Adaption of SYBR Green-based reagent kit for real-time PCR quantitation of GC-rich DNA.

In the mammalian genome, approximately 50% of all genes are controlled by promoters with high GC contents. Analyzing the epigenetic mechanisms regulating their expression is difficult. Hence, we examined a method for stable quantification of such GC-rich DNA sequences. Quantification of DNA during real-time PCR is often based on reagent kits containing the fluorescent dye SYBR Green. However, these ready-made kits may not be suitable for amplifying DNA samples with a high GC content (>70%). DNA segments with eccentric GC contents are frequently found in proximal promoter areas, and their quantification may be necessary in chromatin accessibility by real-time polymerase chain reaction or chromatin immunoprecipitation analyses of epigenetic mechanisms of gene regulation. We therefore optimized the SYBR Green I FastStart reaction system by supplementing the system with dimethyl sulfoxide, betaine, and increased DNA polymerase content. Here, we describe the development of the assay and demonstrate its effectiveness for two different DNA templates, showing that these modifications allow for the reliable amplification and quantification of DNA with GC contents exceeding >70% using the LightCycler instrument.

Nucleotide sequences of the 26S mRNAs of the viruses defining the Venezuelan equine encephalitis antigenic complex.

Genetic relationships among viruses defining the Venezuelan equine encephalitis (VEE) virus antigenic complex were determined by analyzing the 3'-terminal 561 nucleotides of the nonstructural protein 4 gene and the entire 26S RNA region of the genome. New sequence information is reported for VEE 78V-3531 (VEE subtype-variety IF), Mucambo (IIIA), Tonate (IIIB), 71D-1252 (IIIC), Pixuna (IV), Cabassou (V), and AG80-663 (VI) viruses. The results reported here and by previous investigators largely support the current classification scheme of these viruses, while clearly identifying Everglades (II) as a subtype I virus. A genetic relationship between 78V-3531 (IF) and AG80-663 (VI) viruses contradicted previous serologic results. Mutations near the amino terminus of the E2 envelope proteins of Pixuna and AG80-663 viruses probably account for the previously reported low reactivity of the protective monoclonal antibody 1A2B-10 with these two viruses. Variations in the distribution of potential glycosylation sites in the E2 glycoprotein are discussed.

Nucleotide sequence variation of the envelope protein gene identifies two distinct genotypes of yellow fever virus.

The evolution of yellow fever virus over 67 years was investigated by comparing the nucleotide sequences of the envelope (E) protein genes of 20 viruses isolated in Africa, the Caribbean, and South America. Uniformly weighted parsimony algorithm analysis defined two major evolutionary yellow fever virus lineages designated E genotypes I and II. E genotype I contained viruses isolated from East and Central Africa. E genotype II viruses were divided into two sublineages: IIA viruses from West Africa and IIB viruses from America, except for a 1979 virus isolated from Trinidad (TRINID79A). Unique signature patterns were identified at 111 nucleotide and 12 amino acid positions within the yellow fever virus E gene by signature pattern analysis. Yellow fever viruses from East and Central Africa contained unique signatures at 60 nucleotide and five amino acid positions, those from West Africa contained unique signatures at 25 nucleotide and two amino acid positions, and viruses from America contained such signatures at 30 nucleotide and five amino acid positions in the E gene. The dissemination of yellow fever viruses from Africa to the Americas is supported by the close genetic relatedness of genotype IIA and IIB viruses and genetic evidence of a possible second introduction of yellow fever virus from West Africa, as illustrated by the TRINID79A virus isolate. The E protein genes of American IIB yellow fever viruses had higher frequencies of amino acid substitutions than did genes of yellow fever viruses of genotypes I and IIA on the basis of comparisons with a consensus amino acid sequence for the yellow fever E gene. The great variation in the E proteins of American yellow fever virus probably results from positive selection imposed by virus interaction with different species of mosquitoes or nonhuman primates in the Americas.

Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5'-noncoding region and the E2 envelope glycoprotein.

The virulent Trinidad donkey (TRD) strain of Venezuelan equine encephalitis (VEE) virus and its live attenuated vaccine derivative, TC-83 virus, have different neurovirulence characteristics. A full-length cDNA clone of the TC-83 virus genome was constructed behind the bacteriophage T7 promoter in the polylinker of plasmid pUC18. To identify the genomic determinants of TC-83 virus attenuation, TRD virus-specific sequences were inserted into the TC-83 virus clone by in vitro mutagenesis or recombination. Antigenic analysis of recombinant viruses with VEE E2- and E1-specific monoclonal antibodies gave predicted antigenic reactivities. Mouse challenge experiments indicated that genetic markers responsible for the attenuated phenotype of TC-83 virus are composed of genome nucleotide position 3 in the 5'-noncoding region and the E2 envelope glycoprotein. TC-83 virus amino acid position E2-120 appeared to be the major structural determinant of attenuation. Insertion of the TRD virus-specific 5'-noncoding region, by itself, into the TC-83 virus full-length clone did not alter the attenuated phenotype of the virus. However, the TRD virus-specific 5'-noncoding region enhanced the virulence potential of downstream TRD virus amino acid sequences.