K. V. Popov

9-Aminoacridine: An efficient reagent to improve human and plant chromosome banding patterns and to standardize chromosome image analysis

Successful automated chromosome analysis requires the development of new techniques to increase and standardize chromosome length and improve banding patterns.

Genome Comparisons with Chromosomal and Molecular Markers for Three Closely Related Flax Species and Their Hybrids

Chromosome C-banding patterns were analyzed in three closely related flax species (Linum usitatissimumL., 2n = 30; L. angustifolium Huds., 2n = 30; and L. bienneMill., 2n = 30) and their hybrids. In each case, the karyotype included metacentrics, submetacentrics, and one or two satellite chromosomes. Chromosomes of the three flax species were similar in morphology, size (1–3 μm), and C-banding pattern and slightly differed in size of heterochromatic regions. In all accessions, a large major site of ribosomal genes was revealed by hybridization in the pericentric region of a large metacentric. A minor 45S rDNA site was observed on a small chromosome in L. usitatissimum and L. bienne and on a medium-sized chromosome in L. angustifolium. Upon silver staining, a nucleolus-organizing region (NOR) was detected on a large chromosome in all species. InL. angustifolium, an Ag-NOR band was sometimes seen on a medium-sized chromosome. In the karyotypes of interspecific hybrids, silver-stained rDNA loci were observed on satellite chromosomes of both parental species. RAPD analysis with 22 primers revealed a high similarity of the three species. The greatest difference was observed between L. angustifolium and the other two species. The RAPD patterns of L. bienne and L. usitatissimum differed in fewer fragments. A dendrogram of genetic similarity was constructed for the three flax species on the basis of their RAPD patterns. Genome analysis with chromosome and molecular markers showed thatL. bienne must be considered as a subspecies of L. usitatissimum rather than a separate species. The three species were assumed to originate from a common ancestor, L. angustifoliumbeing closest to it.

Antisense CD11b integrin inhibits the development of a differentiated monocyte/macrophage phenotype in human leukemia cells

Macrophage-like development of myeloid leukemia cells which can be induced by agents such as phorbol esters (TPA) is accompanied by integrin expression and cell adhesion. Thus, in differentiating myeloid leukemia cells CD11b is predominantly expressed which can associate with CD18 to form the functional heterodimeric integrin Mac-1. To elucidate the role of cell adhesion during macrophage-like differentiation, we transfected human U937 myeloid leukemia cells with a vector containing the CD11b gene in antisense orientation. Expression of the CD11b antisense gene in stably transfected U937 cells (as-CD11b cells) resulted in an attenuated response to TPA. As-CD11b cells demonstrated poor adhesion to solid substrate upon TPA treatment in contrast to U937 control cells. Constitutive expression of c-myc in as-CD11b transfectants was higher than in control cells and failed to be repressed by TPA treatment. Moreover, unlike control cells, antisense transfectants failed to induce expression of early response genes such as c-jun and the redox factor ref-1 upon TPA stimulation. Consequently, the induction of monocytic differentiation markers such as the activity of alpha-naphthyl acetate esterase, the capacity to reduce nitroblue tetrazolium and the expression of the vimentin gene was much lower in antisense transfectants than in control U937 cells. According to the failure to undergo a monocytic differentiation program, TPA treatment of as-CD11b cells resulted in a progressively increasing amount of apoptotic cells whereas the differentiated population of U937 control cells remained alive. Taken together, these data suggest that the integrin-mediated (particularly CD11b-mediated) adhesion of myeloid leukemia cells in the course of induced monocytic differentiation is crucial for cell attachment, development of a monocytic phenotype and subsequent survival.

Chromosome Localization of 5S and 45S Ribosomal DNA in the Genomes of Linum L. Species of the Section Linum (Syn. Protolinum and Adenolinum)

Fluorescence in situ hybridization (FISH) was for the first time used to study the chromosomal location of the 45S (18S–5.8S–26S) and 5S ribosomal genes in the genomes of five flax species of the section Linum (syn. Protolinum and Adenolinum). In L. usitatissimum L. (2n = 30), L. angustifolium Huds. (2n = 30), and L. bienne Mill. (2n = 30), a major hybridization site of 45S rDNA was observed in the pericentric region of a large metacentric chromosome. A polymorphic minor locus of 45S rDNA was found on one of the small chromosomes. Sites of 5S rDNA were colocalized with those of 45S rDNA, but direct correlation between signal intensities from the 45S and 5S rDNA sites was observed only in some cases. Other 5S rDNA sites mapped to two chromosomes in these flax species. In L. grandiflorum Desf. (2n = 16) and L. austriacum L. (2n = 18), large regions of 45S and 5S rDNA were similarly located on a pair of homologous satellite-bearing chromosomes. An additional large polymorphic site of 45S and 5S rDNA was found in the proximal region of one arm of a small chromosome in the L. usitatissimum, L. angustifolium, and L. bienne karyotypes. The other arm of this chromosome contained a large 5S rDNA cluster. A similar location of the ribosomal genes in the pericentric region of the pair of satellite-bearing metacentrics confirmed the close relationships of the species examined. The difference in chromosomal location of the ribosomal genes between flax species with 2n = 30 and those with 2n = 16 or 18 testified to their assignment to different sections. The use of ribosomal genes as chromosome markers was assumed to be of importance for comparative genomic studies in cultivated flax, a valuable crop species of Russia, and in its wild relatives.

Comparative Genome Analysis in Two Flax Species by C-Banding Patterns

C-banding patterns of the karyotypes of two closely related wild flax species, Linum austriacumL. (2n= 18) and Linum grandiflorumDesf. (2n= 16), were studied. The karyotypes of both species were similar in the chromosome morphology and size. In each species, metacentric and acrocentric chromosomes (1.7–4.3 μm) and one satellite chromosome were observed. In the karyotypes of the species studied, all homologous chromosome pairs were identified, and quantitative idiograms were constructed. Eight chromosome pairs in the two species had similar C-banding patterns. A low level of intraspecific polymorphism in the intercalary and telomeric C-bands was shown in both species. The results indicate that the genomes of two flax species originated from one ancestral genome with the basic chromosome number of 8 or 9. Apparently, the duplication or loss of one chromosome with subsequent redistribution of the chromosome material in the ancestral form resulted in the divergence into two species,L. austriacumL. and L. grandiflorumDesf. A considerable similarity of chromosomes in these species provides evidence for their close phylogenetic relatedness, which makes it possible to place them in one section within the Linumgenus.

Peculiarity of the Analysis of Heterochromatic Regions in Small Chromosomes of Plants

Visual analysis of small chromosomes of plants is difficult in most cases, because the chromosomes are similar in morphology and banding patterns. The computer software applied to chromosome analysis extended the capabilities of karyotypic analysis of small-chromosome species, because precise mathematical parameters of chromosomes and chromosome bands became available [1‐4]. This approach made it possible to completely identify chromosomes of some small-chromosome species and compare their karyotypes with the use of C-banding, which is commonly accepted in plant chromosome analysis [3‐7]. However, standard C-banding of chromosome preparations is often ineffective, and the banding pattern does not enable chromosome identification and measurements. To elongate small chromosomes, DNA intercalators are usually used [5, 6, 8], which enhances the resolution of C-banding patterns, and, hence, the possibility of chromosome identification. However, variation in C-banding patterns increases as the chromosome lengths increase after treatment with DNA intercalators. At the same time, there are no exact principles determining the range of chromosome length enabling stable C-banding and accurate morphometry to ensure not only chromosome identification and construction of idiograms, but also the possibility of comparative cytogenetic analysis. In this study, we aimed to develop these criteria. We demonstrated that, in principle, genomic polymorphism of the small-chromosome plant species can be studied by means of morphometric chromosome analysis, and the basic rules of the latter were formulated. We studied changes in the sizes of heterochromatic regions in both large and small mitotic chromosomes during their shortening. Chromosome selection for further measurements was based on the following criteria: the average size in the karyotype, the presence of intercalary heterochromatic bands, the absence of the secondary constriction, and the simplest identification. We finally selected chromosomes of the fourth groups in the karyotypes of the Djau-Kabutac cultivar of barley ( Hordeum vulgare L.) [9] and large-flower flax ( Linum grandiflorum Desf.) [6]. We examined all chromosomes identifiable from C-banding patterns that did not overlap in the same preparation meristem from a single plant to exclude the possibility of individual C-banding polymorphism. Barley chromosome preparations were obtained using the standard technique described previously [9]. To obtain flax preparations, the intercalator 9-aminoacridine was used [6]. On every barley or flax metaphase plate, one of a pair of homologous chromosomes was analyzed morphometrically. Numerous preliminary measurements (at least 30) of the same barley or flax chromosome were made to determine the limits of error for the morphometry. Similar results were obtained with both types of chromosomes (0.056 μ m).

Localization of Human Ribosomal Gene DNA Probes on Barley Chromosomes

To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S–28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barleyHordeum vulgareL. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 70% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.

Assessment of the Extent of the Necessary Clinical Testing of New Biotechnological Products Based on the Analysis of Scientific Publications and Clinical Trials Reports

To estimate patients risks and make clinical decisions, evidence based medicine (EBM) relies upon the results of reproducible trials and experiments supported by accurate mathematical methods. Experimental and clinical evidence is crucial, but laboratory testing and especially clinical trials are expensive and time-consuming. On the other hand, a new medical product to be evaluated may be similar to one or many already tested. Results of the studies hitherto performed with similar products may be a useful tool to determine the extent of further pre-clinical and clinical testing. This paper suggests a workflow design aimed to support such an approach including methods for information collection, assessment of research reliability, extraction of structured information about trials and meta-analysis. Additionally, the paper contains a discussion of the issues emering during development of an integrated software system that implements the proposed workflow.