Novel vectors for co-expression of two proteins in E. coli

Two new vectors, pAC28 and pEGST, for the co-expression of recombinant genes in E. coli were developed. This two-plasmid system allows for an efficient expression and purification of large amounts of protein-protein complexes formed in bacterial cells. We have utilized this new system to express and isolate a stable complex of two human proteins, hematopoietic cell tyrosine phosphatase (HePTP) and mitogen-activated proteins kinase Erk2. This approach is useful for biochemical and structural studies of protein-protein interactions.     

Suppression of Nonsense Mutations in the Dystrophin Gene by a Suppressor tRNA Gene

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10–15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing -galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.     

Molecular changes in inbred mice with individual vulnerability vs resilience to stress-induced depressive-like state

The C57BL/6 mice were subjected to a chronic combined stress which resulted in the induction of a depressive-like state. The occurrence of a depressive-like state was defined by a decrease in sensitivity to the reward determined by the diminished preference of sweetened solutions over regular drinking water. Such decrease is generally considered as a sign of an unhedonic-like state: one of the key features of clinical depression. Applied here, the paradigm in mice allows unhedonia induction in a subpopulation of stressed animals (54% in the current study); remaining mice are regarded as resilient to stress-induced hedonic deficit. The resilient subgroup is taken, therefore, as a "functional control" for those effects of stress that are not accompanied by development of the stress-induced depressive-like state in mice. The analysis of the mRNA extracted from the hippocampi of stress-subjected and home-cage control mice enabled the assessment of gene expression level of over 13 000 genes. This study showed that unhedonic mice are characterized by an up-regulation of 278 and down-regulation of 174 genes related mostly to the CNS development and functions, inter-cellular interactions and signalling, neurological disorders, apoptosis and behavioural regulation. Resilient animals demonstrated up-regulation of 924 and down-regulation of only 29 genes that control formation of cell assemblies, molecular transport, CNS functioning, neurological disorders and various biochemical reactions. Thus, gene expression profiles in the hippocampus of susceptible vs resilient to stress-induced unhedonia inbred subgroups of animals are strictly distinct in both quantity and quality.     

Population genetic study of Russian cosmonauts and test subjects: genetic demographic parameters and immunogenetic markers

Genetic demographic characteristics and immunogenetic markers (blood groups ABO, Rhesus, MNSs, P, Duffy, Kidd, and Kell) have been studied in a group of 132 Russian cosmonauts and test subjects (CTSG). Analysis of pedigrees has shown a high exogamy in the preceding generations: almost half of the subjects have mixed ethnic background. According to the results of genetic demographic analysis, a sample from the Moscow population was used as control group (CG). Comparison between the CTSG and CG has demonstrated significant differences in genotype frequencies for several blood group systems. The CTSG is characterized by a decreased proportion of rare interlocus genotypic combinations and an increased man heterozygosity. Analysis of the distributions of individual heterozygosity for loci with codominant expression of alleles has shown that highly heterozygous loci are more frequent in the CTSG. Taking into account that the CTSG has been thoroughly selected from the general population, it is concluded that heterozygosity is related to successful adaptation to a space flight.     

A New Method to Measure the Functional Activity of Class-1 Translation Termination Factor eRF1

Termination of protein synthesis (hydrolysis of the last peptidyl-tRNA on the ribosome) takes place when the ribosomal A site is occupied simultaneously by one of the three stop codons and by a class-1 translation termination factor. The existing procedures to measure the functional activity of this factor both in vitro and in vivo have serious drawbacks, the main of which are artificial conditions for in vitro assays, far from those in the cell, and indirect evaluation of activity in in vivo systems. A simple reliable and sensitive system to measure the functional activity of class-1 translation termination factors could considerably expedite the study of the terminal steps of protein synthesis, at present remaining poorly known, especially in eukaryotes. We suggest a novel system to test the functional activity in vitro using native functionally active mRNA, rather than tri-, tetra-, or oligonucleotides as before. This mRNA is specially designed to contain one of the three terminating (stop) codons within the coding nucleotide sequence. Plasmids have been generated that carry the genes of suppressor tRNAs each of which is specific toward one of the three stop codons. They were shown to support normal synthesis of a reporter protein, luciferase, by reading through the stop codon within the coding mRNA sequence. We have demonstrated that human class-1 translation termination factor eRF1 is able to compete with suppressor tRNA for a stop codon and to completely prevent its suppressive effect at a sufficient concentration. Forms of eRF1 with point mutations in functionally essential regions have lower competitive ability, demonstrating the sensitivity of the method to the eRF1 structure. The enzymatic reaction catalyzed by the full-size reporter protein is accompanied by emission of light quanta. Therefore, competition between suppressor tRNA and eRF1 can be measured using a luminometer, and this allows precise kinetic measurements in a continuous automatic mode.     

Variation of anthropometric traits in normal newborns]

Variation of the set of anthropometric traits was studied in 302 normal singleton newborns (174 boys, 128 girls). Birth order has a significant effect on birth weight, body length and circumference of head and breast. The number of minor deviations from development per newborn (stigma) is negatively correlated with body weight and size. The number of stigma is significantly higher in a group of newborns deviated in normal body proportions. The index of fluctuating asymmetry is negatively correlated with body weight and size.     

Influence of mother's heterozygosity on the variation of anthropometric traits in newborns]

The relationship between mother's heterozygosity as revealed by 14 polymorphic loci and variation of the set of anthropometric traits were studied in normal singleton newborns (174 boys, 127 girls). Statistically significant negative correlation between mother's heterozygosity and the index of fluctuating asymmetry was found in girls. Variances of four traits--body weight, body length, circumference of head and breast measured by first principal component, and mean number of minor deviations from development (stigma) are shown to be minimal in the children born by mothers with the average level of heterozygosity. The groups of newborns with different level of mother's heterozygosity are characterized by different combinations of first principal component, stigma, and the index of fluctuating asymmetry. It is concluded from the whole set of data that children born by mothers with the average level of heterozygosity have the highest level of viability.     

On the origin of Solovetskaya vendace <Emphasis Type="Italic">Coregonus albula</Emphasis> and of the syamozero smelt <Emphasis Type="Italic">Osmerus eperlanus</Emphasis>

The genetic analysis is made on the population of the European smelt Osmerus eperlanus occasionally introduced to Lake Syamozero (Karelia) and of the vendace Coregous albula supposedly acclimatized in Solovetskie Islands as a result of fish cultural activities of the Solovetskii Monastery. They are compared with several natural populations—possible donors for both introductions. Genetic variation in a sample of smelt was estimated by means of restrictase analysis of mtDNA (fragment ND1/ND2) in samples of vendace—by means of allozyme analysis of six isoenzyme systems. The probable population for the Syamozero smelt is the population of Onega Lake in spite of the previously noted greater morphological similarity of colonizers with the smelt of Ladoga Lake. A high level of genetic variation in the Syamozero smelt in comparison with native populations indicates that, beside the introduction from Onega Lake, there were repeated introductions from neighboring water bodies. The genetic analysis of the Solovetskaya vendace does not prove that the vendace appeared on islands due to acclimatization. Frequencies of alleles of allozyme loci in the Solovetskaya population significantly differ from frequencies in continental populations. Still, it is compared with some populations of the Arkhangelsk oblast. Estimations of genetic diversity of the Solovetskaya vendace turned out to be comparable with those in native populations.