Immune proteasomes in the developing rat thymus.

The age dynamics of the content of the immune proteasome subunits LMP2 and LMP7 in rat thymus during prenatal and early postnatal ontogeny was studied. The LMP2 and LMP7 immune subunits were detected by Western blotting already by the 18th day of embryonic development, their amount increased to the 21st day to the level characteristic of the postnatal state. Double immunofluorescent labeling showed that in the thymus tissue the largest amount of LMP2 and LMP7 is localized in epithelial cells, whereas the level of their expression in thymocytes is lower. The results suggest that the establishment in thymus of selection processes, which depend on activity of immune proteasomes, can take place already in prenatal ontogeny. Analysis of age dynamics of the natural apoptosis level in thymocytes also favors this supposition. The presence of immune proteasomes in thymocytes during perinatal ontogeny suggests that, besides the antigen presentation, immunoproteasomes may possess other important functions.

[Formation of immune proteasomes and development of the immune system in mammalian ontogenesis].

Current concepts of the structure of immune proteasomes and their role in immune response have been considered. The main attention has been paid to the formation of immune proteasomes in secondary lymphoid and nonlymphoid organs during ontogenesis of mammals. The causes of ineffective formation of immune system in early postnatal development have been discussed.

Peculiarities of proteasome pool formation in rat spleen and liver during postnatal development.

Changes in the specific activity and amounts of 26S and 20S proteasome pools in rat spleen and liver during postnatal development and appearance in them of immune subunits were studied. Two decreases in chymotrypsin-like activity of the proteasome pools were recorded during the first three weeks after birth. The activity minimum fell on the 11th and 19th days, and the first decrease was more prolonged and pronounced than the second. The decrease in the specific activity of the 26S proteasome pools was associated with a reduction of their quantity. The 20S proteasome pools displayed no such decreases. Noticeable quantities of immune subunits LMP7 and LMP2 were revealed by Western blotting in the spleen on the 7th day and on the 19th day in the liver, concurrently with the beginning of the decrease in the proteasome activity. It was concluded that during the first three weeks of postnatal development the proteasome pools in rat spleen and liver were replaced twice, and in the spleen (a lymphoid organ) a qualitatively new pool containing immune subunits appeared nearly two weeks earlier than in the liver (a non-lymphoid organ). The appearance of immune proteasomes in different organs and tissues during some weeks after birth seems to explain the immune system inefficiency during embryogenesis and early postnatal development.

[The peculiarities of repair DNA-polymerases in the loach embryogenesis]. / Osobennosti reparativnykh DNK-polimeraz v émbriogeneze v'iuna.

The current concept of eukaryotic DNA polymerases is considered, which are involved in nuclear DNA repair. The data are given on a new group of DNA polymerases that maintain the integrity of DNA structures without preliminary excision of damaged regions. A special attention is paid to specific features of the functioning of repair DNA polymerases in embryogenesis of the loach. A possible existence is discussed of the previously unknown pathway of DNA repair with participation of DNA polymerase delta as independent from the nuclear antigen of proliferating cells.

Interaction of loach DNA polymerase delta with DNA duplexes with single-strand gaps.

The interaction of DNA polymerase delta purified from eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes with single-strand gaps of 1-13 nucleotides was studied. In the absence of template-restricting DNA, the enzyme elongated primers on single-stranded DNA templates in a distributive manner. However, in the presence of the proximal 5;-terminus restricting the template, the enzyme activity significantly increased. In this case, the enzyme was capable of processive synthesis by filling gaps of 5-9 nucleotides in DNA duplexes. These data indicate that DNA polymerase delta can interact with both the 3;- and 5;-termini located upstream and downstream from the gap. Analysis of the complexes formed by DNA polymerase delta and different DNA substrates by electrophoretic mobility shift assay confirmed the assumption that this enzyme can interact with the proximal 5;-terminus restricting the gap. DNA polymerase delta displayed much higher affinity in duplexes with gaps of approximately 10 nucleotides compared to the standard template-primer complexes. Maximal affinity was observed in experiments with DNA substrates containing unpaired 3;-tails in primers. The results of this study suggest that DNA polymerase delta exerts high activity in the cell nuclei during repair of DNA intermediates with single-strand gaps and unpaired 3;-termini.

Identification of DNA polymerase delta in eggs of a teleost fish (loach).

DNA polymerase found in an extract from eggs of the teleost fish Misgurnus fossilis (loach) has been identified as an enzyme of the delta type. The enzyme was purified 4000- to 5000-fold from the extract by liquid chromatography. The DNA polymerase activity was sensitive to the inhibiting action of aphidicolin but resistant to N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP). The enzyme activity correlates with the presence of a polypeptide with molecular mass of 120-130 kD that interacts specifically with polyclonal antibodies against calf thymus DNA polymerase delta as revealed by Western blotting and is presumably the catalytic subunit of the enzyme. The loach DNA polymerase possesses the 3'-->5'-exonuclease activity specific to single-stranded DNA and catalyzes distributive elongation of primers in primer-template complexes.

Preferential interaction of loach DNA polymerase delta with DNA duplexes containing single-stranded gaps.

We studied the interaction of DNA polymerase delta (pol delta) purified from the eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes containing single-stranded gaps of 1-13 nucleotides (nt). In the absence of processivity factors (PCNA, RF-C, and ATP), pol delta elongated primers on single-stranded DNA templates in a distributive manner. However, the enzyme was capable of processive synthesis by filling gaps of 5-9 nt in DNA duplexes. These data suggest that, upon filling a small gap, pol delta interacts with the 5'-terminus downstream of the gap as well as with the 3'-terminus of the primer. Interaction of pol delta with the proximal 5'-terminus restricting the gap was confirmed by electrophoretic mobility shift assay. Analysis of the enzyme binding to DNA duplexes containing gaps of various sizes showed a much higher affinity of pol delta for duplexes with gaps of about 10 nt than for DNA substrates with primers annealed to single-stranded templates. The most efficient pol delta binding was observed in experiments with DNA substrates containing unpaired 3'-tails in primers. The data obtained suggest that DNA molecules with small gaps and single-stranded tails may serve as substrates for direct action of pol delta in the course of DNA repair.