamily. Ty1-copia, Ty3-gypsy and Athila LTR retroelements were also prevalent within the region. The WM1 gene cluster is present on 3DS and on barley 3HS but missing from the A and B genomes of hexaploid wheat. This suggests either recent generation of the cluster or specific deletion of the cluster during wheat polyploidisation. The evolutionary significance of the cluster, its possible roles in disease response or floral and early meiotic development and its location at or near the Ph2 locus are discussed.