Acetyl-CoA carboxylase mRNA species with or without inhibitory coding sequence for Ser-1200 phosphorylation

Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in the biogenesis of long chain fatty acids. The phosphorylation of the Ser-1200 residue by cyclic AMP-dependent protein kinase transforms ACC from a citrate-independent form to a citrate-dependent form (10, 16). We have isolated ACC cDNA clones with and without 24 bases which code for 8 additional amino acids located 4 residues upstream to the Ser-1200. The presence of the 8 extra amino acids inhibits the in vitro phosphorylation of the Ser-1200 by the catalytic subunit of cyclic AMP-dependent protein kinase. The S1 nuclease protection experiments indicate that the corresponding two ACC mRNA species occur in vivo. Furthermore, the occurrence of the two forms of ACC mRNA is regulated under different physiological conditions for lipogenesis in a tissue-specific manner. The existence of two forms of ACC mRNA provides the basis for the existence of isozymes of ACC whose Ser-1200 can be selectively phosphorylated. The location of this regulatory sequence for a specific phosphorylation site represents a new regulatory mechanism for protein phosphorylation.     

Multiple splice forms of ribonuclease-inhibitor mRNA differ in the 5'-untranslated region

We report the sequence of the cDNAs representing five independent splice forms of human placental RNase inhibitor (RI) mRNA. RI mRNAs differ principally in the 5'-untranslated region, which may include or lack a 68-nucleotide (nt) exon inserted at a splice site located only 20 nt upstream from the initiator AUG. At least three other exons may also abut the same splice site. This unusual and variable feature of the mRNA would suggest that secondary structure in the region of the start codon may differ among RI messages. A single copy of the RI gene exists in the human genome.     

伤处理和乙烯对桃ACC氧化酶基因表达的影响

桃果实不耐贮运,特别是溶质水蜜桃、珐I大11＿程技术的发展使番茄果实实现延熟保鲜'－'、人f［］也试图通过乙烯合成的关键酶的］i义HNA技术来延长桃果实的贮运期。：ICC氧化酶是乙烯生物合成的关键酶之一,ACC氧化酶4peJL从番茄'、矮牵牛。。。桃卜'、苹果、'等许多植物...     

The 3'-untranslated region of apolipoprotein II mRNA contains two independent domains that bind distinct cytosolic factors

The 3'-untranslated region of apolipoprotein II (apoII) mRNA contains target sites for mRNA breakdown (Binder, R., Hwang, S.-P. L., Ratnasabapathy, R., and Williams, D. L. (1989) J. Biol. Chem. 264, 16910-16918). Degradation occurs via endonucleolytic cleavage at 5'-AAU-3'/5'-UAA-3' elements in single-stranded loop domains of the 3'-untranslated region. Degradation target sites occur in two clusters that are localized within two larger domains of secondary structure. In this study, gel shift and label transfer assays were used to identify liver cytosolic factors that recognize the 3'-untranslated region of apoII mRNA. The results show preferential binding of cytosolic factors to the 3'-untranslated region as compared to the coding region. UV cross-linking experiments confirmed that cytosolic factors labeled by the 3'-untranslated region are a subset of proteins labeled by the entire mRNA. Two distinct binding domains were identified within the 3'-untranslated region. The upstream domain encompassing nucleotides 400-547 extends from the translation stop codon through the complex stem-loop D structure described previously. This domain labeled primarily a 34-kDa protein in UV cross-linking experiments. The downstream binding domain encompassing nucleotides 568-643 includes another region of secondary structure and terminates within the universal polyadenylation signal. The downstream domain labeled primarily a 60-kDa protein in UV cross-linking experiments. The upstream and downstream binding domains did not compete with each other in gel shift or cross-linking experiments. These results indicate that the 3'-untranslated region can form two independent messenger ribonucleoprotein complexes localized to domains that include target sites for apoII mRNA degradation. We speculate that these messenger ribonucleoprotein complexes may play a role in the degradation of apoII mRNA or in the regulation of this process.     

Regulation of ferritin and transferrin receptor mRNAs

Iron regulates the synthesis of two proteins critical for iron metabolism, ferritin and the transferrin receptor, through novel mRNA/protein interactions. The mRNA regulatory sequence (iron-responsive element (IRE)) occurs in the 5'-untranslated region of all ferritin mRNAs and is repeated as five variations in the 3'-untranslated region of transferrin receptor mRNA. When iron is in excess, ferritin synthesis and iron storage increase. At the same time, transferrin receptor synthesis and iron uptake decrease. Location of the common IRE regulatory sequence in different noncoding regions of the two mRNAs may explain how iron can have opposite metabolic effects; when the IRE is in the 5'-untranslated region of ferritin mRNA, translation is enhanced by excess iron whereas the presence of the IREs in the 3'-untranslated region of the transferrin receptor mRNA leads to iron-dependent degradation. How and where iron actually acts is not yet known. A soluble 90-kDa regulatory protein which has been recently purified to homogeneity from liver and red cells specifically blocks translation of ferritin mRNA and binds IRE sequences but does not appear to be an iron-binding protein. The protein is the first specific eukaryotic mRNA regulator identified and confirms predictions 20 years old. Concerted regulation by iron of ferritin and transferrin receptor mRNAs may also define a more general strategy for using common mRNA sequences to coordinate the synthesis of metabolically related proteins.     

Extrathymic TCR gene rearrangement in human small intestine: identification of new splice forms of recombination activating gene-1 mRNA with selective tissue expression

Two new 5'-untranslated region (5'UTR) exons were identified in the human gene for the lymphocyte-specific endonuclease recombination activating gene-1 (RAG1) required for the somatic recombination yielding functional Ag receptors. These 5'UTR exons were used in three different splice forms by jejunal lymphocytes of the T cell lineage. RAG1 mRNA containing the previously described 5'UTR exon was not expressed in these cells. Conversely, one of the new 5'UTR exons was not expressed in thymus. The new RAG1 mRNA splice forms were all expressed in immature T cells (CD2(+)CD7(+)CD3(-)). This cell population also expressed high levels of mRNA for the pre-T alpha-chain. In situ hybridization demonstrated jejunal cells expressing the new splice forms of RAG1 mRNA, both intraepithelially and in lamina propria. Pre-T alpha-chain mRNA-expressing cells were detected at the same sites. These results strongly suggest ongoing TCR gene rearrangement in human small intestinal mucosa, yielding T cells specially adapted for this environment. This seems to be achieved by two parallel processes, extrathymic T cell development and peripheral Ag-driven TCR editing.     

Complete structure of the porcine pro-opiomelanocortin mRNA derived from the nucleotide sequence of cloned cDNA

Polyadenylated RNA isolated from porcine pituitary neurointermediate lobes was used to construct a cDNA library. The library was screened with a rat genomic DNA fragment specific for pro-opiomelanocortin sequences. Two positive clones, pJA-19 and pJA-20, containing respectively 850 bp and 550 bp were characterized. Sequence analysis of the cDNA inserts revealed the complete structure of the porcine pro-opiomelanocortin mRNA. This mRNA would include 129 5'-untranslated nucleotides, 801 nucleotides coding for the 267 amino acids precursor and 162 3'-untranslated nucleotides. Comparison with pro-opiomelanocortin mRNA sequences from other species shows regions of high homology not only in the coding sequences but also in the 5'untranslated region where the first 50 nucleotides are over 80% purines.     

Complete nucleotide and deduced amino acid sequences of rat L-type pyruvate kinase

Four overlapping cDNA clones for L-type pyruvate kinase (PK-L) were isolated from carbohydrate-induced rat liver cDNA libraries. They contained all the coding sequence of the enzyme from the 7th codon and the entire 3'-untranslated extension up to the poly(A) tail. The sequence of the first 7 codons and that of the 5'-untranslated region were determined by primer extension. The analyzed PK-L mRNA has 19 5'-untranslated bases, 1629 coding bases and 1281 3'-untranslated bases without the poly(A) tail; it corresponds to the heavier, 3.2 kb species of the L-type mRNAs. The codons for the phosphorylatable site are located at the 5'-end of the messenger. The unusually long 3'-untranslated extension contains a repetitive element complementary to the 'brain-specific' identifier sequence described by Sutcliffe et al. [(1982) Proc. Natl. Acad. Sci. USA 79, 4942-4946].     

Rainbow trout has two genes for growth hormone

We report the primary structures of two mRNA species (GH1 and GH2), each predicted from the cloned cDNA and genomic gene sequences, that encode growth hormone in rainbow trout (Salmo gairdneri). Both GH1 and GH2 mRNA contain open reading frames comprising 630 nucleotides and encode 210 amino acid residues, of which 11 are variant. The translated regions of mRNA are flanked by a short 5'-untranslated sequence, which is highly conserved, and a relatively long 3'-untranslated sequence, which is highly divergent. The differences at the 3'-untranslated regions suggest that the GH1 and GH2 mRNA originate from different loci. RNA blot analysis of trout pituitary RNA using an oligonucleotide probe specific for the GH2 sequence indicates that the cloned gene is expressed. The GH1 and GH2 mRNA likely are transcribed from two distinct loci, which were duplicated during tetraploidization of the salmonid genome between 50 and 100 million years ago.     

Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.     

Rat IGF-I cDNA's contain multiple 5'-untranslated regions

DNA sequencing of several independent rat IGF-I cDNA clones has revealed three different 5'-untranslated region sequences which contain multiple, upstream, in-frame initiation codons. Use of these codons could generate N-terminal heterogeneity in IGF-I precursor proteins. One of these 5'-untranslated region sequences contains a 40-bp segment which is an inverted repeat of a region in the common 3'-untranslated region. The ends of the IGF-I mRNA corresponding to this cDNA could form a stable duplex structure. Such a complex could prevent ribosomal access to the AUG codons preceding the coding region for the pre-pro-IGF-I peptide, suggesting the possibility of translational regulation of this form of IGF-I mRNA. The 3'-untranslated region inverted repeat sequence also is present in human and mouse IGF-I cDNA's, and, intriguingly, is more highly conserved than the rest of the 3'-untranslated region.