Biological activity and intracellular location of the Tat protein of equine infectious anemia virus

The Tat protein of equine infectious anemia virus (EIAV) was synthesized in Escherichia coli using the inducible expression plasmid, pET16b, which contains a His.Tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. Yields of up to 20 mg of Tat were obtained from 10¹¹ bacterial cells. The recombinant Tat protein was shown to potently trans-activate the EIAV long terminal repeat (LTR) following its introduction into canine cells by ‘scrape loading’. The EIAV Tat protein was found to localize predominantly within the cytoplasm, in contrast to HIV-1 Tat. The availability of large amounts of purified functional EIAV Tat protein should greatly facilitate detailed structure-function analyses.     

Highly efficient de novo mutant identification in a Sorghum bicolor TILLING population using the ComSeq approach

Screening large populations for carriers of known or de novo rare single nucleotide polymorphisms (SNPs) is required both in Targeting induced local lesions in genomes (TILLING) experiments in plants and in screening of human populations. We previously suggested an approach that combines the mathematical field of compressed sensing with next‐generation sequencing to allow such large‐scale screening. Based on pooled measurements, this method identifies multiple carriers of heterozygous or homozygous rare alleles while using only a small fraction of resources. Its rigorous mathematical foundations allow scalable and robust detection, and provide error correction and resilience to experimental noise. Here we present a large‐scale experimental demonstration of our computational approach, in which we targeted a TILLING population of 1024 Sorghum bicolor lines to detect carriers of de novo SNPs whose frequency was less than 0.1%, using only 48 pools. Subsequent validation confirmed that all detected lines were indeed carriers of the predicted mutations. This novel approach provides a highly cost‐effective and robust tool for biologists and breeders to allow identification of novel alleles and subsequent functional analysis.     

DNA replication origin of polyoma virus: early proximal boundary.

We constructed a series of deleted polyoma genomes by Bal 31 nuclease digestion from the unique Bg/I site at nucleotide 86 on the "early" side of the origin of DNA replication. The ability of the cloned deleted genomes to replicate was tested after transfection into mouse 3T6 fibroblasts or into the polyomatransformed C127 (COP5) mouse cell line (Tyndall et al., Nucleic Acids Res. 9:6231-6251, 1981). Deletions up to nucleotide 64-had no effect on the amount of replicated DNA accumulated, but larger deletions, extending up to nucleotide 42, decreased this amount 7- to 10-fold. By nucleotide 38, the quantity of detected DNA was down 100-fold, and by nucleotide 20, no replication could be detected. The minimum origin segment does not contain any known high-affinity, large tumor antigen binding site.     

Serological Analysis of the Deoxyribonucleic Acid Polymerase of Avian Oncornaviruses I. Preparation and Characterization of Monospecific Antiserum with Purified Deoxyribonucleic Acid Polymerase

Monospecific antiserum was prepared against purified deoxyribonucleic acid (DNA) polymerase from avian myeloblastosis virus (AMV). Immunodiffusion assay with purified DNA polymerase revealed that the anti-DNA polymerase serum formed one precipitation band, whereas no reaction with any of the seven major structural proteins of AMV was observed. The antiserum also demonstrated enzyme-neutralizing antibody activity that was associated with the immunoglobulin G fraction. There was no difference in the neutralization of DNA polymerase activity directed by ribonucleic acid (RNA), DNA, or RNA-DNA hybrid templates.