No abstract available

[en] Nanopores are being hailed as a potential next-generation DNA sequencer that could provide cheap, high-throughput DNA analysis. In this review we present a detailed summary of the various sensing techniques being investigated for use in DNA sequencing and mapping applications. A crucial impasse to the success of nanopores as a reliable DNA analysis tool is the fast and stochastic nature of DNA translocation. We discuss the incorporation of biological motors to step DNA through a pore base-by-base, as well as the many experimental modifications attempted for the purpose of slowing and controlling DNA transport. (paper)

No abstract available

[en] Ectromelia virus is the causative agent of mousepox, an acute exanthematous disease of mouse colonies in Europe, Japan, China, and the U.S. The Moscow, Hampstead, and NIH79 strains are the most thoroughly studied with the Moscow strain being the most infectious and virulent for the mouse. In the late 1940s mousepox was proposed as a model for the study of the pathogenesis of smallpox and generalized vaccinia in humans. Studies in the last five decades from a succession of investigators have resulted in a detailed description of the virologic and pathologic disease course in genetically susceptible and resistant inbred and out-bred mice. We report the DNA sequence of the left-hand end, the predicted right-hand terminal repeat, and central regions of the genome of the Moscow strain of ectromelia virus (approximately 177,500 bp), which together with the previously sequenced right-hand end, yields a genome of 209,771 bp. We identified 175 potential genes specifying proteins of between 53 and 1924 amino acids, and 29 regions containing sequences related to genes predicted in other poxviruses, but unlikely to encode for functional proteins in ectromelia virus. The translated protein sequences were compared with the protein database for structure/function relationships, and these analyses were used to investigate poxvirus evolution and to attempt to explain at the cellular and molecular level the well-characterized features of the ectromelia virus natural life cycle

[en] Microsatellites, or simple sequence repeats, are considered to be junk DNA. Yet their exploitation in DNA profiling techniques has expanded the repertoire of useful sequences for plant biologists. Some old and new techniques involving microsatellites are portrayed, such as microsatellite fingerprinting, microsatellite primed polymerase chain reaction (PCR), anchored microsatellite primed PCR, random amplified (microsatellite) polymorphic DNA (RAPD) analysis, the generation of microsatellite fingerprints in RAPDs and the production of sequence tagged microsatellite sites. (author). 8 refs

No abstract available

[en] Full text: Livestock improvement greatly depends on the exploitation of DNA level polymorphisms. Specific sequences of DNA are being used as genetic markers to identify loci responsible for expression of complex traits both in man and animals. Presently several classes of markers are available namely RFLPs, AFLPs, VNTRs, STRs, SNPs etc. DNA Sequences with basic repeat motifs of two to six nucleotides can be synthesized and hybridized to genomic sequences from a variety of species to produce multilocus band patterns. Several such oligonucleotide sequences have been reported to be useful in producing highly polymorphic DNA fingerprints in a variety of species. These markers have short-range uses such as parentage determination, individual identification, detection of twin zygosity, etc., and long-range applications such as gene mapping and marker assisted selection The degree of polymorphism elucidated from a probe or a marker may differ from species to species depending on probe-species combination. It is important to screen DNA markers for their informativeness and polymorphism for various domestic species of animals before considering them for further use. In literature several synthetic probes having the core sequences of (AT) (GT), (GC), (CAC), (GAA), (GGAT), (GACA), (TGG), and (GATA) have been reported for DNA fingerprinting of a variety of species of animals. However, the indigenous Zebu cattle, which constitute major proportion of Indian cattle population has poorly been explored with DNA-based markers. In this study, four different oligonucleotide markers were screened for their usefulness as markers in Zebu cattle. The investigations were carried out on genomic DNA of randomly selected unrelated (15 animals) and from two sire families (11 animals) of Sahiwal breed of Zebu cattle maintained in a herd at National Dairy Research Institute, Karnal. Oligonucleotide probes were custom synthesised and used after radio-isotopic labelling with (γ''3''2P) dATP ''3''2P using the enzyme polynucleotide kinase by the standard procedure. Hybridization of labelled oligonucleotide probes to genomic DNA on Nylon membranes was carried out at 45 deg. C for probes (GTG)₅ and (TCC)₅, 43 deg. C for (GT)₈ and 65 deg. C for (GT)₁₂. Post-hybridization treatments and autoradiography were carried out and size of each fragment on X-ray film, i.e. DNA fingerprint, was estimated using computer software GelBase (UVP, UK). Number of total bands and shared bands in the fingerprints of each individual were recorded in the range of 2.5 to 23.0 KB. Number of bands, average band sharing rate (BS), mean allelic frequencies (a) and heterozygosity (h) level were calculated. All four probes used produced multilocus fingerprints with differing levels of polymorphism. Means of number of bands per individual, band sharing rate, allele frequencies and heterozygosity was calculated. The probes (GT)₈, (GT)₁₂ and (TCC)₅ produced fingerprinting patterns of medium to low polymorphism whereas the probe (GTG)₅ produced highly polymorphic pattern. The probe (GT)8 probe produced as many as 32 bands in resolvable portion of the gel. However, nearly 40% of the bands were shared by all the individuals hence, the average bands sharing rate was found to be high. High band sharing rate in this study indicate that the animals examined might be genetically more homogeneous with respect to (GT)_n sequences. Comparison of average number of bands obtained between different probes reveal that the probe GT₈ hybridized to more number of fragments than the other probes. This result indicates that GT_n are more abundant in zebu cattle genome compared to other sequences studied. The probe (GT)₁₂ produced a multilocus fingerprints with lower level of polymorphism in comparison with (GT)₈ fingerprints. Mean number of bands and polymorphism were low as compared to (GT)₈ fingerprints. Variation in the nucleotide constitution of repeat sequences and differences in hybridization and stringency conditions could be the reason for variation in banding pattern of same core sequences of differing length. The probe TCC₅ produced multilocus polymorphic fingerprints. The level of polymorphism was low as revealed by high mean band sharing values of 0.75. The reason for this deviation from the present observation could be the variation between genome of Bos taurus and Bos indicus. Alternatively it is possible that HinfI restriction sites are adjacent to TCC_n sequences are conserved while there may be variation in HaeIII restriction sites. The probe GTG₅ produced highly polymorphic DNA fingerprints. The number of bands ranged between 9-17 with average band sharing of 0.48. The probe GTG₅ or its complementary sequences CAC₅ produced highly polymorphic fingerprints. High heterozygosity level obtained in this study and low level of mutation rate associated with the sequences indicate that the probe can be used for analyzing population structure, parentage verification and as a marker to identify loci controlling quantitative traits, disease resistance, fertility etc. (author)

[en] Cloned DNA sequences were hybridized to salivary gland polytene chromosomes of the medfly, Ceratitis capitata, establishing molecular markers for 55 sites on all (ten) autosome arms. Fourteen of the markers correspond to characterized medfly transcription units, while the function of the remaining clones is unknown. Five additional markers were identified as repetitive elements that hybridized to a large number of autosomal sites and also to the granular network that represents the X chromosome. Some of the clones were also hybridized to polytene chromosomes from the orbital bristle cells of C. capitata to align the two types of polytene chromosomes, which differ significantly in their banding pattern. (author)

[en] Molecular techniques were used to characterize sixteen Newcastle Disease (ND) Virus (NDV) isolates from ND outbreaks in chickens in Uganda in 2001 and to evaluate ND epidemiology. Virus isolation and was made out in SPF eggs. RNA was extracted from the virus isolates using QIAgen kit. Reverse and forward primers covering the cleavage site of the fusion (F) protein gene and a specific HN gene segment were designed and used in a single tube Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR). The resultant complimentary DNA fragment products were visualized on agarose gel and extracted using QIAgen kit. The purified amplified F and HN gene products were purified by microspin column technique. The PCR primers were used to sequence the products in BigDye Terminator cycle sequencing. The cycle sequencing products were precipitated by ethanol/Na-acetate method and loaded onto acrylamide gel for analysis in the ABI 377 automated DNA sequencer (Perkin Elmer Applied Biosystems). Comparative genetic and phylogenetic tree analyses were performed on the HN genes of the isolates and 17 NDV strains selected from the GenBank. ClustalX 1.81 and phylip software were used for gene alignment and the final phylogeny was produced by neighbour-joining method. Results showed that all the Ugandan NDV isolates were closely related. F gene cleavage site sequence analysis had the amino acid sequence ¹¹²RRQKRF¹¹⁷ at the C-terminus of the F2 protein and F (phenylalanine) at residue 117. The amino acid sequence at the C-terminus and around the cleavage site is shown. There is therefore a pair of basic amino acids R, arginine and K lysine at residues 116 and 115 respectively and a phenylalanine, F at residue 117 as well as a basic amino acid R, arginine at residue 113 indicating a high virulence for the NDV isolates from Ugandan ND outbreaks. The ¹¹²RRQKRF¹¹⁷ motif is majority/consensus sequence around the F2/F1 cleavage site. All the NDV isolated from Uganda were highly virulent and none of them was related to the vaccine strain. Upon phylogenetic analysis, all isolates formed separate clades from the currently known 8 genotypes suggesting that they are a novel genotype, unrelated to those that have caused previous outbreaks worldwide

[en] Full text: I-2 is an avirulent, thermostable strain of Newcastle disease virus (NDV) of Australian origin, used as a vaccine for village-based flocks of chickens in developing countries. There have been many studies on the biological properties of strain I-2, but none until now on the molecular basis of these properties. A single tube RT-PCR technique was conducted and generated 387 bp and 300 bp cDNA fragments encoding a portion of F0 gene of NDV strain I- 2, respectively. The amplicons were directly sequenced by the dye-terminator cycle method. The resulting nucleotide sequences were used to deduce amino acid sequences surrounding the cleavage site of F protein. This was compared with sequence for other NDV strains. The cleavage-activation site of strain I-2 had a pair of basic residues followed by a single basic residue with an intervening glutamine (¹¹²RKQGR¹¹⁶) whereas the corresponding site of other avirulent strains has a sequence of ¹¹²GKQGR¹¹⁶. The obtained sequence motif for strain I-2 was unique amongst the lentogenic strains of NDV by having a substitution of arginine (R) for glycine (G) at position 112 at the C-terminus of F2 protein. At the N terminus of F1 protein, strain I-2 had a sequence pattern of ¹¹⁷LIG¹¹⁹, which was similar to other lentogenic strains and not the ¹¹⁷FIG¹¹⁹ motif of virulent strains. On the basis of these findings, it was concluded that strain I-2 is an avirulent strain having a non-basic residue (position 115) at the cleavage activation site and leucine (L) at position 117 of the N-terminus of the fusion inducing domain. It is noteworthy that the sequence pattern of strain I-2 at the cleavage activation site was different from that of emerging virulent strains causing clinical outbreaks of Newcastle disease (ND) in Australia. The determination and analysis of amino acid sequences provides useful information on NDV. This information may contribute towards the understanding of molecular epidemiology of ND clinical outbreaks and determination of the suitability of using strain I-2 as a vaccine to control ND. (author)