The influence of rifampicin, streptolydigin, tetracycline and chloramphenicol on phage DNA transport from the T7 virion into the E. coli cell was studied. It has been found that the DNA transport proceeds in at least three stages. During the initial stage the phage injects into the host cell the left approximately 10 per cent of its DNA molecule. The entrance of the next 50 per cent of 17 DNA molecule is blocked by inhibitors which block transcription but not translation. Moreover, the entrance time of this part of the T7 DNA increases in the case of the T7 mutant D111 (which contains a deletion of the A2 and A3 promoters) and decreases in the case of the D53 mutant (which contains a deletion in the region of the early gene transcription terminator). It would appear, that the second stage of the phage DNA transport is tightly coupled with its transcription and that a mechanical function is carried out by RNA polymerase. The translation inhibitors completely block the entrance of the remaining 40 per cent of the 17 DNA molecule (class III genes) into the host cell. It would appear that some class I and (or) II gene product(s) are obligatory components of the final stage of 17 DNA transport. Some probable consequences of this virus DNA transport model as well as its agreement with the functional structure of T7 chromosome and with T7 development are discussed.

The polyprotein of Cocksfoot mottle virus (CfMV ; genus Sobemovirus) is translated from two overlapping open reading frames (ORFs) 2a and 2b by a N1 ribosomal frameshifting mechanism.In this study, a 12 kDa protein was purified from viral RNA-derived samples that appears to correspond to the CfMV genome-linked protein (VPg). According to the determined N-terminal amino acid sequence, the VPg domain is located between the serine proteinase and replicase motifs and the N terminus of VPg is cleaved from the polyprotein between glutamic acid and asparagine residues. Western blot analysis of infected plant material showed that the polyprotein is processed at several additional sites. An antiserum against the ORF 2a product recognized six distinct proteins, whereas, of these, the VPg antiserum clearly recognized only a 24 kDa protein. This indicates that the fully processed 12 kDa VPg detected in viral RNA-derived samples is a minor product in infected plants. An antiserum against the ORF 2b product recognized a 58 kDa protein, which indicates that the fully processed replicase is entirely or almost entirely encoded by ORF 2b. The origin of the detected cleavage products and a proposed polyprotein processing model are discussed.

The immuno-PCR (iPCR) method combines advantages of enzyme-linked immunosorbent assay and polymerase chain reaction, which is used in iPCR as a method of "visualization" of antigen-antibody interaction. The use of iPCR provides classical PCR sensitivity to objects traditionally detected by ELISA. This method could be very sensitive and allow for detection of quantities of femtograms/ml order. However, iPCR is still not widely used. The aim of this review is to highlight the special features of the iPCR method and to show the main aspects of its development and application in recent years.

Immuno-PCR (iPCR) is one of the methods used for the detection of a wide range of analytes and features the high sensitivity of the polymerase chain reaction (PCR) method. iPCR uses antibodies coupled to DNA, followed by the amplification of the attached DNA using RT-PCR. Two major types of antibody-DNA conjugates are currently used, which are obtained as a result of non-covalent (biotin-streptavidin) or covalent interactions. Using a strain-promoted azide-alkyne cycloaddition (SPAAC), we synthesized covalent DNA-antibody conjugates, optimized the reaction conditions, and developed an efficient protocol for the purification of conjugates, with which all unreacted antibodies and oligonucleotides are separated. Covalent DNA-antibody conjugates were tested with iPCR assays that were previously developed for the detection of IgE and IgM antibodies with the use of the supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin. The results show that the modification of antibodies with amino groups did not allow us to obtain monolabeled antibodies or antibodies with a strictly defined number of DNA-labels. The degree of labeling determined by the dyes introduced through the azido group reflects the actual labeling degree statistically. If the average labeling degree for azido groups is 1.1, the conjugates contain 25% mono-labeled antibodies, 50% double-labeled antibodies, and 25% unlabeled ones. The specificity of the monoclonal antibody to human IgE (BE5) changed after conjugation with the oligonucleotide. The sensitivity of iPCR in the detection of IgM antibodies produced against the LeC disaccharide using a covalent conjugate was similar to that of a supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin, but the new procedure is two steps shorter.

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