The presence of two types of prorenin converting enzymes in the mouse submandibular gland

We have recently demonstrated, by protein and cDNA sequence analyses, that prorenin converting enzyme (PRECE) in the ICR mouse submandibular gland is identical to the epidermal growth factor-binding protein (EGF-BP) type B, the mGK-13 gene product identified in Balb/c mouse. However, in the course of cDNA cloning, we noticed the presence of the other cDNA type highly homologous but not identical to the PRECE cDNA. The sequence of the newly identified cDNA was identical to that of the pSGP-2 cDNA cloned from NMRI mice, which also encodes EGF-BP type B different at 9 out of 261 amino acids from the mGK-13 product. Although this difference has been explained by strain polymorphism, our results indicate that these two proteins are distinct gene products. The product of the newly identified cDNA also had a prorenin converting activity. Thus, the products of both cDNAs identified in previous and present studies are involved in maturation of two bioactive polypeptides, renin and EGF.     

Epidermal growth factor binding protein: identification of a different protein

Partial amino acid sequence analysis of epidermal growth factor binding protein (EGF-BP), an arginine esteropeptidase that specifically associates with EGF to form a high molecular weight complex in male mouse submandibular glands, has revealed a single, distinct protein that is different from three previously reported forms of EGF-BP. This protein shows substantial sequence homology with these other putative forms of EGF-BP as well as with a large family of kallikreins expressed in the mouse submandibular gland. Purified EGF-BP contains three polypeptide chains as a result of two internal cleavages at residues 87-88 and 140-141. These modifications may represent processing events that are critical for determining the binding specificity of EGF-BP, since they occur within regions surrounding the substrate binding site.     

Prediction of the three-dimensional structures of the nerve growth factor and epidermal growth factor binding proteins (kallikreins) and an hypothetical structure of the high molecular weight complex of epidermal growth factor with its binding protein.

We have predicted the three-dimensional structures of the serine protease subunits (gamma-NGF, alpha-NGF, and EGF-BP) of the high molecular weight complexes of nerve growth factor (7S NGF) and epidermal growth factor (HMW-EGF) from the mouse submandibular gland (from the X-ray crystal structures of two related glandular kallikreins). The conformations of three of the six loops surrounding the active site are relatively well defined in the models of gamma-NGF and EGF-BP, but three other loops are likely to have flexible conformations. Although the amino acid sequence of alpha-NGF is closely related to those of gamma-NGF and EGF-BP, it is catalytically inactive. Model-building studies on alpha-NGF suggested that mutations (in alpha-NGF) just prior to the active site serine (195) and an unusual N-terminal sequence are consistent with alpha-NGF having a zymogen-like conformation (similar to that in chymotrypsinogen). An hypothetical model of the quaternary structure of HMW-EGF has been constructed using this model of EGF-BP and the NMR structure of murine EGF. The C-terminal arm of EGF was modeled into the active site of EGF-BP based on data indicating that the C-terminal arginine of EGF occupies the S1 subsite of EGF-BP. Data suggesting one of the surface loops of EGF-BP is buried in the HMW-EGF complex and symmetry constraints were important in deriving a schematic model. A molecular docking program was used to fit EGF to EGF-BP.     

Complexity and sequence identification of 24 rat V beta genes

Twenty-four TCR V beta genes were cloned by anchored PCR from the Lewis rat strain and identified by nucleotide and amino acid sequence comparisons to known mouse V beta genes. Rat V beta genes exist in 17 single-member and 3 multimember subfamilies and exhibit 86 to 94 and 72 to 92% nucleotide and amino acid sequence similarities, respectively, to their mouse counterparts. A single rat gene, designated V beta 20, having no previously known mouse counterpart was identified; a closely related gene was subsequently isolated from the C57BL/6 mouse strain. Characterization of the rV beta genes provides the basis for addressing rat T cell tolerance mechanisms and V beta gene usage in immunologically mediated rat disease models.     

Substrate specificities of growth factor associated kallikreins of the mouse submandibular gland

The kinetic constants for the hydrolysis of a series of tripeptide p-nitroanilide substrates by mouse epidermal growth factor binding protein (EGF-BP), the gamma-subunit of mouse nerve growth factor (gamma-NGF), bovine pancreatic trypsin (BPT), and porcine pancreatic kallikrein (PPK) have been evaluated. These substrates correspond to the carboxyl-terminal three amino acids of the mature forms of epidermal growth factor (EGF) and beta-nerve growth factor (beta-NGF), as well as various substitutions in the penultimate and antepenultimate positions, and, as such, represent potential recognition sites for precursor processing. The mouse kallikreins (EGF-BP and gamma-NGF) preferentially hydrolyze the substrates with the sequences of their specifically associated growth factors; however, the constants derived from these reactions do not account for the association constants observed with the mature growth factors, and additional significant binding interactions between EGF-BP and EGF and between gamma-NGF and beta-NGF are predicted to exist outside of the catalytic binding site, i.e., the P3 to P1 positions. A comparison of the kinetic constants of BPT, PPK, and the mouse kallikreins indicates that EGF-BP and gamma-NGF display a hybrid catalytic character. A favorable substrate P1 arginine guanidinium group interaction exists for the mouse kallikreins, similar to that of BPT, but a preference for a hydrophobic side chain in the substrate P2 position makes the mouse kallikreins, especially EGF-BP, more closely resemble PPK than BPT. These findings have significant implications with regard to molecular modeling of the mouse kallikreins.     

Concerted evolution of human amylase genes.

Cosmid clones containing 250 kilobases of genomic DNA from the human amylase gene cluster have been isolated. These clones contain seven distinct amylase genes which appear to comprise the complete multigene family. By sequence comparison with the cDNAs, we have identified two pancreatic amylase genes and three salivary amylase genes. Two truncated pseudogenes were also recovered. Intergenic distances of 17 to 22 kilobases separate the amylase gene copies. Within the past 10 million years, duplications, gene conversions, and unequal crossover events have resulted in a very high level of sequence similarity among human amylase gene copies. To identify sequence elements involved in tissue-specific expression and hormonal regulation, the promoter regions of the human amylase genes were sequenced and compared with those of the corresponding mouse genes. The promoters of the human and mouse pancreatic amylase genes are highly homologous between nucleotide -160 and the cap site. Two sequence elements thought to influence pancreas-specific expression of the rodent genes are present in the human genes. In contrast, similarity in the 5' flanking sequences of the salivary amylase genes is limited to several short sequence elements whose positions and orientations differ in the two species. Some of these sequence elements are also associated with other parotid-specific genes and may be involved in their tissue-specific expression. A glucocorticoid response element and a general enhancer element are closely associated in several of the amylase promoters.     

Class II major histocompatibility complex genes of the sheep

The class II genes of the sheep major histocompatibility complex (MHC) have been cloned from two unrelated heterozygous sheep into cosmid vectors. By restriction mapping and hybridization with a number of class II probes of human and mouse origin, the cloned genetic material has been assigned to seven distinct alpha genes, 10 distinct beta genes and 14 beta-related sequences. It was difficult to identify homologues of specific HLA class II genes because of a tendency for the ovine genes to cross-hybridize between HLA probes representing different loci. Such cross-hybridization was especially marked among the beta genes. While DQ and DR homologues have been tentatively identified by several criteria, no genes corresponding to DP have been identified. Cosmids containing class II alpha and beta genes have been transfected into mouse LTK- cells, and surface expression of a sheep class II molecule has been obtained.     

The class II genes of the rat MHC

Genes that encode class II Ag from the MHC of the rat, the RT1 region, have been isolated as a series of cosmid clones. The cosmids define two clusters, each of which contains three identifiable sequences; one homologous to alpha-chain and two to beta-chain genes. Both the serologically identified rat class II Ag have been expressed in mouse L cell fibroblasts after the introduction of each alpha-chain gene along with a beta-chain gene from the same cluster. There are substantial homologies to the I region of the mouse H-2 complex in the presence, location, orientation, and expression of the six identified sequences from the rat RT1, supporting the view that the overall organization of the two gene complexes has remained conserved since the species separated.     

Connexin genes in the mouse and human genome

Gap junctions serve for direct intercellular communication by docking of two hemichannels in adjacent cells thereby forming conduits between the cytoplasmic compartments of adjacent cells. Connexin genes code for subunit proteins of gap junction channels and are members of large gene families in mammals. So far, 17 connexin (Cx) genes have been described and characterized in the murine genome. For most of them, orthologues in the human genome have been found (see White and Paul 1999; Manthey et al. 1999; Teubner et al. 2001; S?hl et al. 2001). We have recently performed searches for connexin genes in murine and human gene libraries available at EMBL/Heidelberg, NCBI and the Celera company that have increased the number of identified connexins to 19 in mouse and 20 in humans. For one mouse connexin gene and two human connexin genes we did not find orthologues in the other genome. Here we present a short overview on distinct connexin genes which we found in the mouse and human genome and which may include all members of this gene family, if no further connexin gene will be discovered in the remaining non-sequenced parts (about 1-5%) of the genomes.     

The heavy chain genes of a lupus anti-DNA autoantibody are encoded in the germ line of a nonautoimmune strain of mouse and conserved in strains of mice polymorphic for this gene locus

The variable region of the heavy chain of a prototypic anti-DNA autoantibody from the lupus-prone mouse, MRL-lpr/lpr, was cloned and sequenced. The VH and JH genes expressed by this antoantibody were found to be identical to germ line genes from the nonautoimmune mouse strain, BALB/c. The D gene of this autoantibody differed by one nucleotide from several members of the germ line SP2 family, but has been found in expressed D genes from several strains of mice. These results show that a normal mouse strain contains all of the structural information necessary for the expression of the heavy chain variable region of a lupus autoantibody. A fragment that is present in both BALB/c and MRL mice is highly homologous in both coding and flanking sequences to the autoantibody VH gene (VH130) and is the same size as the BALB/c germ line gene. This suggests that these two strains may share the same allele of this VH gene, despite the fact that they are polymorphic for this VH gene family. Other mouse strains that are polymorphic for this locus contained one to three VH genes that were highly related to VH130 in both coding and flanking regions. Thus, VH genes that may be allelic to the antibody VH gene or that may have arisen by gene conversion, unequal crossing over or gene duplication, are conserved in many mouse strains.     

Members of a novel gene family, Gsdm, are expressed exclusively in the epithelium of the skin and gastrointestinal tract in a highly tissue-specific manner

Gasdermin (Gsdm) was originally identified as a candidate causative gene for several mouse skin mutants. Several Gsdm-related genes sharing a protein domain with DFNA5, the causative gene of human nonsyndromic hearing loss, have been found in the mouse and human genomes, and this group is referred to as the DFNA5-Gasdermin domain family. However, our current comparative genomic analysis identified several novel motifs distinct from the previously reported domain in the Gsdm-related genes. We also identified three new Gsdm genes clustered on mouse chromosome 15. We named these genes collectively the Gsdm family. Extensive expression analysis revealed exclusive expression of Gsdm family genes in the epithelium of the skin and gastrointestinal tract in a highly tissue-specific manner. Further database searching revealed the presence of other related genes with a similar N-terminal motif. These results suggest that the Gsdm family and related genes have evolved divergent epithelial expression profiles.     

Molecular evolution of the zinc-containing long-chain alcohol dehydrogenase genes

Phylogenetic relationships and rates of nucleotide substitution were studied for alcohol dehydrogenase (ADH) genes by using DNA sequences from mammals and plants. Mammalian ADH sequences include the three class I genes and a class II gene from humans and one gene each from baboon, rat, and mouse. Plant sequences include two ADH genes each from maize and rice, three genes from barley, and one gene each from wheat and two dicots, Arabidopsis and pea. Phylogenetic trees show that relationships among ADH genes are generally consistent with taxonomic relationships: mammalian and plant ADH genes are classified into two distinct groups; primate class I genes are clustered; and two dicot sequences are clustered separately from monocot sequences. Accelerated evolution has been detected among the duplicated ADH genes in plants, in which synonymous substitutions occurred more often within the coenzyme-binding domain than within the catalytic domains.     

Genomic organization of mouse Dishevelled genes

We report the isolation and characterization of two new genomic loci corresponding to the mouse Dishevelled (Dvl) genes Dvl2 and Dvl3. The Dvl genes are homologs of the Drosophila dsh segment polarity gene, and are involved in the Wnt/wingless signal transduction pathway. Dvl2 and Dvl3 genomic clones were isolated from a mouse 129 strain λFIXII genomic library and have identical exon/intron organization to Dvll. All three Dishevelled genes span 15 exons and 14 introns and have a number of conserved splice junction sites.     

Molecular cloning of two distinct renin genes from the DBA/2 mouse.

We report the molecular cloning of cDNA copies of DBA/2 mouse submaxillary gland (SMG) renin mRNA, which were used to probe Southern transfers of mouse genomic DNA. The results suggested either that there is a single renin gene containing a large intron in that part of the gene corresponding to the probe, or that there are two distinct renin genes. We have shown that the latter is the case by cloning and isolating two similar but distinct renin genes from DBA/2 mouse DNA. Restriction maps of the regions containing the two renin genes are presented, together with nucleotide sequence data locating a complete exon coding for amino acids 268-315 of mouse SMG renin.     

Selection and validation of microarray candidate genes from subregions and subnuclei of the brain

DNA array technology now allows an enormous amount of expression data to be obtained. For large-scale gene profiling enterprises, this is of course welcome. However, the scientist interested in follow-up studies of a handful of differentially expressed genes may find it hard to sift through the vast datasets to pinpoint genes with the most desirable and reliable behaviors. Here, we present the methodology we have employed to discover genes differentially expressed in the adult mouse brain. We first used Affymetrix microarrays to compare gene expression from five different brain regions: the amygdala, cerebellum, hippocampus, olfactory bulb, and periaqueductal gray. Second, we identified genes differentially expressed within three distinct amygdala subnuclei. In this case, the tissue was microdissected by laser-capture to minimize contamination from adjacent subnuclei, and extracted RNA was subjected to three rounds of linear amplification prior to hybridization to the microarrays. To select candidate genes, we developed a custom algorithm to identify those genes with the most robust changes in expression across different replicate samples. Confirmation of expression patterns with in situ hybridization uncovered further criteria to consider in the selection process.     

Genomic structure, mapping, and expression analysis of the mammalian Lunatic, Manic, and Radical fringe genes

The three members of the mammalian fringe gene family, Manic fringe (Mfng), Radical fringe (Rfng), and Lunatic fringe (Lfng), were identified on the basis of their similarity to Drosophila fringe (fng) and their participation in the evolutionarily conserved Notch receptor signaling pathway. Fringe genes encode pioneer secretory proteins with weak similarity to glycosyltransferases. Both expression patterns and functional studies support an important role for Fringe genes in patterning during embryonic development and an association with cellular transformation. We have now further characterized the expression and determined the chromosomal localization and genomic structure of the mouse Mfng, Rfng, and Lfng genes; the genomic structure and conceptual open reading frame of the human RFNG gene; and the refined chromosomal localization of the three human fringe genes. The mouse Fringe genes are expressed in the embryo and in adult tissues. The mouse and human Fringe family members map to three different chromosomes in regions of conserved synteny: Mfng maps to mouse Chr 15, and MFNG maps to human Chr 22q13.1 in the region of two cancer-associated loci; Lfng maps to mouse Chr 5, and LFNG maps to human Chr 7p22; Rfng maps to mouse Chr 11, and RFNG maps to human Chr 17q25 in the minimal region for a familial psoriasis susceptibility locus. Characterization of the genomic loci of the Fringe gene family members reveals a conserved genomic organization of 8 exons. Comparative analysis of mammalian Fringe genomic organization suggests that the first exon is evolutionarily labile and that the Fringe genes have a genomic structure distinct from those of previously characterized glycosyltransferases. Received: 19 February 1999 / Accepted: 22 February 1999