New comparative genome hybridization technology on NotI-microarrays is presented (Karolinska Institute International Patent WO02/086163). The method is based on comparative genome hybridization of NotI-probes from tumor and normal genomic DNA with the principle of new DNA NotI-microarrays. Using this method 181 NotI linking loci from human chromosome 3 were analyzed in 200 malignant tumor samples from different organs: kidney, lung, breast, ovary, cervical, prostate. Most frequently (more than in 30%) aberrations--deletions, methylation,--were identified in NotI-sites located in MINT24, BHLHB2, RPL15, RARbeta1, ITGA9, RBSP3, VHL, ZIC4 genes, that suggests they probably are involved in cancer development. Methylation of these genomic loci was confirmed by methylation-specific PCR and bisulfite sequencing. The results demonstrate perspective of using this method to solve some oncogenomic problems.

The biology of the past century was, undoubtedly, reductionist. Its general trend was expressed in the appearance and burst of scientific disciplines, such as biochemistry, biophysics, molecular biology, molecular genetics, virology, cell biology, and bioorganic chemistry. All these disciplines are united by an aspiration to explain the phenomena of life through the description of the properties of molecules, first of all, biopolymers, which constitute the living organisms. In current science, all these directions are often united under the general heading "physicochemical biology", while classical biological disciplines, such as zoology, botany, embryology, etc., are referred to as "general biology".

Highly purified (>99%) bovine tryptophanyl-tRNA synthetase (bWRS) incubated with [gamma-P-32]ATP incorporated the radioactive phosphate into proteins of 54 kDa and 40 kDa corresponding, respectively, to the full-length bWRS polypeptide and to the enzymatically active truncated bWRS lacking the N domain. Endogenous ATP:protein phosphotransferase in bWRS preparations phosphorylated histones but was inactive against casein. As shown by phosphoamino acid analysis of HCl hydrolysates, in both bWRS and histones only Ser residues were phosphorylated; phosphorylated Ser-471 was identified in bWRS. In gel filtration and high performance ion-exchange chromatography, histone kinase activity was coeluted with bWRS. The Am2 monoclonal antibodies directed against an epitope within the 40-kDa core of the bWRS molecule affected neither histone kinase activity nor self-phosphorylation of bWRS preparations. However, the Aml monoclonal antibodies directed against an epitope of the bWRS N domain inhibited self-phosphorylation of bWRS as well as phosphorylation of histones and kemptide by bWRS. The bWRS-Am2 complex adsorbed on Protein A-Sepharose phosphorylated histones under standard conditions. These results suggest association of endogenous protein kinase activity in bWRS preparations with the N domain of the bWRS molecule. The N domain contains sequences of putative ATP binding and Ser/Thr-protein kinase catalytic domains. Therefore, bWRS appears to be a multifunctional enzyme with both tryptophanyl-tRNA synthetase and protein kinase activities.

Nine microsatellite motifs were studied in DNA inserts of 150 NotI linking and jumping clones of human chromosome 3, and also in the contig of 22 overlapping cosmids related to the region of homozygous deletions in small-cell lung carcinoma (locus 3p21.3, 570 kbp). Compared with the human genome in total, the NotI clones had three times less CA repeats and more GAG, GGA, AAT, CAC, AAAG, AATG, and GATG motifs, More than 11 kbp of subcloned DNA fragments from clones NL1-212, NLM-216, NL-026, J32-135H and cosmids 6, 7, and 16 were sequenced. Allele polymorphism was studied in 10 microsatellite loci identified on human chromosome 3 and in 8 CAG/CTG(n), loci of the brain cDNA (data from GenBank). Polymorphic loci were rather rare near the NotI sites (one of seven) and in cDNA (one of eight). This phenomenon and the distribution patterns of some microsatellites suggest that the NotI clones of human chromosome 3 and the cosmids related to the 3p21.3 region are rich in genes. Two polymorphic and nine STS markers were generated for the human chromosome 3; three of them were located in regions frequently affected in various solid tumors and four were related to genes. Polymorphic markers were related to the murine neuropeptide gene encoding receptor Y-YI gene (93.3% homology, 62 bp) and to the gene EST04896 expressed in brain.

A new DNA fragment from the human chromosome 3 was studied. Fluorescence in situ hybridization showed that this fragment is located mainly in the unstable regions of the chromosome. The known repeated elements in the fragment were rearranged, it contained matrix attachment regions (MAR) and numerous sequences known to be associated with recombination sites. More than 60% of the fragment was composed of nonrandomly distributed direct repeats. The distribution of direct repeats suggested their origin from unequal recombination of homologous chromosomal fragments. The DNA structure and distribution of direct repeats are supposed to be related with the propensity to recombination within and between the chromosomes.

The disadvantages of the NotI libraries available for the human chromosome 3 were analyzed. An improved procedure to generate the NotI libraries is suggested, allowing one to clone almost all NotI restriction sites, A representative NotI linking library of the total human genome was generated containing about 20,000 clones. Some genomic DNA fragments flanking the NotI restriction sites were sequenced. The genomic NotI sites were shown to be clustered, with the distance between the neighboring sites sometimes as small as 24-180 bp.

Observations were made on the aggregation of bovine pancreatic tryptophanyl-tRNA synthetase (EC 6.1.1.2) by the small-angle x-ray scattering method at temperatures close to physiological. The initial enzyme preparation, which was homogeneous when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol, was inhomogeneous in terms of particle size even when held at low temperature (4-8-degrees-C). The proportion of oligomers and their size increased on heating over the temperature range 30-45-degrees-C, with the change depending on the temperature and incubation time; formation of large aggregates that were an order of magnitude greater in size than the initial particles was observed. Subsequent lowering of the temperature to 20-degrees-C did not lead to oligomer dissociation, as a consequence of formation of disulfide crosslinks between the aggregated tryptophanyl-tRNA synthetase subunits. It has been hypothesized that the aggregation found to occur in vitro for bovine tryptophanyl-tRNA synthetase but not for any aminoacyl-tRNA synthetase from unicellular organisms is one mechanism by which this enzyme is compartmentalized in the pancreas.

Several nucleoside 5'-triphosphate analogs were investigated as inhibitors of human hepatitis B virus replication. Different analogs inhibited DNA synthesis differently, 3'-azido-2',3'-dideoxythymidine 5'triphosphate being the most active compound. This inhibitor blocked DNA synthesis by 50% at inhibitor: substrate molar ratio 1:8, and by 80% - at 1:1. The hypothesis is formulated that 3'-azido-2',3'-dideoxythymidine 5'-triphosphate inhibits RNA directed viral DNA replication due to incorporation of this compound into 3'-termini of newly synthesized DNA chains. The phenomenon observed opens new possibilities for chemotherapy of acute and chronic human hepatitis B.

: Data concerning the regions of tRNA molecules recognized by cognate aminoacyl-tRNA synthetases and obtained recently by the new methods (the synthesis of mutant tRNA genes and their transcription in vitro, chimeric suppressor tRNA, RNA engineering) are briefly discussed. The results of several laboratories are in full agreement with the hypothesis proposed earlier on the role of a tRNA anticodon as a specific region by which the enzyme identifies various tRNAs. More than half of tRNAs belong to the group recognized by the aminoacyl-tRNA synthetases by this mechanism. The behavior of natural and artificially constructed tRNAs which contain modified anticodons, but conserve amino acid specificity in the reaction of aminoacylation, indicates that in some tRNAs the role of the anticodon in the tRNA identity is minor if any. This group of tRNA is recognized by the aminoacyl-tRNA synthetases predominantly via the double helical region of the molecule. The authors discuss the significance of the absence of topographic conservatism in the mechanism by which tRNAs are recognized by the aminoacyl-tRNA synthetases.