Nucleosome positioning pattern derived from oligonucleotide compositions of genomic sequences.

Availability of nucleosome positioning pattern(s) is crucial for chromatin studies. The matrix form of the pattern has been recently derived (I. Gabdank, D. Barash, E. N. Trifonov. J Biomol Struct Dyn 26, 403-412 (2009), and E. N. Trifonov. J Biomol Struct Dyn 27, 741-746 (2010)). In its simplified linear form it is described by the motif CGRAAATTTYCG. Oligonucleotide components of the motif (say, triplets GRA, RAA, AAA, etc.) would be expected to appear in eukaryotic sequences more frequently. In this work we attempted the reconstruction of the bendability patterns for 13 genomes by a novel approach-extension of highest frequency trinucleotides. The consensus of the patterns reconstructed on the basis of trinucleotide frequencies in 13 eukaryotic genomes is derived: CRAAAATTTTYG. It conforms to the earlier established sequence motif. The reconstruction, thus, attests to the universality of the nucleosome DNA bendability pattern.

The mode of reproduction in natural populations of ascomycetous fungus, Emericella nidulans, from Israel.

The mode of reproduction of the soil ascomycetous fungus Emericella nidulans of Israeli populations was studied using 15 microsatellite (simple sequence repeats or SSR) trinucleotide markers. The study was performed in three canyons: two located in the northern part of Israel (Mount Carmel and western Upper Galilee) and one in the southern Negev desert. In each canyon, E. nidulans strains were isolated from the opposite slopes and (in the desert canyon) the valley bottom. Testing the reproductive structure of the populations indicated the presence of sexuality in the northern population and predominant clonality in the desert population. The predominantly clonal character of the desert population of E. nidulans was explained by the assumption that for relevant multilocus systems of a fungus, only several haplotypes can survive in the rather constant, extremely stressful desert conditions. Additionally, the very low density of E. nidulans populations in the soil of the desert canyon, which reduces the probability of finding a sexual partner, might favour predominant clonality via selfing. Increasing sexuality in E. nidulans populations on the north-facing slopes of the northern canyons may be a result of biotic stress (pressure of competitive fungal species), due to the more mild ecological conditions in these canyons.

Fractioned DNA pooling: a new cost-effective strategy for fine mapping of quantitative trait loci.

Selective DNA pooling (SDP) is a cost-effective means for an initial scan for linkage between marker and quantitative trait loci (QTL) in suitable populations. The method is based on scoring marker allele frequencies in DNA pools from the tails of the population trait distribution. Various analytical approaches have been proposed for QTL detection using data on multiple families with SDP analysis. This article presents a new experimental procedure, fractioned-pool design (FPD), aimed to increase the reliability of SDP mapping results, by "fractioning" the tails of the population distribution into independent subpools. FPD is a conceptual and structural modification of SDP that allows for the first time the use of permutation tests for QTL detection rather than relying on presumed asymptotic distributions of the test statistics. For situations of family and cross mapping design we propose a spectrum of new tools for QTL mapping in FPD that were previously possible only with individual genotyping. These include: joint analysis of multiple families and multiple markers across a chromosome, even when the marker loci are only partly shared among families; detection of families segregating (heterozygous) for the QTL; estimation of confidence intervals for the QTL position; and analysis of multiple-linked QTL. These new advantages are of special importance for pooling analysis with SNP chips. Combining SNP microarray analysis with DNA pooling can dramatically reduce the cost of screening large numbers of SNPs on large samples, making chip technology readily applicable for genomewide association mapping in humans and farm animals. This extension, however, will require additional, nontrivial, development of FPD analytical tools.

Molecular-genetic biodiversity in a natural population of the yeast Saccharomyces cerevisiae from "Evolution Canyon": microsatellite polymorphism, ploidy and controversial sexual status.

The yeast S. cerevisiae is a central model organism in eukaryotic cell studies and a major component in many food and biotechnological industrial processes. However, the wide knowledge regarding genetics and molecular biology of S. cerevisiae is based on an extremely narrow range of strains. Studies of natural populations of S. cerevisiae, not associated with human activities or industrial fermentation environments, are very few. We isolated a panel of S. cerevisiae strains from a natural microsite, "Evolution Canyon" at Mount Carmel, Israel, and studied their genomic biodiversity. Analysis of 19 microsatellite loci revealed high allelic diversity and variation in ploidy level across the panel, from diploids to tetraploids, confirmed by flow cytometry. No significant differences were found in the level of microsatellite variation between strains derived from the major localities or microniches, whereas strains of different ploidy showed low similarity in allele content. Maximum genetic diversity was observed among diploids and minimum among triploids. Phylogenetic analysis revealed clonal, rather than sexual, structure of the triploid and tetraploid subpopulations. Viability tests in tetrad analysis also suggest that clonal reproduction may predominate in the polyploid subpopulations.

[The nature of the phosphorylation of retinoblastoma gene product in stable mouse and human cell lines]. / Kharakter fosforilirovaniia produkta gena retinoblastomy v stabil'nykh liniiakh kletok myshi i cheloveka.

The retinoblastoma gene product mediates the interaction between transcriptional factors and cyclin-kinase complexes, which perform a regulatory and effector function in the process of cell division. The activity of the retinoblastoma gene product is regulated by phosphorylation, which results in the appearance of additional protein molecules migrating in the region of 105--116 kDa during electrophoresis. Stereochemical analysis has established a direct correspondence between the extent of phosphorylation of the retinoblastoma gene product and its electrophoretic mobility. The results obtained permit an estimate of the phosphorylation of this protein in cells of different tissues by immunoblotting analysis of their lysates. The results of this study demonstrate that the degree of phosphorylation of retinoblastoma gene product in a series of stable cell lines increases as we go from monolayer to multilayer cultures, and further to cell cultures in suspension. Human cells produce more phosphorylated proteins compared to homologous mouse cells. The phosphorylation pattern of retinoblastoma gene product is probably tissue-specific. Much like mouse fibroblasts, HeLa cells may contain a hypophosphorylated protein with a molecular weight of 105 kDa. Phosphorylation of the retinoblastoma gene product in cells of embryonic mouse adenocarcinoma (line p19) changes in response to cloning or stimulation of differentiation by retinoic acid. The formation of all forms of retinoblastoma gene product, which pre-exist during asynchronous growth, is increased in the course of cloning. Differentiation is associated with the synthesis of an increased amount of hypophosphorylated protein. The results obtained lead to the hypothesis that even though the phosphorylation pattern of the retinoblastoma gene product is tissue-specific, it can show significant variation under the conditions of either cloning or differentiation and can be maintained for extended period of time at a new level that was not characteristic of the initial cell population.

[Growth of stable clones of mouse fibroblast cell line C3H10T1/2 expressing the human retinoblastoma gene product]. / Izuchenie rosta stabil'nykh klonov myshinykh fibroblastov linii C3H10T1/2, ékspressiruiushchikh produkt gena retinoblastomy cheloveka]

Phosphorylation and dephosphorylation of the retinoblastoma gene product (pRB) are recognized as necessary events in the cell cycle progression. To study the role of pRB in regulation of cell proliferation, the stable cell lines with constitutive expression of the exogenous RB gene can be employed. In order to obtain such cell lines in this work C3H10T1/2 mouse fibroblasts were infected with defective retrovirus encompassing the RB and Neo gene conferring resistance to geniticine (G418). The pRB production and its phosphorylation pattern were analyzed by immunoblotting in cell lysates considering well known data on correlation between pRB phosphorylation pattern and its electrophoretic mobility. Cell lines subjected to G418 selection with the following cloning procedure were identical to the control cells expressing beta-galactosidase, when compared for pRB production and phosphorylation in the cell cycle stages characterized by hyperphosphorylated pRB. However, cells of the experimental cell lines hypophosphorylated pRB much faster and accumulated much more underphosphorylated protein compared to the control cell lines. The doubling time of the cells was not affected either by changes in the pRB phosphorylation pattern or by its overproduction during separate cell cycle stages. These results suggest that maintaining of the physiological level of pRB phosphorylation in cycling cells is strictly controlled and is considered to be a more important condition of the cell cycle progression than pRB dephosphorylation.

Patterns of urinary 17-ketosteroid fractions in hirsutism.

Fractionated 17-ketosteroids were determined in 60 females with hirsutism. The chromatographic method was adapted in the Oncological Institute of Cluj-Napoca. In 32 patients the idiopathic form of hirsutism was confirmed with normal results for all the fractions and only sometimes a slight rise of androsterone. In 18 patients an ovarian origin was affirmed; all of them had androsterone and etiocholanolone over normal limits. In ten patients, who had an adrenal origin of hirsutism the great values of DHEA (dehydroepiandrosterone) were observed. In one patient with virilizing adrenal tumour, both total 17-ketosteroids, and all fractions were much elevated DHEA being over 100 mg/24 hours.

Some aspects of the endocrine status in the inflammatory breast carcinoma (Mastitis carcinosa).

The main endocrine features in a group of 87 patients with inflammatory breast carcinoma with very rapid evolution (group I) and those of 400 patients with breast cancer with no inflammatory signs (group II) have been compared. The mean age at menarche showed a significantly higher degree in group I, where the later menarche was reported in 36.8% of cases (greater than or less than 16 years). The mean number of abortions has been decreased in the same groups. No significant differences regarding to parity, lactation and mean age of the menopause have been found in these two groups. The mean values for total urinary estrogens were significantly higher in group I. The main fractions of 17-ketosteroids were estimated in 60 women of group I and 61 women of group II. The mean levels of these hormones were more decreased in group I, the most significantly differences in these two groups were registered for ethiocholanolone. In the most cases with decreased levels of ethiocholanolone, the survival time was also shorter. Such data showed a more prominent endocrine imbalance in the inflammatory breast carcinoma in comparison with breast cancer patients of normal evolution.

Urinary 17-ketosteroid fractions in young patients with breast cancer.

The main fractions of 17-ketosteroids were estimated in 30 young women (under 40 years) with breast cancer and 20 controls of the same age. The determination of the fractionated ketosteroids was made by thin layer chromatography on silicagel. The mean levels of etiocholanolone, androsterone, total ketosteroids and the sum of the fractions of 17-ketosteroids were significantly decreased in patients with breast cancer in comparison with the controls. No significant differences between the mean levels of dehydroepiandrosterone and 11-oxygenated-17-ketosteroids were noted in these groups.

Levels of principal 17-ketosteroid fractions in urine of breast cancer patients with or without malignant evolutive push.

The main fractions of 17-ketosteroids were estimated in 102 women with breast cancer. Sixty two patients were cases with rapid evolution (malignant evolutive push) or very rapid evolution (inflammatory breast carcinoma). The mean ethiocholanolone levels and partially androsterone levels were significantly decreased in patients with rapid evolution in comparison with the corresponding values found in patients with breast cancer of slow growth.

Clinical and hormonal aspects in breast cancer with special reference to malignant evolutive push.

It is tried to differentiate between various stages of malignant evolutive push and normal mammary tumors to establish an individual therapeutic procedure. Besides mammography and thermometry, investigations of urinary excretions of estrogenic and androgenic steriods are qualified for this purpose. Patients with malignant evolutive push showed an increased excretion of estrogens which reached its highest levels in patients suffering from inflammatory mammary carcinomas. The excretion of 17-ketosteroids shows a behaviour vice versa. Altogether mammary tumors with malignant evolutive push are characterized by a state of absolute and relative hyperestrogenity respectively. This phenomenon preferably depends on the clinical stage of the disease than on the age of the patients.

[Hormonal patterns in breast cancers]. / Hormonale Aspekte beim Mammakarzinom

Urinary outpout of oestrogens and 17-ketosteroids were estimated in 200 women with breast cancer; 60 of them were cases with rapid evolution (malignant evolutive push). The majority of cases analysed had abnormal hormonal levels, especially of oestrogens. The average oestrogenic levels are higher than the normal values; the highest values correspond to the most severe and rapidly evolutive type of breast cancer, the inflammatory breast carcinoma. In the patients without evolutive push, the proportion of cases with absolute or relative hypooestrogeny is approximately equal toor exceeds the proportion of hyperoestrogenic cases. In the presence of the evolutive push and especially of his most severe type, inflammatory carcinoma, the cases with hyperoestrogeny, both absolute and relative, by hypoandrogeny, are dominant. The proportion of cases with higher 17-ketosteroid urinary outputs than the normal mean is greater in the patients without evolutive push, while in those with evolutive push, the majority of cases have lower values of 17-ketosteroids than the normal mean.