Oligomeric structure of bAE3 protein.

The "brain" form of the anion exchanger protein 3 (bAE3) has been purified to homogeneity from the rabbit kidney by differential centrifugation and immunoaffinity chromatography. A single protein band of approximately 165 kDa was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Monomers, dimers (a major component), and a higher oligomeric form (apparently tetramers) were found after oxidative cross-linking of purified bAE3. The largest form of bAE3 was separated from dimers and monomers by sucrose gradient centrifugation and was studied by transmission electron microscopy to confirm a tetrameric structure. Two main types of bAE3 images were detected, round (approximately 11-14 nm) and square-shaped (approximately 12 x 12 nm). Image analysis revealed fourfold rotational symmetry of both the round and square-shaped images, indicating that bAE3 consists of multiples of 4 subunits. We conclude that bAE3 in Triton X-100 solution is predominantly a mixture of dimers and tetramers with a smaller amount of monomers.

Tail length determination in bacteriophage T4.

This report identifies a protein that regulates tail length in bacteriophage T4. Earlier work (Duda et al., 1990) suggested that the gene 29 protein could be involved in T4 tail length determination as a "template" or "tape-measure", similar to that proposed for the gene H protein in bacteriophage lambda. We have altered the length of a recombinant gene 29 by constructing deletions and duplications in different parts of the gene. Each of these constructs was incorporated into the high-level expression vector, pET-11d. Seven plasmids with different lengths of gene 29 were made and used in complementation studies. We have found that the length of the tail can be decreased by deleting the C-terminal part of gene 29 or increased by forming duplications in different parts of this gene, and that the length of the tail can be proportional to the size of the engineered protein. Unlike phage lambda, plasmids with deletions in the middle of gene 29 or from the N-terminal end produced correspondingly smaller but inactive gene 29 protein and no viable phage were formed. Our results show that alterations in the length of gene 29 protein proportionately alters tail length, and argue strongly for a scheme in which 29 protein is a ruler or template that determines tail length during tail assembly.

[Attachment of long fibrils at various stages of assembly of the virus particle of a bacteriophage]. / Prisoedinenie dlinnykh fibrill na raznykh étapakh sborki virusnoi chastitsy bakteriofaga.

Bacteriophage T4 fibrillar structural elements were obtained when the basal plates and their complexes with the core were complemented in vitro with long tail fibrils. The organisation and functioning of the complexes were studied using PAAG electrophoresis, electron microscopy and sedimentation analysis. About 80% of the particles attached fibrils and were biologically active structures, i.e. could participate in the process of infection. Apparently, the organisation of virus particles is not a strictly regulated and consistent process at all the steps, but only at certain stages of their formation.

Formation of complexes between long tail fibres and substructural elements of phage T4D.

Complexes of substructural elements of bacteriophage T4 (baseplates, baseplate-core complexes) with long tail fibres were obtained for the first time by complementation in vitro. A study of the organization of the complexes was carried out by PAGE, electron microscopy and sedimentation analysis. About 90% of baseplates and baseplate-core complexes were combined with fibres. However, the number of the attached fibres varied from one to six. On the basis of the data obtained, we proposed that the attachment of long tail fibres can occur before the assembly of the whole bacteriophage.

[Structural changes in proteins of the bacteriophage T4 basal plate resulting from the attachment of long fibrils]. / Strukturnye perekhody belkov bazal'noi plastinki bakteriofaga T4 pri prisoedinenii dlinnykh fibrill.

The effect of the attachment of long tail fibers on the structure of proteins of the bacteriophage T4 baseplate was studied by digital processing of electron microscopic images. The attachment of the long fibers was found to result in dramatical changes of the proteins of the baseplate plag, while the wedges, to which the long fibers are attached, undergo only slight changes. We studied the baseplates with one to six attached fibers and found that the attachment of one fiber resulted in the change of the entire baseplate, although the wedge located in the vicinity of the fiber attachment changed to a greater extent. Only after the attachment of three and more fibers the changes of the same kind occurred through the entire baseplate.

[Proteins of the basal plate of bacteriophage T4 participating in the contractile impulse]. / Belki bazal'noi plastinki bakteriofaga T4, uchastvuiushchie v peredache dvigatel'nogo impul'sa.

Reversible linking of proteins with Cu2+ and Ni2+ ions was used to study the topography of the structural proteins of bacteriophage T4 basal plate. Gene products (GP) 9 and 10 were found to directly contact the proximal part of long fibrils (GP34). GP27, GP54 and GP5 interact with the lower disk of the contractive sheath (GP18), while GP48 and GP54 are in contact with the core (GP19). The proteins of the sheath (GP18) and the core (GP19) were found to have contact over the whole tail length.