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At least 973 records · Page 54Linked to original sources
PubMed1980

Selection and sequence analysis of a cDNA clone encoding a known chorion protein of the A family.

Using as criteria the size, abundance and developmental specificity of hybridizing mRNA sequences, we have selected from our chorion cDNA library a clone corresponding to a specific chorion protein, A4--cl. Comparison between the clone sequence and the largely known sequence of A4--cl validates the use of the cDNA library for sequence analysis of the chorion multigene families. The two major chorion protein families, A and B, share certain structural similarities.

Amino Acid Sequence↗
PubMed1989

Identification of a putative isoform of the Na,K-ATPase beta subunit. Primary structure and tissue-specific expression.

We have isolated cDNA clones from rat brain and human liver encoding a putative isoform of the Na,K-ATPase beta subunit. The rat brain cDNA contains an open reading frame of 870 nucleotides coding for a protein of 290 amino acids with a calculated molecular weight of 33,412. The corresponding amino acid sequence shows 98% identity with its human liver counterpart. The proteins encoded by the rat and human cDNAs exhibit a high degree of primary sequence and secondary structure similarity with the rat Na,K-ATPase beta subunit. We have therefore termed the polypeptides these cDNAs encode a beta 2 subunit with the previously characterized rat cDNA encoding a beta 1 subunit. Analysis of rat tissue RNA reveals that the beta 2 subunit gene encodes a 3.4-kilobase mRNA which is expressed in a tissue specific fashion distinct from that of rat beta 1 subunit mRNA. Cell lines derived from the rat central nervous system shown to lack beta 1 subunit mRNA sequences were found to express beta 2 subunit mRNA. These results suggest that different members of the Na,K-ATPase beta subunit family may have specialized functions.

Amino Acid Sequence↗
PubMed2001

Cap-independent translation conferred by the 5'-untranslated region of human thymidine kinase mRNA.

Translational control is one of the mechanisms that regulate thymidine kinase (TK) expression in the cell cycle. Evidence for the TK mRNA sequence that is involved in its own translation has been lacking. In this report, we show that TK-deficient mouse fibroblasts transfected with pFLAG-TK express a TK mRNA containing the 5'-untranslated region (5'UTR) and produce two polypeptides, FLAG-TK and TK, resulting from an alternative initiation of translation. Most interestingly, the 5'UTR of TK allowed the translation of FLAG-TK mRNA to become cap-independent in an in vitro translation system. Furthermore, this 5'UTR sequence decreased significantly the efficiency of translation from the AUG codon of FLAG when the concentration of FLAG-TK RNA was low. Here, we also show that in normal human IMR-90 fibroblasts the induction of TK polypeptide by serum stimulation is insensitive to rapamycin treatment, which is known to inhibit the translations of transcripts of some growth-controlled genes by affecting the cap-binding efficiency. Taken together, we propose that the 5'UTR in TK mRNA might actually confer a secondary structure to regulate ribosome binding during translation in a cap-independent manner.

5' Untranslated Regions↗
PubMed1993

Apolipoprotein B mRNA editing: the sequence to the event.

Editing of the mRNA coding for apolipoprotein B involves a cytidine to uridine transition at nucleotide 6666 and enables translation of two protein variants. The development of in vitro editing systems has led to the identification of three sequence requirements in this process. The mechanism for C-->U editing requires specific sequences for editing site recognition, positioning of the catalytic activity and enhancement of catalytic efficiency. The dependence of in vitro editing on extract proteins has focused future research in this field on the identification of factors involved in apoB mRNA editing and the role of these factors in the assembly of ribonucleoprotein editosomes.

Animals↗
PubMed1992

Metabolic compartmentation of vertebrate glutamine synthetase: putative mitochondrial targeting signal in avian liver glutamine synthetase.

The evolution of uricoteley as a mechanism for hepatic ammonia detoxication in vertebrates required targeting of glutamine synthetase (GS) to liver mitochondria in the sauropsid line of descent leading to the squamate reptiles and archosaurs. Previous studies have shown that in birds and crocodilians, sole survivors of the archosaurian line, hepatic GS is translated without a transient, N-terminal targeting signal common to other mitochondrial matrix proteins. To identify a putative internal targeting sequence in the avian enzyme, the amino acid sequence of chicken liver GS was derived by a combination of sequencing of cloned cDNA, direct sequencing of mRNA, and sequencing of polymerase chain reaction (PCR) products amplified from reverse-transcribed mRNA. Analysis of the first 20 or so N-terminal amino acids of the derived sequence for the chicken enzyme shows that they are devoid of acidic amino acids, contain several hydroxy amino acids, and can be predicted to form a positively charged, amphipathic helix, all of which are characteristic properties of mitochondrial targeting signals. A comparison of the N-terminus of chicken GS with the N-termini of cytosolic mammalian GSs indicates that at least three amino acid replacements may have been responsible for converting the N-terminus of the cytosolic mammalian enzyme into a mitochondrial targeting signal. Two of these, His15 and Lys19, result in additional positive charges, as well as in changes in hydrophilicity. Both could have resulted from third-base-codon substitutions. A third replacement, Ala12, may contribute to the helicity of the N-terminus of the chicken enzyme. The N-terminus of the cytosolic chicken brain GS (positions 1-36) was found to be identical to that of the liver enzyme. The complete sequence of chicken retinal GS is also identical to that of the liver enzyme. GS is coded by a single gene in birds, so these sequence data suggest that, unlike the situation in other tissue-specific compartmental isozymes, differential targeting of avian GS to the mitochondrial or cytosolic compartments is not dependent on the sequence of the primary translation product of its mRNA but may involve some other tissue-specific factor(s).

Amino Acid Sequence↗
PubMed1991

Rat pancreatic stone protein messenger RNA. Abundant expression in mature exocrine cells, regulation by food content, and sequence identity with the endocrine reg transcript.

We used a cDNA encoding the human pancreatic stone protein (PSP-S), the secretory inhibitor of CaCO3 crystal growth, as a probe for cloning rat PSP-S messenger RNA. Overlapping clones gave a mRNA sequence of 783 nucleotides encoding a preprotein of 165 amino acids including a prepeptide of 21 amino acids. Rat and human PSP-S showed 70% identity, and the mature proteins had the same length. PSP-S mRNA concentration was measured in the pancreas of rats adapted to diets containing 15, 25, or 70% protein. Compared with the 15% protein diet, concentration increased 3 and 12 times with the diets with 25 and 70% protein, respectively, which is 3 times higher than for serine proteases. A complete sequence identity was observed between the rat PSP-S transcript and the reg mRNA described by Terazono et al. (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114), which is expressed in regenerating pancreatic islets but not in mature islets. A specific role of the reg protein in islet regeneration was suggested. We found that PSP-S (reg) mRNA concentration was indeed increased in isolated regenerating islets. Yet, a transient increase was also observed in exocrine tissue during the initial phase of regeneration following pancreatectomy or acute pancreatitis, suggesting increased expression during cell dedifferentiation. It is concluded that, in mature pancreas, expression of the reg/PSP-S gene occurs primarily in acinar cells. The gene product, which encodes a secretory protein inhibiting CaCO3 crystal growth in juice, is unlikely to play a specific role in islet regeneration.

Amino Acid Sequence↗
PubMed1994

N6-adenosine methylation in mRNA: substrate specificity and enzyme complexity.

The N6-methylation of internal adenosine residues is a common post-transcriptional modification of eukaryotic pre-mRNA sequences. An in vitro methylation system which retains the precise selectivity of in vivo methylation sites has been used to further define the nature of RNA site recognition. In addition to short consensus sequences, other structural features or context effects contribute to the selection of methylation sites in pre-mRNAs. Partial purification of the mRNA N6-adenosine methyltransferase revealed unexpected levels of complexity. The methyltransferase is composed of three separate components with molecular masses of 30, 200 and 875 kDa, respectively. These components are readily separated under non-denaturing conditions and each is required for mRNA methylation activity.

Adenosine↗
PubMed1995

Identification of an mRNA element promoting rate-limiting cleavage of the polycistronic puf mRNA in Rhodobacter capsulatus by an enzyme similar to RNase E.

We have identified an mRNA element that is involved in the initial cleavage of the pufBALMX mRNA species in Rhodobacter capsulatus. This endoribonuclease recognition site, the first to be identified in a bacterial species other than Escherichia coli, shows strong similarities to mRNA sequences cleaved by the endoribonuclease E in E. coli. The presence of an RNase E-like enzyme in R. capsulatus is further supported by in vitro cleavage of E. coli transcripts by R. capsulatus extracts at sites attributed to RNase E and by the cross-reaction of a polypeptide from R. capsulatus with antisera against E. coli RNase E. Our data provide evidence that mRNAs are degraded in different bacterial species by enzymes with similar recognition sequences and activities. We present a model that attributes the segmental differences in stability of the polycistronic puf transcript to a specific distribution of mRNA decay-promoting and mRNA decay-impeding elements.

Base Sequence↗
PubMed1996

Genomic organization of the human beta-catenin gene (CTNNB1).

The cytoplasmic beta-catenin protein is implicated in signal transduction and associates with both the cell-cell adhesion protein E-cadherin and the tumor suppressor gene product APC. We determined the primary structure of the human beta-catenin gene (CTNNB1) by analysis of cDNA and genomic clones. The size of the complete gene was determined to be 23.2 kb. Restriction mapping and partial sequence analysis revealed 16 exons. All splice donor and acceptor sites were conformable to the GT/AG rule. The exon size ranged from 61 to 790 bp. Half of the introns were smaller than 550 bp, with the smallest being 84 bp and the longest being 6700 bp. The intron-exon boundaries did not coincide either with conserved sites in the 12 armadillo repeat sequences of beta-catenin or with intron-exon boundaries in the armadillo gene of Drosophila. A major site for transcription initiation was identified as an A residue 214 nucleotides upstream of the ATG initiation codon. The resulting transcript is 3362 nucleotides long. Compared to the previously published mRNA sequence, additional residues were identified, 16 at the 5' end and 766 at the 3' end of the mRNA. An alternative splice acceptor site within exon 16 reduced the 3' UTR sequence by 159 bp. Polymerase chain reaction on cDNA from 14 human cell lines demonstrated the general occurrence of both splice variants. The 5'-flanking region is highly GC-rich and lacks a CCAAT box, but contains a TATA box and potential binding sites for several transcription factors, such as NF kappa B, SP1, AP2, and EGR1. Both a 437-bp fragment and a 6-kb fragment, containing about 4.7 kb of the 5'-flanking region in addition to the noncoding exon 1 and 1 kb of intron 1, showed clear promoter activity when these fragments were linked to a secreted alkaline phosphatase reporter gene and transfected into a mouse epithelial cell line.

Alternative Splicing↗
PubMed1990

Identification of chicken calbindin D28K pre-messenger RNA sequences by polymerase chain reaction.

A transcribed RNA sequence encompassing the junction between the first intron and the second exon of the chicken calbindin D28K gene was copied in a cDNA fragment and subsequently amplified by polymerase chain reaction. When intestinal RNA is used as template, the appearance of the 161 bp amplified fragment is strictly dependent on the vitamin D status of the animal. In fact no amplified fragment is obtained when the RNA is extracted from the intestine of vitamin D-deficient chickens, while it is easily detected when the RNA is extracted only 30 min after injection with 1,25-dihydroxycholecalciferol. Conversely, the amplified fragment is obtained, irrespectively of the vitamin D status of the animal, when the RNA template is extracted from the brain. The appearance of unspliced RNA sequences upon vitamin D induction is followed, after a 30 min lag, by the appearance of the corresponding mature mRNA sequences.

Animals↗
PubMed1983

Growth hormone acts at a pretranslational level in hepatocyte cultures.

We have examined the effects of ovine growth hormone and recombinant DNA synthesized human growth hormone on hepatocytes maintained in serum free cultures. Both growth hormone preparations augmented or attenuated 3 specific mRNA sequences as revealed by two-dimensional gel electrophoresis of [35S] methionine labeled products synthesized in vitro in an mRNA dependent rabbit reticulocyte lysate system. The results clearly indicate that growth hormone, free of potential pituitary contaminants, acts directly on hepatocytes at a pretranslational level.

Animals↗
PubMed2001

Targeting exported substrates to the Yersinia TTSS: different functions for different signals?

Many Gram-negative pathogens utilize a type III secretion system (TTSS) to inject toxins into the cytosol of eukaryotic cells. Previous studies have indicated that exported substrates are targeted to the Yersinia TTSS via the coding regions of their 5' mRNA sequences, as well as by their cognate chaperones. However, recent results from our laboratory have challenged the role of mRNA targeting signals, as we have shown that the amino termini of exported substrates are crucial for type III secretion. Here, we discuss the nature of these amino-terminal secretion signals and propose a model for the secretion of exported substrates by amino-terminal and chaperone-mediated signals. In addition, we discuss the roles of chaperones as regulators of virulence gene expression and present models suggesting that such regulation can occur independently of the delivery of their substrates to the secretion apparatus.

Bacterial Outer Membrane Proteins↗
PubMed1975

Sequence homology of the mitochondrial leucyl-tRNA cistron in different organisms.

Sequence divergence of the mitochondrial leucytl-tRNA cistron in several eukaryotes has been examined by RNA-DNA hybridized. Rat mitochondria Leucyl-tRNA was hybridized with rat, mouse, guinea pig, monkey, chicken, and yeast mitochondrial DNAs (mtDNA) immobilized onfilters. Hybridization was carried out in 50% formamide (Tm -12degrees) or in 20% fromamide (Tm -21degrees). melting profiles of the hybrids were obtained for evaluation of the extent of base sequence micmatching. Under the more stringent hybridization conditions (50% formamide, Tm -12degrees), only mouse and quines pig mtDNAs hybridized with rat mitochondrial leucyl-tRNA. The Tm's of the heterohybrids were depressed by 2 and 9 degrees, respectively. Under less stringent hybridization conditions (Tm-21 degrees), monkey mtDNA also hybridized, and the Tm was depressed by about 15 degrees. Chicken and yeast mtDNAs did not form specific hybrids with rat mibochondrial leucyl-tRNA under these hybridization conditions. Mitochondrial leucyl-tRNA sequences in different eukaryotes appear to be conserved to a less extent than cytoplasmic rRNA, 5S RNA, or hemoglobin mRNA sequences.

Animals↗
PubMed1988

Isolation of cDNA clones for human erythrocyte membrane sialoglycoproteins alpha and delta.

We have isolated almost full-length cDNA clones corresponding to human erythrocyte membrane sialoglycoproteins alpha (glycophorin A) and delta (glycophorin B). The predicted amino acid sequence of delta differs at two amino acid residues from the sequence determined by peptide sequencing. The sialoglycoprotein delta clone we have isolated contains an interrupting sequence within the region that gives rise to the cleaved N-terminal leader sequence for the protein and represents a product that is unlikely to be inserted into the erythrocyte membrane. Comparison of the cDNA sequences of alpha and delta shows very strong homology at the DNA level within the coding regions. The two mRNA sequences are closely related and differ by a number of clearly defined insertions and deletions.

Amino Acid Sequence↗
PubMed1976

Short-lived methylated messenger RNA in mouse kidney.

In experiments originally designed to examine selective turnover of methylated "caps" in renal mRNA, we observed that [3H]methyl label decayed from mRNA containing poly(A) with a half-life of 1-2 hr. (Caps are blocked, methylated mRNA sequences of the general structure m7GpppNm p(1 or 2)Np.). To distinguish between metabolism of short-lived mRNA and discriminate turnover of "caps", we compared residual [3H]methyl label in 5' and 3'mRNA fragments prepared from mRNA isolated during the decay period. Hydrolysis of mRNA at 0 degrees with dilute KOH before oligo(dT)-cellulose selection produced 5' mRNA fragments enriched with an alkali-resistant oligonucleotide with a -5 charge; the 3' mRNA fraction was correspondingly reduced in oligonucleotide content. Since methyl label disappeared at the same rate from both fractions, we conclude that mouse kidney contains short-lived mRNA and that the "caps" of these labile mRNAs turn over with the rest of the mRNA molecule.

Animals↗
PubMed2004

[Gene expression of heparanase in peripheral blood leukocytes from rats with nephrotic syndrome and its relationship to proteinuria].

OBJECTIVE: The beta-D-endoglycosidase heparanase (Hpa) is HS-specific which leads to heparan sulfate (HS) degradation. An increased permeability of the glomerular basement membrane (GBM) for proteinuria is related with a decreased HS side chains of the GBM. However, whether an up-regulated expression of Hpa exists in nephrotic syndrome (NS) remains unknown. The purpose of this study was to evaluate the role of Hpa in the development of NS and the relevance to proteinuria. METHODS: NS was induced in SD rats by tail intravenous injection of 5 mg/kg Adriamycin. Rats were housed in the metabolic cages for 24 hours to collect urine prior to induction of disease and at day 7 and 14 following disease induction. By using the method of reverse transcriptase-PCR (RT-PCR), Hpa gene from the peripheral blood leukocytes of these rats was assayed. RESULTS: The resulting cDNA fragment was consistent with rat heparanase mRNA sequence in GenBank (accession number AF184963). The rats at day 7 and 14 showed significantly up-regulated Hpa mRNA expression in contrast to the normal rats. There was a significant correlation between the level of Hpa mRNA and the quantification of urinary protein (r=0.925, P<0.001). CONCLUSION: Up-regulated expression of Hpa mRNA may be an important contributor to loss of glomerular negative charge in GBM and hence may lead to proteinuria in nephrotic syndrome.

Animals↗
PubMed1986

Primary structure of the mouse glycerol-3-phosphate dehydrogenase gene.

We have isolated from a BALB/c genomic library a 17.5-kilobase (kb) DNA fragment containing the entire mouse sn-glycerol-3-phosphate dehydrogenase (glycerol-P dehydrogenase) gene, and a substantial portion of the flanking regions. The DNA sequence of 10.8 kb of this fragment was determined. Using this information, together with the DNA sequence of several partial glycerol-P dehydrogenase cDNA clones and the rabbit glycerol-P dehydrogenase amino acid sequence, we determined the structural organization of the mouse glycerol-P dehydrogenase gene. The gene is 7.3 kb long and contains 8 exons. Transcription starts 19 base pairs 5' of the ATG initiation codon. Sequence analysis and S1 nuclease mapping indicated that the 8th exon contains coding sequences for the last 31 amino acids of glycerol-P-dehydrogenase, followed by 1.7 kb of 3' untranslated mRNA sequence. The mouse glycerol-P dehydrogenase amino acid sequence determined from the DNA sequence is 90% homologous with the rabbit enzyme. An examination of the exon/intron organization of the mouse glycerol-P dehydrogenase gene shows that intron 3 precisely separates the NAD-binding and the catalytic domains and intron 2 separates the adenine- and nicotinamide-binding regions.

Amino Acid Sequence↗