Two polymorphicAvaI andHhaI sites in a differentially methylated region of the human H19 gene

Summary The H19 gene is paternally imprinted both in the human and mouse (Bartolomeiet al., 1991; Zhang and Tycko, 1992), although its expression pattern seems somewhat different between the two species (Jinno,et al., 1995). DNA-methylation is a promising candidate for a parent-of-origin mark of the gene, and a paternal allele-specific methylation imprint was recently identified at the mouse H19 locus (Tremblayet al., 1995). We found a 50% methylated region in the human H19 gene (Jinno, unpublished data). A search for polymorphisms in this region revealed two novelAvaI andHhaI RFLPs, which contribute to the detection of allele-specific methylation at the human H19 locus.PCR primers for the AvaI-site PANL2 5-GAGCCTGCCAAGCAGAGCG-3 - PANR2 5-CACATAAGTAGGCGTGACTTGA-3 PCR primers for the HhaI-site ASMA 5-CAATGAGGTGTCCCAGTTCCA-3 - PANR2 5-CACATAAGTAGGCGTGACTTGA-3     

Genetic Analysis of Pestiviruses at the 3′ End of the Genome

Specific PCR primers were selected for each pestivirus genotype which flanked the 3-part of the NS5B gene and more than three quarters of the 3-UTR. PCR products were sequenced in both directions using an automatic sequencing device and analyzed by computer package program DNASTAR. A comparative analysis of the 3 untranslated region (3-UTR) of 82 viruses, representing the four genotypes of the Pestivirus genus, provided a similar phylogenetic grouping as other genomic regions. Intertypic recombination was not observed, but Border disease virus (BDV) and Bovine viral diarrhoea virus type 1 (BVDV I) showed great intragenotypic variability. In most pestiviruses the stop codon is TGA, but BDV isolates were found to have either a TAG or a TAA stop codon. Various deletions and insertions were observed in the 3-UTR region. Viruses of the BVDV Ib group contained a characteristic deletion of 41 nucleotides. Compared to the 5-UTR, the 3-UTR was less conserved. The first 50–60 nucleotides were particularly variable, whilst the most conserved part was found at the 3 end of the studied region. All 82 viruses contained AT-rich stretches, the positions and sizes of which differed between the genotypes.     

Nucleotide sequence and determination of the extremities of the 26S ribosomal RNA gene in wheat mitochondria: evidence for sequence rearrangements in the ribosomal genes of higher plants

Summary The nucleotide sequence of the wheat mitochondrial 26S ribosomal RNA gene and flanking regions was determined and compared with mitochondrial 26S rRNA genes from maize and Oenothera. All three genes exhibit a high degree of homology except within two variable regions. When the plant mitochondrial 26S rRNA genes are compared with Escherichia coli 23S rRNA and chloroplast 23S and 4.5S rRNA genes, a third variable region is apparent close to the 3 end of the gene. The 5 and 3 ends of the wheat mitochondrial gene were determined by S1 nuclease mapping. Computer analysis of the wheat mitochondrial gene revealed several small sequences present either in the 5 region of the 26S rRNA gene or in the 18S rRNA gene.     

Bean yellow mosaic,clover yellow vein,and pea mosaic are distinct potyviruses: evidence from coat protein gene sequences and molecular hybridization involving the 3′ non-coding regions

Summary The sequences of the 3 1019 nucleotides of the genome of an atypical strain of bean yellow mosaic virus (BYMV-S) and of the 3 1018 nucleotides of the clover yellow vein virus (CYVV-B) genome have been determined. These sequences contain the complete coding region of the viral coat protein followed by a 3 non-coding region of 173 and 178 nucleotides for BYMV-S and CYVV-B, respectively. When the deduced amino acid sequences of the coat protein coding regions were compared, a sequence identity of 77% was found between the two viruses, and optimal alignment of the 3 untranslated regions of BYMV-S and CYVV-B gave a 65% identity. However, the degree of homology of the amino acid sequences of coat proteins of BYMV-S with the published sequences for three other strains of BYMV ranged from 88% to 94%, while the sequence homology of the 3 untranslated regions between the four strains of BYMV ranged between 86% and 95%. Amplified DNA probes corresponding to the 3 non-coding regions of BYMV-S and CYVV-B showed strong hybridization only with the strains of their respective viruses and not with strains of other potyviruses, including pea mosaic virus (PMV). The relatively low sequence identities between the BYMV-S and CYVV-B coat proteins and their 3 non-coding regions, together with the hybridization results, indicate that BYMV, CYVV, and PMV are distinct potyviruses.     

Nucleotide sequence of the coat protein genes of <Emphasis Type="Italic">Alstroemeria mosaic virus</Emphasis> and Amazon lily mosaic virus,a tentative species of genus <Emphasis Type="Italic">Potyvirus</Emphasis>

Summary. The nucleotide sequences of the 3 terminal region of the genomes of Alstroemeria mosaic virus (AlsMV) and the Amazon lily mosaic virus (ALiMV) have been determined. These sequences contain the complete coding region of the viral coat protein (CP) gene followed by a 3-untranslated region (3-UTR). AlsMV and ALiMV share 74.9% identity in the amino acid sequence of the CP, and 55.6% identity in the nucleotide sequence of the 3-UTR. Phylogenetic analysis of these CP genes and 3-UTRs in relation to those of 79 potyvirus species revealed that AlsMV and ALiMV should be assigned to the Potato virus Y (PVY) subgroup. AlsMV and ALiMV were concluded to have arisen independently within the PVY subgroup.     

Translation enhancer in the 3′-untranslated region of rotavirus gene 6 mRNA promotes expression of the major capsid protein VP6

Summary. The eleven rotavirus mRNAs contain 5-cap structures and most end with the 3-consensus sequence 5-UGACC-3. The UGACC functions as a common translation enhancer (3-TE-con) that upregulates viral protein expression through a process mediated by the nonstructural protein NSP3. To address the possibility that gene-specific enhancers are also contained in the untranslated regions (UTRs) of the rotavirus mRNAs, we used rabbit reticulocyte lysates to investigate the translation efficiencies of analog RNAs containing viral-specific 5-and 3-UTRs and the open reading frame for chloramphenicol acetyltransferase. These experiments combined with the analysis of full-length viral RNAs and RNAs containing 3-truncations showed that a highly active enhancer was present near the 5-end of the 139-nucleotide 3-UTR of the gene 6 mRNA (3-TEg6). The 3-TEg6 represents a functionally independent enhancer, as no other portion of the gene 6 mRNA was required for its activity. The 3-TEg6 differs significantly from the 3-TE-con in that the gene 6-specific enhancer does not require viral protein for activity and is formed by a sequence unique to only one of the eleven viral mRNAs. Together, our findings suggest that the 3-UTR of the gene 6 mRNA contains two TEs, one is gene-specific (3-TEg6) and the other is common to nearly all rotavirus genes (3-TE-con). The activity of the 3-TEg6 is likely important for directing the efficient translation of the gene 6 mRNA at levels sufficient to provide the 780 copies of VP6 necessary for the assembly of each progeny virion.     

Adenosine 3′,5′-cyclic monophosphate in rabbit cerebrospinal fluid during fever induced byE. coli-endotoxin

In rabbit cerebrospinal fluid during fever induced byE. coli-endotoxin adenosine 3,5-cyclic monophosphate concentrations are about 2-fold higher in comparison to normal values. In addition to prostaglandins of the E series adenosine 3,5-cyclic monophosphate might act as another mediator in the genesis of fever during infectious diseases.This investigation was supported by the Deutsche Forschungsgemeinschaft.     

Molecular localization of human IgG anti-F(ab′)2 reactivity with variable- and constant-region λ light-chain epitopes

Human IgG antibodies reacting with antigenic determinants on F(ab)₂ fragments represent generic antiidiotypic antibodies present in the serum of normal individuals. Additionally, the titers of these antibodies in the sera of patients with systemic lupus erythematosus (SLE) are inversely related to disease activity. Because these autoantibodies recognize predominantly light chain-related epitopes, especially type, we synthesized constant (C)- and variable (V)-related overlapping 7-mer peptides on polypropylene pins to determine anti-F(ab)₂-reactive epitopes on human light chains. ELISA reactivity of affinity-purified anti-F(ab)₂ antibodies obtained from normal individuals and from patients with SLE, as well as murine anti-human light-chain monoclonal antibodies specific for C and V subgroup-related determinants, was tested using the overlapping 7-mers of human light-chain sequence. The patterns of reactivity against C-related peptides were similar in both normal and SLE-derived anti-F(ab)₂ antibodies. However, reactivity profiles against V-related peptides were distinctively different between the normal and the SLE-associated anti-F(ab)₂ autoantibodies. A decrease in reactivity among the SLE IgG anti-F(ab)₂ antibodies was noted for particular amino acid V complementarity-determining region (CDR) residues, including glycine at positions 27 and 54, alanine at 16 and 37, and tyrosine at 28 and 91. This different pattern of reactivity from normal may indicate that in SLE there is a failure of antiidiotypic control mechanisms, as reflected by a defect in production of antibody to immunodominant V CDR residues.     

High-resolution SNP map of ASPN, a susceptibility gene for osteoarthritis

Osteoarthritis (OA) is a very common bone and joint disease characterized by breakdown of cartilage in the joint. We recently found that an aspartic-acid repeat polymorphism of the asporin gene (ASPN) on chromosome 9 is associated with susceptibility to OA in Japanese. We provide here a high-resolution single nucleotide polymorphism (SNP) map within a 33.4-kb genomic region containing ASPN. A total of 19 SNPs were isolated from the region by systematic screening using 48 Japanese patients with OA: 7 SNPs in the 5 flanking region, 8 in introns, and 4 in the 3 untranslated region. Nine SNPs were novel. This high-resolution SNP map will be a useful resource for analyzing genes associated with OA and other bone and joint diseases.     

Rhinovirus detection using probes from the 5′ and 3′ end of the genome

Summary This study investigated the abilities of cDNA probes from the 5 and 3 ends of the genome of human rhinoviruses (HRV-) 14, 9, and 1B to detect RNA from 59 rhinovirus serotypes. The results show that probes from the 5 end of the genomes of HRV-14, 9, and 1B detected a large number of serotypes but the detection rate was variable and depended on the degree of homology with the particular probe. In contrast, all the 3 end probes were specific for the homologous virus. However, along HRV-9 probe detected a large number of serotypes.It was concluded that such cDNA probes would not detect all serotypes with equal efficiency. Synthetic oligonucleotides corresponding to short but highly conserved regions in the 5 non coding region may overcome this problem.     

Proteins specifically binding to the 3′ untranslated region of hepatitis A virus RNA in persistently infected cells

Summary Establishment of persistency is the common result of hepatitis A virus (HAV) infection in most HAV/cell culture systems. Previous studies provided evidence that shortly before or concomitantly with establishment of persistent infections synthesis of viral RNA is down-regulated. This may be an effect of regulating factors. Using RNA/protein binding assays it was shown that, at the critical time during virus replication, proteins accumulate which interact specifically with a distinct nucleotide sequence (HPE) within the 3 non-coding region of the HAV genome and/or (HME) within the 5 terminal region of the HAV antigenome. The sequences consist of 23 nucleotides (HPE: 5-AAAUUUUCUUAAAAUUUCUGAGG-3; HME: 5-CCUCAGAAAUUUUAAGAAAAUUU-3). A sequence with 79% similarity was found in the corresponding 3 non-coding region of poliovirus type I (Sabin) RNA. The latter sequence was shown to bind proteins from HAV infected cells but comparable proteins were absent in cells infected poliovirus.     

Repetitive sequences in the ITS1 region of ribosomal DNA in congeneric microphallid species (Trematoda: Digenea)

In searching for species-specific DNA sequences of microphallid species (Digenea, Trematoda) we examined the ribosomal internal transcribed spacer regions (ITS) of three closely related species (Levinseniella group) hosted by mud snails (first intermediate host) and marine crustaceans (second intermediate host). In the ITS1 region we found consistent patterns of repeating sequences of 130 bp. Within each main repeat there was a varying number of subrepeats specific for each of the species. All repeats including subrepeats were identified by a similar starting sequence: 5-CCTGTGG-3. As this sequence has close resemblance to the chi sequence 5-GCTGGTGG-3 found in phage we speculate if it serves the same function as a recombination hotspot. Alternatively but less likely, it could be an inactive, mutational relic of a sequence that once served this purpose.     

A Matrix Associated Region Localizes the Human SOCS-1 Gene to Chromosome 16p13.13

The MarFinder algorithm was applied to a newly sequenced segment of 16p13.13 abutting the 3 end of the human PRM1 PRM2 TNP2 locus. A candidate region of matrix attached was identified. Subsequent biophysical analysis showed that this region was attached to the somatic nuclear matrix. Nucleotide sequence analysis also revealed the presence of a CpG island. Data base queries showed that this region contained the SOCS-1 gene. Thus, the SOCS-1 gene is bounded by a somatic MAR and is just 3 of the spermatid-expressed PRM1 PRM2 TNP2 domain at position 16p13.13.     

Allele frequencies of intragenic,and 5′ and 3′ markers of the dystrophin gene in Japanese families afflicted with Duchenne or Becker muscular dystrophy

Summary Using the polymerase chain reaction method (PCR), we examined the allele frequencies and heterozygosities of 7 polymorphic sites (pERT87, and CA polymorphisms in the 5 and 3 regions) of the dystrophin gene in 20 Japanese Duchenne muscular dystrophy and Becker muscular dystrophy (DMD or BMD) families consisting of 36 males, including 23 cases of DMD and BMD, and 28 females. The allele frequencies of three primer and enzyme sets in the pERT87 locus were well comparable to those in the previously reported Japanese female cases but different from in other countries. The frequencies of 5 markers of the dystrophin gene in Japanese were different from the reported Caucasian frequencies. As for 5DYS-I and 5DYS-II, the numbers of alleles in our cases were less than in Caucasians, and the heterozygosities of all three markers (5DYS-I, II and III) were lower than in Caucasians. However, the 3CA polymorphisms showed almost the same frequencies and heterozygosities as in Caucasians. All of our females showed a heterozygous pattern for at least one locus, with the combination of the seven markers. The usefulness of linkage analysis involving PCR methods with these intragenic, and 5 and 3 markers of the dystrophin gene in the carrier and prenatal diagnosis of DMD and BMD was confirmed by the successful prenatal diagnoses in 15 fetuses, the exception being one case considered to have a new mutation.     

Attenuated Lapinized Chinese Strain of Classical Swine Fever Virus: Complete Nucleotide Sequence and Character of 3′-Noncoding Region

The complete nucleotide sequence including precise 5- and 3-terminal non-coding regions (NCRs) of the attenuated lapinized Chinese strain (HCLV) of Classical Swine Fever Virus (CSFV) was determined from overlapping cDNA clones constructed by separated RT-PCR and rapid amplification of cDNA ends (RACE) methods. The genomic RNA of the HCLV strain consists of 12,310 nucleotides (nts) including 374nts and 242nts in the 5- and 3-NCRs, respectively. It contains one large open reading frame (ORF) encoding a polyprotein of 3,898 amino acids with a calculated molecular weight of 437.6kDa. There is one notable insertion of 12 continuous nts, CTTTTTTCTTTT in the 3-NCR of HCLV genomic cDNA when compared with its parental virulent Shimen strain. Sequence alignment of partial 3-NCR reveals two groups of CSFV vaccine strains carrying similar T-rich insertions at different positions in this region. Computer-predicted secondary structures suggest that T-rich insertion greatly change the structures and thus decrease the promoter functions of 3-NCRs during the replications of these two groups of CSFV vaccine strains.     

3′-Deoxy-3′-fluoroinosine as a potent antileishmanial agent

We studied the antileishmanial activity of 3-deoxy-3-fluoroinosine (3-FI) againstLeishmania tropica andL. donovani. In in vitro cultivation, the EC₅₀ values (the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value) obtained for 3-FI against the promastigotes ofL. tropica andL. donovani were 2.3×10^–7 and 1.0×10^–6 M, respectively. It was less toxic toward mouse mammary-tumor FM3A cells, a model host; the EC₅₀ value was 1.9×10^–4 M. Leishmania promastigote metabolized 3-FI to 3-deoxy-3-fluoroadenosine 5-triphosphate (3-FATP) but FM3A cells did not. 3-FI was effective againstL. donovani amastigotes in J774.1 cells in an in vitro cultivation system under conditions similar to those used in the in vivo assay. 3-FI (50 mg/kg, given i.v.)showed a cytotoxic effect against the amastigotes ofL. donovani in mice.     

In wheat ctDNA,segments of ribosomal protein genes are dispersed repeats,probably conserved by nonreciprocal recombination

Summary Some dispersed repeated sequences and their flanking regions from wheat and maize ctDNAs have been characterized. Two sets of wheat ctDNA repeats were found to be the chloroplast ribosomal protein genesrpl2 andrpl23, plus nonfunctional segments of them, designatedrpl2 andrpl23. Pairwise comparisons were made between the wheatrp123 andrpl23, and the maizerp123 sequences. The precise patterns of homology suggest that the divergence of the wheat and maize nonfunctional (rpl23) sequences is being retarded by nonreciprocal recombination, biased by selection for individuals with functional (rpl23) sequences. The implied involvement of these sequences in mechanisms of homologous recombination, and therefore in the creation and spread of new ctDNA variants, is discussed.     

The oligodendroglial reaction to brain stab wounds: An immunohistochemical study

Summary Myelin/oligodendrocyte specific protein was compared to glial fibrillary acidic protein and 23-cyclic nucleotide 3-phosphodiesterase expression in normal rat brains and following stab wounds to the cerebral cortex, corpus callosum and hippocampus. Animals with stab wounds were allowed to recover for 5,15,28, 45 and 70 days post-operation before fixation by perfusion. Sections were reacted with antibodies against myelin/oligodendrocyte specific protein, glial fibrillary acidic protein and 23-cyclic nucleotide 3-phosphodiesterase, and observed by light and electron microscopy. Normal cerebral cortex had very few myelin/oligodendrocyte specific protein-positive and 23-cyclic nucleotide 3-phosphodiesterasepositive cells, but some glial fibrillary acidic protein-positive cells. The myelinated fibres of the corpus callosum were heavily stained for myelin/oligodendrocyte specific protein but unstained by glial fibrillary acidic protein or 23-cyclic nucleotide 3-phosphodiesterase antibodies. Some immunopositive cells were present in the corpus callosum and hippocampus with all three antibodies. After stab wound myelin/oligodendrocyte specific protein-positive reactive cells had more and longer processes and stained more intensely than equivalent cells in normal brain. These cells were distributed along the wound track, including within the cerebral cortex. The numbers of these cells increased until 28 days post-operation and then decreased so that very few were found at 70 days post-operation except in the corpus callosum. Where demyelination occurred myelin/oligodendrocyte specific protein-staining was lost. Staining for 23-cyclic nucleotide 3-phosphodiesterase revealed a similar pattern. Glial fibrillary acidic protein-positive reactive cells, which were also more robust than the normal cells, were more widely distributed. They increased in number throughout the time periods studied and gliosis was evident on the contralateral side. The glial fibrillary acidic protein-positive astrocytes were also different from the myelin/oligodendrocyte specific protein-positive and 23-cyclic nucleotide 3-phosphodiesterase-positive oligodendrocytes in terms of cell shape. With electron microscopy myelin/oligodendrocyte specific protein-positive cells showed features typical of immature oligodendrocytes. We conclude that the injury caused a numerical increase in oligodendrocytes and that myelin/ oligodendrocyte specific protein is a good marker for the oligodendroglial response and demyelination in pathological conditions.     

DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA

Summary We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3 termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I fromEscherichia coli, which has 35 exonuclease and replacement activities as well as its main 53 polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.     

Dissociation of acetylcholine- and cyclic GMP-induced currents inXenopu oocytes

InXenopus follicular oocytes, activation of muscarinic receptors evokes a slow potassium current (H-response); a similar current is evoked by intracellular injection of cyclic guanosine 3,5-monophosphate, cGMP (Dascal et al. 1984). We have tested the hypothesis that cGMP may be the second messenger that mediates the opening of K channel by acetylcholine (ACh). ACh elevated the intracellular level of cGMP with a time course similar to that of the development of the muscarinic H-response; maximal increase in cGMP concentration above the control was about 0.2 pmole/oocyte. The amount of injected cGMP that produced a detectable K current (threshold dose) varied between 0.5 and 3 pmole/oocyte. At low doses of cGMP, the slope of log dose-log response curve was about 2.5, suggesting involvement of a biochemical process with a positive cooperativity of at least 3. Higher doses of cGMP evoked, in addition to the outward current, an irregular, rapidly developing, long-lasting inward current, that never reached amplitudes comparable to those of ACh-evoked Cl currents. The K current elicited by cGMP was insensitive to elevation or depletion of external Ca. It was potentiated by isobutylmethylxanthine (IBMX). ACh strongly inhibited the cGMP-evoked K current when applied at the plateau of the latter. 4-Phorbol 12,13-dibutyrate (PDBu) (1 M) rapidly and completely inhibited the cGMP response. It is concluded, that most of the results presented in this report contradict the hyothesis that cGMP is the intracellular mediator of ACh-induced changes in membrane conductance in the oocytes.Abbreviations ACh acetylcholine - cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5-monophosphate - EGTA ethylenediaminetetraacetic acid - Hepes N-2-hydroxyethyl-piperazinc-N-2-hydroxypropanesulphonic acid - IBMX 3-isobutyl-l-methylxanthine - IP₃ inositol 1,4,5-trisphosphate - PDBu 4-phorbol 12,13-dibutyrate