Preparation of yeast mitochondrial DNA for direct sequence analysis

We describe two simple protocols for preparation of templates for direct sequencing of yeast mitochondrial DNA (mtDNA) by automatic DNA analyzers. The protocols work with a range of yeast species and yield a sufficient quantity and quality of the template DNA. In combination with primer-walking strategy, they can be used either as an alternative or a complementary approach to shot-gun sequencing of random fragment DNA libraries. We demonstrate that the templates are suitable for re-sequencing of the mtDNA for comparative analyses of intraspecific variability of yeast strains as well as for primary determination of the complete mitochondrial genome sequence.     

Ambiguity of the effect of high and low doses of amino-acid preparations on the immune response and phagocytosis in mice

It is shown that the effect of amino-acid preparations (levamine-70–70, cerebrolysin, and aviamine) is dose-dependent. Thus, levamine-70 and cerebrolysin at 65 mg/kg do not affect the immune response but stimulate phagocytosis. Aviamine at 65 mg/kg inhibits the immune response but stimulates phagocytosis and in a dose of 6.5×10⁻² mg/kg boosts both processes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 5, pp. 500–501, May, 1994 Presented by A. D. Ado, Member of the Russian Academy of Medical Sciences     

Mutations in the highly conserved SLQYLA motif of Vif in a simian-human immunodeficiency virus result in a less pathogenic virus and are associated with G-to-A mutations in the viral genome

The simian-human immunodeficiency virus (SHIV)/macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of accessory genes in lentiviral pathogenesis. In this study, we introduced two amino acid changes in the highly conserved SLQYLA domain (to AAQYLA) of the SIV Vif protein. The resulting virus, SHIV_VifAAQYLA, was used to infect three macaques, which were followed for over six months. Plasma viral loads and circulating CD4⁺ T cell levels were assessed during the course of infection. The three macaques inoculated with SHIV_VifAAQYLA did not develop significant CD4⁺ T cell loss over the course of their infection, had plasma viral RNA loads that were over 100-fold lower than macaques inoculated with parental SHIV_KU-1bMC33, and developed no histological lesions in lymphoid tissues. DNA and RT-PCR analysis revealed that only a select number of tissues were infected with this virus. Sequence analysis indicates that the site-directed changes were stable during the first three weeks after inoculation but thereafter the S147A amino acid substitution changed to a threonine in two of three macaques. The L148A substitution remained stable in the vif amplified from the PBMC of all three macaques. Sequence analysis of vif, vpu, env and nef genes revealed G-to-A mutations in the genes amplified from macaques inoculated with SHIV_VifAAQYLA, which were higher than in a macaque inoculated with parental SHIV_KU-1bMC33. We found that the majority (> 85%) of the G-to-A mutations were in the context of 5′-TC (minus strand) and not 5′-CC, suggestive that one or more of the rhesus APOBEC3 proteins may be responsible for the observed mutational patterns. The data also suggest that rhesus APOBEC3G probably accounted for a minority of the mutations since its GG-to-AG mutational pattern was infrequently detected. Finally, macaques inoculated with SHIV_VifAAQYLA developed immunoprecipitating antibody responses against the virus. The results from this study provide the first in vivo evidence of the importance of the SLQYLA domain in viral pathogenesis and show that targeted mutations in vif can lead to a persistent infection with G-to-A changes accumulating in the viral genome.     

A single amino-acid substitution in the beta-tubulin gene of Neurospora confers both carbendazim resistance and diethofencarb sensitivity

Summary Two MBC-resistant mutants of Neurospora crassa, F914 and F939, were sensitive to diethofencarb at a concentration of 0.1 g/ml, while the wild-type strain and other MBC-resistant mutants showed resistance to diethofencarb at a concentration of 100 g/ml. Genetic analysis suggested that the mutations in these two strains were closely linked to the Bml locus which codes for beta-tubulin. When the wild-type strain was transformed by the cloned beta-tubulin gene of the F914 strain, the transformants showed both MBC resistance and diethofencarb sensitivity. On the other hand, the diethofencarb sensitivity of the F914 strain was cancelled by transformation with the wild-type beta-tubulin gene. DNA sequencing of F914 beta-tubulin revealed that glycine was substituted for glutamic acid at position 198 in the F914 strain. Therefore, a single base change in the betatubulin gene was proved to confer both MBC resistance and diethofencarb sensitivity.     

Cloning of a DNA fragment from Cephalosporium acremonium which functions as an autonomous replication sequence in yeast

Summary A fragment of DNA which functions as an autonomous replication sequence in yeast was cloned from Cephalosporium acremonium. Mitochondrial DNA (mtDNA) was isolated from an industrial strain of C. acremonium (08G-250-21) highly developed for the production of the antibiotic, cephalosporin C. Size, 27 kb, and restriction pattern indicated this DNA was identical to mtDNA previously isolated (Minuth et al. 1982) from an ancestral strain (ATTC 14553) which produces very low amounts of cephalosporin C. A 1.9 kb Pst1 fragment of the Cephalosporium mtDNA was inserted into a Pst1 site of the yeast integrative plasmid, Ylp5, to produce a 7.5 kb plasmid, designated pPS1. The structure of pPS1 was verified by restriction analysis and hybridization.PS1 transformed Saccharomyces cerevisiae (DBY-746) to uracil prototrophy at a frequency of 272 transformants/g DNA. Transformation frequencies of 715 transformants/g DNA and zero were obtained for the replicative plasmid, YRp7, and the integrative plasmid YIp5, respectively. Southern hybridization and transformation of E. coli by DNA from yeast transformed by pPS1 verified that pPS1 replicates autonomously in yeast.The uracil-independent pPS1-yeast transformants were mitotically unstable. The average retention of pPS1 after three days growth in selective and non-selective medium was 4.5% and 0.4%, respectively, compared to retentions of 4.6% and 0.5% for YRp7. The properties of pPS1 were compared to those of a related plasmid, pCP2. pCP2 was constructed (Tudzynski et al. 1982) by inserting the C. acremonium 1.9 kb Pst1 fragment into the yeast integrative plasmid, pDAM1.     

Feasibility of fetal-derived hypermethylated RASSF1A sequence quantification in maternal plasma — Next step toward reliable non-invasive prenatal diagnostics

We determined the feasibility of universal fetal marker detection in maternal circulation. Using real-time PCR, we compared the levels of fetal (SRY and hypermethylated RASSF1A) and total (GLO gene and total RASSF1A) extracellular DNA and fractions of extracellular fetal DNA (SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A) in maternal circulation. Sensitivity and specificity reached 100% as the fetal-specific hypermethylated RASSF1A sequence was detected in all 151 examined plasma samples derived from 70 normal pregnancies with a singleton male (n = 51) or female (n = 19) fetus sampled throughout gestation and absent in non-pregnant individuals (n = 29). A strong positive correlation was observed between fetal-derived hypermethylated RASSF1A and SRY (ρ = 0.66, P < 0.001), total RASSF1A and GLO (ρ = 0.65,P < 0.001), SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A ratio (ρ = 0.62, P < 0.001) in maternal plasma. The results indicate that a universal fetal marker could be useful not only for the confirmation of the presence of fetal cell-free DNA in maternal plasma but could enable quantification of cell-free fetal DNA in pregnancy associated disorders, independently of the sex of the fetus.     

Cloning and sequence analysis of the glucoamylase gene of Neurospora crassa

A 1.0-kb DNA fragment, corresponding to an internal region of the Neurospora crassa glucoamylase gene, gla-1, was generated from genomic DNA by the polymerase chain reaction, using oligonucleotide primers which had been deduced from the known N-terminal amino-acid sequence or from consensus regions within the aligned amino-acid sequences of other fungal glucoamylases. The fragment was used to screen an N. crassa genomic DNA library. One clone contained the gene together with flanking regions and its sequence was determined. The gene was found to code for a preproprotein of 626 amino acids, 35 of which constitute a signal and propeptide region. The protein and the gene are compared with corresponding sequences in other fungi.     

Localization of a vertebrate telomeric sequence in the chromosomes of two marine worms (phylum Annelida: class polychaeta)

Using the fluorescencein situ hybridization (FISH) technique, the presence of the vertebrate telomeric sequence (TTAGGG) _n was found in the chromosomes of two marine polychaetes belonging to two separate orders: one errant,Platynereis dumerilii (family Nereidae), and the other sessile,Pomatoceros lamarckii (family Serpulidae). This sequence was exclusively present at the ends of the chromosomes in both species.     

Detection of phosphotyrosine-containing proteins in the nuclear matrix of some tumors

Tyrosine-containing proteins were detected by the immunoperoxidase method in the nuclear matrix of the liver and some tumors of mice. Two strips with molecular weights of about 180 and 170 kD are characteristic of hepatoma 22a and Ehrlich's ascitic carcinoma. Immunoelectron study with colloid gold showed that tyrosine-containing proteins and fibronectin are commonly present in the nucleus and cytoplasm. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 9, pp. 279–281, September, 1995     

Novel and deviant Walker A ATP-binding motifs in bacteriophage large terminase-DNA packaging proteins

Bacteriophage terminases constitute a very interesting class of viral-coded multifunctional ATPase "motors" that apparently drive directional translocation of DNA into an empty viral capsid. A common Walker A motif and other conserved signatures of a critical ATPase catalytic center are identified in the N-terminal half of numerous large terminase proteins. However, several terminases, including the well-characterized lambda and SPP1 terminases, seem to lack the classic Walker A in the N-terminus. Using sequence alignment approaches, we discovered the presence of deviant Walker A motifs in these and many other phage terminases. One deviation, the presence of a lysine at the beginning of P-loop, may represent a 3D equivalent of the universally conserved lysine in the Walker A GKT/S signature. This and other novel putative Walker A motifs that first came to light through this study help define the ATPase centers of phage and viral terminases as well as elicit important insights into the molecular functioning of this fundamental motif in biological systems.     

Complete genomic RNA sequence of the Polish <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> isolate belonging to the US2 strain

We have determined the complete nucleotide and amino acid sequences of the Polish Pepino mosaic virus (PepMV) isolate marked as PepMV-PK. The PepMV-PK genome consists of a single positive-sense RNA strand of 6412-nucleotide-long that contains five open reading frames (ORFs). ORF1 encodes the putative viral polymerase (RdRp), ORFs 2–4 the triple gene block (TGB 1–3), and ORF5-coat protein CP. Two short untranslated regions flank the coding ones and there is a poly (A) tail at the 3′ end of the genomic RNA. Thus, the genome organization of PepMV-PK is that of a typical member of the genus Potexvirus. Phylogenetic analysis based on full-length genomes of PepMV sequences showed that PepMV-PK was most closely related to the Ch2 isolate from Chile. Comparison of PepMV-PK and Ch2 showed the following nucleotide identities: 98% for the RdRp, 99% for the CP genes, and 98, 99, and 98% for the TGB1, TGB2, and TBG3, respectively. This high level of nucleotide sequence identity between the Chilean and Polish PepMV-PK isolates suggest their common origin.     

Immunochemical identification of some high-molecular proteins in tumor nuclear matrix

Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 116, N ^o 10, pp. 418–421, October 1993     

Synthesis of plasma membrane proteins in cells of the regenerating liver

Protein synthesis was studied by determining incorporation of [³H]glycine in cells of the regenerating rat liver. The rate of incorporation of [³H]glycine into total liver proteins, proteins of the microsomal fraction and of the hyaloplasm, and proteins of the plasma membranes, soluble and insoluble in 0.05 M K₂CO₃ was determined. The rate of incorporation of [³H]glycine into soluble proteins of the plasma membranes reached a maximum 1 h after partial hepatectomy. The maximal rate of synthesis of proteins of the other fractions occurred at the end of the g₁ period. The sharp increase in the rate of synthesis of plasma membrane proteins soluble in 0.05 M K₂CO₃ is evidently one of the earliest biochemical events in cells of the regenerating liver preparing to divide.Laboratory of Chemical Factors of Regulation of Growth and Cell Division, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 5, pp. 589–591, May, 1978.     

Role of thyroid hormones in stress-induced synthesis of heat-shock proteins in the myocardium

Thyroxine in near-physiological doses increased the content of heat-shock proteins in the myocardium and stimulated their accumulation during immobilization stress. Blockade of thyroid functions with methimazole decreased the content of heat-shock proteins in rat myocardium during stres and heat shock and prevented their accumulation during adaptation to short-term immobilizations. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 12, pp. 617–619, December, 2000     

Heat-shock proteins and cardioprotection

Here we summarized the results of our studies and the data on the role and protective effects of heat-shock proteins, mechanisms of activation of their synthesis, and the role of nitric oxide in this process. The role of heat-shock proteins in preconditioned and adaptive cardioprotection and the possibility of their use as prognostic criteria in cardiology are discussed Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 12, pp. 604–611, December, 1998