Gene-targeted and site-directed mutagenesis. Reverse genetics methods (loss of function)Using transgenic plant with insertion/deletion or site-specific mutations.Host gene is replaced with mutant allele.The most conventional approach to the analysis of gene function is loss-of-function mutagenesis by chemicals or fast neutrons that introduce random mutations or deletions in the genome (Ostergaard and Yanofsky 2004).www.intechopen.com

BACKGROUND:Psoriasis is complex inflammatory skin pathology of autoimmune origin. Several cell types are perturbed in this pathology, and underlying signaling events are complex and still poorly understood.RESULTS:In order to gain insight into molecular machinery underlying the disease, we conducted a comprehensive meta-analysis of proteomics and transcriptomics of psoriatic lesions from independent studies. Network-based analysis revealed similarities in regulation at both proteomics and transcriptomics level. We identified a group of transcription factors responsible for overexpression of psoriasis genes and a number of previously unknown signaling pathways that may play a role in this process. We also evaluated functional synergy between transcriptomics and proteomics results.CONCLUSIONS:We developed network-based methodology for integrative analysis of high throughput data sets of different types. Investigation of proteomics and transcriptomics data sets on psoriasis revealed versatility in regulatory machinery underlying pathology and showed complementarities between two levels of cellular organization.

Psoriatic and atherosclerotic plagues were examined using the real-time polymerase chain reaction. Expression of the FOSL1 gene proved to substantially increase in both psoriatic lesions of the skin and atherosclerotic lesions of vessels as compared with nonlesion samples.

Методом полимеразной цепной реакции в реальном времени проанализированы псориатические и атеросклеротические бляшки. Показано существенное повышение экспрессии гена FOSL1 как в пораженной псориазом коже, так и в пораженных атеросклерозом сосудах по сравнению с непораженными образцами.

Psoriasis was used as a model to analyze the pathogenetic pathways of immune-mediated inflammatory diseases, and the results of bioinformatic, molecular-genetic and proteomic studies are provided. Cell mechanisms, common for the pathogenesis of psoriasis, as well as Crohn’s disease, are identified. New approaches for immune-mediated diseases are discussed.

Проведен сравнительный биоинформатический анализ молекулярно-генетических процессов в патогенезе многофакторных заболеваний – псориаза и болезни Крона – на основании обработки данных биологических микрочипов базы данных GEO DataSets. Установлены общие гены и общие молекулярно-генетические процессы для двух патологий. Сделано предположение об участии ряда транскрипционных факторов, включая систему транскрипционных факторов АР-1, в патогенезе как псориаза, так и болезни Крона.

A comparative bioinformatics analysis of the molecular genetic processes in the pathogenesis of multifactorial diseases-psoriasis and Crohn's disease-was performed using the results of microarray experiments deposited with the GEO DataSets database. A number of genes and molecular genetic processes common to both pathologies were found. The involvement of several transcription factors, including AP-1 transcription factors, in the pathogeneses of both psoriasis and Crohn's disease was postulated.

A new strategy for creating experimental models for functional genomics has been proposed. It is based on the expression in transgenic plants of genes from thermophilic bacteria encoding functional analogues of plant proteins with high specific activity and thermal stability. We have validated this strategy by comparing physiological, biochemical and molecular properties of control tobacco plants and transgenic plants expressing genes of beta-glucanases with different substrate specificity. We demonstrate that the expression of bacterial beta-1,3-1,4-glucanase gene exerts no significant influence on tobacco plant metabolism, while the expression of bacterial beta-1,3-glucanase affects plant metabolism only at early stages of growth and development. By contrast, the expression of bacterial beta-1,4-glucanase has a significant effect on transgenic tobacco plant metabolism, namely, it affects plant morphology, the thickness of the primary cell wall, phytohormonal status, and the relative sugar content. We propose a hypothesis of beta-glucanase action as an important factor of genetic regulation of metabolic processes in plants.

Psoriasis is a multifactorial autoimmune skin disease widely spread in the Caucasoid population. Similar to other complex genetically determined pathologies, the success in treating psoriasis can be reached with the discovery of the molecular genetic mechanisms triggering the pathogenesis, including the search for the candidate target genes for drugs. This work, utilizing bioinformatics analysis of transcriptomics data, demonstrated that the genes for transcription factor AP-1 components can be regarded as psoriasis candidate genes.

Продукт гена NAT2 (N-ацетилтрансфераза 2) участвует в биотрансформации и детоксикации ряда ароматических аминов (в частности, 2-аминофлуорена, 4-аминобифенила и 4-нафтиламина), которые являются сильными мутагенами и канцерогенами, а также влияет на метаболизм лекарственных препаратов, ацетилируемых этим ферментом. Разработан биологический микрочип для обнаружения 16 точечных мутаций гена NAT2, сочетания которых определяют 36 аллелей и 660 генотипов гена NAT2. Эти генотипы можно разделить на четыре группы по фенотипу ацетилирования: фенотип “быстрого” (R/R), “промежуточного” (R/S), “медленного” (S/S) ацетилирования и группа, в которой могут быть аллели с фенотипом как промежуточного, так и медленного ацетилирования (R/S или S/S). Группа “R/S или S/S” включает аллели, определяемые сочетанием 7 мутаций (191G/A, 282C/T, 341T/C, 481C/T, 590G/A, 803A/G, 857G/A), цис-транс-положение которых можно установить с помощью рестрикционного анализа. Однозначно определен NAT2-генотип 37 из 71 образца ДНК, а 34 образца охарактеризованы более чем двумя генотипами. По фенотипу ацетилирования в группу “R/S или S/S” из этих 34 образцов ДНК вошли 16, содержащих шесть мутаций: 282C/T, 341T/C, 481C/T, 590G/A и 803A/G. Таким образом, данный биочип позволяет проводить первичный анализ большинства полиморфных замен в гене NAT2.

The product of gene NAT2 (N-acetyltransferase 2) is involved in the biotransformation system and participates in detoxication of some arylamine derivatives (in particular 2-aminofluorene, 4-aminobiphenyl and 4-naphthylamine) which are strongly mutagenic and carcinogenic. It also renders toxicological and pharmacological influence on a metabolism of medical products metabolized by the enzyme. We developed a microchip for detection of 16 functionally significant mutations coding 36 alleles of gene NAT2. Combinations of these alleles allow us to reveal more than 660 genotypes, which can be divided into four groups according acetylation phenotype: "fast" (R/R), "intermediate" (R/S), "slow" (S/S) and group with average or slow acetylating (R/S or S/S) alleles. The groups "R/S or S/S" include alleles, formed by a combination of 7 mutations (191G/A, 282C/T, 341T/C, 481C/T, 590G/A, 803A/G, 857G/A), theirs cis-trans position can be revealed by restriction analysis. In 37 of 71 DNA samples we unequivocally defined NAT2-genotypes, and other 34 samples have been characterized by more than two genotypes. 16 samples out of 34 had acetylation phenotype of group "R/S or S/S", which is characterized by the following combination of mutations: 282C/T, 341T/C, 481C/T, 590G/A and 803A/G. Thus, the developed biochip is a convenient screening method for primary detection of the majority of polymorphic replacements in gene NAT2.

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.

The review is dedicated to molecular-and-genetic aspects of psoriasis. The paper contains a general characterization of this skin disease and a range of hypotheses concerning its appearance, clinical manifestations, and maintenance. Its autoimmune nature with the central role played by T-cells is considered to be the basic theory of the appearance of psoriasis. According to the results of analysis of experimental data on psoriasis genetics, including family background, psoriasis is defined as a complex multigene multifactor disease, whose manifestations are determined by a combination of certain genes as well as by various environmental trigger factors. The authors consider ethnic features of psoriasis, and genes that determine predisposition to the disease. Prospects of the search of predisposition genes, including analysis of the polymorphism of unit nucleotides, are outlined in the article.

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.

The functional analysis of plant genes employs various comprehensive approaches that include transcriptome analysis using microchips, gene knockout, RNA interference, and various experimental models. We have proposed a novel experimental model approach for plant functional genomics. It is based on creating and studying transgenic plants that express bacterial genes which are functionally similar to plant genes. The validity of this approach owes to the similarity of basic pro- and eukaryotic metabolic pathways and gene networks controlling the functioning of these organisms under normal conditions and in response to abiotic and biotic stresses.Our studies using molecular biology, physiology and biochemistry methods have demonstrated adequateness of the proposed strategy. It allows the modeling of processes taking place in the plant cell and differential assessment of contribution of individual enzymes. Our results indicate that the proposed approach is highly effective for functional genomics, namely, for determining the function of a gene product in vivo. The experimental data can be further used as a basis for elaboration of gene networks controlling particular physiological processes and stress responses of plants to biotic and abiotic environmental factors.

Psoriasis, a multifactorial disease with genetic predisposition, has been used as an example to study the role of the ethnic background in multifactorial diseases in the Dagestan population. The individual information card (IIC) is proposed as the main tool for correct collection and processing of information. The results of the study demonstrate that the Dagestan population is a convenient and adequate model population for studying multifactorial diseases, such as psoriasis, and may serve as an object for studying the role of heredity in the etiologies and pathogeneses of this and other multifactorial diseases.

Expression of the bacterial gene for thermostable β-1,4-glucanase (cellulase) from Clostridium thermocellum in transgenic tobacco plants was shown to produce significant changes in tobacco plant structure and activities. The transgenic plants differed in their growth rate and morphology, and their hormonal status was affected. Thus, the transgenic plants expressing the gene for thermostable bacterial cellulase are a convenient model to study the role of β-1,4-glucanases in plant physiological processes.

The bacterial gene of the thermostable endo-β-1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased bushiness and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.