Cloning and sequencing of winged bean (Psophocarpus tetragonolobus) basic agglutinin (WBA I): presence of second glycosylation site and its implications in quaternary structure

We report cloning of the DNA encoding winged bean basic agglutinin (WBA I). Using oligonucleotide primers corresponding to N- and C-termini of the mature lectin, the complete coding sequence for WBA I could be amplified from genomic DNA. DNA sequence determination by the chain termination method revealed the absence of any intervening sequences in the gene. The DNA deduced amino acid sequence of WBA I displayed some differences with its primary structure established previously by chemical means. Comparison of the sequence of WBA I with that of other legume lectins highlighted several interesting features, including the existence of the largest specificity determining loop which might account for its oligosaccharide-binding specificity and the presence of an additional N-glycosylation site. These data also throw some light on the relationship between the primary structure of the protein and its probable mode of dimerization.

Characterization of a local isolate of Bombyx mori nuclear polyhedrosis virus

Polyhedral bodies of Bombyx mori nuclear polyhedrosis virus, BmNPV (BGL) isolated from infected silkworms around Bangalore were propagated either in the cultured B. mori cell line, BmN or through infection of larvae. Electron microscopic (EM) observations of the polyhedra revealed an average length of 2 mu m and a height of 0.5 mu m. The purified polyhedra derived virions (PDV) showed several bands in sucrose gradient centrifugation, indicating the multiple nucleocapsid nature of BmNPV. Electron microscopic studies of PDV revealed a cylindrical, rod-shaped nucleocapsid with an average length of 300 nm and a diameter of 35 nm. The genomic DNA from the PDV was characterized by extensive restriction analysis and the genome size was estimated to be 132 kb. The restriction pattern of BmNPV (BGL) resembled that of the prototype strain BmNPV-T3. Distinct differences due to polymorphic sites for restriction enzyme HindIII were apparent between BmNPV (BGL) and the virus isolated from a different part of Karnataka (Dharwad area), BmNPV (DHR).

Expression of Human Growth Hormone in Silkworm Larvae through RecombinantBombyx moriNuclear Polyhedrosis Virus

We have generated a recombinantBombyx morinuclear polyhedrosis virus, vBmhGH, harboring the full-length human growth hormone gene (2.4-kb genomic DNA, with four introns and the signal peptide sequences) under the control of the polyhedrin promoter. BmN cells in culture infected with the recombinant virus showed the presence of RNA corresponding to the authentic growth hormone mRNA as well as its incompletly processed precusor. Electrophoretic analysis and immunoprecipitation of proteins of recombinant virus-infected BmN cells revealed the presence of the growth hormone protein. Infection of silkworm larvae with vBmhGH led to the synthesis and efficient secretion of the protein into hemolymph. The recombinant human growth hormone was biologically active in a radioreceptor competition binding assay. The secreted protein was isolated and purified to homogeneity by a single step immunoaffinity chromatography, to a specific activity of 2.4 × 104U/mg. The recombinant hGH retained the immunological and biolological properties of the native peptide. We conclude that BmNPV vectors can be used successfully for expressing chromosomal genes harboring multiple introns.

Human parvovirus B19: tissue persistence and prevalence of prototypic and new variants

Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.

Association of FOXE1 Polyalanine Repeat Region with Papillary Thyroid Cancer

CONTEXT: Polyalanine tract variations in transcription factors have been identified for a wide spectrum of developmental disorders. The thyroid transcription factor forkhead factor E1 (FOXE1) contains a polymorphic polyalanine tract with 12-22 alanines. Single-nucleotide polymorphisms (SNP) close to this locus are associated with papillary thyroid cancer (PTC), and a strong linkage disequilibrium block extends across this region. OBJECTIVE: The objective of the study was to assess whether the FOXE1 polyalanine repeat region was associated with PTC and to assess the effect of polyalanine repeat region variants on protein expression, DNA binding, and transcriptional function on FOXE1-responsive promoters. DESIGN: This was a case-control study. SETTING: The study was conducted at a tertiary referral hospital. PATIENTS AND METHODS: The FOXE1 polyalanine repeat region and tag SNP were genotyped in 70 PTC, with a replication in a further 92 PTC, and compared with genotypes in 5767 healthy controls (including 5667 samples from the Wellcome Trust Case Control Consortium). In vitro studies were performed to examine the protein expression, DNA binding, and transcriptional function for FOXE1 variants of different polyalanine tract lengths. RESULTS: All the genotyped SNP were in tight linkage disequilibrium, including the FOXE1 polyalanine repeat region. We confirmed the strong association of rs1867277 with PTC (overall P = 1 × 10(-7), odds ratio 1.84, confidence interval 1.31-2.57). rs1867277 was in tight linkage disequilibrium with the FOXE1 polyalanine repeat region (r(2) = 0.95). FOXE1(16Ala) was associated with PTC with an odds ratio of 2.23 (confidence interval 1.42-3.50; P = 0.0005). Functional studies in vitro showed that FOXE1(16Ala) was transcriptionally impaired compared with FOXE1(14Ala), which was not due to differences in protein expression or DNA binding. CONCLUSIONS: We have confirmed the previous association of FOXE1 with PTC. Our data suggest that the coding polyalanine expansion in FOXE1 may be responsible for the observed association between FOXE1 and PTC.

Next-generation sequencing: A frameshift in skeletal dysplasia gene discovery

In the last decade, huge breakthroughs in genetics - driven by new technology and different statistical approaches - have resulted in a plethora of new disease genes identified for both common and rare diseases. Massive parallel sequencing, commonly known as next-generation sequencing, is the latest advance in genetics, and has already facilitated the discovery of the molecular cause of many monogenic disorders. This article describes this new technology and reviews how this approach has been used successfully in patients with skeletal dysplasias. Moreover, this article illustrates how the study of rare diseases can inform understanding and therapeutic developments for common diseases such as osteoporosis. © International Osteoporosis Foundation and National Osteoporosis Foundation 2013.

A male-specific nuclease-resistant chromatin fraction in the mealybug Planococcus lilacinus

In mealybugs, chromatin condensation is related to both genomic imprinting and sex determination. The paternal chromosomal complement is condensed and genetically inactive in sons but not in daughters. During a study of chromatin organization in Planococcus lilacinus, digestion with micrococcal nuclease showed that 3% to 5% of the male genome is resistant to the enzyme. This Nuclease Resistant Chromatin (NRC) apparently has a nucleosomal organization. Southern hybridization of genomic DNA suggests that NRC sequences are present in both sexes and occur throughout the genome. Cloned NRC DNA is A+T-rich with stretches of adenines similar to those present in mouse alpha-satellite sequences. NRC DNA also contains sequence motifs that are typically associated with the nuclear matrix. Salt-fractionation experiments showed that NRC sequences are matrix associated. These observations are discussed in relation to the unusual cytological features of mealybug chromosomes, including the possible existence of multiple centres of inactivation.

Detection,amplification and sequence homology of sodC in clinical isolates of Salmonella sp

Background & objectives: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified, cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were compared with the standard strain Salmonella Typhimurium 14028. Methods: Salmonella enterica serovar Typhi (S. Typhi) and Salmonellaenterica serovar Paratyphi (S. Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC) was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were compared using CLUSTAL X.Results: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons respectively. Single mutational difference at position 489 was observed between thesodC of S. Typhi and S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that of standard strain S. Typhimurium 14028.Interpretation & conclusions: The presence of SodC in pathogenic bacteria could be a novel candidate as phylogenetic marker.

Hiekkatympösen (Hebeloma cylindrosporum) L-aminohappo-oksidaasi -geenin eristäminen

Pohjoisella havumetsävyöhykkeellä typpi on usein kasvien kasvua rajoittava tekijä. Metsämaan typpivarannot koostuvat pääasiassa orgaaniseen ainekseen sitoutuneista typpiyhdisteistä, erityisesti aminohapoista. Ektomykorritsasienet osallistuvat metsämaassa tapahtuvaan typenkiertoon hajottamalla orgaanisia typpiyhdisteitä ja kuljettamalla niitä kasvien käytettäväksi. Sienisolun sisällä tapahtuvasta aminohappojen mineralisaatiosta tiedetään toistaiseksi melko vähän. Aminohappo-oksidaasit katalysoivat aminohappojen mineralisaatiota. Eräissä ektomykorritsaa muodostavien kantasienten suvuissa on osoitettu L-aminohappo-oksidaaseja (LAO). Toistaiseksi LAO-geeniä ei tunneta kantasienistä. Työssä kuvattiin ensimmäistä kertaa LAO-geeni kantasienistä. Hiekkatympösen LAO1- geenin cDNA:n 5´ ja 3´ päiden emäsjärjestykset määritettiin RACE-PCR -menetelmällä, josta saatujen sekvenssien perusteella suunniteltiin alukkeet koko geenin cDNA:n ja genomisen DNA:n monistamiseksi. Genomisen DNA ja cDNA -sekvenssien perusteella määritettiin hiekkatympösen LAO1-geenin rakenne. Hiekkatympösen LAO1-geeni koostuu viidestä eksonista ja neljästä intronista. Hiekkatympösen LAO1-geenin yläpuoliselta alueelta löydettiin typpimetabolian säätelyyn osallistuvan proteiinin sitoutumiskohta. LAO1-geeniä edeltävä geenin osittainen genominen DNA-sekvenssi määritettiin. Kangaslohisienen genomissa LAO1-geeniä edeltävä geeni oli ennustettu pyruvaattidekarboksylaasiksi. Lisäksi työssä määritettiin hiekkatympösen toisen LAOhomologin cDNA:n osittainen emäsjärjestys. Työssä tunnistettiin myös toisen kantasienen, kangaslohisienen, LAO-geeni. LAO-geeniksi tunnistettu kangaslohisienen geenimalli oli aiemmin ennustettu NCBI:n tietokannassa toiminnaltaan tuntemattomaksi proteiiniksi. Proteiinien sukupuun perusteella hiekkatympösen ja kangaslohisienen LAO:n kantamuoto on kahdentunut. Työstä saatu tutkimustulos tuo täysin uutta tietoa molekyylibiologian tasolla ektomykorritsasienten aminohappojen katabolisista reaktioista. Aminohappojen mineralisaation seurauksen muodostuneet ammoniumionit saattavat olla merkittävä typen lähde myös maan muille mikrobeille ja kasveille. On mahdollista, että ektomykorritsasienten LAO-entsyymi on yksi merkittävä tekijä metsämaan typenkierrossa.

Expression of cyclin E in endomitotic silk-gland cells from mulberry silkworm

The silk glands of mulberry silkworm Bombyx mori are endoreplicating tissues in which the genomic DNA undergoes multiple rounds of replication without mitosis and nuclear division. In the absence of normal mitotic division, the cell cycle essentially alternates between the G1 and S phases. Cyclin E is crucial for the G1/S transition in both mitotic and endoreplicating cycles. We have cloned and characterized cyclin E (cyclin box) from B. mori, which is nearly identical to the Drosophila cyclin E box except for an insertion of 21 amino acids. Two distinct cyclin E transcripts (1.7 and 2.1 kb) were detected in the silk-gland cells of B. mori and in the B. mori-derived embryonic cell line, BmN. Using anti Cyclin E antibodies two protein bands of 52 and 44 kDa were detected in silk glands and BmN cells at Comparable levels. Both BmN- and the silk-gland cells showed the presence of the interacting kinase Cdk2. Transcripts of the mitotic cyclin, cyclin B, were barely detectable in the endoreplicating silk-gland cells and amounted to only 4-7% of that seen in the mitotically dividing BmN cells. The near absence of cyclin B transcripts and the abundant expression of cyclin E in the silk glands correlate well with the alternation of only G1 and S phases without the intervening mitosis in these cells. (C) 2000 Elsevier Science B.V. All rights reserved.

Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris

Expression of genes involved in methanol metabolism of Pichia pastoris is regulated by Mxr1p, a zinc finger transcription factor. In this study, we studied the target gene specificity of Mxr1p by examining its ability to bind to promoters of genes encoding dihydroxyacetone synthase (DHAS) and peroxin 8 (PEX8), since methanol-inducible expression of these genes is abrogated in mxr1-null mutant strains of P. pastoris. Different regions of DHAS and PEX8 promoter were isolated from P. pastoris genomic DNA and their ability to bind to a recombinant Mxr1p protein containing the N-terminal 150 amino acids, including the zinc finger DNA-binding domain, was examined. These studies reveal that Mxr1p specifically binds to promoter regions containing multiple 5'-CYCC-3' sequences, although all DNA sequences containing the 5'-CYCC-3' motif do not qualify as Mxr1p-binding sites. Key DNA-binding determinants are present outside 5'-CYCC-3' motif and Mxr1p preferably binds to DNA sequences containing 5'-CYCCNY-3' than those containing 5'-CYCCNR-3' sequences. This study provides new insights into the molecular determinants of target gene specificity of Mxr1p, and the methodology described here can be used for mapping Mxr1p-binding sites in other methanol-inducible promoters of P. pastoris. Copyright (C) 2010 John Wiley & Sons, Ltd.

Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3'-UTR of the BCL2 gene and are mainly clustered in the major breakpoint region (MBR). Recently, we found that the BCL2 MBR has a non-B DNA character in genomic DNA. Here, we show that single-stranded DNA modeled from the template strand of the BCL2 MBR, forms secondary structures that migrate faster on native PAGE in the presence of potassium, due to the formation of intramolecular G-quadruplexes. Circular dichroism shows evidence for a parallel orientation for G-quadruplex structures in the template strand of the BCL2 MBR. Mutagenesis and the DMS modification assay confirm the presence of three guanine tetrads in the structure. 1H nuclear magnetic resonance studies further confirm the formation of an intramolecular G-quadruplex and a representative model has been built based on all of the experimental evidence. We also provide data consistent with the possible formation of a G-quadruplex structure at the BCL2 MBR within mammalian cells. In summary, these important features could contribute to the single-stranded character at the BCL2 MBR, thereby contributing to chromosomal fragility.

Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3'-UTR of the BCL2 gene and are mainly clustered in the major breakpoint region (MBR). Recently, we found that the BCL2 MBR has a non-B DNA character in genomic DNA. Here, we show that single-stranded DNA modeled from the template strand of the BCL2 MBR, forms secondary structures that migrate faster on native PAGE in the presence of potassium, due to the formation of intramolecular G-quadruplexes. Circular dichroism shows evidence for a parallel orientation for G-quadruplex structures in the template strand of the BCL2 MBR. Mutagenesis and the DMS modification assay confirm the presence of three guanine tetrads in the structure. 1H nuclear magnetic resonance studies further confirm the formation of an intramolecular G-quadruplex and a representative model has been built based on all of the experimental evidence. We also provide data consistent with the possible formation of a G-quadruplex structure at the BCL2 MBR within mammalian cells. In summary, these important features could contribute to the single-stranded character at the BCL2 MBR, thereby contributing to chromosomal fragility.

Genome analysis: A new approach for visualization of sequence organization in genomes

In this article we describe and demonstrate the versatility of a computer program, GENOME MAPPING, that uses interactive graphics and runs on an IRIS workstation. The program helps to visualize as well as analyse global and local patterns of genomic DNA sequences. It was developed keeping in mind the requirements of the human genome sequencing programme, which requires rapid analysis of the data. Using GENOME MAPPING one can discern signature patterns of different kinds of sequences and analyse such patterns for repetitive as well as rare sequence strings. Further, one can visualize the extent of global homology between different genomic sequences. An application of our method to the published yeast mitochondrial genome data shows similar sequence organizations in the entire sequence and in smaller subsequences.

Structural proteins of mycobacteriophage I3: cloning, expression and sequence analysis of a gene encoding a 70-kDa structural protein

The structural proteins of mycobacteriophage I3 have been analysed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), radioiodination and immunoblotting. Based on their abundance the 34- and 70-kDa bands appeared to represent the major structural proteins. Successful cloning and expression of the 70-kDa protein-encoding gene of phage I3 in Escherichia coli and its complete nucleotide sequence determination have been accomplished, A second (partial) open reading frame following the stop codon for the 70-kDa protein was also identified within the cloned fragment. The deduced amino-acid sequence of the 70-kDa protein and the codon usage patterns indicated the preponderance of codons, as predicted from the high G+C content of the genomic DNA of phage I3.