[Characteristics of δ-Aminolevulinic Acid Dehydratase of the Cold-Water Sponge Halisarca dujardinii].

δ-Aminolevulinic acid dehydratase (ALAD) is a key enzyme of the cytoplasmic heme biosynthesis pathway. The primary structure of the ALAD gene, the multimeric structure of the ALAD/hemB protein, and ALAD expression during the annual reproductive cycle were studied in the cold-water marine sponge Halisarca dujardinii. The results implicated the GATA-1 transcription factor and DNA methylation in regulating ALAD expression. Re-aggregation of sponge cells was accompanied by a decrease in ALAD expression and a change in the cell content of an active ALAD/hemB form. Further study of heme biosynthesis and the role of ALAD/hemB in morphogenesis of basal animals may provide new opportunities for treating pathologies in higher animals.

[Structure of Neuroglobin from Cold-Water Sponge Halisarca dujardinii].

The iron-containing protein neuroglobin (Ngb) involved in the transport of oxygen is generally considered the precursor of all animal globins. In this report, we studied the structure of Ngb of the cold-water sponge Halisarca dujardinii. In sponges, the oldest multicellular organisms, the Ngb gene contains three introns. In contrast to human Ngb, its promoter contains a TATA-box, rather than CG-rich motifs. In sponges, Ngb consists of 169 amino acids showing rather low similarity with its mammalian orthologues. It lacks Glu and Arg residues in positions required for prevention of hypoxia-related apoptosis. Nevertheless, Ngb contains both proximal and distal conserved heme-biding histidines. The primary structure of H. dujardinii neuroglobin predicted by sequencing was confirmed by mass-spectrometry analysis of recombinant Ngb expressed in E. coli. The high level of Ngb expression in sponge tissues suggests its possible involvement in the gas metabolism and presumably in other key metabolic processes in H. dujardinii.

The Role of Epibionts of Bacteria of the Genus Pseudoalteromonas and Cellular Proteasomes in the Adaptive Plasticity of Marine Cold-Water Sponges.

It was found that cells of different color morphs of the cold-water marine sponges Halichondria panicea (Pallas, 1766) of the class Demospongiae differ in the content of epibionts of bacteria of the genus Pseudoalteromonas. The sponge cells with elevated levels of epibionts of bacteria of the genus Pseudoalteromonas showed an increased expression of Hsp70 proteins but had a reduced level of the proteasomal catalytic beta 5 subunit, which was accompanied by a change in their activity. Probably, epibionts of bacteria of the genus Pseudoalteromonas may affect the ubiquitin-proteasome system in the cells of cold-water marine sponges and, thereby, ensure their adaptive plasticity.

Role of proteasomes in non-specific immune response of marine annelids.

We investigated functioning of proteasomes and chaperones in Arenicola marina coelomocytes in conditions of lipopolysaccharide-induced inflammation. We observed the increase of chymotrypsin-like proteasome activity in coelomocytes 1 h after induction. Amount of proteasome subunits alpha- and beta-5 types increased as well. We also detected appearance of a new form of Hsp70 chaperone in infected coelomocytes. Our results allow us to consider the changes in proteasome structure and induction of chaperones as principle mechanisms in stress adaptation and defensive reactions development in annelids.

[A Simple and Efficient Method of Inducing Targeted Deletions in the Drosophila Genome].

Deletion mutagenesis is one of the most efficient approaches to studying gene function. However, conventional methods of inducing targeted mutations in the drosophila genome are time- and labor-consuming. This work proposes a new, simple, and effective method of producing drosophila mutants with gene deletions. The method involves the insertion of I-Scel and I-CreI recognition sites and a fragment homologous to the target sequence into the chromosome region of interest by means of an attB-containing construct, the induction of double-strand DNA breaks by the appropriate meganuclease, and their repair by homologous recombination. The procedure results in a deletion extending from the attP-site to the target locus. A cassette was designed to enable single-step construct production for the deletion of any given genomic region. A set of markers facilitates the selection of recombination events. The efficacy of the proposed technique was confirmed by the induction of a 47-kb deletion containing the qtc gene.

[Plasticity of nervous and immune systems in different species: the role of proteasomes].

Nervous and immune systems have many general features in their organization and functioning in various animal species from insects to mammals. These systems are capable to regulate effectively each other by exchange of information through rather small molecules like oligopeptides, cytokines, and neuropeptides. For many such molecules, that function as transmitters or signaling peptides, their origin and receptors are common within nervous and immune systems. Development of nervous and immune systems during ontogenesis and their functions in various species are controlled by the ubiquitous HYPERLINK "http://slovari.yandex.ru/proteolytic/en-ru/Medical/" \1 "longvo/" proteolytic ubiquitin-proteasome system (UPS). UPS regulates key biochemical processes in both systems by providing formation of synaptic connections and synaptic plasticity, and governs immune responses. In the review, the molecular mechanisms of functioning and interaction between nervous and immune systems are considered in different species of invertebrats and vertebrats. The role of UPS in these processes in the main subject of this review.

[Affinity capture of specific DNA fragments with the use of short synthetic sequences].

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine and 5-propynyl-2'-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments from mixtures.

[Replication of the baculovirus genome]. / Replikatsiia genoma bakulovirusov.

The review describes the current state of studying the baculovirus DNA replication. The structural organization of replication initiation sites and replication intermediates are considered. Attention is focused on virus replication factors, including DNA polymerase, helicase, IE-1, LEF-1, LEF-2, and LEF-3.

[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.

Helix-destabilizing properties of the baculovirus single-stranded DNA-binding protein (LEF-3).

The helix-destabilizing properties of a single-stranded DNA-binding protein, LEF-3, of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Partial duplexes of DNA containing single-stranded (ss) tails of different sizes and orientations were used as substrates for assay of the unwinding ability of LEF-3. Upon noncooperative binding to ssDNA, LEF-3 was capable of unwinding the duplexes with 5' ss tails. However, it did not cause melting of the duplexes containing 3' ss tails, even at oversaturation of ssDNA adjacent to the duplexes. Upon cooperative binding to long ss tails, LEF-3 also produced the polar melting effect; it unwound the duplexes with long 5' ss tails, but not those with long 3' ss tails. These data suggest that LEF-3 has a preferential direction for entry into duplex DNA, namely 5' to 3' with respect to the bound DNA strand. In agreement with its polarity, LEF-3 efficiently melted the primer-template complexes which serve as substrates for DNA polymerases. However, the formation of a complex with viral DNA polymerase before addition of LEF-3 protected the primer-templates from the destabilization effect of LEF-3. Although the destabilization effect of LEF-3 was highly sensitive to monovalent and divalent salts, the protein was capable of melting DNA duplexes in a polar manner at physiological conditions, i.e., 30 degrees C in 0.15 M NaCl. Therefore, the polar destabilization effect of LEF-3 seems to be physiologically important and may be connected, in particular, with the polar action of viral helicase holoenzyme during baculovirus replication.

Colocalization of baculovirus IE-1 and two DNA-binding proteins, DBP and LEF-3, to viral replication factories.

We have recently identified a DNA-binding protein (DBP) from the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) which can destabilize double-stranded DNA (V. S. Mikhailov, A. L. Mikhailova, M. Iwanaga, S. Gomi, and S. Maeda, J. Virol. 72:3107-3116, 1998). DBP was found to be an early gene product that was not present in budded or occlusion-derived virions. In order to characterize the localization of DBP during viral replication, BmNPV-infected BmN cells were examined by immunostaining and confocal microscopy with DBP antibodies. DBP first appeared as diffuse nuclear staining at 4 to 6 h postinfection (p.i.) and then localized to several specific foci within the nucleus at 6 to 8 h p.i. After the onset of viral DNA replication at around 8 h p.i., these foci began to enlarge and eventually occupied more than half of the nucleus by 14 h p.i. After the termination of viral DNA replication at about 20 h p.i., the DBP-stained regions appeared to break down into approximately 100 small foci within the nucleus. At 8 h p.i., the distribution of DBP as well as that of IE-1 or LEF-3 (two proteins involved in baculovirus DNA replication) overlapped well with that of DNA replication sites labeled with bromodeoxyuridine incorporation. Double-staining experiments with IE-1 and DBP or IE-1 and LEF-3 further confirmed that, between 8 and 14 h p.i., the distribution of IE-1 and LEF-3 overlapped with that of DBP. However, IE-1 localized to the specific foci prior to DBP or LEF-3 at 4 h p.i. In the presence of aphidicolin, an inhibitor of DNA synthesis, immature foci containing IE-1, LEF-3, and DBP were observed by 8 h p.i. However, the subsequent enlargement of these foci was completely suppressed, suggesting that the enlargement depended upon viral DNA replication. At 4 h p.i., the number of IE-1 foci correlated with the multiplicity of infection (MOI) between 0.4 and 10. At higher MOIs (e.g., 50), the number of foci plateaued at around 15. These results suggested that there are about 15 preexisting sites per nucleus which are associated with the initiation of viral DNA replication and assembly of viral DNA replication factories.

Bombyx mori nucleopolyhedrovirus encodes a DNA-binding protein capable of destabilizing duplex DNA.

A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived from Bombyx mori) infected with the B. mori nucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no. L33180). This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3'-->5' exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.

Termination within oligo(dT) tracts in template DNA by DNA polymerase gamma occurs with formation of a DNA triplex structure and is relieved by mitochondrial single-stranded DNA-binding protein.

Xenopus laevis DNA polymerase gamma (pol gamma) exhibits low activity on a poly(dT)-oligo(dA) primer-template. We prepared a single-stranded phagemid template containing a dT41 sequence to test the ability of pol gamma to extend a primer through a defined oligo(dT) tract. pol gamma terminates in the center of this dT41 sequence. This replication arrest is abrogated by addition of single-stranded DNA-binding protein or by substitution of 7-deaza-dATP for dATP. These features are consistent with the formation of a T.A*T DNA triplex involving the primer stem. Replication arrest occurs under conditions that permit highly processive DNA synthesis by pol gamma. A similar replication arrest occurs for T7 DNA polymerase, which is also a highly processive DNA polymerase. These results suggest the possibility that DNA triplex formation can occur prior to dissociation of DNA polymerase. Primers with 3'-oligo(dA) termini annealed to a template with a longer oligo(dT) tract are not efficiently extended by pol gamma unless single-stranded DNA-binding protein is added. Thus, one of the functions of single-stranded DNA-binding protein in mtDNA maintenance may be to enable pol gamma to successfully replicate through dT-rich sequences.

Effects of Xenopus laevis mitochondrial single-stranded DNA-binding protein on primer-template binding and 3'-->5' exonuclease activity of DNA polymerase gamma.

Mitochondrial DNA (mtDNA) is replicated by DNA polymerase gamma by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol gamma. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3'-portion of a 71-mer template containing 40 dT residues at its 5'-end as a binding site for mtSSB. DNA pol gamma bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5'-dT40-tail in the 18:71-mer primer-template reduced the binding of DNA polymerase gamma and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 3'-->5' exonuclease of DNA polymerase gamma. The size of fragments protected from 3'-->5' exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.

[Analysis of the conformational mobility of DNA in transcriptionally-active chromatin]. / Analiz konformatsionnoi podvizhnosti DNK v transkriptsionno-aktivnom khromatine.

An attempt is made to estimate the degree of conformational flexibility of DNA in different fractions of nucleosomes of transcriptionally active chromatin of higher eukaryotes, and in yeast nucleosomes as well. We have used the circular dichroism method to estimate conformational changes in DNA induced by temperature shift. The DNA conformational potential was shown to be approximately the same in all the fractions of nucleosomes under study.