Cloning and characterization of Disc1, the mouse ortholog of DISC1 (Disrupted-in-Schizophrenia 1)

We cloned the mouse ortholog of DISC1 (Disrupted-in-Schizophrenia 1), a candidate gene for schizophrenia. Disc1 is 3163 nucleotides long and has 60% identity with the human DISC1. Disc1 encodes 851 amino acids and has 56% identity with the human protein. Disc1 maps to the DISC1 syntenic region in the mouse, and genomic structure is conserved. A Disc1 splice variant deletes a portion of Disc1 beginning at amino acids orthologous to the human truncation. Bioinformatic analysis and cross-species comparisons revealed sequence conservation distributed across the genes and conservation of leucine zipper and coiled-coil domains in both orthologs. In situ hybridization in adult mouse brain revealed a restricted expression pattern, with highest levels in the dentate gyrus of the hippocampus and lower expression in CA1-CA3 of the hippocampus, cerebellum, cerebral cortex, and olfactory bulbs. Identification of Disc1 will facilitate the study of DISC1's function and creation of mouse models of DISC1 disruption.     

Isolation and characterization of the mouse ortholog of the Fukuyama-type congenital muscular dystrophy gene

Fukuyama-type congenital muscular dystrophy (FCMD) is a severe autosomal-recessive muscular dystrophy accompanied by brain malformation. Previously, we identified the gene responsible for FCMD through positional cloning. Here we report the isolation of its murine ortholog, Fcmd. The predicted amino acid sequence of murine fukutin protein encoded by Fcmd is 90% identical to that of its human counterpart. Radiation hybrid mapping localized the gene to 2.02 cR telomeric to D4Mit272 on chromosome 4. Northern blot analysis revealed ubiquitous expression of Fcmd in adult mouse tissues. Through in situ hybridization, we observed a wide distribution of Fcmd expression throughout embryonic development, most predominantly in the central and peripheral nervous systems. We also detected high Fcmd expression in the ventricular zone of proliferating neurons at 13.5 days post-coitum. Brain malformation in FCMD patients is thought to result from defective neuronal migration. Our data suggest that neuronally expressed Fcmd is likely to be important in the development of normal brain structure.     

Cloning and characterization of mouse homolog of the CXC chemokine receptor CXCR1

CXCR2/IL-8RB was the only receptor previously reported in mice for ELR+ CXC chemokines, whereas the receptors for these chemokines in human include both CXCR1 and CXCR2. In this study, we cloned the full length cDNA of the mouse CXCR1 (mCXCR1) gene. The deduced amino acid of mCXCR1 was 77% and 58% identical to the rat and human CXCR1, respectively. RT-PCR and Northern blot analysis showed that mCXCR1 mRNA was expressed in lung, spleen, thymus, peripheral blood leukocytes, as well as in the isolated neutrophils. In a mouse respiratory inflammation model induced by lipopolysaccharide, a large number of neutrophils infiltrated into the lung and, meanwhile, the mCXCR1 expression was significantly increased in the recruited neutrophils, suggesting that mCXCR1 may mediate the recruitment of neutrophils to the inflammation site under certain infections.     

Identification and functional characterization of the AGO1 ortholog in maize

Eukaryotic Argonaute proteins play primary roles in miRNA and siRNA pathways that are essential for numerous developmental and biological processes. However, the functional roles of the four ZmAGO1 genes have not yet been characterized in maize (Zea mays L.). In the present study, ZmAGO1a was identified from four putative ZmAGO1 genes for further characterization. Complementation of the Arabidopsis ago1‐27 mutant with ZmAGO1a indicated that constitutive overexpression of ZmAGO1a could restore the smaller rosette, serrated leaves, later flowering and maturation, lower seed set, and darker green leaves at late stages of the mutant to the wild‐type phenotype. The expression profiles of ZmAGO1a under five different abiotic stresses indicated that ZmAGO1a shares expression patterns similar to those of Argonaute genes in rice, Arabidopsis, and wheat. Further, variation in ZmAGO1a alleles among diverse maize germplasm that resulted in several amino acid changes revealed genetic diversity at this locus. The present data suggest that ZmAGO1a might be an important AGO1 ortholog in maize. The results presented provide further insight into the function of ZmAGO1a.     

Molecular cloning and characterization of rat CCL9 (MIP-1gamma), the ortholog of mouse CCL9

We identified an EST sequence that was up-regulated during osteoclast formation in the rat. Investigating further, we cloned the cDNA from rat long bone and found it to be highly homologous to the mouse CC chemokine, CCL9, both at the nucleotide and amino acid levels. The rat CCL9 amino acid sequence is 74% identical to the mouse sequence, with an additional 11% similar amino acids. Recombinant rat CCL9 was used in chemotaxis assays of rat bone marrow cells and it was found to have a strong and dose-dependent effect. In addition, CCL9 mRNA was very highly up-regulated during osteoclast differentiation of rat bone marrow-derived mononuclear cells, increasing by over 100-fold when stimulated by colony stimulating factor-1 and the TNF superfamily member, RANKL. Together, these results establish that, like the mouse, the rat also uses CCL9 to promote the complex process of osteoclast formation.     

Cloning and characterization of a putative GS3 ortholog involved in maize kernel development

The GS3 gene was the first identified gene controlling the grain size in rice. It has been proven to be involved in the evolution of grain size during domestication. We isolated the maize ortholog, ZmGS3 and investigated its role in the evolution of maize grain size. ZmGS3 has five exons encoding a protein with 198 amino acids, and has domains in common with the rice GS3 protein. Compared with teosinte, maize has reduced nucleotide diversity at ZmGS3, and the reduction is comparable to that found in neutrally evolving maize genes. No positive selection was detected along the length of the gene using either the Hudson–Kreitman–Aguadé or Tajima’s D tests. Phylogenetic analysis reveals a distribution of maize sequences among two different clades, with one clade including related teosinte sequences. The nucleotide polymorphism analysis, selection test and phylogenetic analysis reveal that ZmGS3 has not been subjected to selection, and appears to be a neutrally evolving gene. In maize, ZmGS3 is primarily expressed in immature ears and kernels, implying a role in maize kernel development. Association mapping analysis revealed one polymorphism in the fifth exon that is significantly associated with kernel length in two environments. Also one polymorphism in the promoter region was found to affect hundred kernel weight in both environments. Collectively, these results imply that ZmGS3 is involved in maize kernel development but with different functional polymorphisms and thus, possibly different mechanisms from that of the rice GS3 gene.     

Cloning and characterization of the maize An1 gene.

The Anther ear1 (An1) gene product is involved in the synthesis of ent-kaurene, the first tetracyclic intermediate in the gibberellin (GA) biosynthetic pathway. Mutations causing the loss of An1 function result in a GA-responsive phenotype that includes reduced plant height, delayed maturity, and development of perfect flowers on normally pistillate ears. The an1::Mu2-891339 allele was recovered from a Mutator (Mu) F2 family. Using Mu elements as molecular probes, an An1-containing restriction fragment was identified and cloned. The identity of the cloned gene as An1 was confirmed by using a reverse genetics screen for maize families that contain a Mu element inserted into the cloned gene and then by demonstrating that the insertion causes an an1 phenotype. The predicted amino acid sequence of the An1 cDNA shares homology with plant cyclases and contains a basic N-terminal sequence that may target the An1 gene product to the chloroplast. The sequence is consistent with the predicted subcellular localization of AN1 in the chloroplast and with its biochemical role in the cyclization of geranylgeranyl pyrophosphate, a 20-carbon isoprenoid, to ent-kaurene. The semidwarfed stature of an1 mutants is in contrast with the more severely dwarfed stature of GA-responsive mutants at other loci in maize and may be caused by redundancy in this step of the GA biosynthetic pathway. DNA gel blot analysis indicated that An1 is a single-copy gene that lies entirely within the deletion of the an1-bz2-6923 mutant. However, homozygous deletion mutants accumulated ent-kaurene to 20% of the wild-type level, suggesting that the function of An1 is supplemented by an additional activity.     

Cloning and molecular characterization of three ubiquitin fusion degradation 1 (Ufd1) ortholog genes from Xenopus laevis, Gallus gallus and Drosophila melanogaster.

The yeast ubiquitin fusion degradation 1 (Ufd1) protein is involved in a degradation pathway for ubiquitin fused products. The human ortholog gene (UFD1-like, UFD1L) is deleted in patients affected by the DiGeorge/velocardiofacial syndromes. We report the cloning of UFD1L orthologs from Drosophila melanogaster (dufd1l), Xenopus laevis and Gallus gallus. The 1,125-bp Drosophila cDNA encodes a protein of 316 amino acids, showing 60% identity with the human and murine proteins. The identity to the G. gallus, X. laevis, C. elegans and S. cerevisiae proteins is 95%, 83%, 32%, and 36%, respectively. Northern expression data in Drosophila indicate that dufd1l is expressed through embryonic, larval and pupal development, as well as in the adult fly.