Tandem arrangement of human genes for interleukin-4 and interleukin-13: resemblance in their organization.

The genes encoding human interleukin-4 (IL-4) and interleukin-13 (IL-13) are located on segment q23-31 of chromosome 5 and encode two multifunctional lymphokines with some common functions. We have cloned 72 kb of human genomic DNA that contain IL-4 and IL-13 and their flanking sequences, and constructed a restriction map of this region. Using Southern analysis, we have shown that IL-13 is located 12 kb 5' to IL-4 and linked in a 'tail-to-head' fashion. We have also determined the complete nucleotide sequence of the DNA fragment (about 4.8 kb) containing IL-13 and its 5' flanking regulatory region (2.1 kb) with a 'CpG island'. We identified potential binding sites for a different transcription factors in the 5' flanking region and in the first intron of IL-13. Comparison of IL-13 and IL-4 revealed considerable similarity in the structural organization of these genes and also many potential binding sites for transcription factors common to both genes: AP1, AP2, AP3, PEA3, HRE, TCF-1, GATA-3 and the interferon-inducible and enhancer elements. These results, along with the similarity in functional activity of IL-4 and IL-13 suggests that their expression may be coregulated.

Determination of binding site of anti-tumour necrosis factor-alpha monoclonal antibody using hybrid and mutant proteins.

In order to map the immunogenic epitope for the monoclonal antibody E7H2 on the human tumour necrosis factor (hTNF-alpha) molecule, a number of chimeric proteins were developed by in-frame joining segments of the human genes encoding TNF-alpha and lymphotoxin (TNF-beta) as well as by coupling appropriate coding regions for human and mouse TNF-alpha. High level expression of these chimeric genes was achieved in Escherichia coli by placing the coding sequences under control of either E. coli trp-promoter or a tandem of bacteriophage T7 constitutive promoters A2 and A3. As revealed by Western blot analysis with monoclonal antibody E7H2 directed against human TNF-alpha, the region involved in the binding of this antibody includes sequence ValGluLeuArg in the N-terminal part of the TNF-alpha molecule.

Reverse transcription, amplification and sequencing of poliovirus RNA by Taq DNA polymerase.

A model for virion RNA of the poliomyelitis virus, which does not pass the stage of DNA copies during biogenesis, demonstrates that Taq DNA polymerase is capable of synthesizing 960-bp cDNA with the specific primer. When comparing the nucleotide sequence of the starting virion RNA and recombinant DNAs, isolated from several independent clones, copying and amplification of virion RNA appear accurate (one substitution per 960 bp). A comparison of Taq and Tth DNA polymerases in RT/PCR indicates that the sensitivity of Taq polymerase seems to be two orders of magnitude higher than that of Tth polymerase. The RNA detection level under the chosen conditions approached 10(4) RNA copies per test. The present investigation indicates the great versatility of Taq polymerase, which promoted the reverse transcription reaction of RNA, cDNA amplification, screening of recombinant clones as well as sequencing of recombinant DNA. Thus application of Taq polymerase is rather promising not only to detect nucleic acids in biological samples, but also for isolating and cloning individual genes, encoded on DNA and/or on RNA templates.

DNA rearrangements generating artificial promoters.

The promoter-cloning plasmid pBRH4 (a derivative of pBR322 with a partially deleted promoter of the tet gene) is shown to contain a sequence which is located near the EcoRI site and can operate as an effective Pribnow box, but is not the remainder of the deletion-inactivated tet promoter of pBR322. If there is a sequence homologous to the '-35' promoter region at the border of the DNA fragment inserted at the EcoRI site, then a compound promoter arises and activates the tet gene. Point mutations in the nonfunctional--35 region of pBRH4 also activate the cryptic Pribnow box. Several compound promoters were obtained through deleting small portions of DNA around the HindIII site of pBR322; the deletions moved various sequences that could operate as Pribnow boxes towards the -35 region of the tet promoter.

Deoxyribonuclease treatment improves the homogeneity of single-stranded DNA preparations.

The isolation of single-stranded (ss) phagemid DNA using standard protocols often results in impure preparations, which contain undesirable quantities of chromosomal and/or double-stranded (ds) phagemid DNA. Here we report a simple and efficient method for elimination of virtually all dsDNA by incubation of phagemid viral particles with deoxyribonuclease I. In addition to analyzing the ratio of linear-to-circular topological forms of ssDNA after deoxyribonuclease I treatment, we verified that no decrease in transformation efficiency occurred and demonstrated that ssDNA molecules covered by capsid proteins remained intact following such treatment.

Differences in the biological activity of TNF alpha and TNF beta correlate with their different abilities for binding to the target cells.

TNF alpha and TNF beta were compared regarding their binding to different types of target cells, cytotoxic/cytostatic activity against murine and human tumor cell lines as well as human capillary endothelial cells, their ability to induce differentiation in myeloid leukemia cell lines, and induction of hemorrhagic tumor necrosis and tumor regression as well as lethal toxicity in tumor-bearing mice. The results show considerable quantitative differences in the biological activity between TNF alpha and TNF beta depending on the type of target cell which has been used. TNF beta was 3 fold more cytotoxic than TNF alpha against murine L929 fibroblasts and 3-5 times more active concerning the induction of hemorrhagic tumor necrosis, complete tumor regression and more toxic in tumor-bearing mice. In contrast to this, TNF beta was markedly less cytotoxic against human capillary endothelial cells and the human mammary carcinoma cell line MCF7 and much less cytostatic against the human myeloid leukemia cell lines HL60 and U937. The lesser antiproliferative effect of TNF beta correlated with a lower ability for induction of differentiation in these cell lines. Competitive radioligand binding assays showed that TNF beta was about 4 fold more effective than TNF alpha in competing with 125I-labeled TNF alpha for the binding to murine L929 fibroblasts. But it was 15-20 times less effective in binding to the human MCF7 cells and the human myeloid leukemia cell lines HL60 and U937. This revealed that, at least for these targets, the differences in the biological activity between TNF alpha and TNF beta are due to different abilities for binding to the target cells. Possible mechanisms for these different binding abilities are discussed.

[Design of chimeric proteins based on a pentameric superhelical fragment of COMP. II. Properties of a hybrid, containing an VNTR (MUC1) antigen of human tumors]. / Konstruirovanie khimernykh belkov na osnove pentamernogo superspiral'nogo fragmenta COMP. II. Svoistva gibrida, soderzhashchego antigen VNTR (MUC1) opukholei cheloveka.

An oligomeric chimeric protein DB-2 was constructed, to design drugs with antitumor activity and to develop highly sensitive immunospecific tests for the diagnostics of a wide variety of malignant epithelial cells in humans. DB-2 contains an immunodominant site of tetanus toxin and a fragment of the locus of the human tumor-associated antigen MUC1 with a variable number of tandem repeats. A pentameric superhelical fragment of the human cartilage oligomeric matrix protein was used as an oligomerization matrix. The expression of the protein in Escherichia coli cells was studied, a method for its purification was developed, and its main biochemical properties were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

[Design of chimeric proteins based on pentameric superhelical fragments of COMP. I. Properties of a hybrid containing an immunodominant segment of the surface protein of Plasmodium falciparum]. / Konstruirovanie khimernykh belkov na osnove pentamernogo superspiral'nogo fragmenta COMP. I. Svoistva gibrida, soderzhashchego immunodominantnyi uchastok osnovnogo poverkhnostnogo belka plazmodiia Plasmodium falciparum.

The oligomeric recombinant protein DB-1 containing the immunodominant sites of the circumsporozoite protein of Plasmodium falciparum and tetanus toxin was constructed to optimize the schemes of presentation of B-cell epitopes during vaccination with chimeric proteins without the use of adjuvants. A fragment of the pentameric coiled-coil human cartilage oligomeric matrix protein was used as an oligomerization matrix. The expression of the protein in Escherichia coli cells was studied, a method for its purification was developed, and it was biochemically characterized. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

[Recombinant plasmids containing hybrid protein genes with antigenic determinants of the foot and mouth virus]. / Rekombinantnye plazmidy, soderzhashchie geny gibridnykh belkov s antigennoi determinantoi virusa iashchura.

Plasmids have been constructed which contain genes coding for fused proteins including beta-galactosidase or human leukocyte interferon alpha 2 and monomeric or pentameric form of the main antigenic determinant of the foot-and-mouth disease virus (FMDV) serotype 01K. Expression of the hybrid genes has been studied. It is shown that fused proteins, containing beta-galactosidase and the antigenic determinant (monomer or pentamer), interact specifically with anti-FMDV anti-sera and with antibodies against peptide 141-160 of FMDV VP1 coat protein.

[Cloning and expression of the Escherichia coli thioredoxin gene]. / Klonirovanie i ékspressiia gena tioredoksina Escherichia coli.

The gene encoding thioredoxin (trxA) was isolated from chromosomal DNA of E. coli HB101 strain using the polymerase chain reaction. The cloned structural gene with a synthetic Shine-Dalgarno sequence was placed under the control of either inducible tac-promoter or a tandem of two strong constitutive promoters A2 and A3 from early region of bacteriophage T7. Both constructions were shown to provide high levels of biosynthesis of recombinant thioredoxin.

[Primary structure of cDNA of Bos taurus kappa-casein macropeptide]. / Pervichnaia struktura kDNK makropeptida kappa-kazeina Bos taurus.

By means of nucleotide sequencing in a library of clones, containing cDNA of Bos taurus mammary gland, a clone corresponding to kappa-casein has been identified. In addition, the region encoding for the complete nucleotide sequence of cDNA of kappa-casein B macropeptide has been detected. The nucleotide changes in the DNA sequence have been identified which mean that the isolated clone corresponds to the genetic variant B of kappa-casein.

[Selectively cleaved synthetic oligodeoxyribonucleotides for reverse immobilization of DNA]. / Spetsifichno otshchepliaemye sinteticheskie oligodezoksiribonukleotidy dlia obratimoi immobilizatsii DNK.

A streptavidin-coated TSK-gel support, with the loading capacity of 50-70 nmol of biotinylated substance per gram of dry support, and biotinylated oligonucleotides, containing the 4,9-dithiadodecane-6,7-dihydroxy-1,12-diphosphate insert, were prepared for the reversible immobilization of DNA. A non-nucleotide link can be located either at 5'- or 3'-end of the DNA fragment between the biotin moiety and the nucleotide sequence and is subjected to the selective periodate cleavage at the glycol group, which takes 45 min in solution and 3 h in heterophase. For the incorporation of the cleavable and biotin moieties into synthetic oligonucleotides, the corresponding phosphoramidite reagents and biotinylated CPG support were synthesized.

[Design of recombinant Escherichia coli strains, determining the secretory expression of artificial human granulocyte-macrophage colony-stimulating factor genes]. / Konstruirovanie rekombinantnykh shtammov Escherichia coli, determiniruiushchikh sekretornuiu éksprssiiu iskusstvennykh genov granulotsitarno-makrofagal'nogo koloniestimuliruiushchego faktora cheloveka.

A number of recombinant plasmids for expression of artificial genes encoding human granulocyte-macrophage colony stimulating factor (GM-CSF) were constructed. A hybrid gene was obtained that contains a sequence encoding the leader peptide and a tandem of two IgG-binding domains of protein A from Staphylococcus aureus coupled, through an enteropepdidase linker, to a synthetic gmcsf gene. The construction enables Escherichia coli to carry out biosynthesis of the hybrid protein and its subsequent transport into the periplasmic space of bacteria. Another hybrid gene, combining sequences for the signal peptide of the E.coli outer membrane protein OmpA and GM-CSF, was obtained using polymerase chain reaction. The localization of the mature protein produced by the hybrid gene was found to depend on the strength of the promoter used.

[Enzymatic ligation of DNA fragments containing phosphoamide modification of the internucleotide bonds]. / Enzimaticheskoe ligirovanie fragmentov DNK, soderzhashchikh fosfamidnuiu modifikatsiiu mezhnukleotidnykh sviazei.

DNA fragments with the point amidophosphate (cyclohexylamido- or morpholido-) modification in the sugar-phosphate backbone were synthesized and separated into individual diastereoisomer. The isomers were separated by the reversed-phase HPLC (RPC), and chirality at phosphorus was assigned by a stereochemical correlation scheme using phosphorothioate standards. The RPC-retention time values for Rp-isomers were found to be lower than for Sp-analogues. Amidophosphate DNA fragments were used as P- and OH-components in the T4 DNA-ligation. The enzyme does not ligate amidated fragments with modified internucleotide linkage near 5'- or 3'-end, independently of the amidophosphate chirality. When an unmodified phosphodiester linkage separates the amidophosphate group from 3'-end in O-component, the ligation occurs only with Sp-isomer, whereas Rp-analogue does not give the ligation product. In the P-component of the ligation, configuration of the modified linkage separated from 5'-phosphate by an unmodified linkage does not affect the result of the enzymatic reaction: both Sp-and Rp-stereomers do take part in the ligation. As a result of the ligation of the modified fragments on unmodified templates a set of 31-mers was obtained. They contain FokI and EcoRI recognition sites with the cleavage points of both endonucleases coinciding and being amidated. Upon treatment of duplex DNA consisted of unmodified and amidated strands with these endonucleases Sp-configuration did not hinder the cleavage of the unmodified strand, whereas Rp-configuration inhibited the EcoRI and did not affect the FokI cleavage.

[Construction of a gene coding for the precursor of human tumor necrosis factor]. / Konstruirovanie gena, kodiruiushchego predshestvennik faktora nekroza opukholei cheloveka.

The cDNA sequence for human tumor necrosis factor (hTNF) was reconstructed in vitro from genomic sequence. Using the oligonucleotide directed mutagenesis, a site for restriction endonuclease ClaI was introduced into the end of the first exon. The nucleotide sequence representing the second and third exons flanked with restriction sites ClaI and XhoI was obtained by means of chemical enzymatic synthesis. Assembly of the total gene coding for precursor of hTNF was accomplished in pTNF33 plasmid containing semisynthetic gene for mature hTNF with appropriate restriction sites.

[The vector containing a signal for specific degradation of chimeric proteins. Synthesis of [Leu5]enkephalin using enteropeptidase]. / Vektor, soderzhashchii signal dlia spetsificheskogo rasshchepleniia khimernykh belkov. Poluchenie [Leu5]énkefalina s pomoshch'iu énteropeptidazy.

A plasmid vector (pEK1) coding, in framework of beta-galactosidase gene, for the amino acid sequence (Asp)4Lys which is recognized by bovine enteropeptidase has been constructed. Using this vector and chemically synthesized DNA coding for the [Leu5]-enkephalin, a plasmid (pEK-ENK) has been obtained in which the beta-galactosidase gene is fused, through the enteropeptidase linker, with the gene for [Leu5]enkephalin. The chimeric protein produced by expression of this plasmid has been isolated and then cleaved by the enteropeptidase to give [Leu5]enkephalin with the yield 74%.

[Cloning and expression of the gene for Escherichia coli thermostable enterotoxin]. / Klonirovanie i ékspressiia gena termostabil'nogo énterotoksina Escherichia coli.

Gene estA coding for thermostable enterotoxin of Escherichia coli has been cloned. It is shown that in the E. coli strain SA162 this gene is located on the chromosome. Using polymerase chain reaction a site-directed mutagenesis of the cloned gene has been carried out, resulted in a recombinant strain--producer of the thermostable enterotoxin.

[Location of the initiating codon AUG in relation to the 5'-end of mRNA mediates the effectiveness of translation in E. coli cells]. / Polozhenie initsiiruiushchego kodona AUG otnositel'no 5'-kontsa mRNK vliiaet na éffektivnost' translitsii v kletkakh E. coli.

A semisynthetic gene for beta-galactosidase (lacZ) and a synthetic DNA fragment containing the "ideal" promoter sequence were used for construction of an artificial operon including translation initiation codon ATG and no SD sequence. Cloning this artificial operon into pBR322 vector resulted in a number of pV plasmids; ATG positions were varied by insertions of synthetic oligonucleotides between lacZ coding sequence and starting point of transcription. It was found that efficiency of beta-galactosidase synthesis in E. coli cells harbouring pV plasmids strongly depended on the relative position of AUG and mRNA 5'-end. High level of the synthesis was provided by translation of mRNA with AUG codon in 5'-terminal position. Amounts of synthesized beta-galactosidase diminished with increase of the distance (2, 4, and 5 nucleotides) between 5'-end of lacZ mRNA and AUG codon.

[A new approach to DNA synthesis from synthetic oligonucleotides. Synthesis of DNA coding for the repeated antigenic determinant of foot and mouth disease virus]. / Novyi podkhod k polucheniiu DNK iz sinteticheskikh oligonukleotidov. Sintez DNK, kodiruiushchikh povtoriaiushchuiusia antigennuiu determinantu virusa iashchura.

A rapid method for assembly of DNA from synthetic oligodeoxynucleotides has been developed which involves separate ligation of top- and bottom-strand oligonucleotides followed by filling in 3'-ends of the duplex formed, blunt end cloning into a specialized vector pBBV, and recovery of the synthetic DNA from the recombinant plasmid by means of restriction nuclease BbvII. The method allows for many oligonucleotides to be ligated at once, with no intermediates being isolated, and any DNA to be recovered on cloning, no matter what the sequences of its termini are. Ten oligodeoxynucleotides (I)-(X) have been chemically synthesised and used to prepare, by this method, a 60-membered duplex with complementary tetranucleotide 5'-protrusions (DNA I) which comprises the cDNA sequence 3397-3456 of foot and mouth disease virus (FMDV) strain O1K. Self-ligation of the duplex in the head-to-tail manner yielded 120 to 900 bp long synthetic DNAs (DNA II-DNA XV) coding for oligomers of the major antigenic determinant (the amino acid sequence 141-160 of protein VP1) of FMDV. The synthetic hexamer (DNA VI) was fused to gene lacZ' on plasmid pBBV21 and expressed in E. coli. The fusion was found to complement the lacZ deletion M15, from which it follows that the fused protein associated with the alpha-deficient beta-galactosidase to yield a tetramer carrying, on its N-termini, 24 antigenic determinants of FMDV.

[Chemical-enzymatic synthesis and cloning of DNA, coding the signal for secretion of proteins in gram-negative bacteria]. / Khimiko-fermantativnyi sintez i klonirovanie DNK, kodiruiushchei signal dlia sekretsii belkov v gramootritsatel'nykh bakteriaiakh.

Chemical-enzymatic synthesis of an artificial gene encoding leader peptide and 22 N-terminal amino acids of mature carboxypeptidase G2 from Pseudomonas sp. followed by enterokinase signal sequence (Asp4Lys) has been accomplished. The resulted DNA was fused with semi-synthetic gene coding for polypeptide 4-157 of mature human tumour necrosis factor (TNF) and then placed under control of early promoters of T7 bacteriophage. The expression products of the construct obtained was analysed using anti-TNF anti-serum. In E.coli leader peptide was cleaved off during translocation through inner membrane and the resultant product was found in membrane fraction.