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Nucleic Acids Research Volume 16 Number 20 1988 Nucleic Acids Research Sigma region located between C,, and C6 genes of human immunoglobulin heavy chain: possible involvement of tRNA-like structure in RNA splicing Yasushi Akahori, Hiroshi Handal, Kenji Imai, Masumi Abe, Kohzoh Kameyama, Makoto Hibiya, Hisashi Yasui, Kazuhiko Okamura, Morihiro Naito, Hiroshi Matsuoka2 and Yoshikazu Kurosawa Institute for Comprehensive Medical Science, Fujita-Gakuen Health University, Toyoake, Aichi 470-11, 'Department of Bacteriology, Tokyo University School of Medicine, Hongo, Bunkyo-ku, Tokyo 103 and 2Department of Pediatrics, Nagoya University School of Medicine, Tsurumai, Showa-ku, Nagoya 466, Japan Received August 2, 1988; Revised and Accepted September 19, 1988 Accession nos X12842, X12843 ABSTRACT Noncoding regions within the cluster of immunoglobulin heavy chain constant genes in the human genome contained a number of repeats. In the P-6 intron, two repeating units were contained. One 442-base-long fragment located JH- intron ( defined as "sigma i(op)") occupied the position in the p-5 intron. The other 1166-base-long fragment located somewhere in front of S (class switch) region of Cy gene was also found in the p-6 intron. We defined the repeats in the p-6 intron as "SIGMA (E)". The polaxities of the longer repeats in the genome were opposite between the p-6 intron and the upstreams of Cy genes. These inverted copies (deiined as Gy3 and 0y4), located 6 kb upstream of their respective Cy's, were apparently transcribed in vitro, via RNA polymerase III and transcripts should have contained tRNA-like structures. Small DNA fragments capable of encoding tRNA-like structures were also found in corresponding regions of mouse Ig Cy cluster. INTRODUCTION Immunoglobulin (Ig) heavy (H) chain genes on human chromosome 14 are organized into the following gene clusters: VH, DH1 JH and constant(CH) (For a review see ref.1). The order of the CH genes is p-6-y3-yl-*P-al- *y-y2-y4-c-a2 (2, 3, 4). Characteristic repetitive sequences referred to as class switch(S) sequences (5) exist upstream of each CH gene; only exception being C . During B cell ontogeny, these genes undergo two kinds of DNA rearrangements: DH-JH and then VH-DH joinings (For a review see ref.6 ), resulting in the formation of a complete H chain gene; subsequently, p-class H chain gene undergoing class switch via recombination between two S sequences; e.g., S of p and S of y1 (7). Both rearrangements usually involve the loss of intervening sequences (7, 8). However, aside from the classical switch via S-S recombination, there have been reports of B cells expressing two isotypes such as IgM and IgG (9). In these instances, DNA rearrangements have not been detected in CH gene loci. Accordingly, simultaneous expression of two different © JR L Press Limited, Oxford, England. 9497 Nucleic Acids Research isotypes is thought to be mediated by alternative RNA splicing from an extreamly long primary transcript (10). While trying to find new DH gene families utilizing a JH-containing fragment as a probe, we identified an unexpected DNA segment on human germline genome. Apparently, a copy of the DNA segment located between the enhancer (11) and the S sequence (12) upstream of C gene has become inserted into the p-6 intron. Moreover, one more DNA fragment seemingly derived from somewhere upstream of one of the C genes was embedded in the p-6 intron. We determined the nucleotide sequences upstream of C 3 and Cy4 genes. By comparison of nucleotide sequences of JH- introns, i-6 introns and upstream of y genes between human and mouse, we suggest a possible mechanism of alternative splicing of primary transcripts for expression of two isotypes. We also identified a promoter activity for transcription by RNA polymerase III upstream of constant y genes, and discuss the possibility of discontinuous transcription followed by trans- RNA splicing. MATERIALS AND METHODS Clones CH4-38 and CH4-51 containing human C gene (13), were provided by P.Leder ( Harvard Medical School ). Clones 5A and 5D containing human C 2 and C 4 (14), respectively, were provided by L.Hood ( Cal Tech ). Clone MEP12 containing mouse y2b-y2a intron (15) was donated by S.Tonegawa ( MIT ). To prepare a human C 1 probe, the 6 kb HindIII fragment containing C 1 gene (16) was isolated with mouse C 1 gene as a probe. The human genomic library (17) was donated by T.Maniatis ( Harvard University ). Clone ARAJH1 was isolated by the ordinary cloning procedure as described by Sakano et al. (18). Clones 1, 2, 3 and 6 were isolated by screening the Maniatis' human genomic library with ARAJH1 probe according to Benton-Davis' method (19). Nucleotide sequence was determined by the chain termination technique of Sanger et al. (20), using Bluescript M13 vectors ( STRATAGENE ). Southern hybridization was carried out by the published procedure (21). A 66 mer: 5'-CACACATGGCAGTTTGAGCAGCAGAACTCTGTTCTTTCCCAGTTCTAGAGGCCAGAAGTGTGGGCC-3', which corresponds to 3589-3654 in Figure 2 of the paper by Richards et al.(22), was chemically synthesized by DNA synthesizer ( Applied Biosystems Inc.). In vitro transcription mixture was prepared from Hela cells according to the procedure described by Dignam et al. (23). As DNA 9498 Nucleic Acids Research were templates, the EcoRI-HindIII fragments containing ay3 and a 4 recloned in pUC9, resulting in pSG3 and pSG4, respectively. RESULTS AND DISCUSSION Identification of Sigma(E) region With JH gene-containing probe ( JH probe ) as shown in Figure la, we identified a very faint band at 3.8 kb, in addition to the germline band I_____IEE:- a v ~v %V IV I - v - (J'H - probe) (probe 1) (probe 2) (probe 3) v V Clone 3 VW v la IV I I1 gv _ Clone 6 ~~~-Umimd~ ARAJH 1 CYS3 t b s v v I*l v St |C3C 1 v I a vv V*vv 11111111111 Clone 2 °14 I SF21 I C,2 I 1*1 I SF4 I-C4 (probe 4) ikb i 1 v Clone I Figure 1. Physical maps of the human immunoglobulin heavy chain constant gene loci and the clones (thick line) isolated in this study : ARAJH1, clones 1, 2, 3, 6. Details of cloning process are described in the text. In brief, ARAJH1 was first isolated by homology to J probe. Clones 1, 2, 3 and 6 were isolated from Maniatis' human phage Yibrary ( 17 ) with thte 3.8 kb insert of ARAJH1 as the probe. Clone 3 was a JH gene- containing clone. Clone 6 covered from the downstream of J gene cluster to 5' end of C6 genes. Clone 2 covered the upstream region of Cy3 gene. Clone 1 covered from Cy2 to the upstream of C 4 gene. aY ' ay4 and their homologous region in E are indicated by box with thick black arrows, and oy and its homologous region in £ by black box with thick white arrows. Direction of arrows indicates sequence polarity. Probes used in this study are indicated. The EcoRI-HindIII fragments containing CYY3 and 0Y4 indicated by horizontal double arrows were sequenced in this study. Restriction enzymes : Y HindIII, V EcoRI, + BamHl. Restriction sites of regions are not covered by the above clones are referred from published papers ( 2, 13, 14 ). 9499 Nucleic Acids Research at 10 kb in HindIII-digested placenta DNA, cloned the 3.8 kb band, and named it ARAJH1. In Southern hybridization of HindIII-digested placenta DNA with the 3.8 kb DNA of ARAJH1 as the probe, six bands: 10 ( double ), 8, 4.5 and 3.8 kb ( double ) were detected ( date not shown ). We screened a human DNA phage library provided by T.Maniatis (17) using the 3.8 kb DNA as the probe. Restriction maps of the four kinds of clones obtained are shown in Figure 1. The 3.8 kb-containing clone was named clone 6, and a JH gene-containing clone was named clone 3. The other two clones, 1 and 2, are different. Homologous regions to the 3.8 kb probe in clones 1 and 2 were mapped by Southern hybridization as shown in Figure lbc. Clones 1 and 2 contained, respectively, 10 and 4.5 kb HindIII fragments detected by the probe. The size of the HindIII fragment containing JH region was also 10 kb. We compared the restriction maps of clones 1, 2 and 6 with the published maps of Ig CH gene loci (2, 13, 14) and noticed that the maps of clones 1, 2 and 6 almost the same as those of the upstream regions of C 4 and C 3 genes and p-6 intron, respectively. Comparison of restriction maps among clones 6, CH4-38 and CH4-51 (13) indicated that they have overlapping regions ( data not shown ). The complete nucleotide sequence of the region from p to 6 was published by Milstein et al. (24). Strikingly, the 442-nucleotide-long sequence in p-6 intron ( from 6387 to 6828 in Figure 2 of the 1984 paper by Milstein et al.(24)) was 97% homologous to the downstream of JH gene ( from 661 to 1102 in Figure 3 of the 1983 paper by Mills e- al.(25)). Independently, we determined the nucleotide sequence of the relevant region in clone 6, with the same results. Comparison among clones 1, 5A and 5D (14), and Southern hybridization of clones 1 and 2 DNAs with human C 1 probe indicated that clones 1 and 2 were located upstream of C 4 and C 3 genes, respectively ( data not shown ). Figure 1 shows the location of clones 1, 2, 3 and 6 in the CH chain gene loci and their physical maps.
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