Bernhard Hirt

Relaxed circular, covalently closed simian virus 40 DNA molecules were associated with the four histones that are present in virions. In electron micrographs the resulting complexes appear twisted, with globular structures (nucleosomes) along the DNA. Incubation with an untwisting extract converts the twisted complexes to relaxed structures. Extraction of the DNA from the relaxed complexes yields supercoiled molecules. The number of superhelical turns in these molecules corresponds to the number of nucleosomes per DNA molecule in the complexes.

The large conductance voltage- and Ca2+-activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming alpha-subunit coexpressed with the auxiliary beta1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel alpha-(BKalpha-/-) and beta1-subunit (BKbeta1-/-) knockout mice, we demonstrate normal hearing function and cochlear structure of BKbeta1-/- mice. During the first 4 postnatal weeks also, BKalpha-/- mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BKalpha-/- mice only from approximately 8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations.

The genome of the human parvovirus B19 contains a transcriptional promoter (BP06) at map position 6, upstream from the nonstructural protein genes. By cotransfecting HeLa cells with this promoter cloned before the chloramphenicol acetyltransferase (CAT) gene together with a plasmid containing almost the whole B19 genome, we showed that BP06 is transactivated by a B19 gene product. The transactivating viral protein was identified as the nonstructural protein NS-1. NS-1 synthesized in a wheat germ extract specifically stimulates transcription from BP06 in vitro. NS-1 of the minute virus of mice (MVM) activates the analogous MVM promoter, MP04. NS-1, therefore, has a positive feedback effect on the activity of its own promoter. Moreover, NS-1 of MVM activates the human BP06. We have identified, in the genome of B19, a second transcriptional promoter activity at map position 44, before the capsid protein genes. This promoter, BP44, was identified by cloning fragments of B19 DNA upstream of the CAT gene, transfecting the DNA into HeLa cells, and measuring CAT expression. The strength of the BP44 promoter is similar to that of the capsid gene promoter, MP39, of MVM. In (nonpermissive) HeLa cells, the BP44 promoter is not activated by NS-1. Thus, the BP06 promoter apparently does not determine the tissue specificity of B19 virus but BP44 could do so.

We have characterized an immunosuppressive parvovirus related to the minute virus of mice (MVM). The parvovirus, MVM(i), grew efficiently on the murine lymphoma cell line EL-4 and not on the A-9 strain of L-cells which is a host for the prototype MVM. MVM(i) was immunosuppressive for allogeneic mixed leukocyte cultures, inhibiting the generation of cytolytic T lymphocytes. MVM had no effect on mixed leukocyte cultures. MVM and MVM(i) particles were similar in buoyant density, sedimentation rate, appearance in the electron microscope, and polypeptide composition. We present restriction enzyme maps of the DNAs of MVM and MVM(i) which show that they are closely related. Out of 109 restriction endonuclease cleavage sites (representing together about 10% of the nucleotide sequence), 86 sites were shared by MVM and MVM(i), whereas 22 sites were absent from one of the two viruses. MVM(i) DNA had an apparent deletion of about 60 nucleotides relative to MVM, located near the 5' terminus of viral DNA.