Karl H. Brackmann

Antibody directed to a synthetic peptide encoding the NH2-terminal 16 amino acids of the adenovirus type 2 E1B-53K tumor antigen recognizes the E1B-20K tumor antigen.

A peptide, H2N-Glu-Arg-Arg-Asn-Pro-Ser-Glu-Arg-Gly-Val-Pro-Ala-Gly-Phe-Ser-Gly-(Cys )COOH, containing the amino acid sequence at the NH2 terminus of the adenovirus type 2 (Ad2) E1B-coded large T antigen (E1B-53K) has been synthesized. Anti-peptide antibody was generated in rabbits and used to immunoprecipitate Ad T antigens from Ad2 early infected cell extracts. In addition to the expected E1B-53K T antigen, anti-peptide antibody precipitated the Ad2 E1B-20K T antigen that was previously shown to be related to E1B-53K (M. Green, K.H. Brackmann, M.A. Cartas, and T. Matsuo, J. Virol. 42, 30-41, 1982). Anti-peptide prepared against the COOH terminus of the E1B-53K T antigen or against the NH2 terminus of the E1B-19K T antigen did not precipitate the E1B-20K T antigen. These data suggest that the Ad2 E1B-20K T antigen initiates translation at nucleotide 2016 in reading frame 3, as does E1B-53K. The viral mRNA that encodes the E1B-20K T antigen has not been identified.

Recombinant JC viral DNA: Verification and physical map of prototype

Abstract Genomic DNA of the human polyomavirus JC was molecularly cloned from DNA extracted from primary human fetal glial cells infected with prototype (MAD-1) virus and from diseased brain tissue from which JC virus originally was isolated. This report documents that these recombinant MAD-1 DNAs are identical, are prototypical, and may serve as unambiguous references to distinguish the variant DNAs produced by independent isolates of JC virus. Possible ambiguity in other recombinants of MAD-1 DNA is discussed. A new restriction endonuclease cleavage map of MAD-1 DNA was derived for 52 sites cleaved by eight enzymes.

The application of high-performance liquid chromatography for the resolution of proteins encoded by the human adenovirus type 2 cell transformation region.

Abstract The human adenovirus 2 (Ad2) transformation genes are located in early region E1a (map position (mp) 1.3–4.5) and E1b (mp 4.6–11.2) on the linear duplex Ad2 DNA genome of M r 23 × 10 6 (viral DNA is divided into 100 map units). E1b codes for three major proteins of apparent molecular weights 53,000 (53K), 19K, and 20K; smaller quantities of 21K, 22K, and 23K proteins that are related to 53K are also synthesized in Ad2-infected cells. Because the resolution and purification of these Ad2 candidate transformation proteins proved very difficult by conventional protein purification methods, the applicability of high-performance liquid chromatography (HPLC) methodology was examined. Starting with a crude cytoplasmic S100 fraction of Ad2-infected human cells, the resolution of the Ad2 E1b-coded 19K, 20K, 21K, 22K, and 23K proteins by reverse-phase HPLC using a C 8 column and a linear 0–60% 1-propanol gradient in 0.5 m pyridine formate was achieved, E1b proteins purified under these conditions retained their immunological reactivity. By anion-exchange HPLC using a linear 10 m m to 1 m NaCl gradient in 10 m m 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.6, the same five Ad2 E1b-coded 19K–23K proteins were separated, with improved resolution of the 19K protein. Based on these findings, protocols for the extensive purification of the E1b-19K and E1b-20K proteins have been developed. These results illustrate the potential of HPLC methodology for the rapid purification of biologically interesting proteins from complex cellular mixtures of proteins.

Introduction of cloned human papillomavirus genomes into mouse cells and expression at the RNA level

The entire DNA genomes of five different human papillomaviruses (HPVs) were cloned into the BamHI site of pBR322 (HPV-1a, HPV-3, HPV-4, and HPV-9) or the EcoRI site of pBR325 (HPV-2), using as starting materials virus preparations isolated from papillomas of individual patients. Under stringent hybridization conditions (Tm-28 degrees), the five cloned HPVs exhibited less than 10% homology with one another. To establish model cell systems that may be useful for the identification of HPV genes and HPV gene products, mouse thymidine kinase negative (tk-) cells were cotransformed to the tk+ phenotype with the herpesvirus thymidine kinase gene and each of the five HPV cloned DNAs (either as intact recombinants or excised HPV DNA without removal of pBR). In most tk+ cell clones, a complex pattern of multiple high molecular weight inserts of HPV DNA were present in high copy number. Most of the HPV DNA sequences in the cotransformed cells were not present as unit-length episomal viral DNA. Analyses of the integration pattern (DNA blot) and RNA expression (RNA blot) of several HPV-1a and HPV-3 transformed cell lines suggest that some copies of the viral genome are integrated in a similar manner in different cell lines leading to the expression of identical viral RNA-containing species. Two of the cell lines transformed by the intact HPV-1a/pBR322 recombinant synthesized substantial amounts of four discrete viral polyadenylated cytoplasmic RNA species of 1.9, 3.2, 3.8, and 4.5 kb. Two cell lines transformed by the intact HPV-3/pBR322 recombinant synthesized 4-5 polyadenylated cytoplasmic viral RNA species ranging from 0.8 to 4.6 kb. The analysis shows that each viral RNA species appears to be a hybrid RNA molecule containing both HPV and pBR322 sequences. Based on these findings and the molecular organization of the HPV-1a genome (O. Danos, M. Katinka, and M. Yaniv (1982). EMBO J. 1, 231-237), it is possible that transcription of each of the HPV-1a RNA species is initiated using the HPV early promoter and terminated in pBR322.

Antibodies to Synthetic Peptides Targeted to the Transforming Genes of Human Adenoviruses: An Approach to Understanding Early Viral Gene Function

Adenoviruses (Ads) are DNA tumor viruses which contain double–stranded linear DNA genomes of molecular weight 20–25 million (GREEN et al. 1967). There are 31 well–defined human adenoviruses, many of which are ubiquitous in the human population. Adenoviruses commonly cause latent infections of lymphoid tissue and are mainly associated with respiratory disease, which can reach epidemic proportions in closed populations. By DNA homology measurements, human Ads 1–31 are classified into five groups, A through E, containing homologous transforming gene sequences (GREEN et al. 1979 a; MACKEY et al. 1979 a). Although probably all human Ads can transform cells and many can induce tumors in laboratory animals, there is no evidence that they play a significant role in human carcinogenesis. An extensive search was made for the presence of DNA sequences representing each of the five human Ad groups in human tumors representing about 90% of the cancer incidence in the United States. Significant Ad genetic information was not detected under conditions that would have detected less than one transforming gene per tumor cell in most cases (Mackey et al. 1976; Green and Mackey 1977; Green et al 1979b; Mackey et al. 1979b; Wold et al. 1979; Green, unpublished data). Thus, although the human Ads are widespread and have oncogenic potential, their oncogenic potential does not appear to be manifested in their native human host species.

Adenovirus DNA: Transcription During Productive Infection, Integration in Transformed Cells, and Replication in Vitro

The human adenoviruses provide excellent model systems for studying the molecular biology of the mammalian cell, i.e., the mechanism of DNA replication and RNA transcription and translation, and for analyzing the integration and function of viral genes in transformed cells. Adenoviruses are icosahedral particles that are 80 nm in diameter, weigh 175 million daltons, and contain 9–10 polypeptides and 12–13% DNA (Green, 1970). Adenovirus genomes are linear, duplex DNA molecules of molecular weight 20–25 x 106 (Green, 1970). The interaction of the adenovirus genome with the cell can result in either (i) productive infection (usually human cells) in which thousands of virus particles are replicated and the cell is killed, or (ii) cell transformation (usually rodent cells) in which no virus is formed, but a portion of the viral genome is integrated into cellular DNA, and growth properties and macromolecular synthesis are controlled in an unknown way by information from viral genes.