G. M. Dymshits

Cytoplasmic male sterility and restoration of pollen fertility in higher plants

The review deals with cytoplasmic male sterility (CMS) in higher plants: impairment of the pollen formation resulting from interaction of the nuclear and mitochondrial genomes. The information on the known nuclear restorer-of-fertility genes and their effects on the expression of CMS-associated mitochondrial loci are considered. Heteroplasmy of mtDNA in plants and its potential association with CMS inheritance, as well as possible mechanisms of the observed direct and reverse association between altered expression of the CMS-inducing mitochondrial genome, metabolic defects, and pollen sterility are discussed.

Polymorphism of the Aggrecan Gene in Families with Idiopathic Scoliosis

493 Idiopathic scoliosis (IS) is a lateral deformity of the spinal column, which manifests itself and grows progressively worse during the growth of a patient. The deformity stages III and IV result in a disability for children and adolescents. Although IS is classed as a hereditary disease (OMIM 181,800), its genetic nature still remains unknown. In 1999 we showed the essential role of one of the genes controlling IS stages II–IV [1]. This allowed us to begin a search for this gene.

Detection of Single-Base Substitutions in Amplified Fragments via Ligation of a Tandem of Short Oligonucleotides in Solution and on a Solid Carrier

Ligation of a tandem of short oligonucleotides was proposed for detecting single-base substitutions in amplified DNA fragments. An octamer–tetramer–octamer tandem was ligated on a 20-mer template with T4 DNA ligase. As shown with radiolabeled oligonucleotides, the efficiency and selectivity of ligation did not change with an octamer linked to a water-soluble carrier based on polyethylene glycol (MPEG), while ligation was somewhat lower with the octamer immobilized on methacrylate beads (DMEG). In both cases, polymer attachment improved the discrimination of 20-mer templates with single-base substitutions in the binding site for the tetramer or for the immobilized octamer. Tandems with a radiolabeled or biotinylated component were also efficiently ligated on amplified DNA fragments. The data obtained with DNA fragments of HIV-1 strains bru and rf demonstrate the possibility of reliable detection of single-base substitutions via ligation of a tandem and colorimetric detection of the immobilized ligation product with the streptavidin–alkaline phosphatase technique.

Short Oligonucleotide Tandem Ligation Assay for Genotyping of Single-Nucleotide Polymorphisms in Y Chromosome

We propose a novel universal methodology, Short Oligonucleotide Tandem Ligation Assay (SOTLA), for SNP genotyping. SOTLA is based on using a tandem of short oligonucleotide (TSO) probes consisting of three fragments: the core oligonucleotide and two flanking oligomers, one of which is immobilized onto a solid support and another one contains the biotin label. TSO is self-associated on a complementary DNA template, forms the complex containing two nicks, which are efficiently ligated with DNA ligase giving biotinylated oligonucleotide covalently bound to polymer beads. No ligation of TSO on an imperfect DNA template bearing the base substitution in the core binding site is occurred. We used SOTLA for the highly selective SNP analysis in different DNA fragments of human Y chromosome. Comparison of SOTLA results with those of PCR-RFLP and allele-specific PCR techniques demonstrates that SOTLA ensures the univocal reliable SNP analysis in different PCR fragments varying in length and base composition. The fundamental difference between SOTLA and well known OLA approaches while using T4 DNA ligase is that the accuracy of SNP analysis in OLA is ensured only by the specificity of ligase while that in SOTLA is provided by the specificity of both ligation and hybridization of TSO probes.

Renin System of the Kidney in ISIAH Rats with Inherited Stress-Induced Arterial Hypertension

The renal renin system was studied in ISIAH rats with inherited stress-induced arterial hypertension. The expression of genes for renin (Ren1) and cyclooxygenase (Cox-2) was evaluated in renal tissue of ISIAH and WAG rats (normotensive control). Basal gene expression for Ren1 and Cox-2 in ISIAH rats was much lower than in WAG rats. Water deprivation for 11 h was followed by a 4-fold increase in Cox-2 gene expression in ISIAH rats. The increase in gene expression was insignificant in WAG rats (by 30%). Renin gene expression in renal tissue of ISIAH and WAG rats remained practically unchanged after water deprivation. We conclude that a change in Cox-2 gene expression after short-term water deprivation serves as a reliable criterion for functional strain of the renal renin system in hypertensive ISIAH rats.

Sequencing of the 5′ region of the Lngfr gene associated with stress-induced arterial hypertension in ISIAH rats

163 Inbred animal strains are broadly used in molecular studies of essential arterial hypertension (HTN). Different strains can be regarded as models of different HTN forms, whose development involves various systems and mechanisms of arterial blood pressure regulation. A strain of rats with inherited stressinduced arterial hypertension (ISIAH strain) has been developed at the Institute of Cytology and Genetics for years [1, 2]. The basal blood pressure in ISIAH rats exceeds the conventional standard value, but HTN manifests itself in full measure under slight emotional stress.

Cytoplasmic Male Sterility-Associated Structural Variation of the Mitochondrial Genome Regions Containing rps3 and orf215 in Sugar Beet Beta vulgaris L.

Several 5′-degenerate primers were selected by computer analysis and used for mtDNA typing in sugar beet cultivars with cytoplasms of the S (typical for cytoplasmic male sterility) or N (normal) type. A number of N- or S-specific markers were found to correspond to transcribed mitochondrial genes. One was from the orf215 region of the N-type mtDNA. A physical map of the corresponding region was constructed for the S-type mtDNA, and a substantial difference observed for the two genome types. One N-specific marker proved to contain a rearranged rps3 region and a truncated atp9 copy. With the known nucleotide sequence of this marker, three-primer PCR was designed and showed that both variants of the rps3 region simultaneously take place in the mtDNA pool, the new one occurring in a substochiometric proportion.

Expression of the renin angiotensin system genes in the kidney and heart of ISIAH hypertensive rats

The content of mRNA of renin-angiotensin system (RAS) genes was measured in the kidney and heart of hypertensive ISIAH and normotensive WAG rats using the real-time PCR. A statistically significant decrease in the mRNA level of RAS genes was registered in the kidney of ISIAH rats, including Ren (by 45%), Ace (43%), AT1A (34%), COX-2 (50%). The level of myocardial expression of AT1A decreased by 28% while Ace expression increased by 80%. These results suggest reduction of renal RAS basal activity in the hypertensive ISIAH rats, and therefore this strain of rats may be referred to the group of models of low-renin hypertension. The ISIAH rats were also characterized by a two-fold increase in the connective tissue sodium concentration and also by a small (but statistically significant) increase in plasma sodium concentration (139 ± 0.3 mmol/l versus 136 ± 0.25 mmol/l in WAG rats). These results together with a tendency to a decrease of plasma aldosterone level also support existence of a classical low-renin hypertension in the ISIAH rats. It is suggested that altered function of renal ion channels represents a basis for the development of low renin hypertension in the ISIAH rats. In addition, impairments in renal system of NO synthesis may also contribute to the pathogenesis of arterial hypertension in the ISIAH rats.

[The mitochondrial genome of higher plants: gene expression regulation].

The multistep regulation of mitochondrial gene expression in higher plants is considered. Data are summarized that concern the structure and function of the transcription system, posttranscriptional changes in the mRNA structure, and other levels of expression regulation.

Expression of Renin–Angiotensin System Genes in Brain Structures of ISIAH Rats with Stress-Induced Arterial Hypertension

We studied the expression of genes encoding angiotensinogen (Agt), renin (Ren), angiotensinconverting enzyme (ACE), and type 1 angiotensin receptor (AT1A) in the hypothalamus and medulla oblongata of hypertensive ISIAH rats and normotensive WAG rats. The amount of Agt mRNA in the hypothalamus of young ISIAH rats was increased by 30% compared to WAG controls. In the medulla oblongata of young ISIAH rats, the levels of mRNA of Agt and AT1A receptor were enhanced by 60% and 24%, respectively, compared to WAG rats. In adult animals, the expression of the studied genes did not differ from the control. It is concluded that changes in gene expression of the renin—angiotensin system in brain structures of ISIAH rats may contribute to the development of hypertension.