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PubMed1986

Isolation and characterization of human glycophorin A cDNA clones by a synthetic oligonucleotide approach: nucleotide sequence and mRNA structure.

In an effort to understand the relationships among and the regulation of human glycophorins, we have isolated and characterized several glycophorin A-specific cDNA clones obtained from a human erythroleukemic K562 cell cDNA library. This was accomplished by using mixed synthetic oligonucleotides, corresponding to various regions of the known amino acid sequence, to prime the synthesis of the cDNA as well as to screen the cDNA library. We also used synthetic oligonucleotides to sequence the largest of the glycophorin cDNAs. The nucleotide sequence obtained suggests the presence of a potential leader peptide, consistent with the membrane localization of this glycoprotein. Examination of the structure of glycophorin mRNA by blot hybridization revealed the existence of several electrophoretically distinct mRNAs numbering three or four, depending on the size of the glycophorin cDNA used as a hybridization probe. The smaller cDNA hybridized to three mRNAs of approximately 2.8, 1.7, and 1.0 kilobases. In contrast, the larger cDNA hybridized to an additional mRNA of approximately 0.6 kilobases. Further examination of the relationships between these multiple mRNAs by blot hybridization was conducted with the use of exact-sequence oligonucleotide probes constructed from various regions of the cDNA representing portions of the amino acid sequence of glycophorin A with or without known homology with glycophorin B. In total, the results obtained are consistent with our hypothesis that the three larger mRNAs represent glycophorin A gene transcripts and that the smallest (0.6 kilobase) mRNA may be specific for glycophorin B.

Amino Acid Sequence↗
PubMed1980

The 3' untranslated regions of the duplicated human alpha-globin genes are unexpectedly divergent.

DNA sequence analysis of a cloned partially deleted human alpha-thalassemia globin gene revealed a novel 3' untranslated region displaying at least nineteen differences when compared with previously published alpha mRNA sequences. Restriction enzyme mapping established the origin of the alpha-thalassemia gene as the more 3' of the normal, duplicated alpha genes (alpha 1). DNA sequencing of a previously isolated alpha 1 gene revealed a 3' untranslated region identical to that of the alpha-thalassemia gene. The sequence of the corresponding region of the more 5' alpha gene (alpha 2) was consistent with published mRNA sequences except in three probably polymorphic positions. Therefore the 3' untranslated regions of the highly homologous alpha-globin genes differ significantly. The recognition that the duplicated alpha genes differ in a region expressed in mature mRNA should not permit direct assessment of relative gene output in various normal and pathologic states. The divergence of the alpha gene 3' untranslated regions in the face of minimal coding sequence differences must be reconciled with current models for matching homologous gene sequences by recombination events.

Base Sequence↗
PubMed1985

The stability of bacteriophage T4 gene 32 mRNA: a 5' leader sequence that can stabilize mRNA transcripts.

In T4-infected cells, the gene 32 monocistronic mRNA is very stable. To study the molecular basis for this stability, we have constructed chimeric plasmids containing the monocistronic promoter and the gene 32 translation initiation sequence fused to either most of the E. coli lac operon or only a segment of the lacZ gene, followed by the gene 32 transcription terminator. The resulting hybrid transcripts are unstable in uninfected cells. In phage-infected cells, however, the hybrid mRNAs are at least as stable as gene 32 mRNA itself. Analysis of other plasmid constructs indicates that the sequences on the gene 32 mRNA from its 5' end to slightly beyond the initiation codon suffice to stabilize these hybrids. Studies with a series of deletions of the gene 32 leader sequence suggest that an RNA sequence near the gene 32 initiation codon is involved. Various models to explain this mRNA stabilization are discussed.

DNA Helicases↗
PubMed1978

Nucleotide sequence of DNA template for the 3' ends of SV40 mRNA. II. The sequence of the DNA fragment EcorII-F and a part of EcorII-H.

The nucleotide sequence for two-thirds of restriction endonuclease fragment EcoRII-F and part of RII-H of SV40 DNA is presented. This segment of SV40 DNA is complementary to the sequence near the 3' end of early mRNA. This sequence could be translated in one reading frame to form a large protein. However, in a second translational frame there are four AUG codons followed by 91 sense triplets, followed by a termination codon. These results provide the sequence for the entire 3' untranslated ends of SV40 early and late mRNAs and for the DNA beyond the 3' ends of the mRNAs. The ends of early and late mRNA are transcribed from the opposite strands of the same segment of DNA. At or beyond the 3' ends of both early and late mRNA are sequences whose transcripts would include uridylic acid-rich products.

Base Sequence↗
PubMed1996

DRPLA gene (atrophin-1) sequence and mRNA expression in human brain.

Dentatorubral pallidoluysian atrophy (DRPLA, Smith's disease) is one of five disorders currently known to result from expansion of a CAG trinucleotide repeat encoding glutamine. The reported full length cDNA sequence encodes a serine repeat and a region of alternating acidic and basic amino acids, as well as the glutamine repeat. We now report the nucleic acid and deduced amino acid sequences of the open reading frame of this gene, obtained from a series of independently isolated and sequenced cDNA clones. Eight nucleotide differences from the originally published sequence result in a change of 34 amino acids, most prominently in the region of alternating acidic and basic residues. Northern analysis and in situ hybridization indicate that atrophin-1 mRNA is expressed in multiple brain regions. The level of mRNA expression as determined by in situ hybridization in a DRPLA-diseased brain is indistinguishable from the level observed in a matched control brain. These results indicate that the correlation between atrophin-1 expression and regions of pathology in DRPLA is at best partial, and that the expanded allele does not cause a major loss of mRNA expression. The pathology of the disorder may therefore arise from the altered structure and function of the abnormal protein.

Amino Acid Sequence↗
PubMed1997

Neural network prediction of translation initiation sites in eukaryotes: perspectives for EST and genome analysis.

Translation in eukaryotes does not always start at the first AUG in an mRNA, implying that context information also plays a role. This makes prediction of translation initiation sites a non-trivial task, especially when analysing EST and genome data where the entire mature mRNA sequence is not known. In this paper, we employ artificial neural networks to predict which AUG triplet in an mRNA sequence is the start codon. The trained networks correctly classified 88% of Arabidopsis and 85% of vertebrate AUG triplets. We find that our trained neural networks use a combination of local start codon context and global sequence information. Furthermore, analysis of false predictions shows that AUGs in frame with the actual start codon are more frequently selected than out-of-frame AUGs, suggesting that our networks use reading frame detection. A number of conflicts between neural network predictions and database annotations are analysed in detail, leading to identification of possible database errors.

Amino Acid Sequence↗
PubMed1984

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat.

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.

Animals↗
PubMedDate not supplied

[Hormonal control of the expression of alpha fetoprotein in newborn rats. Evidence for a selective action of glucocorticoids on gene transcription].

Administration of glucocorticoid hormones to the newborn rat results in a rapid decrease in the synthesis of alpha-fetoprotein (AFP) by the liver. The molecular basis of this hormonal action was investigated by examining the steady-state levels of AFP mRNA and albumin mRNA sequences in polysomal and total RNA preparations isolated from dexamethasone-treated and control animals. Following dexamethasone treatment the number of polysomal and total mRNA sequences hybridizable to specific (32P) cDNA probes was drastically decreased for AFP while it was unchanged for albumin. These data indicate that glucocorticoids exert a selective action on AFP mRNA levels and suggest that dexamethasone operate at the transcriptional level.

Animals↗
PubMed1979

Effect of estrogen on ovalbumin gene expression in differentiated nontarget tissues.

By use of cloned DNA fragments as probes, low levels of ovalbumin RNA sequences (structural and intervening sequences) were detected in nuclear RNA extracts of nontarget tissues, such as liver, spleen, brain, and heart of chicks. The expression of the ovalbumin gene sequences was hormone dependent. In estrogen-stimulated chicks, a low level of ovalbumin RNA sequences, ranging from 0.2 to 0.7 molecule per cell, was present in nontarget tissues while less than 0.01 molecule per cell could be found in the same tissues of unstimulated chicks. A significant amount of the ovalbumin mRNA sequences was also found in polysomes of liver and brain. The ovalbumin mRNA sequences could be translated into proteins which were only localized in a few cells among the entire population of liver cells as determined by an immunocytochemical assay. These results suggest that there are some cells in liver, spleen, heart, and brain which can respond to hormone stimulation and produce ovalbumin mRNA and its translational product.

Animals↗
PubMed1985

Characterization of two highly diverged but developmentally co-regulated cysteine proteinase genes in Dictyostelium discoideum.

The cysteine proteinase 1 and 2 mRNA sequences of Dictyostelium discoideum encode proteins with a high degree of homology to plant and animal sulphydryl proteinases. The two mRNA sequences are co-ordinate in their regulation, both being first expressed late during cellular aggregation, prematurely induced in response to exogenous cAMP and several-fold enriched in prestalk over prespore cells. The two proteins are considerably diverged, with only 43% overall homology but all residues known to be important in catalysis are conserved and both contain a hydrophobic leader peptide which forms part of an N-terminal domain of just over 100 amino acids not found in the mature form of known cysteine proteinases. We have determined the sequence organization of both genes and find differences both in the number and position of introns. The close co-regulation of these two genes suggests that they may play a common role in Dictyostelium development, presumably in the autodigestion of cellular protein which occurs during differentiation. However, the low degree of sequence homology and major differences in gene organization indicate that they have undergone a considerable period of separate evolution and that they may differ in their precise function.

Base Sequence↗
PubMed1984

Rapid regulation of L-type pyruvate kinase mRNA by fructose in diabetic rat liver.

The effect of fructose on the induction of L-type pyruvate kinase mRNA in diabetic rat liver was studied by using a cloned cDNA probe. Fructose feeding resulted in a 5- to 6-fold increase in the L-type enzyme mRNA level after 1 to 3 days. These changes were approximately proportional to the changes in the level of translatable mRNA of this enzyme. A significant increase in total cellular L-type enzyme mRNA level was observed within 2 h after fructose feeding and the level reached a maximum after 8 h. Dietary glycerol also markedly increased the L-type mRNA level. These alterations were essentially due to the changes in the cytosolic mRNA. Northern blot analysis of total cellular RNA revealed that two L-type enzyme mRNA species with molecular sizes of 2.1 and 3.6 kilobases were proportionally increased during the fructose induction. The two mRNA forms were found in immunopurified L-type enzyme mRNA and directed synthesis of the L-type subunit in vitro; they are therefore functional mature forms. In contrast, analysis of nuclear RNA showed five putative precursor RNA species for the enzyme, up to 9.4 kilobases in length, in the liver of fructose-fed rats, while no band of the RNA species was found in the nuclei of control liver. The changes in the number of bands of these RNA species and their intensities after fructose feeding preceded the changes in the level of total cellular L-type enzyme mRNA sequences. These results indicate that dietary fructose causes a rapid increase in the level of L-type pyruvate kinase mRNA sequences by acting at the nuclear level.

Animals↗
PubMed1990

[Expression of ceruloplasmin gene in various organs of the rat].

The contribution of different rat organs to the synthesis of ceruloplasmin (Cp) was studied. Dot hybridization with the use of the Cp cDNA probe revealed Cp mRNA sequences in RNA preparations from liver, heart, kidney as well as from different divisions of brain, the concentration of Cp mRNA sequences being maximal in the liver. Polyribosomes isolated from these organs effectively synthesized Cp in a cell-free system derived from rabbit reticulocytes. After in vivo pulse labeling, the newly formed radioactive Cp was detected in the membrane fractions from all these organs. The newly formed Cp was concentrated within the membranes of the Golgi complex of various organs where it was revealed by different immunochemical techniques. Experiments with isolation of the liver from the systemic circulation showed that the liver is the only organ secreting Cp into the blood stream. It was suggested that mammalian tissues contain at least two molecular forms of Cp, i. e., circulatory and intracellular ones.

Animals↗
PubMed2001

Genetic variation in mRNA coding sequences of highly conserved genes.

The frequency and distribution of genetic polymorphism in the human genome is a question of major importance. We have studied this in highly conserved genes, which encode crucial functions such as DNA replication, mRNA transcription, and translation. Evolutionary comparisons suggest that these genes are under particularly strong selective pressure, and their frequency of nucleotide sequence polymorphism would be expected to represent a minimum estimate for sequence variation throughout the genome. We have analyzed the complete coding sequence and the 3'-untranslated region (3'-UTR) of 22 human genes, most of which have homologs in all cellular organisms and all of which are at least 25% amino acid identical to homologs in yeast. Comparisons with similar studies of less conserved human disease genes indicate that 1) evolutionarily conserved genes are, on average, less polymorphic than disease related genes; 2) the difference in polymorphism levels is attributable almost entirely to reduced levels of variation in protein coding sequences, whereas noncoding sequences have similar levels of polymorphism; and 3) the character of polymorphism, in terms of the spectrum and frequency of mutational changes, is similar.

Cell Line↗
PubMed1989

Distribution of neurons expressing calcitonin gene-related peptide mRNAs in the brain stem, spinal cord and dorsal root ganglia of rat and guinea-pig.

In situ hybridization histochemistry was used to localize calcitonin gene-related peptide mRNAs in spinal cord, brain stem and dorsal root ganglion neurons of the rat and guinea-pig. A 32P-labeled 23-base-long (23mer) oligodeoxyribonucleotide (oligomer) complementary to calcitonin gene-related peptide mRNA sequences encoding residues 23-30 of calcitonin gene-related peptide was used primarily as a probe (CGRP I probe). A 32mer complementary to mRNA sequences for residues 10-20 of calcitonin gene-related peptide (CGRP II probe) was also used as a positive control for specificity of the 23mer for calcitonin gene-related peptide mRNA. In both the guinea-pig and rat calcitonin gene-related peptide mRNA was localized specifically to neurons of the dorsal root ganglion, to spinal motoneurons and to motoneurons of the hypoglossal, facial and accessory facial motor nuclei. Differences in the distribution of calcitonin gene-related peptide mRNA between the rat and guinea-pig included a higher proportion of rat dorsal root ganglion neurons containing calcitonin gene-related peptide mRNA and the localization of calcitonin gene-related peptide mRNA to motoneurons of the ambiguus motor nucleus, parabrachial and peripeduncular nucleus of the rat but not the guinea-pig. In the guinea-pig, in contrast, calcitonin gene-related peptide mRNA was localized also to motoneurons of the abducens, trigeminal, trochlear and oculomotor nerves. The neuronal groups in the intact rat found here to contain calcitonin gene-related mRNA have also been shown previously to contain calcitonin gene-related peptide immunoreactivity in colchicine-treated rats. Colchicine-treated rats, however, have been found to contain additional groups of calcitonin gene-related peptide immunoreactive neurons which, in the intact rats used in the present study, showed no detectable hybridization with the calcitonin gene-related peptide probe.

Animals↗
PubMed1994

Gamma-aminobutyric acidA-receptor messenger ribonucleic acid (alpha-1 subunit) detection by in situ hybridization.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is most likely involved in the efferent cochlear neurotransmission. In situ hybridization (ISH) results in specific annealing of a labelled nucleic acid probe to complementary sequences in fixed tissue and allows subsequent visualization of the location of the probe. We used the ISH technique to localize messenger ribonucleic acid (mRNA) sequences of the alpha-1 subunit of the GABAA receptor with an S-35 labeled oligonucleotide probe. Experiments were performed in rat and guinea pig brain sections and surface tissue preparations of the guinea pig cochlea. Positive signals were obtained for the alpha-1 probe in cortical and hippocampal regions of the rat brain and had weaker expression in the guinea pig brain. Alpha-1 subunit mRNA was localized in Purkinje cells and in stellate and basket cells of the stratum moleculare in the rat and guinea pig cerebellum. In surface tissue preparations of the guinea pig cochlea mRNA sequences of the alpha-1 subunit were detectable with high signal expression. Positive signals were seen on both sides of the tunnel of Corti, predominantly in the region of the outer hair cells. The results indicate expression of GABAA-receptor mRNA in cochlear tissue, supporting the importance of GABAA receptors in cochlear neurotransmission.

Animals↗
PubMed1984

Effects of chronic renal failure on protein synthesis and albumin messenger ribonucleic acid in rat liver.

Previously we reported that chronic renal failure in rats leads to preferential disaggregation of liver membrane-bound polysomes associated with a decrease in albumin synthesis. To determine whether reduced albumin synthesis results from reduced cellular levels of albumin messenger RNA (mRNA) or some other molecular mechanism, we have employed mRNA-DNA hybridization in conjunction with cell-free protein synthesis to determine albumin mRNA sequence content and biological activity in subcellular fractions from control and uremic rat liver. Using high specific activity albumin [3H]-complementary DNA prepared from purified-albumin mRNA, we found that total liver polysomes and albumin mRNA sequence content are increased in uremic animals. The extra polysomes are located within the membrane-bound subcellular fraction. These polysomes, however, have reduced ability to synthesize albumin in the cell-free system, and mRNA isolated from membrane-bound polysomes of uremic liver showed reduced albumin synthesis. Evaluation of albumin mRNA size by hybridization analysis revealed a reduced content of intact albumin mRNA molecules per microgram of RNA in the liver of uremic animals. This was associated with increased ribonuclease activity in uremic cytosol. The diminished albumin synthesis by membrane-bound polysomes of uremic rat liver can, therefore, be explained by enhanced degradation of albumin mRNA.

Albumins↗
PubMed1982

Regulation of the relative abundances of mRNAs in hepatoma and liver.

Comparison of the polysomal poly(A)+ RNAs of normal rat liver and hepatoma (HTC) cells have shown that, while few sequences are specific to either hepatocytes or hepatoma cells, some of the abundant liver mRNAs (perhaps 20% by weight) are much rarer in the polysomal RNA from HTC cells. In contrast, these mRNA sequences are at quite similar abundances in nuclear poly(A)+ RNA from hepatocytes and hepatoma cells. The use of cloned cDNAs to measure the relative abundances of individual liver mRNA sequences in hepatocytes and HTC cells has shown that these changes occur in both directions, and indicated that post-transcriptional modulations, which are cell-type-specific and sequence-specific, play a significant role in establishing steady-state levels of polysomal poly(A)+ mRNAs. To investigate post-transcriptional regulation of mRNA abundance, a cell-free system consisting of isolated HTC-cell nuclei has been developed. This system supports the in vitro processing and/or transport of rRNA and a complex mixture of poly(A)+ RNA sequences which resembles polysomal poly(A)+ RNA from HTC cells. The pattern of relative abundances of the sequences in released poly(A)+ RNA is intermediate between that of polysomal and nuclear poly(A)+ RNAs, and indicates that some degree of sequence-specific selection of processing and/or transport is maintained in isolated nuclei. Sequence-specific selection of individual poly(A)+ RNA sequences in vitro has been detected with cloned cDNAs.

Animals↗
PubMed1978

Amplification and characterization of eukaryotic structural genes.

An approach to the study of eukaryotic structural genes which are differentially expressed during development is described. This approach involves the isolation and amplification of mRNA sequences by in vitro conversion of mRNA to double-stranded cDNA followed by molecular cloning in bacterial plasmids. This procedure provides highly specific hybridization probes that can be used to identify genes and their contiguous DNA sequences in genomic DNA, and to detect specific RNA transcripts during development. The nature of the method allows the isolation of individual mRNA sequences from a complex population of molecules at different stages of development.

Animals↗