Immunization by Replication-Competent Controlled Herpesvirus Vectors.

Replication-competent controlled virus vectors were derived from the virulent herpes simplex virus 1 (HSV-1) wild-type strain 17 by placing one or two replication-essential genes under the stringent control of a gene switch that is coactivated by heat and an antiprogestin. Upon activation of the gene switch, the vectors replicate in infected cells with an efficacy that approaches that of the wild-type virus from which they were derived. Essentially no replication occurs in the absence of activation.

Production of a high-efficiency TILLING population through polyploidization.

Targeting Induced Local Lesions in Genomes (TILLING) provides a nontransgenic method for reverse genetics that is widely applicable, even in species where other functional resources are missing or expensive to build. The efficiency of TILLING, however, is greatly facilitated by high mutation density. Species vary in the number of mutations induced by comparable mutagenic treatments, suggesting that genetic background may affect the response.

Clinical and laboratory diagnostic characteristics and cytotoxigenic potential of Hafnia alvei and Hafnia paralvei strains.

A collection of 68 Hafnia strains previously identified to the species level by 16S rRNA gene sequencing were investigated for simple phenotypic properties that could aid in their recognition in the clinical laboratory. Four tests, including malonate utilization, fermentation of salicin and d-arabinose, and expression of β-glucosidase activity, correctly assigned each strain to either Hafnia alvei or H. paralvei.

Discovery of rare mutations in populations: TILLING by sequencing.

Discovery of rare mutations in populations requires methods, such as TILLING (for Targeting Induced Local Lesions in Genomes), for processing and analyzing many individuals in parallel. Previous TILLING protocols employed enzymatic or physical discrimination of heteroduplexed from homoduplexed target DNA. Using mutant populations of rice (Oryza sativa) and wheat (Triticum durum), we developed a method based on Illumina sequencing of target genes amplified from multidimensionally pooled templates representing 768 individuals per experiment.

The rapidly evolving centromere-specific histone has stringent functional requirements in Arabidopsis thaliana.

Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes.

A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat.

Background: Wheat (Triticum ssp.) is an important food source for humans in many regions around the world. However, the ability to understand and modify gene function for crop improvement is hindered by the lack of available genomic resources.

Tissue-specific variation in DNA methylation levels along human chromosome 1.

Background: DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters.

Results: Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands.

Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription.

Cytosine methylation, a common form of DNA modification that antagonizes transcription, is found at transposons and repeats in vertebrates, plants and fungi. Here we have mapped DNA methylation in the entire Arabidopsis thaliana genome at high resolution. DNA methylation covers transposons and is present within a large fraction of A.

Tissue-specific splicing of the herpes simplex virus type 1 latency-associated transcript (LAT) intron in LAT transgenic mice.

To study the regulation of herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) expression and processing in the absence of other cis and trans viral functions, a transgenic mouse containing the region encompassing the LAT promoter (LAP1) and the LAT 5' exon through the 2.0-kb intron was created. LAT expression was detectable by reverse transcriptase PCR (RT-PCR) in a number of tissues, including the dorsal root ganglia (DRG), trigeminal ganglia (TG), brain, skin, liver, and kidney.

Chromatin and siRNA pathways cooperate to maintain DNA methylation of small transposable elements in Arabidopsis.

Background: DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs.

DNA methylation profiling identifies CG methylation clusters in Arabidopsis genes.

Cytosine DNA methylation in vertebrates is widespread, but methylation in plants is found almost exclusively at transposable elements and repetitive DNA. Within regions of methylation, methylcytosines are typically found in CG, CNG, and asymmetric contexts. CG sites are maintained by a plant homolog of mammalian Dnmt1 acting on hemi-methylated DNA after replication.

Specific histone tail modification and not DNA methylation is a determinant of herpes simplex virus type 1 latent gene expression.

During herpes simplex virus type 1 (HSV-1) latency, gene expression is tightly repressed except for the latency-associated transcript (LAT). The mechanistic basis for this repression is unknown, but its global nature suggests regulation by an epigenetic mechanism such as DNA methylation. Previous work demonstrated that latent HSV-1 genomes are not extensively methylated, but these studies lacked the resolution to examine methylation of individual CpGs that could repress transcription from individual promoters during latency.

Overlapping subdeletions within a 348-bp in the 5' exon of the LAT region that facilitates epinephrine-induced reactivation of HSV-1 in the rabbit ocular model do not further define a functional element.

A previous study identified a 348-bp region at the 5' end of the 8.5-kb latency-associated transcript (LAT) of HSV-1 strain 17Syn+ that is necessary for maximum adrenergically induced reactivation following transcorneal iontophoresis of epinephrine (D.C.

Effects of hydrogen bonding within a damaged base pair on the activity of wild type and DNA-intercalating mutants of human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase "flips" damaged DNA bases into its active site where excision occurs. Tyrosine 162 is inserted into the DNA helix in place of the damaged base and may assist in nucleotide flipping by "pushing" it. Mutating this DNA-intercalating Tyr to Ser reduces the DNA binding and base excision activities of alkyladenine DNA glycosylase to undetectable levels demonstrating that Tyr-162 is critical for both activities.

Altering the expression kinetics of VP5 results in altered virulence and pathogenesis of herpes simplex virus type 1 in mice.

While many herpes simplex virus (HSV) structural proteins are expressed with strict-late kinetics, the HSV virion protein 5 (VP5) is expressed as a "leaky-late" protein, such that appreciable amounts of VP5 are made prior to DNA replication. Our goal has been to determine if leaky-late expression of VP5 is a requirement for a normal HSV infection. It had been shown previously that recombinant viruses in which the VP5 promoter was replaced with promoters of other kinetic classes (including a strict late promoter) exhibited no alterations in replication kinetics or virus yields in vitro.