The murine gene encoding the highly conserved Sm B protein contains a nonfunctional alternative 3' splice site.

The cDNA and partial genomic nucleotide (nt) sequences were derived for the mouse Sm B polypeptide and compared to the cDNA and genomic sequences encoding human Sm B. The deduced amino acid (aa) sequences from the mouse and human genes are identical with the exception of a single conserved aa substitution, accounting for the ability of anti-Sm antibodies to recognize the Sm polypeptides from a broad range of species. The genomic sequence of mouse B gene is similar to the human B genomic locus that extends from exon 6 to exon 7. These loci include conservation of both 3' alternative splice sites and putative branch points required to process B and B' mRNAs in human cells. However, the nt sequence downstream from the putative distal 3' splice junction and single nt flanking the 3' splice site consensus sequence, differ between mouse and human B. This results in a murine mRNA with a different predicted secondary structure around the distal 3' splice site when compared to humans. Thus, secondary structural constraints in the mRNA or changes in the exon sequence might prevent recognition of this alternative splice site to form B' mRNA in murine tissues.     

Mouse C-reactive protein. Generation of cDNA clones, structural analysis, and induction of mRNA during inflammation.

A full-length C-reactive protein (CRP) cDNA clone has been isolated from a CBA/J-strain-mouse acute-phase liver library. The 1614-nucleotide cDNA specifies mRNA 5' and 3' untranslated regions of 81 and 858 bases respectively that flank 675 bases encoding mouse pre-CRP. The derived amino acid sequence predicts a 19-residue leader peptide followed by a 206-residue mature mouse CRP that shows considerable sequence identity with both human and rabbit CRP. Northern-blot analysis of mouse liver CRP mRNA concentrations after inflammatory stimuli and comparison with hepatic induction of mRNA for the major mouse acute-phase protein serum amyloid P component established that CRP, a major acute-phase reactant in human and rabbit, is a minor acute-phase reactant in mouse. The size and organization of the mouse CRP mRNA 5' and 3' untranslated regions are significantly different from those of human and rabbit CRP mRNA and may have implications for its anomalous minimal induction during acute inflammation.     

Cloning and characterization of the rabbit POU5F1 gene.

The product of the POUSF1 gene, Oct4, plays an important role both in embryonic development and in the self-renewal and differentiation of totipotent cells. To understand the function of Oct4 in rabbit ES cells, we cloned and sequenced the rabbit POU5F1 gene, as well as the cDNA encoded by the gene. The Oct4 cDNA contains a 1083 bp ORF encoding a 360 aa protein and a 241 bp 3' UTR sequence. Oct4 mRNA was expressed at a high level in rabbit ES cells and was barely detectable in the adult spleen, kidney, brain and muscle tissues. The POU5F1 gene is approximately 6 kb in length and includes five exons and four introns. Gene organization is similar to that of the mouse, human and bovine orthologs. Sequencing of the gene revealed an 82% (mouse), 90% (human) and 89% (bovine) overall identity at the protein level. The rabbit POUSF1 gene was mapped to chromosome 12q1.1 by PCR amplification of DNA from two putative POU5F1-containing BAC clones, which were previously mapped to chromosome 12q1.1. The cloning of the rabbit POU5F1 gene will facilitate studies on its roles in rabbit embryogenesis and ES cells.     

Isolation and characterization of the rat proenkephalin gene

The rat proenkephalin gene has been isolated by molecular cloning and characterized by DNA-sequence analysis. The gene exhibits a structural organization similar to that of the human gene. The nucleotide sequence encoding the biologically active opioid peptides which are generated from the proenkephalin precursor as well as the 3' untranslated region of the mRNA are found on a large exon at the 3' end of the gene (Exon III). The nucleotide sequence encoding the N terminus of the mature protein and its signal peptide are located on Exon II while Exon I encodes the 5' untranslated region of the mRNA. The nucleotide sequence of these exons and their flanking regions has been determined and compared to the human proenkephalin gene. Analysis of the nucleotide sequence homology between the human and rat proenkephalin gene reveals the presence of highly conserved regions within both the coding and noncoding portions of the genes. Enkephalin-coding sequences as well as 5' flanking sequences appear to be the most highly conserved. The importance and possible function of these sequences are discussed.     

Cloning and mapping of 5' exons from the gene encoding chicken beta nerve growth factor.

An NGF cDNA containing the 5' exons of the nerve growth factor (NGF) messenger was obtained from chicken heart mRNA using the anchored polymerase chain reaction technique. Alignment of the chicken with the corresponding murine and human sequences reveals interspecies similarities. A sequence corresponding to an exon found only in the NGF messenger, which is abundant in the submaxillary gland of the male mouse, is present in the chicken NGF cDNA. The first non-coding exons of the NGF gene are much less conserved between chicken and mouse or human than the region of the last exon encoding the mature protein. After the cloning of the chicken NGF gene from a cosmid library, the chicken NGF exons have been located within 20 kb of DNA. The chicken NGF gene is therefore shorter than its murine counterpart which spans more than 43 kb. Furthermore, the organization of the chicken and murine NGF genes markedly differs in their 5' portion.     

Isolation and characterization of the CRIPTO autosomal gene and its X-linked related sequence.

We have previously reported on the identification of a cDNA clone encoding a novel human growth factor, named "CRIPTO," that is abundantly expressed in undifferentiated human NTERA-2 clone D1 (NT2/D1) and mouse (F9) teratocarcinoma cells. We now report the organization and nucleotide sequence of two related genomic sequences. One (CR-1) corresponds to the structural gene encoding the human CRIPTO protein expressed in the undifferentiated human teratocarcinoma cells, and the other (CR-3) corresponds to a complete copy of the mRNA containing seven base substitutions in the coding region representing both silent and replacement substitutions. The 440 bp 5' to the CAP site of CR-1 are preserved in CR-3. CR-1 maps to chromosome 3, and CR-3 maps to Xq21-q22. Southern blot analysis reveals that multiple CRIPTO-related DNA sequences are present in the human as well as in the mouse genome.     

cDNA cloning and chromosomal assignment of the endothelin 2 gene: vasoactive intestinal contractor peptide is rat endothelin 2.

Four members of the endothelin family of vasoactive and mitogenic peptides have been identified: human endothelins 1, 2, and 3 (ET1, ET2, and ET3, respectively) and mouse vasoactive intestinal contractor (VIC). To characterize the mRNA encoding ET2, a 192-bp fragment of the ET2 gene, amplified by the polymerase chain reaction from human genomic DNA, was used to screen cell lines and tissues for ET2 gene expression. ET2 mRNA was detected in a cell line (HTB119) derived from a human lung small cell carcinoma, and an ET2 cDNA was cloned from a cDNA library prepared from HTB119 mRNA. DNA prepared from human-mouse somatic hybrid cell lines was used to assign the gene encoding ET2 (EDN2) to the 1p21----1pter region of chromosome 1, demonstrating that EDN2 is not linked to genes encoding ET1 (EDN1; chromosome 6) and ET3 (EDN3; chromosome 20). Southern blot hybridization revealed a single gene in human and rat genomes that hybridized with the ET2 gene fragment, and the rat gene was cloned. The endothelin peptide encoded by the rat gene differed from ET2 at 1 of 21 residues and was identical to mouse VIC. We conclude that VIC is the mouse and rat analogue of the human ET2 gene.     

Identification of a previously unrecognized production site of human renin.

We found expression of the renin gene in the intestine of human, mouse and the transgenic mouse in which the 3' flanking sequences of the human renin gene function as a tissue-specific promoter. A cotransfection analysis showed that the promoter is activated by the product of adenovirus E1A 13S mRNA in cells originated from extrarenal tissues.     

Murine ortholog of the novel glycosyltransferase, B3GTL: primary structure, characterization of the gene and transcripts, and expression in tissues

Glycosylation of proteins and lipids is important in cellular communication and maintenance of tissues. B3GTL (beta3-glycosyltransferase-like) is a novel glycosyltransferase that is found in multicellular animals ranging from mammals to insects and nematodes. The aim of this work was to identify and characterize the B3GTL gene in the mouse and to study its expression in various tissues. The murine gene codes for a protein which shares 84% amino acid sequence identity with its human ortholog, and contains all the primary structural features that characterize B3GTL proteins. The murine and human B3GTL genes share an identical exon/intron organization, and both genes utilize multiple polyadenylation signals. Their promoter regions show extensive conservation, implying that the two genes also share regulatory similarities. This notion was reinforced by Northern hybridization analysis of mouse tissues, which showed the tissue distribution of B3GTL mRNA to be similar to that previously found in human tissues, with the heart, kidney, and brain being major sites of expression in both species. The localization of B3GTL mRNA was studied by in situ hybridization in an extensive collection of mouse tissues, of which the granular cells of the olfactory bulb and the epithelium of the seminal vesicle displayed particularly strong signals. Together, these analyses indicate that the B3GTL mRNA is subject to strong tissue-specific and developmental regulation. The findings reported here make possible the design of a B3GTL "knock-out" mouse, provide a framework for analyzing the regulation of the gene, and provide an extensive catalog of tissues in which this novel protein acts.