Robert C. Ting

Correlation of cellular aggregation of transformed cells with their growth in soft agar and tumorigenic potential.

Summary Formation of cellular aggregates by rat, mouse, hamster, and human transformed cells was compared to aggregate formation by untransformed control cells. Viability and proliferation of cells in the aggregate form were found to correlate well with tumorigenic potential regardless of the method of transformation (spontaneous, chemical, or virus-induced). This assay has the potential for a fast and accurate means of evaluating in vitro transformation.

Direct PCR amplification and sequence analysis of extrachromosomal Plasmodium DNA from dried blood spots.

The Plasmodium parasite possesses two extrachromosomal genomes; the mitochondrial genetic element and the extrachromosomal plastid-like DNA. The latter has only been fully described for one culture strain of P. falciparum. In this study, a rapid procedure for amplifying plastid DNA from dried blood spots of blood infected with different malaria species was developed. PCR amplification of a 595 bp fragment within the plastid-like large subunit ribosomal-RNA (LSU-rRNA) gene was achieved using primers derived from the P. falciparum sequence. The PCR product was observed in all Plasmodium species examined. Sequence analysis of amplified products homologous to an LSU-rRNA fragment of the plastid-like extrachromosomal circle revealed extensive conservation between Plasmodium species including P. falciparum, P. vivax, P. malariae and P. berghei.

Mechanism of translation of the bicistronic mRNA encoding human papillomavirus type 16 E6-E7 genes

The transforming genes E6 and E7 of human papillomavirus (HPV) type 16 and other HPV types are expressed from a bicistronic mRNA with a characteristic spacing of 3 to 6 bp between the termination codon of E6 and the initiation codon of E7. Plasmid pSP64E6E7 which contains the reading frames of both E6 and E7 was constructed in order to study the expression of both proteins in a coupled transcription/rabbit reticulocyte translation system. Both E6 and E7 proteins were expressed simultaneously. This translation could be interfered with by antisense oligonucleotides corresponding to various regions of the transcript. Antisense oligonucleotides targeted at sequences flanking either side of the translation initiation codon of the E6 open reading frame were effective in inhibiting the synthesis of both proteins, whereas oligonucleotides complementary to the coding regions downstream of the first start codon showed either a considerably reduced effect or none at all. In particular, there was limited inhibition of E7 translation by antisense oligonucleotides flanking the translation start region of the E7 gene. In the presence of RNase H, it was possible to selectively inhibit the synthesis of either E6 or E7 by several gene-internal antisense oligonucleotides. We conclude that HPV16 E6-E7 bicistronic mRNA is fully functional and that both proteins are translated with equal efficiency via the scanning mechanisms with reinitiation at the second open reading frame. In addition, both AE6 and AE7 may have therapeutical potential as they are capable of inhibiting the proliferation of CaSki cells which contain the HPV16 genome.

Partial nucleotide sequence and organisation of extrachromosomal plastid-like DNA in Plasmodium berghei.

The murine malaria parasite Plasmodium berghei contains a plastid-like extrachromosomal genome. This genome is 30.7 kb in size and is transcriptionally active as shown by RT-PCR. DNA sequence analysis of the genome reveals 69.9-95.5% homology to sequences of the 35-kb extrachromosomal circle found in the human malaria species Plasmodium falciparum. Homologous sequences include regions of genes for the ssu-rRNA, lsu-rRNA, rpo B and clusters of t-RNAs. Sequence variation between the two Plasmodium species exists in the non-coding interspacing regions. A physical map has been constructed for the P. berghei circle, indicating the EcoRI and HindIII restriction sites as well as the arrangement of the rRNA, rpo B and tRNA genes. Arrangement of these genes is similar to that found on the P. falciparum 35-kb circle. The P. berghei circular element is distinct from the mitochondrial 6-kb DNA of both the murine and the human Plasmodium species. Preliminary results indicate that the circle may be a useful target for drug therapy.

ELISA for the detection of serum and saliva IgA against the BMRFI gene product of Epstein‐Barr virus

The BMRF1 protein is an Epstein‐Barr virus (EBV) DNA polymerase accessory protein that forms part of the early antigen diffuse (EA‐D) component. An enzyme‐linked immunosorbent assay (ELISA) was developed for the detection of IgA antibody to the BMRF1 protein of EBV in saliva and serum samples. The assay was shown to be specific for nasopharyngeal carcinoma (NPC) patients and, when used with saliva alone, to have a sensitivity comparable to an existing indirect immunoperoxidase assay for early antigens. The sensitivity of the assay could be significantly enhanced to 86% by the use of paired saliva and serum samples.

Identification of a Plasmodium berghei antigen sharing common features with P. falciparum and P. chabaudi parasitophorous-vacuole membrane antigens.

Abstract On the basis of immunological cross-reactivity, we identified a 43-kDa Plasmodium berghei antigen with homology to the exp-1 antigen from P. falciparium. The P. berghei antigen was recognized by an antibody directed against an epitope on the C-terminus of the P. falciparum exp-1 protein. This antigen is localized on the surface of the parasite and shares peptide sequence homology with the P. chabudi antigen Ag3008. To investigate further the role of the P. berghei antigen, we designed antisense phosphorothioate oligodeoxynucleotides (PS oligos) complementary to sequences of the exp-1 mRNA from P. falciparum. The PS oligos were capable of inhibiting the development of P. falciparum in vitro by 47%. In vivo, experiments in mice showed that the same PS oligos had the potential to extend the life span of mice infected with P. berghei by a factor of 2–4. The immunological cross-reactivity and the antisense inhibition of P. berghei parasite development in vivo indicate that this antigen may be a homologue of exp-1 from P. falciparum that has functional importance for parasite survival.

Diagnosis of parasites

Microscopic Diagnosis of the Parasites of ManBy Dr. Robert B. Burrows. Pp. xii + 328. (New Haven and London: Yale University Press, 1965.) 15 dollars; 105s. net.