European Journal of Molecular Biotechnology

118-130

Authors: Firas Rashad Al-Samarai, Abdulkareem A. Al-Kazaz

The dramatic development of molecular genetics has laid the groundwork for genomics. It has introduced new generations of molecular markers for use in the genetic improvement of farm animals. These markers provide more accurate genetic information and better understanding of the animal genetic resources. Scientists, unfamiliar with the different molecular techniques tend to get lost as each has its own advantages and disadvantages. This review represents a trail to shade alight on the different types of molecular markers by introducing a brief summary on the development of genetic markers including both the classical genetic markers and more advanced DNA-based molecular markers. This review could be helpful to better understand the characteristics of different genetic markers and the genetic diversity of animal genetic resources.

156-173

Authors: Oleg Mosin, Ignat Ignatov

This paper deals with studying biosynthetic pathways of 2H-labeled purine ribonucleoside inosine excreted into liquid microbial culture (LC) by Gram-positive chemoheterotrophic bacterium Bacillus subtilis B-3157 while growing of this bacterium on heavy water (HW) medium with 2% (v/v) hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of 2H-labeled growth substrates. Isolation of 2H-labeled inosine from LC was performed by adsorption/desorption on activated carbon with following extraction by 0,3 M ammonium–formate buffer (pH = 8,9), crystallization in 80% (v/v) EtOH, and ion exchange chromatography (IEC) on a column with AG50WX 4 cation exchange resin equilibrated with 0,3 M ammonium–formate buffer and 0,045 M NH4Cl. The investigation of deuterium incorporation into the inosine molecule by FAB method demonstrated incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment – 65,5 atom% 2H) with 3 deuterium atoms being included into the ribose and 2 deuterium atoms – into the hypoxanthine residue of the molecule. Three non-exchangeable deuterium atoms were incorporated into the ribose residue owing to the preservation in this bacterium the minor pathways of de novo glucose biosynthesis in 2H2O-medium. These non-exchangeable deuterium atoms in the ribose residue were originated from HMP shunt reactions, while two other deuterium atoms at C2, C8-positions in the hypoxanthine residue were synthesized from [2H]amino acids, primarily glutamine and glycine, that originated from deuterated hydrolysate. A glycoside proton at -N9-glycosidic bond could be replaced with deuterium via the reaction of СО2 elimination at the stage of ribulose-5-monophosphate formation from 3-keto-6-phosphogluconic acid with subsequent proton (deuteron) attachment at the С1-position of ribulose-5-monophosphate. Two other protons at C2(C3) and C4 positions in ribose residue could be replaced with deuterium via further enzimatic isomerization of ribulose-5-monophosphate into ribose-5-monophosphate.

56-62

Authors: Grigory A. Gerashchenkov, Gulnar R. Yasybaeva, Nataliya A. Rozhnova, Aleksey V. Chemeris

Apomixis is asexual seed reproduction without both meiosis and fertilization based on the complex developmental processes such as apomeiosis, parthenogenesis and specific endosperm development. This investigation is aimed at engineering of apomixis in Arabidopsis thaliana with sexual seed reproduction. The fragments of known genes of endosperm formation MEA, FIE, FIS2 and gene of apomeiosis DYAD (as control) were isolated using Q5 high fidelity DNA polymerase. These gene fragments of interest at the antisense orientation were fused with isolated constitutive and meiosis specific promoters of Arabidopsis at NcoI sites. The fused promoter-gene fragment modules were cloned in pCambia1301 at SalI cites. The engineered constructions will be used for the floral dip transformation of Arabidopsis and down regulation of these genes at engineering of apomixis.

80-87

Authors: Da´ad Ali Hussain

One of these major genes is growth hormone and growth hormone receptor (GH, GHR) genes which are related to production and reproduction traits in livestock. Associations were analysed between the bovine growth hormone (GH) as well as growth hormone receptor (GHR) combined with their genotypes (GH/AluI& GHR/AluI). There is extensive literature on the genetic polymorphism of GH and GHR in cattle, but perusal of literature has indicated paucity of information on these two genes in buffalo. This study aimed to evaluate the genetic polymorphism within growth hormone and its receptor genes in Iraqi buffalo using PCR-RFLP technique. Genomic DNA extracted from 100 healthy buffaloes and amplified using primers that were designed from the cattle GH and GHR gene sequences. All tested buffalo DNA amplified fragments at 428bp for GH 5th exon. The digestion with AluI endonuclease gave three types of genotypes. The Distribution of the three genotypes and allele frequency was calculated according to Hardy-Weinberg equation, were LL=94 (94.09%), LV=6 (5.82%) and VV=0 (0.09%), so that most of buffaloes was homozygous (LL) due to the presence of the restriction site at position 52^53 (AG^CT) and less heterozygous was for the valine allele as compared with leucine allele homozygous. The amplified fragments of GHR gene obtained from all tested buffalo DNA at 342-bp were digested with AluI endonuclease. The result showed that all tested buffaloes are genotyped as GG for GHR gene, where amplified fragments were digested into two digested fragments at 241and 101-bp due to the presence of AluI restriction site at position (AG^CT).

103-114

Authors: Sajedeh Saeedfar, Marzieh Negahban, Mohammad Mahmoodi Soorestani

Plot trials were carried out in a research field in Tehran (Iran) to determine the effect of drought stress on the essential oil content and some of the plant biochemical characteristics of Anise Hyssop (Agastache foeniculum [Pursh] Kuntze), which is a valuable medicinal plant. Drought stress was conducted at different levels including: well-water (100% FC), mild drought stress (85% FC), moderate drought stress (70% FC), severe drought stress (55% FC), 100% FC (vegetative stage) 85% FC (reproductive stage), 100% FC (vegetative stage) 70% FC (reproductive stage), and 85% FC (vegetative stage) 100% FC (reproductive stage). The experiment was arranged as a RCBD with three replications. The output results showed that, water deficit stress significantly (P≤0.05) increased activities of antioxidant enzymes (Superoxide Dismutase, Catalase, Glutathione Peroxidase) as well as Essential Oil yield and Abscisic Acid content. Lipid and protein oxidation (malondialdehyde and dityrosine contents) also increased significantly under severe water deficit stress. According to the results, severe drought conduction (55% FC) is the optimum level of soil moisture to plant Anise Hyssop under water deficit stress.

4-11

Authors: Rabah Chadli, Aman Bouzid, Khadidja Bouzid, Hamida Nader

This research concerns the study of antibacterial properties of different aqueous extracts of the bark of the pomegranate (Punica granatum L.). Three bacterial strains were used in this test: Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella. Very interesting bactericidal properties of aqueous extracts of the bark of the pomegranate were found on bacteria. The inhibition zones have a very large diameter up to 20 mm and the MIC and MBC are low, of the order of 0.78 mg/ml. This work has shown antibacterial activity against three bacteria may contribute to the fight against infectious diseases, and possibly offer the possibility of using pomegranate peel pharmaceutical and food industries.

27-36

Authors: Ali Saeed Atiyah AL-Janabi

In this study gene expression for DGAT1 gene was analyzed. Diacylglycerol acyltransferases (DGATs) catalyze the final step of the triacylglycerol (TAG) biosynthesis of the Kennedy pathway. Two major gene families have been shown to encode DGATs, DGAT1 (type-1) and DGAT2 (type-2). Gene expression were analyzed for 10 Olive cultivars (Olea europaea L.) (Khaderi, Qaysi, Manzenillo, Baashiqi, Arabqween, Nabali, Labeeb, Dahkan, Shami and Sorani). Different plant organs as plant materials (mature leaves, mesocarp and seeds for drups) used for analysis. Two methods for analysis gene expression were used, first method was called semi – quantitative and second method was called relative – quantitative, used in relative method (Real time PCR and Actine gene as Housekeeping gene). On the other hand chemical analysis was used on fruits like moisture % and oil % of dry and fresh weigh. The results revealed the following: DGAT1 gene expression in leaves, mesocarp and seeds by two methods (semi- quantitative and relative quantity) were the convergent results and clear, also if this results compared with chemical analysis shows that the best cultivars were Arabqween, Khaderi, Qaysi and Labeeb. The cultivars Shami and Khaderi then were contain in fruits desirable qualities of olive oil, low moisture and high oil percentages ratios. While Nabali, Manzanello, and Sorani cultivars middle desirable quantity, and Baashiqi and Dahkan cultivars had undesirable because of low oil quantity and high moisture in contain fruits. Some cultivars have low intensity in semi- quantitative and little fold in relative quantity but it have high oil in contain fruits that may be indicate that these cultivars were complete gene expression and begin to accumulation and save oil in tissue. Therefore particular emphasis was given to the temporal regulation of olive DGATs during drupe development. In olive fruit, TAGs are formed and stored in both the mesocarp and the seed .Two drupe compartments that have different physiological functions and roles and also display difference in the mode of TAG accumulation. DGATI share an overlapping expression pattern after 28 WAF, suggesting that they probably function at those stages. However, following maximal mRNA levels at 22 WAF, DGAT1 transcription declined substantially.

160-168

Authors: Khadidja Bouzid, Fouzia Toumi Benali, Rabah Chadli, Mohamed Bouzouina, Aman Bouzid, Amal Benchohra, Mustapha Mahmoud Dif

The aim of the current study was to evaluate the total phenolic, flavonoid content and to investigate the antioxidant capacities of the fruit extracts of Arbutus unedo L. that grows in Tiaret area (Western Algeria). First we have extracted the fruit by some non-polar solvent (chloroform, ethyl acetate, 1-butanol). Total phenolic content and total flavonoid content were evaluated according to the Folin-Ciocalteu procedure, and a colorimetric method, respectively. Extracts content was determined by using a high-performance liquid chromatography (HPLC)-UV method. The total phenolic contents of A.unedo L. varied between 12.75±0.06 to 34.17±1.36 mg gallic acid equivalent/g of dry weight of extract. The total flavonoid varied from 2.18±0.10 to 6.54±1.14 mg catechin equivalent/g. The antioxidant potential of all extracts was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, the IC50 of acetate ethyl was the best by 0,009 mg/ml may due to the phenolic compound, in the second was the chloroform extract by IC50=0,015mg/ml, in the third was butanol extract by IC50= 0,022 mg/ml and in the last was water extract by IC50= 0,048mg/ml. the antioxidant activity of all extracts was better than ascorbic acid. The extract obtained under optimum conditions was analyzed by HPLC and five flavonoid compounds were identified; they are catechin, apiginin, silybin, fisetine and naringin.

180-209

Authors: Oleg Mosin, Ignat Ignatov, Dmitry Skladnev, Vitaly Shvets

Biological influence of deuterium on cells of various taxonomic groups of prokaryotic and eucaryotic microorganisms realizing methylotrophic, chemoheterotrophic, photo-organotrophic, and photosynthetic ways of assimilation of carbon substrates (methylotrophic bacteria Brevibacterium methylicum, chemoheterotrophic bacteria Bacillus subtilis, photo-organotrophic halobacteria Halobacterium halobium, and green micro algae Chlorella vulgaris) was investigated at the growth on media with heavy water (2H2O). For investigated microorganisms are submitted the data on growth and adaptation on the growth media containing as sources of deuterated substrates 2H2O, [2H]methanol and hydrolisates of deutero-biomass of methylotrophic bacteria B. methylicum, obtained after multistage adaptation to 2H2O. The qualitative and quantitative composition of intra- and endocellular amino acids, proteins, carbohydrates and fatty acids in conditions of adaptation to 2H2O is investigated. It is shown, that the effects observed at adaptation to 2H2O, possess a complex multifactorial character and connected to cytological, morphological and physiological changes – the magnitude of the lag- period, time of cellular generation, output of biomass, a parity ratio of synthesized amino acids, proteins, carbohydrates and lipids, and also with an evolutionary level of the organization of the investigated object and the pathways of assimilation of carbon substrates as well.

223-228

Authors: Ajay Tomer, Ramji Singh, Manoj Kumar Maurya

Deoiled cakes of Neem and Jatropha served as source of diversified nutrition for Pseudomonas flourescens when used as substrate for mass culturing of antagonist. The present investigation were undertaken to test the suitability of neem and Jatropha cakes for liquid Concentration and longevity of Pseudomonas flourescens in vitro. Increasing the dilution of stock of initial inoculums resulted in decrease of total viable counts of Pseudomonas flourescens was comparatively highest population (64.67×105) and (201.67×105) lesser after 15 days than after 7 days of inoculation on Neem and Jatropha cake concentration respectively.

104-119

Authors: Ignat Ignatov, Oleg Mosin

This review article views predominately the structure and function of animal and bacterial photoreceptor pigments (rhodopsin, iodopsin, bacteriorhodopsin) and their aspects of nano- and biotechnological usage. On an example of bacteriorhodopsin is described the method of its isolation from purple membranes of photo-organotrophic halobacterium Halobacterium halobium by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, the solubilization with 0,5 % (w/v) SDS-Na and subsequent fractionation by methanol and gel filtration chromatography on Sephadex G-200 Column balanced with 0.09 M Tris-borate buffer (pH = 8,35) with 0,1 % (w/v) SDS-Na and 2,5 mM EDTA. Within the framework of the research the mechanism of color perception by the visual analyzer having the ability to analyze certain ranges of the optical spectrum, as colors was studied along with an analysis of the additive mixing of two colors. It was shown that at the mixing of electromagnetic waves with different wavelengths, the visual analyzer perceive them as separate or average wave length corresponding to mix color.

131-148

Authors: Oleg Mosin, Ignat Ignatov

Methylotrophs – aerobic chemoheterotrophic microorganisms submitted by cocci and bacilli mobile forms, are inhabitants of reservoirs and soils of various type, where there are going on various processes of decomposition of organic substances with formation of the one-carbon С1-compounds and some С2-, and С3-compounds, capable to be assimilated by methylotrophs. These microorganisms assimilating carbon on ribuloso-5-monophospate and serine pathways, are allocated from soil ground, the sewage containing decomposing vegetative remainss, from ruminant paunch and other sources. Methylotrophic bacteria recently draw the increasing attention of biotechnology as feasible sources of natural biologically active compounds – fodder fibers and irreplaceable amino acids, carotenoid pigments, lipids and policarbohydrates. For preparation of these compounds are used genetically marked strains of methylotrophic bacteria, obtained via genetic engineering approaches and selection. Recently developed gene-engineering methods of manipulation with the methylotrophic genom allow create on the basis of microbic DNA of methylotrophs expression vectors of eukaryotic proteins for medical and veterinary purposes, as human insulins. In this review article there are submitted data including the results of the authors’ own research on evolution of methylotrophic bacteria, the metabolism and their biotechnological usage.