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PubMed1992

Two types of murine CD34 mRNA generated by alternative splicing.

To characterize and clarify the function of CD34 antigen experimentally, we isolated two types of CD34 mRNA from a cDNA library of murine stromal cell line, PA-6 stimulated with lipopolysaccharide (LPS) and 12-o-tetra-decanoylphorbol 13-acetate (TPA) using a human CD34 probe. In addition to the clone (open reading frame [ORF]:1149bp) reported by Brown et al, a novel clone (ORF:978 bp) was obtained. The difference between the two clones was in the cytoplasmic portion of CD34; the former has 73 amino acids, while the latter has 16. We investigated the genomic sequence of cytoplasmic portion and found conserved nucleotide sequences at the exon-intron junction (GT ... AG). Thus, it was concluded that alternative splicing gave two types of CD34 mRNA. A novel clone contains the longer cDNA, including a insert of 156 bp, but results in a shorter predicted coding sequence because of the introduction of an inframe stop codon. Northern blot analysis using a murine cDNA probe (HindIII fragment, 900 bp) showed that CD34 was highly expressed in the brain and testis, and moderately in the thymus, spleen, and bone marrow, but not in adult liver. However, day 12 to 14 fetal liver cells showed significant expression of CD34. Quantitative reverse transcription polymerase chain reaction showed that spleen, thymus, bone marrow, and testis RNA gave two bands of almost equal intensity, but in the brain a novel clone was expressed three times more than the other clone. Furthermore, Northern blot analysis using a probe (156 bp) specific for the spliced intracellular region confirmed the significant mRNA expression of a novel clone. Although the biologic significance of alternative splicing remains to be elucidated, it is suggested that a different carboxyterminal tail causes a change in signal transduction.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the rat Bmyc gene.

We have cloned and sequenced the rat Bmyc gene. The rat Bmyc gene contains sequences related to the central part of c-myc, namely the first intron, the second exon, and the noncoding part of the third exon. The homology drops in the 3' part of the c-myc second exon, but continues in the noncoding part of the third exon. We have sequenced the total predicted coding region of the Bmyc. The longest open reading frame in Bmyc suggests a protein of 178 amino acids, which is only 41% of the c-myc protein size. To confirm the putative open reading frame, we have produced a trpE-Bmyc protein that is detected with a pan-myc antibody. We discuss these findings in the context of potential functional domains and the possibility of overlapping and distinct activities of myc-family proteins.

Amino Acid Sequence↗
PubMed1985

Design verification on a speech synthesizer using electron beam testing techniques.

The use of Electron Beam Testing (EBT) techniques in the verification both of the circuit and of electric simulation tools turns out to be very powerful and promises to improve the reliability and performance of VLSI circuit design by providing an effective feedback from measurements to circuit design. That is true within the traditional design environment and, more effectively, in a standard cell design approach, characterized by a fast development and update time. In the latter case it is really possible to take strong advantage of tools and circuit improvements. The design tool taken into account is the world-wide used electrical simulator SPICE. The EBT system used, allows in sampling mode 1 ns time resolution and applies an oscilloscope with programmable digitizer to display the acquired waveforms. The actual example is carried out on a 2-phase generator inside a VLSI circuit: a speech synthesizer (Linear Predicting Code technique) designed in CSELT and implemented in a N-MOS 4 micron process by SGS-ATES.

Communication Devices for People with Disabilities↗
PubMed1988

Tactile communication of speech: comparison of two computer-based displays.

Two methods of encoding speech for tactile displays were compared in discrimination experiments using speech segments. One display represented the short-term speech spectrum in time-swept mode and used vibration amplitude to encode spectral amplitude. The other represented the linear predictive coding (LPC)-derived vocal tract shape as a filled bar graph in which the number of active vibrators was used to encode cross sectional area. The displays were applied to the thigh via a matrix of vibrators. The vibrators were driven at 250 Hz during voiced segments, and by random noise during unvoiced segments. Overall results show a slight superiority for the spectral display in vowel discrimination. Detailed results were analyzed in terms of an articulatory description of the speech stimuli, a multidimensional scaling (MDS) analysis of confusions, and an ideal receiver analysis. The results of these analyses suggest that the detailed characteristics of the tactile patterns were only crudely discriminated.

Computer Systems↗
PubMed1985

Electrodiagnostic model for motor unit action potential (MUAP) generation.

The paper presents the development of inverse analysis of EMG, in terms of a Linear Prediction Coding (LPC) model and its parametric simulation of recorded single motor-unit action potential, for electrodiagnosis of neuromuscular disorders due to motor neuron degeneration, regeneration after lesion, myopathy, etc.

Action Potentials↗
PubMed1988

Identification and characterization of a varicella-zoster virus DNA-binding protein by using antisera directed against a predicted synthetic oligopeptide.

We have identified, in varicella-zoster virus (VZV)-infected cells, the product of the gene predicted to code for the VZV analog of the herpes simplex virus major DNA-binding protein. The open reading frame of the VZV gene has the potential to code for a protein with a predicted molecular weight of 132,000 (a 132K protein). To detect the protein, a 12-amino-acid oligopeptide corresponding to the carboxyl terminus of the putative open reading frame was synthesized and used to prepare antisera in rabbits. The resulting antibodies reacted specifically in Western immunoblot analysis and immunoprecipitation with a single 130K polypeptide found in VZV-infected cells. The specific reactivity of the antisera with the 130K polypeptide was inhibited by the addition of synthetic peptide. Immunofluorescence studies with the antisera as probe for the 130K polypeptide suggested that this peptide is located predominantly within the nuclei of infected cells. Analysis of proteins that bind to single-stranded DNA immobilized on cellulose matrices indicated that 30 to 50% of the 130K polypeptide is capable of interacting with single-stranded DNA and that this interaction is overcome with 0.5 M NaCl. Thus, we have prepared a specific polyclonal antiserum that identifies a VZV DNA-binding protein whose properties are similar to those of the herpes simplex virus ICP8 (Vmw130) DNA-binding protein.

Amino Acid Sequence↗
PubMed1983

The erbB gene of avian erythroblastosis virus is a member of the src gene family.

The erbB gene of an avian erythroblastosis virus, AEV-H, was determined to be 1812 nucleotides long and was predicted to code for a protein of 67,638 daltons. Unexpectedly, a sequence of 285 amino acids in the middle of the protein showed a significant homology (38%) with the sequence in the carboxy terminus of p60src. The nucleotide sequence of a mutant of AEV-H, td-130, which induces sarcomas but not erythroblastosis in chicken, was also analyzed. A deletion of 169 nucleotides was identified in the 3' half of the erbB gene, indicating that the gene codes for a truncated protein with the predicted molecular weight of 46,667. These findings suggest that the homologous domain of erbB protein with its N-terminal portion is sufficient for the transformation of fibroblasts and that one-third of the carboxy-terminal domain has a key role for the transformation of erythroid cells.

Alpharetrovirus↗
PubMed1985

Structural homologies between RNA gene segments 10 and 11 from UK bovine, simian SA11, and human Wa rotaviruses.

The nucleotide sequences of gene segments 10 and 11 from UK bovine rotavirus have been determined. Gene 10 is 751 nucleotides long and contains a single long open reading frame capable of coding for a protein of 175 amino acids. When compared with the published data for gene 10 of the simian rotavirus SA11 and human Wa strains it was found to be more closely related to the SA11 structure (92% nucleotide sequence homology; 97% amino acid sequence homology) than to the human Wa structure (84% nucleotide, 86% amino acid sequence homology). All three strains have two potential N-glycosylation sites in the hydrophobic N terminus of the gene 10 protein. Gene 11 from UK bovine rotavirus is 667 nucleotides long with a single long open reading frame capable of coding for a protein of 198 amino acids. When compared with the published sequence of gene 11 from the human rotavirus Wa, the UK bovine rotavirus gene 11 was found to be one nucleotide longer in the 5'-noncoding region and three nucleotides longer in the coding region. The nucleotide sequence homology was 86%. The predicted proteins coded by segment 11 in UK and Wa rotaviruses are both rich in serine and threonine (23%) and very hydrophilic, but differ appreciably in amino acid sequence (83% homology).

Amino Acid Sequence↗
PubMed1990

A mutation of the transactivation gene of satellite bacteriophage P4 that suppresses the rpoA109 mutation of Escherichia coli.

Satellite bacteriophage P4 requires the products of the late genes of a helper such as P2 in order to grow lytically. The Escherichia coli rpoA109 mutation, which alters the alpha subunit of RNA polymerase, prevents transcription of the late genes of bacteriophage P2. Suppressor mutations that define the P2 ogr gene overcome this block. We found that P4 lytic growth using a P2 ogr+ prophage helper was prevented by the rpoA109 mutation but that this block was overcome when the P2 helper carried the suppressor mutation in the ogr gene. Furthermore, we isolated and characterized four independent mutations in P4, called org, that suppress the E. coli rpoA109 mutation by allowing P4 lytic growth using a P2 ogr+ helper. DNA sequence analysis revealed that the four independent org mutations are identical and that they occur in the P4 delta gene, which codes for a factor that positively regulates the transcription of the P2 and P4 late genes. delta is predicted to code for a basic 166-amino-acid residue protein. Each 83-residue half of the predicted delta gene product is similar to the predicted 72-residue proteins encoded by the ogr gene of P2 and the B gene of phage 186.

Alleles↗
PubMed1991

Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor is a member of the guanine nucleotide binding protein-coupled receptor superfamily with seven predicted hydrophobic transmembrane domains. In vitro transcripts from cloned cDNA templates encompassing the predicted protein coding domain, when injected into Xenopus oocytes, resulted in expression of functional GRP receptors. The predicted amino acid sequence of the open reading frame in cDNA clones matches the amino-terminal sequence as well as the sequence of four tryptic fragments isolated from the purified protein. Expression of the GRP receptor cDNA in model systems potentially provides a powerful assay for the development of subtype-specific receptor antagonists that may prove to be of therapeutic importance in human small cell lung carcinoma.

Amino Acid Sequence↗
PubMed1991

Amino acid sequence similarities of the mitochondrial short chain delta 3, delta 2-enoyl-CoA isomerase and peroxisomal multifunctional delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase enzyme in rat liver. The proposed occurrence of isomerization and hydration in the same catalytic domain of the multifunctional enzyme.

We report the isolation and characterization of a cDNA encoding the mitochondrial short chain delta 3, delta 2-enoyl-CoA isomerase from rat liver. Tryptic fragments of the purified protein were generated, purified, and sequenced. A rat liver cDNA library, constructed in the plasmid vector pUEX1 was screened with oligonucleotides synthesized on the basis of peptide sequences. The obtained clone contained 783 bases predicting to code the entire mature protein of 261 amino acids. The molecular weight of 29,300 predicted from cDNA-derived sequences was consistent with the subunit size determined earlier. A high degree of similarity was noted between the amino acid sequence of isomerase and that of the amino-terminal half of peroxisomal multifunctional isomerase-hydratase-dehydrogenase enzyme and mitochondrial 2-enoyl-CoA hydratase in rat liver. These similarities also appeared at the level of predicted secondary structural elements, suggesting that hte rat multifunctional enzyme has both the isomerization and hydration activities in the amino-terminal domain. This idea is further supported by the proposed existence of only one CoA-binding site in the amino-terminal half of the multifunctional enzyme and by previous studies suggesting that the transfer of the substrate from the isomerization site to the hydration site occurs without aqueous bulk phase (Palosaari P.M., and Hiltunen, J. K. (1990) J. Biol. Chem. 265, 2446-2449).

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1982

Cytochrome oxidase subunit III gene in Neurospora crassa mitochondria. Location and sequence.

We have located and sequenced the gene for cytochrome oxidase subunit III (CoIII) in Neurospora crassa mitochondria. The CoIII gene is located downstream from the small rRNA gene within a cluster of tRNA genes and is coded by the same strand as the tRNA and the rRNA genes. Like the tRNA and the rRNA genes, the CoIII gene is also flanked by the GC-rich palindromic DNA sequences which are highly conserved in N. crassa mitochondria. The CoIII coding sequence predicts a protein 269 amino acids long including 8 tryptophan residues. All 8 tryptophan residues are coded for by UGA. This supports our previous conclusion based on the anticodon sequence of N. crassa mitochondrial tryptophan tRNA and provides evidence for the notion that use of UGA as a codon for tryptophan rather than chain termination may be a feature common to most mitochondrial protein synthesis systems. The close correspondence between the amino acid composition of N. crassa CoIII and that of the protein predicted by the CoIII gene sequence suggests that unlike in mammalian mitochondria, AUA is a codon for isoleucine and not for methionine in N. crassa mitochondria. The N. crassa CoIII sequence shows strong homologies to the corresponding yeast and human proteins (53 and 47%, respectively). The overall hydrophobic character of the protein is consistent with suggestions that most of CoIII is embedded in the mitochondrial inner membrane.

Amino Acid Sequence↗
PubMed1992

A vir-repressed gene of Bordetella pertussis is required for virulence.

Coordinate regulation of gene expression in Bordetella pertussis is controlled by the products of the vir locus, BvgA and BvgS. In the presence of modulating signals such as MgSO4 and nicotinic acid, expression of vir-activated genes (vag) is reduced, while expression of vir-repressed genes (vrg) is maximal. We have cloned one of these vir-repressed genes, vrg-6, in Escherichia coli. DNA sequencing has shown that vrg-6 is contained on a single EcoRI restriction endonuclease fragment and is predicted to code for a protein of 105 amino acids with a molecular weight of 11,441. The predicted protein product appears to have two domains, one consisting of seven hydrophobic proline-rich pentameric repeats and the other consisting of five alkaline trimeric repeats. Southern blot analysis has revealed vrg-6-homologous sequences in the chromosomes of Bordetella bronchiseptica and Bordetella parapertussis, but, unlike Bordetella pertussis, these species do not express vrg-6-homologous RNA when grown under modulating conditions. In order to assess the role of vrg gene products in B. pertussis pathogenesis, two 18323 derivatives which harbor TnphoA insertions in vrg genes were analyzed in a mouse model of respiratory infection. Strain SK6, which carries a vrg-6::TnphoA mutation, failed to induce lymphocytosis and was significantly less able to colonize lungs and trachea than its parent strain 18323 or than SK18, which harbors a TnphoA fusion in the vrg-18 locus. This is the first evidence that a vir-repressed gene may play an important role in the virulence of B. pertussis and the pathogenesis of whooping cough.

Amino Acid Sequence↗
PubMed1991

The fission yeast dis3+ gene encodes a 110-kDa essential protein implicated in mitotic control.

The fission yeast mutant dis3-54 is defective in mitosis and fails in chromosome disjunction. Its phenotype is similar to that of dis2-11, a mutant with a mutation in the type 1 protein phosphatase gene. We cloned the dis3+ gene by transformation. Nucleotide sequencing predicts a coding region of 970 amino acids interrupted by a 164-bp intron at the 65th codon. The predicted dis3+ protein shares a weak but significant similarity with the budding yeast SSD1 or SRK1 gene product, the gene for which is a suppressor for the absence of a protein phosphatase SIT4 gene or the BCY1 regulatory subunit of cyclic AMP-dependent protein kinase. Anti-dis3 antibodies recognized the 110-kDa dis3+ gene product, which is part of a 250- to 350-kDa oligomer and is enriched in the nucleus. The cellular localization of the dis3+ protein is reminiscent of that of the dis2+ protein, but these two proteins do not form a complex. A type 1 protein phosphatase activity in the dis3-54 mutant extracts is apparently not affected. The dis3+ gene is essential for growth; gene disruptant cells do not germinate and fail in cell division. Increased dis3+ gene dosage reverses the Ts+ phenotype of a cdc25 wee1 strain, as does increased type 1 protein phosphatase gene dosage. Double mutant dis3 dis2 is lethal even at the permissive temperature, suggesting that the dis2+ and dis3+ genes may be functionally overlapped. The role of the dis3+ gene product in mitosis is unknown, but this gene product may be directly or indirectly involved in the regulation of mitosis.

Amino Acid Sequence↗
PubMed1984

Molecular basis of the glycoprotein-C-negative phenotype of herpes simplex virus type 1 macroplaque strain.

The basis for the inability of the macroplaque (MP) strain of herpes simplex virus type 1 to express mature glycoprotein C (gC) was examined. RNA transfer (Northern) blot analysis with hybridization probes from the region of the herpes simplex virus type 1 DNA known to encode the gC gene indicated that gC mRNA was produced in MP-infected HeLa cells at levels relative to other mRNAs comparable with that seen in KOS-infected cells. Comparative nucleotide sequence analysis of the gC gene from the MP and KOS strains, coupled with the results of recently reported marker rescue experiments, indicates that the inability of MP to produce gC is due to a frameshift mutation in the gC-coding sequence. Because two different (out-of-phase) open reading frames overlap the gC-coding sequence in the region of the mutation, MP mRNA can encode two gC-related polypeptides. Two polypeptides of the predicted size and precipitable by anti-gC antibodies were produced by in vitro translation of MP mRNA. These polypeptides have not been detected in extracts from infected cells with the same antibodies. Comparative nucleotide sequence analyses led to several corrections in the published sequence for the gC gene and the 17,800-molecular-weight polypeptide gene just to the right in KOS DNA. These relatively minor effects on the predicted amino code sequence of gC are tabulated.

Amino Acid Sequence↗
PubMed1988

Characterization of the full length cDNA for murine beta-1,4-galactosyltransferase. Novel features at the 5'-end predict two translational start sites at two in-frame AUGs.

We have isolated overlapping cDNA clones representing the full length (4038 base pairs) transcript for murine beta-1,4-galactosyltransferase. The coding sequence predicts a membrane-bound glycoprotein with three distinct structural features, a large COOH-terminal domain (355 amino acids), a single transmembrane domain (20 amino acids), and a short NH2-terminal domain. Primer extension analysis, S1 protection analysis, and RNA blotting demonstrate the presence of two sets of mRNA transcripts which differ in length by about 200 base pairs. The 5' boundary of the long transcripts maps upstream of two in-frame ATGs. The 5' boundary of the short transcripts maps between these two ATGs. These results predict that two related forms of beta-1,4-galactosyltransferase of 399 and 386 amino acids are synthesized as a consequence of alternative translation initiation. The long form (399 amino acids) has an NH2-terminal extension of 13 amino acids.

Amino Acid Sequence↗
PubMed1992

Configurational pattern discrimination responsible for dishabituation in common toads Bufo bufo (L.): behavioral tests of the predictions of a neural model.

Recently, a neural model of visual pattern discrimination for stimulus-specific habituation was developed, based on previous behavioral studies which demonstrated that toads exhibit a dishabituation hierarchy for different worm-like stimuli. The model suggests that visual objects are represented by temporal coding and predicts that the dishabituation hierarchy changes when the stimulus/background contrast direction is reversed or the stimulus size is varied. The behavioral experiments reported in this paper were designed to test these predictions. (1) For a pair of stimuli from the contrast reversal prediction, the experimental results validated the theory. (2) For a pair of stimuli from the size reduction prediction, the experimental results failed to validate the theory. Further experiments concerning size effects suggest that configural visual pattern discrimination in toads exhibits size invariance. (3) Inspired by the Groves-Thompson account of habituation, we found that dishabituation by a second stimulus has a separate process from habituation to a first stimulus. This paper serves as an example of a fruitful dialogue between experimentation and modeling, crucial for understanding brain functions.

Animals↗
PubMed1990

Computer prediction of the exon-intron structure of mammalian pre-mRNAs.

A novel approach to the problem of prediction of protein-coding regions is suggested. This approach combines the site prediction methods to predict splicing sites and the global coding region prediction methods to choose the best variant of spliced mRNA. One of the advantages of the suggested algorithm is that the resulting mRNA or protein sequence may then be immediately analyzed further. The true mRNA either coincides with the predicted one or ranks high in the list of variants. In the latter situation the predicted mRNA usually differs from the true one in only one or two of several exons. The combined approach allows the use of a priori information (e.g. the putative protein length or the number of exons). It is possible to use additional parameters not considered here, such as the preferred lengths of exons and introns, and particularly the preferred position of introns in the reading frame and the preferred codon position of exon termini.

Actins↗