1. Structure of the guinea pig neutrophil cationic peptide gene
I Nagaoka, A Someya, K Iwabuchi, T Yamashita FEBS Lett. 1992 May 25;303(1):31-5. doi: 10.1016/0014-5793(92)80471-r.
Guinea pig neutrophils contain the antimicrobial cationic peptides GNCP-1 and GNCP-2 in the granules. In this study, the GNCP gene was isolated, and the structure was characterized. Using cDNA probes, one phage clone was isolated from a guinea pig genomic library. The gene spanned greater than 3 kb, and comprised three exons and two introns. Sequence analysis revealed that the gene encoded GNCP-2. Exon 1 mainly coded for the 5' untranslated region, exon 2 coded for the prepro-peptide region of GNCP-2, and exon 3 coded for the mature peptide region of GNCP-2 and the 3' untranslated region. Primer extension analysis indicated that the transcription initiation site was located to a thymidine residue, 93 bp upstream of the ATG initiation codon of GNCP-2 mRNA. A possible TATA box was located 24 bp upstream of the transcription start site. Interestingly, the pyrimidine-rich sequences identified in the promoter regions of the human neutrophil elastase and myeloperoxidase genes were also found in the 5' flanking region of the GNCP-2 gene.
2. Cloning and characterization of the guinea pig neutrophil cationic peptide-1 and -2 genes
I Nagaoka, A Nonoguchi, T Yamashita DNA Seq. 1993;4(2):123-8. doi: 10.3109/10425179309020151.
Guinea pig neutrophils contain the antimicrobial cationic peptides GNCP-1 and GNCP-2 in the granules. Using cDNA probes, four different GNCP gene clones were isolated from a guinea pig genomic library. Two clones encoded GNCP-1 and other two clones encoded GNCP-2. The nucleotide sequence of GNCP-1 and GNCP-2 gene clones were highly homologous (> 97%) in the region sequenced. The GNCP-1 and GNCP-2 genes spanned 3 kb, and consisted of three exons and two introns. Exon 1 encoded the 5' untranslated region, exon 2 encoded the prepro-peptide region, and exon 3 encoded the mature peptide region and the 3' untranslated region. The transcription start site was located to a thymidine residue 93 bp upstream of the start codon (ATG) of GNCP-1 or GNCP-2 mRNA, and TATA-like sequence was located 24 to 30 bp upstream of the transcription start site. Glucocorticoid regulating element-like sequence was found in the 5' flanking region of the GNCP-1 or GNCP-2 gene, and the GNCP gene transcription rate of bone marrow cells was upregulated by dexamethasone treatment. Together these observations indicate that GNCP-1 and GNCP-2 are encoded by the very homologous but different genes, and that GNCP gene expression is regulated by glucocorticoid.
3. Characterization of the promoters of the guinea pig neutrophil cationic peptide-1 and -2 genes
I Nagaoka, N Ishihara, T Yamashita FEBS Lett. 1994 Dec 12;356(1):33-8. doi: 10.1016/0014-5793(94)01229-6.
Guinea pig neutrophils contain the antimicrobial cationic peptides GNCP-1 and GNCP-2 in the granules. To understand the regulation of the gene expression, the promoters for the GNCP-1 and GNCP-2 genes were characterized. Sequencing analysis of the genomic clones showed that the nucleotide sequences of the 5'-upstream regions (1.7 kb) from exon 1 were homologous (90-93%) between the GNCP-1 and GNCP-2 genes. However, transient transfection assays using luciferase reporter gene constructs revealed that the promoter activity of GNCP-1 was 2-fold greater than that of GNCP-2. Furthermore, DNase I footprint analysis demonstrated that three regions (I, II and III) were protected on the GNCP-1 promoter, whereas only two protected regions (II and III) were identified on the GNCP-2 promoter. Together these observations indicate that GNCP-1 and GNCP-2 are encoded by homologous genes, but the expression of the GNCP-1 and GNCP-2 genes is likely to be different at the level of transcription.
