Differential structural properties and expression patterns suggest functional significance for multiple mouse desmoglein 1 isoforms

The four isoforms of desmosomal cadherin desmogleins (Dsg1-4) are expressed in epithelial tissues in a differentiation-specific manner. Extensive sequencing of the human genome has revealed only one copy of the Dsg1 gene. However, we recently cloned two novel additional mouse Dsg1 genes, Dsg1-beta and -gamma, which flank the original Dsg1-alpha on chromosome 18. Sequence conservation between the Dsg1 isoforms diverged significantly at exon 11, particularly in the region that encodes for the extracellular anchoring (EA) domains. Computational analysis revealed very low hydrophilic potential of the Dsg1-gamma EA compared with the corresponding sequences of Dsg1-alpha and -beta, suggesting that the Dsg1-gamma EA domain may have a stronger affinity to the cell membrane. We generated antibodies using synthetic peptides or recombinant proteins localized within the EA domains. These antibodies were tested for their specificity and were then used to demonstrate expression of Dsg1 isoforms in various tissues. In the epidermis, all Dsg1 isoforms were differentially expressed in the differentiating cell layers. In the hair follicle, all Dsg1 isoforms were present throughout the entire process of its development and cycling but the expression of Dsg1 isoforms is subject to significant hair cycle-dependent changes. Dsg1-beta and -gamma, but not Dsg1-alpha, were detected in the sebaceous gland epithelium and the stratified epithelium of the stomach. Finally, Dsg1-alpha and Dsg1-beta, but not Dsg1-gamma, are proteolytically cleaved by exfoliative toxin A. These results suggest that the developmental complexity of mouse tissues, including skin and hair, may play a significant role in the evolutionary driving force to maintain multiple Dsg1 genes in mouse.     

Immunohistochemical localization of thrombomodulin in the stratified epithelium of the rat is restricted to the keratinizing epidermis

The expression and function of thrombomodulin (TM), an endothelial cofactor protein for thrombin-mediated protein C activation, in the epithelium are not fully characterized. This report describes the distribution and localization of TM in the various types of epithelia in the rat by light and electron microscopic immunocytochemistry. TM showed a limited distribution and was expressed by the keratinizing stratified epithelia of the skin, tongue, and esophagus, but was not present on the non-keratinizing epithelia of the vagina, ureter, trachea, stomach, or gut. An identical pattern of TM expression was seen in mucocutaneous junctions, transitional zones from a non-keratinizing stratified epithelium to a keratinizing epithelium at the edge of the eyelid and in the anal canal. As the keratinization of the stratified epithelia proceeded, the staining intensity increased in the transitional zones. Within the keratinizing stratified epithelia, TM staining was limited to the keratinocytes of the spinous layer, the spinous cells. The subcellular localization of TM on the spinous cells was restricted to the plasma membrane facing the intercellular spaces. TM was not detectable on the desmosomes or the two membranes making up the junction, presumably the nexus. The functional significance of TM in keratinizing epithelia is discussed. Accepted: 14 October 1999     

Characterization of the type I interferon locus and identification of novel genes

The human type I interferon (IFN) genes are clustered on human chromosome 9p21 and the mouse genes are located in the region of conserved synteny on mouse chromosome 4. We have identified two novel mouse Ifna genes (Ifna12, Ifna13) and Ifnl2 (IFN-like 2, a homologue of Limitin/IFN-like 1). Another type I IFN gene was designated Ifne1. Mouse Ifne1 was expressed in ovaries and uterus but not in tissues of hematopoietic origin. IFN-epsilon1 has general structural characteristics of a type I IFN. These studies represent the first detailed annotation of the mouse type I IFN locus, and the products of these novel genes may have important functions in reproduction and host defense.     

The murine GABA(B) receptor 1: cDNA cloning, tissue distribution, structure of the Gabbr1 gene, and mapping to chromosome 17

GABA (gamma-aminobutyric acid) is a major inhibitory neurotransmitter in the central nervous system (CNS) which activates both ionotropic (GABA(A)/GABA(C)) and metabotropic (GABA(B)) receptor systems. We identified two alternatively spliced cDNA variants of the murine GABA(B) receptor 1 that are predominantly expressed in the CNS. Deduced protein structures are highly homologous to the previously characterized rat and human receptors. Comparison of the genomic structures of mouse and human revealed that alternative splicing occurred at the same position, whereas the mouse gene has an additional 5' exon. Radiation hybrid mapping, combined with database searches, indicated that the GABA(B) receptor gene (Gabbr1) is located on mouse chromosome 17, adjacent to the marker D17Mit24 in a region homologous to human chromosome 6p21.3.     

Oracle, a novel PDZ-LIM domain protein expressed in heart and skeletal muscle

In order to identify novel genes enriched in adult heart, we performed a subtractive hybridization for genes expressed in mouse heart but not in skeletal muscle. We identified two alternative splicing variants of a novel PDZ-LIM domain protein, which we named Oracle. Both variants contain a PDZ domain at the amino-terminus and three LIM domains at the carboxy-terminus. Highest homology of Oracle was found with the human and rat enigma proteins in the PDZ domain (62 and 61%, respectively) and in the LIM domains (60 and 69%, respectively). By Northern hybridization analysis, we showed that expression is highest in adult mouse heart, low in skeletal muscle and undetectable in other adult mouse tissues. In situ hybridization in mouse embryos confirmed and extended these data by showing high expression of Oracle mRNA in atrial and ventricular myocardial cells from E8.5. From E9.5 low expression of Oracle mRNA was detectable in myotomes. These data suggest a role for Oracle in the early development and function of heart and skeletal muscle.     

Cloning and Characterization of the Human Lectin P35 Gene and Its Related Gene

We previously cloned a novel human lectin, designated P35, with both collagen-like and fibrinogen-like domains. P35 recognizes GlcNAc residues and is opsonic toward microorganisms. The overall structure of P35 closely resembles those of two pig ficolins that are putative TGF-β1-binding proteins. In this study, we analyzed the exon–intron structure and chromosomal location of the P35 gene as well as its structural relationship to splicing variants. In addition, we isolated another distinct genomic clone corresponding to the upstream region of a P35-related gene that has an exon organization closely resembling that of the P35 gene. The sequences of exons in the P35-related gene were identical to the cDNA sequence reported for “human ficolin.” Northern blotting revealed that the P35 gene is expressed mainly in liver, whereas the P35-related gene is expressed in lung and peripheral blood leukocytes, demonstrating tissue-specific expression of these two genes. Both genes were assigned to a closely related region of chromosome 9 at 9q34.     

Characterization and expression of the gene encoding membralin, an evolutionary conserved protein expressed in the central nervous system

We have identified a novel gene that encodes an evolutionary conserved protein that we name membralin since it contains multiple transmembrane regions. The human gene C19orf6 localizes to chromosome 19p13.3. Splice variant membralin-1 is encoded by 11 exons, translating into 620 amino acids. In addition, we found evidence for two additional splice variants in the human. The mouse gene ORF61 localizes to chromosome 10. We cloned two splice variants in mouse: membralin-1, which is encoded by 12 exons, translating into 574 amino acids, and membralin-2, which translates into 598 amino acids. The existence of rat membralin-1 (574 amino acids long) is, so far, only supported by in situ hybridization result, whereas the existence of rat membralin-2 (598 amino acids long) is strongly supported by overlapping ESTs. Gene homologues were also identified in fruit-fly (CG8405, chromosome 2R 52; two splice variants), nematode (chromosome III), and Arabidopsis thaliana (chromosome 1). Sequence analysis revealed no closely related genes, suggesting that membralin represents the sole member of a unique protein family.     

Alternative splicing in the dyslexia-associated gene <Emphasis Type="Italic">KIAA0319</Emphasis>

The KIAA0319 gene in chromosome 6p22 has been strongly associated with developmental dyslexia. In this article we show a wide expression pattern of this gene in human adult brain by Northern blot analysis. We also performed RT-PCR analysis to detect alternative splicing variants in human brain. Most of the detected variants involve alternative splicing of the exons at the 5′ and the 3′ ends. Two main forms differing in the length of the 5′ UTR are detected at approximately the same rate. Two variants (B and C) lacking exon 19, which encodes the transmembrane domain, are the main alternative forms detected among those predicted to encode protein. These two variants could be secreted and might be involved in signaling functions. A similar RT-PCR analysis performed in mouse and rat adult brains showed that only some of the alternative splicing variants are equivalent to those found in the human gene. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mapping of the KHSRP gene to a region of conserved synteny on human chromosome 19p13.3 and mouse chromosome 17

The K homology-type splicing regulatory protein, KSRP, activates splicing through intronic splicing enhancer sequences. It is highly expressed in neural cells and is required for the neural-specific splicing of the c-src N1 exon. In this study, we mapped the gene (gene symbols KHSRP and Khsrp) to human chromosome 19 by using radiation hybrid panels and to mouse chromosome 17 by studying an interspecific backcross panel. Human KHSRP is a positional candidate gene for familial febrile convulsion and Cayman type cerebellar ataxia. Comparative analysis of the human and mouse genomes indicates that the KHSRP gene is located in regions of conserved synteny between the two species.     

The paralemmin protein family: identification of paralemmin-2, an isoform differentially spliced to AKAP2/AKAP-KL, and of palmdelphin, a more distant cytosolic relative

Paralemmin is a protein implicated in plasma membrane dynamics. Here we describe the identification of two new paralemmin-related proteins. A partial paralemmin homolog, palmdelphin, is predominantly cytosolic, unlike paralemmin which is lipid-anchored to the plasma membrane through a C-terminal CaaX motif. We have mapped the mouse palmdelphin gene to distal chromosome 3 between Amy2 and Abcd3, in a region homologous to human chromosome 1p22-p21 where the human palmdelphin gene is located. We have also identified a second paralemmin isoform, paralemmin-2. It is expressed from a gene on human chromosome 9q31-q33 which ends only 33 kb upstream of the gene encoding the protein kinase A-binding protein,AKAP2/AKAP-KL. The closely adjacent paralemmin-2 and AKAP2 genes are functionally linked in a very unusual manner. Chimeric mRNAs are expressed, apparently by RNA readthrough and differential splicing, that encode natural fusion proteins in which either the N-terminal coiled-coil region or nearly the complete sequence of paralemmin-2 except its C-terminal CaaX motif is fused to AKAP2/AKAP-KL. The N-terminal coiled-coil region is conserved in paralemmin-1, paralemmin-2/AKAP2, palmdelphin and a fourth, uncharacterized gene, suggesting that it is a modular functional domain.