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P-450NF is the major enzyme in human liver involved in the metabolism of the calcium-channel blocker nifedipine. By screening a bacteriophage lambda gt11 expression library, a cDNA clone designated NF 10 with an insert length of 2.8 kilobases (kb) was isolated. This clone was sequenced and found to be highly similar in its overlapping section with sequences of two other cDNA clones previously isolated from the same expression library, NF 25 (Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S., and Guengerich, F. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8064-8068) and HLp (Molowa, D. T., Schuetz, E. G., Wrighton, S. A., Watkins, P. B., Kremers, P., Mendez-Picon, G., Parker, G. A., and Guzelian, P. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5311-5315). However, clone NF 10 had an extra 814 or 813 bases of 3'-noncoding sequence relative to NF 25 or HLp, respectively, and this additional sequence contained a second consensus polyadenylation signal. Specific oligonucleotides were synthesized to differentiate between these three clones at the mRNA level. Oligonucleotides specific to the protein coding region of each clone were found to hybridize to mRNAs of 2.2 and 3.0 kb in size at a ratio of approximately 10:1. The major species of hybridizable mRNA was specific to clone NF 25, and levels of this mRNA could be correlated with levels of immunochemically detectable P-450NF and nifedipine oxidase activity in individual human liver samples. In addition, an oligonucleotide specific to the 3'-noncoding region of clone NF 10 hybridized only with the 3.0-kb mRNA. We conclude that alternative use of the second polyadenylation signal present in clone NF 10 results in production of the 3.0-kb mRNA species and that a pretranslational control mechanism is primarily involved in the regulation of nifedipine oxidase activity.
The 5'-end of the bovine adrenodoxin gene contains unique structural characteristics. Exons 1 and 2 appear to encode different presequences for this mitochondrial precursor protein. However, exon 1 contains a stop codon (TAA) in place of amino acid 15. Thus, while two species of bovine adrenodoxin mRNA arise from a single gene, only the abundant mRNA species (90%) is translated into the adrenodoxin precursor which contains the presequence encoded by exon 2. The minor mRNA species (10%) contains the sequence encoded by exon 1 and cannot be translated into an adrenodoxin precursor. Presumably the sequence within exon 2 is removed from this minor mRNA species by alternative splicing. Furthermore, the initiation of transcription of the major adrenodoxin mRNA (exon 2) lies within intron 1 of this unusual gene, while that for the other mRNA (exon 1) lies in the 5'-flanking region. Thus, the adrenodoxin gene is the first example of a gene encoding a mitochondrial protein which falls into the category of genes having alternate promoters utilizing a pattern of alternative splicing.
Bovine adrenodoxin mRNA is found to consist of several distinct mRNA species which can be divided into two sets. Each set utilizes at least three of four separate poly(A)+ addition sites providing an explanation of the three sizes of adrenodoxin mRNA (1.75, 1.4, and 0.95 kilobases) previously observed in bovine adrenocortical RNA by this laboratory (Okamura, T., John, M.E., Zuber, M.X., Simpson, E.R., and Waterman, M.R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5705-5709). The two sets are distinguished from one another by a unique 5' sequence leading to two different amino acid sequences (approximately 10% homology) for the precursor portion of this nuclear encoded, mitochondrial protein. A common mature adrenodoxin sequence is encoded by both sets of mRNA. One set of RNAs is 10-fold more abundant than the other, but the levels of both sets can be induced by treatment of primary bovine adrenocortical cell cultures with adrenocorticotropin. The biological significance of these two types of adrenodoxin precursor sequences remains obscure.
Time-dependent and cell-specific changes in concentrations of total RNA, of poly A+ RNA, and of specific mRNAs have been measured throughout silkmoth choriogenesis. Levels of total RNA and of poly A+ RNA are maintained throughout much of choriogenesis, but decrease at least fourfold during the very late period, in parallel with a decrease in overall protein synthesis. Very late period changes in total RNA and in poly A+ RNA are less pronounced in the aeropyle crown region, where a subset of chorion proteins is preferentially synthesized, than in the flat region. Maximal accumulation of the E1 and E2 chorion mRNAs occurs preferentially in the aeropyle crown region during the very late period. Uridine pulse-labeling studies suggest that E1 and E2 transcription is similarly aeropyle crown region-specific and immediately precedes the time of maximal E1 and E2 RNA accumulation and protein synthesis. Sequences from the 5' flanking regions of E1 and E2 genes have been compared. Several oligonucleotide sequences are present in both genes, and some are duplicated. These are potential cis-acting, regulatory elements.
Rat liver malic enzyme (ME) synthesis is known to be regulated by 3,5,3'-triiodo-L-thyronine (T3). Hybridization of 32P-labeled ME cDNA with RNA extracted from normal and T3-induced livers (15 or 50 micrograms/100 g body weight for 10 days) showed an increase in the ME mRNA concentration by approximately 11-fold in T3-treated rats. ME activity and ME mass were stimulated to the same degree as ME mRNA. Northern blot analysis of either total or poly(A+) RNA revealed two distinct ME mRNAs (21 and 27 S) which were equally induced by T3 treatment. Both mRNAs were shown by in vitro translation assay to program the synthesis of the same immunoprecipitable protein corresponding to full-sized ME. From all the above, we concluded that both messages code for active enzyme. ME activity and ME mRNA were also detected in nonhepatic tissues for which different responses to T3 induction were observed without direct correlation with their respective content of T3 nuclear receptor. Increases in ME activity and level of hybridizable ME mRNA were seen 48 h after a single administration of T3 (200 micrograms/100 g body weight) in liver, kidney, and heart (10.3- and 15.5-, 1.7- and 2.6-, and 1.72- and 3.4-fold above basal values, respectively). Lower levels of induction could already be detected after 24 h, liver being the most stimulated tissue. ME was not affected in brain, lung, testis, and spleen. Northern blot analysis showed that both ME mRNAs are present in all tissues tested, although in different relative proportions.(ABSTRACT TRUNCATED AT 250 WORDS)
The integrin alpha 6 beta 4 is a heterodimer predominantly expressed by epithelia. While no definite receptor function has yet been assigned to it, this integrin may mediate adhesive and/or migratory functions of epithelial cells. We have determined the complete primary structure of both the alpha 6 and beta 4 subunits from cDNA clones isolated from pancreatic carcinoma cell line libraries. The deduced amino acid sequence of alpha 6 is homologous to other integrin alpha chains (18-26% identity). Antibodies to an alpha 6 carboxy terminus peptide immunoprecipitated alpha 6 beta 4 complexes from carcinoma cells and alpha 6 beta 1 complexes from platelets, providing further evidence for the association of alpha 6 with more than one beta subunit. The deduced amino acid sequence of beta 4 predicts an extracellular portion homologous to other integrin beta chains, and a unique cytoplasmic domain comprised of greater than 1,000 residues. This agrees with the structures of the beta 4 cDNAs from normal epithelial cells (Suzuki, S., and Y. Naitoh. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:757-763; Hogervost, F., I. Kuikman, A. E. G. Kr. von dem Borne, and A. Sonnenberg. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:765-770). Compared to these structures, however, the beta 4 cDNAs that we have cloned from carcinoma cells contain extra sequences. One of these is located in the 5'-untranslated region, and may encode regulatory sequences. Another specifies a segment of 70 amino acids in the cytoplasmic tail. Amplification by reverse transcription-polymerase chain reaction of mRNA indicated that multiple forms of beta 4 may exist, possibly due to cell-type specific alternative splicing. The unique structure of beta 4 suggests its involvement in novel cytoskeletal interactions. Consistent with this possibility, alpha 6 beta 4 is mostly concentrated on the basal surface of epithelial cells, but does not colocalize with components of adhesion plaques.
A retrovirus promoter-trap vector (U3LacZ) has been developed in which Escherichia coli lacZ coding sequences were inserted into the 3' long terminal repeat (LTR) of an enhancerless Moloney murine leukemia virus. The U3LacZ virus contains the longest reported LTR (3.4 kbp); nevertheless, lacZ sequences did not interfere with the ability of the virus to transduce a neomycin resistance gene expressed from an internal promoter. Duplication of the LTR placed lacZ sequences in the 5' LTR just 30 nucleotides from the flanking cellular DNA. Approximately 0.4% of integrated proviruses expressed beta-galactosidase as judged by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, and individual clones expressing lacZ were isolated by fluorescence-activated cell sorting. In all clones examined, beta-galactosidase expression resulted from the fusion of lacZ sequences to transcriptional promoters located in the flanking cellular DNA. Furthermore, by differential sorting of neomycin-resistant cell populations, clones were isolated in which lacZ expression was induced and repressed in growth-arrested and log phase cells, respectively.
NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase catalyzes the oxidation of many prostaglandins at C-15, resulting in a subsequent reduction in their biological activity. We report the isolation of the cDNA for this enzyme. A human placental lambda gt11 cDNA library was screened using polyclonal antibodies prepared against the human placental enzyme. A 2.5-kilobase cDNA containing the entire coding region for the enzyme was isolated. The cDNA encodes for a protein of 266 amino acids with a calculated Mr of 28,975. Identification of the cDNA as that coding for 15-hydroxyprostaglandin dehydrogenase was based on the comparison of the deduced amino acid sequence with the amino acid sequence of two peptides, one from the rabbit lung enzyme and the other from the human placental enzyme. This cDNA hybridizes with two species of poly(A+) RNA isolated from human placenta: one of 3.4 kilobases and the other of 2.0 kilobases. Isolation of the cDNA for 15-hydroxyprostaglandin dehydrogenase should facilitate studies on the structure, function, and regulation of this enzyme.
Mouse embryo-derived AKR-2B fibroblasts and murine fibrosarcoma cells (the 1591 cell line) were transfected with a murine transforming growth factor-beta 1 (TGF beta 1) cDNA under the transcriptional control of either the simian virus-40 early promoter or the cytomegalovirus promoter/enhancer. Selected clones secreted 2- to 4-fold more TGF beta-competing activity into their media than the parental cell line or neomycin-transfected controls. The TGF beta 1 released into the cell-conditioned medium was latent. Despite the latency of the overexpressed TGF beta 1, TGF beta 1-transfected cells exhibited phenotypic features of TGF beta 1-treated cells. When confluent, the TGF beta 1-transfected cells had the morphological characteristics of the parental cells that have been treated with active TGF beta 1. AKR-2B cells that expressed higher levels of TGF beta 1 also expressed high levels of c-sis and c-myc mRNAs and decreased TGF beta 2 and TGF beta 3 mRNAs in the same manner as parental AKR-2B cells that had been treated with active TGF beta 1. The transfected 1591 cells that overexpressed TGF beta 1 bound less [125I]TGF beta 1 than did parental 1591 cells, but after a mild acid wash demonstrated an increase in [125I]TGF beta 1 binding. Our results suggest that these TGF beta 1-transfected fibroblast and fibrosarcoma cells have the capacity to activate TGF beta; however, as very little activated TGF beta is detected in the medium, it is hypothesized that these cells activate latent TGF beta 1 and bind the activated TGF beta 1, thus acquiring a phenotype consistent with TGF beta 1-treated cells.
Renal basement membranes are believed to contain five distinct type IV collagens. An understanding of the specific roles of these collagens and the specificities of their interactions will be aided by knowledge of their comparative structures. Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been cloned and the deduced peptide sequences compared. A fifth chain, alpha 4(IV), has been identified in glomerular and other basement membranes. Using a polymerase chain reaction-based strategy and short known peptide sequences from the noncollagenous domain (NC1), we have cloned and characterized partial bovine cDNAs of alpha 4(IV). Sequence analysis shows that this molecule has characteristic features of type IV collagens including an NH2-terminal Gly-X-Y domain which is interrupted at several points and a COOH-terminal NC1 domain with 12 cysteine residues in positions identical to those of other type IV collagens. Within the NC1 domain bovine alpha 4(IV) has 70, 59, 58, and 53% amino acid identity with human alpha 2(IV), alpha 1(IV), alpha 5(IV), and alpha 3(IV), respectively. Alignment of the peptides also shows that alpha 4(IV) is most closely related to alpha 2(IV). Nevertheless, in the extreme COOH-terminal region of the NC1 domain there are structural features that are unique to alpha 4(IV). Cloning of the region of alpha 4(IV) that encodes the NC1 domain allows comparison of all five type IV collagens and highlights certain regions that are likely to be important in the specificities of NC1-NC1 interactions and in other discriminant functions of these molecules.