We investigated the cis-acting sequences that function in the B-cell-specific and interferon-y-inducible expression of the HLA-DRa gene, a human class II major histocompatibility complex gene. The effects of 5' deletions on the activity of the DRa promoter and the influence of upstream DRa promoter elements on the activity of the herpes simplex virus thymidine kinase promoter were examined by a transient transfection assay in human B-, T-, and fibroblast cell lines. We show that the DRa gene is regulated by positive and negative cis-acting sequences between positions -1300 and +31 from the site of initiation of transcription. We also demonstrate that the DRa promoter sequences from positions -116 to -92 and from -136 to -80 are the minimal sequences required for conferring B-cell specificity and interferon-y inducibility upon the Herpes simplex virus thymidine kinase promoter, respectively. (9). These studies strongly suggest that the cis-acting elements involved in the response to IFN-y lie in the upstream promoter region of the class II genes. Sequences from positions -109 to +31 in the DRa were reported to be sufficient for B-cell-specific expression (10). However, DNase I hypersensitivity studies in B cells revealed two additional DNase I hypersensitive sites (9). They were mapped into the first intron of the DRa gene where a lymphoid cell-specific transcriptional enhancer is located. Therefore, B-cell-specific expression of the DRa gene is likely to result from cooperative interactions between the promoter and the intronic transcriptional enhancer.Three conserved upstream sequences (CUS), which are observed in all class II genes and may play an essential role in the transcriptional regulation of these genes, have been described (11,12). In the DQ83 promoter, they are called the W-, X-, and Y-boxes and are located between positions -142 to -127, -113 to -100, and -80 to -67, respectively (13). In the DRa promoter, they are located from positions -135 to -61. We call them the Z-, X-, and Y-boxes. In this study, the function of the CUS in the B-cell-specific and IFN-yinducible expression of the DRa promoter was analyzed. We show here that sequences from positions -136 to +31 are sufficient for the appropriate expression ofthe DRa promoter in human B, T, and IFN-'y-inducible cells. Furthermore, sequences from positions -116 to -92 and from -136 to -80 confer B-cell specificity and IFN-y inducibility upon the Herpes simplex virus thymidine kinase (TK) promoter (14), respectively.
The unusual electron microscopic appearance of a hybrid formed between 9S mouse P-globin mRNA and its corresponding cloned gene segment is caused by at least one, and possibly two, intervening sequences of DNA We have recently cloned a segment of mouse DNA containing a (B-globin gene and its surrounding nucleotide sequences (1). When hybridized to 9S globin mRNA and visualized in the electron microscope, this segment of DNA has an unusual appearance which, together with a paradoxical restriction site pattern, can best be explained by assuming that a large interruption occurs within the globin coding sequence (1, 2). Because the notion that structural genetic information is encoded in a continuous linear fashion seemed most straightforward and reasonable, this interpretation required circumspection. This is so despite the important discovery that some-but not allreiterated 28S ribosomal genes of Drosophila melanogaster contain internal sequences not present in mature 28S ribosomal RNA (3-6) and the more recent and equally important discovery that the 5' or "leader" segments of adenovirus and simian virus 40 (SV40) mRNAs are encoded at sites remote from the structural viral genes (7-12).In this paper we present electron microscopic and restriction endonuclease analyses that indicate that the mouse ,3-globin gene is interrupted within its amino acid coding sequence by an -550-base-pair segment of DNA whose sequence does not appear in mature 9S globin mRNA. To reach this conclusion, we have determined the nucleotide sequence of a portion of the globin gene and the adjacent intervening sequence. * In addition, using the electron microscope, we have observed a second, smaller, looped-out structure close to the 5' end of the globin coding sequence, suggesting the presence of a second intervening sequence and the possibility that the 0-globin gene is encoded in three discontinuous segments of DNA. The fact that structural genes may be encoded in discontinuous segments of DNA provides a new perspective from which to consider such questions as gene regulation and mRNA biosynthesis as well asThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.processes that might be involved in generating diversity in multigene families such as the immune and histocompatibility systems. MATERIALS AND METHODSPreparation of DNA. The plasmid pMB9-#lG2, which contains a segment of mouse DNA originally cloned from the plasmacytoma MOPC-149, was constructed in vitro from recombinant phage XgtWES-/3G2 DNA (1) for 36 hr and used to transform the Ek2 Escherichia coli strain, X1776. Transformed colonies were selected on LB agar plates in the presence of diaminopimelic acid (100 ,g/ml), thymidine (40 mg/ml), and tetracycline (12.5 ,gg/ml). Those containing the 7.0-kilobase (kb) mouse genomic DNA fragment were identified by the Grunstein and Hogness procedure (13) with 32P...
The complexity of cotranscriptional splicing is reflected in the coordinated interplay between various cis-elements and transacting factors. In this report, we demonstrated that a cis-element in intron 1 of the equine b-casein gene (intronic splicing enhancer 1, ISE1) increases the inclusion of all weak exons in its pre-mRNA. The ISE1 also functioned on a hybrid transcript, which was transcribed from the a-globin promoter, where it increased the inclusion of the human fibronectin EDA exon and the b-casein exon 5. The region of ISE1 necessary for its function included the same sequence as is found in some exonic splicing enhancers. Since the ISE1 influenced the splicing of the entire transcript from intron 1, we propose a model for the cotranscriptional splicing of b-casein mRNA, where the 5¢ end of the growing transcript remains associated with the C-terminal domain of RNA polymerase II. Thus, the ISE1 remains in close proximity to the mRNA exit groove throughout transcription and influences all weak exons as soon as they are copied.
We mapped cis-acting regulatory elements in the HLA-DR alpha gene, which encodes the monomorphic subunit of the HLA-DR heterodimer. Genomic fragments of HLA-DR alpha were placed 5' or 3' to the chloramphenicol acetyltransferase reporter gene, the transcription of which was initiated from the Herpes simplex thymidine kinase promoter. In transient expression assays, fragments from the body of the HLA-DR alpha gene were able to increase chloramphenicol acetyltransferase activity in a position-, orientation-, and promoter-independent yet tissue-specific fashion. These HLA-DR alpha cis-acting regulatory elements contain previously identified DNase I-hypersensitive sites and DNA sequences homologous to those found in other eukaryotic transcriptional enhancers.
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