Biomedical subjects
Publications and source records attributed to F Calvo-Riera.
We have devised an easy non-radioactive assay to evaluate the activity of six different DNA polymerases: the Klenow fragment of E.coli DNA Polymerase I, Taq DNA Polymerase, Sequenase, Moloney Murine Reverse Transcriptase, T7 DNA Polymerase and E.coli DNA Polymerase I. The method is based on the differential mobility of single stranded DNA compared to that of double-stranded DNA in agarose gel electrophoresis.
By means of a radioimmunoassay a substance excreted in feces could be detected in patients with hepatitis non-A,non-B (HNANB). Feces extracts of patients with sporadic and posttransfusion HNANB as well as of healthy persons were precipitated with PEG, digested with RNase and DNase and separated on CsCl. In HNANB-patients a RIA-positive material with a density of 1.3 g/ml CsCl could be detected which contained a partially double-stranded circular DNA. Cloning of this DNA in lambda-phase resulted in DNA of about 5 Kb, which hybridized with feces DNA under stringent conditions. The 5 Kb-DNA were mapped with different restriction enzymes. A 1.5 Kb EcoRi-fragment cross-hybridizes with HBV-DNA. No hybridization and sequence homologies were found with human, viral and procaryotic DNA as well as with plasmid and phage DNA (data base EMBL, Heidelberg). It is assumed that the DNA excreted in feces of HNANB-patients represents a viral genome not detected so far.
An anti-D-immunoglobulin preparation implicated in a hepatitis non-A,non-B transmission was analyzed for the presence of a DNA, which was originally isolated, cloned and sequenced from feces of a patient with posttransfusion HNANB. The investigation was performed by a DNA polymerase chain reaction using synthetic oligoprimers. Commercially available immunoglobulin preparations served as controls. The demonstration of identical DNA sequences in the infectious material speaks in favour of this up to now unknown circular and partially double-stranded DNA to be a virus genome involved in hepatitis non-A,non-B.