Structural and functional characterizations of the 5'-flanking region of the mouse glucagon receptor gene: comparison with the rat gene

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5'-rapid amplification of cDNA ends from mouse liver tissue. The 5'-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5' untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5' region of the rat GR gene. The mouse distal promoter region, extending up to -1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5'-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.     

Structural organization of the proopiomelanocortin gene in Xenopus laevis. 5'-end homologies within the toad and mammalian genes

This study reports the isolation and characterization of the entire proopiomelanocortin (POMC) gene of the amphibian Xenopus laevis. The Xenopus POMC gene consists of three exons of which the main exon 3 codes for all of the bioactive domains of the precursor protein. Intron A (2.6 kb) separates the segments encoding the 5'-untranslated mRNA region and intron B (2.5 kb) interrupts the protein-coding sequence near the signal peptide coding region. In that this structural organization of the Xenopus POMC gene is similar to those of the mammalian genes, apparently the POMC gene has been remarkably stable during 350 million years of vertebrate evolution. A comparative analysis of the 5'-flanking sequences of the Xenopus and mammalian POMC genes reveals the presence of several conserved regions. One of these regions is homologous with sequences located upstream of the capping sites of other glucocorticoid-regulated genes and another region contains a segment reminiscent of a viral enhancer consensus sequence.     

Conserved regulatory elements in the promoter region of the N-CAM gene.

Genomic clones containing 5'-flanking sequences, the first exon, and the entire first intron from the chicken N-CAM gene were characterized by restriction mapping and DNA sequencing. A > 600-bp segment that includes the first exon is very G + C-rich and contains a large proportion of CpG dinucleotides, suggesting that it represents a CpG island. SP-1 and AP-1 consensus elements are present, but no TATA- or CCAAT-like elements were found within 300 bp upstream of the first exon. Comparison of the chicken promoter region sequence with similar regions of the human, rat, and mouse N-CAM genes revealed that some potential regulatory elements including a "purine box" seen in mouse and rat N-CAM genes, one of two homeodomain binding regions seen in mammalian N-CAM genes, and several potential SP-1 sites are not conserved within this region. In contrast, high CpG content, a homeodomain binding sequence, an SP-1 element, an octomer element, and an AP-1 element are conserved in all four genes. The first intron of the chicken gene is 38 kb, substantially smaller than the corresponding intron from mammalian N-CAM genes. Together with previous studies, this work completes the cloning of the chicken N-CAM gene, which contains at least 26 exons distributed over 85 kb.     

Isolation and characterization of the mouse CD8 beta-chain (Ly-3) genes. Absence of an intervening sequence between V- and J-like gene segments

We have isolated and determined the nucleotide sequence and genomic organization of the genes encoding Ly-3.1 and Ly-3.2. These genes span approximately 14 kb on chromosome 6 and consist of six exons and five introns. The exons correlate roughly with the putative functional domains, namely, a leader exon, a variable and joining region-like exon, a hinge region-like exon, a transmembrane exon, and two intracytoplasmic exons. There is no intervening sequence between V- and J-like gene segments, indicating that rearrangement is not necessary for the expression of the Ly-3 gene. In the 5'-flanking region there is no "TATA" box nor "CAAT" box; however, three "GC" boxes are located upstream of the ATG initiator codon. There are short stretches of sequence homologous to 5'-flanking sequences of the Ly-2 gene. In addition, the sequences CTCTGTGGCA at -748 exhibits homology to the enhancer core sequence of the human Ig H chain and TCR genes. Comparison of the nucleotide sequence corresponding to the extracellular portion between Ly-3.1 and Ly-3.2 revealed a single base difference which results in an amino acid substitution. Therefore it is likely that this amino acid difference is responsible for the previously defined Ly-3 allotypes.     

Structure and evolution of the Xenopus laevis albumin genes

The 68K and 74K albumin genes of Xenopus laevis arose by duplication approximately 30 million years ago. Electron microscopic analysis showed that both genes contain 15 coding sequences. The lengths of corresponding coding sequences are almost identical and are extremely similar to those of mammalian albumin genes. A block of four coding sequences, which in mammals codes for one protein domain, is repeated three times. The corresponding introns are usually different in length and have therefore diverged as a result of insertion/deletion events. The extensive homology between these gene sequences is neither confined to nor most extensive in the coding sequences and similar amounts of homologous sequences are found in the flanking DNAs as in the gene regions. Various structures were formed in the 5'-flanking DNA by mutually exclusive pairing of different homology regions. Analysis of the two 74K albumin gene sequences isolated suggests that the X. laevis genome may contain one 68K albumin gene and two very closely related 74K albumin genes.     

Comparative analysis of sequences at the 5' end of the human and mouse apolipoprotein B genes

A comparison was made between the DNA sequences in two regions of the mouse and the human apolipoprotein B genes: the 5'-flanking sequence and the region between the first exon and the second intron. Considerable homology was observed, particularly in the immediate 5' region and in the second intron. Because promoter and enhancer elements have been previously localized to these regions in the human apolipoprotein B gene, it is proposed that regions of conserved base sequence delineate binding regions for regulatory proteins. In some cases, contiguous regions of homology are longer than expected for regions designed as recognition sites for individual nuclear proteins, and may define regions recognizable by a cluster of interacting proteins. Both the human and mouse genes contain repetitive elements and a hypervariable dinucleotide repeat.