Consolidating software tools for DNA microarray design and manufacturing.

As the human genome project progresses and some microbial and eukaryotic genomes are recognized, a novel technology, DNA microarray (also called gene chip, biochip, gene microarray, and DNA chip) technology, has attracted increasing number of biologists, bioengineers and computer scientists recently. This technology promises to monitor the whole genome at once, so that researchers can study the whole genome on the global level and have a better picture of the expressions among millions of genes simultaneously. Today, it is widely used in many fields - disease diagnosis, gene classification, gene regulatory network, and drug discovery. We present a concatenated software solution for the entire DNA array flow exploring all steps of a consolidated software tool. The proposed software tool has been tested on Herpes B virus as well as simulated data. Our experiments show that the genomic data follow the pattern predicted by simulated data although the number of border conflicts (quality of the DNA array design) is several times smaller than for simulated data. We also report a trade-off between the number of border conflicts and the running time for several proposed algorithmic techniques employed in the physical design of DNA arrays.

Characterization of B virus glycoprotein antibodies induced by DNA immunization.

Genes encoding glycoproteins gB, gC, gD, gE, and gG of herpes B virus (species Cercopithecine herpesvirus 1) were cloned into mammalian expression vector pcDNA3.1/V5-His. Abilities of the plasmid constructs to express recombinant glycoproteins were confirmed by Western blot analysis of transfected CHO-K1 and COS-7 cells. Antibody production was induced in rabbits by intramuscular injections with the expression constructs at four-weekly intervals. Antibodies to gB were detected after the second DNA inoculation, while it took an additional plasmid injection to induce responses to gC, gD and gE. The gG plasmid failed to stimulate antibody production. Antisera ELISA titers varied greatly depending on the gene, with gB inducing highest (21,000) and gE inducing lowest (60) antibody titer. The induced antibodies were predominantly conformation-dependent. The gB, gC, and gD antisera contained HSV cross-neutralizing antibodies, but only gB antisera contained B virus neutralizing antibodies. The gB antisera cross-reacted with HSV antigens in Western blot, ELISA, dot-blot, plaque immunostaining and immunoprecipitation assays, whereas gD and gC antisera were mostly B virus-specific. Thus, polyclonal antibodies to B virus glycoproteins can be generated by DNA immunization and used as diagnostic and research reagents.

Expression of Drosophila melanogaster gene encoding transcription factor GAGA is tissue-specific and temperature-dependent.

The GAGA factor (GAF) of Drosophila melanogaster encoded by the Trithorax-like gene is known to maintain expression of many Drosophila genes including homeotic ones, through configuration remodeling of local chromatin. The complicated transcript pattern of the GAF gene has been revealed at all stages of development. The study of GAF gene expression in whole flies and in salivary glands and in the brains with adjacent imaginal disks of the third instar larvae showed tissue-specific variations in transcript patterns and dependence of these patterns on the temperature of development (14-37 degrees C).

A new DNA marker, U15557, is linked to protease inhibitor and adenylate kinase-1 in the laboratory opossum, Monodelphis domestica.

A 323-bp DNA fragment (U15557) was isolated, cloned, and sequenced after polymerase chain reaction (PCR) amplification from Monodelphis domestica genomic DNA. A HindIII restriction fragment length polymorphism was identified in this species using the U15557 PCR, fragment as a hybridization probe. DNA samples exhibited either a 6.4 kb band, a 7.2 kb band, or both bands simultaneously. Behaviour of these two variants in family studies was consistent with codominant autosomal inheritance. Linkage between this marker and the loci encoding protease inhibitor (PI) and adenylate kinase 1 (AK1) was found in M. domestica.

[The evolutionarily conserved gene Nc70F, expressed in nerve tissue of Drosophila melanogaster, encodes a protein homologous to the mouse delta transcription factor]. / Evoliutsionno-koncervativnyi gen Nc70F, ékspressiruiushchiisia v nervnoi tkani Drosophila melanogaster, kodiruet belok gomologichnyi delta transkriptsionnomu faktoru myshi.

A cDNA copy of the Nc70F gene which is specifically expressed in Drosophila neural tissue was cloned and characterized. The gene has an open reading frame for the protein of 384 amino acids. The protein contains dimerization, DNA binding, activation and repression domains which are common for the eucaryotic transcription factors. However, the domain organization of the Nc70F protein has some peculiarities. The primary structure of the Nc70F product and other transcription factors were compared. High level of homology of Nc70F protein with the mouse delta transcription factor was found. The in situ hybridization on tissue section showed that the Nc70F gene expression is restricted to the central nerve system at all stages of Drosophila ontogenesis. By using Drosophila genomic and cDNA clones of Nc70F genes as probes, homologous transcripts were identified in the human poly(A) +RNA. The evolutionary conservative portion of this gene was localized in the 5-exons.

[Molecular-genetic analysis of expression features of the evolutionary-conservative neurogene Nc73EF of Drosophila melanogaster]. / Molekuliarno-geneticheskii analiz osobennostei ékspressii évoliutsionno-konservativnogo neirogena Nc73EF Drosophila melanogaster.

Molecular-genetic analysis of the Nc (neural conserved) genome sequence of Drosophila melanogaster located at the position 73EF of Drosophila melanogaster was performed. The Nc73EF sequence was shown to be expressed in the nervous system of Drosophila. We constructed the restriction map of this sequence and revealed the main RNA-coding fragment in the 5'-3' orientation. The RNA-dot analysis data demonstrated that expression of the Nc73EF transcripts took place mainly in the nervous system. Hybridization with the human brain poly(A)+RNA confirmed the basic RNA-coding fragment to be evolutionary conservative. Southern blot analysis showed this fragment to be unique in the Drosophila genome. Northern blots detected three transcripts of this DNA fragment.

[Improvement in the forms of control over the knowledge of interns in a department of polyclinical therapy]. / K voprosu o sovershenstvovanii form kontrolia znanii u subordinatorov na kafedre poliklinicheskoi terapii.

The authors summarize their experience gained with the methods of perfection the subinterns' knowledge control at the chair of polyclinical therapy. While taking the course of polyclinical therapy, the subinterns are to take 3 tests: the first one after the 4-day studies of urgent conditions which the district physician faces, the second one after the 5-day studies into expert medical evaluation of the working capacity in the activity of the district internist, and the third one after a cycle of studies into polyclinical therapy. During such tests, the knowledge may often be checked in writing.

Multi-site binding of human nuclear protein to the Alu-family repeated DNA.

Nuclear protein which selectively binds to the Alu-family DNA repeat (AFR, Blur8) is partially purified from human HeLa cells using a gel retention assay. At low protein concentrations only a single complex of the protein with AFR is formed (CII). Increasing protein concentrations lead to the gradual disappearance of CII, being replaced by complexes with higher (CI) and lower (CIII, CIV) electrophoretic mobilities. Differential binding of AFR restriction subfragments indicates that multiple protein-binding sites are present within AFR. We discuss two models explaining the anomalous electrophoretic mobility of CII by DNA bending or looping upon cooperative multi-site binding of the protein to AFR.

[Purification and properties of a protein from the nuclear fraction of HeLa cells binding to a cloned fragment of human satellite DNA III]. / Ochistka iz iadernoi fraktsii kletok HeLa i nekotorye svoistva belka, sviazyvaiushchego klonirovannyi fragment satellitnoi DNK III cheloveka.

In nuclear extract of HeLa cells two proteins were identified having the specific binding activity to cloned 1.8kb fragment of human satellite DNA III (HS3). One of the satellite binding proteins (SBP1) purified by column chromatography using DEAE-, phospho- and DNA-cellulose steps interacted also with adenovirus 5 replication enhancer (ARE), another protein (SBP2) was separated during phosphocellulose chromatography from ARE-binding protein. It is suggested that SBP1 is possibly identical to the nuclear factor I purified earlier from the nuclear extract of HeLa cells by other authors.

[Various characteristics of proteins from HeLa cella specifically binding to the human ALU sequence]. / Nekotorye kharakteristiki belkov iz kletok HeLa, spetsificheski sviazyvaiushchikh ALU-posledovatel'nost' cheloveka.

Two proteins with molecular weights of 40 and 80 kDa which are able to bind human Alu-repeat in a sequence-specific manner were found in HeLa nuclear extracts. The proteins were partially purified by column chromatography on DEAE-cellulose, phosphocellulose and FPLC MonoQ sorbent. One of the Alu-binding proteins (ABP2 with m. w. of 80 kDa) was found to bind the sequence within the Alu-repeat that has a homology with the T-antigen binding site of SV40, suggesting that ABP2 is the cellular analog of SV40 T-antigen.

Alu-family repeat binding protein from HeLa cells which interacts with regulatory region of SV40 virus genome.

Using a gel retardation assay the protein which binds selectively to the Alu-family repeat (AFR) has been identified and partially purified from HeLa cell nuclear extract. The protein (AFR-binding protein, ABP) forms multiple discrete complexes with AFR even in the presence of 200 to 2000-fold excess of non-specific (E. coli) DNA. The most stable complex has a relative mobility in 4% polyacrylamide gel (as compared to the free Alu-fragment) of 0.54. Heterogeneity of protein-DNA bands seen in the polyacrylamide gel suggests that ABP is able to form multimeric complexes with AFR. Competition experiments show that ABP does not interact with the RNA polymerase III promoter and with the TGGCA-sequence, but a high affinity binding site for ABP was found within a 660 bp restriction fragment containing the SV40 virus promoter and replication origin.

[Heterogeneity of nuclear proteins from HeLa cells specifically binding to the Alu sequence]. / Geterogennost' iadernykh belkov kletok HeLa, spetsificheski sviazyvaiushchikh Alu-posledovatel'nost'.

Nuclear proteins from HeLa cells specifically binding to the Alu-repeat cloned in the plasmid Blur8 have been studied. 0.35 M nuclear extract proteins have been separated on DEAE-cellulose. The presence of DNA-binding proteins has been found in all fractions by the technique of DNA-binding on nitrocellulose filters. The labelled restricted DNA of the plasmid Blur8 was incubated with the proteins of different fractions with the subsequent identification of specific Alu-protein complexes in polyacrylamide gel at low ionic strength. At least two proteins have been found to have the different affinity to Alu-repeat. Various functions of Alu-repeats and the possibility of their participation in the initiation of DNA replication are discussed.