Highly efficient expression of Escherichia coli heat-labile enterotoxin B subunit in plants using potato virus X-based vector.

A synthetic gene of the B-subunit of Escherichia coli heat-labile toxin, optimized for expression in plants, was designed and synthesized. The recombinant viral vector was constructed on the basis of potato virus X containing the LTB gene instead of the removed triple block of transport genes and the coat protein gene, which provides for LTB expression in plants. The vector is introduced into the plant cells during cell infiltration by agrobacteria incorporating a binary vector, the T-DNA region of which contains a cDNA copy of the recombinant viral genome. Under conditions of posttranscriptional gene silencing inhibition, the LTB yield in Nicotiana benthamiana plants is 1-2% of total soluble protein; in this case, LTB synthesized in plants forms pentameric complexes analogous to those found in the native toxin. The designed viral system of LTB transient expression can be used to obtain in plants a vaccine against enteropathogenic Escherichia coli.

[The 5' untranslated region of the maize alcohol dehydrogenase gene provides efficient translation of mRNA in plants under stress conditions].

Reduced level of expression of most cell proteins under stress conditions is determined by low efficiency of cap-dependent translation of corresponding mRNAs. The maize gene encoding alcohol dehydrogenase, adh1, is an example of a gene which mRNA is efficiently translated under hypoxia. Using reporter gene assay we showed that the leader sequence of adh1 mRNA, provides efficient translation of reporter gene gfp in Nicotiana benthamiana cells under hypoxia and heat shock. The presence of this leader sequence in 5' UTR of mRNA does not change the level of expression in aerobic conditions, but under hypoxia and heat shock the levels of reporter gfp expression were reduced about 5-10 fold in the absence of leader and remained unaffected in its presence in 5'UTR. We found that this leader sequence does not change the level of mRNA stability and does not exhibit promoter activity. Consequently, leader sequence acts as translational enhancer providing efficient translation of mRNA in plant cells under stress conditions. Introduction of this sequence into standard expression cassettes may be used for development of new systems of expression of target proteins in plants, efficient under stress conditions.

Induction and repression of genes of the rat small intestine mucosa with excess and insufficiency of alimentary iron.

Three-month-old Wistar rats were fed with either an iron-deficient diet for 21 days or an iron-excessive diet for 10 days. Fifty thousand clones of a cDNA library of small intestine mucosa were hybridized with two radioactive samples synthesized on mRNA from the small intestine of rats fed with iron-excessive or iron-deficient diets. As a result, genes were found with mRNA level depending on the content of alimentary iron. Iron deficiency increased the mRNA level of genes of apolipoprotein AIV and class 1 antigen and decreased the mRNA level of the apolipoprotein AI gene and of an unknown gene, while no change was found in the mRNA level of the gene of fatty acid-binding protein. Excess of dietary iron resulted in the increase in mRNA of this gene and in a decrease in the apolipoprotein AI gene and in the unknown gene. The mRNA of apolipoprotein AIV did not change. The data indicate that changes in the level of alimentary iron influence the expression of genes involved in metabolism of lipids.

[Anti-nucleic acid antibodies in children with systemic lupus erythematosus and systemic scleroderma and in their relatives living with the probands]. / Antitela k nukleinovym kislotam u detei, bol'nykh sistemnoi krasnoi volchankoi i sistemnoi sklerodermiei, i u ikh rodstvennikov, zhivushchikh sovmestno s probandami.

The nature of antibodies to nucleic acids was studied in children of a Caucasian region suffering from systemic lupus erythematosus (SLE) and systemic sclerodermia (SSD). Two types of antibodies (to two- and one-filament DNA) were revealed in SLE, and in SSD mainly--to one-filament DNA. Antibodies to RNA in patients with SLE were specific for two-filament conformation. Immunogenesis disorders of different degree with respect to DNA and RNA were found; this expressed itself in the synthesis of various classes of immunoglobulins to these antibodies (IgG to DNA and IgM to RNA). High incidence of the RNA-binding activity of relatives living together with probands is noted. These data point to a high occurrence of antibodies to denatured DNA and low incidence of the cases of DNA-binding activity in the blood sera of the patients' fathers and brothers. Thus a combination of three factors: viral, genetic and hormonal in the pathogenesis of this disease is possible.

[Superoxide dismutase in liver ischemia]. / Superoksiddismutaza pri ishemii pecheni.

The properties of Cu,Zn-superoxide dismutase (SOD) from rat liver after 2-hour total ischemia or after ischemia with subsequent 24-hour reperfusion were studied. Two hours after ischemia the specific activity of SOD decreases drastically (about 3-fold) - from 510 +/- 11 u./mg in normal tissue and 196 +/- 33 u./mg after ischemia showing a further increase after reperfusion (276 +/- 40 u./mg). Using competitive immunoenzymatic analysis, the relative contents of SOD in the cytosol were determined. After ischemia the SOD content in the cytosolic fraction decreased (approximately 3-fold) but returned to the initial level after reperfusion. Polyacrylamide gel electrophoresis revealed that in control samples active SOD is heterogeneous and produces 3-4 bands, similar to the purified SOD from rat liver. After the ischemia the intensity of minor fast band IV increased and a new band V of a still higher mobility appeared. After the reperfusion the electrophoretic patterns were similar to control. Two or three times more SOD antigen from ischemia liver cytosol was absorbed to the surface of polystyrol plate in a direct sorption enzyme immunoassay procedure as compared to that from intact liver cytosol. It is suggested that the decreases of amount and the activity as well as changes of properties of SOD could be due to its oxidative modification and degradation of the modified enzyme.

Antibodies to nucleic acids in systemic lupus erythematosus and rheumatoid arthritis patients and their relatives.

Sera of systemic lupus erythematosus (SLE) patients contain antibodies to double-stranded and single-stranded DNA while antibodies found in rheumatoid arthritis sera are specific mainly to single-stranded DNA. Anti-RNA antibodies in the both cases are directed against double-stranded RNA and belong to the IgM class while anti-DNA antibodies are IgG. Genetic variance analysis based on family correlations suggests that the synthesis of antibodies to DNA is subject to different modes of gene regulations in systemic lupus erythematosus and rheumatoid arthritis patients. In the case of SLE, a high degree of genetic determination is demonstrated, due mainly to the X-chromosomal component of the general phenotypic variance. The failures in the immunoregulation system that are responsible for anti-RNA antibody production are rather similar in the two groups of patients and involve a combination of environmental and autosomal factors.

[Chemical and physico-chemical properties of the DNA of Newcastle group bacteriophages]. / Khimicheskie i fiziko-khimicheskie svoistva DNK bakteriofagov gruppy N'iukasl.

DNA of H-1, H-5, H-10, H-17, and H-22 phages were studied, their nucleotide composition, their buoyant densities in cesium chloride and cesium sulphate, curves of melting and spectra of circular dichroism were determined 5-oxymethylcytosine in the composition of H-I and H-22 phage DNA was shown immunologically to be glucozylated.

[Functional interactions of the genomes of Shigella sonnei phages and Escherichia coli phage T4 in mixed infection]. / Funktsional'nye vzaimodeistviia genomov fagov Shigella sonnei i faga Escherichia coli T4 pri smeshannoi infektsii.

A comparative study of Shigella sonnei phages U and G and Escherichia coli phage T4 has shown that enzymes coded for by the Sh. sonnei phages can functionally substitute for some T4-coded products. This finding in indicative of an evolutionary relationship between T-even phages and disenteric phages U and G. The U phage is uncapable to compensate amber mutants for the genes that control the conversion of cytosine into 5-hydroxymethyl cytosine (5-HMC) and the glucosylation of the latter, which agrees with our earlier finding that the U phage DNA contains no 5-HMC. U and G phages are also found to exclude the T4 phage in the course of mixed infection.

[Immunogenic DNA of Shigella sonnei bacteriophage]. / Immunogennaia DNK faga Shigella sonnei.

Immunogenic and some other properties of Sh, sonnei "Ufa" phage DNA were investigated. This DNA has been shown to possess high immunogenic activity indicating the presence of an unusual base. The base is distinct from 5-OmC, glucosylated 5-OmC, 5-OmU or dihydroxypentyluracil. The phage DNA is double stranded and has the molecular weight of 1.11.10(8) daltons and Tm=85.5 degrees C. Base sequences common with T4 DNA have been found using the competition DNA-DNA hybridization technique. Experiments on cross neutralization of intact phage with the corresponding antisera confirm the existence of common antigenic determinants in T4 and Sh, sonnei "Ufa" phage particles.

[Immunochemical and molecular biological characteristics of dysentery phages]. / Immunokhimicheskaia i molekuliarno-biologicheskaia kharakteristika nekotorykh dizenteriinykh fagov

Molecular organization of DNA of six dysentery phages was studied for their classification. The pattern of glucosilation of their DNA was determined by an immunochemical method and the test of competitive hybridization established the degree of homology of these DNA in relation to T4 phage DNA. Four of the phages (DDVI, Zonne "Gorkyi" Zonne "Czechoslovakia", Zonne "Poland") were shown to belong to the group of T-even phages by their pattern of DNA glucosilation and by their capacity to inhibit DNA T4X3H DNA T4 hybridization. According to these criteria, two other phages (Zonne "Ufa" and Zonne "Khabarovsk") do not belong to the group of T-even phages.

[The influence of proteins bound to single-stranded DNA on RNA and poly(A) synthesis. I. Antibodies against DNA]. / Vliianie na sintez RNK i poli(A) belkov, sviazyvaiushchikhsia s odnonitchatoi DNK. I. Antitela protiv DNK

The influence of antibodies to DNA of T4 and SPOI phages as well as to DNA of E. coli on RNA and poly(A) synthesis by E. coli RNA polymerase has been studied. Antibodies to the phage DNA specifically inhibit RNA and poly(A) synthesis by 30 to 60% only in case of homologous DNA, the stage of elongation being inhibited predominantly. A comparison between the effects of the antibodies upon different stages of RNA synthesis on the native DNA and on the synthetic oligonucleotide template (dT)12 allows to draw a conclusion that upon inhibition of the synthesis the antibodies interact with a single-stranded site of the template not covered with RNA polymerase molecule, this site being formed during transcription of double-stranded DNA.

[Immunogenic properties of DNA from E. coli and T4 phage, containing 5-bromouracil]. / Immunogennye svoistva DNK E. coli i faga T4, soderzhashchikh 5-bromdezoksiuridin

5-BUDR incorporated into E. coli or T4 phage DNAs is an immunogenic determinant. Immunodominance of 5-BUDR is determined by the chemical structure of DNA molecule into which it is incorporated. In E. coli DNA, 5-BUDR inhibits immunogenic activity of all other bases. One can conclude that in this DNA 5-BUDR is the dominant antigenic determinant. In T4 DNA, 5-BUDR acts as an additional antigenic determinant. Anti-5-BUDR-T4-DNA contains antibodies to 5-BUDR and glucosylated 5-HMC.