The multiple untranslated first exons and promoters system of the oestrogen receptor gene in the brain and peripheral tissues of the rat and monkey and the developing rat cerebral cortex

Recent studies on the human oestrogen receptor (ER) gene have revealed the complex system with the multiple untranslated first exons and promoters in the ER gene expression. Little information is however available on the system in the ER gene of the rat or nonhuman primate. The rat genomic library was first screened by the rat ER cDNA (0–1) probe. One of the four positive clones (λ rEgEl) was subcloned and sequenced. The nucleotide sequence was found to contain the exon 0, the intron 0, and the exon 1 with its 3′-ends. The novel untranslated first exons, the exon ON and the exon OS, were further identified. These results indicated the presence of at least four subtypes of the rat ER mRNAs; the messages transcribed from promoter P-0 (ER mRNA (0–1)), putative promoter P-1 (ER mRNA (1–1)), promoter P-ON (ER mRNA (ON-1)) and promoter P-OS (ER mRNA (OS-1)). The P-O- or P-1 driven message (0–1) or (1–1) appeared to be expressed most strongly in major oestrogen central- (anterior pituitary, AP, hypothalamus–preoptic area, HPOA, and amygdala, AMG) and peripheral targets (uterus and ovary). The message (ON-1) was strongly expressed in the liver and kidney, but not in the HPOA, AMG, cerebral cortex, CC, and cerebellum, Ce. The OS-1 message was expressed variably but generally in the tissues examined except for the CC and Ce. Thus, the region- and tissue specific expression of the rat ER gene is likely to be regulated by the multiple untranslated exons and promoters system. Furthermore, when the ER mRNA subtypes were examined in the rat neonatal CC where the ER protein level rose transiently, considered as a model for the development of the ER or progestin receptor A and B isoforms, the expression of the ER mRNAs seemed to be differential postnatally, implicating some stage dependent usage of the promoters in the development. In the monkey, we identified the untranslated first exon OS, the homologue of the rat exon OS. Interestingly, the exon C was found to consist of two different exons, the exon OK and the exon OG. By the alternative usage of the promoters and the alternative splicing, at least six ER mRNA subtypes, that is, ER mRNAs (0–1), (1–1), (OS-1), (OS-OG-1), (OK-1) and (OK-OG-1) were identified in the monkey tissues. These messages were also differentially distributed in the monkey brain and other tissues. It was noteworthy that the P-OK driven messages were expressed almost exclusively in the monkey liver. These results have suggested that the systems of the multiple untranslated first exons and promoters and the alternative splicing are involved in the regulation of the region- and tissue specific expression of the ER gene in the brain and peripheral tissues of the rat and monkey. Stage-related usage of the promoters was also suggested in the ER gene expression in the CC of the postnatal rat in development.     

Cloning and characterization of a third type of human α-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human α-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229–235; Tomita et al., Gene 76 (1989) 11–18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human α-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively.Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197–1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5′ region; hence the promoter lies far upstream relative to the other two AMY genes.     

Cloning and characterization of a novel upstream untranslated exon of the mouse Fgf-1 gene

Fibroblast growth factor 1 (FGF-1 or aFGF), is the prototype member of the heparin-binding growth factors which are capable of angiogenesis in vivo. FGF-1 has been implicated in atherosclerosis, cancer, wound repair and inflammatory autoimmune diseases. As part of an effort to understand the role of FGF-1 in the etiopathogenesis of inflammation and cancer, we have undertaken steps to isolate and characterize the mouse Fgf-1 gene. Southern blotting and sequence analysis displayed considerable conservation within the coding and upstream untranslated regions of Fgf-1 in human, mouse, hamster, rat and bovine. By using primers derived from the 5′-untranslated exon of a rat prostate-specific Fgf-1 cDNA, a 220-bp product was amplified from mouse genomic DNA via PCR. Sequence analysis of this amplicon showed that there was 80% similarity with the corresponding region of the rat FGF-cDNA sequence. Primers designed from this amplicon and the Fgf-1 coding region were used to isolate multiple overlapping genomic clones spanning the entire mouse Fgf-1 gene. Sequencing analysis of the genomic sequence upstream from this novel 5'-untranslated exon did not reveal typical TATA, CCAAT sequences. It appears that the occurrence of multiple untranslated exons for FGF-1 is a highly conserved theme for this gene across species.     

A novel snoRNA (U73) is encoded within the introns of the human and mouse ribosomal protein S3a genes

The mouse ribosomal protein S3a-encoding gene (mRPS3a) was cloned and sequenced in this study. mRPS3a shares identical exon/intron structure with its human counterpart. Both genes are split to six exons and exhibit remarkable conservation of the promoter region (68.8% identity in the 250 bp upstream of cap site) and coding region (the proteins differ in two amino acids). mRPS3a displays many features common to other r-protein genes, including the CpG-island at 5′-end of the gene, cap site within an oligopyrimidine tract and no consensus TATA or CAAT boxes. However, mRPS3a represents a rare subclass of r-protein genes that possess a long coding sequence in the first exon. Comparison of human and mouse S3a genes revealed sequence fragments with striking similarity within introns 3 and 4. Here we demonstrate that these sequences encode for a novel small nucleolar RNA (snoRNA) designated U73. U73 contains C, D and D′ boxes and a 12-nucleotide antisense complementarity to the 28S ribosomal RNA. These features place U73 into the family of intron-encoded antisense snoRNAs that guide site-specific 2′-O-ribose methylation of pre-rRNA. We propose that U73 is involved in methylation of the G1739 residue of the human 28S rRNA. In addition, we present the mapping of human ribosomal protein S3a gene (hRPS3a) and internally nested U73 gene to the human chromosome 4q31.2–3.