Human adenovirus cloning vectors based on infectious bacterial plasmids

By making use of the fact that human adenovirus DNA circularizes in infected cells, and that circular forms of the viral genome are infectious, we have developed an improved adenovirus-based cloning system. A deletion mutant of adenovirus type 5 (Ad5) with deletions in early regions 1 (E1) and 3 (E3) was converted to a bacterial plasmid which can regenerate infectious virus following transfection into human 293 cells. A single XbaI recognition site in the deleted E3 region serves as a site for the insertion of foreign DNA. We have used this system to clone a number of genes into the Ad5 genome and describe the insertion of the neomycin/G418 resistance marker into Ad5 as an example.     

Monoclonal antibody with specificity to a conserved epitope in the C-terminal domain of histone H1 variants.

A monoclonal type M-immunoglobulin (IgM) was generated in mice against a nuclease-urea extract of HeLa metaphase chromosomes. This antibody stains metaphase chromosomes from a variety of mammalian cultured cell types by indirect immunofluorescence. Antibody 12C7 reacts by western transfer technique with histone H1 in all the cell lines tested. The antibody cross-reacts with H1, and H1(0) in human cells. Proteolytic digestions of H1 suggest that the epitope is localized in the carboxy-terminal domain of the histone H1 molecule. Digestion with trypsin demonstrates that the antibody 12C7 does not react with the globular domain of histone H1. The C-terminal domain of H1 subtypes therefore seems to have a conserved determinant which does exist in H1, H1(0), and probably in H5. This antibody has applications in studying the role of that domain of H1 in processes like chromosome condensation and variations in chromatin structure which influence gene expression.