Duplication detection in Japanese Duchenne muscular dystrophy patients and identification of carriers with partial gene deletions using pulsed-field gel electrophoresis

DNA samples from 21 unrelated Japanese patients with Duchenne muscular dystrophy (DMD) with nondeletion-type abnormality in the dystrophin gene and three samples from possible deletion carriers were analyzed using pulsed-field gel electrophoresis (PFGE). Among the 21 patients, 7 were found to carry partial duplications of the dystrophin gene spanning 50–400 kb. Of these 7 patients, 4 carried duplications corresponding to the major hot-spot regions for deletions (7.5–8.5 kb from the 5 end of cDNA), whereas two cases contained duplications in a region about 10 kb from the 5 end of cDNA, where causative mutations are reported to be rare. Only 1 case was found to contain a duplication of a region about 1 kb from the 5 end of cDNA, which is the reported duplication prone region. A combination of Southern blot analyses of conventional agarose gel electrophoresis and PFGE was confirmed to be useful, not only for detecting duplications and deletions, per se, but also for identifying carriers in the affected family.     

Molecular cloning,nucleotide sequence and expression of a cDNA encoding an intracellular protein tyrosine phosphatase,PTPase-2, from mouse testis and T-cells

The PTP-2 cDNA encoding an intracellular protein tyrosine phosphatase (PTPase-2) was isolated and sequenced from mouse testis and T-cell cDNA libraries. This PTP-2 cDNA was found to be homologous to human PTP-TC and rat PTP-S, and contained 1,551 nucleotides, including 1,146 nucleotides encoding 382 amino acids as well as 5 (61 nucleotides) and 3 (344 nucleotides) non-coding regions. Northern blot analysis indicated that PTP-2 mRNA of 1.9 Kb was most abundant in testis and kidney, although it was also present in spleen, muscle, liver, heart and brain.Abbreviations PTPase Protein Tyrosine Phosphatase (EC3.1.3.48) - PTKase Protein Tyrosine Kinase (EC2.7.1.112)     

Conserved and variable repeat structures in the Balbiani ring gene family in Chironomus tentans

Summary The four Balbiani ring (BR) genes, BR1, BR2.1, BR2.2, and BR6 in the midge Chironomus tentans constitute a gene family encoding secretory proteins with molecular weights of approximately 10⁶ daltons. The major part of each gene is known to consist of tandemly organized composite repeat units resulting in a hierarchic repeat arrangement.Here, we present the sequence organization of the 5 part of the BR2.2 and BR6 genes and describe the entire transcribed part of the two genes. As the BR1 and BR2.1 genes were also fully characterized recently, this allows the comparison of all genes in the BR gene family.All four genes share the same exon-intron structure and have evolved by gene duplications starting from a common ancestor, having the same overall organization as the BR genes of today.The genes encode proteins that have an approximately 10,000-amino acid residue extended central domain, flanked by a highly charged, 200-residue amino-terminal domain and a globular 110-residue carboxy-terminal domain. Exons 1–3 and the beginning of exon 4 encode the amino-terminal domain, which throughout contains many regions built from short repeats. These repeats are often degenerate as to repeat unit and sequence and are present in different numbers between the genes. In several instances these repeat structures, however, are conserved at the protein level where they form positively or negatively charged regions.Each BR gene has a 26–38-kb-long exon 4, which consists of an array of 125–150 repeat units and encodes the central domain. The number of repeat units appears to be largely preserved by selection and all repeat units in the array are very efficiently homogenized. Occasionally variant repeats have been introduced, presumably from another BR gene by gene conversion, and spread within the array.Introns 1–3 at the 5 end of the genes have diverged extensively in sequence and length between the genes. In contrast, intron 4 at the 3 end is virtually identical between three of the four genes, suggesting that gene conversion homogenizes the 3 ends of the genes, but not the 5 ends. Offprint requests to: L. Wieslander     

Reaction on alternating GC oligonucleotide templates

Summary The condensation reactions of activated nucleotides, ImpN or 2-MeImpN, with the selfcomplementary ribo-octanucleotide GCGCGCGC or with the partially self-complementary heptanucleotide GCGCGCG were studied. The templatedirected reaction of 2-meImpC with the heptamer yields the 3–5 octamer as the main product. All other reactions yield 2–5-linked octamers and pyrophosphates as major products. Surprisingly, 2-MeImpG facilitates the reaction of 2-MeImpC with the heptamer.Procedures for the analysis by gel electrophoresis of the oligomeric products obtained in reactions of this kind are described.     

Abiotic formation of oligonucleotides on basalt surfaces

The complication and further evolution of abiotic syntheses products occurred under environmental influences at the prebiological stage. From this point of view, the influence of some types of irradiation on the organic molecules adsorbed on the surfaces of volcanic rocks, appeared to be of great importance. In this connection, the effect of gamma rays on the AMP molecules adsorbed on mineral surfaces such as cinders and ashes has been studied. It has been shown that they can polymerize with the formation of oligonucleotides. The treatment of oligomers obtained by venom phosphodiesterase has shown that a polymeric product has mainly 3–5 and 2–5 bonds between nucleotides. The results obtained have been discussed from the evolutionary aspect.     

Biological activities of human interferon and 2′–5′ oligoadenylates in plants

Exogenous human interferon 2 (IFN) and 2–5 oligoadenylates (2–5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1–1 u/ml and 2–5A at 1–10 nM.     

Oligomerization of deoxynucleoside-bisphosphate dimers: Template and linkage specificity

Evidence is presented that a poly(U) template selectively favors the oligomerization of the activated, 3–5 pyrophosphate-linked dimer pdAppdAp, in comparison with the 3–3 and 5–5 linked dimers. In the absence of poly(U), the 5–5linked dimer is the most reactive, and chains are formed which are more than 60 monomer units in length.Nucleic Acid-Like Structures V. For the previous paper in this series see Visscher and Schwartz (1988).     

Age Dependence of the Level ofcopia Retrotransposon Expression in the Testes of Drosophila melanogaster

Expression of the lacZ reporter gene controlled by various deletion derivatives of the regulatory region of the copia retrotransposon was studied in the testes of adult transgenic males of the Drosophila melanogaster y ¹ w ^67c23(2) strain at the age of 3, 6–9, 12–15, 18–21, and 24–27 days. When the construct contained the full-length regulatory region, which included the 5-long terminal repeat (LTR) and the 5-untranslated region (UTR), expression was the lowest in males aged 12–15 days and the highest in males aged 3 or 24–27 days. A similar V-shaped age dependence was previously observed for the copia transposition rate and RNA content in a strain with a high rate of copia transposition. Thus, the V-shaped age dependence of expression, which is unusual for Drosophila, proved to be characteristic of copiaregardless of its transposition rate. Deletion of the 5 or 3 end of the LTR, but not of the UTR, changed the age dependence of the level of reporter gene expression. In this case, expression was the highest in 3-day-old males and gradually decreased with age, as typical for many Drosophila genes. It was assumed that the 5- and 3-terminal regions of the copiaLTR contain regulatory elements responsible for the V-shaped age dependence of expression, while the expression level depends to a greater extent on the regulatory elements of UTR.     

Influence of theophylline on concentrations of cyclic 3′,5′-adenosine monophosphate and cyclic 3′,5′-guanosine monophosphate of rat brain

The influence of theophylline (2.5–100 mg/kg p.o.) on cyclic 3,5-adenosine monophosphate (cAMP) and cyclic 3,5-guanosine monophosphate (cGMP) in brain of Sprague-Dawley rats (0.5–3.0 hr after administration of theophylline) was investigated. It was found that theophylline increases cAMP and cGMP levels when administered in a dose of 25 mg/kg or higher. A significant decrease of cGMP level was observed after administration of 10 mg/kg. The results of this study suggest that the influence of theophylline on cyclic nucleotide levels of rat brain is the result of two factors: (a) inhibitory properties of theophylline on cAMP and cGMP phosphodiesterases and (b) competition of theophylline with adenosine.     

Isolation and characterization of cDNAs encoding vacuolar H+-pyrophosphatase isoforms from rice (Oryza sativa L.)

The vacuolar H⁺-pyrophosphatase (V-PPase) is an electrogenic H⁺ pump, which was found in the plant vacuolar membrane. Two cDNA clones (OVP1 and OVP2) encoding the V-PPase were isolated from cultured rice (Oryza sativa L.) cells and subsequently sequenced. The sequence analysis has revealed thatOVP1 contains 2316 nucleotides of open reading frame (ORF) and 362 nucleotides of the 3-untranslated region, whereasOVP2 comprises 2304 nucleotides of ORF and 312 nucleotides of the 3-untranslated region. The nucleotide sequences of ORF ofOVP1 andOVP2 are 80.7% identical, and their 5- and 3-untranslated regions have 39.4% and 48.4% identity, respectively. The polypeptides encoded by the ORF ofOVP1 andOVP2 contain 771 and 767 amino acids, respectively, and the sequences of the OVP proteins are very similar to those of other V-PPases, which are shown to have 85–91% homology. Chromosomal mapping by RFLP techniques demonstrates that OVP1 and OVP2 are isoforms encoded by different genes. BothOVP1 andOVP2 are mapped on the same chromosome (chromosome 6) to a distance of ca. 90 cM. Northern analysis indicates that theOVP1 andOVP2 are also expressed in intact rice plants andOVP2 shows higher expression in the calli than the roots and shoots, compared toOVP1. These results show that at least two genes encoding the V-PPases are present in rice genome and their expressions are probably regulated in a different manner.     

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase synthesis by antisense oligodeoxynucleotides

Oligodeoxynucleotides 18 nucleotides in length having sequences complementary to regions spanning the initiation codon regions of ornithine decarboyxlase or S-adenosylmethionine decarboxylase mRNAs were tested for their ability to inhibit translation of these mRNAs. In reticulocyte lysates, a strong and dose dependent reduction of ornithine decarboyxlase synthesis in response to mRNA from D-R L1210 cells was brought about by 5-AAAGCT GCTCATGGTTCT-3 which is complementary to the sequence from - 6 to + 12 of the mRNA sequence but there was no inhibition by 5-TGCAGCTTCCATCACCGT-3. Conversely, the latter oligodeoxynucleotide which is complementary to the sequence from – 6 to + 12 of the mRNA of S-adenosyl methionine decarboxylase was a strong inhibitor of the synthesis of this enzyme in response to rat prostate mRNA and the antisense sequence from ornithine decarboxylase had no effect. The translation of ornithine decarboxylase mRNA in a wheat germ system was inhibited by the antisense oligodeoxynucleotide at much lower concentration than those needed in the reticulocyte lysate suggesting that degradation of the hybrid by ribonuclease H may be an important factor in this inhibition. These results indicate that such oligonucleotides may be useful to regulate cellular polyamine levels and as probes to study control of mRNA translation.Abbreviations ODC ornithine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO difluoromethylornithine     

Montmorillonite Catalysis of 30–50 Mer Oligonucleotides: Laboratory Demonstration of Potential Steps in the Origin of the RNA World

Elongation of the primer ³²pdA(pdA)₈pA proceeds by thereaction of the 5-phosphorimidazolides of adenosine and uridine in the presence of montmorillonite clay. Daily addition of the activated nucleotides for up to 14 days results in theformation of 40–50 mers using the 5-phosphorimidazolide of adenosine (ImpA) and 25–30 mers using the 5-phosphorimidazolide of uridine (ImpU). The limitation on thelengths of the chains formed is not due to the inhibitors formedsince the same chain lengths were formed using 2–3 times the amount of montmorillonite catalyst. The shorter oligomers formedby the addition of U monomers is not due to its greater rate ofdecomposition since it was found that both the A and the U adducts decompose at about the same rates. Alkaline phosphatase hydrolysis studies revealed that some of the oligomers are cappedat the 5-end to form, with ImpA,Ap³²pdA(pdA)₈pA(pA)_n. The extent of capping depends on the reaction time and the purine or pyrimidine base inthe activated mononucleotide. Hydrolysis with ribonuclease T₂ followed by alkaline phosphatase determined the sites ofthe 3, 5- and 2, 5-phosphodiester bonding to the primer. The potential significance of the mineral catalyzed formation of 50 mer oligonucleotides to the origin of life basedon RNA (the RNA world scenario) is discussed.     

Molecular cloning,characterization and expression of Mn-superoxide dismutase from the rubber tree (Hevea brasiliensis)

The gene and the RNA from Arabidopsis thaliana for the plastid-located glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) and their encoded product have been studied. The gene (designated ATS1) was isolated by screening a DASH genomic library for cross-hybridization with a radiolabeled probe prepared from cDNA for GPAT from squash. cDNA clones representing the mRNA were isolated by screening a ZAPII cDNA library for hybridization with a radiolabeled probe prepared from a DNA fragment of ATS1. The nucleotide sequences of the gene and the cDNA were determined, and the 5 end of the RNA was mapped by primer extension. Sequences similar to the TATA box, polyadenylation sequences and intron-splicing sequences were found at the expected locations. The pre-mRNA was 3288 nucleotides long and contained 5 and 3-untranslated sequences of 57 and 442 nucleotides, respectively. The coding sequence of 1377 nucleotides was interrupted by 11 introns of 1412 nucleotides in total and the 3-untranslated sequence contained another intron of 94 nucleotides. The open-reading frame encoded a polypeptide of 459 amino acid residues, the amino acid sequence of which was highly homologous to those of precursors to plastid-located GPATs from squash and pea. The enzymatic activity of a gene product that was over-produced in Escherichia coli confirmed the indentity of the gene.Abbreviations ACP acyl carrier protein - GPAT glycerol-3-phosphate acyltransferase - IPTG isopropyl--thiogalactopyranoside.     

Observation of a distinct transition in the mode of interconversion of ring pucker conformers in non-crystalline d-ribose-2'-d from 2H NMR spin-alignment

Internal motions of d-ribose selectively ²H-labeled at the 2 position were measured using solid state ²H NMR experiments. A sample of d-ribose-2 -d was prepared in a hydrated, non-crystalline state to eliminate effects of crystal-packing. Between temperatures of –74 and –60°C the C2–H2 bond was observed to undergo two kinds of motions which were similar to those of C2–H2/H2 found previously in crystalline deoxythymidine (Hiyama et al. (1989) J. Am. Chem. Soc., 111, 8609–8613): (1) Nanosecond motion of small angular displacement with an apparent activation energy of 3.6 ± 0.7 kcal mol^–1, and (2) millisecond to microsecond motion of large amplitude with an apparent activation energy 4 kcal mol^–1. At –74°C, the slow, large-amplitude motion was best characterized as a two-site jump with a correlation time on the millisecond time scale, whereas at –60°C it was diffusive on the microsecond time scale. The slow, large-amplitude motions of the C2–H2 bond are most likely from interconversions between C2-endo and C3-endo by way of the O4-endo conformation, whereas the fast, small-amplitude motions are probably librations of the C2–H2 bond within the C2-endo and C3-endo potential energy minima.     

Differential methylation at the 5′ and the 3′ CCGG sites flanking the X chromosomal hypervariable DXS255 locus

Summary The degree of methylation at the 5 and 3 CCGG sequences flanking the variable number of tandem repeat (VNTR) region of the DXS255 locus at Xp11.22 was analysed separately in several haematopoietic cell lineages. The 5 CCGG site on active chromosomes was found to be completely methylated in B and T lymphocytes and granulocytes. Methylation of the 5 site on inactive X chromosomes differed between females (0%–60%), but was consistent in different cell lineages obtained from individual females. In contrast, methylation at the 3 CCGG site on active chromosomes was found to vary in B lymphocytes (40%–100%), whereas complete methylation was found in T lymphocytes and granulocytes. The extent of methylation on inactive X chromosomes was found to differ significantly between B lymphocytes (17%), T lymphocytes (54%) and granulocytes (82%). Thus, methylation at the 5 CCGG site seems to be primarily related to the status of X chromosome inactivation, whereas methylation at the 3 CCGG site is mainly subject to cell-lineage-specific influences.     

Nucleotide sequence of the 3′-terminal region of chilli vein-banding mottle potyvirus isolated from pepper in Thailand

The sequence of the 2227 nucleotides of the 3-terminus of chilli vein-banding mottle virus-Chiangmai isolate (CVbMV-CM1) genome was determined. This sequence encodes a putative 287 amino acid coat protein. Downstream of coat protein coding region is a 288 nucleotide untranslated sequence terminated by a polyadenylate tract. The amino acid sequence of coat protein contained aspartic acid–alanine–glycine tripeptide, which may correlate with the high frequency of aphid transmissibility. The cleavage site between large nuclear inclusion protein (NIB) and coat protein was glutamine/serine. Comparison of 3 non-coding region nucleotide and coat protein amino acid sequences of CVbMV-CM1 with those of other 18 potyviruses showed 44–56% and 54–65% homology respectively, suggesting CVbMV should be regarded as new potyvirus.     

Sequence-specific interactions of wound-inducible nuclear factors with mannopine synthase 2′ promoter wound-responsive elements

A 318 bp mannopine synthase 2 (mas2) promoter element from the T-DNA of Agrobacterium tumefaciens can direct wound-inducible and root-preferential expression of a linked uidA gene in transgenic tobacco plants. Wound inducibility is further enhanced by sucrose in the medium. Promoter deletion analysis indicated that the sucrose enhancement is conferred by a region extending from –318 to –213. DNase I footprinting indicated that an A/T-rich DNA sequence in this region is protected by tobacco nuclear factors. Regions extending from –103 to +66 and from –213 to –138 directed wound-inducibile expression of a linked uidA gene when placed downstream of a CaMV 35S enhancer or upstream of a truncated (–209) CaMV 35S promoter, respectively. DNase I footprinting analyses indicated that proteins from wounded tobacco leaves specifically bound to three contiguous motifs downstream of the mas2 TATA box. In addition to a common retarded band formed by the upstream wound-responsive element complexed with proteins from either wounded or unwounded tobacco leaves, two unique retarded bands were observed when this element was incubated with protein from wounded leaves. Methylation interference analysis additionally identified an unique motif composed of promoter elements and nuclear factors derived specifically from wounded tobacco leaves. We propose a model to describe the involvement of nuclear factors with mas2 promoter elements in wound-inducible gene expression.     

Changes in brain components during the development of mice homozygous for the locus “dwarf” (dw)1,5

Brain composition and developmental changes were investigated in mice homozygous for the locus dwarf, and characterized by a reduced level of growth hormone, thyroid stimulating hormone, and prolactin, and by secondary hypothyroidism. The difference in adult brain weight (–32%) between the dwarf and the normal mice was not found to parallel the difference in body weight (–71%), whereas the differences in the weight of the liver (–79%) and that of the kidney (–75%) did. Several biochemical parameters of brain development were assayed in dwarf and normal mice between the ages of 15 and 210 days. Levels of cerebrosides, sulfatides, gangliosides, phospholipids, cholesterol, protein, and RNA (per gram wet weight) were the same for the dwarf and the controls, but the net difference in total brain DNA was less than the net total brain RNA difference (–11% vs. –27%). Total brain lipids (absolute quantities) were the same at 15 days. The difference was –37% by the 50th day, and remained constant thereafter. No change in the specific activity of 2,3-cyclic nucleotide 3-phosphohydrolase or 3-phosphoadenosine-5-phosphosulfate: galactocerebroside sulfotransferase was observed. These data suggest that the regulation of the development of brain structures is maintained, but the level of the synthesis of the various brain constituents is reduced in proportion to the brain weight. The development of the dwarf brain seems to proceed harmoniously.Abbreviations used PAPS 3-phosphoadenosine-5-phosphosulfate - PAPS-CST 3-phosphoadenosine-5-phosphosulfate:galactocerebroside sulfotransferase - CNP 2, 3-cyclic nucleotide 3-phosphohydrolase - Neu NAc N-acetylneuraminic acid This paper is part of the Doctorat d'Etat thesis of L. L. Sarliève.