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PubMed2000

Distribution of the histamine H(2) receptor in monkey brain and its mRNA localization in monkey and human brain.

The distribution of histamine H(2) receptor mRNA was determined by in situ hybridization histochemistry in human and monkey brain. In the case of monkey brain, we combined this technique with receptor ligand autoradiography to compare the distribution of mRNA and receptor binding sites. [(125)I]Iodoaminopotentidine ([(125)I]-APT), a reversible, high specific activity antagonist with high affinity and selectivity for the H(2) receptor, was used for receptor autoradiography. Radiolabeled oligonucleotides derived from the human mRNA sequence encoding this receptor were used as hybridization probes. The highest density of the H(2) receptor mRNA in human and monkey brain was found in caudate and putamen nuclei and external layers of cerebral cortex. Moderate levels were seen in the hippocampal formation and lower densities in the dentate nucleus of cerebellum. Areas such as globus pallidus, amygdaloid complex, cerebellar cortex, and substantia nigra were devoid of hybridization signal. The distribution of H(2) receptor mRNA in monkey brain is generally in good agreement with that of the corresponding binding sites: prominent in caudate, putamen, accumbens nuclei, and cortical areas. The hippocampus showed lower densities of receptors and low levels were detected in the globus pallidus pars lateralis. No binding sites were seen in amygdaloid complex and substantia nigra. The distribution of histaminergic innervation is in good correlation with the areas of high density for H(2) receptors: caudate, putamen, and external layers of cerebral cortex in monkey and human brain. The presence of mRNA in caudate and putamen nuclei, together with its absence from substantia nigra, suggests that the H(2) receptors found in the striatum are synthesized by intrinsic cells and not by nigral dopaminergic cells. These striatal H(2) receptors may be located on short circuit striatal interneurons or somatodendritically on striatal projection neurons which project to the globus pallidus pars lateralis. In conclusion, the present results, which constitute, to our knowledge, the first report of the regional distribution of mRNA encoding H(2) receptors detected by in situ hybridization, define the sites of synthesis of H(2) receptors and are the basis for future, more detailed studies that should result in a better understanding of H(2) receptor function.

Adult↗
PubMed1988

Angiotensinogen gene expression in neuronal and glial cells in primary cultures of rat brain.

Neuronal and glial cells in primary culture prepared from the hypothalamic-brain stem areas of one-day-old rat brains were analyzed for the presence of angiotensinogen messenger RNA (mRNA) to further confirm our previous conclusion that the brain contains an exclusive angiotensin system. Angiotensinogen mRNA was quantitated by Northern analysis using nick-translated angiotensinogen cDNA as the hybridization probe (Kunapuli and Kumar, 1987). Angiotensinogen mRNA sequences were present in the RNA isolated from both neuronal and glial cultures. Quantitative measurements of the mRNA using dot-blot analysis revealed that the level of angiotensinogen mRNA was three times higher in neuronal cultures compared with glial cultures. These observations provide the first evidence for the synthesis of angiotensinogen in neuronal as well as in glial cells form the brain.

Angiotensinogen↗
PubMed1992

Localization of luteinizing hormone beta-mRNA by in situ hybridization in the sheep pars tuberalis.

The localization of luteinizing hormone beta (LH beta)-mRNA was studied by in situ hybridization in the pars tuberalis of sheep using a homologous sheep double-stranded 32P- or 35S-cDNA. The labelled cDNA probe detected one mRNA sequence in the pars tuberalis by Northern blot analysis; this sequence was similar to that detected in the pituitary. In situ, the labelling of LH beta-mRNA in the horizontal and sagittal tissue sections was found throughout the pars tuberalis. This labelling was prevented by adding an excess of cold probe or treating the sections by ribonuclease before in situ hybridization. Controls showed a labelling in the pars distalis, but not in the median eminence, hypothalamus, cerebral cortex and liver sections. Double labelling by in situ hybridization followed by immunohistochemistry using a specific LH beta-antiserum indicated that the labelling of LH beta-mRNA appeared more intense in LH-containing cells that were found only in the ventral part of the pars tuberalis. These results suggest that the entire pars tuberalis is able to produce the LH beta subunit, but that the level of translation greatly varies according to the location of the cells.

Animals↗
PubMed1987

Nucleotide sequence comparisons of turnip yellow mosaic virus isolates from Australia and Europe.

The genomic sequences of four isolates of turnip yellow mosaic virus (TYMV-Cd) from Australia, and three TYMV-1 (type) and three TYMV-2 (cauliflower) isolates from Europe were compared by cDNA-RNA hybridization tests, by analysis of the fragments produced from cDNA-RNA hybrids by restriction endonuclease treatment, and by determining the 3' terminal nucleotide sequences of their coat protein mRNAs. All three methods showed only slight differences (ca. 1%) between the mRNA sequences of different TYMV-1 and TYMV-Cd isolates, and did not distinguish between those groups of isolates. By contrast, the nucleotide sequences of TYMV-2 isolates differed from those of the other TYMVs by ca. 5% (sequence analysis) to 11% (restriction fragment analysis). Published biogeographic evidence has indicated that the TYMV-Cd and TYMV-1 populations probably separated more than 12,000 years ago. This implies that these TYMV genomes have changed at a rate of, at most, 1% in 10,000 years.

Australia↗
PubMed1979

Analysis of the genetic complexity and abundance classes of messenger RNA in human liver and leukemic cells.

In an effort to determine the number of genes expressed as messenger RNA in disparate human tissues we have analyzed the genetic complexity of the polyribosome-associated poly(A)-containing RNA population obtained from liver and lymphoblastic leukemic cells. This was accomplished by measuring the kinetics of hybridization of mRNA to a complementary DNA probe synthesized by avian myeloblastosis virus reverse transcriptase in vitro. The results obtained from such an analysis revealed the presence of two major abundance classes of mRNA with a total genetic complexity of approximately 10,000 diverse mRNA species in both of these cell types. Diversity of mRNA species in these unrelated human cells was studied by heterologous hybridization reactions between the cDNA of one cell type and a vast excess of poly(A)-containing mRNA from another. These types of studies indicated that extensive homology (more than 80%) exists in the mRNA sequences of disparate human cell types and suggest that the vast majority of genetic information expressed as mRNA is required for the maintenance of cellular functions common to functionally different human tissues.

Avian Myeloblastosis Virus↗
PubMed1983

Seasonal variation in the level of antifreeze protein mRNA from the winter flounder.

Livers from winter flounder collected at monthly intervals throughout the year were analyzed for their content of antifreeze protein mRNA. Putative mRNA was detected in liver RNAs from summer fish by liquid hybridization to kinetically purified antifreeze protein cDNA. These mRNA sequences were shown by Northern blot analysis to be of the same length as mature antifreeze protein mRNA isolated from winter fish, and were able to direct the incorporation of alanine into a translation product which comigrated with antifreeze preproprotein. The build-up of antifreeze protein mRNA levels in the autumn and their decline in the spring to summer levels were measured by liquid hybridization. These seasonal fluctuations match closely, but slightly precede, the rise and fall in plasma osmolality due to the presence of antifreeze protein. In mid-winter 0.5% of the total liver RNA is antifreeze protein mRNA, and although by late August the level of this mRNA has declined to 0.0007% of the total RNA, at no time during the summer is the mRNA undetectable. These results suggest that antifreeze protein production is more likely to be regulated by changing the rate of transcription of their genes than by switching them between active and inactive states.

Acclimatization↗
PubMed1993

Identification of unusual RNA folding patterns encoded by bacteriophage T4 gene 60.

A 50-nucleotide (nt) untranslated region (coding gap sequence) that interrupts the amino acid coding sequence in T4 gene 60, plus an additional 5 nt upstream and another 3 nt downstream from the gap sequence, shows unusual folding patterns according to RNA structure prediction. A predicted highly stable and significant hairpin structure in the 5' half of the gap sequence and a plausible tertiary structural element computed in the 3' part of the gap sequence seem significant by statistical tests on the wild-type (wt) sequence. This feature is absent in insertion, deletion and substitution variants of the gap sequence, in which template activities are markedly lower than that of the wt. The proposed feature is consistent with currently available data showing that the translational bypass of the coding gap is correlated with a stop codon involved in a stem-loop structure folded in the gap sequence. We suggest that the role of this segment in 'ribosomal bypass' of a portion of the mRNA sequence is a property of its special folded structure.

Bacteriophage T4↗
PubMed2004

Molecular basis for complement component 6 (C6) deficiency in rats and mice.

Complement component C6 is a part of the lytic membrane attack complex formed during complement activation. Animal modeling to define the role of C5a vs. C5b-9 in human disease has used rodents deficient in C6, yet the molecular basis for the deficiencies has not been ascertained. Oligonucleotides derived from a 493 bp EST sequence of the rat C6 gene were used to isolate full-length transcripts of rat C6 mRNA. Sequence analysis confirmed that the derived amino acid sequence for rat C6 is highly homologous to human and mouse. We identified a 31 bp deletion in exon 10 of the C6 gene that leads to C6 deficiency in a strain of PVG rats (PVG/c-) and developed a PCR-based genotyping test. In addition, we identified four point mutations in the mouse C6 gene that may result in C6 deficiency observed in the Peru-Coppock mouse strain. A serendipitous finding from this study was a coagulation defect in the C6 deficient mice and rats. C6 deficient mice or rats demonstrated prolonged tail bleeding times that was reversed by treatment with purified rat C6 protein. Further, adenosine diphosphate induced platelet aggregation were markedly reduced in C6 deficient rats. The molecular basis for these coagulations defects is unknown at present.

Amino Acid Sequence↗
PubMed1988

Structural similarity of bovine lung prostaglandin F synthase to lens epsilon-crystallin of the European common frog.

Cloned cDNA sequences specific for prostaglandin F (PGF) synthase have been isolated from a cDNA library of bovine lung mRNA sequences. Nucleotide-sequence analyses of cloned cDNA inserts have revealed that PGF synthase consists of a 969-base pair open reading frame coding for a 323-amino acid polypeptide with a Mr of 36,666. The sequence analysis indicates that bovine lung PGF synthase shows 62% identical plus conservative substitutions compared with human liver aldehyde reductase [Wermuth, B., Omar, A., Forster, A., Francesco, C., Wolf, M., Wartburg, J.P., Bullock, B. & Gabbay, K.H. (1987) in Enzymology and Molecular Biology of Carbonyl Metabolism: Aldehyde Dehydrogenase, Aldo-Keto Reductase, and Alcohol Dehydrogenase, eds. Weiner, H. & Flynn, T.G. (Liss, New York), pp. 297-307], which is similar to PGF synthase in molecular weight and substrate specificity. However, comparison of the amino acid sequence of PGF synthase with the National Biomedical Research Foundation protein data base reveals that the sequences of 225 amino acids from C termini of epsilon-crystallin of the European common frog (Rana temporaria) [Tomarev, S.I., Zinovieva, R.D., Dolgilevich, S.M., Luchin, S.V., Krayev, A.S., Skryabin, K.G. & Gause, G.G. (1984) FEBS Lett. 171, 297-302] and of PGF synthase show 77% identical and conservative substitutions without deletions/additions. The result suggests that European common frog lens epsilon-crystallin is identical to bovine lung PGF synthase.

Aldehyde Oxidoreductases↗
PubMed2000

The MAPK kinase kinase TAK1 plays a central role in coupling the interleukin-1 receptor to both transcriptional and RNA-targeted mechanisms of gene regulation.

Mechanisms of fulminant gene induction during an inflammatory response were investigated using expression of the chemoattractant cytokine interleukin-8 (IL-8) as a model. Recently we found that coordinate activation of NF-kappaB and c-Jun N-terminal protein kinase (JNK) is required for strong IL-8 transcription, whereas the p38 MAP kinase (MAPK) pathway stabilizes the IL-8 mRNA. It is unclear how these pathways are coupled to the receptor for IL-1, an important physiological inducer of IL-8. Expression of the MAP kinase kinase kinase (MAPKKK) TAK1 together with its coactivator TAB1 in HeLa cells activated all three pathways and was sufficient to induce IL-8 formation, NF-kappaB + JNK2-mediated transcription from a minimal IL-8 promoter, and p38 MAPK-mediated stabilization of a reporter mRNA containing IL-8-derived regulatory mRNA sequences. Expression of a kinase-inactive mutant of TAK1 largely blocked IL-1-induced transcription and mRNA stabilization, as well as formation of endogenous IL-8. Truncated TAB1, lacking the TAK1 binding domain, or a TAK1-derived peptide containing a TAK1 autoinhibitory domain were also efficient in inhibition. These data indicate that the previously described three-pathway model of IL-8 induction is operative in response to a physiological stimulus, IL-1, and that the MAPKKK TAK1 couples the IL-1 receptor to both transcriptional and RNA-targeted mechanisms mediated by the three pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Localization of N-acetyltransferases NAT1 and NAT2 in human tissues.

Human acetyl coenzyme A-dependent N-acetyltransferase (EC 2.3.1.5) (NAT) catalyzes the biotransformation of a number of arylamine and hydrazine compounds. NAT isozymes are encoded at 2 loci; one encodes NAT1, formerly known as the monomorphic form of the enzyme, while the other encodes the polymorphic NAT2, which is responsible for individual differences in the ability to acetylate certain compounds. Human epidemiological studies have suggested an association between the "acetylator phenotype" and particular cancers such as those of the bladder and colon. In the present study, NAT1- and NAT2-specific riboprobes were used in hybridization histochemistry studies to localize NAT1 and NAT2 mRNA sequences in formalin-fixed, paraffin-embedded human tissue sections. Expression of both NAT1 and NAT2 mRNA was observed in liver, gastrointestinal tract tissues (esophagus, stomach, small intestine, and colon), ureter, bladder, and lung. In extrahepatic tissues, NAT1 and NAT2 mRNA expression was localized to intestinal epithelial cells, urothelial cells, and the epithelial cells of the respiratory bronchioles. The observed heterogeneity of NAT1 and NAT2 mRNA expression between human tissue types may be of significance in assessing their contribution to known organ-specific toxicities of various arylamine drugs and carcinogens.

Arylamine N-Acetyltransferase↗
PubMed1991

cDNA sequence and deduced amino acid sequence of human preprocolipase.

Complementary DNA clones for human pancreatic colipase were identified in human pancreatic cDNA libraries by hybridization with a pool of synthetic oligonucleotides containing all possible coding sequences for amino acids 75 to 80 of the partial human colipase protein sequence (Sternby, et al. Biochim Biophys Acta 1984;784:75). Alignment of overlapping cDNA clones yielded an mRNA sequence of 504 nucleotides [not including the poly(A) tail] encoding a polypeptide of 112 amino acids. The prepeptide comprised 17 amino acids, with an amino-terminal cluster of charged residues followed by a hydrophobic core of 12 residues typical of leader sequences. The deduced human procolipase sequence comprised 95 residues, including a propeptide of 5 residues. It was in complete agreement with the partial sequence previously obtained by protein sequencing. Northern blot analysis revealed that the polyadenylated preprocolipase transcript had a length of approximately 680 nucleotides.

Amino Acid Sequence↗
PubMed1994

Posttranscriptional regulation of ferritin during nodule development in soybean.

During soybean (Glycine max) nodule development, induced ferritin mRNA concentration remains elevated while the protein concentration decreases 4- to 5-fold (M. Ragland and E.C. Theil [1993] Plant Mol Biol 21: 555-560). Investigation of posttranscriptional regulation of nodule ferritin during development showed that ferritin mRNA was efficiently translated based on polyribosome size in vivo, protein synthesis (0.8% of total protein) in vitro, and protein synthesis in intact nodules. Ferritin, a plastid protein, was processed in both immature and mature nodules. In chimeric mRNA, soybean ferritin mRNA sequences blocked the function of the iron regulatory element (IRE), the cis regulatory element of animal ferritin mRNA; the IRE regulates chimeric animal mRNAs. The absence of translational regulation of ferritin in plants contrasts with ferritin regulation in animals. Thus, ferritin regulation has diverged during evolution, whereas structure of the mature protein has been conserved. Ferritin in mature soybean nodules is apparently regulated after translation, possibly in analogy with such plastid proteins as chlorophyll-binding proteins D1, CP43, LHCI, and LHCII, the small subunit of ribulose-bisphosphate carboxylase, and apoplastocyanin. An autocatalytic mechanism observed in vivo for degradation of plastid protein D1 and in vitro for pea ferritin during iron release could explain the ferritin decreases in mature nodules.

Animals↗
PubMed1987

Structural variations in the alanine-rich antifreeze proteins of the pleuronectinae.

The sequence and activity of antifreeze proteins from two right eye flounder species were compared to assess the influence of structural variations on antifreeze capacity. The cDNA encoding the major serum antifreeze protein in the yellowtail flounder (Limanda ferruginea) was cloned from liver tissue. Its DNA sequence shows that the precursor to the antifreeze is a 97-residue preproportion. Edman degradation identified the N-terminus of the 48-amino-acid mature serum antifreeze protein and confirmed the sequence of the first 36 residues. A comparison with the previously determined winter flounder antifreeze protein and mRNA sequences shows strong homology through the 5' and 3' untranslated regions and in the peptide region. The mature protein section has the greatest sequence variation. Specifically, the yellowtail antifreeze protein, in contrast to that of the winter flounder, contains a fourth 11-amino-acid repeat and lacks several of the hydrophilic residues that have been postulated to aid in the binding of the protein to ice crystals. Intramolecular salt bridges are present in the antifreeze proteins from both species but in different registries with respect to the 11-amino-acid repeats. On a mass basis the yellowtail flounder antifreeze, though longer than that of the winter flounder, is only 80% as effective at depressing the freezing temperature of aqueous solutions. This lower activity might be due to the reduced number of hydrophilic ice-binding residues per molecule.

Alanine↗
PubMed1983

Molecular cloning of human adenosine deaminase gene sequences.

Using a mixture of synthetic 17-mer oligonucleotides encoding the 64 possible sequences for a peptide of adenosine deaminase as probe, we have isolated a clone for adenosine deaminase mRNA sequences from a collection of T-cell cDNA recombinants. This cDNA clone, phADA-1, contains an insert of 0.8 kilobase. In addition to the peptide chosen for synthesis of the oligonucleotide probe, the complete DNA sequence predicts 16 other experimentally determined peptides. Mapping of total cellular human DNAs with several restriction enzymes revealed relatively simple patterns of hybridization with phADA-1 as probe, including a polymorphism for PvuII cleavage. In agreement with previous studies, the adenosine deaminase gene was localized by blot hybridization to chromosome 20 in a hybrid cell mapping panel. Using the cDNA as probe, an 18-kilobase EcoRI fragment of human cellular DNA was also cloned in bacteriophage Charon 4A. These adenosine deaminase clones will prove valuable in the full characterization of the cellular gene, molecular analysis of inherited enzyme deficiency associated with immunodeficiency, and regional mapping of human chromosome 20.

Adenosine Deaminase↗
PubMed1980

Codon frequencies in 119 individual genes confirm consistent choices of degenerate bases according to genome type.

The poor printing of our previous Figure 2 (1) is corrected. Codon usage in mRNA sequences just published is also given. A new correspondence analysis is done, based on simultaneous comparison in all mRNA of use of the 61 codons. This analysis reinforces our claim that most genes in a genome, or genome type, have the same coding strategy; that is, they show similar choices among synonymous codons, or among degenerate bases (2). Like analysis on frequency variation in the amino acids coded reveals an entirely different pattern.

Animals↗
PubMed1991

Identification of sites of pre-MRNA/spliceosome association.

RNase H and synthetic DNA oligonucleotides were used to analyze the ribonucleoprotein (RNP) structure of the yeast spliceosome and to assay the pre-mRNA sequence requirements for step 1 of splicing. The data suggest that tight, stable contacts between the pre-mRNA and the spliceosome may be limited to the 5' splice site and branch point regions of the intron. A 30 nucleotide segment 3' of the branch point was found to be necessary for spliceosome maturation and essential for step 1 of splicing. Somewhat surprisingly, the 3' splice site was sensitive to nuclease digestion and completely dispensable for step 1 of splicing.

Base Sequence↗
PubMed2006

Recognition and positioning of mRNA in the ribosome by tRNAs with expanded anticodons.

Mutant tRNAs containing an extra nucleotide in the anticodon loop are known to suppress +1 frameshift mutations, but in no case has the molecular mechanism been clarified. It has been proposed that the expanded anticodon pairs with a complementary mRNA sequence (the frameshift sequence) in the A site, and this quadruplet "codon-anticodon" helix is translocated to the P site to restore the correct reading frame. Here, we analyze the ability of tRNA analogs containing expanded anticodons to recognize and position mRNA in ribosomal complexes in vitro. In all cases tested, 8 nt anticodon loops position the 3' three-quarters of the frameshift sequence in the P site, indicating that the 5' bases of the expanded anticodon (nucleotides 33.5, 34, and 35) pair with mRNA in the P site. We also provide evidence that four base-pairs can form between the P-site tRNA and mRNA, and the fourth base-pair involves nucleotide 36 of the tRNA and lies toward (or in) the 30 S E site. In the A site, tRNA analogs with the expanded anticodon ACCG are able to recognize either CGG or GGU. These data imply a flexibility of the expanded anticodon in the A site. Recognition of the 5' three-quarters of the frameshift sequence in the A site and subsequent translocation of the expanded anticodon to the P site results in movement of mRNA by four nucleotides, explaining how these tRNAs can change the mRNA register in the ribosome to restore the correct reading frame.

Anticodon↗