Characterization of 13 microsatellites for Lahontan cutthroat trout (Oncorhynchus clarki henshawi) and cross-amplification in six other salmonids.

Thirteen newly developed tri- and tetranucleotide repeat microsatellite markers were developed for Lahontan cutthroat trout (Oncorhynchus clarki henshawi), a threatened subspecies endemic to the Lahontan hydrographic basin in the western USA. These loci are highly polymorphic with five to 30 alleles per locus and observed heterozygosities ranging from 0.4 to 0.7. Cross-species amplification of these markers was most successful in the closely related rainbow trout, Oncorhynchus mykiss, with only three loci amplifying in brown trout, Salmo trutta. Nonoverlapping allelic distributions for many of these loci among the six salmonid species screened suggest these markers may be useful for hybrid determination.

Polymorphic microsatellite markers for the Mojave desert tortoise, Gopherus agassizii.

We describe primers and polymerase chain reaction (PCR) conditions to amplify 14 tri- and tetranucleotide microsatellite loci for the Mojave desert tortoise (Gopherus agassizii). Across three populations (87 individuals) located in the Mojave Desert, USA, the markers yielded a range of four to 33 alleles and an average observed heterozygosity of 0.733 (range 0.433 to 0.933). We neither detected linkage disequilibrium between any pair of loci nor did we find a consistent pattern of deviation from Hardy-Weinberg equilibrium. These microsatellites are designed for PCR multiplexing, and provide higher throughput capacity to aid in conservation genetics studies for this threatened species.

Generation of a life-expanded rhesus monkey fibroblast cell line for the growth of rhesus rhadinovirus (RRV).

RRV, the rhesus macaque equivalent to HHV-8 or kaposi's sarcoma-associated herpesvirus (KSHV) was recently isolated from a simian immunodeficiency virus (SIV) infected macaque with a lymphoproliferative disorder. The growth of RRV in tissue culture requires propagation of primary rhesus monkey fibroblasts (RFs). In an effort to extend the life of these primary cells in tissue culture, the catalytic subunit of telomerase (hTERT) was introduced into RF cells using a recombinant retrovirus. This new cell line, Telo-RFs, have currently been passed in tissue culture over 80 times compared to a maximum passage number of 38 for wild type RFs, remain fully permissive for RRV DNA replication and production of infectious virus. Viral gene expression of immediate-early and early RNA transcripts was virtually identical to that observed in wild-type (wt) RFs. In addition, transfection experiments show that telo-RFs are easily and more efficiently transfected than wtRFs.

Identification of the rhesus macaque rhadinovirus lytic origin of DNA replication.

We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8.