A Very Strong Enhancer Is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus

A strong transcription enhancer was identified in the genomic DNA (235 kb) of human cytomegalovirus (HCMV), a ubiquitous and severe pathogen of the herpesvirus group. Cotransfection of enhancerless SV40 DNA with randomly fragmented HCMV DNA yielded two SV40-HCMV recombinant viruses that had incorporated overlapping segments of HCMV DNA to substitute for the missing SV40 enhancer. Within HCMV, these enhancer sequences are located upstream of the transcription initiation site of the major immediate-early gene, between nucleotides -118 and −524. Deletion studies with the HCMV enhancer, which harbors a variety of repeated sequence motifs, show that different subsets of this enhancer can substitute for the SV40 enhancer. The HCMV enhancer, which seems to have little cell type or species preference, is severalfold more active than the SV40 enhancer. It is the strongest enhancer we have analyzed so far, a property that makes it a useful component of eukaryotic expression vectors

Turning gold into 'junk': transposable elements utilize central proteins of cellular networks

The numerous discovered cases of domesticated transposable element (TE) proteins led to the recognition that TEs are a significant source of evolutionary innovation. However, much less is known about the reverse process, whether and to what degree the evolution of TEs is influenced by the genome of their hosts. We addressed this issue by searching for cases of incorporation of host genes into the sequence of TEs and examined the systems-level properties of these genes using the Saccharomyces cerevisiae and Drosophila melanogaster genomes. We identified 51 cases where the evolutionary scenario was the incorporation of a host gene fragment into a TE consensus sequence, and we show that both the yeast and fly homologues of the incorporated protein sequences have central positions in the cellular networks. An analysis of selective pressure (Ka/Ks ratio) detected significant selection in 37% of the cases. Recent research on retrovirus-host interactions shows that virus proteins preferentially target hubs of the host interaction networks enabling them to take over the host cell using only a few proteins. We propose that TEs face a similar evolutionary pressure to evolve proteins with high interacting capacities and take some of the necessary protein domains directly from their hosts

A Family of Developmentally Excised DNA Elements in \u3cem\u3eTetrahymena\u3c/em\u3e is under Selective Pressure to Maintain an Open Reading Frame Encoding an Integrase-Like Protein

Tlr1 is a member of a family of ~20-30 DNA elements that undergo developmentally regulated excision during formation of the macronucleus in the ciliated protozoan Tetrahymena. Analysis of sequence internal to the right boundary of Tlr1 revealed the presence of a 2 kb open reading frame (ORF) encoding a deduced protein with similarity to retrotransposon integrases. The ORFs of five unique clones were sequenced. The ORFs have 98% sequence conservation and align without frameshifts, although one has an additional trinucleotide at codon 561. Nucleotide changes among the five clones are highly non-random with respect to the position in the codon and 93% of the nucleotide changes among the five clones encode identical or similar amino acids, suggesting that the ORF has evolved under selective pressure to preserve a functional protein. Nineteen TIC transitions in T/CAA and T/CAG codons suggest selection has occurred in the context of the Tetrahymena genome, where TAA and TAG encode Gin. Similarities between the ORF and those encoding retrotransposon integrases suggest that the Tlr family of elements may encode a polynucleotide transferase. Possible roles for the protein in transposition of the elements within the micronuclear genome and/or their developmentally regulated excision from the macronucleus are discussed

454-Pyrosequencing: A Molecular Battiscope for Freshwater Viral Ecology

Viruses, the most abundant biological entities on the planet, are capable of infecting organisms from all three branches of life, although the majority infect bacteria where the greatest degree of cellular diversity lies. However, the characterization and assessment of viral diversity in natural environments is only beginning to become a possibility. Through the development of a novel technique for the harvest of viral DNA and the application of 454 pyrosequencing, a snapshot of the diversity of the DNA viruses harvested from a standing pond on a cattle farm has been obtained. A high abundance of viral genotypes (785) were present within the virome. The absolute numbers of lambdoid and Shiga toxin (Stx) encoding phages detected suggested that the depth of sequencing had enabled recovery of only ca. 8% of the total virus population, numbers that agreed within less than an order of magnitude with predictions made by rarefaction analysis. The most abundant viral genotypes in the pond were bacteriophages (93.7%). The predominant viral genotypes infecting higher life forms found in association with the farm were pathogens that cause disease in cattle and humans, e.g. members of the Herpesviridae. The techniques and analysis described here provide a fresh approach to the monitoring of viral populations in the aquatic environment, with the potential to become integral to the development of risk analysis tools for monitoring the dissemination of viral agents of animal, plant and human diseases

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate

Genomic Variation in the Canine Adenoviruses

Considerable differences exist in the pathogenicity of wild-type and vaccine strains of canine adenovirus. An analysis of their genome structure was undertaken in orrder to investigate the molecular basis of their differing pathogenicities. Six vaccine strains and two wild-type strains were examined. Preliminary investigations revealed significant variation near the genome termini

Herpes Simplex Virus Ribonucleotide Reductase DNA and Requirements for mRNA 3' End Formation

This study was undertaken to examine the organisation and structures of mRNAs mapping at the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase locus. From comparisons between equivalent HSV-2 and HSV-1 nucleotide sequences, putative transcription control signals were identified, and functional analysis of certain control elements was performed. The positions of translated regions within the mRNAs encoding ribonucleotide reductase were identified as were conserved amino acid domains between viral and cellular reductases. During the course of these comparisons, a conserved DNA sequence, YGTGTTYY (Y = pyrimidine), located 30nuc downstream from the polyadenylation (AATAAA) signal of both viral and cellular genes, was identified. Functional tests showed that the YGTGTTYY signal was required for efficient formation of mRNA 3' termini. Arrangement of mRNAs and DNA Sequence Comparisons at the HSV-2 Ribonucleotide Reductase Locus. Four overlapping mRNAs have been identified at the ribonucleotide reductase locus. These mRNAs are unspliced and share common 5' and 3' termini. Two early mRNAs with sizes of 4.5kb and 1.2kb have a common 3' terminus and encode HSV-2 polypeptides Vmw138 and Vmw38 respectively which are almost certainly components of the viral ribonucleotide reductase. The other two mRNAs are late, 5' co-terminal species with sizes of 6.4kb and 1.7kb which appear to encode an identical 54,000 mol. wt. protein; the 6.4kb transcript is 3' co-terminal with the early mRNAs. As a consequence of this mRNA arrangement, the 5' termini of the 4.5kb and 1.2kb mRNAs and the 3' terminus of the 1.7kb mRNA are located within larger, overlapping transcripts. The genome region adjacent to the 6.4kb and 1.7kb mRNAs specifies three late transcripts which are synthesised in the opposite orientation; the 5' terminal region of one of these species overlaps with the 6.4kb and 1.7kb mRNAs. The 3' terminus of the late 1.7kb mRNA is located within the transcribed region of the 6. 4kb species. Previously, it has been proposed that read-through of HSV poly A sites is due to inefficient processing at certain 3' termini. The ability of sequences flanking the 1.7kb mRNA 3' terminus to produce functional mRNA was tested using a plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) protein coding sequences fused to an HSV-2 immediate early promoter. CAT activities produced in HeLa cells by a plasmid carrying the 1.7kb mRNA 3' terminal sequences were comparable to activities obtained with a plasmid containing the 3' processing signals from a non-internal poly A site. Read-through of HSV poly A sites may therefore reflect a general reduction in mRNA 3' processing efficiency rather than differences in the 3' processing signals of individual genes. The HSV-1 transcripts equivalent to the 4.5kb and 1.2kb mRNAs have sizes of 5. 0kb and 1. 2kb and encode HSV-1 polypeptides Vmw136 and Vmw38 respectively. Nucleotide sequence comparisons between the 5' flanking regions of these equivalent HSV-2 and HSV-1 mRNAs have revealed the following homologies: 1) upstream from the 4.5kb and 5.0kb mRNA 5' termini, blocks of conserved sequences are present which resemble transcription control signals at the promoter regions of other genes; TATA box homologues, C-rich tracts and A+C-rich elements have been identified. (Abstract shortened by ProQuest.)