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PubMed1982

Rat preprocarboxypeptidase A: cDNA sequence and preliminary characterization of the gene.

Rat carboxypeptidase A cDNA clones have been isolated from a cDNA library prepared from pancreatic mRNA. An almost complete mRNA sequence has been deduced that predicts a polypeptide having 78% amino acid sequence homology with bovine carboxypeptidase A. The amino acid sequence of the activation and signal peptides of the carboxypeptidase A precursor were inferred from the nucleotide sequence. The cDNA was used as a probe to identify DNA fragments containing carboxypeptidase A sequences in a bacteriophage lambda library of rat genomic DNA. Heteroduplexes revealed that the DNA coding sequence occupies 5.5 kilobases and is interrupted by nine intervening sequences. The nucleotide sequence of the 5' end of the gene and the adjacent flanking region provides information on the site of initiation of transcription and the putative control regions. There is no evident relationship between the localization of intervening sequences in the gene and functional/structural domains of the protein.

Animals↗
PubMed1988

Discriminant analysis of promoter regions in Escherichia coli sequences.

We have previously developed a general method based on the statistical technique of discriminant analysis to predict splice junctions in eukaryotic mRNA sequences [Nakata, K., Kanehisa, M. and DeLisi, C. (1985) Nucleic Acids Res., 13, 5327-5340]. In order to evaluate further applicability of this method, we now analyze the promoter region of Escherichia coli sequences. The attributes used for discrimination include the accuracy of consensus sequence patterns measured by the perceptron algorithm, the thermal stability map, the base composition and the Calladine-Dickerson rules for helical twist angle, roll angle, torsion angle and propeller twist angle. When applied to selected E. coli sequences in the GenBank database, the method correctly identifies 75% of the true promoter regions.

Algorithms↗
PubMed1986

Bovine adrenocortical cytochrome P-450(17 alpha). Regulation of gene expression by ACTH and elucidation of primary sequence.

In the steroidogenic pathway of the adrenal cortex, 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha)) is a major regulatory enzyme. Previous studies have shown that stimulation of 17 alpha-hydroxylase activity occurs upon treatment of bovine adrenocortical cells in culture with adrenocorticotropic hormone (ACTH), via cAMP, due to an increase in the enzyme concentration. Alterations in the levels of this enzyme result in pronounced changes in the pattern of steroids produced, with the potent glucocorticoid cortisol, as well as the sex steroids, being products of 17 alpha-hydroxylated steroid precursors. In the present study, the identification and sequencing of two cloned DNA molecules, pB17 alpha-1 and pcD17 alpha-2, which are complementary to mRNA sequences encoding bovine adrenal cortex P-450(17 alpha), are reported. Clone pcD17 alpha-2 contains an open reading frame coding for the complete amino acid sequence of P-450(17 alpha) which consists of 509 amino acids and has a molecular weight of 57,251. By comparison to other forms of cytochrome P-450, we conclude that P-450(17 alpha) is a member of a previously unidentified family within the P-450 multigene superfamily. Single bovine and human mRNA species of approximately equal to 1850 bases in length hybridize to these clones. Regulation of the concentration of this RNA has been studied using bovine adrenocortical cells in culture. Treatment with ACTH or dibutyryl cAMP causes an increase in the content of P-450(17 alpha) RNA in as little as 2 h, and its concentration increases greater than 20-fold by 8 h. In contrast, other steroidogenic enzymes that have been studied respond more slowly to ACTH. Actinomycin D and cycloheximide block induction of P-450(17 alpha) RNA, indicating that regulation of 17 alpha-hydroxylase activity is controlled at the level of transcription and requires ongoing protein synthesis.

Adrenal Cortex↗
PubMed1995

Interleukin-2 transcripts in human and rodent brains: possible expression by astrocytes.

Cytokines have been suggested to be involved in the cross talk between the immune and the nervous systems, under normal and pathological conditions. For example, the cytokine interleukin-2 was suggested to be involved in response to CNS trauma and spontaneous regeneration. Here, we examined whether mammalian CNS has an intrinsic potential to produce interleukin-2 and, if so, what its cellular origin is. mRNA sequences encoding for interleukin-2 were detected in brains of humans and rodents. Northern blot analysis revealed the presence of several interleukin-2 transcripts of different sizes in the brain, all recognized by lymphocyte-derived interleukin-2 cDNA probes. One of the transcripts, a high molecular weight form of approximately 5 kb, appeared to be unique to the brain. Reverse transcription and amplification by PCR of human fetal brain mRNA revealed one cDNA product that, upon sequence analysis, showed a high degree of homology with the human lymphocyte-derived interleukin-2 coding sequence. To identify the possible cellular source of the interleukin-2 transcripts within the mammalian brain, we similarly analyzed mRNA of rat brain cells in culture. Northern blot analysis revealed that astrocytes contain transcripts that hybridize with interleukin-2 cDNA probe. These findings point to the astrocytes as a possible source of brain interleukin-2.

Animals↗
PubMed2002

Computational analysis of alternative splicing using EST tissue information.

Expressed sequence tags (ESTs) from normal and tumor tissues have been deposited in public databases. These ESTs and all mRNA sequences were aligned with the human genome sequence using LEADS, Compugen's alternative splicing modeling platform. We developed a novel computational approach to analyze tissue information of aligned ESTs in order to identify cancer-specific alternative splicing and gene segments highly expressed in particular cancers. Several genes, including one encoding a possible pre-mRNA splicing factor, displayed cancer-specific alternative splicing. In addition, multiple candidate gene segments highly expressed in colon cancers were identified.

Alternative Splicing↗
PubMed1996

The mouse gene for vascular endothelial growth factor. Genomic structure, definition of the transcriptional unit, and characterization of transcriptional and post-transcriptional regulatory sequences.

We describe the genomic organization and functional characterization of the mouse gene encoding vascular endothelial growth factor (VEGF), a polypeptide implicated in embryonic vascular development and postnatal angiogenesis. The coding region for mouse VEGF is interrupted by seven introns and encompasses approximately 14 kilobases. Organization of exons suggests that, similar to the human VEGF gene, alternative splicing generates the 120-, 164-, and 188-amino acid isoforms, but does not predict a fourth VEGF isoform corresponding to human VEGF206. Approximately 1. 2 kilobases of 5'-flanking region have been sequenced, and primer extension analysis identified a single major transcription initiation site, notably lacking TATA or CCAT consensus sequences. The 5'-flanking region is sufficient to promote a 7-fold induction of basal transcription. The genomic region encoding the 3'-untranslated region was determined by Northern and nuclease mapping analysis. Investigation of mRNA sequences responsible for the rapid turnover of VEGF mRNA (mRNA half-life, <1 h) (Shima, D. T. , Deutsch, U., and D'Amore, P. A. (1995) FEBS Lett. 370, 203-208) revealed that the 3'-untranslated region was sufficient to trigger the rapid turnover of a normally long-lived reporter mRNA in vitro. These data and reagents will allow the molecular and genetic analysis of mechanisms that control the developmental and pathological expression of VEGF.

Alternative Splicing↗
PubMed1996

Characteristics of major structural protein coding gene and leader-body sequence in subgenomic mRNA of porcine reproductive and respiratory syndrome virus isolated in Japan.

Nucleotide sequence, 1713nt in length, of porcine reproductive and respiratory syndrome virus (PRRSV) isolated in Japan was determined. The sequence encompassed 3 overlapping open reading frames (ORFs), ORF5 to ORF7. These ORFs encodes major structural proteins of PRRSV. The deduced amino acid sequence of each ORF showed higher than 87.5% identity with an American isolate, and lower (54.6 to 80.5%) identity with an European isolate. This result supported a previous report about antigenic characteristics of the EDRD-1 strain. Leader-body junction sequence in subgenomic mRNA of the EDRD-1 strain was determined by sequencing cDNA clones of subgenomic RNAs. A common sequence motif of 5 nucleotide, represented by UA(A/G)CC, was identified as the junction sequence.

Amino Acid Sequence↗
PubMed2002

Nitric oxide and peroxynitrite promote complete disruption of the [4Fe-4S] cluster of recombinant human iron regulatory protein 1.

Iron regulatory protein 1 (IRP1) is a metalloprotein which regulates several proteins involved in mammalian iron homeostasis at a post-transcriptional level, by binding to specific mRNA sequences termed iron responsive elements (IREs). IRP1 exhibits two mutually exclusive activities, either aconitase or mRNA-binding protein, depending on the intactness of a versatile [4Fe-4S] cluster. Here we asked whether NO and peroxynitrite act directly on IRP1 and how they modify its functions. Recombinant human IRP1 was purified from Escherichia coli as a [4Fe-4S] cluster-containing protein, and exposed to 3-morpholinosydnonimine (SIN-1), which was used either as a peroxynitrite donor or as an NO donor when added with an excess of superoxide dismutase (SOD). We showed that, in both settings, IRP1 lost aconitase activity and iron concomitantly. Iron release reached 3.5-4 iron atoms per IRP1 molecule, proving that the Fe-S cluster was completely disrupted. An increase in IRP1 IRE-binding was observed upon the sequential addition of SIN-1/SOD and low concentrations of 2-mercaptoethanol, whereas SIN-1 alone induced a decrease in binding capacity which was not reversed by 2-mercaptoethanol, even at high concentrations. Moreover, nitrotyrosine adducts were detected on SIN-1-treated IRP1 by Western blot analysis.

Aconitate Hydratase↗
PubMed1983

Human beta and delta globin messenger RNAs turn over at different rates.

We have tested the hypothesis of Winslow & Ingram (1966) that the steady-state levels of human beta and delta globin proteins are determined, in part, by the stabilities of their respective messenger RNAs. Nucleated bone marrow cells were cultured under different RNA "chase" conditions, and samples were harvested at intervals thereafter. The quantities of beta and delta globin mRNA sequences in total RNA from these cells were measured by solution hybridization with specific 32P-labeled DNA probes. The average half-lives of beta and delta globin mRNAs are 16.5 and 4.5 hours, respectively. Both mRNAs are polyadenylated. The rapid turnover of delta globin mRNA accounts, at least in part, for the low level of delta globin mRNA in non-nucleated peripheral blood reticulocytes. The possibility that the rate of mRNA decay is determined by nucleotide sequence signals located in the 3' untranslated region is discussed.

Adult↗
PubMed1988

Identification of a transcriptionally active pseudogene in the chorion locus of the silkmoth Bombyx mori. Regional sequence conservation and biological function.

We have determined the primary structure of a 3500 base-pair part of the silkmoth chorion locus mapping in a region containing genes of late developmental specificity. This part of the locus was found to harbour a pseudogene related to one of the families of chorion genes encoding high cysteine proteins, HcB. The pseudogene exhibits an overall sequence identity of 84% to the consensus coding region of HcB chorion genes. A 95% identity was observed over a length of 190 base-pairs of its immediate 5' upstream sequences and the corresponding part of the consensus 5'-intergenic sequences of Hc gene pairs, normally encompassing 270 base-pairs. Thus, the pseudogene has retained part of the promoter region that includes sequence elements whose presence is thought to be necessary for transcriptional competence of HcB genes. The pseudogene is also characterized by the elimination of part of its first exon containing most of the 5' untranslated region, the ATG translation initiation codon and part of the signal peptide sequences. Its intron is longer than that of other HcB genes due to the insertion of a copy of a repetitive element that appears to be transcribed by RNA polymerase III. A previously characterized chorion cDNA clone, m2282, representing a rare mRNA sequence of late developmental specificity, was found to be identical to the pseudogene over its entirety spanning 65% of the pseudogene's second exon. Hybridizations of clones spanning a 260,000 base-pair domain of the chorion locus of Bombyx mori and of total genomic DNA to a subfragment of the cDNA clone containing relatively unique sequences, coupled to primer extension experiments, have demonstrated that m2282 mRNA originated from the pseudogene and that the pseudogene transcripts are initiated at the chorion cap site consensus sequence. We conclude that the 5'-flanking sequences retained by the pseudogene encompass elements necessary and adequate for correct transcriptional activation, but may not include those required for quantitative expression of the promoter. Possible reasons for the observed lack of random drift in the 5'-upstream sequences of the pseudogene and the maintenance of a functional promoter in a non-functional gene are discussed on the basis of the observation that elements resembling scaffold attachment sites are present in these sequences.

Amino Acid Sequence↗
PubMed1998

Partial sequencing of Reissner's fiber glycoprotein I (RF-Gly I).

The bulk of the secretion of the subcommissural organ is formed by glycoproteins that appear to be derived from two precursor forms of 540 and 320 kDa. Upon release into the ventricle, these glycoproteins aggregate to form Reissner's fiber. We report the isolation of three cDNA clones from a cDNA library prepared from bovine subcommissural organ RNA, by using an anti-Reissner's fiber serum for immunoscreening. Inserts of 0.7, 1.2, and 2.5 kb were amplified by the polymerase chain reaction, subcloned into pUC18 vector, and sequenced. Although restriction mapping of the three inserts initially suggested that all of them were derived from the same mRNA, sequence analysis showed that a short non-homologous region was present in the 0.7-kb insert when compared with the 1. 2-kb and 2.5-kb inserts, suggesting that they corresponded to two different, although highly homologous, mRNAs. Northern analyses showed a single mRNA species of approximately 9.5 kb present in the subcommissural organ and missing in the choroid plexus, brain cortex, and liver. In situ hybridization confirmed that the expression of the RNA was restricted to cells of the bovine subcommissural organ. Polyclonal antibodies raised against a synthetic peptide, whose amino-acid sequence was deduced from the 2.5-kb cDNA, reacted specifically with the bovine and rat subcommissural organ-Reissner's fiber complex. In immunoblots of bovine subcommissural organ, this antibody revealed the precursor 540-kDa form and its putative processed form of 450 kDa. It is concluded that the cloned cDNA encodes for the major constitutive glycoprotein of Reissner's fiber, here designated as RF-Gly I. The sequenced region of RF-Gly I displays a high degree of homology with some regions of the von Willebrand factor and certain mucins; it also displays two motifs homologous with repeats present in proteins of the spondin family and other proteins. A core sequence of the RF-Gly I repeats suggests that this molecule displays protein-binding properties.

Amino Acid Sequence↗
PubMed1994

Nitric oxide synthase: mRNA expression of different isoforms in human monocytes/macrophages.

To detect mRNA expression of nitric oxide synthase (NOS) isoforms in human monocytes/macrophages reverse transcription polymerase chain reaction (RT-PCR) was used. mRNA was isolated from stimulated or unstimulated monocytes/macrophages and RT-PCR was performed using oligonucleotide primers derived from mRNA sequences of either human endothelial constitutive (c) or human hepatocyte inducible (i) NOS. RT-PCR of mRNA isolated from resting monocytes and macrophages resulted in the amplification of a cNOS specific mRNA fragment. When the cells were stimulated with lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) prior to mRNA extraction, RT-PCR yielded an iNOS-specific amplification product. Whereas the activation of both cell types was accompanied by expression of iNOS mRNA, the cNOS signal seemed to be diminished upon immunostimulation. Not only in purified human monocytes but also in the human monocytoid cell lines MonoMac 6, THP-1, and U937 cNOS mRNA was detected. The data clearly demonstrate the presence of iNOS and cNOS mRNA in human monocytes/macrophages and provide the necessary tools to investigate the regulation of NO synthesis in these cell populations.

Amino Acid Oxidoreductases↗
PubMed1992

Regulation of the Escherichia coli uncH gene by mRNA secondary structure and translational coupling.

The uncH gene is one of the most poorly-expressed genes of the proton-translocating ATPase (unc) operon of Escherichia coli. We constructed in-frame lacZ fusions to uncH and used site-directed mutagenesis to decrease the stability of the putative mRNA secondary structure in the Shine and Dalgarno region for this gene. These mutations significantly increased the expression of uncH. We also used the unc-lac fusions to show that the insertion of stop codons and a frameshift mutation in uncF, the gene preceding uncH, caused a 10-fold reduction in uncH expression. Hybridization of total cellular RNA with a lacZ-specific probe indicated that transcriptional polarity could not account for the observed decrease in gene expression. These results demonstrate that uncH expression is controlled by mRNA sequences around the translational initiation region, and is translationally coupled to uncF, even in cases where the putative mRNA secondary structure is weakened or eliminated.

Bacterial Proteins↗
PubMed1985

Isolation and characterization of the glucose-6-phosphate dehydrogenase gene of Drosophila melanogaster.

To investigate the molecular basis of dosage compensation in Drosophila, a recombinant lambda phage containing the Drosophila melanogaster glucose-6-phosphatase dehydrogenase (G6PD) gene was isolated by differential screening of a Drosophila genomic lambda library with poly(A)+RNA obtained from polyribosomes enriched for or depleted of G6PD mRNA sequences. Of 44 000 plaques screened, a single phage, lambda DmG21, showed hybridization with the enriched poly(A)+RNA but not the depleted one. Confirmation that the Drosophila DNA fragment cloned in lambda DmG21 contains the G6PD gene sequence is based on the following observations. lambda DmG21 DNA shows hybridization only to the 18D region of the salivary gland X-chromosome, which is the known cytological locus for the G6PD gene. In vitro translation of the poly(A)+mRNA selected by hybridization to lambda DmG21 DNA sequences shows a polypeptide product of apparent Mr 55 000, identical to that of the monomeric unit of G6PD. When the putative coding sequence of G6PD is cloned into the expression vector lambda gt11, recombinant plaques are recognized by anti-G6PD immunoglobulin. A transcriptional map of the G6PD gene shows that it is divided into two exons, 0.9 kb (exon I) and 1.8 kb (exon II) long, which are separated by a 2.4-kb intron. The G6PD mRNA is 2.0 kb in length and the steady-state level of the mRNA is similar in both sexes. Measurement of the copy number of the G6PD gene in males and females shows the gene to be present once per X-chromosome in both sexes. No amplification of the gene sequence was observed in males. These results are, therefore, in agreement with the previous suggestion that dosage compensation is the result of enhanced transcription of X-linked genes in males.

Animals↗
PubMed2000

Ribosome traffic in E. coli and regulation of gene expression.

The ribosome traffic during translation of E. coli coding sequences was simulated, assuming that the rate of translation of individual codons is limited by the cognate tRNA availability. Actual translation rates were taken from Solomovici et al. (J. theor. Biol. 185, 511-521, 1997). The mean translation rates of the 4271 sequences cover a broad, two-fold range, whereas the local rate of translation along messengers varies three-fold on average. The simulation allows one to sketch the ribosome traffic on the polysome, in particular by providing the extent of mRNA sequences uncovered between consecutive ribosomes and the time during which these sequences are exposed. These parameters may participate in the control of mRNA stability and transcriptional polarity. By averaging the translation rates in a 17-codon window, assumed to be the sequence covered by a translating ribosome, and sliding this window along a given coding sequence, the addresses KMAX and KMIN, and the times TMAX and TMIN of respectively the slowest and the fastest translated window were determined. It is shown that under the assumptions made, TMAX sets the number of proteins translated from a given mRNA molecule per unit time, in case the delay between consecutive translation starts is below TMAX. Both windows display two strong biases, one as expected on the usage of codon frequencies, and the other surprisingly on the occurrence of amino acids.

Amino Acids↗
PubMed1978

Homologous nucleotide sequences between prokaryotic and eukaryotic mRNAs: the 5'-end sequence of the mRNA of the lipoprotein of the Escherichia coli outer membrane.

The sequence of the first 89 nucleotides at the 5' end of the mRNA for the lipoprotein of the Escherichia coli outer membrane is: GCUACAUGGAGAUUAACUCAAUCU-AGAGGGUAUUAAUAAUGAAAGCUACUAAACUGGUACU-GGGCGCGGUAAUCCUGGGUUCUACUCUG. The sequence of the first 72 nucleotides was established by direct sequencing methods and was extended to 89 residues on the basis of the known sequences of oligonucleotides obtained from complete digestion of the mRNA by ribonuclease T1 or A and the known amino acid sequence of the prolipoprotein. The mRNA has an untranslated region of 38 residues before the initiation codon, AUG. A unique feature of the 5'-end sequence of the mRNA is that the sequence of 12 nucleotides (GUAUUAAUAAUG) prior to, and including, the initiation codon is the same as that found at the ribosome-binding site for 80S ribosomes in brome mosaic virus RNA4, a eukaryotic mRNA [Dasgupta, R., Shih, D., Saris, C. & Kaesberg, P. (1975) Nature 256, 624-628].

Bacterial Proteins↗
PubMed1988

cDNA cloning and characterization of three genes uniquely expressed in cerebellum by Purkinje neurons.

The characteristics that distinguish the different neuronal cell types of an organism are believed to be primarily determined by unique patterns of cellular gene expression. The identification of cell-type specific molecules should therefore provide a good basis for understanding the biology of specific neuron types. In this paper, we describe the isolation of cDNA clones corresponding to mRNA uniquely expressed by Purkinje cells in mature mouse cerebellum. Three cDNA clones were selected from a library of normal mouse cerebellar cDNA by virtue of their failure to hybridize to mRNA sequences from the cerebella of Purkinje cell degeneration (pcd) mice. The cDNA clones were shown by in situ and Northern hybridization to correspond with mRNA present in Purkinje cells but absent or at low levels in other cell types of the cerebellum. By sequence analysis, clone PCD29 was determined to encode the calcium-binding protein calbindin-D28K. Clones PCD5 and PCD6 encode previously undescribed proteins of 99 and greater than 500 amino acids, respectively. All 3 PCD clones hybridized to mouse mRNA from sources other than cerebellum; clone PCD5 was found to have the most restricted expression, as it hybridized only to mRNA from cerebellum and eye. To define potential correlations between the PCD clones and mutations in the mouse genome known to affect Purkinje cells, clones PCD5, PCD6, and PCD29 were localized to mouse chromosomes 8, 6, and 4, respectively.

Animals↗
PubMed1992

The fusion protein gene of phocine distemper virus: nucleotide and deduced amino acid sequences and a comparison of morbillivirus fusion proteins.

The nucleotide sequence of the gene encoding the fusion protein of phocine distemper virus has been determined. The mRNA is 2206 nucleotides in length and contains one major open reading frame (ORF) of 1893 nucleotides encoding a potential protein of 631 amino acid residues. However, analogy with canine distemper virus (CDV) suggests that translation of the F protein starts at the sixth AUG codon in the mRNA sequence which is located at position 461, resulting in an F0 protein of exactly the same size (537 aa) as that of CDV. The overall homology at nucleotide level between the CDV and PDV F genes is 66%. The homology between the two F proteins of these respective viruses is 83%.

Amino Acid Sequence↗