Characterization of changes in the short unique segment of pseudorabies virus BUK-TK900 (Suivac A) vaccine strain

Mutant strains of pseudorabies virus (PRV) of reduced virulence, such as Bartha or BUK-TK900, have been used for vaccination purposes for many years. In contrast to the Bartha strain, BUK-TK900 has not been well characterised at the molecular level. The detailed analysis of this vaccine strain was urged by the fact of the isolation in Poland of field strains which were suspected to originate from BUK-TK900. We characterised changes in the US region of this strain, focusing our attention on gE and gI genes. The only deletion, about 300bp, found in BamHI 7 fragment (covering most of the US region) was located in the 28K (US2) gene. BUK-TK 900 produced small plaques on all cell lines tested in our laboratory (SK6, Vero, MDBK, 3T3). The plaque size was restored to about 70% of wild type virus plaque size when growing BUK-TK900 virus on 3T3 complementing cell line expressing PRV gE and up to 100% when cell line producing gE and gI was used. Both gE and gI genes from BUK-TK900 and from some derivative field isolates have been amplified by PCR reaction but no deletions in these genes have been found. Molecular weight of gene products differed from wild type proteins: gE was bigger than wild type gE while gI was smaller. Both proteins were correctly recognised by all tested polyclonal and monoclonal antibodies. Radioimmunoprecipitation study showed that BUK-TK900 gE and gI interact forming a complex. The whole ORF of BUK-TK900 gE was sequenced and only few point mutations were found; only two of them led to changes of amino acids in the polypeptide chain. These were: methionine at position 124 replaced by threonine and glutamine at position 162 replaced by arginine. The introduction of first of these mutations (Met to Thr) to PRV wild type strain NIA-3 resulted in 22% reduction of plaque size. This result confirms the importance of this domain of gE for its function; it was found previously by others that deletion of amino acids 125 and 126 reduced virulence and neurotropism of PRV. More changes were found in BUK-TK900 gI sequence. Over 80% of these changes were located in the terminal 1/3rd of the sequence. Some of these mutations may have significant effect on the secondary structure of gI glycoprotein. The change of the secondary structure may be responsible for the decrease of gI stability and the observed reduction of gI molecular mass

Nucleotide sequence of a cluster of early and late genes in a conserved segment of the vaccinia virus genome.

The nucleotide sequence of a 7.6 kb vaccinia DNA segment from a genomic region conserved among different orthopox virus has been determined. This segment contains a tight cluster of 12 partly overlapping open reading frames most of which can be correlated with previously identified early and late proteins and mRNAs. Regulatory signals used by vaccinia virus have been studied. Presumptive promoter regions are rich in A, T and carry the consensus sequences TATA and AATAA spaced at 20-24 base pairs. Tandem repeats of a CTATTC consensus sequence are proposed to be involved in the termination of early transcription

Trichinella spiralis thymidylate synthase : cDNA cloning and sequencing, and developmental pattern of mRNA expression

Part 2International audienceThe persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequenceddagger, allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35 582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar V-max value, but higher K-m(app) values describing interactions with dUMP (28.8 mum vs. 3.9 mum) and (6RS, alphaS)-N-5,N-10-methylenetetrahydrofolate (383 mum vs. 54.7 mum). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle

DNA computing: implementation of data flow logical operations

Self-assembly of DNA is considered a fundamental operation in realization of molecular logic circuits. We propose a new approach to implementation of data flow logical operations based on manipulating DNA strands. In our method the logic gates, input, and output signals are represented by DNA molecules. Each logical operation is carried out as soon as the operands are ready. This technique employs standard operations of genetic engineering including radioactive labeling as well as digestion by the second class restriction nuclease and polymerase chain reaction (PCR). To check practical utility of the method a series of genetic engineering experiments have been performed. The obtained information confirms interesting properties of the DNA-based molecular data flow logic gates. Some experimental results demonstrating implementation of a single logic NAND gate and only in one vessel calculation of a tree-like Boolean function with the help of the PCR are provided. These techniques may be utilized in massively parallel computers and on DNA chips. 2001 Elsevier Science B.V. All rights reserved