An RNA microchip containing immobilized oligoribonucleotides with protective groups at 2'-O-positions

To analyze RNA interactions with RNA binding molecules an RNA microchip containing immobilized oligoribonucleotides with protective groups [t-butyldimethylsilyl (tBDMS)] at 2'-O- positions was developed. The oligonucleotides were immobilized within three-dimensional (3-D) hydrogel pads fixed on a glass support. The protective groups preserved the oligoribonucleodes from degradation and were suitable to be removed directly on the microchip when needed, right before its use. These immobilized, deprotected oligoribonucleotides were tested for their interaction with afluorescently labeled oligodeoxyribonucleotide and analyzed for their availability to be cleaved enzymatically by the RNase binase. Stability of tBDMS-protected immobilized oligoribonucleotides after 2.5 years of storage as well as after direct RNase action was also tested. Melting curves of short RNA/DNA hybrids that had formed into gel pads of the microchip were found to exhibit clearly defined S-like shapes, with the melting temperatures in full accordance with those theoretically predicted for the same ionic strength. This approach, based on keeping the protective groups attached to oligoribonucleotides, can be applied for manufacturing any RNA microchips containing immobilized oligoribonucleotides, including microchips with two-dimensional (2-D) features. These RNA microchips can be used to measure thermodynamic parameters of RNA/RNA or RNA/DNA duplexes as well as to study ligand- or protein-RNA interactions.     

Sex and rank in competitive brood hierarchies influence stress levels in nestlings of a sexually dimorphic bird

Studies of sibling competition within brood hierarchies have rarely assessed simultaneously the effects of sex and rank in the brood hierarchy on traits other than offspring mortality and differential growth. We studied the expression of heat-shock proteins (Hsps) to assess the physiological stress response to different combinations of sex and position within competitive brood hierarchies in the black kite Milvus migrans (Bodd.), a sexually dimorphic raptor showing facultative siblicide. Senior males showed higher stress levels than did senior females and younger siblings of each sex as revealed by Hsp60 values. The analysis of Hsp70 levels indicated that nestlings from broods in which the senior chick was a male showed higher stress levels than did nestlings from broods in which the senior chick was a female. In addition, levels of Hsp60 were related negatively to nutritional condition expressed as levels of plasmatic albumin. This suggests that the sex of senior chicks may be key in determining their stress level and that of their siblings, which is probably associated with sibling competition by fighting within brood hierarchies. The comparatively higher stress levels of senior males (and their siblings) may be a consequence of their ability to exploit their potential advantage from being the head start while avoiding a possible competitive disadvantage from being the smaller sex, independent of environmental conditions determining the probability of brood reduction. Differential stress levels depending on sex and rank in the brood hierarchy may be a consequence of parental control of offspring behaviour through differential resource allocation (e.g. yolk androgens) or it may reflect adaptations of particular chicks (senior males) to enhance their competitive ability within brood hierarchies.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 383–390.     

The Effect of the Structure of Fluorescently Labeled Nucleotide Derivatives on the Efficiency of Their Incorporation in DNA in the Polymerase Chain Reaction

The efficiency of fluorescence DNA labeling was estimated for four fluorescent 2′-deoxyuridine 5′-triphosphate derivatives differing in the orientation of the main dye axis, which passes through the polymethine chain, relative to the linker connecting the dye to the nucleotide. To estimate the polymerase chain reaction (PCR) rate, real-time PCR was run with two commercial hot-start DNA polymerases possessing 5′→3′ exonuclease activity in the presence of an intercalating dye. The efficiency of the test compound incorporation in the PCR product was estimated via a quantitative analysis of the amplification product by agarose gel electrophoresis. The fluorescently labeled product was then hybridized on a biological microchip and the ratio of signals from perfect match and mismatch duplexes was determined. The incorporation efficiency and discrimination between perfect match and mismatch duplexes were found to depend on the relative orientation of the dye and the linker between the dye and pyrimidine base, as well as on the presence of hydrophilic groups in the dye. Compounds that are efficiently incorporated in a growing DNA strand and show a high specificity in hybridization analysis were identified using biochips.     

Comparative analysis of N-acetylation polymorphism in humans as determined by phenotyping and genotyping

The N-acetylation polymorphisms of volunteers from the Moscow population analyzed by phenotyping and genotyping have been compared. The ratios between the proportions of fast acetylators (FAs) and slow acetylators (SAs) estimated by phenotyping and genotyping do not differ significantly from each other (47 and 44%, respectively). The absolute acetylation rate widely varies in both FAs and SAs. The NAT2 genotype and allele frequencies in the population sample have been calculated. The most frequent alleles are NAT2*4 (a “fast” allele), NAT2*5, and NAT2*6 (“slow” alleles); the most frequent genotypes are NAT2*5/*5, NAT2*4/*6, and NAT2*4/*5. Comparative analysis of N-acetylation polymorphism estimated by phenotyping and genotyping in the same subjects has shown a complete concordance between the phenotype and genotype in only 62 out of 75 subjects (87%). Comparative characteristics and presumed applications of the two approaches (quantitative estimation of acetylation rate and qualitative determination of the acetylator genotype) to the identification of individual acetylation status are presented.     

Analysis of NAT2 point mutations with biological microchips

The NAT2 product, N-acetyltransferase 2, is involved in biotransformation and detoxification of several aromatic amines (in particular, 2-aminofluorene, 4-aminobiphenyl, and 4-naphthylamine), which are strongly mutagenic and carcinogenic, and acetylates some drugs, affecting their metabolism. A biological microchip was developed to detect 16 point mutations, which determine 36 alleles and 660 genotypes of NAT2. The genotypes can be divided into four groups according to the acetylator phenotype: groups with rapid (R/R), intermediate (R/S), or slow (S/S) acetylation and a group combining intermediate and slow alleles (“R/S or S/S”). The last group includes the alleles determined by combinations of seven mutations (191G/A, 282C/T, 341T/C, 481C/T, 590G/A, 803A/G, and 857G/A), whose cis or trans position is detectable by restriction enzyme analysis. The NAT2 genotype was unequivocally established for 37 out of 71 DNA specimens, while the other 34 specimens were characterized by more than two genotypes. By the acetylator phenotype, 16 out of the 34 genotypes were assigned to the group “R/S or S/S,” combining mutations 282C/T, 341T/C, 481C/T, 590G/A, and 803A/G. Thus, the biochip allows primary analysis of most NAT2 polymorphic substitutions, the acetylator genotype being important to know in predictive medicine and individualized therapy.     

Identification of genetically modified vegetable sources in food and feed using hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by the hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in feed and food products. The biochip contained 22 immobilized probes intended for (i) detection of plant DNA; (ii) plant species determination (soybean, maize, potato, rice); (iii) identification of transgenic elements, including 35S CaMV, 35S FMV, rice actine gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, nptII marker genes. The limit of detection was 0.5% of genetically modified (GM) soybean and maize in analyzed samples. Identification of transgenic DNA in food and feed products using either the developed approach or real-time PCR led to virtually identical results. The assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of the GM component based on the identified transgene.     

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na⁺] and [K⁺] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na⁺‐selective class I‐HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single‐nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near‐isogenic lines (NILs): 157‐14 (double homozygote for the cheesmaniae alleles) and 157‐17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157‐14 in comparison with 157‐17. A significant difference between NILs was also found for [K⁺] and the [Na⁺]/[K⁺] ratio in leaf and stem but not for [Na⁺] arising a disagreement with the corresponding RIL population. Their association with leaf [Na⁺] and salt tolerance in tomato is also discussed.     

Identification of plant-derived genetically modified organisms in food and feed using a hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in food and feed products. The biochip contained 22 immobilized oligonucleotide probes that were intended for (1) detection of plant DNA, (2) determination of plant species (soybean, maize, potato, and rice), and (3) identification of transgenic elements, including sequences of 35S CaMV, 35S FMV, rice actin gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, and nptII marker genes. The limit of detection was 0.5% for genetically modified (GM) soybean and maize in the analyzed samples. The tests on food and feed products using the developed approach and real-time PCR showed full agreement in determination of transgenic DNA in the samples. The proposed assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of GM component based on the identified transgene.