A McLachlan

Cloning and expression of a human muscle phosphofructokinase cDNA

The nucleotide sequence of a 2.86-kb cDNA clone containing the complete human muscle phosphofructokinase (PFK) protein-coding region was determined. It comprises 76 bp of 5-untranslated sequence, 2340 bp encoding human muscle PFK polypeptide, and 399 bp of 3-untranslated sequence plus a poly(A) tract. A retroviral vector was utilized to express the product of this coding sequence in mouse fibroblasts. The PFK-coding cDNA was shown to code for an enzymatically active polypeptide by immunoprecipitation analysis and DEAE-Sephadex A-25 chromatography.

Retroviral-mediated transfer and expression of hepatitis B e antigen in human primary skin fibroblasts and Esptein-Barr virus-transformed B lymphocytes☆

Previously, an amphotropic retroviral expression system coding for the neomycin resistance gene was developed and used to synthesize hepatitis B e antigen (HBeAg) and hepatitis B core/e antigen (HBc/eAg) in transfected mouse NIH 3T3 fibroblasts (A. McLachlan et al., 1987, J. Virol. 61, 683-692). In the present study, these transfected cell lines were infected with a helper amphotropic murine leukemia virus resulting in the production of infectious recombinant retrovirus. The recombinant retrovirus was examined for its capacity to transmit resistance to the antibiotic, G418, and to express hepatitis B virus antigens in mouse NIH 3T3 fibroblasts, human primary skin fibroblasts, and Epstein-Barr virus (EBV)-transformed B lymphocytes. A mouse NIH 3T3 fibroblast clone was generated which produced recombinant retrovirus with the capacity to transmit HBeAg expression to these murine and human cell lines. In contrast, it was not possible to transmit HBc/eAg synthesis efficiently to these cell lines by recombinant retroviral infection. The difference between the efficiencies of transmission of HBeAg and HBc/eAg expression by recombinant retroviral-mediated infection was not predicted as the expression vector coding for HBc/eAg synthesis differs only by the deletion of approximately 90 nucleotides of HBV DNA sequence from the vector coding for HBeAg synthesis.

T-Cell Recognition of Pre-S Regions of HBsAg can Bypass Nonresponse to the S Region

The objective of the studies reported herein was to identify and characterize T cell and B cell recognition sites within the pre-S regions of HBsAg/p39, and to analyze functional T-cell-B cell interactions at the level of in vivo antibody production. The results indicate: (1) several peptides within the pre-S(1) region of HBsAg were identified which can induce and elicit HBsAg/p39-specific T-cell proliferation; (2) a 10 amino acid peptide, p12-21, and the 94-117 sequence define pre-S(1)-specific T-cell recognition sites; (3) five distinct, pre-S(1)-specific antibody binding sites and 2 pre-S(2)-specific antibody binding sites were identified; (4) synthetic pre-S(1) region T-cell determinants can prime in vivo antibody production to multiple B-cell epitopes within the pre-S(2) and S regions, as well as within the pre-S(1) region; and (5) specificity of the primed T cell population can influence the specificity of the B-cell response.