The rat peptidylarginine deiminase-encoding gene: structural analysis and the 5'-flanking sequence.

Genomic clones of the rat peptidylarginine deiminase (PAD)-encoding gene (PAD) were isolated, and the gene organization was analyzed by restriction mapping and nucleotide sequencing. The PAD spans more than 50 kb and contains 16 exons and 15 introns. The lengths of the introns from 0.5 kb to more than 16.5 kb. A 1.7-kb sequence in the 5'-flanking region was determined. S1 nuclease mapping revealed two putative cap sites 79 and 81 bp upstream from the N-terminal ATG codon of PAD, which had been determined by amino acid sequence analysis. This ATG was confirmed to be the translation start site, since no other ATG codon was found in the open reading frame downstream from the cap sites. The 5'-flanking sequence contains four potential SP1-binding sites, a putative Pit-1/GHF-1-binding site, four short sequences either identical or homologous to the sequences in the promoter regions of rat or human growth hormone encoding genes, as well as a sequence similar to an estrogen-responsive element. However, neither a typical TATAA box, nor CCAAT box is present. These results provide important clues for elucidating the mechanism of female-specific and/or sex cycle-dependent gene expression.     

Nucleotide sequence of the chicken cardiac alpha actin gene: absence of strong homologies in the promoter and 3'-untranslated regions with the skeletal alpha actin sequence

The entire nucleotide sequence of the chicken cardiac alpha-actin (CC alpha A) gene has been determined. This is the first complete sequence of a cardiac actin gene that includes the promoter region, cap site, all the introns, and the polyadenylation site. The gene contains six introns, five of which interrupt the coding region at amino acids (aa) 41, 150, 204, 267, and 327. The first intron is in the 5'-noncoding region and is 438 bp in length. The CC alpha A gene encodes an mRNA of approx. 1400 bp with 5'- and 3'-untranslated region of 59 and 184 nucleotides (nt), respectively. Like the chicken skeletal alpha-actin gene, the CC alpha A gene has the codon for the aa cysteine between the initiator ATG and the codon for the N-terminal aspartic acid residue of the mature protein. There are no strong homologies (less than 13 consecutive nt) in the promoter or 3'-untranslated regions between the CC alpha A and chicken skeletal alpha-actin genes even though both are expressed in skeletal muscle during development. However, the 3'-untranslated region of the CC alpha A gene demonstrates significant sequence homology (76% over a 200-nt region) with the same region in the partial sequence of the human cardiac gene. The conservation of these sequence homologies between identical isoforms rather than the different alpha actin genes suggests these conserved regions may have a role in regulation rather than tissue-specific expression, as previously proposed.     

Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA

We report the cloning, nucleotide (nt) sequence and expression of the cDNA (pah) encoding phenylalanine hydroxylase (PAH) of Drosophila melanogaster. The strong hybridization signals observed in genomic blots when D. melanogaster DNA was probed with 32P-labeled human pah cDNA, indicated the existence of a high degree of sequence similarity between the pah genes of both species. The length of the pah genomic fragment is about 30 to 40 kb. The cDNA contains 84 bp of the 5'-untranslated region, 1359 bp of the protein-coding region and 87 bp of the 3' region, with only one polyadenylation signal. The isolated cDNA is probably full-length, since the size of the D. melanogaster PAH mRNA is 1.5 kb. At the nt level, the similarity of the D. melanogaster cDNA with human and rat pah cDNAs is 57.9% and 58.1%, respectively. The highest similarities are restricted to the nt sequence coding for the presumed hydroxylation domain. There is no nt sequence similarity between the first three exons of the human pah gene and an equivalent fraction of the D. melanogaster pah gene. At the amino acid (aa) level, the similarity in the presumed hydroxylation domain is 88.5%, in which two motifs of the structure AGLLSSXXXL are found, where X represents any aa. It was interesting to notice the conservation of aa 408, 311 and 280, where mutations are associated with phenylketonuria in humans. We observed, moreover, that, as it occurs in humans and rats, the expression of the D. melanogaster pah gene is tissue-specific and temporally regulated.