Molecular structure of the human albumin gene is revealed by nucleotide sequence within q11-22 of chromosome 4

The human albumin gene spans 16,961 nucleotides from the putative "Cap" site to the first poly(A) addition site. It is split into 15 exons by 14 intervening sequences which are symmetrically placed within the three domains of albumin. The 5' region is highly conserved up to position -250 and contains the putative TATA (-32) and CAT (-88) boxes. A consensus 5' splice sequence reads /GTAGAGT while the 3' splice sequence is pyrimidine rich and contains CTAG/ at the splice junction. The gene contains three polyadenylation signals, and this 3' region presumably arose by triplication of a shorter fragment prior to mammalian radiation. The albumin gene exhibits a high degree of DNA polymorphism and appears to have been recently invaded by Alu repetitive sequences.     

Identification of the promoter sequences involved in the cell specific expression of the rat somatostatin gene.

DNA sequences containing the 5' flanking region of the rat somatostatin gene were linked to the coding sequence of the bacterial chloramphenicol acetyl transferase gene. This recombinant plasmid is active in expressing CAT activity in the neuronally derived, somatostatin producing CA-77 cell line. Deletion analyses of the somatostatin promoter show that the sequences proximal to position -60, relative to the cap site are required for expression of this promoter. A 4 base pair deletion of residues -46 through -43 within the somatostatin promoter results in a down mutation in vivo suggesting the existence of an element critical for the expression of the promoter in CA-77 cells. In addition, the somatostatin recombinant and its 5' deletion constructs preferentially express CAT activity in CA-77 cells, whereas only basal level of expression is observed in HeLa, BSC40, and RIN-5F cell lines, pointing to the cell specific nature of this promoter.     

Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs.