Isolation of the missing 5′-end of the encoding region of the bovine leukemia virus cell receptor gene

Summary The missing 5-end of the encoding region of the bovine leukemia virus (BLV) cell receptor gene (BLVRcp1/5) was isolated from a lambda gt11 cDNA library using the³²P-labeledEcoRI-SamI fragment corresponding to the 5-end of a 2.3 kbp cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor gene (BLVRcp1). The nucleotide and amino acid sequence analysis of the BLVRcp1/5 cDNA revealed that the 1058 bpEcoRI fragment at its 5-end contained a new 114 amino acid long sequence, and at its 3-end contained a completely identical 88 amino acid overlapping region with the 5-end of the BLVRcp1 cDNA. The combined sequences of both cDNAs represent the whole encoding region of the BLV cell receptor gene. The longest open reading frame of the BLV cell receptor gene encodes a protein containing 843 amino acids with a calculated molecular mass of 94.2 kDa which concurs with experimentally detected native BLV receptor protein. Search for homology has shown that about 250 bp of the BLV cell receptor gene is highly homologous to Venter's tag sequences of an unidentified gene from the human brain library.     

Androgenic correlates of genetic variation in the gene encoding 5α-reductase type 1

Androgens determine male secondary sexual characteristics and influence a variety of metabolic pathways. Circulating levels of androgens are highly heritable; however, the genes involved are largely unknown. The 5-reductase enzymes types 1 and 2 responsible for converting testosterone to the more potent androgen dihydrotestosterone are encoded by the SRD5A1 and SRD5A2 genes, respectively. We performed indirect genetic association studies of SRD5A1 and SRD5A2 and the dihydrotestosterone/testosterone ratio that reflects the activity of 5-reductase in 57 males with type 2 diabetes. We found evidence of significant association between a single nucleotide polymorphism in SRD5A1 and the dihydrotestosterone/testosterone ratio (median 0.10, interquartile range 0.08 vs. median 0.06, interquartile range 0.04, P=0.009). The polymorphism was not associated with any diabetic phenotypes. These results suggest that functional genetic variants might exist in or around SRD5A1 that affect the activity of the 5-reductase enzyme type 1 and influence androgen levels.     

Cloning of the 3′-terminal region encoding movement and coat proteins of a Korean isolate of odontoglossum ringspot virus

Summary The 3-terminal 1855 nucleotides (nts) of a Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) were cloned and sequenced. The sequence contained two open reading frames, which encode the cell-to-cell movement protein (MP) and coat protein genes, and are 912 nts and 477 nts long, respectively. The MP gene contained a conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA locating between 1117 nts and 1292 nts from the 3-end. The 3 untranslated region (UTR) of the virus comprises 414 nts, includes nine pseudoknots and a tRNA-like structure domain containing aminoacyl acceptor arm and the anticodon hairpin loop coding for histidine.The nucleotide sequence data for ORSV-Cy reported in this paper will appear in the EMBL, GenBANK and DDBJ nucleotide databases under the accession numbers X78966 and X80053.     

A mitochondrial intron sequence in the 5′-flanking region of a plant nuclear lectin gene

Summary A sequence fragment from the cis-splicing intron between exons a and b of the NADH-dehydrogenase subunit 5 gene (nad5) in plant mitochondria is also present in one of two closely related nuclear-encoded lectin genes of Dolichos biflorus. This sequence of 116 nucleotides is the major difference in the 5-flanking region of two recently described lectin genes (Harada et al. 1990). The stem and leaf lectin DB58 does not contain the insert, while the otherwise more than 90% identical 5-flanking region of the seed lectin is interrupted by this mitochondrial intron sequence.     

Distribution of Δ32 alelle of the CCR5 gene in the population of Poland

The chemokine receptor CCR5 constitutes a major co-receptor for the R5 strains of HIV-1, and a mutant allele of the CCR5 gene, especially in the homozygous form Δ32/Δ32, confers resistance against infection by the virus. The frequency of the Δ32 allele was determined in blood donors from 16 provinces, covering the entire territory of Poland. Among 861 individuals 182 (21.1%) were carriers of the mutated allele; 7 of them (0.8 %) were homozygotes Δ32/Δ32, and 175 (20.3%) were heterozygotes +/Δ32, resulting in a 10.9% frequency of the Δ32 allele. The highest frequencies of the mutated allele were found in the eastern and western provinces, and the lowest frequencies of the Δ32 allele were detected in the provinces in the center of the country. This pattern of distribution may reflect the migration of the population from the eastern territories of Poland to the western part of the country after World War II. Received: March 17, 2000 / Accepted: May 29, 2000     

Cloning of the genome of cricket paralysis virus: sequence of the 3′ end

DNA complementary to the single-stranded RNA genome of the insect picornavirus, cricket paralysis virus (CrPV), was cloned into the plasmid pBR322 by a hybrid RNA-cDNA cloning strategy. Positive CrPV-specific clones were selected by colony hybridization and characterised by restriction enzyme mapping. Overlapping clones spanning 7.5 kb of the estimated 8.5 kb genome were obtained, the largest being 7.0 kb. Comparison of the restriction enzyme map with those of mammalian picornaviruses revealed no conserved pattern of cleavage sites. The CrPV-cDNA was sequenced using the M13-dideoxy chain-terminating method although the chemical method of Maxam and Gilbert was employed to complete gaps in the sequence. The sequence of the 3′-terminal 1600 nucleotides is presented and is compared with those of mammalian picornaviruses. Computer comparisons of the CrPV sequence and those of mammalian picornaviruses revealed no significant homology between either the nucleotide or the predicted amino acid sequence of this region.     

Characterization of the 5′-flanking region and chromosomal assignment of the human brain natriuretic peptide gene

Brain natriuretic peptide (BNP) is a cardiac hormone that occurs predominantly in the ventricle, and synthesis and secretion of BNP are greatly augmented in patients with congestive heart failure and in animal models of ventricular hypertrophy. In order to elucidate the molecular mechanisms underlying the human BNP gene expression in the heart, the human BNP gene was isolated from a size-selected genomic minilibrary. The 1.9-kb human BNP 5-flanking region (–1813 to +110) contained an array of putative cis-acting regulatory elements. Various lengths of the cloned 5-flanking sequences were linked upstream to the bacterial chloramphenicol acetyltransferase (CAT) gene, and their promoter activities were assayed. The 1.9-kb promoter region showed a high-level CAT activity in cultured neonatal rat ventricular cardiocytes. When the CT-rich sequences (–1288 to –1095) were deleted, the high-level activity was reduced to approximately 30%. The 399-bp BNP 5 flanking region (–289 to +110) showed approximately 10% activity of the 1.9-kb region. Furthermore, using human-rodent somatic hybrid cell lines, the BNP gene was assigned to human chromosome 1, on which the atrial natriuretic peptide gene is localized. The present study leads to a better understanding of the molecular mechanisms for the human BNP gene expression in the heart.Abbreviations ANP Atrial natriuretic peptide - AP-1 Activator protein-1 - BNP Brain natriuretic peptide - CAT Chloramphenicol acetyltransferase     

Localization of the gene encoding the α2 subunit of the human VLA-2 receptor to chromosome 5q23-31

The ₂ subunit of the VLA-2 receptor (CD49B) was mapped to human chromosome 5 by several independent approaches. First, the expression of the ₂ subunit at the protein level was investigated in a panel of human-mouse hybrid cell lines. Cell surface expression was detected by indirect immunofluorescence with monoclonal anti-₂ antibody 12F1. Intracellular ₂ antigen was detected by immunostaining of whole cell extracts or of immunoprecipitated 12F1 antigen with the monoclonal antibodies 3H8 and 5C5. Second, the presence of human genomic ₂ sequences in the panel of human-mouse hybrids was detected by PCR, using primers derived from the published ₂ cDNA sequence. The specificity of the amplification product was shown by direct sequencing. The results of the PCR study were confirmed by amplifying aCD14 gene fragment, known to map to chromosome 5. Finally, in situ hybridization with a³H-labeled 1040-bp cDNA probe, also obtained by PCR, confirmed and refined the localization ofCD49B on chromosome 5 at q23-31.     

Regulatory sequences clustered at the 5′ end of the first intron of the human thymidylate synthase gene function in cooperation with the promoter region

A human thymidylate synthase (TS) minigene containing 5- and 3-flanking sequences, all the exons, and only intron 1 showed a normal frequency of stable transformation when transfected into TS-negative mutant cells, whereas minigenes in which intron 1 was replaced by intron 2 or deleted in the above construct showed only a few percent of the above frequency. Introduction of intron 1 into the above intronless or intron 2 minigene restored the transforming activities regardless of its position and orientation. Deletion analysis revealed two positive and one negative regulatory sequences in the 5 end of intron 1, each of which seemed to bind specific proteins as shown by gel shift analysis. Intron 1 also stimulated expression of a TS promoter-CAT gene construct but not that of an SV40 promoter-CAT gene construct. These results indicate that the multiple regulatory sequences clustered in intron 1 stimulate TS gene expression in concert with the 5-flanking sequences.     

Disruption of the 3′ phosphoglycerate kinase (pgk) gene in Aspergillus nidulans

We report the isolation of a pgk ^- mutant strain of Aspergillus nidulans by means of a gene disruption strategy, and demonstrate that the pgk gene is located on chromosome VIII. The pgk ^- mutant conidiates poorly, will only grow on media supplemented with both a glycolytic and a gluconeogenic carbon source, and is inhibited by hexoses.     

A −16C>T substitution in the 5′ UTR of the puratrophin-1 gene is prevalent in autosomal dominant cerebellar ataxia in Nagano

The molecular bases of autosomal dominant cerebellar ataxia (ADCA) have been increasingly elucidated, but 17–50% of ADCA families still remain genetically undefined in Japan. In this study we investigated 67 genetically undefined ADCA families from the Nagano prefecture, and found that 63 patients from 51 families possessed the –16C>T change in the puratrophin-1 gene, which was recently found to be pathogenic for 16q22-linked ADCA. Most patients shared a common haplotype around the puratrophin-1 gene. All patients with the –16C>T change had pure cerebellar ataxia with middle-aged or later onset. Only one patient in a large, –16C>T positive family did not have this change, but still shared a narrowed haplotype with, and was clinically indistinguishable from, the other affected family members. In Nagano, 16q22-linked ADCA appears to be much more prevalent than either SCA6 or dentatorubral-pallidoluysian atrophy (DRPLA), and may explain the high frequency of spinocerebellar ataxia.Takako Ohata and Kunihiro Yoshida have equally contributed to this work     

Sequence variation in 5′ termini of rubella virus genomes: changes affecting structure of the 5′ proximal stem-loop

Summary Variation within a 523 nucleotide region proximal to the 5 terminus of seven rubella virus strains has been anlysed. Compared to the Therien strain twenty sites of nucleotide variation have been identified, three of which are in the 5 untranslated region. Individual strains have between three and nine nucleotide differences, only three of which result in amino acid substitutions. TO-336 has a serine for threonine at amino acid (aa) 42 and CM arginine for histidine at aa 159. RA27/3 has arginine for lysine at aa 3 and serine for threonine at aa 42. Nucleotide differences which affect a stem-loop structure reported to be important for binding of host cell proteins have been identified.     

Completion of the full-length genome sequence of Menangle virus: characterisation of the polymerase gene and genomic 5′ trailer region

Summary. Menangle virus (MenV), isolated in 1997 from stillborn piglets during an outbreak of reproductive disease at a large commercial piggery, is the only new paramyxovirus to be identified in Australia since Hendra virus in 1994. Following partial characterisation of the MenV genome, we previously showed that MenV is a novel member of the genus Rubulavirus. Here we report the characterisation of the large (L) polymerase gene and the adjacent 5′ trailer region of MenV, which completes the full-length genome sequence of this novel paramyxovirus (15,516 nucleotides), and thereby confirm its taxonomic position within the family Paramyxoviridae.     

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.     

A novel Sac I RFLP in the 3′ untranslated region of the myotonin protein kinase gene

We found a novel Sac I polymorphism downstream of CTG repeats in the 3′ untranslated region of the myotonin protein kinase (MT-PK) gene. A C to G transition at nucleotide 13,590 in the gene was revealed by Southern blotting and confirmed by sequencing analyses. The allelic frequency of the C : G polymorphism in 63 unrelated Japanese individuals was estimated to be 0.98: 0.02. When Southern blotting is employed in the analysis of the CTG repeat length in the MT-PK gene, this Sac I polymorphism should be taken into consideration. Received: 15 October, 1998 / Accepted: 31 October, 1998     

Activation of defense-related gene expression and systemic acquired resistance in cucumber mosaic virus-infected tobacco plants expressing the mammalian 2′5′oligoadenylate system

Summary.  Tobacco plants expressing the mammalian 2′5′oligoadenylate system (2-5A system) exhibit resistance to cucumber mosaic virus (CMV). Here, to characterize the molecular aspect of the resistance to CMV in 2-5A system-expressing tobaccos, the activation of defense-related genes and systemic acquired resistance (SAR) as the markers for the hypersensitive resistance (HR), were elucidated. Northern hybridization analysis indicated that the expression of four pathogenesis-related (PR) protein genes and five HR-related genes were induced in CMV-infected tobaccos expressing 2-5A system. Furthermore, the induction of SAR against Pseudomonas syringae pv. tabaci as second challenge, was observed on CMV-inoculated tobaccos expressing 2-5A system. These results suggested that the resistance to CMV in tobacco expressing 2-5A system is associated with the establishment of an HR-like response. Received September 12, 2002; accepted November 20, 2002 Published online February 7, 2003