Isolation of an Arabidopsis homologue of the maize homeobox Knotted-1 gene

The shoot apical meristem (SAM) is responsible for forming most of the above-ground portion of the plant. We sought to isolate regulatory genes expressed in the Arabidopsis SMA by screening a Brassica oleracea (cauliflower) meristem cDNA library with the homeobox fragment from the maize Knotted-1 (Kn1) gene. We isolated and characterized the corresponding clone, Merihb1, from Arabidopsis. Analysis shows that the predicted MERIHB1 protein exhibits strong homology to KN1 and RS1 from maize, SBH1 from soybean, and KNAT1 and KNAT2 from Arabidopsis. Merihb1 is highly expressed in mRNA from cauliflower meristems and also accumulates in stem and flower mRNA. Based on the similarity of the Merihb1 and Kn1 sequences, expression patterns, and in situ hybridizations, we suggest that Merihb1 represents an Arabidopsis homologue of the maize Kn1 gene.     

Expression of rice OSH1 gene is localized in developing vascular strands and its ectopic expression in transgenic rice causes altered morphology of leaf

Transgenic rice plants (Oryza sativa cv. Nipponbare) carrying 1 or 2 copies of a rice homeobox gene, OSH1, under the control of the CaMV 35S promoter were generated. The transgene caused altered morphology of leaf, such as ligule-replacement and abnormal division of sclerenchyma cells. The phenotype of these leaves resembles that of maize leaf morphological mutant, Knotted 1, which is caused by duplication of the KN1 gene (Veit et al., 1990). The in situ hybridization analysis has revealed that the expression of endogenous OSH1 is mainly localized in developing vascular strands of stem. We have discussed the biological roles of OSH1 in rice based on these results.     

Gnarley1 is a dominant mutation in the knox4 homeobox gene affecting cell shape and identity.

Maize leaves have a stereotypical pattern of cell types organized into discrete domains. These domains are altered by mutations in knotted1 (kn1) and knox (for kn1-like homeobox) genes. Gnarley (Gn1) is a dominant maize mutant that exhibits many of the phenotypic characteristics of the kn1 family of mutants. Gn1 is unique because it changes parameters of cell growth in the basal-most region of the leaf, the sheath, resulting in dramatically altered sheath morphology. The strongly expressive allele Gn1-R also gives rise to a floral phenotype in which ectopic carpels form. Introgression studies showed that the severity of the Gn1-conferred phenotype is strongly influenced by genetic background. Gn1 maps to knox4, and knox4 is ectopically expressed in plants with the Gn1-conferred phenotype. Immunolocalization experiments showed that the KNOX protein accumulates at the base of Gn1 leaves in a pattern that is spatially and temporally correlated with appearance of the mutant phenotype. We further demonstrate that Gn1 is knox4 by correlating loss of the mutant phenotype with insertion of a Mutator transposon into knox4.     

A dominant mutation in the COL1A1 gene that substitutes glycine for valine causes recurrent lethal osteogenesis imperfecta

Summary Type I collagen chains of a proband from a family with recurrent lethal osteogenesis imperfecta (OI) migrated as a doublet when submitted to gel electrophoresis. Cyanogen bromide (CNBr) peptide mapping demonstrated that the post-translational over-modifications were initiated in 1ICB7. Chemical cleavage of cDNA-RNA heteroduplexes identified a mismatch in the 1I cDNA; this mismatch was subsequently confirmed by sequencing a 249-bp fragment amplified by the polymerase chain reaction. A G to T transition in the second base of the first codon of exon 41 resulted in the substitution of glycine 802 by valine. This mutation impaired collagen secretion by dermal fibroblasts. The over-modified chains were retained intracellularly and melted at a lower temperature than normal chains. Collagen molecules synthesized by parental fibroblasts had a normal electrophoretic mobility, but hybridization of genomic DNA with allele-specific oligonucleotides revealed the presence of the mutant allele in the mother's leukocytes. The mutation was not detected in her fibroblasts consistent with the protein data. These results support the hypothesis that somatic and germ-line mosaicism in the phenotypically normal mother explain the recurrence of OI.     

Cell-surface changes induced by ectopic expression of the murine homeobox gene Hox-3.3.

Murine homeobox-containing genes (Hox genes) are postulated as playing key roles in the establishment of the anterior-posterior embryonic body axis, possibly providing cells with positional cues. Little is known, however, concerning how cells might respond to homeobox gene expression to interpret these cues. Since changes in the cell-surface are central to many processes in early development we reasoned that cells expressing different complements of Hox genes might have different surface properties. In order to investigate this we have used the sensitive, non-disruptive technique of multiple two-phase aqueous partition, which is able to detect small differences on the surface of intact cells. Using this technique we have found that ectopic expression of the murine Hox-3.3 gene in cultured cells induces reproducible changes in the cell surface. Changes only occurred above a threshold level of gene expression, but above this level a correlation between surface change and gene expression was seen. The implications for the establishment of a 'Hox' code of homeobox genes acting to specifically change cell-surface properties are discussed.     

hairy gene function in the Drosophila eye: normal expression is dispensable but ectopic expression alters cell fates.

The regulatory gene hairy is expressed and required during early embryogenesis to control segmentation gene expression properly and during larval and pupal development to control the pattern of certain adult sensory structures. We have found the hairy protein to be expressed transiently during two stages of eye imaginal disc development, including all cells immediately anterior to the morphogenetic furrow that traverses the developing eye disc, and again in the presumptive R7 photoreceptor cells of the developing ommatidia. This pattern is conserved in a significantly diverged Drosophila species. We show that, surprisingly, ommatidia formed by homozygous hairy- mutant clones are apparently normal, indicating that hairy function in the eye is dispensable. However, we do find that ectopic expression of hairy causes numerous structural abnormalities and the alteration of cell fates. Thus, proper regulation of hairy is still essential for normal eye development. We suggest that the loss of hairy function may be compensated by other regulatory proteins, as has been observed previously for several structurally and functionally related genes involved in sensory organ development. The effects of ectopic hairy expression may result from interactions with proneural genes involved in the development of the eye and other sensory organs.     

A loss-of-function mutation in the rice KNOX type homeobox gene, OSH3

KNOX homeodomain (HD) proteins encoded by KNOTTED1-like homeobox genes (KNOX genes) are thought to work as switches for cells to change from an indeterminate to a determinate state, although their direct functions are not clear. In the process of isolating KNOX genes from rice, we found that one gene, named OSH3, has two amino acid substitutions in three of the invariant amino acid residues in the HD of KNOX proteins. These amino acid substitutions are not universal in rice: two of the cultivars from the Indica variety of rice do not carry those substitutions but two of the cultivars from Japonica variety do. We tested the effect of these amino acid substitutions on their ability to form dimers and to induce abnormal morophologies when overexpressed in transgenic plants. We found that OSH3 without those substitutions can form dimers and can induce an abnormal phenotype in overexpression studies, and that OSH3 with those amino acid substitutions is defective in both. Based on these observations, we concluded that OSH3 from two of the cultivars from the Japonica variety could have lost its original function, or could have acquired a novel function by modifying the action of HD, or both.     

A dominant mutation that alters the regulation of INO1 expression in Saccharomyces cerevisiae.

A dominant, single nuclear gene mutation, CSE1, caused inositol auxotrophy in yeast cells. The inositol requirement was marked when choline was present in the medium. Inositol-1-phosphate synthase, the regulatory enzyme of inositol synthesis, is repressed by inositol, or more profoundly by a combination of inositol and choline in the wild type. In CSE1, the level of inositol-1-phosphate synthase was low and was greatly repressed on the addition of choline alone. In accordance with this, INO1 mRNA encoding the enzyme was low even under the depressed conditions and was profoundly decreased by choline in CSE1. But in the wild type, the addition of choline alone had little effect. An INO1-lacZ fusion was constructed and the control of the INO1 promoter in CSE1 was studied. lacZ expression was repressed not only by inositol, but also by choline in CSE1, whereas it was repressed by inositol, but only slightly by choline in the wild type. CSE1 was unlinked to the INO1 structural gene. Thus CSE1 was thought to be a regulatory mutation. Furthermore, when the CDP-choline pathway was mutationally blocked, choline did not affect INO1 expression, indicating that the metabolism of choline via the CDP-choline pathway is required for INO1 repression.     

A mutation in the SOS1 gene causes hereditary gingival fibromatosis type 1

Hereditary gingival fibromatosis (HGF) is a rare, autosomal dominant form of gingival overgrowth. Affected individuals have a benign, slowly progressive, nonhemorrhagic, fibrous enlargement of the oral masticatory mucosa. Genetic loci for autosomal dominant forms of HGF have been localized to chromosome 2p21-p22 (HGF1) and chromosome 5q13-q22 (HGF2). To identify the gene responsible for HGF1, we extended genetic linkage studies to refine the chromosome 2p21-p22 candidate interval to approximately 2.3 Mb. Development of an integrated physical and genetic map of the interval identified 16 genes. Sequencing of these genes, in affected and unaffected HGF1 family members, identified a mutation in the Son of sevenless-1 (SOS1) gene in affected individuals. In this report, we describe the genomic structure of the SOS1 gene and present evidence that insertion of a cytosine between nucleotides 126,142 and 126,143 in codon 1083 of the SOS1 gene is responsible for HGF1. This insertion mutation, which segregates in a dominant manner over four generations, introduces a frameshift and creates a premature stop codon, abolishing four functionally important proline-rich SH3 binding domains normally present in the carboxyl-terminal region of the SOS1 protein. The resultant protein chimera contains the wild-type SOS1 protein for the N-terminal amino acids 1-1083 fused to a novel 22-amino acid carboxyl terminus. Similar SOS1 deletion constructs are functional in animal models, and a transgenic mouse construct with a comparable SOS1 chimera produces a phenotype with skin hypertrophy. Clarification of the functional role of this SOS1 mutant has implications for understanding other forms of gingival fibromatosis and corrective gingival-tissue management.     

Craniofacial abnormalities induced by ectopic expression of the homeobox gene Hox-1.1 in transgenic mice

Hox-1.1 is a murine homeobox-containing gene expressed in a time- and cell-specific manner during embryogenesis. We have generated transgenic mice that ectopically express Hox-1.1 from the chicken beta-actin promoter. In these mice Hox-1.1 expression was changed to an almost ubiquitous pattern. Ectopic expression of Hox-1.1 leads to death of the transgenic animals shortly after birth and is associated with multiple craniofacial anomalies, such as cleft palate, open eyes at birth, and nonfused pinnae. This phenotype is similar to the effects seen after systemic administration of retinoic acid during gestation. This suggests that retinoic acid embryopathy and the specific developmental defects caused by ectopic expression of a potential developmental control gene share a common pathogenic mechanism.