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In the +27.6 to +36.7 kb downstream region from the transcriptional start site of the rat growth hormone (GH) gene, a gene encoding BAF60b, a component of mammalian SWI/SNF complexes, was found to have the same transcriptional orientation as the GH gene. The 5' end of the BAF60b gene was heterogeneous and the longest gene was 9060 bp long with 13 exons. The largest of all exons was estimated to be 2774 bases. Deduced rat BAF60b protein was made of 531 amino acids and its amino acid sequence was 97% identical with the human counterpart. No TATA box was found up to the -100 bp region but five GC boxes corresponding to the Sp1 binding site were observed up to 640 bp upstream from the transcriptional start site. Sixty-three bases downstream from the BAF60b gene, the polyadenylation site of the gene encoding transcription factor SUG/proteasome p45, whose expression is constant in many tissues, was identified. The BAF60b gene was expressed as 3.0 kb poly(A)-rich RNA in seven tissues and one cell line from rat but its expression varied considerably according to the tissue.

Previously, we used homologous recombination to delete six groups of cell-type-specific DNase I hypersensitive sites (DHSs), potential transcriptional and epigenetic regulators, scattered in and around the Ig-β gene from their natural context in B-lymphocyte-derived chicken DT40 cells. Simultaneous deletion of all six groups completely shut down transcription and epigenetic regulation of the Ig-β gene; therefore, the cooperation of the scattered regulatory regions was essential for transcription and epigenetic regulation. In this study, we regrouped the cell-type-specific DHSs of Ig-β, those in the original six deletions and three additional ones, into three larger regional groups-the long upstream region, the intron, and the long downstream region-and deleted these groups individually or in combination. Combinatorial deletion of all three regional groups decreased Ig-β mRNA levels to 0.4% of the control, which was significantly higher than

DNase I hypersensitive sites (DHSs) and histone acetylation status were examined in the Ig-beta locus of chicken B lymphocyte-derived DT40 cells and liver-derived LMH cells. Twelve DT40-specific DHSs were identified: one in the Ig-beta promoter, one in the first intron of the Ig-beta gene, three in the sodium channel gene located upstream of the Ig-beta gene, two between the sodium channel gene and the Ig-beta gene, four between the Ig-beta gene and a downstream growth hormone (GH) gene, and one in the downstream region of the GH gene. Transient transfection studies show that the DHS in the intron of Ig-beta gene enhances the activity of the Ig-beta promoter fourfold. A 1.6 kb DNA fragment, which includes two DHSs, from the sodium channel gene enhanced promoter activity threefold. The transcription enhancing ability of the intron DHS was dependent on orientation, but was not promoter specific. Electrophoretic mobility shift assays (EMSA) demonstrated that an Ets protein family member binds to the intron DHS. In DT40 cells, a distinguished acetylation of H3 and H4 histones was found at the Ig-beta promoter, in addition to the enhanced acetylation of both histones at DT40-specific DHSs.

Using avian B lymphoma-derived DT40 cells, we disrupted a gene encoding the transcription factor PU.1. The mutant mRNA codes for a protein incapable of functioning as a transcription factor because of the deletion of the protein's DNA-binding domain. The absence of a functional PU.1 protein in the mutant cells was confirmed by Western blotting and electrophoretic mobility shift assay, thereby demonstrating that PU.1 was not essential for the proliferation of DT40 cells. An examination of the expression of several genes known to be PU.1 protein targets revealed almost the same levels of Ig-beta and Ig lambda L chain mRNA in mutant cells as in wild-type cells, indicating that the PU.1 protein plays no essential role in the transcription of these genes. Mutant cell doubling times 1.3 times longer than those of wild-type cells confirmed the PU.1 protein to be involved in the proliferation of B lymphocytes.

In the +9.7 to +21.7 kb downstream region from the transcriptional start site of the rat growth hormone (GH) gene, a gene specifically expressed in the testis was found to have reverse transcriptional orientation to the GH gene. Its exon comprised 2693 bases encoded a protein having 548 amino acids (60 479 Da). The amino acid sequence of the testis-specific protein resembled that of the intercellular adhesion molecules (ICAM) 1 and 3. The gene was thus given the name testicular adhesion molecule (TCAM) 1 gene. The TCAM-1 gene was found to be 12 041 bases with eight exons. Although exon 1 was noncoding, the remaining seven exons corresponded to the domains coding for the signal sequence, five immunoglobulin (Ig) domains, and the transmembrane plus cytoplasmic domain. The organization of TCAM-1 gene was shown to be the same as that of the ICAM-1 gene. The polyadenylation site of TCAM-1 gene was located 7.6 kb downstream of that of the GH gene, whereas the 5′ end of TCAM-1 gene was separated 5.9 kb from that of the gene coding for BAF60b, a component of SWI/SNF complexes known as the chromatin remodeling factor. Six genes were thus mapped in the following order in the 88 kb region of the rat GH locus: Na-channel (5′ to 3′)–B29/Ig-β (5′ to 3′)–GH (5′ to 3′)–TCAM-1 (3′ to 5′)–BAF60b (5′ to 3′)–SUG/p45 (3′ to 5′).

The promoter regions of the genes encoding the rat and chum salmon growth hormones (GH) and rat prolactin (PRL) were combined with a reporter gene and introduced into GH- and/or PRL-producing cells from rat. The rat GH and PRL promoters (pGH and pPRL, respectively) were most active in cells producing GH and PRL, respectively. The activity of the salmon pGH was much less than that of the rat pGH in rat GH-producing cells. The regulatory region required for cell-type-specific gene expression of pituitary hormones thus contains information, not only for cell-type specificity, but possibly for species specificity as well. A reporter plasmid containing the GH or somatolactin (SL) promoter and an effector plasmid having a gene encoding transcription factor Pit-1 (rat or salmon) were cotransfected into HeLa (human) or EPC (carp) cells. Rat and salmon Pit-1 were more active in HeLa and EPC cells, respectively, indicating that Pit-1 appears to interact species specifically with the transcription machinery.

Pit-1/GHF-1, a POU family transcription factor, was originally isolated as a protein that contributes to the pituitary-specific expression of the mammalian growth hormone(GH)-encoding gene. Pit-1/GHF-1 has been recently implicated in the development of three types of hormone-producing cells which secrete GH, prolactin (PRL) and thyroid-stimulating hormone. A presumed pituitary hormone, somatolactin (SL) which belongs to the GH/PRL family, was previously isolated from fish. As the first step for determining the possible involvement of the Pit-1 protein in the pituitary-specific expression of the SL gene and/or in the development of SL-producing cells, chum salmon Pit-1 cDNA clones were isolated. Chum salmon Pit-1 mRNAs were mainly 2 kb and 3 kb in size, and specifically expressed in the pituitaries. They encoded a 365-amino acid (aa) protein, which was 74 aa larger than that found in mammals. The identity of aa sequences between salmon and rat Pit-1 was 69%. These proteins were highly conserved in the region specific for the POU family. Salmon Pit-1 had two additional sequences each consisting of about 30 aa in the N-terminal region. The sequence involved in transcriptional activation is considered to be located in the region including the above sequences, and thus, salmon Pit-1 may possibly have novel functions distinct from those of rat Pit-1.

Two MCH genes coding for melanin-concentrating hormone (MCH) were isolated from a chum salmon liver DNA library and characterized. They were shown to be intronless genes with 0.63-kb exons, each of which commonly consisted of an about 80-bp 5'-untranslated region, a region coding for 132 amino acids (aa) MCH precursor protein and an approx. 160-bp 3'-untranslated region. About 20 bp upstream from the putative cap site, sequences were found corresponding to the TATA box. The two genes were 86% identical at the nucleotide sequence level. Sequences homologous to the chum salmon MCH genes were present in the genomes of other fish such as catfish, carp and Chinese grass carp, whereas no highly homologous sequence could be detected in other vertebrate genomes.