Kirrel2, a novel immunoglobulin superfamily gene expressed primarily in beta cells of the pancreatic islets

A novel immunoglobulin superfamily (Igsf) protein gene was discovered by computational analysis of human draft genomic DNA, and multiple cDNA clones were obtained. The protein encoded by this gene contains five Ig domains, one transmembrane domain, and an intracellular domain. It has significant similarity with several known Igsf proteins, including Drosophila RST (irregular chiasm C-roughest) protein and mammalian KIRREL (kin of irregular chiasm C-roughest), NEPH1, and NPHS1 (nephrin) proteins. All these proteins have multiple Ig domains, possess properties of cell adhesion molecules, and play important roles in organ development. RT-PCR and Northern blot results indicate this gene is predominantly expressed in pancreas, and public sequence databases indicate there is also expression in the nervous system. We have named this gene Kirrel2 (kin of irregular chiasm-like 2), to reflect its similarity to irregular chiasm C-roughest and Kirrel. Four splice forms of Kirrel2 were observed, including two that we cloned from pancreas mRNA as well as two GenBank entries, one from the brain and one from a retinoblastoma cell line. A partial cDNA clone of the mouse orthologue was obtained by RT-PCR from mouse brain, and the inferred protein sequence has 85% sequence identity to the human protein. Immunohistochemical staining results indicate that the KIRREL2 protein is conserved from rodents to primates, and it is highly expressed in pancreatic islets. RT-PCR results on mouse pancreatic cell lines indicate that expression in the pancreas is restricted to beta cells. Thus, KIRREL2 protein is a beta-cell-expressed Ig domain protein and may be involved in pancreas development or beta cell function.     

Immunological evidence for a relationship between the dendrotoxin-binding protein and the mammalian homologue of the Drosophila Shaker K+ channel

Polyclonal antibodies were raised against two synthetic peptides from different parts of the predicted amino acid sequence of the mouse homologue (MBK1) of the Drosophila Shaker K+ channel. The antibodies recognized the toxin-binding subunit of the dendrotoxin-binding proteins from rat and bovine brain. The results suggest that the dendrotoxin-binding protein is related to the expression products of the mammalian homologue of the Shaker gene.     

Molecular cloning and characterization of rat and human calpain-5

Until today, 14 isoforms of mammalian calpains have been identified, including calpain-5. The C. elegans calpain-5 homologue tra-3 is reported to be essential for necrotic neuronal cell death. In this study, we cloned and characterized rat calpain-5, which is highly homologous to human and mouse sequences. The nucleotide sequence is 87% and 93% identical with human and mouse calpain-5, respectively. The protein sequence is well conserved, showing 96% identity in mouse and 92% in human. RT-PCR analysis revealed strong expression of calpain-5 in rat lungs, kidneys, and brain while week expression in heart, whereas in rat brain regions it is ubiquitously expressed. The mRNA expression in different human tissues showed equal expression. However, in human brain regions calpain-5 was strongly expressed in hypothalamus, thalamus, cerebellum, and frontal lobe. Western blot analysis on human neuroblastoma SH-SY5Y cells demonstrated calcium-dependent processing of calpain-5, despite the absence of calmodulin-like domain.     

Molecular cloning and functional characterization of mouse chitotriosidase

Mammalian chitinase and chitinase-like proteins are members of a recently discovered gene family. Thus far, neither chitin nor chitin synthase has been found in mammals. The existence of chitinase genes in mammals is intriguing and the physiologic functions of chitinases are not clear. Human chitotriosidase, also called chitinase 1 (chit1), has been cloned. It has been found that high levels of serum chitotriosidase are associated with several diseases, but the physiologic functions of this enzyme are still unclear. To facilitate the studies in animal models we cloned and characterized a cDNA that encodes the mouse chitotriosidase. The open reading frame of this cDNA predicts a protein of 464 amino acids with a typical chitinase structure, including a signal peptide, a highly conserved catalytic domain and a chitin-binding domain. The predicted amino acid sequence is highly homologous to that of human chitotriosidase and to that of mouse acidic mammalian chitinase. Sequence analysis indicates that the mouse chitotriosidase gene has 12 exons, spanning a 40-kb region in mouse chromosome 1. The constitutive expression of mouse chitotriosidase is restricted to brain, skin, bone marrow, kidney, tongue, stomach and testis. Recombinant expression of the cloned cDNA demonstrated that the encoded protein is secreted and has chitinolytic activity that is sensitive to the specific chitinase inhibitor allosamidin and has the ability to bind to chitin particles. Substitution mutations at the conserved catalytic site completely abolished the enzymatic activity of the recombinant protein. These studies illustrate that mouse chitotriosidase is a typical chitinase that belongs to the mammalian chitinase gene family.     

The alpha-amylase gene in Drosophila melanogaster: nucleotide sequence, gene structure and expression motifs.

We present the complete nucleotide sequence of a Drosophila alpha-amylase gene and its flanking regions, as determined by cDNA and genomic sequence analysis. This gene, unlike its mammalian counterparts, contains no introns. Nevertheless the insect and mammalian genes share extensive nucleotide similarity and the insect protein contains the four amino acid sequence blocks common to all alpha-amylases. In Drosophila melanogaster, there are two closely-linked copies of the alpha-amylase gene and they are divergently transcribed. In the 5'-regions of the two gene-copies we find high sequence divergence, yet the typical eukaryotic gene expression motifs have been maintained. The 5'-terminus of the alpha-amylase mRNA, as determined by primer extension analysis, maps to a characteristic Drosophila sequence motif. Additional conserved elements upstream of both genes may also be involved in amylase gene expression which is known to be under complex controls that include glucose repression.     

Characterization of the adaptor protein ARH expression in the brain and ARH molecular interactions

Previously, pA134 was identified as one of the mRNAs present in the squid giant axon. Comparative sequence analyses revealed that the pA134 gene product manifested significant similarity to the mammalian lipoprotein receptor adaptor protein also known as ARH (autosomal recessive hypercholesterolemia). ARH mRNA and protein displayed very similar pattern of expression throughout the mouse brain. Significant levels of expression were observed in cells with a predominantly neuronal profile in the cerebellum, brainstem, olfactory bulb, hippocampus, and cortex. A yeast two hybrid screen for ARH protein interactions in mouse brain identified the following binders: amyloid precursor-like protein 1, low density lipoprotein receptor-related protein (LRP) 1, LRP8, and GABA receptor-associated protein-like 1. The interactions of ARH with LRP1 and GABA receptor-associated protein-like 1 were subsequently verified by co-immunoprecipitation of the protein complexes from transfected human embryonic kidney cells. The presence of ARH mRNA in axon of primary sympathetic neurons was established by RT-PCR analyses and confirmed by in situ hybridization. Taken together, our data suggest that ARH is a multifunctional protein whose spectrum of function in the brain goes beyond the traditionally known metabolism of lipoproteins, and that ARH may be locally synthesized in the axon.     

Molecular cloning and functional characterization of chicken brain tau: isoforms with up to five tandem repeats

Tau is a major microtubule-associated protein in mammalian brain, where it exists as multiple isoforms that are produced from a single gene by alternative mRNA splicing. Here we present the first report on the structure and function of tau protein from a nonmammalian vertebrate. In the adult chicken brain, five main tau isoforms are expressed. One isoform has three tandem repeats, two isoforms have four repeats each, and two isoforms have five repeats each. Similar to mammalian tau, some chicken tau isoforms contain an amino-terminal insert of 53 amino acids. Unlike mammalian tau, a 34 amino acid insert in the proline-rich region upstream of the repeats is alternatively spliced in chicken tau. It is preceded by a constitutively expressed sequence of 17 amino acids that is absent in tau from human and rodent brains. The expression of chicken tau isoforms and their phosphorylation are developmentally regulated, similar to what has been described in mammalian brain. Functionally, chicken tau isoforms with five repeats have the greatest ability to promote microtubule assembly, followed by isoforms with four and three repeats, respectively. The 34 amino acid insert positively influences both the rate and the extent of microtubule assembly, whereas the 53 amino acid insert only influences the extent of assembly.     

胚胎发育早期头部特异表达的小鼠新基因BSG5的克隆及功能

BrainSpecificGene 5 (BSG5 )基因是用消减差异筛选的方法克隆到的在小鼠胚胎头部特异表达的新基因。它与人的KIAA0 6 2 8基因在氨基酸水平上有 81 9%的同源性。BSG5基因长 2 4 87bp ,定位在小鼠的第 1 5号染色体上 ,包含 2个外显子。它编码的蛋白质全长 4 99个氨基酸 ,含 1 2个C2H2型的锌指结构域。以BSG5基因全长编码区为探针的原位杂交结果显示BSG5基因在小鼠胚胎发育早期头部特异表达 ,在小鼠胚胎发育稍后时期的尾部和肢芽也有表达。此外 ,以鸡胚为模型研究BSG5基因也发现该基因在鸡胚的头部特异表达。这提示BSG5基因与头部发育有密切关系 ,其结构与表达特征预示着它编码的是一个具DNA结合功能的转录调控因子     

An inflammatory role for the mammalian carboxypeptidase inhibitor latexin: relationship to cystatins and the tumor suppressor TIG1

Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 A resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface.     

Conservation of regional gene expression in mouse and human brain

Many neurodegenerative diseases have a hallmark regional and cellular pathology. Gene expression analysis of healthy tissues may provide clues to the differences that distinguish resistant and sensitive tissues and cell types. Comparative analysis of gene expression in healthy mouse and human brain provides a framework to explore the ability of mice to model diseases of the human brain. It may also aid in understanding brain evolution and the basis for higher order cognitive abilities. Here we compare gene expression profiles of human motor cortex, caudate nucleus, and cerebellum to one another and identify genes that are more highly expressed in one region relative to another. We separately perform identical analysis on corresponding brain regions from mice. Within each species, we find that the different brain regions have distinctly different expression profiles. Contrasting between the two species shows that regionally enriched genes in one species are generally regionally enriched genes in the other species. Thus, even when considering thousands of genes, the expression ratios in two regions from one species are significantly correlated with expression ratios in the other species. Finally, genes whose expression is higher in one area of the brain relative to the other areas, in other words genes with patterned expression, tend to have greater conservation of nucleotide sequence than more widely expressed genes. Together these observations suggest that region-specific genes have been conserved in the mammalian brain at both the sequence and gene expression levels. Given the general similarity between patterns of gene expression in healthy human and mouse brains, we believe it is reasonable to expect a high degree of concordance between microarray phenotypes of human neurodegenerative diseases and their mouse models. Finally, these data on very divergent species provide context for studies in more closely related species that address questions such as the origins of cognitive differences.