Structure, expression and mutations of DMD gene, the largest human gene

Summary: Duchenne and Becker muscular dystrophies are allelic forms of disease occurring with an incidence of 1 in 3500 ive male births. The disease is associated with mutation in DMD gene, mapped to Xp21. High rate of mutation involves deletions (60%) comprising even several exons. The remaining are duplications (6%) and point mutations (34%). Molecular-genetic diagnostics includes analysis of deletions of promotor and 19 exons, located in two „hot-spot� regions, by multiplex PCR. Detection of small changes (microdeletion, microinsertion, transition and transversion) is not included in routine analysis, but is necessary for complete diagnostics of the disease especially in cases where identification of carriers is required. New approaches are needed for fast and reliable diagnostics tests which make the analysis of molecular basis of disease possible. Identification of mutation is especially important for females in affected families, who are potential carriers of mutant allele and are able to pass it to their offspring. In case when determination of mutation is not possible, carriers still can be identified by indirect methods, but they inform about inheritance of genetic markers correlated with disease, but not of nature of mutation. 
 

Key Words: Duchenne/Becker muscular dystrophy, DMD gene, dystrophin, analysis of deletion, point mutation, polymorphism, carrying determination

APC gene

Summary: Familial adenomatous polyposis coli is dominantly inherited cancer disease characterized by numerous adenomatous polyps of the colon and rectum. The polyps progressing to malignancy in untreated carriers of mutant gene with a median age at diagnosis of 40 years. FAP is linked with mutations in localized on chromosome 5q21 APC gene. APC gene is a tumor suppressor gene and acts as a negative regulator of the Wnt signal transduction cascade. The mutations of APC gene are inherited, or de novo arise in parental germ line. The most of mutations of the gene are small mutation observed as deletion or insertion a few base pair. In rare cases we can observe substitutions. Most of the APC mutations truncate the 300 kD APC protein product. During lifetime mutation in second copy of APC gene cause absence of functional APC protein that lead to displasia and progression of cancer disease. 

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Structure and function of protein ATM

Summary: TProtein ATM was identified after the positional cloning search and discovery of the the ATM gene. It is a 350,6 kD glycoprotein displaying the activity of protein kinase. The substrates of this activity are: p53, cAbl, p34 and Chk1. Phosphorylation of p53 inhibits binding of this protein to the oncogene MDM2, preventing the degradation of p53. In cells exposed to ionizing radiation the ATM protein enhances p53 DNA binding with the resultant transactivation of the genes. The protein kinase module of ATM is found within the carboxyl end of the protein and displays homology to a family of phosphoinositol-3 kinases. In the central portion of the ATM protein the leu ZIP module is found, which shows homology to leuZIP modules in DNA-PK and ATR/FRB. The ATM protein plays a key role in cells response to ionizing radiation by sensing the double strand breaks of DNA as well as in the control function in relation to the cell cycle.

Słowa kluczowe: ATM protein, ATM gene, PI-3-kinase, p53, p53 phosphorylation, DNA-PK, MDM2, ionizing radiation.
 

Genetic basis of osteoporosis - current state of research

Summary: Current knowledge concerning searching for osteoporosis genes is described. Osteoporosis and subsequent fractures is growing major health problem. Genetic background is believed as the most important factor for development of the disease. However, finding of genes is difficult because of polygenic determination and environmental factors, which influence the phenotype. Understanding the genetic basis of osteoporosis will likely be the starting-point of effective prophylactic and more specific treatment.

Key Words: osteoporosis, polymorphisms, association analysis, polygenic disease

Application of hybridization analysis in molecular diagnostics

Summary: Molecular diagnostics was performed for the first time on the basis of hybridization of examined DNA with molecular probes. In eighties hybridization was the most commonly used procedure in diagnostics of inherited diseases. Hybridization found practical application in studies of genomes organization. With time many analyses based on hybridization have been replaced by PCR technique, which shortened time of analysis and reduced costs. Hybridization analyses are performed wihtout amplification of DNA in vitro and are used in studies related to humans, animals and plants. In this paper examples of practical application of hybridization analysis are presented.

Key Words: DNA, hybridization, DNA repeats, molecular diagnostics, pathogen detection, mutation, polymorphism.
 

The chromosome ends: telomeres, telomerase and telomere binding proteins

Summary: Telomeres were for many years of interest to cytologists, as static elements protecting chromosome ends. Discovery of their involvement in cellular aging and replication, cell cycle checkpoint and first of all in tumorigenesis makes the study of telomeres very popular to many areas of science.

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Molecular analysis of sex development abnormalities: the key to understand genetics mechanism of sex determination

Summary: Sex determination is the process responsible for testis or ovarian development from primary gonad. This process is genetically controlled. In this review the role of SRY, SOX9, WT1, SF1, DAX1 genes in sex determination is described and the current knowledge about interactions of proteins encoded by these genes is summarised. 

Key Words: sex-determination, genes: SRY, SOX9, WT1, SF1, DAX1
 
 

Molecular analysis of Y chromosome in men with defects of spermatogenesis undergoing in vitro fertilization  

Summary: In about 50% of infertile couples the male factor is involved. However, the majority of infertile males who visit centers specialized in treatment of infertility are otherwise healthy. In the last two decades more and more attention is paid to potential genetic background of infertility. Significant progress was made in this field since it was demonstrated that deletions of AZF region on the Y-chromosome cause infertility in some otherwise healthy males. The constantly growing knowledge on the structure of the Y-chromosome was essential for establishing molecular tests of screening of AZF deletions in patients who undergo assisted reproduction procedures, including intra-cytoplasmic sperm injection (ICSI). These methods offer the possibility to infertile males, even to those with severe spermatogenic failure, to conceive children. However, such fertilization process causes the risk of transmission of the genetic defect to the male offspring. Therefore, molecular analysis of the Y-chromosome and genetic counseling are crucial for treated couples.

Key Words: Y chromosome, male infertility, defects of spermatogenesis, oligospermia, azoospermia, in vitro fertilization
 

The role of telomerase in the development of cancer

Summary: Telomerase is a rybonucleoprotein complex, consist of reverse transcriptase, protein component and RNA template for the syntesis of telomeric DNA. Telomerase activity has been found in the majority of cancer, but absent in the most normal cells. Many studies have demonstrate that the presence of telomerase activity can be used to distinguish malignant from normal cells in various organs including prostate, breast, thyroid and liver. Critical review of current literature indicates that telomerase expression may be more accurate then telomerase activity in distinguishing benign from malignant tissue. Our own pilot study suggests that determination of telomerase activity and its expression might be a useful diagnostic and as well prognostic marker for cancer development.

Key Words: telomerase activity and expression, cancer, aging
 

Development of markers� genome map of dog and horse

Summary: Dog and horse breeding have been started long time ago � it was 14.000 years ago in case of the dog and 6.000 years for the horse. In a consequence a large variety of breeds were developed. At present over 340 dog breeds and approximately 250 horse breeds are world wide recognised. A negative side effect of the selection is an accumulation in gene pools, mainly in case of the dog, of mutations responsible for development of hereditary diseases. The main aim to establish marker genome maps for these species is their use for identification of genes causing the diseases. Density of the newest dog genome map, basing on segregation analysis of 268 genetic markers, is 9.3 cM. Moreover, on each chromosome at least one marker was physically assigned. Density of the horse map, including 100 markers, is 12.6 cM, but unfortunately until now there are chromosomes on which no one marker was localised. 

Key Words: marker genom maps, dog, horse, genetic markers, genome
 

The genetic analysis by classical and molecular cytogenetics

Summary: The change of the number or structure of chromosomes may to cause abnormal phenotypic features. The main aim of cytogenetic diagnosis is identification of karyotype of patient. The karyotype has been identified on analysis of metaphase and prometaphase chromosomes (HRBT-high resolution banding technique) of peripheral blood lymphocytes. The methods of classical (GTG, RBG, QFQ, CBG, BAT, Ag-NOR) and molecular cytogenetics (FISH, PRINS, CGH) are usually used in cytogenetics diagnosis.

Key Words: chromosomal aberrations, classical cytogenetics, molecular cytogenetics, fluorescence in situ hybridization, cytogenetic diagnosis.
 

Cytogenetic analysis of human neoplasms - squamous cell carcinoma of the larynx

Summary: Cytogenetic analysis of human neoplasms indicated common incidence of chromosomal aberrations. Complex data, mostly concerning neoplasms of haemopoietic and lymphatic systems, have been derived using "classic" cytogenetic methodology. Because of biological structure of solid tumors and problems in preparations and analysis of this kind of material, new techniques have been developed. Among the others, florescent in situ 
hybridization (FISH) and comparative genomic hybrydization (CGH) became the most important methods. The data described by our group concerned chromosomal aberrations in squamous cell carcinoma of the larynx. To determine copy number changes in analysed group comparative genomic hybrydization has been used. The most striking observations were: a remarkable incidence of deletions of genetic material in the group of larynx carcinomas with local lymph node metastases, and an occurrence of cytogenetic differences between primary tumors and their metastases.

Key Words: cytogenetic, chromosomal aberrations, larynx carcinomas, comparative genomic hybrydization (CGH)

Methodological aspects of molecular analysis in laryngeal cancer

Summary: The review presents techniques applied in molecular biology and genetic background of tobacco smoke-associated laryngeal cancer. The following molecular techniques are described: comet assay, a radiochemical method of carcinogen:DNA adduct identification, and methods used for an analysis of defects of tumour suppressor genes (mutations, loss of heterozygosity) and histochemical detection of markers of cell proliferation. Classical and molecular cytogenetics were applied to estimate chromosome instability, chromosome aberrations and imbalance in DNA copy number. PCR-based genotyping is presented as a useful technique to estimate a significance of genetic factor in relation to defects of detoxifying enzymes. The main message is a need of application of a broad spectrum of molecular and cellular techniques in order to understand the molecular basis of laryngeal cancer. Certain techniques seem to be promising in diagnostic attempts.

Key Words: laryngeal cancer, molecular methods, cytogenetics, genetic factor

Future of gene therapy in neurological disorders

Summary: In the last years gene therapy is the most rapidly developing discipline of medicine. Gene therapy may be defined as experimental treatment of various diseases by the gene transfer technology. Therapeutical genes carried by specific vectors can be introduced into targed cells by means of the ex vivo and in vivo approaches. Many promising results have been obtained in animal models of neurological disorders. Therefore gene therapy is most likely to be applied in the treatment of disorders including Parkinson's disease, Alzheimer's disease, Duchenne muscular dystrophy and multiple sclerosis. In a number of clinical trials using suicide genes transfer gancyclovir regression of brain tumours have been observed. It is a great hope that in the future gene transfer technology will be a common method of neurological disorders treatment and prevention. 

Key Words: gene therapy, gene transfer, neurologic disorders
 

Mammalian cloning: now and future

Summary: The techniques of cloning have been briefly characterized as well as recent advances in mammalian cloning, by using diploid nuclear transfer from somatic, differentiated cell to cytoplast. There are mentioned basic problems resulting from attempts to synchronize the cell cycle in newly reconstructed 'clone' originating from two different types of cells. It is described the cloning efficiency presently obtained for sheep, cow, mouse and Rhezus monkey and relationship between a success ratio and a type of donor cell (source of diploid nucleus). It is outlined a perspective for therapeutic cloning, based on recent advances in generation of omni- (toti) potential stem cells. Due to possible applications of cloned tissues (organs) in medicine and possibility of reproductive cloning in humans - new ethical considerations do emerge and have to be carefully discussed. 

Key Words: cloning, mammals, farm animals, tissue (organ) cloning.

Searching for genes responsible for personality traits in humans and instinctive traits in animals

Summary: The paper presents issues connected with the search for genes responsible for personality traits in humans and instinctive traits in animals. The research focused on selecting appropriate investigation methods. An attempt was made to clarify some problems arising in the process of mapping genes responsible for behaviour.

Key Words: personality traits, instinctive traits, strategies of mapping, methodolgy problems
 

Anther cultures as a method of the induction of rye haploids (Secale Cereale L.)

Summary: Haploids plants are often used in scientific and application research. Androgenesis (anther culture and isolation microspores) is one of the methods of obtaining them. The study presents the most important factors which determine the course of the process. Special attention should be paid to: genotype, physiological condition of donor plants, the stage of microspores development, the initial treatment of spikes and anthers, the medium composition and the culture condition. Inhibitors to the application of this method are: low percentage of regenerated plants and high participation individuals with chlorophile defects.

Key Words: rye, androgenesis, anther culture, haploids
 

Application of flow cytometry in plant molecular studies

Summary: In recent years flow cytometry played important role in understanding of plant genome structure and function. This paper reviews the applications of flow cytometry for the analysis of isolated nuclei and chromosomes. Speed, precision and efficiency of this method found an enormous number of applications which cover basic research, breeding and plant production. The results obtained with sorted chromosomes indicate that the technique might greatly simplify the analysis of plant genomes at the molecular level.

Key Words: chromosome, DNA, flow cytometry, chromosome sorting, molecular markers, FISH, PRINS, DNA library